AU783450B2 - Recombinant vaccine against botulinum neurotoxin - Google Patents

Description

WO 00/67700 PCT/US00/12890 Recombinant Vaccine Against Botulinum Neurotoxin This application is based on U.S. Provisional Application Nos. 60/133,866, 60/133,868, 60/133,869, 60/133,865, 60/133,873, 60/133,867, all filed May 12, 1999, and U.S. Provisional Application No. 60/146,192, filed July 29, 1999, all of which are incorporated herein in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention This invention is directed to preparation and expression of synthetic genes encoding polypeptides containing protective epitopes of botulinum neurotoxin (BoNT). The invention is also directed to methods of vaccination against botulism using the expressed peptides.

Related Art The sporulating, obligate anaerobic, gram-positive bacillus Clostridium produces eight forms of antigenically distinct exotoxins. Tetanus neurotoxin (TeNT) is produced by Clostridium tetani while Clostridium botulinum produces seven different neurotoxins which are differentiated serologically by specific neutralization. The botulinum neurotoxins (BoNT) have been designated as serotypes A, B, CI, D, E, F, and G. Botulinum neurotoxins (BoNT) are the most toxic substances known and are the causative agents of the disease botulism. BoNT exert their action by inhibiting the release of the neurotransmitter acetylcholine at the neuromuscular junction (Habermann, et al., (1986), "Clostridial Neurotoxins: Handling and Action at the Cellular and Molecular Level," Cur. Top. Microbiol.

Immunol., 129:93-179; Schiavo, et al., (1992a), "Tetanus and Botulinum-B Neurotoxins Block Neurotransmitter Release by Proteolytic Cleavage of Synaptobrevin," Nature, 359:832-835; Simpson, (1986), "Molecular Pharmacology of Botulinum Toxin and Tetanus Toxin," Annu. Rev. Pharmacol.

Toxicol., 26:427-453) which leads to a state of flaccid paralysis. Indeed, only a few molecules of toxin can abolish the action of a nerve cell. Polyclonal antibodies derived for a specific neurotoxin can neutralize the toxic effects of that toxin but will not cross-neutralize another toxin serotype. Thus, to protect against all seven toxins, one needs seven vaccines.

WO 00/67700 PCT/US00/12890 Botulinum neurotoxins are translated as a single 150 kDa polypeptide chain and then posttranslationally nicked, forming a dichain consisting of a 100 kDa heavy chain and a 50 kDa light chain which remain linked by a disulfide bond (DasGupta, et al., (1972), "A Common Subunit Structure in Clostridium botulinum Type A, B, and E Toxins," Biophys. Res. Commun., 48:108-112; DasGupta, (1989), "The Structure of Botulinum Neurotoxins," Botulinum Neurotoxin and Tetanus Toxin, (Simpson, pp. 53-67, Academic Press, New York). Most of the clostridial strains contain specific endogenous proteases which activate the toxins at a protease-sensitive loop located approximately one third of the way into the molecule from the amino-terminal end. Upon reduction and fractionation (electrophoretically or chromatographically), the two chains can be separated; one chain has a Mr of -100 kDa and is referred to as the heavy chain while the other has a Mr -50 kDa and is termed the light chain.

The mechanism of nerve intoxication is accomplished through the interplay of three key events, each of which is performed by a separate portion of the neurotoxin protein. First, the carboxy half of the heavy chain (fragment C or He is required for receptor specific binding to cholinergic nerve cells (Black, et al., (1986), "Interaction of 2 5 I-botulinum Neurotoxins with Nerve Terminals. I.

Ultrastructural Autoradiographic Localization and Quantitation of Distinct Membrane Acceptors for Types A and B on Motor Nerves," J. Cell Biol., 103:521- 534; Nishiki, et al., (1994), "Identification of Protein Receptor for Clostridium botulinum Type B Neurotoxin in Rat Brain Synaptosomes," J. Biol. Chem., 269:10498-10503; Shone, et al., (1985), "Inactivation of Clostridium botulinum Type A Neurotoxin by Trypsin and Purification of Two Tryptic Fragments. Proteolytic Action Near the COOH-terminus of the Heavy Subunit Destroys Toxin-Binding Activity, Eur. J. Biochem., 151:75-82). There is evidence suggesting that polysialogangliosides (van Heyningen, (1968), "Tetanus," Sci.

Am., 218:69-77) could act as receptors for the toxins but the data supporting a specific receptor remains equivocal (Middlebrook, J.L, (1989), "Cell Surface Receptors for Protein Toxins," Botulinum Neurotoxins and Tetanus Toxin, (Simpson, Ed.) pp. 95-119, Academic Press, New York). After binding, the toxin is internalized into an endosome through receptor-mediated endocyctosis WO 00/67700 PCT/US00/12890 (Shone, et al., (1987), "A 50-kDa Fragment from the NH 2 -terminus of the Heavy Subunit of Clostridium botulinum Type A Neurotoxin Forms Channels in Lipid Vesicles, Euro. J. Biochem., 167:175-180) The amino terminal half of the heavy chain is believed to participate in the translocation mechanism of the light chain across the endosomal membrane (Simpson, 1986; Poulain, et al., (1991), "Heterologous Combinations of Heavy and Light Chains from Botulinum Neurotoxin A and Tetanus Toxin Inhibit Neurotransmitter Release in Aplysia," J.

Biol. Chem., 266:9580-9585; Montal, et al., (1992), "Identification of an Ion Channel-Forming Motif in the Primary Structure of Tetanus and Botulinum Neurotoxins," FEBS, 313:12-18). The low pH environment of the endosome may trigger a conformational change in the translocation domain, thus forming a channel for the light chain. The final event of intoxication involves enzymatic activity of the light chain, a zinc-dependent endoprotease (Schiavo, 1992a; Schiavo, et al., (1992b), "Tetanus Toxin is a Zinc Protein and its Inhibition of Neurotransmitter Release and Protease Activity Depend on Zinc," EMBO 11:3577-3583), on key synaptic vesicle proteins (Schiavo, 1992a; Oguma, et al., (1995), "Structure and Function of Clostridium botulinum Toxins," Microbiol. Immunol., 39:161-168; Schiavo, et al., (1993), "Identification of the Nerve Terminal Targets of Botulinum Neurotoxin Serotypes A, D, and J. Biol. Chem., 268:23784-23787; Shone, et al., (1993), "Proteolytic Cleavage of Synthetic Fragments of Vesicle- Associated Membrane Protein, Isoform-2 by Botulinum Type B Neurotoxin," Eur. J.

Biochem., 217:965-971) necessary for neurotransmitter release. The light chains of BoNT serotypes A, C 1 and E cleave SNAP-25 (synaptosomal-associated protein of M25,000), serotypes B, D, F, and G cleave VAMP/synaptobrevin (synaptic vesicleassociated membrane protein); and serotype C 1 cleaves syntaxin. Inactivation of VAMP, or syntaxin by BoNT leads to an inability of the nerve cells to release acetylcholine resulting in neuromuscular paralysis and possible death, if the condition remains untreated.

Human botulism poisoning is generally caused by type A, B, E or rarely, by type F toxin. Type A and B are highly poisonous proteins which resist digestion by the enzymes of the gastrointestinal tract. Foodborne botulism poisoning is caused by the toxins present in contaminated food, but wound and infant botulism are caused WO 00/67700 PCT/US00/12890 by in vivo growth in closed wounds and the gastrointestinal tract respectively. The toxins primarily act by inhibiting the neurotransmitter acetylcholine at the neuromuscular junction, causing paralysis. Another means for botulism poisoning to occur is the deliberate introduction of the toxin(s) into the environment as might occur in biological warfare. When the cause of botulism is produced by toxin rather than by in vivo infection the onset of neurologic symptoms is usually abrupt and occurs within 18 to 36 hours after ingestion. The most common immediate cause of death in respiratory failure due to diaphragmatic paralysis. Home canned foods are the most common sources of toxins. The most frequently implicated toxin is toxin A, which is responsible for more than 50% of morbidity resulting from botulinum toxin.

Because even small amounts of botulinal toxin can cause serious illness, persons such as laboratory workers who are exposed to toxin must learn to handle all samples that may contain toxin with extreme care. It is also suggested that such workers be protected from illness by vaccination against the toxins. Furthermore, persons exposed to conditions in which botulism toxins might be in the environment which might be inhaled or ingested, such as military personnel, need to be protected from the toxin.

Agents that abolish the action of BoNT have been investigated since the 1940s. Early work at Fort Detrick in the 1940s lead to the development of a toxoid vaccine to protect against serotypes A, B, C1, D, and E toxins. The toxoid vaccine was manufactured by growing five Clostridium botulinum strains, extracting and precipitating the toxin from the growth media after cell lysis. Formalin was added to the crude preparation to inactivate the neurotoxin. Residual formalin was left in the vaccine product to ensure the toxin remains non-toxic. The product was adsorbed to aluminum hydroxide and blended. Currently, a pentavalent toxoid vaccine against serotypes A through E (Anderson, et al., (1981), "Clinical Evaluation of Botulinum Toxoids," Biomedical Aspects of Botulism, (Lewis, pp. 233- 246, Academic Press, New York; Ellis, R. (1982), "Immunobiologic Agents and Drugs Available from the Centers for Disease Control. Descriptions, Recommendations, Adverse Reactions and Scrologic Response," 3rd ed., Centers for Disease Control. Atlanta, GA; Fiock, et al., (1963), "Studies of Immunities to 4 WO 00/67700 PCT/US00/12890 Toxins of Clostridium Botulinum. IX. Immunologic Response of Man to Purified Pentavalent ABCDE Botulinum Toxoid," J. Immunol., 90:697-702; Siegel, L.S., (1988), "Human Immune Response to Botulinum Pentavalent (ABCDE) Toxoid Determined by a Neutralization Test and by an Enzyme-Linked Immunosorbent Assay," J. Clin. Microbiol., 26:2351-2356), available under Investigational New Drug (IND) status, is used to immunize specific populations of at-risk individuals, scientists and health care providers who handle BoNT and our armed forces who may be subjected to weaponized forms of the toxin. Though serotypes A, B, and E are most associated with botulism outbreaks in humans, type F has also been diagnosed (Midura, et al., (1972), "Clostridium botulinum Type F: Isolation from Venison Jerky," Appl. Microbiol., 24:165-167; Green, et al., (1983), "Human Botulism (Type F) A Rare Type," Am. J. Med., 75:893-895; Sonnabend, et al., (1987), "Intestinal Toxicoinfection by Clostridium botulinum Type F in an Adult. Case Associated with Guillian-Barre Syndrome," Lancet, 1:357-361; Hatheway, (1976), "Toxoid of Clostridium botulinum Type F: Purification and Immunogenicity Studies," Appl. Environ. Microbiol., 31:234-242). A separate monovalent toxoid vaccine against BoNTF is available under IND status. Hatheway demonstrated that the BoNTF toxoid could protect guinea pigs against a homologous challenge (Wadsworth, et al., (1990), "Botulinum Type F Neurotoxin," Biochem. 268:123-128).

Even though toxoid vaccines are available, there are numerous shortcomings with their current use and ease of production. First, because C. botulinum is a sporeformer, a dedicated facility is required to manufacture a toxin-based product. The requirement for a dedicated manufacturing facility is not trivial. It is extremely costly to renovate and upgrade an existing facility or to build a new one and then to maintain the facility in accordance with current Good Manufacturing Practices (cGMP) to manufacture one vaccine. Second, the yields of toxin production from C.

botulinum are relatively low. Third, the toxoiding process involves handling large quantities of toxin and thus is dangerous, and the added safety precautions increase the cost of manufacturing. Fourth, the toxoid product for types A-E consists of a crude extract of clostridial proteins that may influence immunogenicity or reactivity of the vaccine, and the type F toxoid is only partially purified (IND 5077). Fifth, WO 00/67700 PCT/US00/12890 because the toxoiding process involves the use of formaldehyde, which inactivates the toxin, and residual levels of formaldehyde (not to exceed 0.02%) are part of the product formulation to prevent reactivation of the toxin, the vaccine is reactogenic.

An additional component of the toxoid vaccines is the preservative thimerosal which also increases the reactogenicity of the product.

The development of a new-generation, recombinant vaccine could alleviate many of the problems associated with the toxoid. A recombinant vaccine would eliminate the need for a dedicated manufacturing facility. Presently, many cGMP facilities are in existence and available that could manufacture a recombinant product. There would be no need to culture large quantities of a hazardous toxinproducing bacterium. Production yields from a genetically engineered product is expected to be high. There would be no need to treat the vaccine with formalin because the product would be non-toxic from the outset. Recombinant products would be purer, less reactogenic, and more fully characterized. Thus, the cost of a recombinant product would be expected to be much lower than a toxoid because there would be no expenditures required to support a dedicated facility, and the higher production yields would reduce the cost of the vaccine product.

SUMMARY OF THE INVENTION It is an object of this invention to provide immunogenic peptides capable of eliciting protective immunity against botulinum neurotoxin of serotypes A-G.

It is another object of this invention to provide vaccines capable of eliciting protective immunity against botulinum neurotoxin, where the vaccines do not act as neurotoxins themselves.

It is yet another object of this invention to provide methods for preparing non-toxic peptides for use in vaccines against botulinum neurotoxin by growing recombinant organisms which express the peptides.

It is still another object of this invention to provide methods for fast and efficient purification of the non-toxic peptides from cultures of recombinant organisms.

These and other objects are met by one or more of the following embodiments of the present invention WO 00/67700 PCT/US00/12890 In one embodiment, this invention provides a nucleic acid encoding the carboxy-terminal portion of the heavy chain (HC) of botulinum neurotoxin (BoNT), the BoNT being selected from the group consisting of BoNT serotype A, BoNT serotype B, BoNT serotype Cl, BoNT serotype D, BoNT serotype E, BoNT serotype F, and BoNT serotype G, wherein said nucleic acid is expressable in a recombinant organism selected from Escherichia coli and Pichia pastoris.

Preferably, the nucleic acid comprises a nucleic acid sequence selected from SEQ ID No:1 (serotype SEQ ID No:7 (serotype SEQ ID No:9 (serotype CI), SEQ ID No:ll (serotype SEQ ID No:13 (serotpye SEQ ID No:15 (serotype and SEQ ID No:17 (serotype In an alternative preferred embodiment, the nucleic acid encodes an HC amino acid sequence of BoNT selected from SEQ ID No:2 (serotype SEQ ID No:8 (serotype SEQ ID No:10 (serotype Cl), SEQ ID No:12 (serotype SEQ ID No:14 (serotpye SEQ ID No:16 (serotype and SEQ ID No:18 (serotype G).

In another embodiment, this invention provides a nucleic acid encoding the amino-terminal portion of the heavy chain (HN) of botulinum neurotoxin (BoNT), the BoNT being selected from the group consisting of BoNT serotype B, BoNT serotype Cl, BoNT serotype D, BoNT serotype E, BoNT serotype F, and BoNT serotype G, wherein said nucleic acid is expressable in a recombinant organism selected from Escherichia coli and Pichia pastoris. In a prefered embodiment, the nucleic acid comprises a nucleic acid sequence selected from SEQ ID No:21 (serotype SEQ ID No:23 (serotype Cl), SEQ ID No:25 (serotype SEQ ID No:27 (serotpye SEQ ID No:29 (serotype and SEQ ID No:31 (serotype G).

Alternatively, the nucleic acid nucleic acid encodes an HN amino acid sequence of BoNT selected from SEQ ID No:22 (serotype SEQ ID No:24 (serotype Cl), SEQ ID No:26 (serotype SEQ ID No:28 (serotpye SEQ ID No:30 (serotype and SEQ ID No:32 (serotype G).

Preferably, the nucleic acid of this invention is a synthetic nucleic acid. In a preferred embodiment, the sequence of the nucleic acid is designed by selecting at least a portion of the codons encoding HC from codons preferred for expression in a host organism, which may be selected from gram negative bacteria, yeast, and mammalian cell lines; preferably, the host organism is Escherichia coli or Pichia WO 00/67700 PCT/US00/12890 pastoris. In another preferred embodiment, the nucleic acid sequence encoding HC is designed by selecting codons encoding HC which codons provide HC sequence enriched in guanosine and cytosine residues. More preferably, nucleic acid encoding HC or HN is expressed in a recombinant host organism with higher yield than a second nucleic acid fragment encoding the same HC sequence, said second nucleic acid fragment having the wild-type Clostridum botulinum sequence of HC.

In yet another embodiment, this invention provides anexpression vector comprising the nucleic acid of this invention, whereby HC and/or HN is expressed upon transfection of a host organism with the expression vector. Another embodiment of this invention provides a method of preparing a polypeptide comprising the carboxy-terminal portion of the heavy chain (HC) of botulinum neurotoxin (BoNT) or the amino-terminal portion of the heavy chain (HN) of botulinum neurotoxin (BoNT) selected from the group consisting of BoNT serotype A, BoNT serotype B, BoNT serotype C, BoNT serotype D, BoNT serotype E, BoNT serotype F, and BoNT serotype G, said method comprising culturing a recombinant host organism transfected with the expression vector of of this invention under conditions wherein HC or HN is expressed. Preferably, the recombinant host organism is a eukaryote. In another preferred embodiment, the method of this invention further comprises recovering insoluble protein from the host organism, whereby a fraction enriched in HC or HN is obtained. Preferably, the host organism is Pichia pastoris.

In still another embodiment, this invention provides an immunogenic composition comprising the carboxy-terminal portion of the heavy chain (HC) of botulinum neurotoxin (BoNT) selected from the group consisting of BoNT serotype A, BoNT serotype B, BoNT serotype C, BoNT serotype D, BoNT serotype E, BoNT serotype F, and BoNT serotype G. Preferably, the immunogenic composition is prepared by culturing a recombinant organism transfected with an expression vector encoding HC. More preferably, the immunogenic composition is prepared by a method wherein an insoluble protein fraction enriched in HC is recovered from said recombinant organism.

In yet another embodiment, this invention provides an immunogenic composition comprising the amino-terminal portion of the heavy chain (HN) of WO 00/67700 PCT/US00/12890 botulinum neurotoxin (BoNT) selected from the group consisting of BoNT serotype A, BoNT serotype B, BoNT serotype C, BoNT serotype D, BoNT serotype E, BoNT serotype F, and BoNT serotype G. Preferably, the immunogenic composition comprising HN is prepared by culturing a recombinant organism transfected with an expression vector encoding HN. More preferably, the immunogenic composition is prepared from an insoluble protein fraction enriched in HN which is recovered from the recombinant organism.

In still another embodiment, this invention provides an immunogenic composition comprising a polypeptide comprising epitopes contained in the carboxy-terminal portion of the heavy chain (HC) of botulinum neurotoxin (BoNT) and/or the amino-terminal portion of the heavy chain (HN) of botulinum neurotoxin (BoNT) selected from the group consisting of BoNT serotype A, BoNT serotype B, BoNT serotype C, BoNT serotype D, BoNT serotype E, BoNT serotype F, and/or BoNT serotype G, said epitopes eliciting protective immunity toward the respective BoNT serotype. Preferably, the immunogenic composition elicits an ELISA response to the respective BoNT serotype(s) in an animal which is detectable in serum from the animal even when the serum is diluted 100-fold.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype A and the encoded amino acids sequence.

Figure 2 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype A and the encoded amino acids sequence.

Figure 3 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype A and the encoded amino acids sequence.

Figure 4 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype B and the encoded amino acids sequence.

Figure 5 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype C and the encoded amino acids sequence.

Figure 6 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype D and the encoded amino acids sequence.

WO 00/67700 PCT/US00/12890 Figure 7 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype E and the encoded amino acids sequence.

Figure 8 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype E and the encoded amino acids sequence.

Figure 9 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype F and the encoded amino acids sequence.

Figure 10 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype G and the encoded amino acids sequence.

Figure 11 shows the sequence for a synthetic gene encoding the HN fragment of BoNT serotype A and the encoded amino acids sequence.

Figure 12 shows the sequence for a synthetic gene encoding the HN fragment of BoNT serotype B and the encoded amino acids sequence.

Figure 13 shows the sequence for a synthetic gene encoding the HN fragment of BoNT serotype C and the encoded amino acids sequence.

Figure 14 shows the sequence for a synthetic gene encoding the HN fragment of BoNT serotype D and the encoded amino acids sequence.

Figure 15 shows the sequence for a synthetic gene encoding the HN fragment of BoNT serotype E and the encoded amino acids sequence.

Figure 16 shows the sequence for a synthetic gene encoding the HN fragment of BoNT serotype F and the encoded amino acids sequence.

Figure 17 shows the sequence for a synthetic gene encoding the HN fragment of BoNT serotype G and the encoded amino acids sequence.

Figure 18 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype F and the encoded amino acids sequence.

Figure 19 shows AT base content of a putative fragment C region in native C. botulinum DNA. Reduction at AT content after the first design (rBoNTF(Hc)l) of the synthetic gene. AT content of the final gene design (rBoNTF(Hc)2) used to express recombinant rBoNTF(Hc) in P. pastoris.

Figure 20 shows SDS-PAGE and Western blot of samples at various steps along the rBoNTF(Hc) purification. Lanes from both figures are identical except lane 1, where SDS-PAGE shows Novex mark 12 wide-range molecular weight markers and Western blot shows Novex See Blue prestained molecular WO 00/67700 PCT/US00/12890 weight markers. Lane 2 is the cell lysate, lane 3 is the cell extract, lane 4 is the cell extract after dialysis, lane 5 is pool of rBoNTF(Hc) positive fractions after Mono S column chromatography, and lane 6 is pool of rBoNTF(Hc)-positive fractions after hydrophobic interaction chromatography.

Figure 21 shows purification of rBoNTF(Hc) by sequential chromatography.

Mono S cation exchange chromatography of extract from P. pastoris. Proteins were eluted with increasing NaCI gradient. Fractions positive for rBoNTF(Hc) by Western analysis were pooled individually and subjected to hydrophobic interaction chromatography and proteins were eluted with a decreasing ammonium sulfate gradient. In both panels, protein monitored by A280nm is recorded on the left axis and elution conditions are recorded on the right axis, with the gradient trace laid over the chromatogram.

Figure 22 shows CD spectra of purified soluble and resolubilized rBoNTF(Hc) at 30 Ag/ml (0.62 pM) in 10 mM sodium phosphate, pH 7.0 in a 1-cm path length cell. Spectra were the average of four accumulations, scanned from 260 to 200 nm at a scan rate of 10 nm/min with a 2-s response and a 1-nm bandwidth.

The temperature was maintained at 20 0 C using a Peltier thermocontrol device.

DETAILED DESCRIPTION OF THE EMBODIMENTS OF THIS INVENTION The present inventors have determined that animals, including primates, may be protected from the effects of botulinum neurotoxin (BoNT) by immunization with fragments of the botulinum neurotoxin protein expressed by recombinant organisms. Specifically, peptides comprising protective epitopes from the receptor binding domain and/or the translocation domain, found in the carboxy terminal and the amino terminal portions of the heavy chain of the BoNT protein, respectively, are expressed by recombinant organisms transfected with expression vectors encoding the peptides for each serotype of BoNT. Immunization with these recombinantly produced peptides will elicit antibodies capable of protecting animals against intoxication with the BoNT of the respective serotype.

This invention provides a genetically engineered vaccine for protection against botulism. The vaccine comprises fragments of the A and B toxins known as P.\Op\Ej iode1\5OO35 00 usmy.imwddpacs dcO04/3/O3 "C fragments" (He domain). It is now possible to produce the He fragments of the A and B toxins in E. coli using gene segment constructs of the HC fragment or an HC polypeptide fused to E. coli maltose binding protein. It has been found that the fusion product provides excellent protection against the native toxin challenge. The invention provides plasmids and recombinant proteins for use as vaccines to provide protection against toxins of Clostridium botulinum.

Kozaki et al. in "Antibodies against Botulism Neurotoxin", L.L. Simpson, ed., 1989, Academic Press, New York, suggested that a protective epitope might be present in the 50 kDa carboxyl terminus (HC) region of the protein. Thompson et al. (1990, Eur. J. Biochem. 189:73-81) deduced the amino acid sequence for the serotype A botulinum toxin. DasGupta, et al. (1990, Biochemie, 72:661-664), identified the "nick" site for post-translational cleavage of the expressed toxin polypeptide, from which the sequence of the heavy chain are deduced as SEQ ID NO:41 (see also Krieglstein, et al., J. Protein Chem., 13:57).

*o *o go oO* -12- P.OpcarEjdbwd\50035 00 y mwY dpagm dc04103/03 Whelan et al. (Appl. Environ. Microbiol. 58:2345-2354, 1992) have deduced the amino acid sequence for the serotype B botulinum toxin. Schmidt, et al. (1985), Arch. Biochem. Biophys. 238:544-548), provided N-terminal sequence information for the heavy chain resulting from post-transcriptional cleavage of the expressed toxin polypeptide, and the sequence of the heavy chain can be deduced from this information as SEQ ID NO:42.

Analogous post-transcriptional cleavage for all BoNT serotypes produces analogous heavy chain and light chain structures (see Krieglstein, et al., 1994, J.

Protein Chem., 13:49-57, and references cited therein).

Synthetic Gene Construction Preliminary experiments indicated that the DNA sequence found in C botulinum encoding the relevant BoNT fragments are not well expressed in typical recombinant hosts. Therefore, synthetic gene construction was undertaken, based on the amino acid sequence of the respective fragments.

S* *o 13- WO 00/67700 PCT/US00/12890 Synthetic gene construction is a technique used to optimize for expression in heterologous host systems. The base composition percent A+T or percent G+C) as well as the specific codons in a gene sequence play a role in determining whether a gene from one organism will be optimally expressed in a different organism. There is a reason why certain codons are used and why some are not.

Organisms will use the codons in which corresponding tRNAs are present. If the organisms do not use certain codons, they most likely lack those specific tRNAs. As it turns out, codons found in clostridial DNA genes found in the genus of bacterial called Clostridium) are very unique both in terms of base composition very high A+T base composition) and in the use of codons not normally found in E.

coli or yeast.

Table 1 is a chart depicting codon usage in Pichia pastoris. This table was generated by listing the codons found in a number of highly expressed genes in P.

pastoris. The codon data was obtained by sequencing the genes and then listing which codons were found in the genes.

From Table 1, it is clear that the amino acid residues can be encoded for by multiple codons. When constructing synthetic genes using P. pastoris codon usage, it is preferred to use only those codons that are found in the naturally occurring genes in P. pastoris, and it should be attempted to keep them in the same ratio found in the genes of the natural organism. When the clostridial gene has an overall A+T richness of greater than 70% and A+T regions that have spikes of A+T of 95% or higher, they have to be lowered for expression in expression systems like yeast.

(Preferably, the overall A+T richness is lowered below 60% and A+T in spikes is also lowered to 60% or below). It is of course necessary to balance keeping the same codon ratio for glycine GGG was not found, GGA was found 22% of the time, GGT was found 74% of the time, GGC was found 3% of the time) with reducing the high A+T content. In the construction of the genes, it is preferred to keep the A+T spikes about Considering codon usage for a number of organisms including E. coli, it turns out that a synthetic gene using E. coli codon usage also expresses fairly well in P. pastoris. Similarly, a synthetic gene using P. pastoris codon usage also appears to express very well in E. coli.

WO 00/67700 PCT/US00/12890 Synthetic genes for the He fragments of botulinum neurotoxin serotypes A-G are shown in Figures 1-10, along with the amino acid sequences encoded by the synthetic genes. Synthetic genes for the HN fragments of botulinum neurotoxin serotypes A-G are shown in Figures 11-17, along with the amino acid sequences encoded by the synthetic genes. Synthetic genes having alternative gene sequences may be prepared by following the guidance provided herein concerning codon selection. The amino acid sequence encoded by such synthetic genes will preferably be the sequence of one of the BoNT serotype proteins, or a fragment thereof which contains protective epitopes. Suitable fragments include the He fragments of BoNT serotypes A, B CI, D, E, F, and G, and the HN fragments of BoNT serotypes A, B,

C

1 D, E, F, and G. Such alternative gene sequences are within the contemplation of this invention.

Also within the contemplation of this invention are proteins containing protective epitopes from both the N-terminal and the C-terminal domains of the respective serotype BoNT proteins. Such proteins may be prepared by fusing a sequence encoding the translocation domain (HN) to the sequence of the HC region.

This may be accomplished by removing the restriction enzyme site of the 3' end of the translocation domain gene as well as the termination codon, and also removing the initiation codon, restriction enzyme site and any other nucleotides on the 5' end of the gene that are not part of the botulinum toxin gene. Then a common restriction enzyme site not found in either synthetic gene may be inserted on the 3' end of the HN gene and the 5' end of the He gene, and this common restriction site may be used to fuse the two genes together.

Recombinant Peptide Production The nontoxin fragment is very safe, will not require formalin treatment, and has been shown to produce significant immunity against the fully toxic parent molecule. There are two major advantages of the invention over the presently employed vaccine. First, the recombinantly-produced botulinum neurotoxin (rBoNT) protein fragments are completely nontoxic and, is thus, very safe. The fermentation of the host cell harboring the rBoNT gene Escherichia coli or Pichia pastoris) will not require the high biological containment facilities presently needed to ferment the spore-forming Clostridium botulinum required for the WO 00/67700 PCT/US00/12890 production of the toxoid vaccine. Second, the synthetic gene can be placed in high expression systems and used to make much larger quantities of the fragment than toxin produced by the parent organism, Clostridium botulinum. Thus, there will be immense cost savings because it will be easier and safer to produce much larger quantities of the vaccine than is now possible.

Synthetic genes as described herein may be transfected into suitable host organisms to create recombinant production organisms, and cultures of these recombinant organisms can then be used to produce immunogenic peptide fragments capable of conferring protective immunity against BoNT of the respective serotypes.

Exemplary techniques for transfection and production of BoNT fragments are shown in the Examples. Alternative techniques are well documented in the literature (See, Maniatis, Fritsch Sambrook, "Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning: A Practical Approach," Volumes I and II Glover, ed., 1985); "Oligonucleotide Synthesis" Gait, ed., 1984); "Nucleic Acid Hybridization" Hames S.J. Higgins, eds., 1985); "Transcription and Translation" Hames S.J. Higgins, eds., 1984); "Animal Cell Culture" (R.I.

Freshney, ed., 1986); "Immobilized Cells and Enzymes" (IRL Press, 1986); B.

Perbal, "A Practical Guide to Molecular Cloning" (1984), and Sambrook, et al., "Molecular Cloning: a Laboratory Manual" (1989)). Such techniques are explained fully in the literature, and modification of these techniques within the scope of this invention is within the skill in the art.

The synthetic gene for BoNT serotype B fragment He (see Figure 4A) has been inserted into the yeast expression vector pHIL-D4, and integrated into the chromosome of Pichia pastoris strain GS115. The expressed product (see amino acid sequence in Figure 4B) had the expected molecular weight as shown by denaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis using antibodies directed against botulinum neurotoxin serotype B. The expressed recombinant BoNTB (Hc) elicited high antibody titers as judged by the Enzyme Linked Immunosorbent Assay (ELISA) and, more importantly, these circulating serum titers protected mice, guinea pigs, and non-human primates from challenges with active toxin. Industrial scale manufacturing processes (fermentation and WO 00/67700 PCT/US00/12890 purification) have been completed and a pilot lot has been produced in compliance with cGMP.

The synthetic gene for BoNT serotype C fragment He (see Figure 5A) has been inserted into the yeast expression vector pHIL-D4, and integrated into the chromosome of Pichia pastoris strain GS115. The expressed product (see amino acid sequence in Figure 5B) had the expected molecular weight as shown by denaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis using antibodies directed against botulinum neurotoxin serotype C. The expressed recombinant BoNTC (Hc) elicited high antibody titers as judged by the Enzyme Linked Immunosorbent Assay (ELISA) and, more importantly, these circulating serum titers protected mice from challenges with active toxin.

The synthetic gene for BoNT serotype D fragment He (see Figure 6A) has been inserted into the yeast expression vector pHIL-D4, and integrated into the chromosome of Pichia pastoris strain GS115. The expressed product (see amino acid sequence in Figure 6B) had the expected molecular weight as shown by denaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis using antibodies directed against botulinum neurotoxin serotype D. The expressed recombinant BoNTD (He) elicited high antibody titers as judged by the Enzyme Linked Immunosorbent Assay (ELISA) and, more importantly, these circulating serum titers protected mice from challenges with active toxin.

The synthetic gene for BoNT serotype E fragment He (see Figure 7A) has been inserted into the yeast expression vectors pHILD2, pHILD3, and pPIC9K (see Figures 7B). A modified form of the synthetic gene in which an internal EcoRI site was removed and the gene was enlarged (see Figure 8) was inserted into the yeast vector pHIL-D4, and integrated into the chromosome of Pichia pastoris strain GS115. The expressed product (see amino acid sequence in Figure 8) had the expected molecular weight as shown by denaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis using antibodies directed against botulinum neurotoxin serotype E. The expressed recombinant BoNTE (Hc) elicited high antibody titers as judged by the Enzyme Linked Immunosorbent Assay (ELISA) and, more importantly, these circulating serum titers protected mice from challenges with active toxin.

WO 00/67700 PCT/US00/12890 The synthetic gene for BoNT serotype F fragment He (see Figure 9A) has been inserted into the yeast expression vector pHIL-D4, and integrated into the chromosome of Pichia pastoris strain GS115. The initial step in the development of the rBoNTF(Hc) vaccine candidate was to design a gene which could satisfactorily be expressed in a pichia host. A synthetic gene encoding rBoNTF(Hc) was constructed to lower the inherent AT richness of the native clostridial gene and to remove any potentially rare codons. Clostridial genes having an AT content in excess of 65% or having an average AT content but containing AT-rich tracts usually contain multiple terminators/polyadenylation signals, which can result in premature termination of transcripts when expression is attempted in yeast (Romanos, et al., (1995), "Expression of Cloned Genes in Yeast," DNA Cloning 2: Expression Systems," (Glover et al., Eds.), Oxford Univ. Press, London). The synthetic gene in this study required two successive rounds of alterations before the yeast could properly produce full-length antigen. The expressed product (see amino acid sequence in Figure 9B) had the expected molecular weight as shown by denaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis using antibodies directed against botulinum neurotoxin serotype F.

A previous study (Hatheway, 1976) demonstrated that the serotype F toxoid antigen needed to be at least partially purified to be efficacious. The same observation was noted with the rBoNTF(Hc) antigen produced in pichia cells as the crude cell lysate did not protect mice against a BoNTF challenge. The putative receptor-binding domain of BoNTF was purified from yeast and shown to be efficacious in a mouse model. The expressed recombinant BoNTF (Hc) elicited high antibody titers as judged by the Enzyme Linked Immunosorbent Assay (ELISA) and, more importantly, these circulating serum titers protected mice from challenges with active toxin.

The synthetic gene for BoNT serotype G fragment He (see Figure 10A) has been inserted into the yeast expression vector pHIL-D4, and integrated into the chromosome of Pichia pastoris strain GS115. The expressed product (see amino acid sequence in Figure 10B) had the expected molecular weight as shown by denaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis WO 00/67700 PCT/USOO/12890 using antibodies directed against botulinum neurotoxin serotype G. The expressed recombinant BoNTG (Hc) elicited high antibody titers as judged by the Enzyme Linked Immunosorbent Assay (ELISA) and, more importantly, these circulating serum titers protected mice from challenges with active toxin.

When purifying a protein for the first time, it is important to generate a viable means for identifying which fractions contain product. If the protein of interest is not an enzyme or does not absorb at a unique wavelength, there are still suitable assays (for example mass spectrometry) for identifying the product. The inventors chose to monitor the purification of rBoNT(Hc) through immunological detection by Western blot analysis. However, with various polyclonal antibodies against whole toxin available but without an appropriate positive control, the Western blot results can only be interpreted as ambiguous until a purified sample is sequenced or shown to be protective.

There are two major issues of concern when extracting C-fragment, HN, and/or heavy chain (He) antigens from pichia cells. The first concern is the solubility of these proteins can enough product be extracted into the soluble fraction for further processing?). The second concern deals with the effective removal of polynucleic acids and/or other contaminating materials, which strongly interfere with the necessary chromatography.

The zwiterionic detergent, CHAPS, is most notably an effective agent for solubilizing membrane proteins. Membrane proteins exist in a hydrophobic environment, and if removed from that environment, possess strong tendencies to aggregate and ultimately precipitate. CHAPS prevents that aggregation from occurring with membrane bound proteins. The inventors extrapolated this premise to the clostridial proteins noted above. C-fragments, translocation domains (HN), and entire heavy chains are missing their natural partner (the remaining segments of the neurotoxin) and thus, presumably bare exposed hydrophobic regions on their protein surface where the He, HN, or heavy chain normally associates with rest of the neurotoxin. These exposed hydrophobic regions are potential nucleation sources for protein aggregation, because the natural tendency of a protein in an aqueous environment is to bury their hydrophobic surface. When pichia cells are disrupted with CHAPS (on the order of 0.3% W/V) present in the cracking buffer, the amount WO 00/67700 PCT/US00/12890 of fragment C protein isolated in the soluble fraction has been observed to increase from less than 5% to nearly 80% with serotype C 1 Dramatic increases in solubility have been noted with C-fragment serotypes A and F as well.

Once a soluble antigen has been produced, the subsequent task is to separate that antigen from the myriad of pichia host proteins, lipids, and other impurities that exist in the extracted medium. In order for the chemical separations to be feasible by liquid chromatography, it is critical that polynucleic acids be efficiently removed.

Nucleotides will either bind to the C-fragment (serotypes A, E, and F due to their elevated pIs) or will bind to the anion-exchange chromatography resin (as is used in the first purification step of the C 1 process). With either case, the chromatography is rendered futile. The C-fragment product will either fail to bind to the chromatography media or it will elute over an unacceptably large sodium chloride concentration range. Pichia cells possess an abundant amount of DNA.

Polyethyleneimine (PEI) is a polycationic agent that readily precipitates nucleotides.

When pichia cell extracts are treated with PEI, the nucleic acids are efficiently precipitated and removed by centrifugation without significant loss of product.

More importantly, the chromatographic separation of C-fragments from pichia proteins are dramatically improved.

The soluble portion of the cell lysate may typically be purified in two conventional chromatographic steps. The ultimate objective of this work is to obtain FDA licensure of rBoNT as a safe and effective vaccine. Even though separations can be accomplished at extremely high resolution with affinity chemistry, there remains an undesirable effect of hapten leaching from the resin. Thus, a preferred separation employs a cation-exchange step followed by hydrophobic interaction chromatography (HIC). These two steps complement each other as they provide separations based on electrostatic and hydrophobic interactions. The cationexchange step was particularly efficient in increasing the purity of rBoNTF(Hc), as the antigen was estimated to be purified greater than 52-fold. The efficiency of purification is primarily attributed to the significant difference in isoelectric points between most pichia proteins (pls<7 and rBoNT(Hc) (experimental pi=9.4 for rBoNTF(Hc) data not shown) and thus, the pichia proteins were removed in the column flow through. Precipitate that results when the cation-exchange pool is WO 00/67700 PCT/US00/12890 treated with ammonium sulfate contains mostly pichia proteins and very little rBoNT product. The HIC step removes most or all of the remaining impurities. The yield of soluble rBoNTF(Hc) from the total recombinant yeast cell lysate was estimated to be greater than 28% with a purity greater than 98%. Use of similar purification steps for rBoNTA(Hc) produced greater than 95% pure material.

A significant amount of rBoNTF(Hc) product (30-40%) was identified in the insoluble portion of the cell lysate. Also, the antigen was 35% of the total protein present in the pellet; in effect it was more pure than the soluble rBoNTF(Hc) was after the ion-exchange step. This suggests an alternative process whereby insoluble rBoNT product produced in yeast may be resolubilized and purified to homogeneity.

The resolubilization may be performed by resuspending the pellet in urea and subsequently removing the urea by dialyses in nondenaturing buffer. A single chromatographic step using cation-exchange chemistry may be sufficient to purify the resolubilized antigen, in some cases to greater than 98%. The yield of resolubilized rBoNTF(Hc) product from the total cell lysate was estimated to be The overall bench scale yield of purified soluble and resolubilized rBoNTF(Hc) was estimated to be greater than 47% or 240 mg/Kg of the cell paste.

A similar procedure would be suitable for purification of rBoNTA(Hc) and other rBoNT fragment peptides from yeast.

Analysis of CD spectra of both soluble and resolubilized product revealed the presence of significant P-sheet which is in agreement with that predicted for rBoNTF(Hc) using an artificial neural network (Lebeda, et al., (1997), "Predicting Differential Antigen-Antibody Contact Regions Based on Solvent Accessibility," J. Protein Chem., 16:607-618), and that determined by crystal structure of BoNT serotype A (Lacy, et al., (1998), "Crystal Structure of Botulinum Neurotoxin Type A and Implications for Toxicity," Nat. Struct. Biol., 5:898-902). However, even though CD revealed that the two antigens possessed similar folds, there were subtle differences between the two spectra suggesting that the secondary structures, and hence, tertiary structures were not identical.

Immunization The purified soluble and resolubilized antigens appear to be in a folded conformation. However, the bottom line with any potential vaccine is the WO 00/67700 PCT/US00/12890 demonstration of protection. Are the antigens in a conformation that will elicit the production of neutralizing antibody? To answer this question, mice were inoculated with rBoNTF(Hc) and subsequently challenged with a high level of rBoNTF toxin.

The purified soluble rBoNTF(Hc) completely protected mice receiving three inoculations of 0.2 gLg from challenge with 1000 mouse i.p. LD 5 0 of BoNT/F toxin Analysis of the association of dose with survival indicated that dose was associated with the odds of surviving (odds ratio=2.0, meaning that the odds of survival increase twofold per unit increase in dose with a 95% confidence level from 1.3 to The number of inoculations was also associated with survival. Both two inoculations and three inoculations were associated with increased odds of survival relative to a single inoculation (5.3-fold with a 95% confidence level of 1.2-23 for two inoculations and 22-fold with a 95% confidence level of 4.3 -110 for three inoculations). It is apparent that a single shot at higher doses achieved protection comparable to multiple inoculations at lower doses. Also, three doses of 1 Pg of purified resolubilized rBoNTF(Hc) completely protected mice from a challenge of 5000 mouse i.p. LD 5 0 of BoNTF toxin, thus demonstrating that refolded rBoNTF(Hc) from the insoluble fraction of lysate could also be a prosperous source of antigen.

Individual antibody ELISA titer appears to be an excellent predictor of mouse survival. If the antibody titer of a mouse was 100 or greater, that mouse was predicted to and did survive a challenge of 1000 mouse i.p. LDso of BoNTF toxin.

Upon vaccination of mice with 2 or 3 doses of rBoNTA(Hc) or rBoNTB(Hc) vaccine delivered on a specific schedule parental intramuscular injection at 0,4, and 8 weeks), survival of animals challenged with 100,000 or 1,000,000 million LD50 of toxin is very high. Measurement of the antibody levels in these animals via an ELISA shows that the survival rate can be correlated with the measured antibody level. The ELISA is performed by coating a microtiter plate with toxin or fragment C itself, then sera from the vaccinated mice is added at various dilutions sera diluted 1/100, 1/400, 1/1600, 1/6400, etc.). Since fragment C is sufficient to elicit protection in animals, preferably assays for neutralizing antibody titer in sera from animals vacinated with fragment C are performed using microtiter plates coated with fragment C. Antibody in the sera will bind to the toxin or the fragment C, and the WO 00/67700 PCT/US00/12890 bound antibody may be detected by a secondary antibody anti-mouse IgG) that is coupled to horse-radish peroxidase or alkaline phosphatase. The secondary IgG will bind to the anti-BoNT antibody that was raised to the fragment C vaccine. After washing the microtiter wells, a substrate for the peroxidase or phosphatase enzymes is added to the wells. The substrate will give off a color once the enzyme has cleaved the substrate, and the intensity of the color measured at 405 nm.

Typically, a reading of 0.2 is used as the base. Thus, if dilution of the sera by 1/1600 gives a reading of 0.15 at 405 nm and a dilution of 1/400 gives a reading of 0.45 at 405 nm, the antibody titer in the sera in characterized as 1/400 dilution titer of 400 fold). Obviously, if readings of 0.2 are obtained at higher dilutions, better protection is observed. With rBoNTA(Hc) vaccination, for mice which had ELISA titers of less than 100, only 14.3% survival rate was observed under the conditions of vaccination and challenge. With rBoNTF(Hc), for mice which had ELISA titers of 100 fold, under the condition of vaccination and challenge, 100% of the mice were protected.

It also will be well known to one of ordinary skill in the art that a susceptible host may be immunized using the appropriate peptide vaccine formulated in adjuvant to increase the immune response. Such adjuvants include but are not limited to Freund's (complete and incomplete), mineral gels, such as aluminum hydroxide, surface active substances such as keyhole limpet hemocyanin, lysolecithin, pluronic polyols, polyanions, peptides, BCG (Bacille Calmette-Guerin), oil emulsions and dinotrophenols. Immunization can be carried out with additional various presentation and cross-linking permutations. By way of example and not of limitation, such permutations include rBoNT peptides cross-linked to KLH as a carrier, any rBoNT peptide cross-linked to any other rBoNT protein as carrier, rBoNT peptides cross-linked to themselves, and these combinations presented by the various adjuvants listed above. It will become evident that such permutations are available in regard to other peptides and self-assembled peptides disclosed throughout this specification.

It will also be known to one of ordinary skill in the art that use of the term "susceptible host" includes any such mammalian host susceptible to intoxication by BoNT. It will be further evident that any such susceptible host is a candidate for WO 00/67700 PCT/USOO/12890 treatment to promote protection from BoNT utilizing the peptide vaccines and associated methods described in this specification.

EXAMPLES

In order to facilitate a more complete understanding of the invention, a number of Examples are provided below. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.

Example 1. Synthesis and cloning of a synthetic gene encoding rBoNTF(Hc) A synthetic gene encoding a putative fragment C region of botulinum neurotoxin serotype F was designed and constructed for expression in Escherichia coli (Holley et al., submitted to Vaccine). The recombinant BoNTF(Hc,)i, gene was expressed in E. coli as a fusion protein with maltose-binding protein (MBP) with yields of 1 mg/L culture (See Figure 18).

The same gene was used for expression studies in the yeast, P. pastoris. This particular host was chosen because it could produce high levels of recombinant proteins (Cregg, et al., (1993), "Recent Advances in the Expression of Foreign Genes in Pichia pastoris," Bio/Technology, 11:905-909; Romanos, et al., (1992), "Foreign Gene Expression in Yeast: A Review," Yeast, 8:423-488; Sreekrishna, et al., (1988), "High Level Expression of Heterologous Proteins in Methylotrophic Yeast Pichia pastoris," J. Bas. Microbiol., 28:265-278) and because it lacked endotoxins which would facilitate product development. Intracellular expression of the antigen was used to avoid potential glycosylation of the recombinant protein. The rBoNTF(Hc)i, gene was modified at its 3' end for insertion into the unique EcoR I site of the yeast vector, pHILD4. The recombinant construct containing the rBoNTF(Hc)i gene was subsequently linearized with Sac I and the cassette integrated into the chromosomal alcohol oxidase (AOX 1) of Pichia pastoris strain GS 115 (Clare, et al., (1991), "High-Level Expression of Tetanus Toxin Fragment C in Pichia pastoris Strains Containing Multiple Tandem Integrations of the Gene," Bio/Technology, 9:455-460). Yeast transformants expressing the selectable markers histidine dehydrogenase (Cregg, et al., WO 00/67700 PCT/US00/12890 (1985), "Pichia pastoris as a Host System for Transformations," Mol. Cell. Biol., 5:3376-3385) and aminoglycoside phosphotransferase 3' (Scorer, et al., (1994), "Rapid Selection Using G418 of High Copy Number Transformants of Pichia pastoris for High-Level Foreign Gene Expression," Bio/Technology, 12:181- 184) were isolated. These isolates were further characterized for their ability to express rBoNTF(Hc) after induction with methanol. Although the various transformants generated were able to express the selectable markers, no expression of rBoNTF(Hc,) as judged by SDS/PAGE and blot analysis was observed in these isolates (data not shown).

SDS/PAGE, Western blot, and protein assays Total protein concentrations were determined by using the Pierce BCATM (bicinchoninic acid) protein assay kit with BSA as a standard. The purity of the rBoNTF(Hc) product was assessed by SDS/PAGE with Novex (San Diego, CA, gel electrophoresis supplies, reagents, protocols, and National Institutes of Health (NIH) imaging software as previously described (Byrne, et al., (1998), "Purification, Potency, and Efficacy of the Botulinum Neurotoxin Type A Binding Domain from Pichia pastoris as a Recombinant Vaccine Candidate," Infect. Immun., 66:4817-4822). Western blot assays were used to identify FPLC fractions containing rBoNTF(Hc) as previously described (Byrne, 1998) with the following changes. The primary antibody used was a polyclonal protein G sepharose-purified horse anti- BoNTF antibody incubated at 1 p.g/ml for 3 h The secondary antibody used was a horseradish peroxidase-labeled affinity-purified goat anti-horse IgG (Kirkegaard Perry Laboratories, Gaithersburg, MD, assayed at 1 p.g/ml for 2 h.

Example 2. Synthesis and cloning of a synthetic gene encoding rBoNTF(Hc) A second synthetic gene, rBoNTF(Hc)2, was subsequently designed to facilitate expression in P. pastoris. Redesigning the gene was intended to lower specific regions of the rBoNTF(Hc), gene in which spikes of AT-rich tracts still remained. Previous work had shown that rare codons (Makoff, et al., (1989), "Expression of Tetanus Toxin Fragment C in E. coli: High Level Expression by Removing Rare Condons," Nucleic Acids Res., 17:10191-10201) and/or highly enriched AT base compositions (Romanos, et al., (1991), "Expression of WO 00/67700 PCT/US00/12890 Tetanus Toxin Fragment C in Yeast: Gene Synthesis is Required to Eliminate Fortuitous Polyadenylation Sites in AT-rich DNA," Nucleic Acids Res., 19:1461- 1467) in clostridial DNA were incompatible with optimum expression of clostridial genes in E coli and yeast. A second synthetic gene encoding the rBoNTF(Hc) fragment was designed and constructed using P. pastoris codon usage (Sreekrishna, (1993), "Strategies for Optimizing Protein Expression and Secretion in the Methylotrophic Yeast Pichia pastoris," Industrial Micororganisms: Basic and Applied Molecular Genetics, (Baltz, R. et al, Eds.), pp. 119-126, Am. Soc.

Microbiol., Washington, DC). Briefly, complimentary oligonucleotides encoding the amino terminal region of the F(Hc)(423 nucleotides flanked with EcoRl and PstI sites), the central region of the F(Hc) (606 nucleotides flanked by PstI and Sall sites) and the carboxy-terminal region of F(Hc) (336 nucleotides flanked by Sal and EcoRI sites) were annealed and cloned into pUC or PCR zero-blunt plasmid vectors.

The AT base composition in the native clostridial F(Hc) DNA averaged 76% while rBoNTF(Hc)i averaged 58% and rBoNTF(Hc) 2 53% (Figure 19). The synthetic gene sequence of rBoNTF(Hc) 2 and the 432 amino acids it encoded for is shown in Figure 9. After nucleotide sequencing, the cloned fragments were excised by the appropriate restriction endonucleases, separated by agarose gel electrophoresis, and purified. The isolated DNA fragments were ligated simultaneously into EcoR I digested and dephosphorylated plasmid pHILD4. The vector harboring the rBoNTF(Hc) 2 gene was integrated into the chromosomal AOX1 locus of P. pastoris as described above. Transformants expressing selectable markers (histidine dehydrogenase and aminoglycoside phosphotransferase 3' were isolated and tested for their ability to express rBoNTF(Hc). Unlike the rBoNTF(Hc)i gene, rBoNTF(Hc) 2 was expressed after induction with methanol and yielded the expected molecular weight of approximately 50000 daltons as judged by SDS/PAGE and Western blot analysis (Figure 20). The deduced molecular mass of the encoded polypeptide was 50,250 daltons.

Example 3. Expression and Cell Disruption of rBoNTF(Hc) in P.

pastoris Large-scale fermentation conditions and optimal intracellular expression of rBoNTF(Hc) were determined for the yeast strain P. pastoris.

WO 00/67700 PCT/US00/12890 Protein expression A stock seed culture of P. pastoris was grown in shake-flasks containing L of YNB medium (13.4 g/L yeast nitrogen base without amino acids, 20 g/L glycerol, 0.4 mg/L biotin, in 100 mM sodium phosphate, pH Cultures were grown at 30'C until an A 6 oo of 20 absorbance units was achieved, and then used to inoculate a 5-L BioFlo 3000 fermentor (New Brunswick Scientific, Edison, NJ, containing 2.5 L basal-salt medium plus PTM 4 trace mineral salts and 4% glycerol. Dissolved oxygen was maintained at 40% and the pH was maintained at with 30% ammonium hydroxide. After the initial glycerol was consumed, glycerol was added at a rate of 20 g/L/h for 1 h then decreased linearly to 0 g/L/h over 3 h. The medium was enriched with 1.5 g methanol/L of medium.

Methanol feed was started at 4 g/L/h and linearly increased to 9 g/L/h over 10 h. The methanol feed rate was adjusted by using the dissolved oxygen-spike method (Chiruvolu, et al., (1997), "Recombinant Protein Expression in an Alcohol Oxidase-Defective Strain of Pichia pastoris in Feed-Batch Fermentations, Enzyme Microbiol. Technol., 21:277-283). After 10 h of methanol induction, the cells were harvested by centrifugation at 6000 g for 10 min at 4°C with a Beckman JA-10 rotor (Beckman Instruments, Palo Alto, CA, and then stored at Protein expression A stock seed culture of P pastoris was grown in shake-flasks containing L of YNB medium (13.4 g/L yeast nitrogen base without amino acids, 20 g/L glycerol, 0.4 mg/L biotin, in 100 mM sodium phosphate, pH Cultures were grown at 30*C until an A 60 o of 20 absorbance units was achieved, and then used to inoculate a 5-L BioFlo 3000 fermentor (New Brunswick Scientific, Edison, NJ, containing 2.5 L basal-salt medium plus PTM 4 trace mineral salts and 4% glycerol. Dissolved oxygen was maintained at 40% and the pH was maintained at with 30% ammonium hydroxide. After the initial glycerol was consumed, glycerol was added at a rate of 20 g/L/h for 1 h then decreased linearly to 0 g/L/h over 3 h. The medium was enriched with 1.5 g methanol/L of medium.

Methanol feed was started at 4 g/L/h and linearly increased to 9 g/L/h over 10 h. The methanol feed rate was adjusted by using the dissolved oxygen-spike method (Chiruvolu, 1997). After 10 h of methanol induction, the cells were harvested by WO 00/67700 PCT/US00/12890 centrifugation at 6000 x g for 10 min at 4"C with a Beckman JA-10 rotor (Beckman Instruments, Palo Alto, CA, and then stored at Cell disruption and sample preparation Eleven g of frozen cell paste was resuspended in 100 ml of 50 mM Na 2 HP04/2 mM Na 2 EDTA/I mM PMSF, pH 6.8 at 4°C. The suspended cells were disrupted by three successive passes through a microfluidizer device (model 110Y, Microfluidics Corp., Newton, MA, at 21000 psi. The temperature of the disruptate was kept below 10'C throughout the process by cooling the exit line and collection flask with ice. The cells were judged to be greater than 95% disrupted as determined by microscopy. In comparison, 8-10 passes through a Gaulin homogenizer were required to efficiently disrupt the cells in past protocols. SDS- PAGE and Western blot analysis of cell lysate showed that expressed rBoNTF(Hc) represented of the total protein. The resulting cell lysate volume was 105 ml with a protein concentration of 11 mg/ml. Cellular debris and insoluble proteins were removed by centrifugation at 15000 g for 15 min at 4'C with a Sorval SS-34 rotor (Sorval Instruments, Newtown, CT, The resulting extract was noticeably turbid due to the presence of lipids and significant quantities of nucleic acids. As rBoNTF(Hc) possessed a calculated isoelectric point of 9.1 and presumably interacted strongly with DNA, DNase was added to the cell extract in order to digest the polynucleotides and facilitate purification. To remove the polynucleotides, the extract was treated with DNase (100 units/ml, Aldrich) and ZnCI 2 (2 mM, Aldrich) at room temperature for 30 min and then dialyzed extensively with 10 kDa molecular weight cut off (MWCO) Slide-A-Lyzer dialysis cassettes (Pierce) in 50 mM Na 2 HP04/2 mM Na 2 EDTA/1 mM PMSF, pH 6.8 at 4C.

A precipitate developed during dialysis that was separated by centrifugation at 15000 g for 15 min at 4°C with a Sorval SS-34 rotor. The clarified extract contained 7.8 mg/ml of total protein and was used as starting material for the FPLC purification of soluble rBoNTF(Hc) while the pellet was used as starting material for the resolubilized rBoNTF(Hc) purification.

WO 00/67700 PCT/US00/12890 Example 4. Conventional Purification of rBoNTF(Hc) from P. pastoris The rBoNTF(Hc) protein was purified to homogeneity using an FPLC system and two chromatographic steps. First, the material was subjected to cation exchange chromatography (Figure 21A).

FPLC purification of soluble rBoNTF(Hc) Soluble rBoNTF(Hc) was purified by using a Pharmacia model 500 FPLC system (Pharmacia, Uppsala, Sweden) with programmed elution and A 280 monitoring. The starting material was loaded onto a Pharmacia HR 10/10 Mono S cation-exchange column equilibrated with 50 mM Na 2 HP04/2 mM Na 2 EDTA/1 mM PMSF, pH 6.8 (buffer A) at a flow rate of 2 ml/min (150 cm/h) The column was washed with 16 ml (2 bed volumes) of buffer A. Flow through and wash were collected separately and stored for subsequent analysis. Protein was eluted from the column with a linear gradient from 0 to 300 mM NaCI over 80 ml (10 bed volumes), then a linear gradient from 300 to 1000 mM NaCI over 20 ml (2.5 bed volumes), and then an isocratic gradient at 1000 mM over 10 ml (1.25 bed volumes). Four-ml fractions were collected throughout the linear and isocratic gradients. This step was highly efficient as most pichia proteins possess isoelectric points between pH 5 and 7 and, therefore, pass through the column without binding. Fractions eluting between 230 and 260 mM NaCI were positive for rBoNTF(Hc) by Western blot analysis and were pooled. The pooled fractions were adjusted to 1.5 M ammonium sulfate by the slow addition of 2 M (NH 4 2 SO450 mM Na 2 HP04/2 mM Na 2 EDTA/25 mM NaCI, pH 7.5 with stir bar agitation. A protein precipitate formed which consisted primarily of yeast proteins with a small amount of rBoNTF(Hc) product (approximately The precipitate was removed by centrifugation at 6000 g for 10 min at 4"C with a Sorval SS-34 rotor. Fortunately, when the pool of Mono S column fractions was diluted with ammonium sulfate, most of the rBoNTF(Hc) product remained in solution (approximately 90%) while significant quantities of pichia proteins salted out. The first step enriched the desired product from <0.5 to 26% of the total protein (Table 2).

HIC was used as a second chromatographic step (Figure 21B) and separated proteins based on their differences in surface hydrophobicity. It was determined that neopentyl chemistry provided the appropriate hydrophobic interaction with WO 00/67700 PCT/US00/12890 rBoNTF(Hc). The supernatant was loaded onto a Pharmacia alkyl superose 10/10 hydrophobic interaction chromatography (HIC) column equilibrated with 1.5 M (NH4) 2 S04/50 mM Na 2 HPO4/2 mM Na 2 EDTA/25 mM NaCI, pH 7.5 (buffer B) at a flow rate of 1 ml/min (75 cm/h). The column was washed with 8 ml (1 bed volume) of buffer B. Protein was eluted from the column with a linear gradient of decreasing

(NH

4 2 S0 4 from 1.5 to 0 M over 60 mi (7.5 bed volumes). The rBoNTF(Hc) eluted from the HIC column at 0.92 M ammonium sulfate in a volume of 3 ml with a protein concentration of 0.52 mg/ml. Fractions positive by Western blot analysis and which only showed a single band by SDS/PAGE were pooled and dialyzed extensively in 50 mM Na 2 HP0 4 /2 mM Na 2 EDTA, pH 6.8.

The recovery of purified product from cell extract was estimated to be greater than 42%, with a yield of 140 mg/kg of cell paste (Table The resulting rBoNTF(Hc) was judged to be greater than 98% pure as only a single band was detected by SDS-PAGE (Figure 20) even when moderately (4 fig) overloaded.

Capillary isoelectric focusing showed the antigen possessed an isoelectric point of 9.4 (data not shown), which is in reasonable agreement with the calculated pi of 9.1.

TABLE 2. Purification of soluble rBoNTF(Hc) Total protein concentration was determined by Pierce BCATM assay. rBoNTF(Hc) was identified by Western blot analysis and purity was estimated by analysis of individual lanes of SDS/PAGE by pixel densitometry using NIH imaging software.

Concentration Protein rBoNTF(Hc) Purity Fold Recovei Step (mg/ml) (mg) (mg) Purification Lysate 11 1100 5.6 <0.5 3.8 Dialzed extract 7.8 740 3.7 <0.5 66 Mono S 1.2 9.6 2.5 26 >52 Alyl superose 0.52 1.6 1.6 100 3.8 29 CD of purified soluble and resolubilized rBoNTF(Hc) Purified soluble and resolubilized rBoNTF(Hc) were subjected to CD spectroscopy in a Jasco 600 spectropolarimeter (Japan Spectroscopy company, Tokyo, Japan). Experiments were performed at a concentration of 30 [tg/ml (0.62 pM) in a 1 cm path length cell in 10 mM Na 2

HPO

4 pH 7.0. Spectra were obtained WO 00/67700 PCT/US00/12890 as an average of four accumulations, scanned from 260-200 nm, at a scan rate of nm/min, with a 2 sec response, and a 1 nm band width. The temperature was maintained at 20°C with a Peltier thermocontrol device.

Analysis of the far-UV circular dichroism spectrum (Figure 22) of the purified antigen showed a positive peak at 233 nm and a minimum at 214 nm. This suggests the molecule is in a folded conformation and possesses considerable Psheet.

Example 5. Purification of Resolubilized rBoNTF(Hc) Western blot analysis revealed that approximately 30-40% of the total expressed rBoNTF(Hc) was present in the insoluble pellet after cell lysis. To investigate whether this insoluble protein could be recovered, the pellet was extracted in the denaturant urea and then dialyzed in nondenaturing buffer.

FPLC purification of resolubilized rBoNTF(Hc) The cell lysate pellet was resuspended into 20 ml of 3 M urea/50 mM Na 2

HPO

4 pH 7.0 and extracted 15 h at 4°C on a Labquake rotator. The cellular components not solubilized by the denaturing buffer were removed by centrifugation with a Sorval SS-34 rotor at 15000 g for 10 min at 4'C. The supernatant was dialyzed extensively using 10 kDa MWCO Pierce Slide-A-Lyzer dialysis cassettes in buffer A. A slight precipitate formed during the dialysis which was removed by centrifugation as described above. Western blot analysis showed that rBoNTF(Hc) was present only in the supernatant, which was estimated to be about 35% pure by SDS/PAGE. The supernatant was loaded onto a Pharmacia HR 10/10 Mono S cation-exchange column and separated by the same conditions as above. Fractions containing only a single positive rBoNTF(Hc) band SDS/PAGE and Western blot analysis were pooled and dialyzed in 50 mM Na 2 HP04/2 mM Na 2 EDTA, pH 6.8 in kDa MWCO dialysis cassettes. The final resolubilized rBoNTF(Hc) product was judged to be greater than 98% pure as determined by SDS/PAGE.

After a single cation exchange chromatography separation step, the rBoNTF(Hc) was greater than 98% pure as judged by SDS-PAGE. The total yield of purified resolubilized rBoNTF(Hc) was 100 mg/kg of cell paste. The conformation of purified resolubilized antigen showed significant P-sheet as determined by CD spectral analysis (Figure 22). However, the overall fold appeared WO 00/67700 PCT/US00/12890 slightly different than that shown by rBoNTF(Hc) purified from the cell lysate supernatant. The primary difference was the lack of a positive peak at 233 nm, indicating differences in P-sheet. content.

Example 6. Mouse Immunogenicity and Efficacy Studies To assess the immunogenicity of the recombinant rBoNTF(Hc), mice were inoculated with either one, two, or three doses of purified rBoNTF(Hc) from the soluble fraction of lysate at doses ranging from 0.008 to 5 ug per mouse.

Mouse inoculations and BoNTF toxin challenge Mice, Crl:CD-1, ICR mice (Charles River, NC, weighing 16-22 g on receipt, were injected intramuscularly with purified rBoNTF(Hc). Mice were challenged intraperitoneally 21 days after their last rBoNTF(Hc) injection with BoNTF toxin complex (Langeland strain) diluted in gelatin/0.4% (w/v) Na 2

HPO

4 pH 6.2, in 100 [l total volume per mouse. Groups of five naive mice were also used as toxin controls. Mice were observed daily and deaths were recorded five days post challenge. All animal manipulations were in accordance with applicable regulations in AAALAC-accredited facilities.

The efficacy of the purified soluble rBoNTF(Hc) was determined by inoculating groups of five female mice with one, two, or three doses of 0.008, 0.04, 0.2, 1.0, or 5.0 gg rBoNTF(Hc) (diluted in 100 tl of 0.2% Alhydrogel (Superfos Biosector, Kvistgaard, Denmark) in 0.9% saline) per mouse at 14 day intervals. Two days before challenge, mice were bled retroorbitally and serum was collected for ELISA testing Mice were challenged with 1000 mouse i.p. LDso of BoNTF toxin complex.

All of the mice, including five naive controls, were challenged with 1000 mouse ip LDso of BoNTF toxin. The controls all died within 2-4 h. A dose response was observed from groups of mice receiving different numbers of inoculations (Table A single inoculation of 5 jg protected four of five mice, while a dose of 0.2 pg or below protected one or no mice. Two and three inoculations protected four of five and five of five mice at doses of 0.2 and 0.04 pg, respectively. At all dose levels studied, the number of surviving mice increases with the number of inoculations.

PCT/USOO/12879 WO 00/68044 Serum antibody titers for each individual mouse were determined by ELISA, followed by calculation of the geometric mean titers for each group in the study.

Mouse serum ELISA Individual mouse serum ELISAs were performed as previously described (Byrne, 1998) except for the following differences. Botulinum neurotoxin serotype F (Langeland strain, Food Research Institute, University of Wisconsin, Madison, WI, was used as the coating antigen and the positive control for each assay was a mouse IgG monoclonal antibody, 7F8.G2.H3 (Brown, et al., (1997), "Identification and Characterization of a Neutralizing Monoclonal Antibody Against Botulinum Neurotoxin, Serotype F, Following Vaccination with Active Toxin," Hybridoma, 16:447-456).

TABLE 3. Survival, antibody group ELISA titers, and serum neutralization titers of mice after inoculation with purified soluble rBoNTF(H,) Mice were challenged with 1000 i.p. LD 50 BoNTF toxin 21 days after last inoculation Antibody ELISA titers were measured as the reciprocal of the highest dilution having an OD 405 greater than 0.2 AU after correcting for background.

Geometric mean ELISA titers were determined by taking the geometric mean of the logarithm of the individual titers. Standard deviations of the geometric means are also reported. If the ELISA titer was determined to be below the detection limit of the assay the ELISA titer was arbitrarily assigned a value of 25. A geometric mean titer value of 1.4 means that all ELISA titers within that group were below the detection limit.

Vaccination Survival (alive/5 tested) Geometric mean ELISA titers dose (pg) IX 2X 3X IX 2X 3X 0.008 0* 0 2 1.4 1.4 1.6±0.3 0.04 0 1 4 1.4 1.5±0.3 2.4±0.8 0.2 1 4 5 1.4 2.1±0.9 2.9±1.3 2 5 5 1.4 2.8±0.3 4.3±0.3 4 4 5 1.6±0.3 2.8±0.9 4.1+0.5 Only four mice were tested within this group WO 00/67700 PCT/US00/12890 Statistical analysis The logistic regression model was used to test associations of geometric mean ELISA titers and individual titers with survival by using SAS, version 6.10.

Geometric mean titers correlated well with protection (Table The three groups with no survivors had geometric means titers below the detection limit of the assay Similarly, the four groups that showed complete protection had geometric means titers of 2.8 or greater. Individual mouse antibody titers correlated extremely well with protection (Table Only 7 out of 38 mice survived whose titers were below 100. On the other hand, 34 out of 34 survived whose titers were 100 or greater. One mouse in the study could be classified as a "nonresponder." The mouse, receiving two injections at the highest dose level, had an antibody titer below the detection limit and did not survive the BoNTF challenge. The rest of the mice in that particular group had titers of 1600 or greater.

TABLE 4. Correlation of individual antibody ELISA titer with protection after inoculation with purified soluble rBoNTF(H,) Serum was bled from each mouse individually. Titer is reciprocal of the highest dilution having an OD 405 greater than 0.2 AU after correcting for background. Mice were challenged with 1000 i.p. LDso BoNTF toxin 21 days after last inoculation.

Individual Survival ELISA titer (alive/total)* survival <100 7/38 18.4 100 7/7 100 400 4/4 100 1600 11/11 100 6400 3/3 100 25600 9/9 100 The individual antibody titers from three mice were not measured. Two mice did not offer enough serum and one mouse was not challenged.

WO 00/67700 PCT/US00/12890 The resolubilized antigen was also evaluated for immunogencity and protective efficacy by its ability to protect mice from a BoNTF toxin challenge.

Groups of 10 male mice each received three inoculations of either 1 Ig or 5 Lg of rBoNTF(Hc) (diluted in 100 pl 0.2% Alhydrogel in 0.9% saline) per mouse at 14 day intervals. Two days before challenge, mice were bled retroorbitally and serum was collected for ELISA testing Mice inoculated with 1 ig doses were challenged with 5000 mouse ip LDso of BoNTF toxin. Ten of ten mice survived the challenge. Because 100% protection was observed with the group inoculated with 1 pg doses, the group that received three doses of 5 jg were subjected to a challenged level two orders of magnitude greater in order to test the limits of the antigen.

Therefore, the 5 pg dose group was challenged with 500,000 mouse ip LDso of BoNTF toxin. None of the mice survived the challenge; however, a significant delay in time to death was observed (24-48 All the control mice succumbed within 2-4 h after challenge.

Example 7. Synthesis and clonining of a synthetic gene encoding rBoNTA (Hc) The preparation of genetically engineered proteins to provide protection from the toxins produced by Clostridium botulinum was accomplished in E. coli.

Restriction endonucleases and DNA modifying enzymes were obtained from GIBCO BRL (Gaithersburg, Maryland). Polymerase chain reaction (PCR) reagents were purchased from Perkin-Elmer Cetus (Norwalk, CT). SDS PAGE precast gels and running buffers were acquired from Amersham (Arlington Heights, Illinois). All oligonucleotides were synthesized by Macromolectular Resources (Ft. Collins, Colorado). ELISA reagents were obtained in house or from Sigma (St. Louis, Missouri) or Kirkegard and Perry Laboratories (Gaithersburg, Maryland).

The Escherichia coli host was K12DH5a, purchased as competent cells from GIBCO BRL. Expression vectors pMAL from New England Biolabs (Beverly, Massachusetts) and pKK233-2 from Pharmacia LKB (Piscataway, New P.\Op.Ej'b. mwd\OD3S 00 y 1om.dOO3JO 3 Jersey) were used according to the manufacturers' standard protocols. The DNA clone coding of the He domain of c. botulinum toxin serotype A wa pCBA3, kindly provided by Nigel Minton.

Oligonucleotide primers incorporating appropriate terminal restriction enzyme sites were used to PCR amplify the He region of the C.

botulinum clone pCBA3. Gel-purified insert DNA and vector DNA were cleaved with the appropriate restriction enzymes, purified on low melting point agarose, and ligated overnight at room temperature. Competent DH5a host cells were transformed according to suppliers recommendations and plated on LB plates with 100 ug/ml ampicillin. Protein electrophoresis was run on precast 11-20% SDS PAGE at the manufacturer's recommended parameters. ELISA plates were incubated with capture antibody (horse anti-botulinum A polyclonal serum) overnight, then blocked with skim milk prior to application of various dilutions of tests material, signal antibody (rabit antibotulinum A polyclonal serum), signal HRP conjugated anti-(rabbit IgG) and ABTS substrate solution. Plates were read on an automated reader at 405 nm.

The sequence of the C fragment of the A chain was deduced as SEQ ID NO:38.

The C fragment protein sequence was reversed translated using E. coli optimal codon usage. The gene was then altered in many places to insert restriction sites, start codon, stop codon. Other changes were also effected to make the molecule more appropriate for use in the vector. Throughout, the fidelity of the protein sequence generated therefrom was maintained.

The sequence for the synthetic gene is SEQ ID NO:37.

oo *oo *•o *°oo -36- P:OpcrEj'b mwdMs5oofJ.30.00 y mo ~c dc4W03103 This gene has been synthesized using a large number of oligmers of approximately 60-65 bases corresponding to the sequences of the and strands.

The oligomers had overlaps of 7 bases. The oligomers were allowed to anneal and were ligated to form 5 subunits of 250-300 base pairs each. Each subunit had been designed to have restriction sites at their termini which allowed them to be -37- P.Xpcr\Ejb.=wdd\00 0035 sm uy mwddpages dmcc-13/03 assembled in the right order to form the complete gene. On confirmation there was shown that the correct gene had 7 deletion errors. These errors were repaired using in vitro mutagenesis and the repair sites sequenced to confirm.

Example 8. Synthesis and cloning of a synthetic gene encoding rBoNTB (Hc) The C fragment for botulism toxin serotype B of Whelan was studied and the portion of the protein having the sequence of SEQ ID NO:40 was defined as the C fragment.

The synthetic gene for expression in E. coli was produced in the manner described for synthesis of the gene for the C fragment of the A strand, namely, using a large number of oligomers of approximately 60-65 bases corresponding to the sequences of the and strands with overlaps of 7 bases. The oligomers were allowed to anneal and were ligated to form subunits of 250-300 base pairs each.

Each subunit had been designed to have restriction sites at their termini which allowed them to be assembled in the right order to form the complete gene. The synthetic gene encoding the C fragment of the B toxin is SEQ ID NO:39.

go -38- POp Ejkmde5OO35 OOnsy =mde.pq doc-03/03 Cloning: Supematants of sonicated, IPTG-induced recombinant pMAL fusion E. coli cultures were tested for the presence of the botulinum He expression product by ELISA and SDS-PAGE gels stained with coomassie brilliant blue were unsuccessful. Attempts to express He fragment as a non-fusion product were unsuccessful. Initial characterization of plasmid DNA from putative clones in Pkk233-2 demonstrated an insert of the expected size was present. In addition, SDS-PAGE indicated the presence of a protein of approximately 50 kDa after induction. However, the recombinants appeared unstable and further preparations of this and other cultures failed to reproduce these results. This approach was subsequently abandoned in favor of the fusion product expression.

-39- WO 00/67700 PCT/US00/12890 Example 9 Immunization Trials: Although attempts to quantitate expressed Hc fusion products were unsuccessful, limited immunization trials were performed on mice to evaluate the vaccine potential of the product. Initial vaccination employed concentrated, crude E. coli lysate with complete Freund's adjuvant. Two weeks later, animals were boosted with amylose column-purified expression product with Freund's incomplete adjuvant. At this time, a second group of five animals received amylose purified product in Freund's incomplete adjuvant as a single vaccination. After two additional weeks, both groups were challenged intraperitoneally with a dose of 3

LD

5 o of toxin. All eleven animals receiving two immunizations with Hc survived while six of the twelve control animals receiving pMAL vector alone died.

Likewise, all five animals receiving one Hc vaccination survived while animals receiving the pMAL vector alone died.

Four weeks after the initial challenge with 3 LDs 5 o of toxin, nine of the eleven animals who had received two immunizations were exposed to 30, 300, or 1200

LD

5 0 doses of toxin. The animals succumbing to the toxin challenge of 30 and 300 LDs 5 o did not exhibit fatality typical of botulinum toxin poisoning in that they appeared healthy after 18 hours, but were dead a few hours thereafter. In contrast, the animal which died from the 1200 LDs 5 0 dose appeared moribund when examined at 18 hours and remained so until death. This reaction is consistent with symptoms usually observed with botulinum toxin-induced paralysis. Additional data on second challenge is shown on Table 5. Hence, it was shown that immunization with the genetically engineered toxin protected against large doses of the toxin.

It is also possible to produce antibodies using the genetically engineered toxin. Because the toxin is not disease-producing in the animal, it is possible to produce large amounts of antitoxin more cheaply. It is also possible to produce antitoxin using hybridoma technology.

WO 00/67700 PCT/US00/12890 TABLE PROTECTION OF MICE IMMUNIZED WITH He OF A TOXIN DERIVED FROM SYNTHETIC GENE of deaths/total animals) Calculated challenge Control Protected dose (LD 50) (vector without insert) (Vector with insert) 4 2/3 0/3 0/3 3/4 0/3 100 0/3 300 0/3 1000 0/3 3000 0/1 The animals received vaccinations of crude lysated cell material at 0, 2 and 4 weeks.

Challenges were administered intraperitoneally with serotype A toxin at 5 weeks.

Example 10 rBoNTA(Hc) purification and protective effect Recombinant BoNTA(Hc) peptide was produced recombinantly in yeast. The first step in the purification process for BoNTA(Hc) was a Streamline expanded bed chromatography column. The product was eluted by a sodium chloride step gradient.

Product eluted from the expanded bed chromatography column was estimated to be pure with a total protein concentration of 0.92 mg/ml. After dialyzing the salt away, the material was loaded onto a mono S cation exchange column for further purification.

Western blot and ELISA data indicated that BoNTA(Hc) eluted from the column at 110 mM sodium chloride. The Mono S pool was subjected to HIC as a final purification step and thus, the material was adjusted to 1.5 M ammonium sulfate. The Mono S product was loaded onto a HIC column and eluted with a gradient of decreasing ammonium sulfate.

Product eluted at 1.04 M ammonium sulfate and BoNTA(Hc) immunologically positive fractions were combined and dialyzed to remove ammonium sulfate. Only a 50 kDa BoNTA(Hc) band was detected by SDS-PAGE and Western blot analysis and was judged to be greater than 95% pure after the final step. Protective effect of this purified material was measured by immunizing mice with 1 dose followed by challenge with 1000 LD50 of BoNTA(Hc). The results are shown in Table 6 below.

WO 00/67700 PCT/USOO/12890 Table 6 Potency assay: 1 dose followed by challenge with 1000 LD50 of BoNTA(Hc) Dose (pg) survival 10/10 10/10 0.625 10/10 0.156 7/10 0.039 2/10 0.0098 0/10 0.0024 0/10 Example 11 rBoNTB(Hc) purification and protective effect Recombinant BoNTC(Hc) peptide was produced recombinantly in yeast. The first separation technique employed for the purification process for BoNTB(Hc) was Streamline chromatography (Pharmacia), which is a single pass expanded bed adsorption operation where proteins can be recovered from crude feed stock or cell lysate without prior clarification. Significant clean-up was accomplished in this step as the MES buffer system prohibited binding of a large percentage of unwanted proteins to the SP resin.

Protein was loaded onto the column at a concentration of 123 mg/mL-resin, using 20 mM MES buffer, pH 5.7 with 10 mM NaCI. The product pool was eluted in a single step.

Under the conditions investigated, on average 3.9% of the total protein loaded was recovered in the elution peak, and the product pool was approximately 70% BoNTB(Hc) fragment based on SDS-PAGE.

The second chromatography step in the process utilizes Poros HS, another strong cation exchange resin. The buffer system was similar to that used for Streamline SP, however enhanced selectivity of Poros HS enriched the product peak to about 85% purity.

The product peak eluted during the gradient at approximately 130 mM NaCI. Strongly bound proteins were eluted with 1 M NaCI.

The final chromatography step utilized a Poros PI column. Analysis of the PI fractions by SDS-PAGE and IEF revealed that the product band, a single band at 50 kD WO 00/67700 PCT/US00/12890 on SDS-PAGE, was present in the pH 8.0 fraction. Analysis of purified BoNTB(Hc) fragment by 2-D electrophoresis resulted in one major spot and two minor, faint spots from the PI-peak 1 fraction. Peak 2 contained several spots at two different molecular weights corresponding to 50 kD and 47 kD. Presumably these spots represent different isoforms. IEF banding patterns detected in the first dimension are in agreement with those seen in Phast IEF for the two peaks. The protective efficacy of this material was determined by potency assay of 1 dose followed by challenge with 1000 LD50 of BoNTB(Hc). The results are shown in the following Table 7.

Table 7 BoNTB(Hc Dose (gg) survival 10/10 10/10 0.625 10/10 0.156 6/10 0.039 1/10 0.0098 0/10 0.0024 0/10 Example 12 rBoNTC(Hc) purification and protective effect Recombinant BoNTC(Hc) peptide was produced recombinantly in yeast. The initial chromatography step used for the purification process for BoNTCi(Hc) was a Mono Q anion-exchange column. The column was equilibrated with 50 mM sodium phosphate, 0.2% CHAPS, 2 mM EDTA, pH 7.0. The CHAPS was incorporated into the column buffers to allow product to elute from the column over a narrower sodium chloride concentration. Fractions positive for BoNTCI(Hc) by Western analysis were pooled and adjusted to 1 M ammonium sulfate. A moderate precipitate formed which was removed by passing the material through a 0.2 .t filtration unit. The clarified Mono Q product pool was subjected to hydrophobic interaction chromatography using a Pharmacia alkyl superose column. This final step removed the remainder of the impurities liberating BoNTCI(Hc) product which was estimated to be greater than 98% pure as judged by SDS/PAGE. Protective effect of this purified material was measured by immunizing mice WO 00/67700 PCT/US00/12890 with 1 dose followed by challenge with 1000 LD50 of BoNTCi(Hc). The results are shown in Table 8 below.

Table 8. Potency Assay: One dose followed by challenge with 1000 of BoNTCI(Hc) Dose (jig) Survival 8.1 10/10 2.7 10/10 0.9 10/10 0.3 9/10 0.1 4/10 0.033 0/10 0.011 0/10 For purposes of clarity of understanding, the foregoing invention has been described in some detail by way of illustration and example in conjunction with specific embodiments, although other aspects, advantages and modifications will be apparent to those skilled in the art to which the invention pertains. The foregoing description and examples are intended to illustrate, but not limit the scope of the invention. Modifications of the above-described modes for carrying out the invention that are apparent to persons of skill in medicine, immunology, hybridoma technology, pharmacology, and/or related fields are intended to be within the scope of the invention, which is limited only by the appended claims.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

WO 00/67700 WO 0067700PCTUSOO/1 2890 //rem S pieiS AO11.*S *EFORMT of 3 6oul2tU6t efte I has from, 1 to& 1152 2-A-0711:40 iAU 1 C4iii Nifkr Frat 1cg REFORMAT of.

from: From REFORMAT of.: f roam: From REFORMAT of: from: from I REFORMAT of: from: AMAC Id COOO*n p IC11I a A012 loans aou2sttPK.dat thWAk: 0641 1 to: 1002 27-44AY-128T 11:41 Ptchla OAS1 gene aaststruc.dst check: 3101 i to: 211U 22-APR.1987 15:08 PICA14 OAS2 gone aas2struc.dal check: 5479 Ito: 2124 t5SJUN..1287 14:3 PICAIaA gone agaostruc.dat Check: 9059 i to: 1002 IS-JUN-ISS? 1d:38 MaSber fraction lTar Thr Thr The.

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0.00 55.00 197.00 9.00 112.00 60.00 56.00 118.00 10.00 8.00 107.00 87.00 1.00 2S.00 147.00 71.00 2-00 111.00 5.00 3.00 1195.00 3.5.00 18.00 119.00 0.00 o .22 0.*74 0.02 0.05 0.32 0.86 0.04 0.0 0.701 0.00 0.10 0.201

ATC

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ACA

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TGT

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TAC

'rG TrA 'rcf TrC

TCA

TCT,

CGC

CG

CAT

CC

CYC

CTA

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CC

CCA

cC? 0.00 G.00 93.00 71.00 5.0 6.00 U8.00 74.00 20.00.

0.00 35.00 7.00 1.00 4.00 16.00 125.00 120.00 21.00 24.00 104.00 5.00 14.00 89.00.

71.00 2.00 0 .00 26.00 0.00 31 .00 59.00 11.00 77-00 35.00 7.00 A3.00 7.00 0.00 97.00 S8.00 7.00 1.00 0.00 0.56 0.44 0.03 0.05 0.50 0.42 .1;00 0.00 0.83 0.17 0.20 0.60 0.12 0. U 04* 2 0.00 0.19 0.1 0.03 0.97 0.*47 0.27 0.01 0.00 0.161 0.00 0.3' 0.6 0.13 0.88 0.15 0.02 0.18 0.03 0.00 0.57 0.30 0.04 Afq Mg Mrg Mrg Lou Lou Lou no Pro Pro Pro 1rixA LI P I CW A C44~C USG0 rjc=I2K UZACX E P.\OPERUiPM\Unacd Sumc Amsy Madiu Reserd\2465372\2465372 mosd spdocm9JOm Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

a o* oo EDITORIAL NOTE APPLICATION NUMBER 50035/00 The following Sequence Listing pages 1 to 71 are part of the description. The claims pages follow on pages 46 to 47.

PZpcajEjh ffd\5O35 00 os y=Wd sduinngdoc 03=2003 -1- <110> Smith, Leonard A.

Byrne, Michael P.

Middlebrook, John L.

Lapenotiere, Hugh Clayton, Michael A.

Brown, Douglas R.

<120> RECOMBINANT VACCINE AGAINST BOTULINUM NEUROTOXIN <130> A33626 067252.0105 <140> 09/611,419 <141> 2000-07-06 <150> PCT/US00/12890 <151> 2000-05-12 <150> 60/133,865 <151> 1999-05-12 <150> 60/133,866 <151> 1999-05-12 <150> 60/133,867 <151> 1999-05-12 <150> 60/133,868 <151> 1999-05-12 <150> 60/133,869 1999-05-12 <150> 60/146,192 <151> 1999-07-29 <160> 42 <170> FastSEQ for Windows Version <210> 1 <211> 1332 <212> DNA <213> Artificial Sequence o <220> <223> Synthetic construct based on BoNTA He <221> CDS <222> (13) (1326) <400> 1 gaattcgaaa cg atg cgt ctg ctg tct acc ttc act gaa tac atc aag aac 51 Met Arg Leu Leu Ser Thr Phe Thr Glu Tyr Ile Lys Asn 1 5 ate atc aat acc tcc atc ctg aac ctg cgc tac gaa tcc aat cac ctg 99 Ile Ile Asn Thr Ser Ile Leu Asn Leu Arg Tyr Glu Ser Asn His Leu P.\Oper\Ejh =c&dd\5OO335O usy a de"isdini &xc03/0317003 -2gao ctg tct cgo Asp Leu Ser Arg tac Tyr got tco aaa atc Ala Ser Lys Ile aac Asn 40 ato ggt tot aaa Ile Gly Ser Lys gtt Val1 147 195 aao tto gat oog Asn Phe Asp Pro gao aag aat oag Asp Lys Asn Gin oag otg tto aat Gin Leu Phe Asn otg gaa Leu Giu tot too aaa Ser Ser Lys atg tao gaa Met Tyr Glu ato Ile gaa gtt ato otg Glu Val Ile Leu aag Lys 70 aat got ato gta Asn Ala Ile Val tao aao tot Tyr Asn Ser oog aaa tao Pro Lys Tyr aao tto too aoo Asn Phe Ser Thr tto tgg ato ogt Phe Trp Ile Arg ato Ile tto aao Phe Asn too ato tot otg Ser Ile Ser Leu aao As n 100 aat gaa tao aoo Asn Giu Tyr Thr ato Ile 105 ato aao tgo atg Ile Asn Cys Met gaa Giu 110 aao aat tot ggt Asn Asn Ser Giy tgg Trp 115 aaa gta tot otg Lys Vai Ser Leu tao ggt gaa ato Tyr Giy Giu Ile ato Ile 125 tgg aot otg oag Trp Thr Leu Gin aot oag gaa ato Thr Gin Giu Ile aaa Lys 13S oag ogt gtt gta tto aaa Gin Arg Val Val Phe Lys 339 387 435 483 531 tao tot oag Tyr Ser Gin gtt aoo ato Val Thr Ile 160 atg Met 145 ato aao ato tot Ile Asn Ile Ser gao Asp 150 tao ato aat ogo Tyr Ile Asn Arg tgg ato tto Trp Ile Phe 155 tao ato aao Tyr Ile Asn aoo aao aat ogt Thr Asn Asn Arg aat aao too aaa Asn Asn Ser Lys ato Ile 170 ggo ogt Gly Arg 175 otg ato gao oag Leu Ilie Asp Gin aaa Lys 180 oog ato too aat Pro Ilie Ser Asn otg Leu 185 ggt aao ato oao Gly Asn Ile His got Ala 190 tot aat aao ato Ser Asn Asn Ile atg Met 195 tto aaa otg gao Phe Lys Leu Asp tgt ogt gao aot Cys Arg Asp Thr oao His 205 ogo tao ato tgg Arg Tyr Ilie Trp aaa tao tto aat Lys Tyr Phe Asn otg Leu 215 tto gao aaa gaa Phe Asp Lys Giu otg aao Leu Asn 220 579 627 675 723 771 gaa aaa gaa Giu Lys Giu otg aaa gao Leu Lys Asp 240 ato Ile 225 aaa gao otg tao Lys Asp Leu Tyr gao Asp 230 aao oag too aat Asn Gin Ser Asn tot ggt ato Ser Gly Ile 235 oog tao tao Pro Tyr Tyr tto tgg ggt gao Phe Trp Gly Asp tao Tyr 245 otg oag tao gao Leu Gin Tyr Asp aaa Lys 250 M p.\Ejbh -wdd50035.00 .w y- d. Iisvig d.~-03/03/2003 atg ctg aat ctg tac gat ccg aac aaa Met Leu Asn Leu Tyr Asp Pro Asn Lys 255 260 ggt Gly 270 act Thr atc Ile aat Asn ctg Leu ctg Leu 350 tcc Ser gac Asp aat Asn cgt Arg atc Ile ac c Thr atc Ile gat Asp gct Ala 335 gaa Giu aag Lys aac Asn atc Ile tcc Ser 415 cgc Arg aac Asn aag Lys cgt Arg 320 acc Thr atc Ile aac Asn aat Asn gct Ala 400 tct Ser ggt Gly at c Ile aaa Lys 305 gta Val1 aat Asn ccg Pro gac Asp ggt Gly 385 aaa Lys cgc Arg tac Tyr tac Tyr 290 tac Tyr tac Tyr gc t Al a gac Asp c ag Gin 370 aac Asn ctg Leu act Thr atg Met 275 ctg Leu gcg Al a atc Ile tct Ser gtt Vali 355 ggt Giy gat Asp gt t Val1 ctg Leu tac Tyr aac As n tct Ser aat Asn c ag Gin 340 ggt Giy atc Ile atc Ile gct Aia ggt Gly 420 ctg Leu tct Ser ggt Gly gtt Val1 325 gc t Ala aat Asn act Thr ggt Giy tcc Ser 405 tgc Cys aaa Lys tcc Ser aac Asn 310 gta Val1 ggt Gly ctg Leu aac Asn ttc Phe 390 aac As n tct Ser tac Tyr ggt Giy ctg Leu 295 aag Lys gtt Vali gta Val1 tct Ser aaa Lys 375 atc Ile tgg Trp tgg Trp gtt Vali ccg Pro 280 tac Tyr gac Asp aag Lys gaa Glu c ag Gin 360 tgc Cys ggt Giy tac Tyr gag Glu gac Asp 265 cgt Arg cgt Arg aat Asn aac As n aag Lys 345 gta Vali aaa Lys ttc Phe aat Asn ttc Phe 425 gtc Vali ggt Gly ggt Giy atc Ile aaa Lys 330 atc Ile gtt Vali atg Met cac His cgt Arg 410 atc Ile aac Asn tct Ser acc Thr gtt Val1 315 gaa Glu ttg Leu gta Vali aat Asn cag Gin 395 c ag Gin ccg Pro aat As n gtt Vali aaa Lys 300 cgc Arg tac Tyr tct Ser atg Met ctg Leu 380 ttc Phe atc Ile gtt Vali gta Vai atg Met 285 ttc Phe aac Asn cgt Arg gct Ala aaa Lys 365 c ag Gin aac Asn gaa Glu gat Asp 819 867 915 963 loll 1059 1107 1155 1203 1251 1299 gac ggt tgg ggt gaa cgt ccg ctg taa gaattc Asp Giy Trp Gly Giu Arg Pro Leu 430 435 <210> 2 <211> 437 <212> PRT <213> Artificial Sequence <220> <223> Encoded poiypeptide of a synthetic construct based 1332 P QpwEjh modcd50035. 00 m dcsNisu doc.03/03/2003 -4on BoNTA Hc <400> 2 Met 1 Thr Ser Pro Ile Asn Ile Ser Gin Met 145 Thr Ile Asn Trp Ile 225 Phe Leu Giy Ile Lys 305 Vali Asn Pro Asp Gly 385 Lys Arg Ser Arg Ile Giu Phe Ser Gly Asp 130 Ile Asn Asp Ile Ilie 210 Lys Trp Tyr Tyr Tyr 290 Tyr Tyr Ala Asp Gin 370 Asn Leu Leu Ilie Tyr Asp Val1 Ser Leu Trp 115 Thr Asn Asn Gin Met 195 Lys Asp Gly Asp Met 275 Leu Al a Ile Ser Val1 355 Gly Asp Val Leu Leu Al a Lys Ile Thr As n i00 Lys Gin Ile Arg Lys 180 Phe Tyr Leu Asp Pro 260 Tyr As n Ser Asn Gin 340 Gly Ilie Ilie Ala Gly 420 Ser 5 Asn Ser Asn Leu Ser Asn Val1 Giu Ser Leu 165 Pro Lys Phe Tyr Tyr 245 Asn Leu Ser Gly Val1 325 Ala Asn Thr Gly Ser 405 Thr Leu Lys Gin Lys 70 Phe Giu Ser Ile Asp 150 Asn Ile Leu Asn Asp 230 Leu Lys Lys Ser Asn 310 Val1 Gly Leu Asn Phe 390 Asn Phe Arg Ile Ile 55 Asn Trp Tyr Leu Lys 135 Tyr Asn Ser Asp Leu 215 Asn Gin Tyr Gly Leu 295 Lys Val Val1 Ser Lys 375 Ile Trp Thr Tyr Asn 40 Gin Ala Ile Thr As n 120 Gin Ile Ser Asn Gly 200 Phe Gin Tyr Val Pro 280 Tyr Asp Lys Giu Gin 360 Cys Gly Tyr Giu Giu 25 Ile Leu Ile Arg Ile 105 Tyr Arg Asn Lys Leu 185 Cys Asp Ser Asp Asp 265 Arg Arg Asn Asn Lys 345 Val1 Lys Phe Asn Phe 425 Tyr 10 Ser Gly Phe Val1 Ile 90 Ile Gly Val1 Arg Ile 170 Gly Arg Lys Asn Lys 250 Val1 Gly Gly Ile Lys 330 Ile Val1 Met His Arg 410 Ile As n Ser As n Tyr 75 Pro Asn Giu Val1 Trp 155 Tyr Asn Asp Giu Ser 235 Pro As n Ser Thr Val1 315 Giu Leu Val1 As n Gin 395 Gin Lys His Lys Leu Asn Lys Cys Ile Phe 140 Ile Ile Ile Thr Leu 220 Gly Tyr Asn Val1 Lys 300 Arg Tyr Ser Met Leu 380 Phe Ile Asn Leu Val1 Giu Ser Tyr Met Ile 125 Lys Phe As n His His 205 Asn Ile Tyr Val1 Met 285 Phe Asn Arg Ala Lys 365 Gin As n Glu Ile Ile Asn Ser Met Phe Giu 110 Trp Tyr Val1 Gly Ala 190 Arg Giu Leu Met Gly 270 Thr Ile Asn Leu Leu 350 Ser Asp Asn Arg Ile Asp Phe Ser Tyr Asn Asn Thr Ser Thr Arg 175 Ser Tyr Lys Lys Leu 255 Ile Thr Ile Asp Ala 335 Giu Lys Asn Ile Ser 415 Asn Leu Asp Lys Giu Ser As n Leu Gin Ile 160 Leu Asn Ile Giu Asp 240 Asn Arg Asn Lys Arg 320 Thr Ile As n Asn Ala 400 Ser S. Arg Thr Leu Cys Ser Trp Glu Ile Pro Val Asp Asp Gly Trp 430 P.NOperEjb =mcd\30035 00 y mmdeswisig dow-033/20O3 Gly Glu Arg Pro Leu 435 <210> 3 <211> 1323 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA Hc <221> CDS <222> (13)..(1314) <400> 3 gaattcgaaa cg atg tct acc ttc act gaa tac atc aag aac atc atc aat 51 Met Ser Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn acc tcc Thr Ser atc ctg aac ctg Ile Leu Asn Leu cgc Arg tac gaa tcc aat Tyr Giu Ser Asn ctg atc gac ctg Leu Ile Asp Leu tct Ser cgc tac gct tcc Arg Tyr Ala Ser aaa Lys 35 atc aac atc ggt Ile Asn Ile Gly tct Ser 40 aaa gtt aac ttc Lys Val Asn Phe gat Asp ccg atc gac aag Pro Ile Asp Lys cag atc cag ctg Gin Ile Gin Leu ttc Phe aat ctg gaa tct Asn Leu Giu Ser tcc aaa Ser Lys 99 147 195 243 291 atc gaa gtt Ile Glu Val aac ttc tcc Asn Phe Ser atc Ile ctg aag aat gct Leu Lys Asn Ala atc I le 70 gta tac aac tct Val Tyr Asn Ser atg tac gaa Met Tyr Giu ttc aac tcc Phe Asn Ser acc tcc ttc tgg Thr Ser Phe Trp cgt atc ccg aaa Arg Ile Pro Lys tac Tyr atc tct Ile Ser 95 ctg aac aat gaa Leu Asn Asn Giu tac Tyr 100 acc atc atc aac Thr Ile Ile Asn tgc Cys 105 atg gaa aac aat Met Glu Asn Asn tct Ser 110 ggt tgg aaa gta Gly Trp Lys Val tct Ser 115 ctg aac tac ggt Leu Asn Tyr Gly atc atc tgg act Ile Ile Trp, Thr ctg Leu 125 339 387 435 cag gac act cag gaa atc aaa cag cgt Gin Asp Thr Gin Glu Ile Lys Gin Arg 130 gt t Val1 135 gta ttc aaa tac Val Phe Lys Tyr tct cag Ser Gin 140 atg atc aac Met Ile Asn atc Ile 145 tct gac tac atc Ser Asp Tyr Ile aat Asn 150 cgc tgg atc ttc Arg Trp Ile Phe gtt acc atc Val Thr Ile 155 acc aac aat cgt ctg aat aac tcc aaa atc tac atc aac ggc cgt ctg P.'OporEjh momded5OO35 OOuymdiqiig&.3O20 -6- Thr Asn Asn 160 Arg Leu Asn Asn Ser 165 Lys Ile Tyr Ile Asn Gly Arg Leu 170 atc gac Ile Asp 175 cag aaa ccg atc Gin Lys Pro Ile tcc Ser 180 aat ctg ggt aac Asn Leu Gly Asn atc Ile 185 cac gct tct aat His Ala Ser Asn aac Asn 190 atc atg ttc aaa Ile Met Phe Lys gac ggt tgt cgt Asp Giy Cys Arg act cac cgc tac Thr His Arg Tyr atc Ile 205 579 627 675 tgg atc aaa tac Trp Ile Lys Tyr ttc Phe 210 aat ctg ttc gac Asn Leu Phe Asp aaa Lys 215 gaa ctg aac gaa Giu Leu Asn Giu aaa gaa Lys Giu 220 atc aaa gac Ile Lys Asp ttc tgg ggt Phe Trp Gly 240 ctg Leu 225 tac gac aac cag Tyr Asp Asn Gin tcc Ser 230 aat tct ggt atc Asn Ser Giy Ile ctg aaa gac Leu Lys Asp 235 atg ctg aat Met Leu Asn gac tac ctg cag Asp Tyr Leu Gin tac Tyr 245 gac aaa ccg tac Asp Lys Pro Tyr tac Tyr 250 ctg tac Leu Tyr 255 gat ccg aac aaa Asp Pro Asn Lys tac Tyr 260 gtt gac gtc aac Vai Asp Val Asn aat Asn 265 gta ggt atc cgc Vai Gly Ile Arg ggt Gly 270 tac atg tac ctg Tyr Met Tyr Leu ggt ccg cgt ggt Gly Pro Arg Giy gtt atg act acc Val Met Thr Thr aac Asn 285 atc tac ctg aac Ile Tyr Leu Asn tct Ser 290 tcc ctg tac cgt Ser Leu Tyr Arg ggt Gly 295 acc aaa ttc atc Thr Lys Phe Ile atc aag Ile Lys 300 819 867 915 963 1011 aaa tac gcg Lys Tyr Ala gta tac atc Val Tyr Ilie 320 tct Ser 305 ggt aac aag gac Gly Asn Lys Asp aat Asn 310 atc gtt cgc aac Ile Vai Arg Asn aat gat cgt Asn Asp Arg 315 ctg gct acc Leu Ala Thr aat gtt gta gtt Asn Val Val Val aag Lys 325 aac aaa gaa tac Asn Lys Giu Tyr cgt Arg 330 aat gct Asn Ala 335 tct cag gct ggt Ser Gin Ala Gly gaa aag atc ttg Glu Lys Ile Leu gct ctg gaa atc Ala Leu Glu Ile ccg Pro 350 gac gtt ggt aat Asp Val Gly Asn ctg Leu 355 tct cag gta gtt Ser Gin Val Val gta Val1 360 atg aaa tcc aag Met Lys Ser Lys 1059 1107 1155 gac cag ggt atc Asp Gin Gly Ile act Thr 370 aac aaa tgc aaa Asn Lys Cys Lys atg Met 375 aat ctg cag gac Asn Leu Gin Asp aac aat Asn Asn 380 ggt aac gat atc ggt ttc atc ggt ttc cac cag ttc aac aat atc gct Gly Asn Asp Ilie Gly Phe Ile Gly Phe His Gin Phe Asn Asn Ile Ala 1203 P. OpcrEjlhmwd OO35.00. y~ md IIising dom.O3/O3/2003 385 390 aaa ctg gtt gct tcc aac tgg tac aat Lys Leu Val Ala Ser Asn Trp Tyr Asn 400 405 cgc act ctg ggt tgc tct tgg gag ttc Arg Thr Leu Gly Cys Ser Trp Giu Phe 415 420 ggt gaa cgt ccg ctg taagaattc Giy Giu Arg Pro Leu 430 395 cgt cag atc gaa cgt tcc tct Arg Gin Ile Glu Arg Ser Ser 410 atc ccg gtt gat gac ggt tgg Ile Pro Vai Asp Asp Giy Trp 425 1251 1299 1323 <210> 4 <211> 434 <212> PRT <2i3> Artificial Sequence <220> <223> Encoded polypeptide of a synthetic construct based on BoNTA Hc <400> 4 S

SS

S

Met 1 Leu Ala Lys Ile 65 Thr Asn Lys Gin Ile 145 Arg Lys Phe Tyr Leu 225 Asp Ser Asn Ser Asn 50 Leu Ser Asn Val1 Giu 130 Ser Leu Pro Lys Phe 210 Tyr Tyr Thr Leu Lys 35 Gin Lys Phe Giu Ser 115 Ile Asp Asn Ile Leu 195 Asn Asp Leu Phe Arg 20 Ile Ile As n Trp, Tyr 100 Leu Lys Tyr Asn Ser 180 Asp Leu Asn Gin Thr 5 Tyr Asn Gin Ala Ile Thr Asn Gin Ile Ser 165 Asn Gly Phe Gin Tyr 245 Giu Giu Ile Leu Ile 70 Arg Ile Tyr Arg Asn 150 Lys Leu Cys Asp Ser 230 Asp Tyr Ser Gly Phe 55 Val1 Ile Ile Giy Val1 135 Arg Ile Giy Arg Lys 215 Asn Lys Ile Asn Ser 40 Asn Tyr Pro Asn Giu 120 Val Trp Tyr Asn Asp 200 Glu Ser Pro Lys His 25 Lys Leu As n Lys Cys 105 Ile Phe Ile Ile Ile 185 Thr Leu Gly Tyr Asn 10 Leu Val1 Giu Ser Tyr 90 Met Ile Lys Phe Asn 170 His His Asn Ile Tyr 250 Ile Ile Asn Ser Met 75 Phe Giu Trp Tyr Val 155 Gly Ala Arg Giu Leu 235 Met Ile Asp Phe Ser Tyr Asn Asn Thr Ser 140 Thr Arg Ser Tyr Lys 220 Lys Leu As n Leu Asp Lys Giu Ser Asn Leu 125 Gin Ile Leu Asn Ile 205 Giu Asp Asn Thr Ser Pro Ile As n Ile Ser 110 Gin Met Thr Ile Asn 190 Trp Ile Phe Leu Ser Arg Ile Glu Phe Ser Gly Asp Ile Asn Asp 175 Ile Ile Lys Trp Tyr 255 Ile Tyr Asp Val1 Ser Leu Trp Thr As n Asn 160 Gin Met Lys Asp Gly 240 Asp *S 55

S

S S P.\Opa\Ejb mwde"30035 00.u y wewqito doc-03103/2003 Pro Asn Lys Tyr Leu Lys 275 Asn Ser Ser Tyr 260 Gly Val Asp Val Asn As n 265 Val1 Val Gly Ile Arg Pro Arg Gly Ser 280 Thr Met Thr Thr Asn 285 Lys Gly Tyr Met 270 Ile Tyr Leu Lys Tyr Ala Leu Tyr Arg 290 Ser Gly Gly 295 Ile Lys Phe Ile Asn Lys Asp Val Arg Asn 305 As n Asn 315 Leu Arg Val Tyr Ile 320 Val Val Val Lys 325 Giu Lys Giu Tyr Arg 330 Ala Ala Thr Asn Ala Ser 335 Gin Ala Gly Gly Asn Leu 355 Ile Thr Asn Val1 340 Ser Lys Ile Leu Ser 345 Met Leu Giu Ile Gin Val Val Lys Ser Lys Asn 365 Asn Pro Asp Val 350 Asp Gin Gly Gly Asn Asp Lys Cys Lys 370 Ile Gly Met 375 His Leu Gin Asp Asn 380 Ile Phe Ile Gly 385 Ala Phe 390 Asn Gin Phe Asn Ala Lys Leu Val1 400 Ser Asn Trp Tyr 405 Glu Arg Gin Ile Giu 410 Asp Ser Ser Arg Thr Leu 415 Giu Arg Gly Cys Ser Trp 420 Phe Ile Pro Val1 425 Asp Gly Trp Gly 430 Pro Leu SO <210> <211> 1326 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA Hc <221> CDS <222> (13) (1317) <400> gaattcgaaa cg atg gcc tct Met Ala Ser 1 acc ttc act gaa tac atc aag aac atc atc 51 Thr Phe Thr Giu Tyr Ile Lys Asn Ile Ile 5 aat acc tcc atc ctg aac ctg cgc tac gaa tcc aat cac ctg atc gac Asn Thr Ser Ile Leu Asn Leu Arg Tyr Giu Ser Asn His Leu Ile Asp 20 ctg tct cgc tac gct tcc aaa atc aac atc ggt tct aaa gtt aac ttc Leu Ser Arg Tyr Ala Ser Lys Ile Asn Ile Gly Ser Lys Val Asn Phe 30 35 40 gat ccg atc gac aag aat cag atc cag ctg ttc aat ctg gaa tct tcc Asp Pro Ile Asp Lys Asn Gin Ile Gin Leu Phe Asn Leu Giu Ser Ser 55 aaa atc gaa gtt atc ctg aag aat gct atc gta tac aac tct atg tac Lys Ile Giu Val Ile Leu Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr 70 P. pajhEO.-d\5035 00 .smwds~Iisdiq do.03/0312003 gaa aac tic Giu Asn Phe tcc acc tcc tic Ser Thr Ser Phe tgg Trp 85 atc cgt aic ccg Ile Arg Ile Pro aaa Lys tac ttc aac Tyr Phe Asn tcc aic Ser Ile tct cig aac aai Ser Leu Asn Asn gaa Giu 100 tac acc aic aic Tyr Thr Ile Ile aac As n 105 tgc aig gaa aac Cys Met Giu Asn aat Asn 110 ict ggi igg aaa Ser Gly Trp Lys gia Val1 115 tct cig aac tac Ser Leu Asn Tyr gaa atc atc tgg Giu Ile Ile Trp act Thr 125 339 387 435 ctg cag gac act Leu Gin Asp Thr gaa aic aaa cag Glu Ile Lys Gin git gia tic aaa Val Vai Phe Lys iac tct Tyr Ser 140 cag atg atc Gin Met Ile aic acc aac Ile Thr Asn 160 aac Asn 145 atc tct gac iac Ile Ser Asp Tyr atc Ile iSO aat cgc igg atc Asn Arg Trp Ile itc git acc Phe Val Thr 155 aac ggc cgt Asn Giy Arg aat cgt cig aat Asn Arg Leu Asn aac Asn 165 tcc aaa atc tac Ser Lys Ile Tyr ctg aic Leu Ilie 175 gac cag aaa ccg Asp Gin Lys Pro tcc aai cig ggt Ser Asn Leu Giy aac Asn 185 aic cac gct tct Ile His Ala Ser sea*

C.

aat Asn 190 aac aic aig iic Asn Ilie Met Phe aaa Lys 195 cig gac ggi igt Leu Asp Gly Cys cgi Arg 200 gac act cac cgc Asp Thr His Arg atc igg atc aaa Ile Trp Ile Lys tac Tyr 210 ttc aat ctg tic Phe Asn Leu Phe gac Asp 215 aaa gaa cig aac Lys Giu Leu Asn gaa aaa Giu Lys 220 gaa aic aaa Giu Ile Lys gac tic igg Asp Phe Trp 240 gac Asp 225 cig tac gac aac Leu Tyr Asp Asn cag Gin 230 tcc aat ici ggi Ser Asn Ser Giy aic ctg aaa Ile Leu Lys 235 iac aig cig Tyr Met Leu ggi gac tac cig Giy Asp Tyr Leu CC CC C C

CC..

C C

C

CCC.

C**C

cag Gin 245 tac gac aaa ccg Tyr Asp Lys Pro tac Tyr 250 aat ctg Asn Leu 255 tac gat ccg aac Tyr Asp Pro Asn aaa Lys 260 tac git gac gtc Tyr Vai Asp Val aac Asn 265 aai gta ggi aic Asn Val Gly Ile cgc Arg 270 ggi tac atg tac Gly Tyr Mei Tyr ctg Leu 275 aaa ggi ccg cgt Lys Gly Pro Arg ggt Giy 280 ict gti atg act Ser Val Met Thr aac atc tac ctg Asn Ilie Tyr Leu aac Asn 290 tct icc ctg tac Ser Ser Leu Tyr cgi Arg 295 ggt acc aaa tic Giy Thr Lys Phe atc aic Ile Ile 300 P.\Opa\Ejb.mc~odd\30035 OD .y-mdwisig.dc-03032003 aag aaa tac Lys Lys Tyr cgt gta tac Arg Val Tyr 320 gcg Ala 305 tct ggt aac aag Ser Gly Asn Lys aat atc gtt cgc Asn Ile Val Arg aac aat gat Asn Asn Asp 315 cgt ctg gct Arg Leu Ala 963 loll atc aat gtt gta Ile Asn Val Val gtt Val1 325 aag aac aaa gaa Lys Asn Lys Glu tac Tyr 330 acc aat Thr Asn 335 gct tct cag gct Ala Ser Gln Ala ggt Gly 340 gta gaa aag atc Val Glu Lys Ile ttg Leu 345 tct gct ctg gaa Ser Ala Leu Glu atc I le 350 ccg gac gtt ggt Pro Asp Val Gly aat Asn 355 ctg tct cag gta Leu Ser Gln Val gtt Val1 360 gta atg aaa tcc Val Met Lys Ser aag Lys 365 1059 1107 1155 aac gac cag ggt Asn Asp Gin Gly act aac aaa tgc Thr Asn Lys Cys atg aat ctg cag Met Asn Leu Gin gac aac Asp Asn 380 aat ggt aac Asn Gly Asn gct aaa ctg Ala Lys Leu 400 gat Asp 385 atc ggt ttc atc Ile Gly Phe Ile ggt Gly 390 ttc cac cag ttc Phe His Gin Phe aac aat atc Asn Asn Ile 395 gaa cgt tcc Giu Arg Ser 1203 1251 gtt gct tcc aac Val Ala Ser Asn tgg Trp 405 tac aat cgt cag Tyr Asn Arg Gin atc Ile 410 0* S 0 0*O C 0@ *O

C

S

0

OSSC

S

0@S S 0@ 0 0 tct cgc Ser Arg 415 act ctg ggt tgc Thr Leu Gly Cys tct Ser 420 tgg gag ttc atc Trp Glu Phe Ile ccg Pro 425 gtt gat gac ggt Val Asp Asp Gly 1299 tgg Trp 430 ggt gaa cgt ccg Gly Giu Arg Pro ctg Leu 435 taagaattc 1326 *0 *6 0 0 CS 00 5 0

S

0 e.g.

C.

a 0 0*.e

S

0S09 <210> 6 <211> 435 <212> PRT <213> Artificial Sequence <220> <223> Encoded polypeptide of a synthetic construct based on BoNTA Hc <400> 6 Met Ala Ser Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser 1 5 10 Ile Leu Asn Leu Arg Tyr Giu Ser Asn His Leu Ile Asp Leu Ser Arg 20 25 Tyr Ala Ser Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile 40 Asp Lys Asn Gin Ile Gin Leu Phe Asn Leu Glu Ser Ser Lys Ile Giu 55 Val Ile Leu Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe 70 75 P:'Op.'Ej -mdeM0035.00 .s y ds i~.dow-O03/Z003 Ser Thr Ser Phe Trp Ile Arg Ile Pro Leu Trp, Thr Asn 145 Asn Gin Met Lys Asp 225 Giy Asp Met Leu Aia 305 Ile Ser Val1 Giy Asp 385 Val1 Leu Arg Asn Lys Gin 130 Ile Arg Lys Phe Tyr 210 Leu Asp Pro Tyr Asn 290 Ser Asn Gin Giy Ile 370 Ile Ala Gly Pro Asn Val1 115 Giu Ser Leu Pro Lys 195 Phe Tyr Tyr As n Leu 275 Ser Gly Val1 Ala Asn 355 Thr Gly Ser Cys Leu 435 Giu 100 Ser Ile Asp As n Ile 180 Leu As n Asp Leu Lys 260 Lys Ser Asn Val1 Gly 340 Leu Asn Phe Asn Ser 420 Tyr Leu Lys Tyr Asn 165 Ser Asp Leu Asn Gin 245 Tyr Gly Leu Lys Val 325 Val1 Ser Lys Ile Trp 405 Trp Thr Asn Gin Ile 150 Ser Asn Giy Phe Gin 230 Tyr Val1 Pro Tyr Asp 310 Lys Giu Gin Cys Giy 390 Tyr Glu Ile Tyr Arg 135 As n Lys Leu Cys Asp 215 Ser Asp Asp Arg Arg 295 Asn As n Lys Val1 Lys 375 Phe Asn Phe Ile Gly 120 Val1 Arg Ile Gly Arg 200 Lys Asn Lys Vali Giy 280 Gly Ile Lys Ile Val1 360 Met His Arg Ile Asn 105 Giu Vali Trp Tyr Asn 185 Asp Giu Ser Pro Asn 265 Ser Thr Vali Giu Leu 345 Vali Asn Gin Gin Pro 425 Lys 90 Cys Ile Phe Ile Ile 170 Ile Thr Leu Gly Tyr 250 Asn Vali Lys Arg Tyr 330 Ser Met Leu Phe Ile 410 Val1 Tyr Met Ile Lys Phe 155 As n His His Asn Ile 235 Tyr Val1 Met Phe Asn 315 Arg Ala Lys Gin Asn 395 Giu Asp Phe Giu Trp Tyr 140 Val1 Giy Al a Arg Giu 220 Leu Met Giy Th r Ile 300 Asn Leu Leu Ser Asp 380 As n Arg Asp Asn Asn Thr 125 Ser Thr Arg Ser Tyr 205 Lys Lys Leu Ile Thr 285 Ile Asp Ala Giu Lys 365 As n Ile Ser Giy Ser Asn 110 Leu Gin Ile Leu Asn 190 Ile Giu Asp As n Arg 270 Asn Lys Arg Thr Ile 350 Asn As n Ala Ser Trp 430 Ile Ser Gin Met Thr Ile 175 Asn Trp Ile Phe Leu 255 Gly Ile Lys Vai As n 335 Pro Asp Gly Lys Arg 415 Gly Ser Gly Asp Ile Asn 160 Asp Ile Ile Lys Trp 240 Tyr Tyr Tyr Tyr Tyr 320 Ala Asp Gin Asn Leu 400 Thr Glu <210> 7 <211> 1341 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA Hc <221> CDS P XOpffEjb-od,53O35 OO y d1sqkig d..0I3/32003 12 <222> (10) (1329) <400> 7 gaattcacg atg gcc aac aaa tac aat tcc gaa atc ctg aac aat atc atc 51 Met Ala Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile Ile ctg Leu aac ctg cgt tac Asn Leu Arg Tyr gac aac aat ctg Asp Asn Asn Leu gat ctg tct ggt Asp Leu Ser Giy tac Tyr ggt gct aaa gtt Gly Ala Lys Val gaa Giu gta tac gac ggt Val Tyr Asp Gly gtt Val1 40 gaa ctg aat gac Giu Leu Asn Asp aag aac Lys Asn cag ttc aaa Gin Phe Lys aat cag aac Asn Gin Asn ctg Leu acc tct tcc gct Thr Ser Ser Ala aac Asn 55 tct aag atc cgt Ser Lys Ile Arg gtt act cag Val Thr Gin tct gtt tcc Ser Val Ser 195 243 atc atc ttc aac Ile Ile Phe Asn gta ttc ctg gac Val Phe Leu Asp ttc tgg Phe Trp atc cgt atc ccg Ile Arg Ile Pro aaa Lys 85 tac aag aac gac Tyr Lys Asn Asp ggt Gly atc cag aat tac Ile Gin Asn Tyr atc Ile cac aat gaa tac His Asn Giu Tyr ac c Thr 100 atc atc aac tgc Ile Ile Asn Cys atg Met 105 aag aat aac tct Lys Asn Asn Ser ggt Gly 110 tgg aag atc tcc Trp Lys Ile Ser atc Ile 115 cgc ggt aac cgt Arg Gly Asn Arg atc Ile 120 atc tgg act ctg Ile Trp Thr Leu atc gat Ile Asp 125 atc aac ggt Ile Asn Gly gac atc tct Asp Ile Ser 145 aag Lys 130 acc aaa tct gta Thr Lys Ser Val ttc gaa tac aac Phe Giu Tyr Asn atc cgt gaa Ile Arg Giu 140 atc acc aat Ile Thr Asn 291 339 387 435 483 531 579 627 gaa tac atc aat Giu Tyr Ile Asn cgc Arg 150 tgg ttc ttc gtt Trp Phe Phe Val acc Thr 155 aac ctg Asn Leu 160 aac aat gct aaa Asn Asn Ala Lys atc Ile 165 tac atc aac ggt Tyr Ile Asn Gly ctg gaa tct aat Leu Giu Ser Asn acc Thr 175 gac atc aaa gac Asp Ile Lys Asp atc Ile 180 cgt gaa gtt atc Arg Giu Val Ile gct Ala 185 aac ggt gaa atc Asn Gly Glu Ile ttc aaa ctg gac Phe Lys Leu Asp ggt Gly 195 gac atc gat cgt Asp Ile Asp Arg acc Thr 200 cag ttc atc tgg Gin Phe Ile Trp atg aaa Met Lys 205 tac ttc tcc atc ttc aac acc gaa ctg tct cag tcc aat atc gaa gaa Tyr Phe Ser Ile Phe Asn Thr Giu Leu Ser Gin Ser Asn Ile Giu Giu P OorEjb =cdef50O35 OOs~y inwdadwIising doc-0310312003 13 210 cgg tac aag Arg Tyr Lys 225 atc cag tct tac Ile Gin Ser Tyr tcc Ser 230 gaa tac ctg aaa Giu Tyr Leu Lys gac Asp 235 ttc tgg ggt Phe Trp Gly aat ccg Asn Pro 240 ctg atg tac aac Leu Met Tyr Asn aaa Lys 245 gaa tac tat atg Glu Tyr Tyr Met ttc Phe 250 aat gct ggt aac Asn Ala Gly Asn aag Lys 255 aac tct tac atc Asn Ser Tyr Ile aaa Lys 260 ctg aag aaa gac Leu Lys Lys Asp ccg gtt ggt gaa Pro Vai Giy Giu atc Ile 270 771 819 867 ctg act cgt tcc Leu Thr Arg Ser aaa Lys 275 tac aac cag aac Tyr Asn Gin Asn tct Ser 280 aaa tac atc aac Lys Tyr Ile Asn tac cgc Tyr Arg 285 gac ctg tac Asp Leu Tyr cag tcc atc Gin Ser Ile 305 atc Ile 290 ggt gaa aag ttc Gly Giu Lys Phe atc Ile 295 atc cgt cgc aaa Ile Arg Arg Lys tct aac tct Ser Asn Ser 300 atc tac ctg Ile Tyr Leu 915 963 aat gat gac atc Asn Asp Asp Ile cgt aaa gaa gac Arg Lys Glu Asp tac Tyr 315 gac ttc Asp Phe 320 ttc aac ctg aat Phe Asn Leu Asn c ag Gin 325 gaa tgg cgt gta Giu Trp Arg Vai tac Tyr 330 acc tac aag tac Thr Tyr Lys Tyr aag aaa gaa gaa Lys Lys Giu Giu gaa Glu 340 aag ctt ttc ctg Lys Leu Phe Leu gct Aia 345 ccg atc tct gat Pro Ile Ser Asp tcc Ser 350 1011 1059 1107 gac gaa ctc tac Asp Giu Leu Tyr acc atc cag atc Thr Ile Gin Ile gaa tac gac gaa Glu Tyr Asp Giu cag ccg Gin Pro 365 acc tac tct Thr Tyr Ser gaa atc ggt Giu Ile Giy 385 tgc Cys 370 cag ctg ctg ttc Gin Leu Leu Phe aag Lys 375 aaa gat gaa gaa Lys Asp Giu Giu tct act gac Ser Thr Asp 380 ggt atc gta Giy Ile Vai ctg atc ggt atc Leu Ile Giy Ilie cgt ttc tac gaa Arg Phe Tyr Giu tct Ser 395 1155 1203 1251 1299 ttc gaa Phe Giu 400 gaa tac aaa gac Giu Tyr Lys Asp tac Tyr 405 ttc tgc atc tcc Phe Cys Ile Ser aaa Lys 410 tgg tac ctg aag Trp Tyr Leu Lys gtt aaa cgc aaa Val Lys Arg Lys ccg Pro 420 tac aac ctg aaa Tyr Asn Leu Lys ctg Leu 425 ggt tgc aat tgg Gly Cys Asn Trp ttc atc ccg aaa Phe Ile Pro Lys gaa ggt tgg acc Giu Gly Trp Thr gaa Giu 440 tagtaagaat tc 1341 PA\Op\Ejb .wdW\50033 00 ury wwdeseqligi doc.03132003 14- <210> 8 <211> 440 <212> PRT <213> Artificial Sequence <220> <223> Encoded polypeptide of a synthetic construct based on BoNTB He <400> 8 Met Ala Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile Ile Leu Asn 1 5 10 Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly Tyr Gly Ala 25 Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys Asn Gin Phe 40 Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr Gin Asn Gin 55 Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val Ser Phe Trp 70 75 Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gin Asn Tyr Ile His 90 Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser Gly Trp Lys 100 105 110 Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile Asp Ile Asn 115 120 125 Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg Glu Asp Ile 130 135 140 Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr Asn Asn Leu 145 150 155 160 Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser Asn Thr Asp 165 170 175 SIle Lys Asp Ile Arg Glu Val Ile Ala Asn Gly Glu Ile Ile Phe Lys 180 185 190 Leu Asp Gly Asp Ile Asp Arg Thr Gin Phe Ile Trp Met Lys Tyr Phe 195 200 205 Ser Ile Phe Asn Thr Glu Leu Ser Gin Ser Asn Ile Glu Glu Arg Tyr 210 215 220 Lys Ile Gin Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro 225 230 235 240 Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn 245 250 255 Ser Tyr Ile Lys Leu Lys Lys Asp Ser Pro Val Gly Glu Ile Leu Thr 260 265 270 Arg Ser Lys Tyr Asn Gin Asn Ser Lys Tyr Ile Asn Tyr Arg Asp Leu 275 280 285 Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn Ser Gin Ser 290 295 300 Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr Leu Asp Phe 305 310 315 320 Phe Asn Leu Asn Gin Glu Trp Arg Val Tyr Thr Tyr Lys Tyr Phe Lys 325 330 335 Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp Ser Asp Glu 340 345 350 Leu Tyr Asn Thr Ile Gin Ile Lys Glu Tyr Asp Glu Gin Pro Thr Tyr P 'Op.\Ejb mmdd\50035 00 .wy-ddqisaig dcc-03/03/2003 15 355 Gin Ser Cys 370 Gly Leu Leu. Leu. Phe 360 Lys Lys 375 Arg Asp Glu Glu 365 Thr Ser 380 Gly Asp Giu Ile Ile Gly Ile Phe Tyr Glu Ile Val Phe 385 Glu Tyr Lys Asp Tyr 405 Tyr Cys Ile Ser Lys 410 Gly Tyr Leu Lys Giu 400 Vali Ile Lys Arg Lys Pro Lys Asp 435 Pro 420 Giu Asn Leu Lys Cys Asn Trp Gin 430 Giy Trp Thr <210> 9 <211> 1371 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA Hc <221> CDS <222> (10) (1359) <400> 9 gaattcacg atg acc atc cca ttc aac atc ttc tcc tac acc aac aac tcc 51 Met Thr Ile Pro Phe Asn Ile Phe Ser Tyr Thr Asn Asn Ser ctg Leu ttg aag gac atc Leu Lys Asp Ile atc Ile 20 aac gag tac ttc Asn Giu Tyr Phe aac As n aac atc aac gac Asn Ile Asn Asp aag atc ctg tcc Lys Ile Leu Ser ctg Leu.

cag aac cgt aag Gin Asn-Arg Lys acc ttg gtc gac Thr Leu. Vai Asp acc tcc Thr Ser ggt tac aac Gly Tyr Asn atc ttc cca Ile Phe Pro gag gtc tcc gag Giu Val Ser Giu gag Giu ggt gac gtc cag Gly Asp Val Gin ctg aac cca Leu Asn Pro gac aga ggt Asp Arg Gly ttc gac ttc aag Phe Asp Phe Lys ggt tcc tcc ggt Gly Ser Ser Giy aag gtc Lys Val atc gtc acc cag Ile Val Thr Gin aac Asn 85 gag aac atc gtc Giu Asn Ile Val tac Tyr aac tcc atg tac Asn Ser Met Tyr gag Giu tcc ttc tcc atc Ser Phe Ser Ile tcc Ser 100 ttc tgg atc aga Phe Trp Ile Arg atc Ile 105 aac aag tgg gtc Asn Lys Trp Vai aac ttg cca ggt Asn Leu Pro Giy tac Tyr 115 acc atc atc gac Thr Ile Ile Asp gtc aag aac aac Vai Lys Asn Asn tcc ggt Ser Gly 125 P %pcrEjh.=wdo"0035 00 sy mmesNi~ do-03103/2003 16tgg tcc atc Trp Ser Ile aac gag gac Asn Glu Asp 145 ggt Gly 130 atc atc tcc aac Ile Ile Ser Asn ctg gtc ttc acc Leu Val Phe Thr ctg aag cag Leu Lys Gin 140 atc tcc aac Ile Ser Asn 435 483 tcc gag cag tcc Ser Giu Gin Ser aac ttc tcc tac Asn Phe Ser Tyr gac Asp 155 aac gct Asn Aia 160 cct ggt tac aac Pro Giy Tyr Asn tgg ttc ttc gtc Trp Phe Phe Val acc Thr 170 gtc acc aac aac Val Thr Asn Asn atg Met 175 atg ggt aac atg Met Gly Asn Met aag Lys 180 atc tac atc aac Ile Tyr Ile Asn aag ctg atc gac Lys Leu Ile Asp acc Thr 190 531 579 627 atc aag gtc aag Ile Lys Vai Lys gag Giu 195 ttg acc ggt atc Leu Thr Giy Ile aac Asn 200 ttc tcc aag acc Phe Ser Lys Thr atc acc Ile Thr 205 ttc gag atc Phe Giu Ile gac aac atc Asp Asn Ile 225 aac Asn 210 aag atc cca gac Lys Ile Pro Asp ac c Thr 215 ggt ctg atc ac Gly Leu Ile Thr tcc gac tc Ser Asp Ser 220 gcc aag gag Ala Lys Giu aac atg tgg atc Asn Met Trp Ile cgt Arg 230 gac ttc tac atc Asp Phe Tyr Ile ttc Phe 235 ttg gac Leu Asp 240 ggt aag gac atc Gly Lys Asp Ile aac Asn 245 atc ctg ttc aac Ile Leu Phe Asn ttg cag tac acc Leu Gin Tyr Thr aac As n 255 gtc gtc aag gac Val Val Lys Asp tgg ggt aac gac Trp, Gly Asn Asp ctg Leu 265 aga tac aac aag Arg Tyr Asn Lys tac tac atg gtc Tyr Tyr Met Val aac As n 275 atc gac tac ttg Ile Asp Tyr Leu aac Asn 280 aga tac atg tac Arg Tyr Met Tyr gcc aac Ala Asn 285 tcc aga cag Ser Arg Gin gag ggt tac Giu Gly Tyr 305 atc Ile 290 gtc ttc aac acc Val Phe Asn Thr aga Arg 295 cgt aac aac aac Arg Asn Asn Asn gac ttc aac Asp Phe Asn 300 acc aac gac Thr Asn Asp aag atc atc atc Lys Ile Ile Ile aag Lys 310 cgt atc aga ggt Arg Ile Arg Gly aac Asn 315 915 963 1011 1059 acc aga Thr Arg 320 gtc aga ggt ggt Val Arg Gly Giy gac Asp 325 atc ctg tac ttc Ile Leu Tyr Phe gac Asp 330 atg act atc aac Met Thr Ile Asn aac Asn 335 aag gcc tac aac Lys Ala Tyr Asn ttc atg aag aac Phe Met Lys Asn gag Giu 345 acc atg tac gcc Thr Met Tyr Ala gac Asp 350 aac cac tcc acc gag gac atc tac gcc atc ggt ctg cgt gag cag acc 1107 V.Opcr\Ejh.=wod50035 Oow y~mmdaseqfisting dm.3/O3/2OO3 Asn His Ser Thr Giu Asp Ile Tyr Ala 17- Ile 360 cag Gin Giy Leu Arg Glu Gin Thr 365 aag gac atc Lys Asp Ile act tac tac Thr Tyr Tyr 385 aac atc tcc Asn Ile Ser aac Asn 370 tac Tyr aac atc atc Asn Ile Ile ttc Phe 375 ttc Phe atc cag cca Ile Gin Pro gct tcc cag Ala Ser Gin aag tcc aac Lys Ser Asn ttc Phe 395 ttc Phe atg aac aac Met Asn Asn 380 aac ggt gag Asn Gly Giu cgt ctg ggt Arg Leu Giy ggt atc tgt Giy Ile Cys 400 ggt gac Gly Asp tcc Ser 405 aac Asn ggt acc tac Gly Thr Tyr aga Arg 410 act Thr 1155 1203 1251 1299 1347 tgg tac aga Trp Tyr Arg 415 aac Asn cac His 420 ctg Leu tac ttg gtt Tyr Leu Val cca Pro 425 acc Thr gtc aag cag Vai Lys Gin tac gcc tcc ttg Tyr Ala Ser Leu 435 gag tcc act Giu. Ser Thr cac tgg gga His Trp Gly ttc Phe 445 cca gtc tcc Pro Val Ser gag Giu 450 taataggaat tc 1371 <210> <211> 450 <212> PRT <213> Artificiai Sequence <220> <223> Encoded poiypeptide of a synthetic construct based on BoNTC Hc <400> 9* Met 1 Lys Leu Asn Pro Ile Phe Pro Ile Asp Thr Asp Ser Ala 50 Phe Val1 Ser Gly Gly 130 Ser Ile Ile Leu Giu Asp Thr Ile Tyr 115 Ile Glu Phe 5 Asn As n Ser Lys As n Phe Ile Ser Ser Asn Glu Arg Giu Leu 70 Giu Trp Ile Asn Ile Phe Phe Asn Gly Ser Ile Arg Ser 120 Leu Phe Ser Asn 25 Thr Asp Ser Vali Ile 105 Val1 Val Ser Tyr 10 Asn Leu Vali Gly Tyr 90 Asn Lys Phe Tyr Thr Ile Vali Gin Glu Asn Lys Asn Thr Asp Asn Asn Asp Leu Asp Ser Trp Asn Leu 140 Ile Ser Ser Ser Pro Gly Tyr Ser 110 Gly Gin Asn Leu Lys Gly Ile Lys Giu Asn Trp As n As n P.'OpaEjh mlmde\5035.00 OO mmdey dusoIis dmc-03/03/2003 18- 145 150 155 160 Pro Gly Tyr Asn Lys Trp Phe Phe Val Thr Val Thr Asn Asn Met Met 165 170 175 Gly Asn Met Lys Ile Tyr Ile Asn Gly Lys Leu Ile Asp Thr Ile Lys 180 185 190 Val Lys Glu Leu Thr Gly Ile Asn Phe Ser Lys Thr Ile Thr Phe Glu 195 200 205 Ile Asn Lys Ile Pro Asp Thr Gly Leu Ile Thr Ser Asp Ser Asp Asn 210 215 220 Ile Asn Met Trp Ile Arg Asp Phe Tyr Ile Phe Ala Lys Glu Leu Asp 225 230 235 240 Gly Lys Asp Ile Asn Ile Leu Phe Asn Ser Leu Gin Tyr Thr Asn Val 245 250 255 Val Lys Asp Tyr Trp Gly Asn Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr 260 265 270 Met Val Asn Ile Asp Tyr Leu Asn Arg Tyr Met Tyr Ala Asn Ser Arg 275 280 285 Gin Ile Val Phe Asn Thr Arg Arg Asn Asn Asn Asp Phe Asn Glu Gly 290 295 300 Tyr Lys Ile Ile Ile Lys Arg Ile Arg Gly Asn Thr Asn Asp Thr Arg 305 310 315 320 Val Arg Gly Gly Asp Ile Leu Tyr Phe Asp Met Thr Ile Asn Asn Lys 325 330 335 Ala Tyr Asn Leu Phe Met Lys Asn Glu Thr Met Tyr Ala Asp Asn His 340 345 350 Ser Thr Glu Asp Ile Tyr Ala Ile Gly Leu Arg Glu Gin Thr Lys Asp 355 360 365 Ile Asn Asp Asn Ile Ile Phe Gin Ile Gin Pro Met Asn Asn Thr Tyr 370 375 380 Tyr Tyr Ala Ser Gin Ile Phe Lys Ser Asn Phe Asn Gly Glu Asn Ile 385 390 395 400 Ser Gly Ile Cys Ser Ile Gly Thr Tyr Arg Phe Arg Leu Gly Gly Asp o 405 410 415 000 Trp Tyr Arg His Asn Tyr Leu Val Pro Thr Val Lys Gin Gly Asn Tyr 420 425 430 Ala Ser Leu Leu Glu Ser Thr Ser Thr His Trp Gly Phe Val Pro Val 435 440 445 Ser Glu 450 <210> 11 I* <211> 1374 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA He 00.

0*0* *<221>

CDS

<222> (10) (1362) <400> 11 gaattcacg atg cgt ttg aag gct aag gtc aac gag tcc ttc gag aac acc 51 Met Arg Leu Lys Ala Lys Val Asn Glu Ser Phe Glu Asn Thr 1 5 P. DperEjb mmdc\50O35 00 .wy mmodedsqIisting doc.0310312003 19 atg Met atc Ile ttg Leu gag Giu gac Asp aac As n tgg Trp atc Ile aac Asn tcc Ser aac Asn 175 aag Lys c tg Leu aac Asn cca Pro atc Ile c ag Gin gtc Val1 ttc Phe aac Asn atc Ile atc Ile ggt Giy ttg Leu 160 aag Lys ttg Leu gac Asp atc Ile ttc Phe aac Asn aac As n aga Arg aag Lys atc Ile aag Lys aac As n aac As n 145 atc Ile tgg Trp tac Tyr gag Giu gac Asp aac As n gag Giu aag Lys gtc Val1 ttg Leu ttg Leu atc Ile tcc Ser 130 atc Ile ttc Phe ttc Phe atc Ile gtc Val1 210 gag Glu atc Ile tac Tyr aag Lys ggt Gly tcc Ser tac Tyr tcc Ser 115 atc Ile gag Giu gac Asp ttc Phe aac As n 195 aag Lys aac Asn ttc Phe 20 ttc Phe aac As n gac Asp tc t Ser tcc Ser 100 aag Lys gag Giu tgg Trp tac Tyr gtc Val1 180 ggt Gly ctg Leu c ag Gin tcc Ser aac Asn gcc Ala aac Asn tcc Ser 85 gcc Ala gac Asp cag Gin atc Ile tcc Ser 165 acc Thr gag Giu gac Asp atg Met tac Tyr tcc Ser ttg Leu gtc Val1 70 ggt Gly atc Ile ttg Leu aac As n ttg Leu 150 gag Giu atc Ile ttg Leu aag Lys ttg Leu acc Thr atc Ile gt c Val1 55 cag Gin gac Asp tac Tyr acc Thr tcc Ser 135 cag Gin tcc Ser acc Thr aag Lys acc Thr 215 tgg Trp aac Asn aac Asn 40 gac Asp ttg Leu aag Lys gag Giu aac Asn 120 ggt Gly gac Asp ttg Leu aac Asn cag Gin 200 atc Ile atc Ile aac Asn 25 gac Asp acc Thr aac Asn atc Ile aac As n 105 tcc Ser tgg Trp gtc Val1 tcc Ser aac Asn 185 tcc Ser gtc Val1 cgt Arg tcc Ser tcc Ser tcc Ser acc Thr atc Ile tcc Ser cac His aag Lys aac Asn cac His 170 atc Ile cag Gin ttc Phe gac Asp ttg Leu aag Lys ggt Gly atc Ile gtc Val1 tct Ser aac Asn t tg Leu cgt Arg 155 acc Thr atg Met aag Lys ggt Gly ttc Phe 235 ttg Leu atc Ile tac Tyr tac Tyr aac Asn gtc Val gag Giu tgt Cys 140 aag Lys ggt Gly gg t Gly atc Ile atc Ile 220 aac As n aag Lys t tg Leu a ac As n acc Thr ttg Leu tcc Ser tac Tyr 125 atc Ile tac Tyr tac Tyr tac Tyr gag Giu 205 gac Asp atc Ile gac Asp tcc Se r gcc Ala aac Asn aac As n ttc Phe 110 acc Thr cgt Arg aag Lys acc Thr atg Met 190 gac Asp gag Giu ttc Phe 99 147 195 243 291 339 387 435 483 531 579 627 675 723 0* @0 0 0000 0000 0000 000 0 00000 225 230 tcc aag gag ctg tcc aac gag gac atc aac atc gtc tac gag ggt cag P:XOparEjb wmded\30035 00 .wy .mwdwsqiu do.03/2003 20 Ser Lys Giu Leu Ser Asn Giu Asp Ile Asn IleVai 250 Tyr Giu Gly Gin ctg agg aac gtc Leu Arg Asn Vai atc Ile 260 aag gac tac tgg Lys Asp Tyr Trp ggt Giy 265 aac cca ctg aag Asn Pro Leu Lys ttc Phe 270 819 867 gac acc gag tac Asp Thr Giu Tyr atc atc aac gac Ile Ile Asn Asp aac Asn 280 tac atc gac cgt Tyr Ile Asp Arg tac atc Tyr Ile 285 gcc cca gag Aia Pro Giu aag ctg tac Lys Leu Tyr 305 tcc Ser 290 aac gtc ctg gtc Asn Val Leu Val ctg Leu 295 gtc cag tac cct Vai Gin Tyr Pro gac cgt tcc Asp Arg Ser 300 tcc gac aag Ser Asp Lys acc ggt aac cct Thr Giy Asn Pro atc Ile 310 acc atc aag tcc Thr Ile Lys Ser gtc Vai 315 aac cct Asn Pro 320 tac tcc cgt atc Tyr Ser Arg Ile aac ggt gac aac Asn Giy Asp Asn atc ctg cac atg Ile Leu His Met ctg Leu 335 tac aac tcc cgt Tyr Asn Ser Arg aag Lys 340 tac atg atc atc Tyr Met Ile Ile cgt Arg 345 gac acc gac acc Asp Thr Asp Thr i011 1059 1107 tac gcc acc cag Tyr Aia Thr Gin 5 ggt Gly 355 ggt gac tgt tcc Giy Asp Cys Ser cag Gin 360 aac tgt gtc tac Asn Cys Vai Tyr gcc ctg Aia Leu 365 aag ctg cag Lys Leu Gin aag aac atc Lys Asn Ile 385 tcc Ser 370 aac ctg ggt aac Asn Leu Giy Asn ggt atc ggt atc Giy Ile Gly Ile ttc tcc atc Phe Ser Ile 380 ttc tcc tcc Phe Ser Ser 1155 1203 gtc tcc aag aac Vai Ser Lys Asn aag Lys 390 tac tgc tcc cag Tyr Cys Ser Gin atc Ile 395 ttc cgt Phe Arg 400 gag aac acc atg Giu Asn Thr Met ctg Leu 405 ctg gcc gac atc Leu Aia Asp Ile tac Tyr 410 aag cct tgg cgt Lys Pro Trp Arg

S

eSSO S. 55 S S ttc Phe 415 tcc ttc aag aac Ser Phe Lys Asn tac act cct gtc Tyr Thr Pro Vai gcc Aia 425 gtc acc aac tac Vai Thr Asn Tyr gag Giu 430 1251 1299 1347 acc aag ctg ctg Thr Lys Leu Leu tcc Ser 435 Thr Ser Ser Phe taataggaat tc tgg Trp 440 aag ttc atc tcc Lys Phe Ile Ser cgt gac Arg Asp 445 cca ggt tgg Pro Giy Trp gtc gag Vai Giu 450 1374 <210> 12 P.\0pakEjb.wmmdea\5035 Wrmny mdeliiu doc-03/0312003 -21- <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Encoded polypeptide of a synthetic construct based on BoNTD He <400> 12 Met Arg Leu Lys Ala Lys Val Asn Glu Ser Phe Glu Asn Thr Met Pro 1 5 10 Phe Asn Ile Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile 25 Asn Glu Tyr Phe Asn Ser Ile Asn Asp Ser Lys Ile Leu Ser Leu Gin 40 Asn Lys Lys Asn Ala Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val 55 Arg Val Gly Asp Asn Val Gin Leu Asn Thr Ile Tyr Thr Asn Asp Phe 70 75 Lys Leu Ser Ser Ser Gly Asp Lys Ile Ile Val Asn Leu Asn Asn Asn 90 Ile Leu Tyr Ser Ala Ile Tyr Glu Asn Ser Ser Val Ser Phe Trp Ile 100 105 110 Lys Ile Ser Lys Asp Leu Thr Asn Ser His Asn Glu Tyr Thr Ile Ile 115 120 125 Asn Ser Ile Glu Gin Asn Ser Gly Trp Lys Leu Cys Ile Arg Asn Gly 130 135 140 Asn Ile Glu Trp Ile Leu Gin Asp Val Asn Arg Lys Tyr Lys Ser Leu 145 150 155 160 Ile Phe Asp Tyr Ser Glu Ser Leu Ser His Thr Gly Tyr Thr Asn Lys 165 170 175 Trp Phe Phe Val Thr Ile Thr Asn Asn Ile Met Gly Tyr Met Lys Leu 180 185 190 Tyr Ile Asn Gly Glu Leu Lys Gin Ser Gin Lys Ile Glu Asp Leu Asp 195 200 205 Glu Val Lys Leu Asp Lys Thr Ile Val Phe Gly Ile Asp Glu Asn Ile 210 215 220 Asp Glu Asn Gin Met Leu Trp Ile Arg Asp Phe Asn Ile Phe Ser Lys 225 230 235 240 Glu Leu Ser Asn Glu Asp Ile Asn Ile Val Tyr Glu Gly Gin Ile Leu 245 250 255 Arg Asn Val Ile Lys Asp Tyr Trp Gly Asn Pro Leu Lys Phe Asp Thr 260 265 270 Glu Tyr Tyr Ile Ile Asn Asp Asn Tyr Ile Asp Arg Tyr Ile Ala Pro 275 280 285 Glu Ser Asn Val Leu Val Leu Val Gin Tyr Pro Asp Arg Ser Lys Leu 290 295 300 Tyr Thr Gly Asn Pro Ile Thr Ile Lys Ser Val Ser Asp Lys Asn Pro 305 310 315 320 0.o Tyr Ser Arg Ile Leu Asn Gly Asp Asn Ile Ile Leu His Met Leu Tyr 325 330 335 Asn Ser Arg Lys Tyr Met Ile Ile Arg Asp Thr Asp Thr Ile Tyr Ala 340 345 350 Thr Gin Gly Gly Asp Cys Ser Gin Asn Cys Val Tyr Ala Leu Lys Leu 355 360 365 Gin Ser Asn Leu Gly Asn Tyr Gly Ile Gly Ile Phe Ser Ile Lys Asn 370 375 380 P:%Op 1 rEjh =mde\50035 OO.u~y mmddseqki g dom-O3/0320O3 22 Ile 385 Glu Val Ser Lys Asn Lys 390 Leu Tyr Cys Ser Gin Ile 395 Lys Phe Ser Ser Phe Asn Thr Met Leu 405 Tyr Ala Asp Ile Tyr 410 Val1 Pro Trp Arg Phe Ser 415 Phe Lys Asn Leu Leu Ser 435 Trp Vai Giu 450 Ala 420 Thr Thr Pro Val Aia 425 Lys Thr Asn Tyr Giu Thr Lys 430 Asp Pro Gly Ser Ser Phe Trp 440 Phe Ile Ser Arg 445 <210> i3 <211> i400 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA Hc <221> CDS <222> (10) (1356) <400> 13 gaattcacc atg gga gag agt cag caa gaa cta aat tct atg gta act gat 51 Met Gly Giu Ser Gin Gin Giu Leu Asn Ser Met Val Thr Asp S S S. eS

S*

acc Thr cta aat aat agt Leu Asn Asn Ser cct ttt aag ctt Pro Phe Lys Leu tct tat aca gat gat Ser Tyr Thr Asp Asp aaa att tta att Lys Ile Leu Ile tcc Ser 35 tac ttc aac aag Tyr Phe Asn Lys ttc Phe 40 ttc aag aga att Phe Lys Arg Ile aag tct Lys Ser tct tcc gtt Ser Ser Val tcc ggt tac Ser Gly Tyr 65 tta Leu aac atg aga tac Asn Met Arg Tyr aat gat aaa tac Asn Asp Lys Tyr gtc gac act Val Asp Thr tac aag tac Tyr Lys Tyr gac tcc aat atc Asp Ser Asn Ile aac Asn 70 att aac ggt gac Ile Asn Gly Asp gtg Val1 S. *S 9 05 S S *555 .5.5 0 cca act Pro Thr 80 aac aaa aac caa Asn Lys Asn Gin ttc Phe 85 ggt atc tac aac Gly Ile Tyr Asn gac Asp aag ctt act gag Lys Leu Thr Giu ctg Leu aac atc tct caa Asn Ile Ser Gin gac tac att atc Asp Tyr Ile Ile gac aac aag tac aag Asp Asn Lys Tyr Lys aac ttc tct att Asn Phe Ser Ile tct Ser 115 ttc tgg gtc agg Phe Trp Val Arg att Ile 120 cct aac tac gac Pro Asn Tyr Asp aac aag Asn Lys 125 atc gtc aac gtt aac aac gag tac act atc atc aac tgt atg aga gac P %Opa\Ejb od f'35.00 lghgx-3O3OO3 23 Ile Val Asn aac aac tcc Asn Asn Ser 145 Val 130 Asn Asn Glu Tyr Thr 135 Ile Ile Asn Cys Met Arg Asp 140 ggt tgg aag gtc Gly Trp Lys Val ctt aac cac aac Leu Asn His Asn gag Glu 155 atc att tgg Ile Ile Trp acc ttg Thr Leu 160 caa gac aac gca Gin Asp Asn Ala ggt Gly 165 att aac caa aag Ile Asn Gin Lys tta Leu 170 gca ttc aac tac Ala Phe Asn Tyr ggt Gly 175 aac gca aac ggt Asn Ala Asn Gly tct gac tac atc Ser Asp Tyr Ile aag tgg att ttc Lys Trp Ile Phe gtc Val1 190 531 579 627 act atc act aac Thr Ilie Thr Asn gac Asp 195 aga tta ggt gac Arg Leu Gly Asp tct Ser 200 aag ctt tac att Lys Leu Tyr Ile aac ggt Asn Gly 205 aac tta atc Asn Leu Ile tct gac aac Ser Asp Asn 225 gac Asp 210 caa aag tcc att Gin Lys Ser Ile tta Leu 215 aac tta ggt aac Asn Leu Gly Asn att cac gtt Ile His Val 220 acc aga tac Thr Arg Tyr 675 723 atc tta ttc aag Ile Leu Phe Lys gtt aac tgc agt Val Asn Cys Ser tac Tyr 235

S.

5 5

S

S@ OS 0 0

SO'S

5

OS..

S

5555 0G 0 0 005 S att ggc Ile Gly 240 att aga tac ttc Ile Arg Tyr Phe aac Asn 245 att ttc gac aag Ile Phe Asp Lys gag Giu 250 tta gac gag acc Leu Asp Glu Thr gag Glu 255 att caa act tta Ile Gin Thr Leu tac Tyr 260 agc aac gaa cct Ser Asn Glu Pro acc aat att ttg Thr Asn Ile Leu aag Lys 270 771 819 867 gac ttc tgg ggt Asp Phe Trp Gly aac As n 275 tac ttg ctt tac Tyr Leu Leu Tyr gac Asp 280 aag gaa tac tac Lys Glu Tyr Tyr tta tta Leu Leu 285 05 0O S 0

S

@505 0* 00 0 5 5 0

SOS.

5

OS..

S

0055 0 00b5 aac gtg tta Asn Val Leu tta agc att Leu Ser Ile 305 aag Lys 290 cca aac aac ttc Pro Asn Asn Phe att Ile 295 gat agg aga aag Asp Arg Arg Lys gat tct act Asp Ser Thr 300 aac aga tta Asn Arg Leu aac aac atc aga Asn Asn Ilie Arg agc Ser 310 act att ctt tta Thr Ile Leu Leu gct Ala 315 915 963 1011 1059 tac tct Tyr Ser 320 ggt atc aag gtt Gly Ile Lys Val aag Lys 325 atc caa aga gtt Ile Gin Arg Val aac Asn 330 aac tct tct act Asn Ser Ser Thr aac Asn 335 gat aac ctt gtt Asp Asn Leu Val aag aac gat cag- Lys Asn Asp Gin tat att aac ttc Tyr Ile Asn Phe gtc Val1 350 gct agc aag act cac tta ttc cca tta tat gct gat acc gct acc acc Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr Thr 1107 P:'Dpc\Ejh smwdoAS0035 00 u~y =wdcisq~istinS doe-03/03003 24 aac aag gag Asn Lys Giu caa gtc gtc Gin Vai Val 385 acc atc aag atc Thr Ile Lys Ile tcc Ser 375 tcc tct ggc aac Ser Ser Gly Asn aga ttt aac Arg Phe Asn 380 atg aac ttt Met Asn Phe 1i55 1203 gtt atg aac tcc Vai Met Asn Ser ggt aac aac tgt Giy Asn Asn Cys acc Thr 395 aaa aat Lys Asn 400 aat aat gga aat Asn Asn Gly Asn aat Asn 405 att ggg ttg tta Ile Giy Leu Leu ggt Giy 410 ttc aag gca gat Phe Lys Aia Asp act Thr 415 gta gtt gct agt Val Val Aia Ser act Thr 420 tgg tat tat acc Trp Tyr Tyr Thr c ac His 425 atg aga gat cac Met Arg Asp His ac c Thr 430 1251 1299 1347 aac agc aat gga Asn Ser Asn Giy tgt Cys 435 ttt tgg aac ttt Phe Trp Asn Phe tct gaa gaa cat Ser Giu Giu His gga tgg Giy Trp 445 caa gaa aaa Gin Giu Lys taatagggat ccgcggccgc acgcgtcccg ggactagtga 1396 attc i4 <2ii> 449 <2i2> PRT <2i3> Artificiai Sequence <220> <223> Encoded polypeptide of a synthetic construct based on BoNTE Hc 1400 <400> i4 Met Giy Glu i Asn Asn Ser Leu Ile Ser 35 Val Leu Asn Ser Gin Gin Giu Leu Asn Ser Met Val Thr Asp 5 Pro Ile Tyr Phe Lys Leu 'Se r 25 Phe Tyr Thr Asp Phe Asn Lys Thr Leu Phe 40 Asn Lys Arg Ile Lys Asp Asp Lys Ile Ser Ser Ser Thr Ser Giy Met Arg Tyr 50 Lys Ile Asp Lys Tyr Tyr Asp Ser Asn Lys Asn Ile Ser Gin Ser Ilie Ser 115 Asn Vai Asn 130 Ser Giy Trp Asn Ile As n 70 Giy Asn Giy Asp Val1 75 Lys Lys Tyr Pro Thr Gin Phe Asn Asp Ile Tyr Asn Asp 90 Asp Leu Thr Glu Leu Asn Tyr Ile Ile 100 Phe Tyr 105 Pro Asn Lys Tyr Lys Asn Phe Lys Ile Vai Trp Vai Arg Ile 120 Ile Asn Tyr Asp Asn 125 Arg Asn Giu Tyr Lys Vai Ser Ile Asn Cys Asp Asn Asn Asn His Asn Glu As HsAs Gle Trp Thr Leu P:%Op\Ejb.mwdd\53003.0 -sdnmy nmdcdwqli doc-03032003 145 150 155 160 Gin Asp Asn Ala Gly Ile Asn Gin Lys Leu Ala Phe Asn Tyr Gly Asn 165 170 175 Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile 180 185 190 Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn Gly Asn Leu 195 200 205 Ile Asp Gin Lys Ser Ile Leu Asn Leu Gly Asn Ile His Val Ser Asp 210 215 220 Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg Tyr Ile Gly 225 230 235 240 Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu Thr Glu Ile 245 250 255 Gin Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu Lys Asp Phe 260 265 270 Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu Leu Asn Val 275 280 285 Leu Lys Pro Asn Asn Phe Ile Asp Arg Arg Lys Asp Ser Thr Leu Ser 290 295 300 Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg Leu Tyr Ser 305 310 315 320 Gly Ile Lys Val Lys Ile Gin Arg Val Asn Asn Ser Ser Thr Asn Asp 325 330 335 Asn Leu Val Arg Lys Asn Asp Gin Val Tyr Ile Asn Phe Val Ala Ser 340 345 350 Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr Thr Asn Lys 355 360 365 Glu Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe Asn Gin Val 370 375 380 Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn Phe Lys Asn 385 390 395 400 Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr Val a A405 410 415 Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His Thr Asn Ser 420 425 430 Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp Gin Glu 435 440 445 Lys <210> S <211> 1317 <212> DNA S* <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA He *e <221> CDS <222> (10)...(1305) <400> gaattcacg atg tcc tac acc aac gac aag ate ctg atc ttg tac ttc aac 51 Met Ser Tyr Thr Asn Asp Lys Ile Leu Ile Leu Tyr Phe Asn 1 5 P:\D.,Ejb mo.d\50035.0 mdscq1Li.S doc-031031'2003 26 aag ctg tac aag aag atc aag Lys Leu Tyr Lys Lys Ile Lys gaa Giu atc Ile atc Ile atc Ile cgt Arg atc Ile aac Asn cag Gin atc Ile tcc Ser 175 aac Asn ggt Gly gac Asp cct aac Asn aac Asn tac Tyr atc Ile atc Ile atc Ile tac Tyr aag Lys aac Asn 160 aga Arg ttg Leu tgt Cys act Thr gac aat As n ggt Giy tcc Ser tac Tyr cca Pro gac Asp aac Asn ttg Leu 145 aag Lys atc Ile ggt Gly aac Asn gag Giu 225 cca aag Lys gac Asp tcc Ser aac Asn aag Lys tgc Cys aag Lys 130 gtc Val1 tgg Trp tac Tyr gac Asp gac Asp 210 ttg Leu tcc ttc Phe gtc Val1 aag Lys gga Gly tac Tyr atc Ile 115 atc Ile ttc Phe atc Ile atc Ile atc Ile 195 ac c Thr ggt Gly atc atc Ile tac Tyr cct Pro aga Arg ttc Phe 100 cgt Arg atc Ile aac Asn ttc Phe aac Asn 180 cac His cgt Arg aag Lys ctg gac Asp atc Ile tcc Ser tac Tyr 85 aac Asn aac Asn tgg Trp tac Tyr gtC Val1 165 ggt Gly gtc Val1 tac Tyr ac c Thr aag 20 gac aac tcc Asp Asn Ser atc tcc ggt Ile Ser Giy 40 tac tcc acc Tyr Ser Thr 55 gag gtc aac Glu Vai Asn 70 cag aac ttc Gin Asn Phe aag gtc aac Lys Vai Asn aat aac tcc Asn Asn Ser 120 acc ctg cag Thr Leu Gin 135 acc cag atg Thr Gin Met 150 acc atc acc Thr Ile Thr aac ttg atc Asn Leu Ile tcc gac aac Ser Asp Asn 200 gtc ggg atc Vai Gly Ile 215 gag atc gag Giu Ile Giu 230 gac ttc tgg atc Ile 25 tac Tyr aat Asn atc Ile tcc Ser ctg Leu 105 gga Gly gac Asp atc Ile aat Asn gac Asp 185 att Ile cgt Arg acc Thr ggt ttg Leu ggt Gly aga Arg gct Ala atc Ile aat Asn tgg Trp acc Thr tcc Ser aac Asn 170 gag Giu ttg Leu tac Tyr t tg Leu aac gac Asp tcc Ser aac Asn cag Gin tcc Ser aac Asn aag Lys gcc Ala atc Ile 155 cgt Arg aag Lys ttc Phe ttc Phe tac Tyr 235 tac atg Met aac As n cag Gin aac As n ttc Phe gag Giu atc Ile ggt Gly 140 tcc Ser t tg Leu tcc Ser aag Lys aaa Lys 220 tcc Ser ctg aga Arg atc Ile ttc Phe aac Asn tgg Trp tac Tyr tcc Ser 125 aac As n gac Asp gga Gly atc Ile atc Ile 205 gtc Val gac Asp c tg tac Tyr tcc Ser gga Gly gac Asp gtc Val1 acc Thr 110 ctg Leu aat Asn tac Tyr aac Asn tcc Ser 190 gtc Val ttc Phe gag Giu tac 99 147 195 243 291 339 387 435 483 531 579 627 675 723 771 P.%Opc\Ejh mwdod50O35 DO us.y mmdedseq~ing doc.03/03/2003 27 Pro Asp 240 Pro Ser Ile Leu Lys 245 Asp Phe Trp Gly As n 250 Tyr Leu Leu Tyr aac Asn 255 aaa cgt tac tac Lys Arg Tyr Tyr ttg Leu 260 ctg aac ttg ttg Leu Asn Leu Leu cgt Arg 265 acc gac aag tcc Thr Asp Lys Ser acc cag aac tcc Thr Gin Asn Ser ttc ttg aac atc Phe Leu Asn Ile aac As n 280 cag cag aga ggt Gin Gin Arg Giy gtc tac Vai Tyr 285 cag aag cca Gin Lys Pro gtc att atc Vai Ile Ile 305 aac Asn 290 atc ttc tcc aac Ile Phe Ser Asn ac c Thr 295 aga ttg tac acc Arg Leu Tyr Thr gga gtc gag Giy Vai Giu 300 acc gat aac Thr Asp Asn aga aag aac gga Arg Lys Asn Giy tct Ser 310 act gat att tcc Thr Asp Ile Ser aac As n 315 ttc gtc Phe Vai 320 aga aag aac gat Arg Lys Asn Asp ctg Leu 325 gct tac atc aac Aia Tyr Ile Asn gtt Vai 330 gtc gac aga gat Vai Asp Arg Asp gtc Vali 335 gaa tac cgt ctg Giu Tyr Arg Leu tac Tyr 340 gcc gat atc tct Aia Asp Ile Ser gcc aaa cct gaa Aia Lys Pro Giu aag Lys 350 atc atc aag ctg Ile Ilie Lys Leu cgt acc tct aac Arg Thr Ser Asn tct Ser 360 aac aac tct ctg Asn Asn Ser Leu gga caa Giy Gin 365 1011 1059 1107 1155 1203 atc atc gtc Ie Ile Vai aac aac aac Asn Asn Asn 385 atg Met 370 gac tcc atc ggt Asp Ser Ile Giy aac tgt acc atg Asn Cys Thr Met aac ttc cag Asn Phe Gin 380 tcc aac aac Ser Asn Asn ggt gga aac atc Giy Giy Asn Ile ggt Giy 390 ttg ttg ggt ttc Leu Leu Giy Phe cac His 395 ttg gtc Leu Vai 400 gct tcc tcc tgg Aia Ser Ser Trp tac Tyr 405; tac aac aac atc Tyr Asn Asn Ile cgt Arg 410 aag aac acc tcc Lys Asn Thr Ser 1251 1299 tcc Ser 415 aac ggt tgc ttc Asn Giy Cys Phe tcc ttc atc tcc Ser Phe Ile Ser aag Lys 425 gag cac ggt tgg Giu His Gly Trp cag Gin 430 gag aac Giu Asn taataggaat tc 1317 <210> <211> <212> <213> 16 432

PRT

Artificial Sequence P.%Opcr\Ejh =mmda60035 00 .s~y =cd adisto n dc.03103/2003 -28- <220> <223> Encoded polypeptide of a synthetic construct based on BoNTF He <400> 16 Met Ser Tyr Thr Asn Asp Lys Ile Leu Ile Leu Tyr Phe Asn Lys Leu 1 5 10 Tyr Lys Lys Ile Lys Asp Asn Ser Ile Leu Asp Met Arg Tyr Glu Asn 25 Asn Lys Phe Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn 40 Gly Asp Val Tyr Ile Tyr Ser Thr Asn Arg Asn Gin Phe Gly Ile Tyr 55 Ser Ser Lys Pro Ser Glu Val Asn Ile Ala Gin Asn Asn Asp Ile Ile 70 75 Tyr Asn Gly Arg Tyr Gin Asn Phe Ser Ile Ser Phe Trp Val Arg Ile 90 Pro Lys Tyr Phe Asn Lys Val Asn Leu Asn Asn Glu Tyr Thr Ile Ile 100 105 110 Asp Cys Ile Arg Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Asn Tyr 115 120 125 Asn Lys Ile Ile Trp Thr Leu Gin Asp Thr Ala Gly Asn Asn Gin Lys 130 135 140 Leu Val Phe Asn Tyr Thr Gin Met Ile Ser Ile Ser Asp Tyr Ile Asn 145 150 155 160 Lys Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser Arg 165 170 175 S: Ile Tyr Ile Asn Gly Asn Leu Ile Asp Glu Lys Ser Ile Ser Asn Leu 180 185 190 Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Gly Cys 195 200 205 Asn Asp Thr Arg Tyr Val Gly Ile Arg Tyr Phe Lys Val Phe Asp Thr 210 215 220 Glu Leu Gly Lys Thr Glu Ile Glu Thr Leu Tyr Ser Asp Glu Pro Asp 225 230 235 240 Pro Ser Ile Leu Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asn Lys 245 250 255 Arg Tyr Tyr Leu Leu Asn Leu Leu Arg Thr Asp Lys Ser Ile Thr Gin 260 265 270 Asn Ser Asn Phe Leu Asn Ile Asn Gin Gin Arg Gly Val Tyr Gin Lys 275 280 285 SPro Asn Ile Phe Ser Asn Thr Arg Leu Tyr Thr Gly Val Glu Val Ile 290 295 300 Ile Arg Lys Asn Gly Ser Thr Asp Ile Ser Asn Thr Asp Asn Phe Val S305 310 315 320 Arg Lys Asn Asp Leu Ala Tyr Ile Asn Val Val Asp Arg Asp Val Glu S325 330 335 Tyr Arg Leu Tyr Ala Asp Ile Ser Ile Ala Lys Pro Glu Lys Ile Ile 340 345 350 Lys Leu Ile Arg Thr Ser Asn Ser Asn Asn Ser Leu Gly Gin Ile Ile 355 360 365 Val Met Asp Ser Ile Gly Asn Asn Cys Thr Met Asn Phe Gin Asn Asn 370 375 380 Asn Gly Gly Asn Ile Gly Leu Leu Gly Phe His Ser Asn Asn Leu Val 385 390 395 400 Ala Ser Ser Trp Tyr Tyr Asn Asn Ile Arg Lys Asn Thr Ser Ser Asn 405 410 415 P OpMEjh md\5D035.0 d"soisi.,g dwc03/0312003 29 Gly Cys Phe Trp Ser Phe le Ser Lys Giu His Gly Trp Gin Giu Asn 420 425 430 <210> 17 <211> 1368 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA Hc <221> CDS <222> (10) (1356) <400> 17 gaattcacg atg aag gac acc atc ctg atc cag gtc ttc aac aac tac atc Met Lys Asp Thr Ile Leu Ile Gin Vai Phe Asn Asn Tyr Ile tcc Ser aac atc tcc tcc Asn Ile Ser Ser gcc atc ctg tcc Ala Ile Leu Ser tcc tac cgt ggt Ser Tyr Arg Gly cgt ctg atc gac Arg Leu Ile Asp tcc Ser 35 tcc ggt tac gga Ser Gly Tyr Gly gcc Ala acc atg aac gtc ggt tcc Thr Met Asn Val Gly Ser 99 147 195 243 gac gtc atc Asp Val Ile tcc gag aac Ser Glu Asn ttc Phe aac gac atc ggt Asn Asp Ile Gly aac Asn

SS

ggt cag ttc aag Gly Gin Phe Lys ctg aac aac Leu Asn Asn gtc gtc tac Val Val Tyr tcc aac atc acc Ser Asn Ile Thr cac cag tcc aag His Gin Ser Lys ttc Phe gac tcc Asp Ser atg ttc gac aac Met Phe Asp Asn ttc Phe 85 tcc atc aac ttc Ser Ile Asn Phe tgg Trp gtc cgt acc cca Val Arg Thr Pro 0*0.0 tac aac aac aac Tyr Asn Asn Asn gac Asp 100 atc cag acc tac Ile Gin Thr Tyr ctg Leu 105 cag aac gag tac Gin Asn Glu Tyr acc Thr 110 291 339 387 435 atc atc tcc tgt Ile Ilie Ser Cys atc Ile 115 aag aac gac tcc Lys Asn Asp Ser ggt Giy 120 tgg aag gtc tcc Trp Lys Val Ser atc aag Ile Lys 125 gga aac cgt Gly Asn Arg tcc atc ttc Ser Ile Phe 145 atc tgg acc ctg Ile Trp Thr Leu atc Ile 135 gac gtc aac gcc Asp Val Asn Ala aag tcc aag Lys Ser Lys 140 gac tac atc Asp Tyr Ile ttc gag tac tcc atc aag gac aac atc Phe Glu Tyr Ser Ile Lys Asp Asn Ile 150 tcc Ser 155 aac aag tgg ttc tcc atc acc atc acc aac gac cgt ctg ggt aac gcc P IOM\Ejh mmdd5OO35 00 u.ywdeslisting doc.0310312003 30 Asn Lys Trp Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu Gly Asn Ala 160 165 170 aac atc tac atc aac ggt tcc ctg aag aag tcc gag aag atc ctg aac 579 Asn Ile Tyr Ile Asn Gly Ser Leu Lys Lys Ser Giu Lys Ile Leu Asn 175 180 185 190 ctg gac cgt atc aac tcc tcc aac gac atc gac ttc aag ctg atc aac 627 Leu Asp Arg Ile Asn Ser Ser Asn Asp Ile Asp Phe Lys Leu Ile Asn 195 200 205 tgt acc gac acc acc aag ttc gtc tgg atc aag gac ttc aac atc ttc 675 Cys Thr Asp Thr Thr Lys Phe Val Trp Ile Lys Asp Phe Asn Ile Phe 210 215 220 ggt cgt gag ctg aac gcc acc gag gtc tcc tcc ctg tac tgg atc cag 723 Gly Arg Giu Leu Asn Ala Thr Giu Val Ser Ser Leu Tyr Trp Ile Gin 225 230 235 tcc tcc acc aac acc ctg aag gac ttc tgg gga aac cca ctg cgt tac 771 Ser Ser Thr Asn Thr Leu Lys Asp Phe Trp, Gly Asn Pro Leu Arg Tyr 240 245 250 gac acc cag tac tac ctg ttc aac cag ggt atg cag aac atc tac atc 819 Asp Thr Gin Tyr Tyr Leu Phe Asn Gin Gly Met Gin Asn Ile Tyr Ile 255 260 265 270 :::aag tac ttc tcc aag gcc tcc atg ggt gag acc gcc cct cgt acc aac 867 Lys Tyr Phe Ser Lys Ala Ser Met Gly Giu Thr Ala Pro Arg Thr Asn *275 280 285 ttc aac aac gcc gcc atc aac tac cag aac ctg tac ctg ggt ctg cgt 915 Phe Asn Asn Ala Ala Ile Asn Tyr Gin Asn Leu Tyr Leu Gly Leu Arg :::290 295 300 ttc atc atc aag aag gcc tcc aac tcc cgt aac atc aac aac gac aac 963 Phe Ile Ile Lys Lys Ala Ser Asn Ser Arg Asn Ile Asn Asn Asp Asn 305 310 315 atc gtc cgt gag ggt gac tac atc tac ctg aac atc gac aac atc tcc 1011 Ile Val Arg Giu Gly Asp Tyr Ile Tyr Leu Asn Ile Asp Asn Ile Ser *320 325 330 gac gag tcc tac cgt gtc tac gtc ctg gtc aac tcc aag gag atc cag 1059 *Asp Giu Ser Tyr Arg Val Tyr Val Leu Val Asn Ser Lys Giu Ile Gin 335 340 345 350 *acc cag ctg ttc ctg gcc cca atc aac gac gac cct acc ttc tac gac 1107 Thr Gin Leu Phe Leu Ala Pro Ile Asn Asp Asp Pro Thr Phe Tyr Asp 355 360 365 gtc ctg cag atc aag aag tac tac gag aag acc acc tac aac tgt cag 1155 Val Leu Gin Ile Lys Lys Tyr Tyr Giu Lys Thr Thr Tyr Asn Cys Gin 370 375 380 atc ctg tgc gag aag gac acc aag acc ttc gga ctg ttc ggt atc ggt 1203 Ile Leu Cys Giu Lys Asp Thr Lys Thr Phe Gly Leu Phe Gly Ile Gly PAOpcEj.=wdeT3035.00 wy .mmesqli9 doc-03/03/2003 -31 385 aag ttc Lys Phe 400 gtc aag gac tac Val Lys Asp Tyr ggt Gly 405 tac gtc tgg gac Tyr Val Trp Asp acc Thr 410 tac gac aac tac Tyr Asp Asn Tyr ttc Phe 415 tgt atc tcc cag Cys Ilie Ser Gin tgg Trp 420 tac ctg cgt cgt Tyr Leu Arg Arg atc Ile 425 tcc gag aac atc Ser Giu Asn Ile aac Asn 430 ggt Gly 1251 1299 1347 aag ctg cgt Lys Leu Arg tgg acc gag Trp Thr Glu ctg gga tgt a.

Leu Gly Cys A: 435 taataggaat tc ac tgg cag sn Trp Gin ttc Phe 440 atc cca gtc gac Ile Pro Val Asp gag Glu 445 1368 <210> 18 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Encoded polypeptide of a synthetic construct based on BoNTG Hc 0 000 0 <400> 18 :00 0 '00000 *0000* Met Ile Ile Ile As n Met As n Ser Arg Phe 145 Trp Tyr Arg Asp Lys Ser Asp Phe Ser Phe Asn Cys Ile 130 Phe Phe Ile Ile Thr 210 Asp Ser Ser Asn Asn Asp Asn Ile 115 Ile Glu Ser Asn Asn 195 Thr Thr Asn 20 Ser Asp Ile Asn Asp 100 Lys Trp Tyr Ile Gly 180 Ser Lys Ile Ala Gly Ile Thr Phe 85 Ile As n Thr Ser Thr 165 Ser Ser Phe Leu Ile Tyr Giy Ala 70 Ser Gin Asp Leu Ile 150 Ile Leu Asn Val1 Gin Ser Ala 40 Gly Gin Asn Tyr Gly 120 Asp Asp As n Lys Ile 200 Ile Val Leu 25 Thr Gin Ser Phe Leu 105 Trp Val Asn Asp Ser 185 Asp Lys Phe 10 Ser Met Phe Lys Trp 90 Gin Lys Asn Ile Arg 170 Giu Phe Asp Asn Tyr As n Lys Phe 75 Val As n Val1 Ala Ser 155 Leu Lys Lys Phe Asn Tyr Arg Gly Val Gly Leu Asn Val Val Arg Thr Giu Tyr Ser Ile 125 Lys Ser 140 Asp Tyr Gly Asn Ile Leu Leu Ile 205 Asn Ile Ile Gly Ser Asn Tyr Pro Thr 110 Lys Lys Ile Ala Asn 190 Asn Phe Ser Arg Asp Ser Asp Lys Ile Giy Ser Asn Asn 175 Leu Cys Gly As n Leu Val Glu Ser Tyr Ile Asn Ile Lys 160 Ile Asp Thr Arg P.%OpcrkEjb inoded\00350 0 y madqitngd-3/32 Giu 225 Thr Gin Phe Asn Ile 305 Arg Ser Leu Gin Cys 385 Vali Ile Arg Giu Leu Asn Tyr Ser Aia 290 Lys Giu Tyr Phe Ile 370 Giu Lys Ser Leu Asn Thr Tyr Lys 275 Aia Lys Giy Arg Leu 355 Lys Lys Asp Gin Giy 435 Aia Leu Leu 260 Aila Ile Aia Asp Vali 340 Ala Lys Asp Tyr Trp 420 Cys Thr Lys 245 Phe Ser Asn Ser Tyr 325 Tyr Pro Tyr Thr Giy 405 Tyr Asn Giu 230 Asp Asn Met Tyr Asn 310 Ile Vali Ile Tyr Lys 390 Tyr Leu Trp Val1 Phe Gin Gly Gin 295 Ser Tyr Leu Asn Giu 375 Thr Val1 Arg Gin Ser Trp Giy Giu 280 Asn Arg Leu Vali Asp 360 Lys Phe Trp Arg Phe 440 Ser Giy Met 265 Thr Leu Asn Asn As n 345 Asp Thr Giy Asp Ile 425 Ile 32 Leu Asn 250 Gin Aia Tyr Ile Ile 330 Ser Pro Thr Leu Thr 410 Ser Pro Tyr 235 Pro Asn Pro Leu Asn 315 Asp Lys Thr Tyr Phe 395 Tyr Giu Val1 Trp Leu Ile Arg Giy 300 Asn Asn Giu Phe Asn 380 Giy Asp Asn Asp Ile Arg Tyr Thr 285 Leu Asp Ile Ile Tyr 365 Cys Ile As n Ile Giu 445 Gin Tyr Ile 270 Asn Arg Asn Ser Gin 350 Asp Gin Giy Tyr As n 430 Gly Ser 240 Thr Tyr Asn Ile Val1 320 Giu Gin Leu Leu Phe 400 Cys Leu Thr 00 0 .00.

.00 0 00 00 0 0 0* @0 0 *009 @000 <210> 19 <2ii> i242 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA Hc <221> CDS <222> (1239) <400> 19 atg gct ctg aac gac ctg tgc atc aaa gtt aac Met Aia Leu Asn Asp Leu Cys Ile Lys Val Asn 1 5 10 ttc tcc ccg tct gaa gac aac ttc act aac gac Phe Ser Pro Ser Giu Asp Asn Phe Thr Asn Asp 25 gaa atc acc tcc gac act aac atc gaa gct gct Giu Ile Thr Ser Asp Thr Asn Ile Giu Ala Ala 40 ctg gac ctg atc cag cag tac tac ctg act ttc aac tgg gac Asn Trp, Asp ctg ttc Leu Phe ctg aac aaa ggc gaa Leu Asn Lys Gly Glu gaa gaa aac atc tct Giu Glu Asn Ile Ser 48 96 144 192 aac ttc gac aac gaa P %Opc6Ejb wde"0035.00vs.y-deodsogising doc-03IO3/2O0fl 33 0* @0 @0 S 0

*OSS

0@*9 000@

S

ego.

0 @0 0 0 Leu ccg Pro ctg Leu gaa Glu gaa Giu ctg Leu aag Lys 145 gaa Giu act Thr gct Ala ctg Leu gct Ala 225 aaa Lys gaa Giu aaa Lys Asp gaa Giu gaa Giu ctg Leu cac His ctg Leu 130 aaa Lys cag Gin gac Asp ctg Leu atc Ile 210 atc Ile gtt Val1 aaa Lys gtt Val1 Leu aac Asn ctg Leu gac Asp ggt Gly 115 aac Asn gt t Val1 ctg Leu aaa Lys aac Asn 195 ttc Phe ccg Pro ctg Leu tgg Trp aac Asn Ile atc Ile atg Met aaa Lys 100 aaa Lys ccg Pro aac Asn gtt Val1 atc Ile 180 atc Ile tct Ser gtt Val1 act Thr gac Asp 260 act Thr Gin tc c Ser ccg Pro tac Tyr tct Ser tc t Ser aaa Lys tac Tyr 165 gct Ala ggt Gly ggC Gly ctg Leu gt t Val1 245 gaa Giu cag Gin Gin atc Ile 70 aac As n acc Thr cgt Arg cgc Arg get Ala 150 gac Asp gac Asp aac Asn gct Al a ggt Gly 230 c ag Gin gtt Val1 atc Ile Tyr 55 gaa Glu atc Ile atg Met atc Ile gtt Val1 135 act Thr ttc Phe atc Ile atg Met gtt Val1 215 acc Thr acc Thr tac Tyr gac Asp Tyr aac Asn gaa Giu ttc Phe gct Ala 120 tac Tyr gaa Giu ac c Thr act Thr ctg Leu 200 atc Ile ttc Phe atc Ile aaa Lys ctg Leu Leu ctg Leu cgc Arg cac His 105 ctg Leu acc Thr gct Ala gac Asp atc Ile 185 tac Tyr ctg Leu gct Ala gac Asp tac Tyr 265 atc Ile Thr tct Ser ttc Phe 90 tac Tyr act Thr ttc Phe gct Ala gaa Giu 170 atc Ile aaa Lys ctg Leu ctg Leu aac Asn 250 atc Ile cgt Arg Phe tcc Ser 75 ccg Pro ctg Leu aac Asn ttc Phe atg Met 155 act Thr atc Ile gac Asp gaa Giu gt t Val 235 gct Ala gtt Val1 aag Lys Asn gac Asp aac Asn cgt Arg tcc Ser tct Ser 140 ttc Phe tct Ser ccg Pro gac Asp ttc Phe 220 tcc Ser ctg Leu act Thr aag Lys Phe atc Ile Gly gct Ala gtt Val1 125 tcc Ser ctg Leu gaa Giu tac Tyr ttc Phe 205 atc Ile tac Tyr tct Ser aac Asn atg Met Asp atc Ile aag Lys c ag Gin 110 aac As n gac Asp ggt Gly gtt Val1 atc Ile 190 gtt Val1 ccg Pro atc Ile aaa Lys tgg Trp 270 aaa Lys Asn ggt Gly aaa Lys gaa Giu gaa Glu tac Tyr tgg Trp tcc Ser 175 ggc Gly ggt Gly gaa Giu gct Ala cgt Arg 255 ctg Leu gaa Glu Giu cag Gin tac Tyr ttc Phe gct Ala gt t Val1 gtt Val1 160 ace Thr ccg Pro gct Ala atc Ile aac Asn 240 aac Asn gct Ala gct Ala 240 288 336 384 432 480 528 576 624 672 720 768 816 864 0@ @0 S 0

S

*000 S S 0 *000

S

0505

C'

0@SS 0005 0 @050 P:)Opor1Ejh~mwde50035 00 w y moddsqIistia do-03/0312003 34 275 ctg gaa Leu Giu 290 aac cag gct gaa Asn Gin Ala Giu gct Ala 295 act aaa gct atc Thr Lys Ala Ile atc Ile 300 aac tac cag tac Asn Tyr Gin Tyr aac Asn 305 cag tac acc gaa gaa gaa aag aac aac Gin Tyr Thr Giu Giu Giu Lys Asn Asn 310 atc Ile 315 aac ttc aac atc Asn Phe Asn Ile gat Asp 320 960 gac ctg tcc tct Asp Leu Ser Ser aaa Lys 325 ctg aac gaa tcc Leu Asn Giu Ser aac aaa gct atg Asn Lys Ala Met atc aac Ile Asn 335 1008 atc aac aaa Ile Asn Lys atg atc ccg Met Ile Pro 355 ttc Phe 340 ctg aac cag tgc Leu Asn Gin Cys tct Ser 345 gtt tcc tac ctg Vai Ser Tyr Leu atg aac tct Met Asn Ser 350 gct tcc ctg Ala Ser Leu 1056 1104 tac ggc gtt aaa Tyr Giy Val Lys ctg gaa gac ttc Leu Giu Asp Phe gac Asp 365 aaa gac Lys Asp 370 gct ctg ctg aaa Ala Leu Leu Lys tac Tyr 375 atc cgt gac aac Ile Arg Asp Asn tac Tyr 380 ggt act ctg atc Gly Thr Leu Ile 1152 1200 ggc Giy 385 cag gtt gac cgt Gin Val Asp Arg ctg Leu 390 aaa gac aag gtt Lys Asp Lys Vai aac As n 395 aac acc ctg tct Asn Thr Leu Ser gac atc ccg ttc Asp Ile Pro Phe c ag Gin 405 ctg tcc aaa tac Leu Ser Lys Tyr gtt Vali 410 gac aac cag taa Asp Asn Gin 1242 <210> <211> 413 <212> PRT <213> Artificial Sequence <220> <223> Encoded poiypeptide of a synthetic construct based on BoNTA Hn <400> Met Aia Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe 1 5 10 Phe Ser Pro Ser Giu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Giu 20 25 Giu Ilie Thr Ser Asp Thr Asn Ile Giu Ala Ala Giu Giu Asn Ile Ser 35 40 Leu Asp Leu Ile Gin Gin Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Giu 55 Pro Giu Asn Ile Ser Ile Giu Asn Leu Ser Ser Asp Ile Ile Gly Gin 70 75 Leu Giu Leu Met Pro Asn Ile Giu Arg Phe Pro Asn Giy Lys Lys Tyr 90 P: Opm\EjbmwddSOO3500 y wda isting doc-0331V2003 Glu Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gin Glu Phe 100 105 110 Glu His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala 115 120 125 Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val 130 135 140 Lys Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val 145 150 155 160 Glu Gin Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr 165 170 175 Thr Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro 180 185 190 Ala Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala 195 200 205 Leu Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile 210 215 220 Ala Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn 225 230 235 240 Lys Val Leu Thr Val Gin Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn 245 250 255 Glu Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala 260 265 270 Lys Val Asn Thr Gin Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala 275 280 285 Leu Glu Asn Gin Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gin Tyr 290 295 300 Asn Gin Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp 305 310 315 320 Asp Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn 325 330 335 Ile Asn Lys Phe Leu Asn Gin Cys Ser Val Ser Tyr Leu Met Asn Ser 340 345 350 Met Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu 355 360 365 Lys Asp Ala Leu Leu Lys Tyr Ile Arg Asp Asn Tyr Gly Thr Leu Ile 370 375 380 Gly Gin Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr 385 390 395 400 Asp Ile Pro Phe Gin Leu Ser Lys Tyr Val Asp Asn Gin 405 410 <210> 21 <211> 1242 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA He <221> CDS <222> <400> 21 atg gct cca gga atc tgt atc gac gtc gac aac gag gac ttg ttc ttc 48 Met Ala Pro Gly Ile Cys Ile Asp Val Asp Asn Glu Asp Leu Phe Phe 1 5 10 P '.Op\Ejb mamd3od\35 00 .wy =mdwdisug doc-03/03/2003 atc gct gac Ile Ala Asp atc gag tac Ile Giu Tyr aag Lys aac tcc ttc tcc Asn Ser Phe Ser gac Asp 25 gac ttg tcc aag Asp Leu Ser Lys aac gag aga Asn Giu Arg ttc cca atc Phe Pro Ile aac acc cag tcc Asn Thr Gin Ser tac atc gag aac Tyr Ile Glu Asn gac Asp aac gag Asn Glu ttg atc ttg gac Leu Ile Leu Asp ac c Thr 55 gac ttg atc tcc Asp Leu Ile Ser aag Lys atc gag ttg cca Ile Giu Leu Pro gag aac acc gag Giu Asn Thr Giu tcc Ser ttg act gac ttc Leu Thr Asp Phe aac As n gtc gac gtc cca Vai Asp Vai Pro tac gag aag caa Tyr Giu Lys Gin gct atc aag aag Aia Ile Lys Lys ttc acc gac gag Phe Thr Asp Giu aac acc Asn Thr atc ttc caa Ile Phe Gin atc tcc ttg Ile Ser Leu 115 ctg tac tct cag Leu Tyr Ser Gin ac c Thr 105 ttc cct ttg gac Phe Pro Leu Asp atc aga gac Ile Arg Asp tcc aac aag Ser Asn Lys acc tct tcc ttc Thr Ser Ser Phe gac Asp 120 gac gcc ctg ctg Asp Aia Leu Leu gtc tac Vai Tyr 130 tcc ttc ttc tcc Ser Phe Phe Ser gac tac atc aag Asp Tyr Ile Lys act Thr i4 0 gct aac aag gtc Ala Asn Lys Val gtc Val1 145 gag gcc ggt ttg Giu Ala Giy Leu ttc Phe 150 gct ggt tgg gtc Ala Giy Trp Val aag Lys 155 cag atc gtc aac Gin Ile Val Asn 432 480 528 ttc gtc atc gag Phe Val Ile Giu gct Ala 165 aac aag tcc aac Asn Lys Ser Asn acc Thr 170 atg gac aag att Met Asp Lys Ile gcc gac Ala Asp 175 atc tcc ttg Ile Ser Leu gag acc gcc Giu Thr Ala 195 att Ile 180 gtc cca tac atc Val Pro Tyr Ile ttg gcc ttg aac Leu Ala Leu Asn gtc ggt aac Val Giy Asn 190 gct ggt gcc Ala Giy Ala 576 624 aag ggt aac ttc Lys Gly Asn Phe gag Glu 200 aac gct ttc gag Asn Ala Phe Giu atc Ile 205 tcc atc Ser Ile 210 ttg ttg gag ttc Leu Leu Giu Phe atc Ile 215 cca gag ttg ttg Pro Glu Leu Leu atc Ile 220 cca gtc gtc ggt Pro Val Val Gly 672 720 gcc Ala 225 ttc ttg ttg gag Phe Leu Leu Glu tcc Ser 230 tac atc gac aac Tyr Ile Asp Asn aag Lys 235 aac aag atc atc Asn Lys Ile Ile aag Lys 240 PAOpM\Ejh mdod\5DO35 DO u y mwWqfsn dm-31032DO3 aco Thr tac Tyr tac Tyr got Ala gag Giu 305 aao Asn ggt Gly gag Giu tac Tyr aag Lys 385 tc Ser atc Ile ggt Gly aco Thr ttg Leu 290 aag Lys gag Giu tgt Cys aag Lys atc Ile 370 tc Ser atc Ile gac Asp ttg Leu atc Ile 275 gag Giu too Ser ggt Gly tcc Ser ttg Leu 3S5 gac Asp aag Lys tao Tyr aac Asn ato Ile 260 aag Lys gag Giu aac Asn att Ile gtC Val1 340 ttg Leu gag Giu gtc Val1 acc Thr gct Ala 245 gtc Val gag Giu atc Ile att Ile aac Asn 325 tco Ser gao Asp aac Asn aac Asn aac Asn 405 ttg Leu gcc Ala ggt Gly ato Ile aac Asn 310 cag Gin tac Tyr ttc Phe aag Lys aag Lys 390 gao Asp acc Thr caa Gin atg Met aag Lys 295 atc Ile gc Aia ttg Leu gac Asp ttg Leu 375 tac Tyr acc Thr aag Lys tgg Trp tac Tyr 280 tac Tyr gao Asp ato Ile atg Met aac Asn 360 tac Tyr ttg Leu ato Ile aga aac Arg Asn 250 ttg too Leu Ser 265 aag gcc Lys Ala aga tao Arg Tyr ttc aac Phe Asn gac aao Asp Asn 330 aag aag Lys Lys 345 acc otg Thr Leu ttg atc Leu Ile aag acc Lys Thr ttg atc Leu Ile 410 gag aag tgg tcc gac atg Giu Lys Trp, Ser Asp Met 255 ac c Thr ttg Leu aao As n gac Asp 315 ato Ile atg Met aag Lys ggt Giy atc Ile 395 gag Glu gto Val aao Asn atc Ile 300 at o Ile aa 0 Asn att Ile aag Lys tcc Ser 380 atg Met atg Met aao As n tac Tyr 285 tac Tyr aao Asn aao Asn oca Pro aao Asn 365 got Ala ooa Pro tto Phe aoo Thr 270 o ag Gin too Ser too Ser tto Phe ttg Leu 350 ttg Leu gag Giu tto Phe taa o aa Gin goo Ala gag Giu aag Lys ato Ile 335 goo Aia ttg Leu tao Tyr gao Asp tto Phe oaa Gin aag Lys otg Leu 320 aao Asn gto Vali aao Asn gag Giu ttg Leu 400 816 864 912 960 1008 1056 1104 1152 1200 1242 <210> 22 <211> 413 <212> PRT <213> Artificiai Sequence <220> <223> Enooded poiypeptide of a synthetio oonstruot based on BoN'TB Hn <400> 22 Met Aia Pro Giy Ile Cys Ile Asp Val Asp Asn Giu Asp Leu Phe Phe 1 5 10 P Op'Ejh d&M0035 00.-y d iq dod-03/O3I2OO3 -38- Ile Ala Asp Lys Asn Ser Phe Ser Asp Asp Leu Ser Lys Asn Glu Arg 25 Ile Glu Tyr Asn Thr Gin Ser Asn Tyr Ile Glu Asn Asp Phe Pro Ile 40 Asn Glu Leu Ile Leu Asp Thr Asp Leu Ile Ser Lys Ile Glu Leu Pro 55 Ser Glu Asn Thr Glu Ser Leu Thr Asp Phe Asn Val Asp Val Pro Val 70 75 Tyr Glu Lys Gin Pro Ala Ile Lys Lys Ile Phe Thr Asp Glu Asn Thr 90 Ile Phe Gin Tyr Leu Tyr Ser Gin Thr Phe Pro Leu Asp Ile Arg Asp 100 105 110 Ile Ser Leu Thr Ser Ser Phe Asp Asp Ala Leu Leu Phe Ser Asn Lys 115 120 125 Val Tyr Ser Phe Phe Ser Met Asp Tyr Ile Lys Thr Ala Asn Lys Val 130 135 140 Val Glu Ala Gly Leu Phe Ala Gly Trp Val Lys Gin Ile Val Asn Asp 145 150 155 160 Phe Val Ile Glu Ala Asn Lys Ser Asn Thr Met Asp Lys Ile Ala Asp 165 170 175 Ile Ser Leu Ile Val Pro Tyr Ile Gly Leu Ala Leu Asn Val Gly Asn 180 185 190 Glu Thr Ala Lys Gly Asn Phe Glu Asn Ala Phe Glu Ile Ala Gly Ala 195 200 205 Ser Ile Leu Leu Glu Phe Ile Pro Glu Leu Leu Ile Pro Val Val Gly 210 215 220 Ala Phe Leu Leu Glu Ser Tyr Ile Asp Asn Lys Asn Lys Ile Ile Lys 225 230 235 240 Thr Ile Asp Asn Ala Leu Thr Lys Arg Asn Glu Lys Trp Ser Asp Met 245 250 255 Tyr Gly Leu Ile Val Ala Gin Trp Leu Ser Thr Val Asn Thr Gin Phe 260 265 270 Tyr Thr Ile Lys Glu Gly Met Tyr Lys Ala Leu Asn Tyr Gin Ala Gin *e 275 280 285 Ala Leu Glu Glu Ile Ile Lys Tyr Arg Tyr Asn Ile Tyr Ser Glu Lys S* 290 295 300 Glu Lys Ser Asn Ile Asn Ile Asp Phe Asn Asp Ile Asn Ser Lys Leu 305 310 315 320 Asn Glu Gly Ile Asn Gin Ala Ile Asp Asn Ile Asn Asn Phe Ile Asn 325 330 335 Gly Cys Ser Val Ser Tyr Leu Met Lys Lys Met Ile Pro Leu Ala Val 340 345 350 Glu Lys Leu Leu Asp Phe Asp Asn Thr Leu Lys Lys Asn Leu Leu Asn 355 360 365 Tyr Ile Asp Glu Asn Lys Leu Tyr Leu Ile Gly Ser Ala Glu Tyr Glu 370 375 380 *Lys Ser Lys Val Asn Lys Tyr Leu Lys Thr Ile Met Pro Phe Asp Leu 385 390 395 400 Ser Ile Tyr Thr Asn Asp Thr Ile Leu Ile Glu Met Phe 405 410 <210> 23 <211> 1200 <212> DNA <213> Artificial Sequence P OM Ejf =mdedMS035 00 fsy mmemlvn doc.03/03/2003 39 <220> <223> Synthetic construct based on BoNTA Hc <221> CDS <222> (1197) <400> 23 atg tcc ctg tac aac aag acc ctt gac Met Ser Leu Tyr Asn Lys Thr Leu Asp 1 5 tgt Cys 10 aga gag ctg ctg Arg Glu Leu Leu gtg aag Val Lys aac act gac Asn Thr Asp atc ttc ctg Ile Phe Leu ctg Leu cca ttc atc ggt Pro Phe Ile Gly atc agt gac gtg Ile Ser Asp Val aag act gac Lys Thr Asp atc tac tac Ile Tyr Tyr cgt aag gac atc Arg Lys Asp Ile aac Asn 40 gag gag act gag Giu Giu Thr Giu gtg Val cca gac Pro Asp aac gtg tca gta Asn Val Ser Val gac Asp caa gtg atc ctc Gin Val Ile Leu aag aac acc tcc Lys Asn Thr Ser gag Giu cat gga caa cta His Gly Gin Leu ctg ctc tac cct Leu Leu Tyr Pro agt Ser 75 atc gac agt gag Ile Asp Ser Giu agt Ser gag atc ctg cca Glu Ile Leu Pro gag aat caa gtc Giu Asn Gin Val ttc Phe 90 tac gac aac cgt Tyr Asp Asn Arg acc cag Thr Gin 192 240 288 336 384 aac gtg gac Asn Vai Asp agt gac aac Ser Asp Asn 11s tac Tyr 100 ctg aac tcc tac Leu Asn Ser Tyr tac Tyr 105 tac cta gag tct Tyr Leu Giu Ser cag aag ctg Gin Lys Leu gag gag gct Giu Giu Ala gtg gag gac ttc Val Glu Asp Phe ttc acg cgt tca Phe Thr Arg Ser atc Ile 125 ctg gac Leu Asp 130 aac agt gca aag Asn Ser Aia Lys gtg Val1 135 tac act tac ttc Tyr Thr Tyr Phe cct Pro 140 acc ctg gct aac Thr Leu Ala Asn 00 00*00* *o *06* aag Lys 145 gtg aat gcc ggt Vai Asn Ala Giy gtg Val1 150 caa ggt ggt ctg Gin Giy Gly Leu ctg atg tgg gca Leu Met Trp Aia aac As n 160 gac gtg gtt gag Asp Val Val Glu ttc act acc aac Phe Thr Thr Asn atc Ile 170 ctg cgt aag gac Leu Arg Lys Asp aca ctg Thr Leu 175 gac aag atc Asp Lys Ile tca Ser 180 gat gtg tca gct Asp Val Ser Ala atc Ile 185 atc ccc tac atc Ile Pro Tyr Ile gga ccc gca Gly Pro Ala 190 ctg aac atc tcc aac tct gtg cgt cgt gga aac ttc act gag gca ttc Leu Asn Ile Ser Asn Ser Vai Arg Arg Gly Asn Phe Thr Giu Ala Phe P:%pcF~jh=mdMS03500 u~y .mmddsef isd dox.03/03/2003 40 gca Ala atc Ile 225 aac Asn aga Arg atc Ile aac As n aag Lys 305 ctg Leu aac Asn ctg Leu gca Ala gag Giu 385 taa gtc Val1 210 cct Pro gag Giu tgg Trp atc Ile tac Tyr 290 tac Tyr aag Lys aag Lys cct Pro aag Lys 370 gtc Val1 195 act Thr gct Al a atc Ile aag Lys ac c Thr 275 c ag Gin tcc Ser aac Asn ttc Phe aag Lys 355 ctg Leu gac Asp ggt Gly c tg Leu atc Ile gac Asp 260 c ag Gin gca Ala gga Gly agt Ser atc Ile 340 gtc Val1 atc Ile gtc Val1 ggt Gly aag Lys 245 tcc Ser ttc Phe ggt Gly agc Ser ctg Leu 325 cga Arg atc Ile aac Asn acc Thr gca Al a 230 ac c Thr tac Tyr aac Asn gca Ala gac Asp 310 gac Asp gag Giu gac Asp ctg Leu atc Ile 215 ttc Phe atc Ile gag Giu aac Asn atc Ile 295 aag Lys gtc Val1 tgC Cys gag Giu atc Ile 375 200 ctg Leu gtg Val gac Asp tgg Trp atc Ile 280 aag Lys gag Giu aag Lys tcc Ser ctg Leu 360 gac Asp ctg Leu atc Ile aac Asn atg Met 265 tcc Ser gct Al a aac Asn atc Ile gtc Val1 345 aac Asn tcc Ser gag Giu.

tac Tyr tgt Cys 250 atg Met tac Tyr aag Lys atc Ile tcg Ser 330 ac c Thr gag Giu cat His gca Ala agt Ser 235 ctg Leu gga Gly c ag Gin atc Ile aag Lys 315 gag Giu.

tac Tyr ttc Phe aac Asn ttc Phe 220 aag Lys gag Giu acg Thr atg Met gac Asp 300 agc Ser gca Ala ctg Leu gac Asp atc Ile 380 205 cct Pro gtc Val1 cag Gin tgg Trp tac Tyr 285 ctg Leu c ag Gin atg Met ttc Phe cga Arg 365 atc Ile gag Glu cag Gin agg Arg ttg Leu 270 gac Asp gag Giu gt t Val aac Asn aag Lys 350 aac As n ctg Leu ttc Phe gag Giu atc Ile 255 tcc Ser tcc Ser tac Tyr gag Giu aac Asn 335 aac Asn ac c Thr gtc Val1 aca Thr cga Arg 240 aag Lys agg Arg ctg Leu aag Lys aac Asn 320 atc Ile atg Met aag Lys ggt Gly 672 720 768 816 864 912 960 1008 1056 1104 1152 0. *o see.

.0 906 0* aag ctg aag gca aag gta aac aac agc ttc cag aac 1197 Lys Leu Lys 390 Ala Lys Val Asn Asn 395 Ser Phe Gin Asn 1200 <210> 24 <211> 399 <212> PRT <213> Artificial Sequence P.%Op\.Ejh -md \5003500 ym-w4isLiui d.,O31312003 -41- <220> <223> Encoded polypeptide of a synthetic construct based on BoNTC Hn <400> 24 Met Ser Leu Tyr Asn Lys Thr Leu Asp Cys Arg Glu Leu Leu Val Lys 1 5 10 Asn Thr Asp Leu Pro Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp 25 Ile Phe Leu Arg Lys Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr 40 Pro Asp Asn Val Ser Val Asp Gin Val Ile Leu Ser Lys Asn Thr Ser 55 Glu His Gly Gin Leu Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser 70 75 Glu Ile Leu Pro Gly Glu Asn Gin Val Phe Tyr Asp Asn Arg Thr Gin 90 Asn Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu Ser Gin Lys Leu 100 105 110 Ser Asp Asn Val Glu Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala 115 120 125 Leu Asp Asn Ser Ala Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn 130 135 140 Lys Val Asn Ala Gly Val Gin Gly Gly Leu Phe Leu Met Trp Ala Asn 145 150 155 160 Asp Val Val Glu Asp Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu 165 170 175 Asp Lys Ile Ser Asp Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala 180 185 190 Leu Asn Ile Ser Asn Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe 195 200 205 Ala Val Thr Gly Val Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr 210 215 220 Ile Pro Ala Leu Gly Ala Phe Val Ile Tyr Ser Lys Val Gin Glu Arg 225 230 235 240 SAsn Glu Ile Ile Lys Thr Ile Asp Asn Cys Leu Glu Gin Arg Ile Lys 245 250 255 Arg Trp Lys Asp Ser Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg 260 265 270 Ile Ile Thr Gin Phe Asn Asn Ile Ser Tyr Gin Met Tyr Asp Ser Leu 275 280 285 Asn Tyr Gin Ala Gly Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys 290 295 300 Lys Tyr Ser Gly Ser Asp Lys Glu Asn Ile Lys Ser Gin Val Glu Asn 305 310 315 320 Leu Lys Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile S325 330 335 Asn Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met 340 345 350 SLeu Pro Lys Val Ile Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys 355 360 365 Ala Lys Leu Ile Asn Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly 370 375 380 Glu Val Asp Lys Leu Lys Ala Lys Val Asn Asn Ser Phe Gin Asn 385 390 395 P:1OpcrkEjh.=mde\035.00 -yymmdedsisenSgdK-0O03/2mD -42- <210> <211> 1161 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA Hc <221> CDS <222> (1158) <400> atg gcc aac tcc cgt gac gac tcc acc tgc atc aag gtc aag aac aac 48 Met Ala Asn Ser Arg Asp Asp Ser Thr Cys Ile Lys Val Lys Asn Asn 1 5 10 aga ctg cca tac gtt gcc gac aag gac tcc atc tcc cag gag atc ttc 96 Arg Leu Pro Tyr Val Ala Asp Lys Asp Ser Ile Ser Gin Giu Ile Phe 25 gag aac aag atc atc acc gac gag acc aac gtt caa aac tac tcc gac 144 Glu Asn Lys Ile Ile Thr Asp Giu Thr Asn Val Gin Asn Tyr Ser Asp 40 aag ttc tct ttg gac gag tcc atc ctg gac ggt cag gtc cca atc aac 192 Lys Phe Ser Leu Asp Giu Ser Ile Leu Asp Giy Gin Val Pro Ile Asn 55 cca gag atc gtc gac cca ctg ttg cca aac gtc aac atg gag cca ttg 240 Pro Giu Ile Val Asp Pro Leu Leu Pro Asn Val Asn Met Glu Pro Leu 70 75 aac ttg cca ggt gag gag atc gtc ttc tac gac gac atc acc aag tac 288 Asn Leu Pro Gly Giu Giu Ile Val Phe Tyr Asp Asp Ile Thr Lys Tyr 85 90 gtc gac tac ttg aac tcc tac tac tac ttg gag tct caa aag ttg tct 336 Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu Giu Ser Gin Lys Leu Ser 100 105 110 aac aac gtc gag aac atc acc ttg acc acc tcc gtc gag gag gcc ttg 384 Asn Asn Vai Giu Asn Ile Thr Leu Thr Thr Ser Val Giu Giu Ala Leu 115 120 125 ggt tac tct aac aag atc tac acc ttc ctg cca tcc ttg gct gag aag 432 Gly Tyr Ser Asn Lys Ile Tyr Thr Phe Leu Pro Ser Leu Ala Giu Lys 130 135 140 gtt aac aag ggt gtt caa gct ggt ttg ttc ctg aac tgg gcc aac gag 480 Val Asn Lys Gly Val Gin Ala Gly Leu Phe Leu Asn Trp Ala Asn Glu 145 150 155 160 gtc gtc gag gac ttc acc acc aac atc atg aag aag gac acc ctg gac 528 Val Val Giu Asp Phe Thr Thr Asn Ile Met Lys Lys Asp Thr Leu Asp 165 170 175 aag atc tcc gac gtc tcc gtc atc atc cca tac atc ggt cca gcc ttg 576 P.\Opa\Ejb mmdcd\0035 00 us~ymwcJg gd-3132 43 see.

.0 0* .006 see* Lys Ile Ser aac atc ggt Asn Ile Gly 195 acc gce ggt Thr Ala Gly 210 eca gcc etg Pro Ala Leu 225 aag atc ate Lys Ile Ile tgg aag gac Trp Lys Asp acc aec caa Thr Thr Gin 275 tac cag gcc Tyr Gin Aia 290 tac tcc ggt Tyr Ser Gly 305 aag aac tee Lys Asn Ser aag tte atc Lys Phe Ile cca aag gte Pro Lys Vai 355 gag ctg ate Giu Leu Ile 370 gtt gac taa Val Asp 385 <z210> 26 Asp 180 aac Asn gtc Val ggt Gly aag Lys tcc Ser 260 ttc Phe gac Asp tcc Ser t tg Leu cgt Arg 340 ate Ile aac Asn Val tcc Ser gce Ala gte Val ace Thr 245 tac Tyr aac As n gcc Ala gac Asp gac Asp 325 gag Giu gac Asp ctg Leu Ser gcc Ala ttc Phe ttc Phe 230 ate Ile c ag Gin c ac His atc Ile aag Lys 310 gtc Val1 tgt Cys gag Glu ate Ile Val ctg Leu ctg Leu 215 acc Thr gag Giu tgg Trp ate Ile aag Lys 295 gag Giu aag Lys tcc Ser ctg Leu gac Asp 375 Ile aga Arg 200 ctg Leu ttc Phe aac Asn atg Met aac Asn 280 gee Ala aac Asn ate Ile gte Val1 aac Asn 360 tcc Ser Ile 185 ggt Gly gag Giu tac Tyr tgc Cys gt t Val1 265 tac Tyr aag Lys atc Ile tc Ser ace Thr 345 aag Lys cac His Pro aac Asn ggt Gly tcc Ser ttg Leu 250 tcc Ser eag Gin ate Ile aag Lys gag Giu 330 tac Tyr ttc Phe aac Asn Tyr ttc Phe ttc Phe tee Ser 235 gag Giu aac Asn atg Met gac Asp tcc Ser 315 gc Ala c tg Leu gac Asp ate Ile Ile aac Asn cca Pro 220 ate Ile cag Gin tgg Trp tac Tyr ctg Leu 300 c ag Gin atg Met ttc Phe ctg Leu atc Ile 380 Gly cag Gin 205 gag Giu cag Gin aga Arg ctg Leu gac Asp 285 gag Giu gtc Val1 aac Asn aag Lys aga Arg 365 ctg Leu Pro 190 gcc Ala ttc Phe gag Giu gte Val1 tee Ser 270 tee Ser tac Tyr gag Giu aac Asn aac As n 350 ac c Thr gt t Val1 Ala ttc Phe ace Thr aga Arg aag Lys 255 aga Arg ctg Leu aag Lys aac Asn ate Ile 335 atg Met aag Lys ggt Gly Leu gcc Ala atc Ile gag Giu 240 aga Arg ate Ile tcc Ser aag Lys ctg Leu 320 aac Asn ctg Leu ace Thr gag Glu 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1161 P 1Op~Ejh cdde%0035 OO.usr y mwmdedseqiios doc-3/OY2003 -44- <211> 386 <212> PRT <213> Artificial Sequence <220> <223> Encoded polypeptide of a synthetic construct based on BoNTD Hn <400> 26 Met Ala Asn Ser Arg Asp Asp Ser Thr Cys lie Lys Val Lys Asn Asn 1 5 10 Arg Leu Pro Tyr Val Ala Asp Lys Asp Ser Ile Ser Gin Glu Ile Phe 25 Glu Asn Lys Ile Ile Thr Asp Glu Thr Asn Val Gin Asn Tyr Ser Asp 40 Lys Phe Ser Leu Asp Glu Ser Ile Leu Asp Gly Gin Val Pro Ile Asn 55 Pro Glu Ile Val Asp Pro Leu Leu Pro Asn Val Asn Met Glu Pro Leu 70 75 Asn Leu Pro Gly Glu Glu Ile Val Phe Tyr Asp Asp Ile Thr Lys Tyr 90 Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu Ser Gin Lys Leu Ser 100 105 110 Asn Asn Val Glu Asn Ile Thr Leu Thr Thr Ser Val Glu Glu Ala Leu 115 120 125 Gly Tyr Ser Asn Lys Ile Tyr Thr Phe Leu Pro Ser Leu Ala Glu Lys 130 135 140 Val Asn Lys Gly Val Gin Ala Gly Leu Phe Leu Asn Trp Ala Asn Glu S* 145 150 155 160 S* Val Val Glu Asp Phe Thr Thr Asn Ile Met Lys Lys Asp Thr Leu Asp *165 170 175 Lys Ile Ser Asp Val Ser Val Ile Ile Pro Tyr Ile Gly Pro Ala Leu 180 185 190 Asn Ile Gly Asn Ser Ala Leu Arg Gly Asn Phe Asn Gin Ala Phe Ala 195 200 205 Thr Ala Gly Val Ala Phe Leu Leu Glu Gly Phe Pro Glu Phe Thr Ile 210 215 220 Pro Ala Leu Gly Val Phe Thr Phe Tyr Ser Ser Ile Gin Glu Arg Glu 225 230 235 240 Lys Ile Ile Lys Thr Ile Glu Asn Cys Leu Glu Gin Arg Val Lys Arg 245 250 255 Trp Lys Asp Ser Tyr Gin Trp Met Val Ser Asn Trp Leu Ser Arg Ile 260 265 270 0-•0*0 Thr Thr Gin Phe Asn His lie Asn Tyr Gin Met Tyr Asp Ser Leu Ser 275 280 285 Tyr Gin Ala Asp Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys 290 295 300 Tyr Ser Gly Ser Asp Lys Glu Asn Ile Lys Ser Gin Val Glu Asn Leu 305 310 315 320 Lys Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn 0* 325 330 335 Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu 340 345 350 Pro Lys Val Ile Asp Glu Leu Asn Lys Phe Asp Leu Arg Thr Lys Thr 355 360 365 Glu Leu Ile Asn Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu 370 375 380 P XOpa\Ejh =wmdd\50035 00v uy mmdisschsg doc-03103/2003 Val Asp 385 <210> 27 <211> 1149 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA He <221> CDS <222> <400> 27 atg tcc atc tgc ate gag atc aac aac ggt gag ctg ttc ttc gtg get 48 Met Ser Ile Cys Ile Glu Ile Asn Asn Gly Glu Leu Phe Phe Val Ala 1 5 10 tec gag aac agt tac aac gat gac aac atc aac act cct aag gag att 96 Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile Asn Thr Pro Lys Glu Ile 25 gac gac acc gtc act tct aac aac aac tac gaa aac gac ctg gac cag 144 Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr Glu Asn Asp Leu Asp Gin 35 40 gtc atc cta aac ttc aac tcc gag tcc gcc cct ggt ctg tcc gac gag 192 S Val Ile Leu Asn Phe Asn Ser Glu Ser Ala Pro Gly Leu Ser Asp Glu 50 55 aag ctg aac ctg acc ate cag aac gac get tac atc cca aag tac gac 240 Lys Leu Asn Leu Thr Ile Gin Asn Asp Ala Tyr Ile Pro Lys Tyr Asp 70 75 tec aac ggt aca tcc gat atc gag cag cat gac gtt aac gag ctt aac 288 Ser Asn Gly Thr Ser Asp Ile Glu Gin His Asp Val Asn Glu Leu Asn 90 gtc ttc ttc tac tta gac get cag aag gtg ccc gag ggt gag aac aac 336 SVal Phe Phe Tyr Leu Asp Ala Gin Lys Val Pro Glu Gly Glu Asn Asn 100 105 110 Sgtc aat ctc acc tct tca att gac aca gcc ttg ttg gag cag cct aag 384 Val Asn Leu Thr Ser Ser Ile Asp Thr Ala Leu Leu Glu Gin Pro Lys 115 120 125 ate tac acc ttc ttc tcc tcc gag ttc atc aac aac gtc aac aag cct 432 Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile Asn Asn Val Asn Lys Pro 130 135 140 gtg cag gcc gca ttg ttc gta agc tgg att cag cag gtg tta gta gac 480 Val Gin Ala Ala Leu Phe Val Ser Trp Ile Gin Gin Val Leu Val Asp 145 150 155 160 ttc act act gag get aac cag aag tcc act gtt gac aag ate get gac 528 P Opa\Ejb mmdeAS0035 00 -y mwcw11m dloc03/0312003 46 Phe ate Ile gag Glu ggt Gly gte Val1 225 gtc Val1 aag Lys ace Thr cag Gin acc Thr 305 atc Ile gac Asp aac Asn Thr tee Ser gca Ala att Ile 210 ttc Phe att Ile gaa Giu cag Gin gtc Val1 290 ctg Leu gag Giu agg Arg gag Giu Thr ate Ile c ag Gin 195 t tg Leu acg Thr aag Lys gte Val1 ttc Phe 275 aac As n gag Giu aac Asn ttc Phe gtc Val1 355 Giu gtc Val1 180 aag Lys ttg Leu atc Ile gce Ala tat Tyr 260 aac Asn gcc Ala gag Giu gag Giu ctg Leu 340 aag Lys Ala 165 gte Val1 Gly gag Giu aag Lys atc Ile 245 tc Ser aag Lys ate Ile aag Lys ctg Leu 325 acc Thr ate Ile Asn cca Pro aac Asn ttc Phe tcc Ser 230 aac Asn ttc Phe cga Arg aag Lys aac As n 310 aac Asn gag Giu aac Asn Gin tac Tyr ttc Phe gaa Giu 215 ttc Phe aac Asn atc Ile aag Lys ace Thr 295 gag Giu cag Gin tcc Ser aag Lys Lys atc Ile aag Lys 200 ccc Pro ctg Leu gee Ala gte Val1 gag Giu 280 atc Ile ctt Leu aag Lys tcc Ser ctg Leu 360 Ser Thr 170 ggt ctg Gly Leu 185 gat gee Asp Ala gag ctg Giu Leu ggt tcc Gly Ser ctg aag Leu Lys 250 teg aac Ser Asn 265 cag atg Gin Met atc gag Ile Giu ace aae Thr Asn gte tc Val Ser 330 atc tee Ile Ser 345 cga gag Arg Giu Vai Asp get etg Ala Leu ctt gag Leu Giu ctg ate Leu Ile 220 tcc gac Ser Asp 235 gag cgt Giu Arg tgg atg Trp Met tac cag Tyr Gin tcc aag Ser Lys 300 aag tac Lys Tyr 315 ate gee Ile Ala tac ctg Tyr Leu tac gac Tyr Asp Lys aac Asn ttg Leu 205 ect Pro aac Asn gac Asp ace Thr get Ala 285 tac Tyr gat Asp atg Met atg Met gag Giu 365 Ile ate Ile 190 ttg Leu ace Thr aag Lys gag Giu aag Lys 270 etg Leu aae Asn ate Ile aae Asn aag Lys 350 aat Asn etg Leu Ala 175 gge Gly ggt Gly ate Ile aac Asn aag Lys 255 ate Ile e ag Gin tee Ser aag Lys aae Asn 335 etc Leu gte Val1 Asp aae Asn gee Ala ctg Leu aag Ly s 240 tgg Trp aa e Asn aae Asn tac Tyr e ag Gin 320 ate Ile ate Ile aag Lys 576 624 672 720 768 816 864 912 960 1008 1056 1104 0 0 *.00.

aeg tac ctg etg aac tac ate ate cag eac Thr Tyr 370 taa Leu Leu Asn Tyr Ile Ile Gin His 375 gga tee ate Giy Ser Ile 380 1146 1149 P.OpaEjh mmded\0035.00 .y -wdodsqioi q dc.03/03f203 -47- <210> 28 <211> 382 <212> PRT <213> Artificial Sequence <220> <223> Encoded polypeptide of a synthetic construct based on BoNTE Hn <400> 28 Met Ser Ile Cys Ile Glu Ile Asn Asn Gly Glu Leu Phe Phe Val Ala 1 5 10 Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile Asn Thr Pro Lys Glu Ile 25 Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr Glu Asn Asp Leu Asp Gin 40 Val Ile Leu Asn Phe Asn Ser Glu Ser Ala Pro Gly Leu Ser Asp Glu 55 Lys Leu Asn Leu Thr Ile Gin Asn Asp Ala Tyr Ile Pro Lys Tyr Asp 70 75 Ser Asn Gly Thr Ser Asp Ile Glu Gin His Asp Val Asn Glu Leu Asn 90 Val Phe Phe Tyr Leu Asp Ala Gin Lys Val Pro Glu Gly Glu Asn Asn 100 105 110 Val Asn Leu Thr Ser Ser Ile Asp Thr Ala Leu Leu Glu Gin Pro Lys 115 120 125 Ile Tyr Thr Phe Phe Ser Ser Glu Phe lie Asn Asn Val Asn Lys Pro *130 135 140 Val Gin Ala Ala Leu Phe Val Ser Trp Ile Gin Gin Val Leu Val Asp S145 150 155 160 S* Phe Thr Thr Glu Ala Asn Gin Lys Ser Thr Val Asp Lys Ile Ala Asp 165 170 175 Ile Ser Ile Val Val Pro Tyr Ile Gly Leu Ala Leu Asn Ile Gly Asn 180 185 190 Glu Ala Gin Lys Gly Asn Phe Lys Asp Ala Leu Glu Leu Leu Gly Ala 195 200 205 Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu Leu Ile Pro Thr Ile Leu 210 215 220 Val Phe Thr Ile Lys Ser Phe Leu Gly Ser Ser Asp Asn Lys Asn Lys 225 230 235 240 Val Ile Lys Ala Ile Asn Asn Ala Leu Lys Glu Arg Asp Glu Lys Trp 245 250 255 Lys Glu Val Tyr Ser Phe Ile Val Ser Asn Trp Met Thr Lys Ile Asn 260 265 270 Thr Gin Phe Asn Lys Arg Lys Glu Gin Met Tyr Gin Ala Leu Gin Asn 275 280 285 Gin Val Asn Ala Ile Lys Thr Ile Ile Glu Ser Lys Tyr Asn Ser Tyr 290 295 300 Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn Lys Tyr Asp Ile Lys Gin 305 310 315 320 Ile Glu Asn Glu Leu Asn Gin Lys Val Ser Ile Ala Met Asn Asn Ile 325 330 335 Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser Tyr Leu Met Lys Leu Ile 340 345 350 Asn Glu Val Lys Ile Asn Lys Leu Arg Glu Tyr Asp Glu Asn Val Lys 355 360 365 Thr Tyr Leu Leu Asn Tyr Ile Ile Gin His Gly Ser Ile Leu

P.)O

1 ~dc535 DO ymme" st dom-O3/O3/20O3 -48 375 380 <210> <211> <212> <213> <220> <223> 29 1227

DNA

Artificial Sequence Synthetic construct based on BoNTA Hc <221> CDS <222> (1224) <400> 29 atg gcc cca cca cgt Met Ala Pro Pro Arg 1 5 ctg tgt att aga Leu Cys Ile Arg aac aac tca gaa Asn Asn Ser Glu tta ttc Leu Phe ttt gtc gct Phe Val Ala aaa gag att Lys Glu Ile tcc Ser gag tca agc tac Glu Ser Ser Tyr aac Asn 25 gag aac gat att Glu Asn Asp Ile aac aca cct Asn Thr Pro cgg aac aac Arg Asn Asn gac gat act acc Asp Asp Thr Thr aac Asn 40 cta aac aac aac Leu Asn Asn Asn tac Tyr ttg gat Leu Asp gag gtt att ttg Glu Val Ile Leu tac aac tca cag Tyr Asn Ser Gln acc Thr atc cct caa att Ile Pro Gin Ile tcc Ser 65 aac cgt acc tta Asn Arg Thr Leu aac As n 70 act ctt gtc caa Thr Leu Val Gin gac Asp 75 aac tcc tac gtt Asn Ser Tyr Val cca Pro aga tac gat tct Arg Tyr Asp Ser aac Asn ggt acc tca gag Gly Thr Ser Glu gag gag tat gat Glu Glu Tyr Asp gtt gtt Val Val gac ttt aac Asp Phe Asn gaa acc aac Glu Thr Asn 115 ttt ttc tat ttg Phe Phe Tyr Leu cat His 105 gcc cag aag gtg Ala Gin Lys Val cca gaa ggt Pro Glu Gly 110 ttg ttg gaa Leu Leu Glu atc tca ttg act Ile Ser Leu Thr tct Ser 120 tcc att gat acc Ser Ile Asp Thr gag tcc Glu Ser 130 aag gat atc ttc Lys Asp Ile Phe tct tcg gag ttt Ser Ser Glu Phe atc Ile 140 gat act atc aac Asp Thr Ile Asn aag Lys 145 cct gtc aac gcc Pro Val Asn Ala gct Ala 150 ctg ttc att gat Leu Phe Ile Asp tgg Trp 155 att agc aag gtc Ile Ser Lys Val aga gat ttt acc act gaa gct act caa aag tcc act gtt gat aag att Arg Asp Phe Thr Thr Glu Ala Thr Gin Lys Ser Thr Val Asp Lys Ile P O j~ wo O3 00 u mme Iitn dom-03I03I2003 49 gct gac atc Ala Asp Ilie att att gag Ile Ile Giu 195 tct Ser 180 ttg att gtc ccc Leu Ile Val Pro tat Tyr 185 gtc ggt ctt gct Val Gly Leu Ala 175 ttg aac atc Leu Asn Ile 190 gaa ttg ttg Giu Leu Leu gca gaa aag ggt Ala Giu Lys Giy aac Asn 200 ttt gag gag gct Phe Giu Giu Ala ttt Phe 205 gga gtt Giy Vai 210 ggt att ttg ttg Gly Ile Leu Leu gag Giu 215 ttt gtt cca gaa Phe Vai Pro Giu acc att. cct gtc Thr Ile Pro Vai tta gtt ttt acg Leu Val Phe Thr aag tcc tac atc Lys Ser Tyr Ile gat Asp 235 tca tac gag aac Ser Tyr Giu Asn aag Lys 240 aat aaa gca att Asn Lys Ala Ile aaa Lys 245 gct att aac aac Ala Ile Asn Asn ttg atc gaa aga Leu Ile Glu Arg gag gct Glu Ala 255 aag tgg aag Lys Trp Lys att aac act Ile Asn Thr 275 gaa Giu 260 atc tac tca tgg Ile Tyr Ser Trp att Ile 265 gta tca aac tgg Val Ser Asn Trp ctt act aga Leu Thr Arg 270 cag gct ctg Gin Ala Leu caa ttt aac aag Gin Phe Asn Lys aga Arg 280 aag gag caa atg Lys Giu Gin Met caa aac Gin Asn 290 caa gtc gat gct Gin Val Asp Ala aag act gca att Lys Thr Ala Ile gaa Giu 300 tac aag tac aac Tyr Lys Tyr Asn 816 864 912 960 1008 aac Asn 305 tat act tcc gat Tyr Thr Ser Asp gag Giu 310 aag aac aga ctt Lys Asn Arg Leu tct gaa tac aat Ser Giu Tyr Asn atc Ile 320 aac aac att gaa Asn Asn Ile Giu gaa Giu 325 gag ttg aac aag Giu Leu Asn Lys aaa Lys 330 gtt tct ttg gct Val Ser Leu Ala atg aag Met Lys 335 o 0:0:: 0 0 o O:ooo o.

0 aat atc gaa Asn Ilie Giu ttg atc aat Leu Ilie Asn 355 aga Arg 340 ttt atg acc gaa Phe Met Thr Giu tct atc tct tac Ser Ile Ser Tyr ttg atg aag Leu Met Lys 350 gat aac cac Asp Asn His gag gcc aag gtt Giu Ala Lys Val ggt Gly 360 aag ttg aag aag Lys Leu Lys Lys tac Tyr 365 1056 1104 1152 1200 gtt aag Val Lys 370 agc gat ctg ctg Ser Asp Leu Leu aac Asn 375 tac att ctc gac Tyr Ile Leu Asp cac His 380 aga tca atc ctg Arg Ser Ile Leu gga Gly 385 gag cag aca aac Giu Gin Thr Asn ctg agt gat ttg Leu Ser Asp Leu act tcc act ttg Thr Ser Thr Leu aac As n 400 P \OpakEjh =dmeOO035. 00-y amwdedsaIifsiig dom-03JO32003 tcc tcc att cca ttt gag ctt tct taa 1227 Ser Ser Ile Pro Phe Glu Leu Ser 405 <210> <211> 408 <212> PRT <213> Artificial Sequence <220> <223> Encoded polypeptide of a synthetic construct based on BoNTF Hn <400> Met Ala Pro Pro Arg Leu Cys Ile Arg Val Asn Asn Ser Glu Leu Phe 1 5 10 Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu Asn Asp Ile Asn Thr Pro 25 Lys Glu Ile Asp Asp Thr Thr Asn Leu Asn Asn Asn Tyr Arg Asn Asn 40 Leu Asp Glu Val Ile Leu Asp Tyr Asn Ser Gin Thr Ile Pro Gin Ile 55 Ser Asn Arg Thr Leu Asn Thr Leu Val Gin Asp Asn Ser Tyr Val Pro 70 75 Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile Glu Glu Tyr Asp Val Val **85 90 Asp Phe Asn Val Phe Phe Tyr Leu His Ala Gin Lys Val Pro Glu Gly 100 105 110 Glu Thr Asn Ile Ser Leu Thr Ser Ser Ile Asp Thr Ala Leu Leu Glu 115 120 125 Glu Ser Lys Asp Ile Phe Phe Ser.Ser Glu Phe Ile Asp Thr Ile Asn 130 135 140 Lys Pro Val Asn Ala Ala Leu Phe Ile Asp Trp Ile Ser Lys Val Ile 145 150 155 160 Arg Asp Phe Thr Thr Glu Ala Thr Gin Lys Ser Thr Val Asp Lys Ile 165 170 175 Ala Asp Ile Ser Leu Ile Val Pro Tyr Val Gly Leu Ala Leu Asn Ile 180 185 190 Ile Ile Glu Ala Glu Lys Gly Asn Phe Glu Glu Ala Phe Glu Leu Leu 195 200 205 Gly Val Gly Ile Leu Leu Glu Phe Val Pro Glu Leu Thr Ile Pro Val 210 215 220 Ile Leu Val Phe Thr Ile Lys Ser Tyr Ile Asp Ser Tyr Glu Asn Lys 225 230 235 240 oo Asn Lys Ala Ile Lys Ala Ile Asn Asn Ser Leu Ile Glu Arg Glu Ala 245 250 255 Lys Trp Lys Glu Ile Tyr Ser Trp Ile Val Ser Asn Trp Leu Thr Arg 260 265 270 Ile Asn Thr Gin Phe Asn Lys Arg Lys Glu Gin Met Tyr Gin Ala Leu 275 280 285 Gin Asn Gin Val Asp Ala Ile Lys Thr Ala Ile Glu Tyr Lys Tyr Asn 290 295 300 Asn Tyr Thr Ser Asp Glu Lys Asn Arg Leu Glu Ser Glu Tyr Asn Ile 305 310 315 320 Asn Asn Ile Glu Glu Glu Leu Asn Lys Lys Val Ser Leu Ala Met Lys P 'OpcrEjb m~dod&5O35 M~-dfom d -O/O/2O3 -51 330 r Ser 325 Phe Asn Ile Giu Leu Ile Asn 355 Val Lys Ser Arg 340 Giu Met Thr Glu Ile Ser Tyr 335 Leu Met Lys 350 Asp Asn His Ser Ile Leu Ala Lys Val Gly 360 Tyr Leu Lys Lys Tyr 365 Arg Asp Leu Leu 370 Gly Giu Asn 375 Ile Leu Asp His 380 Thr Gin Thr Asn 385 Ser Giu Leu Ser 390 Giu Leu Ser Asp Leu Val1 395 Ser Thr Leu Asn 400 Ser Ile Pro Phe 405 <210> 31 <211> i233 <212> DNA <213> Artificiai Sequence <220> <223> Synthetic construct based on BoNTA Hc <221> CDS <222> (1) (1230) <400> 31 atg gcc aaa aat acc Met Aia Lys Asn Thr 1 5 ggt aaa tct gaa Giy Lys Ser Giu tgt att att gtt Cys Ile Ile Val aat aat Asn Asn S S S gag gat tta Giu Asp Leu gct aaa gca Aia Lys Ala ttc ata gct aat Phe Ile Ala Asn aaa Lys gat agt ttt tca Asp Ser Phe Ser aaa gat tta Lys Asp Leu act ata gaa Thr Ile Giu gaa act ata gca Giu Thr Ile Ala tat Tyr 40 aat aca caa aat Asn Thr Gin Asn aat Asn aat aat Asn Asn ttt tct ata gat Phe Ser Ile Asp ttg att tta gat Leu Ile Leu Asp aat Asn gat tta agc agt Asp Leu Ser Ser S S. 55 5 5 5555 ggc Gly 65 ata gac tta cca Ile Asp Leu Pro aat Asn 70 gaa aac aca gaa Giu Asn Thr Giu cca Pro 75 ttt aca aat ttt Phe Thr Asn Phe gac Asp gac ata gat atc Asp Ile Asp Ile cct Pro 85 gtg tat att aaa Vai Tyr Ile Lys tct gct tta aaa Ser Ala Leu Lys aaa att Lys Ile ttt gtg gat Phe Val Asp cct tct aat Pro Ser Asn 115 gat agc ctt ttt Asp Ser Leu Phe gaa Giu 105 tat tta cat gct caa aca ttt Tyr Leu His Ala Gin Thr Phe 110 ata gaa aat cta Ile Giu Asn Leu caa Gin 120 cta acg aat tca Leu Thr Asn Ser tta Leu 125 aat gat gct Asn Asp Ala P.'OpaEjb mmdcd\50035 00 m ddseq1isu.doc-03f03t2003 52 tta Leu gaa Giu 145 aaa Lys ata Ile gct Al a ttt Phe att Ile 225 aaa Lys caa Gin acg Thr tta Leu aat Asn 305 gat Asp ata Ile aga Arg 130 aaa Lys gga Gly gat Asp ttg Leu gaa Giu 210 gta Vai ggg Giy aaa Lys gtt Val1 aat Asn 290 aga Arg ata Ile gat Asp aat Asn gct Ala gta Val1 aaa Lys aat Asn 195 ata Ile cct Pro cat His tgg Trp aat Asn 275 aat As n tat Tyr gat Asp gat Asp aat Asn aat Asn ata Ile gtt Val1 180 gta Val1 ggt Giy ata Ile att Ile ac a Thr 260 act Thr caa Gin agt Ser ttt Phe ttt Phe aat Asn aca Thr gat Asp 165 tca Ser gga Giy gga Gly gtt Val1 att Ile 245 gat Asp caa Gin tca Ser gaa Giu aaa Lys 325 ata Ile aaa Lys gt t Val1 150 gat Asp gat Asp aat Asn gcc Ala gga Giy 230 atg Met atg Met ttt Phe caa Gin gaa Giu 310 ctt Leu aac Asn gtc Val1 135 gta Val ttt Phe gta Val1 gaa Giu gct Ala 215 ttt Phe acg Thr tat Tyr tat Tyr gca Ala 295 gat Asp aat As n caa Gin tat Tyr ggt Gly aca Thr tcc Ser ac a Thr 200 atc Ile ttt Phe ata Ile ggt Giy aca Thr 280 ata Ile aaa Lys caa Gin tgt Cys act Thr gct Aia tct Ser ata Ile 185 gct Al a tta Leu aca Thr tcc Ser ttg Leu 265 ata Ile gaa Glu atg Met agt Ser tct Ser ttt Phe tca Ser gaa Giu 170 att Ile aaa Lys atg Met tta Leu aat Asn 250 ata Ile aaa Lys aaa Lys aat Asn ata Ile 330 ata Ile ttt Phe ctt Leu 155 tcc Ser att Ile gaa Glu gag Glu gaa Glu 235 gct Ala gta Val1 gaa Giu ata Ile att Ile 315 aat Asn tca Ser tct Ser 140 ttt Phe ac a Thr ccc Pro aat Asn ttt Phe 220 tca Ser tta Leu tcg Ser aga Arg ata Ile 300 aa c As n tta Leu tat Tyr aca Thr gta Val1 caa Gin tat Tyr ttt Phe 205 att Ile tat Tyr aag Lys cag Gin atg Met 285 gaa Glu att Ile gca Ala cta Leu aac As n aac As n aaa Lys ata Ile 190 aaa Lys cca Pro gta Val1 aaa Lys tgg Trp 270 tac Tyr gat Asp gat Asp ata Ile atg Met 350 ctt Leu tgg Trp agt Ser 175 gga Gly aat As n gaa Glu gga Gly agg Arg 255 ctc Leu aat Asn caa Gin ttt Phe aac Asn 335 aat Asn gtt Val1 gta Val1 160 act Thr cct Pro gct Ala ctt Leu aat As n 240 gat Asp tca Ser gct Ala tat Tyr aat Asn 320 aat Asn aga Arg 432 480 528 576 624 672 720 768 816 864 912 960 1008 1056 340 345 atg att cca tta gct gta aaa aag tta aaa gac ttt gat gat aat ctt 1104 P %0pcrEjh mmd\5003S 00 u~ny mddulstLsin doc.O3fO312003 53 Met Ile Pro 355 aag aga gat Lys Arg Asp Leu Ala Val Lys tta ttg gag Leu Leu Glu 370 gat gaa Asp Giu tat Tyr 375 aaa Lys Leu Lys Asp Phe Asp Asp Asn Leu 365 gat aca aat gaa cta tat tta ctt Asp Thr Asn Glu Leu Tyr Leu Leu 380 aaa gta aat aga cac cta aaa gac Lys Val Asn Arg His Leu Lys Asp 395 400 tat acc taa Tyr Thr gta aat att Val Asn Ile 385 agt Ser cta Leu 390 ctt Leu tca Ser 1152 1200 1233 ata cca ttt Ile Pro Phe gat Asp 405 tca cta Ser Leu <210> 32 <211> 410 <212> PRT <213> Artificial Sequence <220> <223> Encoded polypeptide of a synthetic construct based on BoNTG Hn <400> 32 Met Glu Ala Asn Gly Asp Phe Pro Leu Glu 145 Lys Ile Ala Phe Ile 225 Lys Al a Asp Lys Asn 50 Ile Ile Val1 Ser Arg 130 Lys Gly Asp Leu Giu 210 Val1 Gly Lys Leu Al a Phe Asp Asp Asp Asn 115 Asn Ala Val1 Lys Asn 195 Ile Pro His Asn Phe Giu Ser Leu Ile Gly 100 Ile Asn Asn Ile Val1 180 Val Gly Ile Ile Thr Phe Thr Ile Pro Pro Asp Glu As n Thr Asp 165 Ser Gly Gly Val Ile Gly Ile Ile Asp Asn 70 Val1 Ser Asn Lys Val1 150 Asp Asp Asn Ala Gly 230 Met Lys Ala Ala Gin 55 Giu Tyr Leu Leu Val 135 Val Phe Val Giu Ala 215 Phe Thr Ser Asn Tyr Leu Asn Ile Phe Gin 120 Tyr Gly Thr Ser Thr 200 Ile Phe Ile Giu Lys 25 Asn Ile Thr Lys Giu 105 Leu Thr Ala Ser Ile 185 Ala Leu Thr Ser Cys Ser Gin Asp Pro 75 Ser Leu Asn Phe Leu 155 Ser Ile Giu Giu Giu 235 Ala Ile Phe Asn Asn Phe Ala His Ser Se r 140 Phe Thr Pro Asn Phe 220 Ser Leu Val1 Lys Thr Leu Asn Lys Gin 110 Asn Asn Asn Lys Ile 190 Lys Pro Val1 Lys Asn Asp Ile Ser Phe Lys Thr Asp Leu Trp Ser 175 Gly As n Giu Gly Arg Asn Leu Glu Ser Asp Ile Phe Ala Val1 Val1 160 Thr Pro Ala Leu As n 240 Asp P:\OpcEj'hmwdeOO35.00u~y m dduIistin doc-0310312003 -54- 245 Asp 250 Ile Gin Lys Trp Thr Val Asn 275 Leu Asn Asn Thr 260 Thr Met Tyr Giy Leu 265 Ile Vai Ser Gin 255 Trp Leu Ser 270 Tyr Asn Aia Asp Gin Tyr Gin Phe Tyr Thr 280 Ile Lys Giu Arg Met 285 Giu Gin Ser Gin 290 Asn Arg Ala 295 Asp Giu Lys Ile Ile 300 Asn Tyr Ser Giu 305 Asp Giu 310 Leu Lys Met Asn Ile Asp Phe Asn 320 Ile Asp Phe Asn Gin Ser Ile 330 Ile Leu Ala Ile Asn Asn 335 Ilie Asp Asp Met Ilie Pro 355 Lys Arg Asp Phe 340 Leu Asn Gin Cys Ser 345 Leu Ser Tyr Leu Aia Vai Lys Lys Asp Phe Asp 365 Leu Met Asn Arg 350 Asp Asn Leu Tyr Leu Leu Leu Leu Giu 370 Asp Giu Tyr 375 Lys Asp Thr Asn Giu 380 Arg Val Asn Ile 385 Ser Leu 390 Leu Ser Lys Vai His Leu Lys Asp 400 Ile Pro Phe Ser Leu Tyr S. 0

SO..

S

<210> 33 <2i1> i3i4 <2i2> DNA <2i3> Artificiai Sequence <220> <223> Synthetic construct based on BoNTA Hc <22i> CDS <222> (10) (1305) <400> 33 gaattcacg atg tct tac act aac gac aaa atc ctg atc ctg tac ttc aac Si Met Ser Tyr Thr Asn Asp Lys Ile Leu Ile Leu Tyr Phe Asn 1 5 S. 55 S S S. 55 S. S 5555 aaa Lys ctg tac aaa Leu Tyr Lys aaa atc Lys Ile 20 ttc atc Phe Ile 35 aaa gac aac tct Lys Asp Asn Ser atc Ile ctg gac atg cgt Leu Asp Met Arg gaa aac aac aaa Giu Asn Asn Lys atc aac ggt gac Ilie Asn Gly Asp atc tac tct tct Ile Tyr Ser Ser gac atc tct Asp Ile Ser ggc tat ggt tct Giy Tyr Giy Ser 40 act aac cgc aac Thr Asn Arg Asn gtc tac atc tac Vai Tyr Ile Tyr tct Ser 55 aac atc tct Asn Ile Ser cag ttc ggt Gin Phe Giy aac aac gac Asn Asn Asp aaa ccg tct Lys Pro Ser gta aac atc gct Val Asn Ile Aia atc atc tac aac ggt cgt tac cag aac ttc tct atc tct ttc tgg gtt Ile Ile Tyr Asn Giy Arg Tyr Gin Asn Phe Ser Ile Ser Phe Trp Vai P.VOperEjb .=.deV\O03SOO s y amwdcdsqii.i doc.03103fl003 55 cgt Arg atc ccg aaa tac Ile Pro Lys Tyr ttc Phe 100 aac aaa gtt aac Asn Lys Vai Asn ctg Leu 105 aac aac gaa tac Asn Asn Giu Tyr act Thr 110 339 387 atc atc gac tgc Ilie Ilie Asp Cys cgt aac aac aac Arg Asn Asn Asn tct Ser 120 ggt tgg aaa atc Gly Trp Lys Ile tct cig Ser Leu 125 aac tac aac Asn Tyr Asn cag aaa ctg Gin Lys Leu 145 aaa Lys 130 atc atc tgg act Ile Ile Trp Thr cag gac act gct Gin Asp Thr Ala ggt aac aac Giy Asn Asn 140 tct gac tac Ser Asp Tyr 435 483 gtt ttc aac tac Val Phe Asn Tyr act Thr 150 cag atg atc tct Gin Met Ile Ser atc Ile 155 att aat Ile Asn 160 aaa tgg atc ttc Lys Trp Ile Phe gt t Val1 165 act atc act aac Thr Ile Thr Asn aac As n 170 cgt ctg ggt aac Arg Leu Giy Asn tct Ser 175 cgt atc tac atc Arg Ile Tyr Ile aac Asn 180 ggt aac ctg atc Gly Asn Leu Ile gaa aaa tct atc Giu Lys Ser Ile tct Ser 190 0O 0@ aac ctg ggt gac Asn Leu Giy Asp cac gtt tct gac His Val Ser Asp aac Asn 200 atc ctg ttc aaa Ile Leu Phe Lys atc gtt Ile Vai 205 531 579 627 675 723 ggt tgc aac Gly Cys Asn gac act gaa Asp Thr Giu 225 gac Asp 210 acg cgt tac gtt Thr Arg Tyr Val ggt Gly 215 atc cgt tac ttc Ile Arg Tyr Phe aaa gtt ttc Lys Val Phe 220 tct gac gaa Ser Asp Giu ctg ggt aaa act Leu Gly Lys Thr gaa Giu 230 atc gaa act ctg Ile Giu Thr Leu tac Tyr 235 ccg gac Pro Asp 240 ccg tct atc ctg Pro Ser Ile Leu aaa Lys 245 gac ttc tgg ggt Asp Phe Trp Giy aac Asn 250 tac ctg ctg tac Tyr Leu Leu Tyr aac As n 255 aaa cgt tac tac Lys Arg Tyr Tyr ctg aac ctg ctc Leu Asn Leu Leu act gac aaa tct Thr Asp Lys Ser atc Ile 270 771 819 867 act cag aac tct Thr Gin Asn Ser aac Asn 275 ttc ctg aac atc Phe Leu Asn Ile aac Asn 280 cag cag cgt ggt Gin Gin Arg Gly gtt tat Val Tyr 285 cag aaa cct Gin Lys Pro gtt atc atc Val Ile Ile 305 aat Asn 290 atc ttc tct aac Ilie Phe Ser Asn cgt ctg tac act Arg Leu Tyr Thr ggt gtt gaa Gly Val Giu 300 act gac aac Thr Asp Asn cgt aaa aac ggt Arg Lys Asn Giy tct Ser 310 act gac atc tct Thr Asp Ile Ser aac Asn 315 P .pOMEjhmde&3035.00 us.y .mmdsqisingdc-3/03/203 56ttc gta Phe Val 320 cgt aaa aac gac Arg Lys Asn Asp ctg Leu 325 gct tac atc aac Ala Tyr Ile Asn gtt Val1 330 gtt gac cgt gac Val Asp Arg Asp gaa tac cgt ctg Glu Tyr Arg Leu tac Tyr 340 gct gac atc tct Ala Asp Ile Ser gct aaa ccg gaa Ala Lys Pro Giu aaa Lys 350 loll 1059 1107 atc atc aaa ctg Ile Ilie Lys Leu cgt act tct aac Arg Thr Ser Asn tct Ser 360 aac aac tct ctg Asn Asn Ser Leu ggt cag Gly Gin 365 atc atc gtt Ile Ilie Val aac aac aac Asn Asn Asn 385 atg Met 370 gac tcg atc ggt Asp Ser Ile Gly aac tgc act atg Asn Cys Thr Met aac ttc cag Asn Phe Gin 380 tct aac aac Ser Asn Asn 1155 1203 ggt ggt aac atc Gly Gly Asn Ile ggt Gly 390 ctg ctg ggt ttc Leu Leu Gly Phe cac His 395 ctg gtt Leu Val 400 gct tct tca tgg Ala Ser Ser Trp tac Tyr 405 tac aac aac atc Tyr Asn Asn Ile cgt Arg 410 aaa aac act tct Lys Asn Thr Ser 1251 1299

S.

S S SS Se S *5*S

S

*5

S

tct Ser 415 aac ggt tgc ttc Asn Gly Cys Phe tgg Trp 420 tct ttc atc tct Ser Phe Ile Ser aaa Lys 425 gaa cac ggt tgg Giu His Gly Trp c ag Gin 430 gaa aac Giu Asn taagaattc 1314 S. S 5*55 5 0 *55* 55.5 <210> 34 <211> 432 <212> PRT <213> Artificial Sequence <220> <223> Encoded poiypeptide of a synthetic construct based on BoNTF Hc <400> 34 Met Ser Tyr Thr Asn Asp Lys Ile Leu Ilie Leu Tyr Phe Asn Lys Leu 1 5 10 Tyr Lys Lys Ile Lys Asp Asn Ser Ile Leu Asp Met Arg Tyr Giu Asn 20 25 Asn Lys Phe Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn 40 Gly Asp Val Tyr Ile Tyr Ser Thr Asn Arg Asn Gin Phe Gly Ile Tyr 55 Ser Ser Lys Pro Ser Giu Val Asn Ile Ala Gin Asn Asn Asp Ile Ile 70 75 Tyr Asn Gly Arg Tyr Gin Asn Phe Ser Ilie Ser Phe Trp Val Arg Ile 90 Pro Lys Tyr Phe Asn Lys Val Asn Leu Asn Asn Giu Tyr Thr Ile Ile P.XOpaEjh mmcd\5035.00 us.~y mmdd1saqfign doc-O3/03f2DO3 57 100 105 110 Asp Cys Ile Arg Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Asn Tyr 115 120 125 Asn Lys Ilie Ile Trp Thr Leu Gin Asp Thr Ala Gly Asn Asn Gin Lys 130 135 140 Leu Val Phe Asn Tyr Thr Gin Met Ile Ser Ile Ser Asp Tyr Ile Asn 145 150 155 160 Lys Trp Ile Phe Vai Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser Arg 165 170 175 Ile Tyr Ile Asn Gly Asn Leu Ile Asp Giu Lys Ser Ile Ser Asn Leu 180 185 190 Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Gly Cys 195 200 205 Asn Asp Thr Arg Tyr Vai Giy Ile Arg Tyr Phe Lys Vai Phe Asp Thr 210 215 220 Giu Leu Giy Lys Thr Giu Ile Giu Thr Leu Tyr Ser Asp Giu Pro Asp 225 230 235 240 Pro Ser Ilie Leu Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asn Lys 245 250 255 Arg Tyr Tyr Leu Leu Asn Leu Leu Arg Thr Asp Lys Ser Ile Thr Gin 260 265 270 Asn Ser Asn Phe Leu Asn Ile Asn Gin Gin Arg Gly Vai Tyr Gin Lys 275 280 285 Pro Asn Ilie Phe Ser Asn Thr Arg Leu Tyr Thr Giy Val Giu Vai Ile 290 295 300 Ile Arg Lys Asn Giy Ser Thr Asp Ile Ser Asn Thr Asp Asn Phe Vai 305 310 315 320 s.Arg Lys Asn Asp Leu Ala Tyr Ile Asn Val Vai Asp Arg Asp Val Giu *325 330 335 0000 Tyr Arg Leu Tyr Ala Asp Ile Ser Ile Ala Lys Pro Giu Lys Ile Ile 00000 340 345 350 0 Lys Leu Ile Arg Thr Ser Asn Ser Asn Asn Ser Leu Gly Gin Ile Ile Vl 3et55 360 365 ValMetAsp Ser Ile Gly Asn Asn Cys Thr Met Asn Phe Gin Asn Asn 370 375 380 Asn Gly Giy Asn Ile Gly Leu Leu Gly Phe His Ser Asn Asn Leu Val 385 390 395 400 Ala Ser Ser Trp Tyr Tyr Asn Asn Ile Arg Lys Asn Thr Ser Ser Asn 405 410 415 Gly Cys Phe Trp Ser Phe Ile Ser Lys Giu His Gly Trp Gin Giu Asn *420 425 430 :<210> *<211> 1278 0.000<212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA Hc <221> CDS <222> (10) (1269) <400> ttcgaaacg atg att tta att tcc tac ttc aac aag ttc ttc aag aga att 51 Met Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile P.\OpM\Ejb omdd\0035 00u.~ny mmewqi~ doc-03/03M203 58 aag Lys tct tct tcc gtt Ser Ser Ser Val tta Leu 20 aac atg aga tac Asn Met Arg Tyr aag Lys aat gat aaa tac Asn Asp Lys Tyr gac act tcc ggt Asp Thr Ser Giy tac Tyr gac tcc aat atc Asp Ser Asn Ile att aac ggt gac Ile Asn Gly Asp gtg tac Val Tyr aag tac cca Lys Tyr Pro act gag ctg Thr Giu Leu act Thr aac aaa aac caa Asn Lys Asn Gin ttc Phe 55 ggt atc tac aac Giy Ile Tyr Asn gac aag ctt Asp Lys Leu gac aac aag Asp Asn Lys aac atc tct caa Asn Ile Ser Gin aac Asn 70 gac tac att atc Asp Tyr Ile Ile tac Tyr tac aat Tyr Asn aac ttc tct att Asn Phe Ser Ile ttc tgg gtc aga Phe Trp Val Arg cct aac tac gat Pro Asn Tyr Asp aac Asn aag atc gtc aac Lys Ile Vai Asn gtt Val1 100 aac aac gag tac Asn Asn Giu Tyr act Thr 105 atc atc aac tgt Ile Ile Asn Cys atg Met 110 aga gac aac aac Arg Asp Asn Asn tcc Ser 115 ggt tgg aag gtc Giy Trp Lys Vai tct Ser 120 ctt aac cac aac Leu Asn His Asn gag atg Giu Met 125 291 339 387 435 483 att tgg acc Ile Trp, Thr aac tac ggt Asn Tyr Giy 145 ttg Leu 130 caa gac aac gca Gin Asp Asn Ala ggt Gly 135 att aac caa aag Ile Asn Gin Lys tta gca ttc Leu Aia Phe 140 aag tgg att Lys Trp Ile aac gca aac ggt Asn Ala Asn Gly att Ile iso tct gac tac atc Ser Asp Tyr Ile aac Asn 155 ttc gtc Phe Vai 160 act atc act aac Thr Ile Thr Asn aga tta ggg gac Arg Leu Gly Asp aag ctt tac att Lys Leu Tyr Ile aac Asn 175 ggt aac tta atc Giy Asn Leu Ile gac Asp 180 caa aag tcc att Gin Lys Ser Ile tta Leu 185 aac tta ggt aac Asn Leu Giy Asn att Ile 190 cac gtt tct gac His Val Ser Asp aac Asn 195 atc tta ttc aag Ile Leu Phe Lys atc Ile 200 gtt aac tgc agt Vai Asn Cys Ser tac aac Tyr Asn 205 aga tac att Arg Tyr Ile gag acc gag Giu Thr Giu 225 ggc Gly 210 att aga tac ttc Ile Arg Tyr Phe att ttc gac aag Ile Phe Asp Lys gag tta gac Giu Leu Asp 220 acc aat att Thr Asn Ile 675 723 att caa act tta Ile Gin Thr Leu tac Tyr 230 agc aac gaa cct Ser Asn Giu Pro aac Asn 235 P \Opa\jl mmdc\5035.00 .s~y mwesq~sn do-03103f2 0 03 59 ttg aag Leu Lys 240 gac ttc tgg ggt Asp Phe Trp Gly aac Asn 245 tac ttg ctt tac Tyr Leu Leu Tyr gac Asp 250 aag gaa tac tac Lys Giu Tyr Tyr tta Leu 255 tta aac gtg tta Leu Asn Val Leu aag Lys 260 cca aac aac ttc Pro Asn Asn Phe att Ile 265 gat agg aga aag Asp Arg Arg Lys gat Asp 270 tct act tta agc Ser Thr Leu Ser att Ile 275 aac aac atc aga Asn Asn Ile Arg act att ctt tta Thr Ile Leu Leu gct aac Ala Asn 285 aga tta tac Arg Leu Tyr tct act aac Ser Thr Asn 305 tct Ser 290 ggt atc aag gtt Gly Ile Lys Val aag Lys 295 atc caa aga gtt Ile Gin Arg Vai aac aac tct Asn Asn Ser 300 tat att aac Tyr Ile Asn gat aac ctt gtt Asp Asn Leu Vai aga Arg 310 aag aac gat cag Lys Asn Asp Gin gtc Val1 315 ttc gtc Phe Vai 320 gct agc aag act Ala Ser Lys Thr tta ttc cca tta Leu Phe Pro Leu gct gat aac gct Ala Asp Asn Ala ac c Thr 335 acc aac aag gag Thr Asn Lys Giu aag Lys 340 acc atc aag atc Thr Ile Lys Ile tcc Ser 345 tcc tct ggc aac Ser Ser Gly Asn ttt aac caa gtc Phe Asn Gin Val gtt atg aac tcc Val Met Asn Ser gtc Val1 360 ggt aac aac tgt Gly Asn Asn Cys acc atg Thr Met 365 loll 1059 1107 1155 1203 1251 aac ttt aaa Asn Phe Lys gca gat act Ala Asp Thr 385 aat Asn 370 aat aat gga aat Asn Asn Gly Asn att ggg tgt tta Ile Gly Cys Leu ggt ttc aag Gly Phe Lys 380 atg aga gat Met Arg Asp gta gtt gct agt Val Val Ala Ser act Thr 390 tgg tat tat acc Trp Tyr Tyr Thr cac His 395 cac acc His Thr 400 aac agc aat gga Asn Ser Asn Gly tgt Cys 405 ttt tgg aac ttt Phe Trp Asn Phe att Ile 410 tct gaa gaa. cat Ser Giu Giu His gga Gly 415 tgg caa gaa aaa taa Trp Gin Giu Lys tagggatcc 1278 <210> 36 <211> 419 <212> PRT <213> Artificial Sequence <220> <223> Encoded polypeptide of a synthetic construct based P.\par\Ejb.=wd5035.00 us~y wd~ssingdoO3O32OO3 60 on BoNTE Hc <400> 36 Met Ile Leu 1 Ser Ser Val Ser Giy Tyr Pro Thr Asn Leu Asn Ile Asn Phe Ser Ile Vai Asn Asn Asn Ser 115 Thr Leu Gin 130 Giy Asn Ala 145 Thr Ile Thr Asn Leu Ile Ser Asp Asn 195 Ile Giy Ile 210 Giu Ile Gin 225 Asp Phe Trp Asn Vai Leu Leu Ser Ile 275 Tyr Ser Giy 290 Asn Asp Asn 305 Aia Ser Lys Asn Lys Giu Gin Vai Vai 355 Lys Asn Asn 370 Thr Val Vai 385 Asn Ser Asn Gin Giu Lys Ile Leu Asp Lys Ser Ile Vali i00 Gly Asp Asn Asn Asp 180 Ile Arg Thr Giy Lys 260 Asn Ile Leu Thr Lys 340 Val1 Asn Aia Giy Ser 5 Asn Ser Asn Gin Ser Asn Trp Asn Giy Asp 165 Gin Leu Tyr Leu Asn 245 Pro Asn Lys Val1 His 325 Thr Met Gly Ser Cys 405 Met Asn Gin Asn 70 Phe Asn Lys Aia Ile i50 Arg Lys Phe Phe Tyr 230 Tyr Asn Ile Val1 Arg 310 Leu Ile Asn Asn Thr 390 Phe Arg Ile Phe 55 Asp Trp Giu Vali Giy 135 Ser Leu Ser Lys Asn 215 Ser Leu Asn Arg Lys 295 Lys Phe Lys Ser Asn 375 Trp Trp, Tyr As n 40 Gly Tyr Vali Tyr Ser 120 Ile Asp Giy Ile Ile 200 Ile Asn Leu Phe Ser 280 Ile Asn Pro Ile Vali 360 Ile Tyr Asn Lys 25 Ile Ile Ile Arg Thr 105 Leu Asn Tyr Asp Leu 185 Vali Phe Giu Tyr Ile 265 Thr Gin Asp Leu Ser 345 Gly Gly Tyr Phe Asn Asn Tyr Ile Ile 90 Ile Asn Gin Ile Ser 170 Asn Asn Asp Pro Asp 250 Asp Ile Arg Gin Tyr 330 Ser Asn Leu Thr Ile 410 Asp Giy Asn Tyr 75 Pro Ile His Lys Asn 155 Lys Leu Cys Lys Asn 235 Lys Arg Leu Vali Val1 315 Al a Ser Asn Leu His 395 Ser Lys Asp Asp Asp Asn Asn Asn Leu 140 Lys Leu Gly Ser Giu 220 Thr Giu Arg Leu Asn 300 Tyr Asp Gly Cys Gly 380 Met Giu Tyr Vali Lys Asn Tyr Cys Giu 125 Aia Trp Tyr Asn Tyr 205 Leu Asn Tyr Lys Aia 285 Asn Ile Thr Asn Thr 365 Phe Arg Glu Val1 Tyr Leu Lys Asp Met 110 Ile Phe Ile Ile Ile 190 Thr Asp Ile Tyr Asp 270 Asn Ser Asn Al a Arg 350 Met Lys Asp His Asp Lys Thr Tyr Asn Arg Ile Asn Phe Asn 175 His Arg Giu Leu Leu 255 Ser Arg Ser Phe Thr 335 Phe Asn Ala His Gly 415 Thr Tyr Giu Lys Lys Asp Trp Tyr Val1 160 Gly Val1 Tyr Thr Lys 240 Leu Thr Leu Thr Val1 320 Thr Asn Phe Asp Thr 400 Trp Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys Ser 10 is P:%Opcr\Ejb omdc"0OO3 my .moudsls~img do-03/03/2003 61 <210> 37 <211> 1338 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTA Hc <221> CDS <222> (1325) <400> 37 ctcgagcc atg gct cgt ctg ctg tct acc ttc act gaa tac atc aag aac Met Ala Arg Leu Leu Ser Thr Phe Thr Glu Tyr Ile Lys Asn atc Ile atc aat acc tcc Ile Asn Thr Ser atc Ile 20 ctg aac ctg cgc Leu Asn Leu Arg tac Tyr 25 gaa tcc aat cac Glu Ser Asn His ctg Leu atc gac ctg tct Ile Asp Leu Ser tac gct tcc aaa Tyr Ala Ser Lys aac atc ggt tct Asn Ile Gly Ser aaa gtt Lys Val aac ttc gat Asn Phe Asp tct tcc aaa Ser Ser Lys 65 ccg Pro atc gac aag aat Ile Asp Lys Asn cag Gin 55 atc cag ctg ttc Ile Gin Leu Phe aat ctg gaa Asn Leu Giu tac aac tct Tyr Asn Ser atc gaa gtt atc Ile Giu Val Ile aag aat gct atc Lys Asn Ala Ile gta Val1 atg tac Met Tyr gaa aac ttc tcc Giu Asn Phe Ser acc Thr 85 tcc ttc tgg atc Ser Phe Trp Ile cgt Arg atc ccg aaa tac Ile Pro Lys Tyr aac tcc atc tct Asn Ser Ile Ser ctg Leu 100 aac aat gaa tac Asn Asn Giu Tyr acc Thr 105 atc atc aac tgc Ile Ile Asn Cys atg Met 110 gaa aac aat tct Giu Asn Asn Ser tgg aaa gta tct Trp Lys Vai Ser aac tac ggt gaa Asn Tyr Giy Giu atc atc Ile Ile 125 tgg act ctg Trp Thr Leu tac tct cag Tyr Ser Gin 145 cag Gin 130 gac act cag gaa Asp Thr Gin Giu atc Ile 135 aaa cag cgt gtt Lys Gin Arg Val gta ttc aaa Val Phe Lys 140 tgg atc ttc Trp Ile Phe atg atc aac atc Met Ile Asn Ile gac tac atc aat Asp Tyr Ile Asn cgc Arg 155 gtt acc Vai Thr 160 atc acc aac aat Ile Thr Asn Asn cgt Arg 165 ctg aat aac tcc Leu Asn Asn Ser aaa Lys 170 atc tac atc aac Ile Tyr Ile Asn P. Op~jl mmod5035.OD ry .mmddsqlisingdoc-0310312003 62 ggc Gly 175 cgt ctg atc gac Arg Leu Ile Asp cag Gin 180 aaa ccg atc tcc aat ctg ggt aac atc Lys Pro Ile Ser Asn Leu Gly Asn Ile 185 c ac His 190 gct tct aat aac Ala Ser Asn Asn atg ttc aaa ctg Met Phe Lys Leu ggt tgt cgt gac Giy Cys Arg Asp act cac Thr His 205 cgc tac atc Arg Tyr Ile gaa aaa gaa Glu Lys Giu 225 tgg Trp 210 atc aaa tac ttc Ile Lys Tyr Phe aat Asn 215 ctg ttc gac aaa Leu Phe Asp Lys gaa ctg aac Giu Leu Asn 220 tct ggt atc Ser Gly Ile atc aaa gac ctg Ile Lys Asp Leu tac Tyr 230 gac aac cag tcc Asp Asn Gin Ser aat As n 23S ctg aaa Leu Lys 240 gac ttc tgg ggt Asp Phe Trp Gly tac ctg cag tac Tyr Leu Gin Tyr aaa ccg tac tac Lys Pro Tyr Tyr atg Met 255 ctg aat ctg tac Leu Asn Leu Tyr gat Asp 260 ccg aac aaa tac Pro Asn Lys Tyr gtt Val1 265 gac gtc aac aat Asp Val Asn Asn ggt atc cgc ggt Giy Ilie Arg Giy tac Tyr 275 atg tac ctg aaa Met Tyr Leu Lys ggt Giy 280 ccg cgt ggt tct Pro Arg Gly Ser gtt atg Val Met 285 act acc aac Thr Thr Asn atc atc aag Ile Ilie Lys 305 atc Ile 290 tac ctg aac tct Tyr Leu Asn Ser tcc Ser 295 ctg tac cgt ggt Leu Tyr Arg Gly acc aaa ttc Thr Lys Phe 300 gtt cgc aac Vai Arg Asn aaa tac gcg tct ggt aac aag gac aat Lys Tyr Ala Ser Gly Asn Lys Asp Asn 770 818 866 914 962 1010 1058 1106 1154 1202 aat gat Asn Asp 320 cgt gta tac atc Arg Vai Tyr Ile aat Asn 325 gtt gta gtt aag Val Val Val Lys aac Asn 330 aaa gaa tac cgt Lys Giu Tyr Arg ctg Leu 335 gct acc aat gct Ala Thr Asn Ala tct Ser 340 cag gct ggt gta Gin Aia Gly Val gaa Giu 345 aag atc ttg ict Lys Ile Leu Ser gct Ala 350 ctg gaa atc ccg Leu Giu Ilie Pro gtt ggt aat ctg Val Gly Asn Leu cag gta gtt gta Gin Val Val Val atg aaa Met Lys 365 tcc aag aac Ser Lys Asn gac aac aat Asp Asn Asn 385 gac Asp 370 cag ggt atc act Gin Gly Ile Thr aac Asn 375 aaa tgc aaa atg Lys Cys Lys Met aat ctg cag Asn Leu Gin 380 cag ttc aac Gin Phe Asn ggt aac gat atc Gly Asn Asp Ile ggt Gly 390 ttc atc ggt ttc Phe Ile Gly Phe cac His 395 P.\Opc\Eb =wdod30035.00 ym ddeqi~n dcc.03/03/2003 63 aat atc Asn Ile 400 cgt tcc Arg Ser gct aaa ctg gtt gct Ala Lys Leu Vai Ala 405 tct cgc act ctg ggt Ser Arg Thr Leu Gly 420 tgg ggt gaa cgt ccg Trp Gly Giu Arg Pro 435 tcc aac tgg tac aat cgt cag atc gaa Ser Asn Trp Tyr Asn Arg Gin Ile Giu 410 tgc tct tgg gag ttc atc ccg gtt gat Cys Ser Trp Giu Phe Ile Pro Vai Asp 425 430 ctg taa cccgggaaag ctt Leu 1250 1298 1338 415 gac Asp ggt Gly 38 <211> 415 <2i2> PRT <213> Clostridium botulinum <400> 38 Arg i Ile Ile As n Trp Tyr Leu Lys Tyr Asn 145 Ser Asp Leu Asn Gin 225 Tyr Gly Leu Tyr As n Gin Ala Ile Thr Asn Gin Ile 130 Ser Asn Giy Phe Gin 210 Tyr Val1 Pro Tyr Giu Ile Leu Ile Arg Ile Tyr Arg 115 Asn Lys Leu Cys Asp 195 Ser Asp Asp Arg Arg Ser Gly Phe Val1 Ile Ile Gly 100 Val1 Arg Ilie Gly Arg 180 Lys Asn Lys Val1 Gly 260 Gly Asn 5 Ser Asn Tyr Pro As n Giu Val Trp Tyr Asn 165 Asp Giu Ser Pro Asn 245 Ser Thr His Lys Leu Asn Lys 70 Cys Ile Phe Ile Ile 150 Ile Thr Leu Giy Tyr 230 Asn Val Lys Leu Val1 Giu Ser 55 Tyr Met Ile Lys Phe 135 Asn His His Asn Ile 215 Tyr Val1 Met Phe Ile Asn Ser 40 Met Phe Giu Trp Tyr 120 Val1 Gly Ala Arg Giu 200 Leu Met Gly Thr Ile 280 Leu 10 Asp Lys Giu Ser Asn 90 Leu Gin Ile Leu Asn 170 Ile Giu Asp Asn Arg 250 Asn Lys Ser Pro Ile Asn Ile 75 Ser Gin Met Thr Ile 155 Asn Trp Ile Phe Leu 235 Gly Ile Lys Arg Ile Giu Phe Ser Gly Asp Ile Asn 140 Asp Ile Ile Lys Trp 220 Tyr Tyr Tyr Tyr Tyr Asp Val1 Ser Leu Trp, Thr As n 125 Asn Gin Met Phe Asp 205 Gly Asp Met Leu Ala 285 Ala Lys Ile Thr Asn Lys Gin 110 Ile Arg Lys Phe Tyr 190 Leu Asp Pro Tyr Asn 270 Ser Ser As n Leu Ser Asn Val1 Giu Ser Leu Pro Lys 175 Phe Tyr Tyr Asn Leu 255 Ser Gly Lys Gin Lys Phe Giu Ser Ile Asp Asn Ile 160 Leu Asn Asp Leu Lys 240 Lys Ser Asn a.

a a a a 275 Lys Asp 290 Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val 295 300 P.%per\Ejh=wd&S0035 00 .s ymaddsqIinzig do.-0O332003 64 Val1 305 Val Lys Asn Lys Giu Tyr 310 Ser Arg Leu Ala Thr Asn 315 Pro Ala Ser Gin Ala Glu Lys Ile Leu 325 Val1 Ala Leu Glu Ile 330 Asn Asp Val Gly Asn Leu 335 Ser Gin Val Lys Cys Lys 355 Ile Giv Phe Val1 340 Met Met Lys Ser Asp Gin Gly Asn Leu Gin Asp 360 Asn Giy Asn Asp 365 Val Ile Thr Asn 350 Ile Gly Phe Ala Ser Asn His Gin Phe 370 Trp Tyr Asn Arg Gin 385 Trp Ile 390 Val Asn Asn Ile 375 Giu Arg Ser Asp Asp Giy Ala Lys Ser Arg 395 Trp, Gly 410 Leu 380 Thr Leu Gly Cys Ser 400 Giu Phe Ile Pro 405 Giu Arg Pro <210> 39 <211> 1348 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct based on BoNTB Hc

C'

<221> CDS <222> (1) <400> 39 atg gcc a Met Ala A 1 (1323) ac aaa tac sn Lys Tyr 5 aat tcc gaa atc Asn Ser Giu Ile aac aat atc atc Asn Asn Ile Ile ctg aac Leu Asn ctg cgt tac Leu Arg Tyr aaa gtt gaa Lys Val Giu gac aac aat ctg Asp Asn Asn Leu atc Ile gat ctg tct ggt Asp Leu Ser Gly tac ggt gct Tyr Gly Ala aac cag ttc Asn Gin Phe gta tac gac ggt Val Tyr Asp Gly gaa ctg aat gac Glu Leu Asn Asp aag Lys aaa ctg Lys Leu acc tct tcc gct Thr Ser Ser Ala aac Asn 55 tct aag atc cgt Ser Lys Ile Arg gtt Val act cag aat cag Thr Gin Asn Gin atc atc ttc aac Ile Ilie Phe Asn tcc Ser gta ttc ctg gac Val Phe Leu Asp ttc Phe 75 tct gtt tcc ttc Ser Val Ser Phe tgg Trp atc cgt atc ccg Ilie Arg Ilie Pro aaa Lys tac aag aac gac Tyr Lys Asn Asp atc cag aat tac Ile Gin Asn Tyr atc cac Ile His aat gaa tac Asn Glu Tyr atc atc aac tgc Ile Ile Asn Cys atg Met 105 aag aat aac tct Lys Asn Asn Ser ggt tgg aag Gly Trp Lys 110 atc tcc atc cgc ggt aac cgt atc atc tgg act ctg atc gat atc aac P.Opeo Ejh mwdo\5O035.0 u wy~awdo1sqIitmg doc-03/03/2003 65 Ile Ser Ile 115 Arg Gly Asn Arg Ile 120 Ile Trp Thr Leu Asp Ile Asn ggt aag Gly Lys 130 acc aaa tct gta Thr Lys Ser Val ttc gaa tac aac Phe Giu Tyr Asn atc Ile 140 cgt gaa gac atc Arg Giu Asp Ile tct Ser 145 gaa tac atc aat Giu Tyr Ile Asn cgc Arg 150 tgg ttc ttc gtt Trp Phe Phe Val ac c Thr 155 atc acc aat aac Ile Thr Asn Asn aac aat gct aaa Asn Asn Ala Lys atc Ile 165 tac atc aac ggt Tyr Ile Asn Gly ctg gaa tct aat Leu Giu Ser Asn acc gac Thr Asp 175 atc aaa gac Ile Lys Asp ctg gac ggt Leu Asp Gly 195 atc Ile 180 cgt gaa gtt atc Arg Giu Vai Ile gct Ala 185 aac ggt gaa atc Asn Gly Giu Ile atc ttc aaa Ile Phe Lys 190 aaa tac ttc Lys Tyr Phe gac atc gat cgt Asp Ile Asp Arg ac c Thr 200 cag ttc atc tgg Gin Phe Ile Trp atg Met 205 tcc atc Ser Ile 210 ttc aac acc gaa Phe Asn Thr Giu ctg Leu 215 tct cag tcc aat Ser Gin Ser Asn gaa gaa cgg tac Giu Giu Arg Tyr aag Lys 225 atc cag tct tac Ile Gin Ser Tyr tcc Ser 230 gaa tac ctg aaa Giu Tyr Leu Lys gac Asp 235 ttc tgg ggt aat Phe Trp Giy Asn ctg atg tac aac Leu Met Tyr Asn aaa Lys 245 gaa tac tat atg Giu Tyr Tyr Met ttc Phe 250 aat gct ggt aac Asn Ala Gly Asn aag aac Lys Asn 255 tct tac atc Ser Tyr Ile cgt tcc aaa Arg Ser Lys 275 ctg aag aaa gac Leu Lys Lys Asp tct Ser 265 ccg gtt ggt gaa Pro Val Gly Giu atc ctg act Ile Leu Thr 270 cgc gac ctg Arg Asp Leu tac aac cag aac Tyr Asn Gin Asn tct Ser 280 aaa tac atc aac Lys Tyr Ile Asn tac Tyr 285 tac atc Tyr Ile 290 ggt gaa aag ttc Gly Giu Lys Phe atc cgt cgc aaa Ile Arg Arg Lys tct Ser 300 aac tct cag tcc Asn Ser Gin Ser 816 864 912 960 1008 atc Ile 305 aat gat gac atc Asn Asp Asp Ilie gta Val1 310 cgt aaa gaa gac Arg Lys Giu Asp tac Tyr 315 atc tac ctg gac Ile Tyr Leu Asp ttc Phe 320 ttc aac ctg aat Phe Asn Leu Asn cag Gin 325 gaa tgg cgt gta Giu Trp Arg Val tac Tyr 330 acc tac aag tac Thr Tyr Lys Tyr ttc aag Phe Lys 335 aaa gaa gaa gaa aag ctt ttc ctg gct ccg atc tct gat tcc gac gaa Lys Giu Giu Giu Lys Leu Phe Leu Ala Pro Ile Ser Asp Ser Asp Giu 1056 P:)OpcEjb.=madd%535.00 OOw y mwddscqiigd-3/3/2O3 66 340 ctc tac aac acc Leu Tyr Asn Thr 355 350 ccg acc tac Pro Thr Tyr atc cag atc aaa gaa tac gac gaa Glu Tyr Asp Glu Ile Gin Ile Lys 360 c ag Gin 365 1104 tct tgc Ser Cys 370 cag ctg ctg ttc Gin Leu Leu Phe aag Lys 375 aaa gat gaa gaa Lys Asp Glu Glu tct Ser 380 act gac gaa atc Thr Asp Giu Ile ggt Gly 385 ctg atc ggt atc Leu Ile Gly Ile cgt ttc tac gaa Arg Phe TIyr Glu ggt atc gta ttc Gly Ile Val Phe gaa Giu 400 1152 1200 1248 1296 gaa tac aaa gac Glu Tyr Lys Asp tac Tyr 405 tac Tyr ttc tgc atc tcc Phe Cys Ile Ser aaa Lys 410 ggt Gly tgg tac ctg aag Trp, Tyr Leu Lys tgc aat tgg cag Cys Asn Trp Gin gaa gtt Giu Val 415 ttc atc Phe Ile aaa cgc aaa Lys Arg Lys ccg aaa gac Pro Lys Asp 435 ccg Pro 420 gaa Giu aac ctg aaa Asn Leu Lys ctg Leu 425 tag ggt tgg acc Gly Trp Thr taacctctag agtcgaggcc 1343 0 00. 0 00* 0 0. tgcag <210> <211> 436 <212> PRT <213> Clostridium botulinum <400> Phe Asn Lys Tyr Asn Ser Giu 1 5 Arg Tyr Lys Asp Asn Asn Leu Val Giu Val Tyr Asp Gly Val Leu Thr Ser Ser Ala Asn Ser 55 Ile Ile Phe Asn Ser Val Phe 70 Arg Ile Pro Lys Tyr Lys Asn Giu Tyr Thr Ile Ile Asn Cys 100 Ser Ile Arg Gly Asn Arg Ile 115 Thr Lys Ser Val Phe Phe Giu 130 135 Tyr Ile Asn Arg Trp Phe Phe 145 150 Ala Lys Ile Tyr Ile Asn Gly 165 Asp Ile Arg Giu Val Ile Ala 1348 Ile Ile Giu 40 Lys Leu Asp Met Trp 120 Tyr Val Lys Asn Leu Asp 25 Leu Ile Asp Gly Lys 105 Thr Asn Thr Leu Gly Asn 10 Leu Asn Arg Phe Ile 90 Asn Leu Ile Ile Giu 170 Glu Asn Ser Asp Vai Ser 75 Gin Asn Ile Arg Thr 155 Ser Ile Ile Gly Lys Thr Val1 Asn Ser Asp Glu 140 Asn Asn Ile Ile Tyr Asn Gin Ser Tyr Gly Ile 125 Asp Asn Thr Phe Leu Gly Gin Asn Phe Ile Trp 110 Asn Ile Leu Asp Lys Asn Al a Phe Gin Trp His Lys Gly Ser Asn Ile 175 Leu Leu Lys Lys Asn Ile Asn Ile Lys Giu As n 160 Lys Asp PlOpa~Ejh mmdef6OO35.00. y.=a.dodwqlisundoc 3 -67- 180 185 190 Gly Asp Ile Asp Arg Thr Gin Phe Ile Trp Met Lys Tyr Phe Ser Ile 195 200 205 Phe Asn Thr Glu Leu Ser Gin Ser Asn Ile Glu Glu Arg Tyr Lys Ile 210 215 220 Gin Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro Leu Met 225 230 235 240 Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr 245 250 255 Ile Lys Leu Lys Lys Asp Ser Pro Val Gly Glu Ile Leu Thr Arg Ser 260 265 270 Lys Tyr Asn Gin Asn Ser Lys Tyr Ile Asn Tyr Arg Asp Leu Tyr Ile 275 280 285 Gly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn Ser Gin Ser Ile Asn 290 295 300 Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr Leu Asp Phe Phe Asn 305 310 315 320 Leu Asn Gin Glu Arg Val Tyr Thr Tyr Lys Phe Lys Lys Glu Glu Glu 325 330 335 Lys Leu Phe Leu Ala Pro Ile Ser Asp Ser Asp Glu Phe Tyr Asn Thr 340 345 350 Ile Gin Ile Lys Glu Tyr Asp Glu Gin Pro Thr Tyr Ser Cys Gin Leu 355 360 365 Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp Glu Ile Gly Leu Ile Gly 370 375 380 Ile His Arg Phe Tyr Glu Ser Gly Ile Val Phe Glu Glu Tyr Lys Asp 385 390 395 400 Tyr Phe Cys Ile Ser Lys Trp Tyr Leu Lys Glu Val Lys Arg Lys Pro S405 410 415 Tyr Asn Leu Lys Leu Gly Cys Asn Trp Gin Phe Ile Pro Lys Asp Glu 420 425 430 Gly Trp Thr Glu 435 <210> 41 <211> 848 <212> PRT <213> Clostridium botulinum <400> 41 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 1 5 10 Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu 25 Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 35 40 Asp Leu Ile Gin Gin Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 50 55 Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gin Leu 70 75 Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 90 Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gin Glu Phe Glu 100 105 110 His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu 115 120 125 P.OperEjb men dcd\50035 00.uuny.am dcdsailis din oc-03/03o2 0 0 3 -68- Leu Lys 145 Gin Asp Leu Phe Pro 225 Leu Trp Asn Asn Tyr 305 Ser Lys Pro Ala Val 385 Pro Phe Arg Ile Ile 465 Asn Trp Tyr Leu Lys 545 Tyr Asn 130 Val Leu Lys Asn Ser 210 Val Thr Asp Thr Gin 290 Thr Ser Phe Tyr Leu 370 Asp Phe Thr Tyr Asn 450 Gin Ala Ile Thr Asn 530 Gin Ile Pro Asn Val Ile Ile 195 Gly Leu Val Glu Gin 275 Ala Glu Lys Leu Gly 355 Leu Arg Gin Glu Glu 435 Ile Leu Ile Arg Ile 515 Tyr Arg Asn Ser Lys Tyr Ala 180 Gly Ala Gly Gin Val 260 Ile Glu Glu Leu Asn 340 Val Lys Leu Leu Tyr 420 Ser Gly Phe Val Ile 500 Ile Gly Val Arg Arg Ala Asp 165 Asp Met Val Thr Thr 245 Tyr Asp Ala Glu Asn 325 Gin Lys Tyr Lys Ser 405 Ile Asn Ser Asn Tyr 485 Pro Asn Glu Val Trp Val Thr 150 Phe Ile Leu Ile Phe 230 Ile Lys Leu Thr Lys 310 Glu Cys Arg Ile Asp 390 Lys Lys His Lys Leu 470 Asn Lys Cys Ile Phe 550 Ile Tyr 135 Glu Thr Thr Tyr Leu 215 Ala Asp Tyr Ile Lys 295 Asn Ser Ser Leu Arg 375 Lys Tyr Asn Leu Val 455 Glu Ser Tyr Met Ile 535 Lys Phe Thr Ala Asp Ile Lys 200 Leu Leu Asn Ile Arg 280 Ala Asn Ile Val Glu 360 Asp Val Val Ile Ile 440 Asn Ser Met Phe Glu 520 Trp Tyr Val Phe Ala Glu Ile 185 Asp Glu Val Ala Val 265 Lys Ile Ile Asn Ser 345 Asp Asn Asn Asp Ile 425 Asp Phe Ser Tyr Asn 505 Asn Thr Ser Thr Phe Met Thr 170 Ile Asp Phe Ser Leu 250 Thr Lys Ile Asn Lys 330 Tyr Phe Tyr Asn Asn 410 Asn Leu Asp Lys Glu 490 Ser Asn Leu Gin Ile Ser Phe 155 Ser Pro Phe Ile Tyr 235 Ser Asn Met Asn Phe 315 Ala Leu Asp Gly Thr 395 Gin Thr Ser Pro Ile 475 Asn Ile Ser Gin Met 555 Thr Ser 140 Leu Glu Tyr Val Pro 220 Ile Lys Trp Lys Tyr 300 Asn Met Met Ala Thr 380 Leu Arg Ser Arg Ile 460 Glu Phe Ser Gly Asp 540 Ile Asn Asp Gly Val Ile Gly 205 Glu Ala Arg Leu Glu 285 Gin Ile Ile Asn Ser 365 Leu Ser Leu Ile Tyr 445 Asp Val Ser Leu Trp 525 Thr Asn Asn Tyr Trp Ser Gly 190 Ala Ile Asn Asn Ala 270 Ala Tyr Asp Asn Ser 350 Leu Ile Thr Leu Leu 430 Ala Lys Ile Thr Asn 510 Lys Gin Ile Arg Val Val Thr 175 Pro Leu Ala Lys Glu 255 Lys Leu Asn Asp Ile 335 Met Lys Gly Asp Ser 415 Asn Ser Asn Leu Ser 495 Asn Val Glu Ser Leu Lys Glu 160 Thr Ala Ile Ile Val 240 Lys Val Glu Gin Leu 320 Asn Ile Asp Gin Ile 400 Thr Leu Lys Gin Lys 480 Phe Glu Ser Ile Asp 560 Asn oooo oo** 565 570 575 Asn Ser Lys Ile Tyr Thr lie Asn Gly Arg Leu Ile Asp Gin Lys Pro P' Ope\Ejl minod50035 00 .mwq~itid& dwO3/020 3 69 Ile Leu Asn 625 Asp Leu Lys Lys Ser 705 As n Val1 Gly Leu Asn 785 Phe Asn Ser Ser Asp 610 Leu As n Gin Tyr Giy 690 Leu Lys Val1 Val1 Ser 770 Lys Ile Trp Trp Asn 595 Giy Phe Gin Tyr Vali 675 Pro Tyr Asp Lys Giu 755 Gin Cys Giy Tyr Giu 835 580 Leu Cys Asp Ser Asp 660 Asp Arg Arg Asn Asn 740 Lys Vali Lys Phe Asn 820 Phe Giy Arg Lys Asn 645 Lys Vali Giy Giy Ile 725 Lys Ile Vali Met His 805 Arg Ile Asn Asp Giu 630 Ser Pro Asn Ser Thr 710 Vai Giu Leu Vali Asn 790 Gin Gin Pro Ile Thr 615 Leu Giy Tyr Asn Vai 695 Lys Arg Tyr Ser Met 775 Leu Phe Thr Val1 585 His Aia 600 His Arg Asn Giu Ile Leu Tyr Met 665 Vai Giy 680 Met Thr Phe Ile Asn Asn Arg Leu 745 Ala Leu 760 Lys Ser Gin Asp Asn Asn Giu Arg 825 Asp Asp 840 Vai Asp Asp Asp 25 Tyr Ile 40 Leu Ile Asp Phe Lys Ile Thr Phe 105 Asp Aia 120 Ser Tyr Lys Lys 650 Ile Ile Thr Ile Asp 730 Aia Giu Lys Asn Ile 810 Ser Giy Asn 10 Leu Giu Ser As n Phe 90 Pro Leu Asn Asn Ile Trp 620 Giu Ile 635 Asp Phe Leu Leu Arg Gly Asn Ile 700 Lys Lys 715 Arg Vai Thr Asn Ile Pro Asn Asp 780 Asn Gly 795 Ala Lys Ser Arg Trp Gly Giu Asp Ser Lys Asn Asp Lys Ile Vai Asp 75 Thr Asp Leu Asp Leu Phe Lys Trp Tyr Tyr 685 Tyr Tyr Tyr Aia Asp 765 Gin Asn Leu Thr Giu 845 Leu Asn Phe Giu Val1 Giu Ile Ser 125 Asp Giy Asp 670 Met Leu Ala Ile Ser 750 Val1 Gly Asp Val1 Leu 830 Arg Phe Giu Pro Leu Pro As n Arg 110 As n Leu Asp 655 Pro Tyr Asn Ser Asn 735 Gin Gly Ile Ile Al a 815 Giy Pro Phe Arg Ile Pro Val1 Thr Asp Lys 590 Ile Met Phe 605 Ile Lys Tyr Lys Phe Tyr 640 Tyr Asn Leu Ser Gly 720 Vali Ala Asn Thr Gly 800 Ser Cys Leu Ile Ile As n Ser Tyr Ile Ile Val1 @0 @0 0000 0 0 00*0 @000 <210> 42 <211> 843 <212> PRT <213> Ciostridium botulinum <400> 42 Ala Pro Gly Ilie Cys Ilie Asp 1 5 Ala Asp Lys Asn Ser Phe Ser 20 Giu Tyr Asn Thr Gin Ser Asn 35 Giu Leu Ile Leu Asp Thr Asp 50 55 Giu Asn Thr Giu Ser Leu Thr 70 Giu Lys Gin Pro Ala Ile Lys Phe Gin Tyr Leu Tyr Ser Gin 100 Ser Leu Thr Ser Ser Phe Asp 115 P:'peOpEjb mmdAM50035 00 urmy mmdeadslising doc0303/032003

OSSS

0O 9 *S Tyr Glu 145 Val Ser Thr Ile Phe 225 Ile Gly Thr Leu Lys 305 Glu Cys Lys Ile Ser 385 Ile Ser Asn Gly Asn 465 Phe Asn Cys Ile Glu 545 Phe Gly Ser 130 Ala Ile Leu Ala Leu 210 Leu Asp Leu Ile Glu 290 Ser Gly Ser Leu Asp 370 Lys Tyr Glu Leu Val 450 Ser Leu Asp Met Trp 530 Tyr Val Lys Phe Gly Glu Ile Lys 195 Leu Leu Asn Ile Lys 275 Glu Asn Ile Val Leu 355 Glu Val Thr Ile Ile 435 Glu Lys Asp Gly Lys 515 Thr Asn Thr Leu Phe Leu Ala Val 180 Gly Glu Glu Ala Val 260 Glu Ile Ile Asn Ser 340 Asp Asn Asn Asn Leu 420 Asp Leu Ile Phe Ile 500 Asn Leu Ile Ile Glu Ser Phe Asn 165 Pro Asn Phe Ser Leu 245 Ala Gly Ile Asn Gin 325 Tyr Phe Lys Lys Asp 405 Asn Leu Asn Arg Ser 485 Gin Asn Ile Arg Thr 565 Ser Met Ala 150 Lys Tyr Phe Ile Tyr 230 Thr Gin Met Lys Ile 310 Ala Leu Asp Leu Tyr 390 Thr Asn Ser Asp Thr 470 Val Asn Ser Asp Glu 550 Asn Asn Asp 135 Gly Ser Ile Glu Pro 215 Ile Lys Trp Tyr Tyr 295 Asp Ile Met Asn Tyr 375 Leu Ile Ile Gly Lys 455 Gin Ser Tyr Gly Ile 535 Asp Asn Thr Tyr Trp Asn Gly Asn 200 Glu Asp Arg Leu Lys 280 Arg Phe Asp Lys Thr 360 Leu Lys Leu Ile Tyr 440 Asn Asn Phe Ile Trp 520 Asn Ile Leu Asp Ile Val Thr Leu 185 Ala Leu Asn Asn Ser 265 Ala Tyr Asn Asn Lys 345 Leu Ile Thr Ile Leu 425 Gly Gin Gin Trp His 505 Lys Gly Ser Asn Ile Lys Lys Met 170 Ala Phe Leu Lys Glu 250 Thr Leu Asn Asp Ile 330 Met Lys Gly Ile Glu 410 Asn Ala Phe Asn Ile 490 Asn Ile Lys Glu Asn 570 Lys Thr Gin 155 Asp Leu Glu Ile Asn 235 Lys Val Asn Ile Ile 315 Asn Ile Lys Ser Met 395 Met Leu Lys Lys Ile 475 Arg Glu Ser Thr Tyr 555 Ala Asp Ala 140 Ile Lys Asn Ile Pro 220 Lys Trp Asn Tyr Tyr 300 Asn Asn Pro Asn Ala 380 Pro Phe Arg Val Leu 460 Ile Ile Tyr Ile Lys 540 Ile Lys Ile Asn Val Ile Val Ala 205 Val Ile Ser Thr Gin 285 Ser Ser Phe Leu Leu 365 Glu Phe Asn Tyr Glu 445 Thr Phe Pro Thr Arg 525 Ser Asn Ile Arg Lys Asn Ala Gly 190 Gly Val Ile Asp Gin 270 Ala Glu Lys Ile Ala 350 Leu Tyr Asp Lys Lys 430 Val Ser Asn Lys Ile 510 Gly Val Arg Tyr Glu Val Asp Asp 175 Asn Ala Gly Lys Met 255 Phe Gin Lys Leu Asn 335 Val Asn Glu Leu Tyr 415 Asp Tyr Ser Ser Tyr 495 Ile Arg Phe Trp Ile 575 Val Val Phe 160 Ile Glu Ser Ala Thr 240 Tyr Tyr Ala Glu Asn 320 Gly Glu Tyr Lys Ser 400 Asn Asn Asp Ala Val 480 Lys Asn Ile Phe Phe 560 Asn Ile 60 0 *0 o P:OpuEjl mmded\5035OD ymosqit dm.-031312003 71 580 585 590 Ala Asn Gly Giu Ile Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr 595 600 605 Gin Phe Ile Trp Met Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser 610 615 620 Gin Ser Asn Ile Giu Giu Arg Tyr Lys Ile Gin Ser Tyr Ser Giu Tyr 625 630 635 640 Leu Lys Asp Phe Trp Gly Asn Pro Leu Met Tyr Asn Lys Giu Tyr Tyr 645 650 655 Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr Ile Lys Leu Lys Lys Asp 660 665 670 Ser Pro Val Giy Giu Ile Leu Thr Arg Ser Lys Tyr Asn Gin Asn Ser 675 680 685 Lys Tyr Ile Asn Tyr Arg Asp Leu Tyr Ile Giy Giu Lys Phe Ile Ile 690 695 700 Arg Arg Lys Ser Asn Ser Gin Ser Ile Asn Asp Asp Ile Vai Arg Lys 705 710 715 720 Giu Asp Tyr Ile Tyr Leu Asp Phe Phe Asn Leu Asn Gin Giu Trp Arg 725 730 735 Val Tyr Thr Tyr Lys Tyr Phe Lys Lys Giu Giu Giu Leu Phe Leu Ala 740 745 750 Pro Ile Ser Asp Ser Asp Giu Phe Tyr Asn Thr Ile Gin Ile Lys Giu 755 760 765 Tyr Asp Giu Gin Pro Thr Tyr Ser Cys Gin Leu Leu Phe Lys Lys Asp 770 775 780 Giu Giu Ser Thr Asp Giu Ile Giy Leu Ile Gly Ile His Arg Phe Tyr *785 790 795 800 *Giu Ser Giy Ilie Vai Phe Giu Giu Lys Asp Phe Cys Ile Ser Trp Tyr *805 810 815 Leu Giu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Giy Cys Asn Trp 820 825 830 *Gin Phe Ile Pro Lys Asp Giu Gly Trp Thr Giu *835 840

Claims (12)

1. A nucleic acid encoding the carboxy-terminal portion of the heavy chain (Hc) of botulinum neurotoxin (BoNT) of BoNT serotype A wherein said nucleic acid is expressed in a recombinant organism selected from Escherichia coli and Pichia pastoris and wherein said nucleic acid comprises a nucleic acid molecule selected from the group consisting of SEQ ID NO:l (serotype SEQ ID NO:3 and SEQ ID

2. The nucleic acid of Claim 1, wherein the nucleic acid encodes an He amino acid sequence of BoNT selected from the group consisting of SEQ ID NO:2 (serotype SEQ ID NO:4 and SEQ ID NO:6. o

3. The nucleic acid of Claim 1, wherein the sequence of the nucleic acid is designed by selecting at least a portion of the codons encoding He from codons preferred for expression in a host organism.

4. The nucleic acid of Claim 1, wherein the nucleic acid sequence encoding He is Sdesigned by selecting codons encoding He which codons provide He sequence enriched in guanosine and cytosine residues.

5. The nucleic acid of Claim 1, wherein said nucleic acid is a synthetic nucleic acid.

6. The nucleic acid of Claim 1, wherein said nucleic acid encoding He is expressed in a recombinant host organism with higher yield than a second nucleic acid fragment encoding the same He sequence, said second nucleic acid fragment having the wild-type Clostridum botulinum sequence of Hc.

7. An expression vector comprising a nucleic acid molecule selected from SEQ ID NOs:1, 3 and

8. A method of preparing a polypeptide comprising the carboxy-terminal portion of 15/09 2005 THU 11:22 [TX/RX NO 6635] 1a004 .LJ iuJ .JJ .LJ. L'LIJ I LU I FV. 01 -vi C P.1PER\H7AhW Uila S4 Ay e Ra t c 2465at2R UlS37O 4$32 a~o u 44h SPAO.d IS 9 -47- the heavy chain (Hc) of botulinum neurotoxin (BoNT) serotype A, said method comprising culturing a recombinant host organism transfected with the expression vector of Claim 7 under conditions wherein Hc is expressed.

9. The method of Claim 8, wherein the recombinant host organism is a eukaryote.

The method of Claim 8, further comprising recovering insoluble protein from said host organism, whereby a fraction enriched in He is obtained.

11. The method of Claim 10, wherein said host organism is Pichia pastoris.

12. A nucleic acid according to any one of Claims 1 to 6 substantially as hereinbefore described with reference to the Figures and Examples, an expression vector according to Claim 7 substantially as hereinbefore described with reference to the Figures and Examples or a method according to any one of Claims 8 to 11 substantially as hereinbefore described with reference to the Figures and Examples. 0* Dated this 15 th day of September, 2005 UNITED STATES ARMY MEDICAL RESEARCH MATERIEL CMD By Its Patent Attorneys S. DAVIES COLLISON CAVE o 15/09 2005 THU 11:22 [TX/RX NO 6635] 1@005