AU616662B2 - Peptide production by protein engineering - Google Patents

Description

.1 I'll _11 1 17,049/88 WORLD INTELLECTUAL PROPERTY ORGANIZATION International Bureau 0

PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 C07K 13/00, 7/08, C12N 15/00 C07H 21/04, C12P 19/34, 21/02 //(C12N 15:00, C12R 1:01 S (11) International Publication Number: WO 88/ 08430 (21) International Application Number: PCT/AU88/00120 (22) International Filing Date: (31) Priority Application Numibr: (32) Priority Date: (33) Priority Country: 27 April 1988 (27.04.88) PI 1595 27 April 1987 (27.04.87) (71) Applicant (for all designated States except US): COM- MONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION [AU/AU]; Limeston Avenue, Campbell, ACT 2601 (AU).

(72) Inventors; and Inventors/Applicants (for US only) MATTICK, John, Stanley [Atl/AU]; Laboratory for Mnoecular Biology, University of Queensland, St.Lucia, QLD 4067 (AU).

JENNINGS, Philip, Anthony [GB/AU]; Commonwealth Scientific and Industrial Research Organisation, Division of Biotechnology, 103 Delhi Road, North Ryde, NSW 2113 (AU).

(74) Agent: F.B. RICE CO.; 28A Mon ague Street, Bal-! main, NSW 2041 (AU).

(81) Designated States: AT (European patent), AU, BE (European ptent), CH (European patent), DE (European patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), SE (European oatent),

US.

Published With international search report, A.OJ.P. 5 JAN 1989

AUSTRALIAN

-2 DEC 1988 PATENT OFFICE (54) Title: PEPTIDE PRODUCTION BY PROTEIN ENGINEERING (57) Abstract The present invention relates to a method of peptide production b' protein engineering. This method involves the culturing of genetically engineered bacteria which produce as extracellular structures .ype imbriae, the peptide being produced in association with these fimbriae. This method is applicable to thle production of virtually any peptide or peptides. The engineered bacteria contains the gene encoding the fimbrial subunit of type fimbriae to which has been added t the C-terminal end the nucleic acid sequence encodir.n the peptide )f choice, together with a system for morphogenetic assembly of type fimbriae such that fimbriae in association with the peptide are produced as extracellular structures.

l -1- Peptide Production By Protein Engineering The present invention relates to a method of peptide production by protein engineering. This method involves the culturing of genetically engineered bacteria which produce as extracellular structures type 4 fimbriae, the peptide being produced in association with these fimbriae. This method is applicable to the production of virtually any peptide or peptides.

Background of the Invention There is a growing demand for biologically-active peptides, either as homologues or analogues cf naturally occurring regulatory, receptor and antv.-nic sequences.

Apart from natural sources, such peptides have to date Sbeen produced by chemical or enzymatic synthesis, or by biosynthesis in recombinant hosts, usually as a fusion product in bacteria. The advent cE protein engineering techniques, which involve a combination of DNA primer-directed mutagenesis with recombinant host-vector cloning and expression systems, has expanded enormously j 20 the scope of, and potential for, the design, production and use of such peptides. These techniques combine precision and flexibility, not only to investigate structure-function relationships in existing peptides and proteins, but also for the expression and evaluation of a large repertoire of novel peptide sequences, which is not limited to that already present in nature. However, there are very few systems for the high level expression and particularly export of such peptides from recombinant hosts. What is -required is a carrier protein system, preferably from a microbial host (for ease of genetic manipulation and industrial fermentation), which has the flexibility to accept grafts of exogenous peptida sequences, either as substitutions or additions, and which may be readily recovered and processed (if necessary) to release the active peptide.

-2- One potential candidate for such a system is the type 4 fimbria (or common pilus) which occurs in a variety of Gram-negative bacteria, including Bacteroides nodosus, Moraxella bovis,.Neisseria gonorrhoeae and Pseudomonas aerug'iosa. These fimbriae are long filamentous surface structures, of about 6nm in diameter and ranging up to several um in length, which are thought to be involved in bacterial colonization of eukaryotic cell surfaces. These fimbriae have a polar or mainly polar location on the cell, and associated with a phenomenon known as twitching motility. They are comprised primarily of a small structural protein subunit, of approximate molecular weight 16,000, which varies in size (145 to 160 amino acids) in different species and serotypes. The subunits from different species share several features in common, most notably a highly conserved and hydrophobic amino-terminal sequence, which begins with the modified amino acid N-methylphenylalanine. The subunits are also all preceded by a similar short (6 to 7 amino acid) positively-charged leader sequence, which is removed prior to incorporation into the mature fimbrial strand. These regions are thought to contain important signals for the export, assembly and structure of the fimbrial strand.

Variation between species and serotypes occurs in the more hydrophilic carboxy-terminal two-thirds of the protein, with most differences clustered in two or three hypervariable regions. A priori it would seem that these regions, due to their conformational flexibility, are the regions most likely to permit substitutio of exogenous peptide sequences. In addition, all subunits end in a series of charged and/or polar amino acid residues, suggesting the carboxy-terminus has a surface location, perhaps suitable for insertion of additional peptide sequences. We have investigated both of these possibilities.

f 3- L- -3- For these experiments we used the fimbrial subunit of D.nodosus as the carrier for peptide grafts, seherichia coli as the host for genetic constructions and P.aeruginosa as the host for morphogenetic expression of modified fimbrial subunits. We have determined the primary sequence of the fimbrial subunit genes representative of all known serogroups existing in the B.nodosus population, allcwing close ;'finition of the conserved and variable regions of the protein, and precise modification (Figure B.nudosus is an anaerobe and the causative agent of ovine footrot. P.aeruginosa is a i genetically well-characterized and easily cultured aerobe, suitable for use in industrial fe,;mentation, and has a compatible type 4 fimbrial system. It has already been demonstrated that high levels of (B.nodosus-type) fimbriae may be obtained from cloned subunit genes under appropriate promoter control in recombinant P.aeruginos.

cells, and that this material is suitable for use as a N vaccine (Australian Patent Application No. 50154/85).

The peptide used in the protype studies was the 16 i amino acid sequence LRGDLQVLAQKVARTL, corresponding to i residues 144-159 of the surface protein VPl of foot-and-mouth disease (FMD) virus (strain 01-BFS).

This particular sequence was chosen for several (related) reasons. Firstly, and importantly, this peptide includes a well-characterized epitope capable of eliciting neutralizing antibodies against the natural FMD virus, thereby providing a potential route for the development and production of a new vaccine against the disease.

Secondly, this peptide appears to contain both thia 3-cell epitope and a helper T-cell determinant, as well as part of a likely binding site important for viral entry into cells. The latter is related to the sequence RGDL, which is similar to the sequence RGDS which forms part of the cell/platelet binding Site identified on the extracellular 1- 2 S// 1 -4matrix protein fibronectin. Thirdly, the peptide may possess a pseudo-autonomous structure, proposed to be alpha-helical, which has the advantage that its important structural and functional features may be maintained in a new environment, but which, on the other hand, may present certain difficulties as a graft in a carrier protein.

This peptide also has a significant length, comparable to that of many other active peptides. In an exploration of the feasibility and (general) utility of the fimbrial expression system for peptide production, this sequence then has the dual advantage that it not only possesses intrinsic biological and immunological importance, but also represents a relatively demanding test case.

The present inventors have found that construction involving substituticn within the hypervariable regions of the type 4 fimbrial subunit of a replacement peptide did not succeed, whilst the alternative construction involving a carboxy-terminal substitution was successful.

Disclosure of Invention Accordingly, in a first aspect the present invention consists in a method of producing a peptide comprising culturing bacteria containing the gene encoding the fimbrial subunit of a bacteria normally producing type 4 fimbriae to which has been added at the C-terminal end the nucleic acid sequence encoding the peptide, and an endogenous compatible system for the morphogenetic S assembly of type 4 fimbrial and/or the genes for the morphogenetic assembly of such fimbriae derived from a type 4 fimbriate species, such that mature type 4 fimbriae in association with the peptide are produced as extracellular structures, harvesting the whole or part of the fimbriae substantially free of the host cells, and optionally separating the peptide from the type 4 fimbriae.

In a second aseact the present invention consists in an antigenic preparation for raising antibodies directed I I C;en i, 1 against the surface protein VPl of foot-and-mouth disease virus (FMDV) comprising the VPl peptides either in isolation or in association with type 4 fimbriae, which has been produced by bacterial host cells containing the gene encoding the fimbrial subunit of a bacteria normally producing type 4 fimbriae to which has been added at the C-terminus the nucleic acid sequence encoding the protein VPl, and an endogenous compatible system for the morphogenetic assembly of type 4 fimbriae and/or those genes for the morphogenetic assembly of such fimbriae derived from a type 4 fimbriate species and in which the VPl peptide has optionally been cleaved from the type 4 fimbriae produced by said host cells.

In a preferred embodiment of the present invention the gene encoding the fimbrial subunit of a bacteria normally producing type 4 fimbriae is derived from a bacteria selected from the group comprising B.nodosus, P.aeruginosa, N.gonorrhoeae, N.meningitidis, M.nonliquefaciens and M.bovis.

In a further preferred embodiment the host bacteria is of the genus Pseudomonas and in particular is the species P.aeruginosa preferably the strain P.aeruginosa IPAK/2Pfs (ATCC No. 53308).

In another preferred embodiment of the present 25 invention the gene encoding the fimbrial structural subunit to which has been added at the C-terminal end .the nucleic acid encoding the peptide is contained within a plasmid or phage.

A preferred embodiment of the present invention is hereinafter described by way of example and with reference to the Figures.

Brief Description of Drawings Fig. 1 is a schematic representation of the B.nodusus fimbrial subunit showing the sites of substitution or addition of peptide sequences. The dark areas indicate 1 C I ~lz u; -6the N-terminal leader and conserved hydrophobic sequences thought to be important in fimbrial assembly and export.

The'shaded (striped) areas indicate segments exhibiting high variability between serotypes. The position of th.

presumptive disulphide loop spanning the centre of the protein is indicated by The total length of the subunit is 158 amino acids. The diagram is drawn approximately to scale. The sites of the various substitutions and C-terminal addition of the FMDV VP1 144-159 peptide are indicated by the arrows.

Fig. 2 shows the engineered insertions and substitutions (DNA and protein sequences) together with the B.nodosus fimbrial subunit gene and protein sequences. The DNA sequence of the DRAl fragment containing the subunit gene is shown for the coding strand. A substitution or insertion at a given position is indicated as a variant (Variant 1 etc.) as explained in the text. Variant 3, not shown above, is a double epitope substitution at the positions used for Variant 1 and Variant 2. The oligonucleotides used for mutagenesis are underlined on the fimbrial subunit gene sequence. FMDV sequences derive from VP1 144-160 (Strain 0 1

-BFS).

Fig. 3 shows the induction and expression of variant fimbrial subunit genes in E.coli as indicated by gel profiles. Panel A shows the coomassie-stained gel profiles of whole E.coli N4830 cells containing the normal t or modified (Variant l-4)fimbrial subunit genes under PL promoter control, either uninduced (30 0 or induced (42 0 The arrowheads point to the position of the induced fimbrial subunit proteins in each case.

Panels B and C show Western Transfer profiles using antisera against native and denatured normal B.nodosus fimbirae, respectively. The different tracks show induced E.coli N4830 cells containing either the normal or modified (Variant 1-4) fimbrial subunit genes. Only the (2 K 7 -7- (relevant) lower portions of the Western Transfer are presented.

Fig. 4 shows a Western Transfer Analysis of Variant fimbrialosubunits using guinea pig anti sera to foot and mouth'disease virus which-was used against Variants 1, 2, i 3 and 4 (reactive bands corresponding to variants carrying i the FMDV-graft shown arrowed).

Fig. 5 shows various gel profiles of the expression in P. aeruginosa of the Variant Fimbrial Subunit containing the C-terminal addition of FMDV VP1 peptide 144-159. Panel A shows the cell pellet and supernatant fimbrial fractions derived by sodium acetate or MgCl 2 precipitations, derived from P.

J aeruginosa cells containing the recombinant plasmid pKT-Var4. Panel B shows a Western Transfer Analysis of i the same display, using anti-B. nodosus fimbrial antiserum. For comparison, Panel C shows an equivalent Western Transfer Analysis of the same fractions obtained o from E. coli containing pKT-Var4.

20 As used throughout the description and claims, the expression "endogenous compatible system" as hereinafter defined refers to a system within the bacterium which enables the assembly of fimbrial structures from fimbrial subunit proteins.

Detailed )escription of Invention I mbrial subunit gene cloning and template preparation The B.nodosus fimbrial subunit gene was derived from the serogroup A prototype strain VCS1001 (ATCC 25539), cloned initially on a 5.5 kilobase Hind III genomic DNA restriction fragment (Australian Patent Application No.

34979/84). The gene was then isolated as a 576 base-pair Dra 1 cartridge, beginning 30 nucleotides upstream of the '^"^fe7

PI

4 'f 7a initiation codon and ending 69 nucleotides be~ond the termination* codon.. This- cartridge also includes a Shine(LDalgarno sequence* for ribosomal docking, and a 'I probable'rho independent[ transcription 0termination signal.- The,'re levant -f eatures and full -sequence are shown in Figures 1 and 2. Sma 1 linker osequences .(-Amersham Cdrp.) were ligated onto the~ends of the Dra 1 cartridge, to allow later, portability of -theslequence during -8subcloning operations required for mutagenesis and expression (see below). The ligation products were then digested with Sma 1 and the modified cartridge subcloned into the corresponding site of the filamentous phage vector M13 mp8. Single stranded DNA was prepared from the recombinant phage, as the template for subsequent mutagenesis. The construction was checked by direct DNA sequencing of this template using the dideoxynucleotide chain termination method, which also established that the orientation of the insert was such that the 5' end of the fimbrial subunit gene was located adjacent to the universal priming site of the vector.

Oligonucleotide-directed mutagenesis The introduction of the PMDV VP1 144-159 peptide sequence into the fimbrial subunit gene was carried out using oligonucleotide primers on the single-stranded recombinarn M13 template. The oligonucleotides were synthesized using an Applied Biosystems DNA Synthesizer, and purified according to the manufacturer's instructions. The basic design of the primers was simila in all cases, consisting of a central cole sequence Iencoding the FMDV peptide (see below) flanked on either side by 12 nucleotide stretches complementary to defined regions of the fimbrial gene template, which specified the exact position of oligonucleotide substitution or addition (see below; Figure The core sequence was TTA CGC GGT I GAT TTA CAA GTT TTA GCT CAA AAA GTT GCT CGC ACT TTA, encoding the peptide LRGDLQVLAQKVARTL, homologous to VPl residues 144-159 of FMDV strain 01-BFS. The oligonucleotide sequence was designed to take into account the codon usage of natural fimbrial subunit genes. In one case (VARl), the first codon (TTA-leucine) was omitted, in order to allow better alignmen' r of the substitute peptide sequence with that originally present in the fimbrial subunit, while still retaining the cysteine residue at -9position 63, required for the presumptive disulphide loop which spans the two major variant regions in the centre of the protein. (There is also a third more dispersed variant region in the carboxy-terminal third of the protein (see Figure 1) which was not modified). In another case (VAR4), two additional glycine codons (GGTGGT) were added to the 5' end of the core sequence to serve as a flexible "hinge" region for the terminal addition of the FMDV epitope. All told, four mutant genes were constructed and analyzed: VAR1, epitope substitution for fimbrial variant region 1, from residues 64-78 inclusive, immediately downstream of cysteine 63; VAR2, epitope substitution for fimbrial variant region 2, from residues 87-102 inclusive, just upstream of cysteine 104; VAR3, double epitope substitution at variant regions 1 and 2, as described above; and VAR4, epitope addition following the terminal asparagine residue at position 158, including the di-glycine "hinge", introduced to minimize Sany interference between the FMDV sequence and the fimbrial subunit. In determining the exact position of epitope substitutions in variant regions 1 and 2, two j factors were taken into account, optimization of the i alignment between the replacement FMDV sequence and the original fimbrial sequence in the region while (ii) 25 limiting the substitution to the segments known to be variant in different serotypes, i.e. to minimize encroachment on conserved regions of the protein. Full Sdetails are shown in Figure 2.

For mutagenesis, oligonucleotide primers were annealed to the single-stranded recombinant Ml3-fimbrial gene template, followed by in vitro synthesis of the complementary strand using the Klenow fragment of DNA polyiferase I. Strand closure was effected by T4 DNA ligase under standard conditions. Following transfection into E.coli TG1 cells and mismatch repair in vivo, mutant i i clones (plaques) were identified by hybridization screening using 3 2 p-end-labelled o ni.~ .eotide as the probe. Positive clones were plaque-purified and their (altered) sequence confirmed by DNA sequencing of the purified recombinant phage DNA using the dideoxy chain termination method.

Subcloning and gene expression Subcloning, of the fimbrial subunit gene for high level morphogenetic expression in P.aeruginosa was carried out by a strategy similar to that described previously (Australian Patent Application No. 50154/85.) Double stranded (replicative form) DNA was prepared from cells infected with recombinant phage carrying the various imutant constructions. The modified fimbrial subunit genes containing FMDV epitope coding sequences were then excised by digestion with Sma 1 and subcloned into the Hpa 1 site of the expression vector plasmid pPL-lambda (Pharmacia-P.L. Biochemicals), using as host the temperature-sensitive lambda lysogen E.coli strain N4830 Clts 857), maintained at the permissive temperature of 31oC. Recombinant clones were identified by DNA hybridization using the Sma 1 fragment (containing the normal fimbrial subunit gene), 32 p-labelled by nick-translation, as the probe. The entire PL promoter-modified timbrial gene construction was th'-n excised on a Bam HI cartridge and subcloned into the corresponding site of the broad host range plaomid vector pKT240, using E.coli N4830 as the host, as described above. Positive clones were again identified by DNA hybridization screening, using 3 2 p-labelled nick-translated Barn H segmenit as probe. These recombinant plasmids are termed pK-VARl, pK-VAR2, pK-VAR3, and pK-VAR4, for each of the constructions, according to the terminology introduced earlier. E. coli cells containing these plasmids were maintained on media -11containing 50 ug ampicillin per ml.

Gene expression was checked in the E.coli host cells by diluting an overnight culture 1:4 into fresh medium shifting to the restrictive temperature of 42 0 °C for 2 I hours. Samples of induced and uninduced cells were i analyzed by sodium dodecyl sulphate (SDS)-urea-gradient polyscrylamide gels and Western transfer immunoblotting, as described in Australian Patent Application No.

50154/85. Control samples of cells containing the equivalent construction with an unmodified fimbrial subunit gene in pKT240 (pJSM202; ATCC40203 Australian Patent Application No. 50154/85) were also included.

The recombinant pKT240-P, promoter-variant subunit gene constructions were then transformed into the multifimbriate P.aeruginosa strain PAK/2Pfs as described in Australian Patent Application No. 50154/85 and deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, USA on 30 October 1985 and given the accession number ATCC 53308. Control transformations with pKT240 itself, as I A I well as pJSM202, were also carried Jut. Transformation were selected and maintained on nutrient plates containing 750 ug carbenicillin per ml.

25 Fimbrial fractions were prepared by standard procedure.: P.aeruginosa cells were resuspended in phosphate-buffered saline, subjected to mechanical blending, and following removal of the cells by centrifugation, fimbriae were recovered from the supernatant by either MgC1 2 (0.1M) precipitation or isoelectric precipitation at pH 4.5 with sodium acetate, as described in Australian Patent Application No.

53154/85. Fimbrial fractions were analysed in SDS-urea-gradient polyacrylamide gels and Western transfer, as above. Diagnostic antisera were raised in I -12rabbits against either native B.nodosus fimbriae or denatured B.nodosus fimbriae (disrupted with 1.6% SDS, 1% 2 -mercaptoethanol at 100 0 C for 3 minutes, prior to emulsification in Freund's incomplete adjuvant).

RESULTS

The general features of the B.nodosus fimbrial subunit and the strategy for peptide subtraction and insertion are outlined in Figure 1. The fimbrial subunit of B.nodosus exhibits particular regions of major sequence variation of which the most prominent are located at either end of the presumptive disulphide loop, which spans the centre one third of the protein. These represented logical candidates as sites for substitution with a replacement peptide such as the FMDV sequence. However (see below) these constructions did not succeed. Since the size of such substitutions at these positions may br limited, and in two cases proved non-viable, we examined the alternative strategy of a carboxy-terminal fusion of the FMDV sequence. The fusion protein thus generated, retains the complete primary sequence of the fimbrial subunit, which may well be important in morphogenetic expression, and also has several other potentially desirable features in the context of a general expression strategy: the peptide addition is presumably exposed as a tail on the carboxy-terminus of the protein where it should be both accessible for interaction with other molecules and/or the immune system and where there is no a prio'ri limit on the size of the peptide. Additionally the peptide should be accessible to cleavage and thus separation from the carrier proteti'.

The details of the normal and mutant sequences, and the oligonucleotides used, are shown in Fig. 2. The mutated genes were placed under PL promoter control and their expression induced in E.coli (Fig. All mutant genes showed good induction and relatively high levels of L i i :cil::i~ T -13expression, directed by the PL promoter in E.coli.

However it is also clear that replacement of the normal sequence with the FMDV sequence had a significant effect on the elecLrophoretic mobility of the protein although the peptide chain lengths were unchanged by the substitution and the molecular weights were approximately the same. Substitutions at both positions 1 and 2 (Figure 1) increased the electrophoretic mobility lower apparent molecular weight) with substitutions at both sites showing an additive effect (evidenced by an approximate doubling in the shift in electrophoretic mobility relative to substitutions at a single site). On the other hand the C-terminal fusion of FMDV sequence showed, as might be expected, a higher molecular weight on gels.

The position of the bands for the B.nodosus fusion proteins was verified and their antigenicity tested by I, Western transfer analysis using antisera raised against CN native and d natured B.nodosus fimbriae (Fig. 3) and against foot-and-mouth disease virus (Fig. All modified subunits were recognised by antisera raised against denatured fimbriae whereas, the substitution at position 2 was associated with a marked reduction in the binding of antibodies against native fimbriae.

Only a minor effect was seen for the substitution at position 1, with no such effect being apparent for the carboxy-terminal addition. This implies that variable region 2 plays an important role in the antigenic profile of the native protein. It also suggests that the carboxy-terminal fusion may not significantly perturb the overall structure of the protein.

Western transfer analysis of the variant fimbrial constructs shows that all FMDV-peptide grafts are recognised by anti-FMDV antisera (neutralising antisera raised in guinea pigs in Fig. 4) with, however, I i i i; I -14significant quantitative differences between them. The carboxy-terminal graft was recognised the most strongly, with VAR1 also showing a high level of reactivity. VAR2 was only relatively weakly recognised and somewhat surpr.singly the tandem construct VAR3 was only weakly recognised as was the substitution at the fimbrial variant region 2 (VAR2), despite VAR 3 it containing the identical graft as the strongly recognised VARl in addition to the substitution at variant region 2. This data strongly indicate local stereochemical effects on antigenicity for this epitope, as is consistent with the synthetic peptide data of other workers and with for a body of data for other continuous epitopes.

The modified fimbrial subunit gene PL promoter constructions in the broad host range vector pKT240 were then purified and transformed into P.aeruginosa.

Transformants were obtained with the C-terminal construction but not with the internal substitutions, despite repeated attempts. A likely explanation for this is that the internal substitutions result in a lethal accumulation of the engineered subunit, presumably because of structural alteration of the protein preventing its normal assembly and export. In this context we note that the normal subunit, under PL promoter control, while not lethal in P.aeruginosa (which correctly processes and exports the protein) is lethal in E.coli (which lacks this ability). The only mutant fimbrial protein which was expressed in P.aeruginosa was the C-terminal addition, which was, however, associated with an impaired growth rate.

Fimbrial fractions were prepared from the viable subunit P.aeruginosa transformants. Fimbriae may normally be derived from the supernatant of cells, following mechanical blending, by either of the two independent methods of MgCl2 precipitation or isoelectric

I

precipitation (at pH 4.5) with sodium acetate. Figure shows Coomassie stain and Western-transfer profiles of the cells and derived fimbrial fractions fiom P.aeruginosa containing the pKT240/fimbrial protein VAR4. In both acetate and MgC12 fimbrial fractions there is clearly evident a protein of the expected size, which was confirmed by antibody recognition in western transfer analysis. This is a similar profile exhibited by normal fimbriate P.aeruginosa cells expressing either the normal endogenous subunit and the cloned wild-type B.nodosus subunit (see Australian Patent Application No. 50154/85), whereas when non-fimbriate cells such as E.coli express this protein there is little or no signal in the supernatant fimbrial fractions (see Figure Thus it appears that the C-terminal fusion of FMDV sequence to the B.nodosus subunit is being assembled and exported on fimbriae in the recombinant host.

DISCUSSION

The results presented herein demonstrate the feasibility of engineering the fimbrial subunit (gene) of B,nodosus to act as a carrier protein for the production and export of exogenous peptide sequences. The FMDV 144-159 VPl peptide used here represents a formidable test case because of its length and higher order structure.

The observation that this peptide, while covalently fused to the carboxy-terminus of the fimbrial subunit, is viably j expressed and exported is indicative of the general utility of the system. While there may be particular w problems with the expression of individual peptides (which may need to be assessed on a case-by-case basis), and there may be certain broad limitations on the size and nature of grafted peptides, there is a strong implication that the system is amenable to substantial manipulation, with clear potential for use with a variety of peptides.

The successful construction described herein involved 0 -16the C-termindl addition of the peptide sequence to the fimbrial subunit. Contrary to what might be expected, substitution at either one of the major variant regions within the protein failed to produce a viable outcome.

This suggests that there are serious constraints on acceptable substitutions in these regions, presumably related to effects on the overall structure of the protein, with consequential interference in the export/assembly process. There are also other limitations on such substitutions as a vehicle for peptide production, related to both the size of the substitution and its environment. The flanking of the substitution by carrier sequences increases interference problems in terms of p;:otein folding (in both directions), which may (at the least) then require removal of the peptide sequence to obtain biological activity. The same considerations and problems apply to internal additions rather than substitutions) of peptide sequences. With terminal additions, on the other hand, there are less likely to be size and structural constraints (as suggested by the present study)- Furthermore, risk of mutual interference is lower and may be further reduced by the addition of neutral linker glycine) residues or a spacer arm to the end of the protein (glycine-proline)n -random coil, or a sequence corresponding to a helical folding domain), and more convenient and practical strategies may be designed for peptide release and purification (see below).

It may prove possible to reduce to a minimum, by deletions of regions of its gene, the size of the fimbrial subunit protein and yet retain assembly and export. This would further limit the range of potential interactions between the subunit carrier and the covalently (and genetically) coupled peptide/protein, and would increase the relative molar yields of the newly expressed ;ir \n

B\

I

-17peptide/protein compared to the fimbrial subunit carrier.

It may also prove possible to utilise the natural propensity for internal cleavage of ceftain serogroups by grafting such that novel expressed sequences are proximal to the C or N terminii generated by protease cleavage of the fimbrial subunit protein. Accordingly we would envisage experimentally testing these possibilities.

In this prototype study we have expressed the sequence of an important neutralizing antigenic determinant of an economically important virus, FMDV. An immediate application of this system is therefore the development and production of an epitope-based vaccine against foot and mouth disease. The recombinant material is while clearly antigenic in an in-vivo assay is to be tested for its antigenicity and its immunogenicity in vivo. Recent studies have indicated that another VPl peptide, covering residues 200-213, plays a significant contributory role in eliciting protective immunity in cattle, and this peptide is therefore a target for analogous or (combinatorial) expression in the fimbrial system, as part of the strategy for vaccine development, as are other peptide epitopes from malaria parasites, polio viruses, hepatitis viruses).

As will be recognised by persons skilled in the art there are a wide range of possible applications and modifications of the fimbrial peptide expression system described herein. Peptides that may be amenable to production by this system include: Peptide hormones, such as luteinizing hormone releasing hormone, somatostatin, growth hormone (or fragments thereof), etc.

Pharmacologically and neurologically active peptides, such as exorphins, endorphins, peptide neurotransmitters, "toxins" and trophic factors.

Peptide epittpes, of which FMDV, malarial repeat sequences, poliovirus and hepatitis B virus and various T cell epitopes are examples. Also included may be peptide epitopes which mimic more complex conformational structures or peptides which while normally non-immunogenic may be rendered immunogenic via presentation alone or with other peptide sequences T cell epitopes) or fimbriae as the carrier species Peptide inhibitors of biological reactions, such as proteolysis, or of biological processes, such as metastasis.

Novel peptides which represent analogues of naturally occurring species, or new sequences with biological activity.

Whole proteins or folding domains of proteins. It is possible that self-contained folding domains or several intact domains (as in many proteins) may interfere less with fimbrial assembly than peptide Ssequences which are free to interact with fimbrial sequences. Many antigenic epitopes the majority of epitopes in the influenza viral haemoggutinin) are discontinuous in nature and in some such cases whole proteins or intact folding domains may be required to present such epitopes in engineered protein constructions, whilst in others it may prove possible to mimic the determinant in a relatively short peptide sequence, Examples of proteins which could be tested in the fimbrial system range from Bovine pancreatic trypsin inhibitor (a small protein) to B-galactsidase (a large protein).

A range of proteins including structural proteins, enzymes and trophic factors trypsin, interleukins, members of the growth hormone family) may prove susceptible to production via the system embodied in this patent.

-19- 7. Peptides which are either partially or completely of random generated amino-acid sequence. This approach would serve to test the scopes of'the fimbrial expression system and would also form the basis of a recombinant DNA approach to the generator/selection of novel peptide specificities.

The system may be modified in several ways. Firstly, more general and flexible cloning vector systems can be developed to improve the speed and efficiency of cloning and mutagenesis operations. One may envisage, for example, the introduction of (multiple) restriction/cloning sites at the 3' end of the fimbrial subunit gene and the construction of mo e sophisticated vectors to enable reduction in the number of steps required for the engineering and expressio of the (modified) fimbrial subunit gene. Secondly, different promoters may be used for gene expression, iicluding regulatable inducible systems which may have significant advantages in circumstances where the mutant cocstruction affects the viability of the host (which may be asy compatible type 4 fimbriate bacteria), or to suit \he particular demands of industrial production. Thirdly, the introduction of coding sequences encoding flexible liker or buffer regions between the fimbrial subunit and the grafted peptide may minimize mutual interference in expression and/or activity. In this context, it may also be desirable to include at such junctions, sequences encoding chemically or proteolytically susceptible cleavage sites, enabling subsequent separation and purification of the graft peptide. Following such cleavage the f mbrial carrier could be conveniently removed by star ard and industrially applicable precipitation procedures. The advantage of the C-terminal peptide addition is that the peptide itself should be accessible as produced or amenable to cleavage and i. 1~ 7: purification. Thus there exists a choice in presenting the peptide as a macromolecular polymer (which may for example confer immunological benefits)'or as a separate entity.

Dependent on the length and nature of allowable additions, it is also possible to envisage a construction and expression of more complex, multi-component peptide sequences with multi-dimensional function, for example, a peptide immunogen containing multiple epitopes together with appropriate signals for effective interaction with various cellular components of the immune system. These various possibilities and modifications of the system will be explored.

I;

Claims (5)

1. A method of producing a peptide comprising culturing bacteria containing a gene for a type 4 fimbrial subunit to which has been added at the C-terminal end the nucleic acid sequence encoding the peptide, and an endogenous compatible system as hereinbefore defined for the morphogenetic assembly of type 4 fimbrial subunit and/or the genes for the morphogenetic assembly of such fimbriae derived from a type 4 fimbriate species, such that mature 10 type 4 fimbriae covalently fused to the peptide are "produced as extracellular structures, harvesting the whole I" or part of the fimbriae substantially free of the host S: cells. i i 2. A method of producing a peptide as defined in claim 1 j 15 wherein the peptide is separated from the type 4 fimbriae. j: 3. A method of producing a peptide as defined in claim 1 wherein the gene encoding the fimbrial subunit of a bacteria normally producing type 4 fimbr.. is derived ee from a bacteria selected from the group comprising Bacteroides nodosus, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella nonliquefaciens and Moraxella bovis.

4. A method of producing a peptide as defined in claim 1 wherein the host cells are bacteria of the genus Pseudomonas and in particular the species P.aeruginosa preferably P.aeruginosa strain PAK/2Pfs (ATCC no. 53308). A method of producing a peptide as claimed in claim 1 wherein the gene encoding the fimbrial structural subunit to which has been added at the C-terminal end the nucleic acid encoding the peptide, is contained within a plasmid or phage.

6. A method of producing a peptide as claimed in claim 1 wherein the peptide is VP1 derived from foot and mouth disease virus strain 01-BFS.

7. An antigenic preparation for raising antibodies

22- directed against the surface protein VP1 of foot and mouth disease virus (FMDV) comprising the VP1 peptides either in isolation or in association with type 4 fimbriae, which has been produced by bacterial host cells containing a gene for a type 4 fimbrial subunit to which has been added at the C-terminal end the nucleic acid sequence encoding the protein VP1, and an endogenous compatible system as hereinbefore defined for the morphogenetic assembly of type 4 fimbrial subunit and/or those genes for the 10 morphogenetic assembly of such fimbriae derived from a type 4 fimbriate species. 8. An antigenic preparation as claimed in claim 7 wherein the VP1 peptide has optionally been cleaved from the type 4 fimbriae produced by said host cells. 9. An antigenic preparation as claimed in claim 7 wherein the gene encoding the fimbrial subunit of a bacteria normally producing type 4 fimbriae is derived from a bacteria selected from the group comprising SB.nodos s, P.aeruainosa, N.gonorrhoeae, N.meningitidis, M.non3-qjuefa-iens and M.bovis. 10. An antigenic preparation as claimed in claim 7 wherein the host cells are bacteria of the genus Pseudomonas and in particular the species is P.aeruginosa Spreferably P.aeruginosa strain PAK/2Pfs (ATCC No. 53308). 11. An antigenic preparation as claimed in claim 7 I wherein the gene encoding the fimbrial structural subunit Sto which hEs been added at the C-terminal end the nucleic acid encoding the peptide is contained wi'thin a plasmid for phage vector. 12. An antigenic preparation as claimed in claim 7 wherein the peptide is VPI derived from foot and mouth disease virus strain 01-BFS. I ~rI.c ABSTRACT PEPTIDE PRODUCTION BY PROTEIN PNGIN ERXNG The present invention relatts to a method of peptide production by protein engineering. This method involves the culturing of genetically engineered bacteria which produce as extracellular structures type 4 fimbriae, the peptide being produced in association with these fimbriae. This method is applicable to the production of virtually any peptide or peptides. The engineered bacteria contains the gene encoding the fimbrial subunit of type 4 fimbriae to which has been added at the C-terminal end the nucleic acid sequence encoding the peptide of choice, together with a system for morphogenetic assembly of type 4 fimbriae such ;dat fimbriae in association with the peptide are produced as extracellular structures. VAR! VAR2 VA, FIG.1 LEADER colv HYDR R 4 00 00 0 0 t'J WO 88/08430 Wa 8808430PCT/AU88/00 120 2/4 FIGURE 2. M K S L QK G F T AAATCTTCACAr'CTTAATAGAAATATCATGAAA.CTTTACAAAAACGTTCA 130 140 150 16C 170 186 1 E LM I V V AI I GI L A A F AlI P CCTTAATCCAA CTCATGATWTAC; TTCCAATTATCGCTATCTTACCGGC=TCC CTATCC 193 200 210 220 230 240 A Y Nl D Y I A R S QA A E G L T L A D G CTGCATATAACGACTACATCGCTCCTTCACACCAGCTCAACGCIE'AACATrIGGCTGATC 250 260 270 280 390 300 VA 1 R G D L QV CCCGTGATIACAAC L K V R I S D Ff L E S C E C K C D A 14 P CGrTAACCTTCGCA=IICTCATCACTTAC AACCCTCAATC CGCAGATGCCAACC 310 320 330 340 350 360 L A Ll K. V A R T L L RC T=rIAGCTCAAAAACTTCCTCCCACTTIA TTA CCC A 3 G S L G N41 DT 'K C K Y A L A T 1 1 C) CAGC'frCAGGATCT=ACCTAATCATCA A.AACCGTAAATACC CTCTTCCTACArAJTGATG 370 380 390 400 410 420 .VAR 2 D L Q V L A QK V A RT L CTGAm7ACAACIIACTCAAAACTTCCTCCCACrITA D Y N K D A K T A rDW-7C C K V V I T GTCATTATAATAAAGACCGAAAACTGCTGATGAAAAATtG~rC TAAAGTTGTAATCA 430 440 450 460 470 480 Y C Q G T A GCE K I1SK L I V G K K L V CTTATGCTCAAGCTACTCCAGGCACAAAAT'ICTAAGTTAATCGTTGGTAAGAAAITCC 490 500 510 520 530 540 L D Q F \V 1 C S Y K, Y N E G E T D L E L TTIACATCAA'ITC7AATCGTI'CATACAAATATAATCA.AGCCAAACTGATTI'CAAC 550 560 570 580 590 600 VAR 4 C G L R C D L QV E, A K V A R T L CCTCCITTACCC:GTGA=1ACA.ACGI-AGCTCAAAAACTTCCTCCCAC=IA K F I P N A ~K N- A_ rI'AArFATTC CGAATC CTTAAAAA CTAATAGC:TACCTCITAAATCGAAACCTCT 610 620 630 640 650 660 CTCTAGAGCCTITIT=ATITGIrCTATCA'I= SUBSTITUTE SHEE,, WO 88/08430 PCT/AU88/00 120 3/4 *V~11l 0% 8 \M %m) MM VVN\mmm \~5 M\NN ,55'5MW I'" ImmV 5'55)S. IRMAVV "W' NNM wN immN\ 'inw *w AV* A"'AN _f_ w VAR I VAR 2 VA R3 VAR 4 3 C 3 FIG. 3 SUBSTITUTE SHEET A WO 88/08430 PCT/AU88/O00120 4 /4 FIG. 4 A FM -FA C FM 4FA C FM FA C SUBSTITUTE SHEiET INTERNATIONAL SEARCH REPORT Int-fmsftonat AVOht,o Picr PCT/AU88/001 1. CLAVSWIICA lioN Of Su EJECT MATTER !I V&W*1~ str.~cIinw,.ooto 12011. o loll ACCOrding 10 11hterhotront Patent Class,.ctialon (IfAC) of I* Coltn Natonal CtaeaafrCotion and tpC Int. Cl 4 C07IK 13/00, 7/08, C12N 15/00, C07H 21/04-, C12P 19/34,21/02 (C12N 15/00, C12R 1:01) A61Y 39/385 If. FIELDS SEARCHILD MAltm~num ocumntation Seercrego C1a ee,(,calsonswoa~m Cli aeAtcatlorn Srnmoot IPC WPI, WPIL Keywords :PILl, PILUS, FIMBRIAE, FIMBRIAL Documrnirtat,oit Seatchod other Imm ininmum OoCuennotton to It's, Eatonthe sn~uchi CUMeItAl Are Included in trt* Fields Searched AU :C12N 15/00, C07K 13/00, 15/12 Chemical Abstracts :Keywords PILI, PILUS, FIMBRIAE,_ FIMBRIAL III, DOCUMENTS CONSIDERED TO 3111 RELIEVANT' Category I CAtuton of ocueont. firi indication. whira acoroorlsti, of the feini o aio, 06141 11 Miterint to CIA,-. to 1t A AU,A,50154/85 (COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION) 15 May 1986 (15.05.86) A AU,A, 52097/86 (BIOTECHNOLOGY AUSTRALIA PTY LTD) 1 July 1986 (01.07.86) A R.W. OLD and 5.8. PRIMROSE, "PRINCIPLES OF GENE MANIPULATION' 3rd Edn, PUBLISHED 1985, BY BLACKWELL SCIENTIFIC PUBLCATIONS (OXFORD) see pages 127-134. *Socal1 ceteorohea of cited docutmenti: to late( document :voliohea After the intrttslitlir Ie of afoirt aits Ad not In Ccnflict -itr, the sooi~c~loo document de(rtihQ the gonole tic of tit am ich is not cIte to 601iii,. ornif o hrfdiel I'th co riideled to oe of Omficiait ioi..ice adI AdI 4o IentofteoynA~jA eeitr docuiment but OU0iihed Oh or am*(er mthu gntOran document at aitticutit feterence; the ctiied 1nnanton (,iing do te con-at be Coimiidered noiet of cannot of comeloared to t.doCuimenlt hicm thit Ito- doubteia O irOrtv Cllirn(s) Or inv01teea o-n l taiie which1 1i Cairte to eiti ni thv puificattini date of Aoflther Y. a~u'n of elculor reterence: theg Claimed meenton ciito o thr cos~ r*&&on tao itsec-t CannOot ilidtfied tom0 fe artON nnetr elseJT1 0 wPen thel O0 docuoenotl referring to en eret disclosure. use, 41hitiion ir documenht 'I CorrOi.o ,ine rir a more other such 4OC. othet moon, eilts. Such Coihotion bosing Goiei0a to a Poison ob.iiid do CuVment Olubbhed Pilot to the mleirmilehit filing dete outl 4 h n titer them the stOr~fI dete CAimed d ocunrt mrember of the sae~i oatetit fameily ly. CEIRIICATION Di4t* of thef ACtual CoM0iotitiM Of the toteretoiehi SmirCM I Dote Ot stin iftO this Iftnetiot Searcht Itege 26 July 1988 (26.07.88) 9 tnetiaihi leChing AW1th0fitt 3ignituri of Authiriei fo AUSTRALIAN PATENT OFFICE S.D. BARKER Par.. oCtttsAio tetcendo shoot) ljii'.osel 18411 ANNEX TO TEfE INT'ERNATIONAL SEARCH REPORT ON flUERNATIONALAPPLICATION NO. PCI'/AU 88/00120 This Annex lists the ]anown publication level patent famnily members relating to the patent docume~nts cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given f~or the purpose of infonnation. Patent Document Cited in Search Patent Family Members Report AU 52097/86 CN 85109506 DK 3701/86 EP 242359 ES 8705033 Fl 863145 WO 8603410 ZA 8509297 AU 50154/85 EP 202260 NZ 214017 WO 8602557 ZA 8508374 END OF ANNX