AU693537B2 - Novel polypeptides - Google Patents

Description

I I COMMONWEALTH OF AUSTRALIA PATENTS ACT 1990 REGULATION 3.2 0 0 000 Name of Applicanti Actual Inventor/s: Address for Service: ASTRA AKTIEBOLAG TANJORE SOUNDARARAJAN BALGANESH ard CHRISTINE MARY TOWN E.F. WELLINGTON CO., Patent and Trade Mark Attorneys, 312 Kilda Road, Melbourne, 3004, Victoria, 0S 0t** 0**0 00 0* .t 0*00 0S9@ Invention Title: "NOVEL POLYPEPTIDES11 Details of Associated Provisional Applications Nos: The following statement is a full description of this inveation incl~uding the best method of perforing it knowu to Us.

-1I- I T1235-1 -l1A- TECHNICAL FIELD The present invention relates to variants of Penicillin Binding Proteins (PBP), which proteins are involved in bacterial peptidoglycan biosynthesis.

Disclosed are also DNA molecules coding for the said PBP variants, as well as vectors and cells harbouring such DNA molecules. The invention is also related to processes for assaying and designing therapeutically useful compounds which have high affinity to PBP, which processes utilize the said PBP variants.

oa BACKGROUND ART 10 Bacteria and most other unicellular organi:ns possess a cell wall, which comprises a cross-linked polysaccharide-peptide complex called peptidoglycan. Peptidoglycan biosynthesis consists of three stages: (1) synthesis of precursors (sugar nucleotides) in the cytosol, precursor transfer across the membrane and formation of the polysaccharide chain, 15 and cross-linking of individual peptidoglycan strands in the cell wall.

9 In the latter stage of peptidoglycan biosynthesis, new bonds must be made between nascent glycan strands and existing peptidoglycan. The newly synthesized chains are about 10 disaccharides long and are extended by transglycosylase enzymes to a final glycan strand of between 100 and 150 disaccharide units. The peptidoglycan is crosslinked by the action of transpeptidases which link the terminal D-ala of one glycan strand to a free e-amino group on a diaminopimelic acid residue on an adjacent region.

A number of antibiotics inhibit bacterial growth by interfering with the formation of the peptidoglycan layer. The cross-linking reaction is the Ii R 1235.1 -2target for action of two important classes of such antibiotics, the penicillins and the cephalosporins. Penicillin is thought to react irreversibly with the transpeptidase that catalyses cross-linking, The penicillin interactive proteins fall into three groups: the p-lactamases, the Low Molecular Weight-Penicillin Binding Proteins (PBPs), which mainly include the carboxypeptidases, and the High Molecular Weight- Penicillin Binding Proteins. Penicillin Binding Proteins are those enzymes which have been shown to bind radiolabelled penicillin G. In Escherichia 10 coli such proteins are called e.g. PBP 1A and PBP 1B, both belonging to the class High Molecular Weight-PBPs. PBP 1A and 18, which are known to be membrane bound proteins, maintain cell integrity and control peptidoglycan side wall extension during growth. Inactivation of either PBP 1A or PBP 1B can be tolerated by the bacteria while the deletion of 15 both the genes, designated ponA and ponB, is lethal (Yousif et al., 1985).

PBP 1B is known to be a bifunctional enzyme possessing both transpeptidase and transglycosylase activity (Ishino et al., 1980). PBP 1A is believed to be bifunctional since it can substitute for PBP 1B. The p-lactam 20 antibiotics, such as penicillin, inhibit only the transpeptidase activity of these proteins.

The transglycosylase reaction is inhibited by e.g. moenomycin, which is a phosphoglycolipid used as a growth promoter in animal nutrition and which has been shown to possess broad spectrum bactericidal activity. The enzyme transglycosylase has been shown to be present in Escherichia coli, Staphylococcus aureus, Bacillus megaterium and Bacillus subtilis. This suggests ,that interference of peptidoglycan biosynthesis by inhibition of transglycosylase could be a lethal event in all clinically important pathogens.

-3- The putative transglycosylase domain of PBP 1B has been assigned to the N-terminal 478 amino acids (Nakagawa et al., 1987). This region includes three conserved stretches of amino acids between the N-terminal half of both PBP 1A and IB and could represent residues involved in the transglycosylase activity.

Preparation of Penicillin Binding Protein 2A from Staphylococcus aureus is disclosed in EP-A-0505151.

SUMMARY OF THE INVENTION In a first aspect, the present invention provides a polypeptide which is a water-soluble active derivative of a bacterial tifunctional penicillin binding protein, said penicillin binding protein being bound to the cell membrane when expressed in a bacterial cell and being capable of exhibiting both transglycosylase and transpeptidase activities and said derivative lacking a membrane anchoring sequence but retaining the capability to exhibit one or both of said enzymic activities, wherein the derivative has an amino acid sequence which is identical to, or 20 substantially similar to, SEQ ID NO: 2, 4, 6, 12 or 13 in the Sequence Listing herein. In preferred embodiments of i: the first aspect of the invention, the polypeptide is characterized in that the bacterial cell is an Escherichia coli cell or a Streptococcus pneumoniae cell.

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In a second aspect, the present invention provides a polypeptide comprising a) a first polypeptide according to the first aspect of the present invention; and -^0 an additional polypeptide which allows binding to an affinity matrix; there being a cleavage site between said polypeptides, preferably the additional polypeptide being glutathione-Stransferase or a polypeptide substantially similar to glutathione-S-transferase, or the additional polypeptide being a polypeptide rich in histidine residues.

In a third aspect, the present invention provides an isolated and purified DNA molecule which has a nucleotide sequence coding for a polypeptide according to the first or second aspects of the present invention, preferably the nucleotide sequence being identical to, or substantially similar to, SEQ ID NO:1, 3 or 5 in the Sequence Listing herein.

In a fourth aspect, the present invention provides a replicable expression vector which carries and is capable of mediating the expression of a DNA molecule according to the third aspect of the present invention, preferably the vector being 0e 20 pARCOS58 (NCIMB No. 40666), 99 pARC0559 (NCIMB No. 40667), pARCOS12 (NCIMB No. 40665), pARC0593 (NCIMB No. 40670), DARC0392 (NCIMB No. 40659), pARC0499 (NCIMB No. 40664), or pARC0400 (NCIMB No. 40660).

In a fifth aspect, the present invention provides a cell harbouring a vector according to the fourth aspect of the present invention.

-3B- In a sixth aspect, the present invention provides a process for production of a polypeptide which is a derivative of penicillin binding protein, comprising growing a cell according to the fifth aspect of the invention in or on a culture medium for expression of the polypeptide and optionally recovering the polypeptide.

In a seventh aspect, the present invention provides a process for the production of a water soluble polypeptide according to the first aspect of the invention, which comprises culturing Escherichia coli cells harbouring an expression vector wherein a DNA coding sequence for said polypeptide is under the control of an isopropyl thiogalactoside (IPTG) inducible promoter, said culturing being carried out in the presence of a sub-optimal concentration of IPTG for induction of the said promoter and at a temperature in the range of 20 to 24°C.

In an eighth aspect, the present invention provides a method of identifying an antibody capable of binding a bacterial bifunctional penicillin binding protein which includes the step of employing a polypeptide according to the first aspect of the invention, in an antibody binding assay and selecting antibodies that bind to the 0polypeptide.

e* In a ninth aspect, the present invention provides a method of assaying for compounds which bind to a penicillin binding protein, said method comprising contacting a polypeptide according to the first or second aspect of the present invention, with a compound to be investigated; and detecting whether said compound binds to the penicillin binding protein.

I -3C- In a tenth aspect, the present invention provides a method of assaying for compounds which bind to a penicillin binding protein, said method comprising culturing cells according to the fifth aspect of the present invention; (b) lysing the said cells and isolating the crude cell extract; exposing the said cell extract to potential inhibitors of a penicillin binding protein; introducing an agent, known to bind a penicillin binding protein, to the said cell extract; removing the unbound fraction of said agent; and assaying the presence of said agent remaining in the cell extract.

In an eleventh aspect, the present invention provides a method of assaying for compounds which bind to a penicillin binding protein, said method comprising exposing a polypeptide according to the first or second aspect of the present invention, immobilised on a solid support, to a potential inhibitor of a penicillin binding protein; (b) exposing an agent, known to bind a penicillin binding protein, to the immobilised polypeptide; removing the 20 unbound fraction of said agent; and assaying the 0 presence of said agent bound to the immobilised polypeptide.

SIn a twelfth aspect, the present invention provides a method of assaying for compounds which bind to a penicillin i binding protein, said method comprising exposing a polypeptide according to the first or cecond aspect of the present invention, to a potential inhibitor of a penicillin binding protein; exposing the said polypeptide to an agent, known to bind a penicillin binding protein, which agent is immobilised on a solid support; and assaying the presence of polypeptide bound to the immobilised agent.

-3D- In a thirteenth aspect, the present invention provides a method of assaying for compounds which bind to the transglycosylase domain of a penicillin binding protein, said method comprising exposing the transglycosylase domain of a polypeptide according to the first or second aspect of the present invention, said polypeptide being immobilised on a solid support, to a potential inhibitor of the transglycosylase activity of a penicillin binding protein; exposing an agent, known to bind the transglycosylase domain of a penicillin binding protein, to the immobilised polypeptide; removing the unbound fraction of said agent; and assaying the presence of sa'd agent bound to the immobilised polypeptide.

In a fourteenth aspect, the present invention provides a method of assaying for compounds which bind to the transglycosylase domain of a penicillin binding protein, said method comprising exposing the transglycosylase S: domain of a polypeptide according to the first or second aspect of the present invention, to a potential inhibitor of a penicillin binding protein; exposing the said polypeptide to an agent, known to bind to the transglycosylase domain of a penicillin binding protein, which agent is immobilised on a solid support; and (c) assaying the presence of polypeptide bound to the :0::25 immobilised agent.

0e In preferred embodiments of the tenth to fourteenth aspects of the present invention, the method is characterized in that the agent known to bind a penicillin binding protein is a monoclonal antibody, or the agent known to bind a penicillin binding protein, is a labelled antibiotic compound.

NTO

-3E- In a fifteenth aspect, the present invention provides a method of determining the protein structure of a penicillin binding protein, characterized in that a polypeptide according to the first aspect of the present invention is utilized in X-ray crystallography.

DESCRIPTION OF THE INVENTION There is a growing number of reports of bacteria which are resistant to antibiotics. There is consequently a need for new compounds which inhibit bacterial growth by means of binding Penicillin Binding Proteins. The present invention provides PBP variants which facilitate processes for assaying and designing therapeutically useful compounds which have high affinity to PBPs.

.615 00 0, 04 *000 %0 0 *0* 0 00 *0 0 0 Accordingly, it is an object of the invention to provide polypeptides which are water-soluble active derivatives of bacterial bifunctional Penicillin Binding Proteins, said Penicillin Binding Proteins being bound to the cell membrane when expressed in a bacterial cell and being capable of exhibiting both transglycosylase and transpeptidase activities and said derivatives lacking a membrane anchoring sequence but retaining the capability to exhibit one or both of said enzymic activities. The "bacterial cell" mentioned above is preferably an Eschenrchin coli cell or a Streptococcus pnenmoniae cell.

0 The soluble PBP variants according to the invention retains transglycosylase activity, indicating that soluble variants of PB3, devoid of membrane anchoring sequences, can recognize lipid linked substrate and polymerise the disaccharide into repeating units. It can thus be assumed

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0ib 1 R 1235-1 -4that other analogues of PBP lacking residues involved in membrane attachment would be enzymatically functional.

Molecules interacting with the penicillin interactive region of soluble PBP variants could be assumed to be capable of interacting identically with wild-type PBPs. Consequently, the soluble PBP variants according to the invention can be used for identifying compounds which are interacting with wild-type Penicillin Binding Proteins.

10 It is furthermore well known that membrane-bound proteins are very difficult to crystallize. The soluble enzymatically active PBP variants can be used for crystallisation and will thereby facilitate a rational design, based on X-ray crystallography, of therapeutic compounds inhibiting High Molecular Weight-PBPs.

A further object of the invention is to provide polypeptides which are truncated water-soluble derivatives of bacterial bifunctional Penicillin Binding Proteins, said Penicillin Binding Proteins being bound to the cell membrane when expressed in a bacterial cell and being capable of 20 exhibiting both transglycosylase and transpeptidase activities and said derivatives lacking the membrane anchoring sequence but retaining the capability to exhibit the transglycosylase activity. The "bacterial cell" mentioned above is preferably an Escherichia coli cell.

Alignment of amino acid sequences of High Molecular Weight-Penicillin Binding Proteins, and the compilation of the motifs involved in the penicillin binding of p-lactamases and carboxypeptidase, have suggested the C-terminal half of PBP 1A and 1B to be the functional domain of the transpeptidase activity and includes the penicillin binding domain. In addition, Nakagawa et al. (1987) showed that a truncated ponB gene encoding the N-terminal 478 amino acids of PBP 1B is capable of the transglycosylase reaction.

On the basis of these findings, it has been suggested that the high molecular weight PBP 1A and 1B proteins are two domain-proteins, with the N-terminal half forming the transglycosylase domain and the C-terminal half the transpeptidase domain. The two domains have been predicted by computer analysis to be joined by a linker or hinge region which does nr-t structurally or enzymatically contribute to the function of the protein. The linker region of E.coli PBP 1B has been predicted to be from position 545-559 while that for E.coli PBP 1A around position 501.

The monofunctional truncated variants of PBP according to the invention will, when used in x-ray crystallography, facilitate obtaining structural information of the transglycosylase domain of penicillin binding proteins.

In addition, the reduced size of the monofunctional variant will facilitate crystallization.

The observation that deletion of the ponA and ponB genes is lethal (Yousif 15 et al., 1985) does not address the question of essentiality of the transglycosylase activity of the encoded PBP 1A proteins, since the deletion results in the loss of both transglycosylation and transpeptidation activities.

In addition, this experiment does not address the possibility that the transglycosylase enzyme activity can be contributed by a Penicillin Binding 20 Protein other than PBP 1A or PBP 1B. It is also possible that hitherto undescribed Penicillin Binding Proteins and/or other proteins that contribute to the transglycosylase activity exist.

0 Alignment c' the anino acids forming the putative transglycosylase domain of PBP 1A and 1B reveals three stretches of 9 out of 12 (Region 1), 9/10 (Region 2) and 8/10 (Region 3) amino acids identical within the NC T 0S

A'

s~: 11235.1 -6- N-terminal half of these two proteins (Broome-Smith et al., 1985) (Fig. 14).

The same 3 regions are identically conserved among two other recently described protein sequences; Streptococcus pneumoniae PBP 1A (Martin et al., 1992) and a 94 kDa protein from Haernophilus influenzae (Tomb et al., 1991). The conservation of these residues in such diverse species suggests their critical requirement in either maintaining structural aspects of the protein, or in the transglycosylation reaction itself.

The overlapping functional transglycosylase and transpeptidase activities of S 10 PBP 1A and 18 also suggests conservation of the catalytic centres and hat molecules designed to interact with the catalytic centre of PBP 1A would be reactive also with PBP 1B.

The functional transglycosylase activity of the expressed protein can be 15 studied either in a direct in vitro assay using appropriate substrates, or in an assay measuring the ability of the protein to complement the deletion of the corresponding genes in the chromosome. It has been shown that a plasmid with a gene encoding the wild type product (PBP 1A or PBP 1B) is capable of maintaining the viability of the E.coli cell (Yousif et al., 1985).

20 This trans-complementation technique can be utilized to assess the functional nature of the mutant gene(s) encoding PBPs with mutations inactivating one of the enzymic (transglycosylation or transpeptidation) functions. The ability of such mutant products to complement in trans the deletion of the chromosomal ponA and ponB genes would define the essential requirement of the individual e, yrnic functions.

There is consequently a need for research tools which will make it possible to study the effects of specific inactivation of the transglycosylase activity of Penicillin Binding Proteins.

Consequently, a further aspect of the invention is a polypeptide which is a transglycosylase deficient derivative of a bacterial bifunctional penicillin binding protein, said penicillin binding protein being bound to t-ie cell membrane when expressed in a bacterial cell and being capable of exhibiting bcth transglycosylase and transpeptidase activities and said derivative lacking the capability to exhibit transglycosylase activity but retaining the capability to exhibit transpeptidase activity. The "bacterial cell" mentioned above is preferably an Escherichia coli cell.

The transglycosylase deficient PBP variants can advantageously be used in X-ray crystallography for the purpose of obtaining structural information of the activity sites of PBPs. Structural analysis of crystal form of soluble transglycosylase deficient PBP variants could allow delineation of the catalytic region and facilitate the design of molecules capable of specifically inhibiting the transglycosylase activity.

In a preferred form, the transglycosylase deficient polypeptide according to invention is a polypeptide which is lacking transglycosylase activity 15 because of a mutation or deletion in the second conserved region of the gene coding for said polvpeptide.

The conventional purification procedure employed for the enrichment of penicillin binding proteins has been the use of a "penicillin" affinity. The binding of the protein to penicillin is covalent and requires harsh conditions to elute the bound protein. This may lead to a certain degree of inactivation of the enzymic activity of the protein. There is consequently a need for alternate affinihy matrices for the efficient purification of the proteins.

NfLT RAO F 1235.1 -8- Included in the invention is consequently a polypeptide comprising a first polypeptide which is a PBP variant according to the invention; and (b) an additional polypeptide which allows binding to an affinity matrix; there being a cleavage site between said polypeptides.

The "additional polypeptide" mentioned above can preferably be glutathione-S-transferase or a polypeptide substantially similar to glutathione-S-transferase. Such an additional polypeptide will enable rapid purification of the protein using Glutathione Sepharose® affinity matrix. In 10 another preferred form, the additional polypeptide is a polypeptide rich in histidine residues, which residues will confer on the protein the ability to bind to an Ni affinity column. The additional polypeptide can be fused S. either to the N-terminus or the C-terminus of the soluble/membrane bound PBP.

The ability of the fusion proteins to bind to an affinity matrix allows immobilisation of the protein. Such immobilised proteins can be used for analysis of competitive binding of different ligands to the bound active protein, and thus for screening of compounds binding to the enzymic 20 domain of interest.

The polypeptides according to the invention are not to be limited strictly to any one of the sequences shown in the Sequence Listing. Rather the invention encompasses polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the biochemical activities of the PBP variants which amino acid sequence is disclosed in the Sequence Listing.

Included in the invention are consequently also polypeptides, the amino acid sequence of which is at least 90% homologous, preferably at least homologous, with the amino acid sequence of any of the PBP variants according to the invention.

A further object of the 'Invention is to provide isolated and purified DNA molecules which have nucleo tide sequences coding for any one of the PBP variants according to the invention.

In a preferred form of the invention, the said DNA molecules have nucleotide sequences identical to SEQ ID NO: 1, 3 or 5 in the Sequence Listing. However, the DNA molecules according to the invention are not to be limited strictly to any of the sequences shown in the Sequence Listing.

Rather the invention encompasses DNA molecules carrying modifications 0 lik~e substitutions, small deletions, insertions or inversions, which nevertheless encode proteins having substantially the biochemical activities of the PBP variants according to the invention.

Included in the invention is also a DNA molecule whicrh nucleo tide 15 sequence is degenerate, because of the genetic code, to the said nucleotide "Oo~o sequence coding for a PBP variant according to the invent-ion. The natural degeneracy of the genetic code is well known in the art, It will thus be appreciated that the DNA sequences shown in the Sequence Listing are 06 99*9"9..Sequence Listing.

A further aspect of the invention is a replicable expression vector which carries and is capable of mediating the expression of a DNA molecule according to the invention. In the present context the term "replicable" means that the vector is able to replicate in a given type of host cell into which is has been introduced. Examples of vectors are viruses such as bacteriophages, cosmidds, plasmids and other recombination vectors.

Nucleic acid molecules are inserted into vector genornes by methods well known in the art. A vector according to the invention can preferably be one of the plasmids listed in Table I. below.

S R'235-1 Included in the invention is also a host cell harbouring a vector according to the invention. Such a host cell can be a prokaryotic cell, a unicellular eukaryotic cell or a cell derived from a multicellular organism. The host cell can thus e.g. be a bacterial, yeast or mammalian cell. The methods employed to effect introduction of the vector into the host cell are wellknown to a person familiar with recombinant DNA methods.

A further aspect of the invention is a process for production of a polypeptide which is a derivative of penicillin binding protein, comprising ts 10 growing a host cell according to the invention in or on a culture medium for expression of the polypeptide and optionally recovering the polypeptide, An appropriate host cell may be any of the cell types 0*e mentioned above, and the medium used to grow the cells may be any conventional medium suitable for the purpose.

The High Molecular Weight-Penicillin Binding Proteins have been shown to be anchored to the membrane, but the majority of the protein is within the periplasmic space of the cell (Edelman et al. 1987). Thus PBP derivatives, devoid of the membrane signal anchoring sequences, are 20 forced to fold into their native state in a heterologous environment, namely 66 the cytosol. This often leads to misfolding, and the majority of the expresse protein aggregates into an inactive form referred to as inclusion bodies.

It has now surp:'singly been found that high yields of an active watersoluble PBP variant can be obtained by regulated transcription of the gene encoding the said PBP variant. Such regulated transcription involves (i) using a suboptimal concentration of the inducer isopropyl thiogalactoside (IPTG); and (ii) culturing the cells expressing the PBP variant at reduced temperature. A cumulative effect of these factors contributes to the overall recovery of the active soluble protein. Consequently, lower rates of expression, achieved through the mentioned combination of sub-optimal 0l'1235.1 -11de-repression of promoter systems and (ii) increased generation time by lowering of the temperature of cultivation, will enhance the solubility of proteins lacking the membrane anchoring segment.

A further important aspect of the invention is a process for the production of a water soluble polypeptide according to the invention which comprises culturing Escherichia coli cells harbouring an expression vector wherein a DNA coding sequence for said polypeptide is under the control of an isopropyl thiogalactoside (IPTG) inducible promoter, said culturing being 10 carried out in the presence of a sub-optimal concentration of IPTG for induction of the said promoter and at a temperature in the range of 20 to *24 0 C, preferably 22 C. The concentration of IPTG can preferably be e approximately 0.01 mM.

15 In the case of expression of ponAdel23, a gene encoding a PBP variant according to the invention, such regulated transcription by controlled de-repression of the T7 promoter by using sub-optimal concentration of the inducer IPTG and (ii) reducing the growth rate by culturing at 22 0

C,

resulted in yields of the active protein which reached nearly 50% of the 20 total induced protein of interest. The growth and induction conditions were critical for the efficient recovery of the soluble protein, as growth at higher temperatures or induction with higher concentrations of IPTG resulted in the majority of the protein becoming inactive and forming inclusion bodies.

I will be appreciated that this method for controlled expression is applicable to other inducible promoter systems, e.g. the tac system, where the inducer is IPTG and the host is a lac Y negative host.

A route to obtain relevant structural information on the active site configuration of an enzyme is the production and characterisation of monoclonal antibodies capable of inhibiting the enzymic reaction. The I Rr235.1 -12antibodies inhibiting the activity represent molecules which block or compete with the substrate for entry into the active site pocket, or can represent molecules which can prevent structural transitions required for catalytic activity. In either case, these antibodies can be used as a tool to quantitate interaction of the target enzyme with binding of radiolabelled inhibitory compounds to judge the affinity of interaction provided the affinity of the inhibiting antibody is known. A further use of mapping the epitopes recognised by the inhibitory antibodies is the ability to delineate residues forming the active site.

Consequently, a further aspect of the invention is a method of identifying an antibody capable of binding a bacterial bifunctional penicillin binding *00.

protein which includes the step of employing a polypeptide according to S.the invention in an antibody binding assay and selecting antibodies that 15 bind to the polypeptide.

oo Also included in the invention are monoclonal antibodies directed to a PBP variant according to the invention. Such a monoclonal antibody is prepared using known hybridoma technology by fusing antibody-producing B-cells 20 from immunized animals with myeloma cells and selecting the resulting hybridoma cell line producing the desired antibody.

Another aspect of the invention is a method of assaying for compounds which bind to a penicillin binding protein, said method comprising (a) Lontacting a polypeptide which is a PBP variant according to the invention with a compound to be investigated; and detecting whether said compound binds to the said PBP variant.

For example, a method of assaying for compounds which bind to a penicillin binding protein can comprise culturing host cells according to the invention; lysing the said cells and isolating the crude cell extract; exposing the said cell extract to potential inhibitors of a penicillin _.2 i R'1235.1 -13binding protein; introducing an agent, known to bind a penicillin binding protein, to the said cell extract; removing the unbound fraction of said agent; and assaying the presence of said agent remaining in the cell extract.

Another method of assaying for compounds which bind to a penicillin binding protein could comprise exposing a polypeptide which is a PBP variant according to the invention, immobilised on a solid support, to a potential inhibitor of a penicillin binding protein; exposing an agent, 10 known to bind a penicillin binding protein, to the immobilised polypeptide; removing the unbound fraction of said agent; and (d) assaying the presence of said agent bound to the immobilised polypeptide.

*e S46 In a preferred form, the said method is a method of assaying for 15 compounds which bind to the transglycosylase domain of a penicillin binding protein, said method comprising exposing the transglycosylase domain of a polypeptide according to the invention, with the proviso that the polypeptide is not a transglycosylase deficient PBP variant, said polypeptide being immobilised on a solid support, to a potential inhibitor 20 of the transglycosylase activity of a penicillin binding protein; exposing an agent, known to bind the transglycosylase domain of a penicillin binding protein, to the immobilised polypeptide; removing the unbound fraction of said agent; and assaying Ole presence of said agent bound to the immobilised polypeptide.

Antibodies specific for transpeptidase can be immobilised on a BIAcore sensor chip surface. The BIAcore system, wherein "BIA" stands for "Biospecific Interaction Analysis", is available from Pharmacia Biosensor, Sweden. Protein binding to the immobilised antibody is detected by the output RU-signal. Screening for TP inhibitors will be possible by a competitive assay wherein soluble protein is preincubated with test compounds. Binding of a test compound to the protein will result in a S R'1235.1 -14decrease in protein binding to TP specific antibody. In the same way, monoclonal antibodies specific for transglycosylase can be used in screening for TG inhibitors.

In a similar way, ampicillin or modified moenomycin can be coupled to the surface and used in an indirect competitive assay whereby protein is preincubated with test ligand prior to introduction in the BIAcore.

Consequently, yet another method of assaying for compounds which bind to a penicillin binding protein could comprise exposing a polypeptide which is a PBP variant according to the invention to a potential inhibitor of a penicillin binding protein; exposing the polypeptide to an agent, '*known to bind a penicillin binding protein, which agent is immobilised on a solid support; and assaying the presence of polypeptide bound to the S. 15 immobilised agent.

In a preferred form, the said method is a method of assaying for S compounds which bind to the transglycosylase domain of a penicillin binding protein, said method comprising exposing the transglycosylase 20 domain of a polypeptide according to the invention, with the proviso that the polypeptide is not a transglycosylase deficient PBP variant, to a potential inhibitor of a penicillin binding protein; exposing the said polypeptide to an agent, known to bind to the transglycosylase domain of a penicillin binding protein, which agent is immobilised on a solid support; and assaying the presence of polypeptide bound to the inunobilised agent.

The "agent known to bind a penicillin binding protein" referred to above can e.g. be a monoclonal antibody or a labelled antibiotic compound such as [3Hllampicillin.

A further aspect of the invention is a method of determining the protein structure of a penicillin binding protein, characterized in that a polypeptide which is a PBP variant according to the invention is utilized in X-ray crystallography.

Some of the features of the preferred PBP variants according to the invention are summarised in Table 1 below. The plasmids listed in the Table have been deposited under the Budapest Treaty at the National Collection of Industrial and Marine Bacteria Limited (NCIMB), Aberdeen, 10 Scotland, UK. The date of deposit is 28 June 1994.

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R 1235-1 -16- TABLE 1 Example Features Plasmid Deposit Fig. SEQ no. no. ID NO: (pARC) (NCIMB) Soluble variants E.coli PBP 1A with at 1-23 deleted 6058 40666 o 00 *D 0 0S 9b *s

S

I

*000 ft *0 9 *r I *0 99 0 9

I

0*09o

SO

4. 4

S

2.1 E.coli PBP 1B with aa 65-87 deleted 0559 40667 9 3, 4 3.1 eS.pn notiae PBP IA with aa 1-38 0512 40665 12 5,6 deleted Transglycosylase deficient variants 4.1 E.coli PBP lB with glutamines 270- 0438 40661 7 271 substituted to alanines E.coli PBP 1B with glutarnines 270- 0468 40662 8 271 substituted to Icucines Ecoli PBP 1B with aa 264-271 0469 40663 9 deleted 4.2 E.coli PBP IA with glutamines 123- 0571 40668 19 124 substituted to alanincs Truncated variants 5.1 aa 1-553 of Exorli PP 1B 0592 4066i9 21 11 aa 1-553 of E-evU P1W 11B with na 0593 40670 22 12 65-87 deleted 5.2 na 21068 of PEcoli PW 113 0392 40659 23 13 Fusion proteins 6.1 Excoli P1BP 1A with 23 la deletion, ligated to glutathione-9transferase 0499 F404 E.coli PBP IA with 23 ai deletion, ligtated to histidin? strtth 000 40660 R'123 5 1 -17- EXAMPLES OF THE INVENTION In the following examples, the terms "standard protocols" and "standard procedures" are to be understood as protocols and procedures found in an ordinary laboratory manual such as the one by Sambrook, Fritsch and Maniatis (1989).

EXAMPLE 1 1.1. Construction of gene encoding soluble form of E.coli PBP 1A The possible amino acid residues involved in the membrane anchoring region of PBP 1A was deduced following the computer program described 15 by Kyte Dolittle (1982). The predicted hydrophobicity of the N-terminal 60 amino acid is shown in Fig. 1. Based on this hydrophobicity profile, it was predicted that the N-terminal 23 amino acids were strongly implicated to contribute to the membrane anchoring domain of the protein, but may not entirely encompass the membrane anchoring domain. This region was 20 then putatively designated as the region involved in "membrane anchoring".

The plasmid p8S98, harbouring the native ponA gene (encoding wild type PBP 1A), was obtained from Prof. B.S. Spratt, Microbial Genetics Group, School of Biological Sciences, University of Sussex, Brighton, UK. The construction of pBS98 is described in Broome-Smith et al. (1985). Plasmid DNA from cells harbouring pBS98 was made following standard protocols.

Oligonucleotide primers for use in the polymerase chain reaction (PCR) were synthesized in Applied Biosystems Model 380 A. The oligonucleotide primer used was TG-82: S R'1235 1 -18- NcoI ACC ATG GGC CTA TAC CGC TAC ATC G-3' M G L Y R Y I 23 24 25 26 27 28 29 (Amino acid No.) TG-82 incorporates the following characteristics: it allows construction of mutant ponA gene whose encoded product would have the 24th amino acid (glycine) of the wild type PBP 1A as the second amino acid of the expressed mutant protein; and it introduces DNA sequences recognized by the restriction enzyme Ncol. This introduces the codon ATG which corresponds to the first amino acid of the mutant PBP 1A when expressed in suitable systems.

15 The 3'-oligonucleotide primer used was TG-64: 5'-CGC GGA TCC GAA TCA CAA CAA TTC CTG TGC-3'

TT

BamHI TG-64 has the following characteristics: it introduces a termination .e codon following the 850th amino acid of the structural protein of PBP lA; it introduces a site for the restriction enzyme BanHI to facilitate cloning into suitable expression vectors.

Using these pr! PCR was carried out using pBS98 DNA as template following stand .irutocols. A DNA fragment of approximately 2.5 kb was amplified. The fragment was digested with the restriction enzyme Ncol followed by digestion with BamHI. This 2.5 kb NcoI BamHI DNA fragment was then ligated to the vector pBR329 (Covarrubias et al., 1982) previously cut with NcoI and BamHI. Ligation of the two DNA fragments were carried out using standard protocols and the ligation mixture transformed into Ecoi DH 5a. The transformed cells were plated on LB agar plates with 50 pg/ml ampicillin. Following overnight incubation at 371C, individual ampicillin resistant colonies were tested for their R1235.1 -19tetracycline sensitivity as insertion into the Nco[ BamMl region renders the plasmid chioramphenicol and tetracycline sensitive. A recombinant plasrnd bearing the 2.5 kb insert was designated pARCO488.

The NcoI BamM-l 2.5 kb DNA fragment was released from pARC0488 and ligated to NcoI BatnHI cleaved and purified pARC038 (Fig. The plasmidd pARCO38 is a derivative of pETld (Studier et 1990) in which the EcoRI and PstI sites were made blunt ended with T4 exonuclease and the EcoRI Pstl 0.75 kb DNA fragment replaced with a blunt ended 10 kanamydn resistance cartridge (Pharmacia Biochemicals). The ligation mnixture was transform~ed into competent cells of E~coli BL 26 (DE3) The transformation mix was plated on LB agar with 50 pig/n-I kanamycin.

Mini-prep plasn-dd DNA was made from several kanamycin resistant colonies and screened by r'estriction endonuclease mapping using standard 15 procedures.

of the colonies harbouring plasmid with expected structure (Fig. 3) was labelled pARC0558 (NCIMB 40666). The DNA sequence of the mutant potiA gene labelled as ponAdel23 is shown as SEQ iD) NO., 1. The amino 20 acid sequence of the soluble P'BP 1Adel23 is shown as 0SErQ ID NO: 2.

Goo& 1.2. Expression of pottdel23 E.coli BL 26 (D83) cells (obtained from Dr. J.J. Duntn, Biology Dept., Brookhaven National Lab., Long Island. NY, USA) harbouring pARCO558 were grown in LB with 50 pig/mI kanainycin till an 0.D3, at 600 nm of 0.6 and induced with 0.01 mMI isopropyl thiogalactoside (IPTG) for 6 hours.

Following 6 hours of induction, cells were harvested and broken by passing through -a French press. After centrifugation at low speed to ietinove unbroken cells and debris, the cytosolic (soluble) fraction was obtained by either of the following two methods: following a procedure R'1235.1 described Page et al. (1982) in which the pellet, membrane and soluble proteins are separated by sucrose gradient centrifugation; or by spinning the obtained supernatant at 200,000 x g for 90 minutes, whereafter the supernatant obtained is taken as the cytosollc soluble protein fraction.

1.3. Penicillin binding of expressed PBP lAdel23 The obtained cytosolic fraction was tested for the presence of mutant PBP 1A Ly following the method of Rojo et al. (1984). This procedure involves using 125 1)cepluhradine as the labelled penicillin as it is specific for PBP 1A.

'Mutant PBP lAdel23 capable of binding the labelled cephradine could be demonstrated in the cytosolic fraction. Approximately 50% of the expressed .00 mutant protein fractionated as a soluble protein, while the remaining .0.

0 15 fractionated into the inclusion body and/or into the membrane associated 0 00 fractions, Consequently, enhanced levels of active mutant PBP lAdel23 004 4 were obtained since the cells were induced with sub-optimal concentration of IPrnG and the since cultuies were grown at 22°C. The penicillin binding profile of the soluble PBP lAdel23 is shown in Fig. 4.

S: 1.4. Purification of soluble PBP lAdel23 ,boo* 0 9. The cell pellet of E.coli BL26 (DE3) pARC0558 obtained following 6 hours of induction at 22°C was washed twice with buffer A (30 mM Tris-Cl, pH 8.0; 10 mM EDTA; 10 pg/ml leupeptin; 10 pg/ml aprotinin; 5 mM DTT) and resuspended in the same buffer. The cell suspension was passed through a French press at 1200 psi. The lysate was spun at 10,000 rpm for minutes and the obtained supernatant centrifuged at 200,000 x g for minutes. The obtained supernatant was then adjusted to 30% saturation with ammonium sulphate. The mixture was centrifuged at 12,000 rpm for min atd the pellet resuspended in buffer A containing 1 M NaCI. The dissolved pellet was then treated with Cephradine-Affigel 10 matrix.

R f235.1 -21- Cephradine was conjugated to Affigel 10 following the instructions of the manufacturers (Biorad Laboratories, USi). The soluble PBP 1Adel23 containing fraction, dissolved in buffer A containing 1 M NaCI, was incubated 16 hrs at 4 0 C. with cephradine-affigel 10 beads. The beads were then washed with Buffer A containing 1 M NaCl until the absorbance at 280 nm was nearly zero. Elution of PBP 1Adel23 was monitored by assaying for penicillin binding activity in the wash. This activity was measured using 125 I]cephradine prepared as described in Rojo et al.

(1984). Bound PBP 1Adel23 was eluted from the beads using 1 M hydroxylamine (pH 8.5) at 25 0 C for 120 minutes. This fraction was concentrated by ultrafiltration using YM 30 filters (Amicon, USA) in Buffer A with 0.25 M NaCI. The ultrafiltration also resulted in the removal of hydroxylamine. The purified fraction containing >85% of the protein species corresponding to PBP 1Adel23 showed both penicillin binding and transglycosylase enzyme activities. The protein profile as seen by Coomassie Brilliant Blue staining and the 1 25 Ilcephradine penicillin binding profile of the different fractions, obtained during the various stages of purification, are shown iit Fig. 5. The N-terminal amino acid sequence of the soluble PBP 1Adel23 was confirmed by sequencing the 20 purified protein.

1.5. Transglycosylase activity of soluble PBP 1Adel23 The transglycosylase activity of the soluble PBP 1Adel23 protein was measured using essentially the method described by Ishino et al. (1980).

The substrate for the detection of the enzymic activity were essentially prepared and purified following the protocols described by leijenoort et al. (1992). The concentration dependent transglycosylase activity of PBP 1Adel23 measured as the amount of peptidoglycan formed was compared to the amounts of peptidoglycan formed by different concentrations of the membrane bound form of native PBP IA. As seen in Fig. 6, the peptidoglycan polymerizing efficiency of the mutant soluble PBP 1Adel23 R 123G51 -22was nearly identical to the enzymic activity of the membrane bound form of the protein.

It has consequently been found that the elimination of the 23 amino acid residue stretch does not interfere with the ability of the protein to assume its native structure capable of both the enzymatic activities, i.e. the transglycosylase and the transpeptidase activities.

EXAMPLE 2 2.1. Construction of gene encoding soluble form of Ecoli PBP 1B e* e :I The ponB gene encoding PBP 1B was obtained on a plasmid pBS96 from Prof. B.S. Spratt, Microbial Genetics Group, School of Biological Sciences, 15 University of Sussex, Brighton, UK. The construction of pBS96, as well as the nucleotide sequence of the wild-type ponB gene and the derived amino acid sequence, are described in Broome-Smith et al. (1985).

The hydropathy plot of the N-terminal approximately 150 amino acids as S 20 derived using the method of Kyte and Doolittle (1982) is shown in Fig. 7.

.Analysis of the hydropathicity plot indicated that the amino acids at positions 65 to 87 of the PBP 1B sequence contributed largely to the hydrophobicity of the N-terminus and can be putatively assigned to be the membrane anchoring domain of the protein. In addition, P-lactamase studies of Edelman et al. (1987) had indicated that amino acids C-terminal to amino acid position 87 were present in the periplasmic space of the Ecoli cell and that amino acids N-terminal to position 65 of PBP 1B were within the cytoplasm of the cell.

The strategy employed to construct a mutant ponB gene encoding a soluble form of PBP 1B is shown in Fig. 8. Initially a DNA fragment of approximately 200 bp of the 5'-end of the ponB gene was amplified by R' 12351 -23- PCR, from the ponB gene on the plasmid pBS96 (Broome-Smith et al., 1985). The oligonucleotide primers used were 5'-primer TG-77 AAA CCA TGG CCG GGA ATG ACC-3') which includes a NcoI restriction enzyme site which also coincides with the start ATG codon of the sequence, and 3'-primer TG-84 (5'-AAG TCG CGA GCC GCG TIT GCC AC-3') which includes a site for the restriction enzyme NrnI and encodes for amino acids corresponding to position 64 of the PBP 1B sequence.

Step 1: The PCR amplified fragment following restriction with the enzymes NcoI and Nrui was cloned into the NcoI NrIl sites of the cloning vector S. pBR 329 (Covarrubias et al., 1982). Ligation, transformation and screening were carried out using standard protocols and the recombinant plasmid with the expected structure labelled pARC0547 (Fig. 8) was obtained.

15 Another DNA fragment of approximately 1.2 kb was amplified by PCR using primer sequences corresponding to amino acid 87 to 480. This DNA fragment encodes the C-terminal half of the TG domain of PBP 1B, The primers used were 5'-primer TG-79 (5'-CGG ATA TCG ATC AAA AAA TTC GTA GCC which included the nucleotide sequence for the O 20 cleavage site for the restriction enzyme EcoRV, and 3'-primer (5'-GCG GAT CCT TAG TCG ACG ACC ACA ATC GCA which included the sequence for BamHI cleavage.

Step 2: The PCR amplification of this fragment was done using the ponB gene on pBS96 (Broome-Smith et al., 1985) DNA as template. The amplified fragment was cloned into the EcoRV BamHI sites of pBR 329 (Covarrubias et al., 1982) using standard protocols. The recombinant plasmid obtained was labelled pARC0534 (Fig. 8).

Step 3: The 200 bp Ncol NruI fragment cloned in pARC0547 was excised as a Ncol Nrul fragment and cloned into Ncol EcoRV cleaved pARC0534 to obtain pARC0551 (Fig. 8).

S R'12351 -24- The mutant ponB gene on pARC0551 has DNA sequences coding for the N-terminal 64 amino acids of PBP 1B fused to the nucleotide sequences encoding the amino acids 88 to 480. A 1.3 kb PstI BamHI DNA fragment of pBS96 was then ligated to PstI BamHI cleaved pARC0551 and the ligation mixture transformed into E.coli DH5ca using standard procedures.

Individual transformants were then screened and colonies harbouring recombinant plasmid with the expected structure identified. The plasmid was labelled pARC0552. A Ncol BamHI fragment from pARC0552 encompassing the entire mutant ponB gene was then excised and ligated to the T7 expression vector pARCO38 to obtain pARC0559 (NCIMB 40667; Fig.

9).

The 3'-end of the cloned fragment of Step 1 has the nucleotide sequence TCG (partial NruI site sequence) while the 5'-end of the fragment cloned in 15 Step 2 has the sequence ATC (partial EcoRV cleavage sequence). The S. junction nucleotide sequence which is the outcome of the fusion of TCG and ATC results in the introduction of the codons for serine and isoleucine. Thus the mutant ponB gene encodes a PBP 1B with the amino acid sequence 1 to 64 corresponding to the wild type PBP 1B fused to the 20 sequence 87 to 844. The two stretches are joined by the amino acids serine and isoleucine, *oo# 6 The nucleotide sequence of the mutant ponB gene is shown as SEQ ID NO: 3 and the derived amino acid as SEQ ID NO: 4.

2.2. Expression of soluble PBP 1B The plasmid DNA of pARC0559 was transformed into the T7 expression host E.coli BL 26 (DE3) and the restriction map profile of the transformed plasmid confirmed using standard procedures. E.coli BL26 (DE3)/pARC0559 were grown at 22CC and induced with 0.01 mM IPTG and the cells allowed to grow for 6 hours. Cells were then harvested and broken by passage through a french press. The lysate was centrifuged at 10,000 rpm for 10 minutes and the supernatant obtained was centrifuged at 200,000 x g for 45 minutes in a Beckman ultracentrifuge.

2.3. Characterization of the expressed soluble PBP 1B The obtained supernatant, i.e. the cytosolic soluble fraction, was tested for the presence of the mutant PBP 1B using 125 1lampicillin as the radio-ligand. The 125 llampicillin was prepared as described by Rojo et al.

(1984) for the preparation of [1 25 1]cephradine. The mutant PBP lB was detected in the soluble fraction and bound radioactive ampicillin.

*o A Soluble PBP 18 could also be purified using Ampicillin Affigel beads by a procedure analogous to the one described in Section 1.4. The protein 1 15 profile of the different fractions seen by Coomassie Blue staining and the Ott. binding of 125 qIja= i illin of the enriched PBF 1B fraction is shown in Fig.

The purified protein was enzymatically active in the peptidoglycan *Ott' 20 transglycosylase assay (Heijenoort et al., 1992) and bound penicillin with -an affinity comparable to that of the membrane bound native PBP 1B.

E~XAMPLE 3 3.1. Construction of gene encoding soluble form of Streptocccis ptieurntotite PBP 1A The molecular architecture of the S.pneimiotiiae I'BPI' A is predicted to be simnilar to that of Exo1i PBP 1A and PBP 1B3 protein in the fact that the protein is anchored to the memnbranie via a N-torminal membrane -Anchoring sequence. The nuclooti'ie sequence of the gene encodinig native niembraire bound Sqj iewtnae PBP 1A and its derived amino acid R 1235.1 -26sequence are described in Martin et al., (1992). The hydropathicity profile of the N-terminal 100 amino acids as derived by the Kyte and Doolittle plot is shown in Fig. 11. A stretch of 38 amino acids contributed significantly to the hydrophobicity of this region and was assumed to be the membrane interacting domain. A mutant gene of S.pneumoniae PBP 1A was constructed by deleting the nucleotide sequence coding for the N-terminal 38 amino acids of S.pneumoniae PBP 1A.

Using standard PCR protocols, sequences encoding the wild type S.pneumoniae PBP 1A gene was amplified as a 2.5 kb DNA fragment from the chromosome of S.pneumoniae strain PM1 (obtained from S.A. Lacks, S. Biology Department, Brookhaven National Laboratory, Upton, New York, USA) (Lacks, 1968) using the primers designed based on the sequence 0. reported by Martin et (1992) and the amplified fragment cloned into the 'i 15 pneumococcal vector pLS 101 (Balganesh and Lacks, 1984).

e The mutant gene encoding a soluble form of Spneumoninae PBP 1A was constructed by using of plasmid DNA harbouring the wild type gene as template and amplifying a 2.3 kb DNA fragment by using PCR following 20 standard procedures. The sequence of the primers used were TG-24 (5'-TAC GTT ACC ATG GCT CCT AGC CTA TCC-3') and 3'-primer TG-25 (5'-GAC AGG ATC CTG AGA AGA TGT CTT CTC The 5'-primer TG-24 includes the sequence for the restriction enzyme Ncol while the 3'-primer TG-25 includes the site for the restriction enzyme BamHI. The Ncol and BanmH digested PCR amplified DNA fragment was ligated to NcoI BamHI cleaved pARC039. The plasmid pARC039 is a derivative of pET 8c (Studier et al., 1990) in which the gene coding for the p-lactamase has been replaced by a kanamycin resistance cartridge.

Following ligation and screening using standard protocols, the structure of the recombinant plasmid was confirmed by detailed restriction mapping -27and transformed into the T7 expression host Excoli BL 21 (DE3) (Studier et al., 1990), The re. ornbinant plasmidd was labelled pARCO512 (NCIMB 40665) and is schematically represented in Fig. 12.

The nucleotide sequence of the mutant S.pneutnoniae PBP IA gene is shown as SEQ ID NO: 5 and the derived amino acid sequence is shown as SEQ ID NO: 6.

3.2. Expression and characterization of soluble form of Streptococcus pnieuton toe PBP IlA The gene coding for soluble S.pneuontiae PBP 1A was expressed by a e~oo procedure analogous to the one described in Section The cytosolic Go*$ fraction of E.coli B3L 21 (DE3)/pARC0512 was isolated and tested for the 15 presence of the soluble form of the S.pneumnoniac PBP 1Adel38. The radioactive ligand used for the binding studies was 1 3 H]benzyl penicillin .:::'(Amersham) which was prepared as described eaflier. Approximately of the expressed protein from the mutant gene was found to be in the soluble fraction and bound ['l 25 1lpenicillin. (Rojo et al., 1984) or 20 ['HIpenicillin (Araersham) when the culture was grown and induced at 22"C with 0.01 mM IPTG. The growth and induction conditions were a. critical for the efficient recovery of the soluble protein, as growth at higher temperatures or induction with higher concen tra tions of IPTG resulted in, the majority of the protein becoming inactive and forming inclusion bodies.

Optimum levels of soluble active protein was found following induction for 6-8 h. (Fig. 13).

The soluble $.pneunmoniae PBP 1Adel38 protein could also be efficiently purified essentially following the protocol used for the purificationx of the soluble E.coli PBP 1B protein.

The efficiency of penicillin binding of the soluble PBP lAdel38 was comparable to that of the native membrane bound S.ptieumoniae PBP IA.

EXAMPLE 4 4.1. Transglycosylase deficient E.coli PBP 1B The conserved amino acids within Region 2 (Fig. 14) were chosen for sitedirected MUtagenesis. W4ithin this stretch of 10 amino acids three different mutations were constructed: the glutamines at position 270 and 271 of the PI3P 1B sequence were too. changed to alanines; the glutamines at position 270 and 271 of the PBP 18 sequence were *0.9 changed to leucines; and a deletion of the nucleotide sequence encoding amino acids from ~:position 264 to 271.

Lo 6 Mutants of 'the pontB gene were constructed essentially following the procedure of K~unkel et al. (1985). A 1.5 kb EcoI~I Sall fragment of the poiz 8 gene of the plasmid pBS96 was excised and cloned into EcoRI Sall .cleaved Ml3znpl9 following standard protocols.

The primer used for mutating the nucleotide sequence coding for glutan-ine residues 270 and 271 into a sequence coding for alanine residues was TG-21: -ATG CS3 ACG VGCC GiCT C'TG G3TI AAA-3~ TI 1, T z% A 1 V The primer used for mutating the sequence coding for the glutarnine, residues 270 and 271 into leucine residues was TG-23: S' -AC CI3 A2Cl -2P ~1X13 GTI; AAA T L T L L L V K The primer used for creating a deletion of the nucleotides encoding amino acids at position 264 to 271, all of which are within the conserved Region 2, was TG-22: 51-CGC ACG GTA CAG CTG GTG.3 AAA AAC-31 R T V Q L V K 260 261 262 263 272 273 274 (amino acid no.) Following mutagenesis, the nucleot-ide sequence of the mutagenized EcoPJ Still fragment was determined following the protocol of Sanger et al.

too 0(1977). The sequencing confirmed the nucleotide changes and also ruled out any extraneous changes. This mutated 1.5 kb DNA fragment was 15 ligated back to EcoRI Still cleaved pBS96 and the ligated DNA transformed in to E.coli DH-5a cells following standard protocols.

Kanamycin resistant transformants were analyzed for their plasmid profiles 44 0 and the plasmnid with the TG-21 mutation was labelled pARCO438 09. (NUIMB 40661). The mutant protein is referred to as P13' 113 QQ-AA (SE.Q *too:: 20 ID NO: 7).

The plasmid with the mutation Mb introduced by TG-23 was labelled pARCO468 (NUIMB 40662). The mutant protein is referred to -is PBP 1B3 QQ-LL (SEQ ID NO: 8) The plasmid Mi the deletion Wc obtained using TG-22 was labelled pARC0469 (NCIMB 40663). The mutant protein is referred to as PBP 113de18 (SEQ ID NO: 9).

Thte four plasmid DNAs of pBS9t6, pARC0458, pAlW0468 and pAI 046'4 were individually transformed into tExoh ponB3:spe; cells (Broome-Sinith et al., 1985) in whlich a deleted poiiB gene had been marked with spectinomnwein resistance mnarker.

E~coli ponB:spcr cells having the individual plasmids pBS96, pARCO438 or pARCO469 were grown and membrane preparations made following the procedure described by Spratt (1977) and the profile of the penicillin binding proteins analyzed on a 8% SDS-PAGE following labelling with radioactive penicillin. The mutant proteins were initially analyzed for in vivo stability and localization into the membrane using anti-PBP 1B sera raised against purified membrane bound native PBP 1B (Fig. The mutant proteins were found to be localized to the membrane and no degraded protein fragments reacting with the antibody could be detected *0 indicating no gross instability. In addition the mutant proteins bound penicillin with an affinity comparable to that of the wild type PBP 113 (Fig, *Soo 4 0. 6 00 15 After assaying for transglycosylase activity as described in Heijenoort et at.

(1978), no activity could be detected in the membranes expressing the *mutant proteins, while the mnembrane with the wild type PBPI 11D showed transgiycosylase activity. This defines the amino acids 263 to 271 as being t* critical for transglycosylase activity.

The ability of the mutant proteins to bind penicillin with anafnt ~comparable to that of the wild type suget tha th rnpeds activity of the mutant proteins would also be comparable to that of thle wild type. K(nowing that the bifunctional protein P131 1B3 expressed onl a plasmid can in trans complement the deletions of both ponA and pon 13 CYousif ot al., 1985) the ability of the tranisglycosylase negative transpeptidlase positive proteins PBP 113 QQ-AA and IPBP llBdel8 to complement the absence of chroinosoinally encoded PBP 1A and lB was tested.

The wild type ponl and the mutant poiff genes were cloned into low Copy vector pMIAK "M5 (Hamilton et al., 1989). The resulting plasmids were 512351I -31designated pARCO462, (wild type ponB, Fig. 16), pARCO463, (ponBdel8, Fig.

17) and pARCO470 (ponB QQ-AA, Fig. 18). The plasmids were individually transformed into E.coli del ponA (E.coli with a deletion of the ponA gene).

E.coli del ponA /pARCO462, E.coli del ponA/pARCO463 and Ecoli del ponA/pARCO470 were used as recipients of the P1 phage for the transduction of the ponB:spcr marker. The transduction was performed as described by Miller (1972) The phage P1 lysate was made on E.coli ponB:spJ strain (Yousif et al., 1985). Following infection, the infected cells were plated on spectinomycin. Integration of the DNA fragment harbouring and pon:spcr transduced into any of the recipients results in the inactivation of the chromosomal pouB gene rendering the chromosome ponA" and ponB". This genotype being lethal for the cell, the E.coli spectinomycin resistant transductants can remain viable only if the plasmid *i 15 encoded ponB or the pon8l mutant can functionally complement in trans.

The following E.coli strains were subject to phage PI transduction analysis of trans-complementation: E.coli AMA 1004 which has chromosomally coded wild type ponA and ponfl; E.coi AMA 1004 which has a chromosomally inactivated ponD and is the host for the plasmid coded mutant ponB genes; E.coli AMA 1004 host bearing the plasmid pARCO462 encoding the wild type ponB gene; Ecoli AMA 1004 host bearing the plasmid pARCO463 encoding PBP 1del8; and E.coli AMA 1004 host bearing the plasmid pARCO470 encoding PBP IB QQ-AA.

-L R '2353.1 -32- Results (Number of Kmr transductants ml) Excoli AMA 1004 3.0 x 104 Excoli AMA 1004, ponB:s per 1 E~coll AMA 1004, ponB:spJr (PBP 113 wt) 1.1 Excoli AMA 1004, potiBspJr (PBP IBdel8) <1I Excoli AMA 1004, po-.:spJr (PBP 1B QQ-AA) <1I A comparable number of transductants were obtained for an internal marker trp transduction using the same P1 phiage lysate.

The above results show that viable transductants could be obtained only with wild type PBP 1B, indicating that the TG' TP+ product encoded by 0*0 poriB QQ-AA or pon~del8 could not complement the loss of chromosomally 15 encoded PUP 1A and lB. However, as these mutant proteins bind penir"n *.and thus can be assumed to have transpeptidase activity, the inability, complement must be the absence of the transglycosylase enzymic activity.

These results confirm the essential nature of the transglycosylase activity of PBP 1A or 113 for the viability of the Exco~i cell.

The mutants described define the Region 2 to be involved in the traznsglycosylase activity of the protein. As this stretch of amino acids is conserved witin the four high molecular weight penicillin binding proteins namely E.coli PBP 1A, 1B and S.pneutoidae 1A and the 94 kDa protein of HWitfluetizae (Fig. 14) it is reasonable to assumne similar catalytic or structural involvement of this region in all the transglycosylase enzymes utilizing substrates similar to that used by PBP 1A and 113 of Exeoli.

4.2. Transglycosylase deficient Ex.ohi PBP 1A The conserved Region 2 was chosen for site-direc; ,d mutagenesis and the nue-otide sequence coding for glutamine at positions 123 and 124 of CEcol n 1236.1 -33- PUP IA was changed to a sequence coding for alanine by PCR mutagenesis as follows. The 5' half of the ponA gene was amplified as 2 fragments, the corresponding to amino acid I to 123 (fragment A) and the 3'fragment corresponding to amino acid 1 74 to 434 (fragment B).

The sequence of the 5'-primer used for the amplification of fragment A was TG-93 GCG CGG ACC ArG CTG AAC TTC OTA AAG rAT-3') while the 3'-primer used for the amplification of fragment A was TG-106 TGC TGC AGT AAT GGT ACT TGC CCC TTG-3'), The 3'-primer for fragment A amplification included the sequence for the restriction enzyme Pstl which allowved the conversion of the sequence encoding the glutamine residues in position 123 and 124 into a nucleotide sequence coding for alanine residues.

Fragment B wvas amplified with the 5'-primer TG-107 (5'-ATT ACT GCA GCA CTC CC AGA AAC TTC TTC-3') and thc 3'-prinier TO-1O$ CGA GAT ATC TGC CCC ATr (.AT CGA CAC-3').

20 The 5' -primier for amplifying fragment B included the sequence for the restriction enzyme Pstl overlapping the sequence with that of 3'-primer for amplifying fragment A. Ligation of the 3'-end of fragment A to the of fragment B recreated the site for Pstl and resulted in the change of the nucleotide sequence encoding glutaniie 123 and 124 into alanine 123 and 29 124. The amplified fragments A and B were individually cloned into pBR 329, and corresponding clones trARCOS65 and pARC0566 were obtained.

Fragment A and B obtained from pARCOS65 and pARCO%16 were ligated to obtain pARC0567. The ponA sequences were completed by introducing an Vil PatnHl fragment of pARCO489 (which is identical to pARCO558 (Fig. 3) except for having additional Lact and Lac operator sequenices) into pARCOt7 to obtain pARCO%68. The A-110 A 111 fragment of pARCO-Io8 R 1235-1 which included the Q13- Q24to A 123 A1 24 mutated region was then used to replace the otherwise identical M10I BgIIH fragmenit of pBS98 to obtain the plasmid pARCO57I (Fig. 19; NCIMB 406658). The mutant protein was labelled Pb?' 1A QQ-AA (SEQ ID NO: Expression studies on the mutant indicated that the mutant protein was localised to the membrane (as detected by anti PBP 1A ant~hodies) and bound penicillin with an affinity comparable to that of the native fBP 1A (Fig. An in vivo complementation assay, similar to that described in the previous **:section, was performed by checking the ability of mutant PBP 1A protein to complement in trans. The in vivo complementation was performed using phage P1 transduction and trzansducing ponB:spL- into the host Ecoli 1. 15 (recipient) del ponA harbouring the plasnid encoding the mutant protein PBP IA QQ-AA.

In order to carry out the complemneittation analysis the wild type ponA t. *gene was cloned into the low copy vector pMAK 705 (Hamilton et 11, 1989) to obtain pARCO583 and the mutant ponA gene encoding PUP 1A QQ-AA *to cloned into pMAK 705 to obtain pARC0582..

6 so The rollowing E.coti strains were subject to phage P1 transduction analysis of trans-complementation: Exco~i AMA 1004 whidch has chromosomally coded ponA and ponB; Excoli AIMA 1004 ponA which has a chrornosomally inactivated ponA and is the host for the plasmid coded mutant ponA genes;- hiost bearing the plasmid pARCO583 encoding the wild type ponA gene;- host bearing the plasmnid pARCO582 encoding PBP 1A QQ.AA.

A 12350 Results (Number of Sp!' transductants mld) Excoli AMA 1004 2.1 Excoli AMA 1004, ponA 1 Excoli AMA 1004, ponA (PBP IA wt) 1.64 Excoli AMA 1004, ponA (PBP 1A QQ-AA) 1 A comparable number of transductants were obtained for internal marker: trp transduction using the same PI. phage lysate, As shown above, no viable transductants could be obtained with Excoli del ponA pARCO582 as recipient indicating that the mutant PBP IA QQ-AA could not complement the absence of chromosomally encoded PBP 1A/lB3.

This indicates that the Q13and Q14of region 2 of PBP 1A also affects trans glycosyl ase activity of the protein as the loss of the complementing function must be a reflection of the loss of transglycosylase activity. The transpeptidlase activity of the protein is unaffected as tested by its affinity to bind penicillin.

o. 20 These results argue in favour of the region 2 as a critical stretch of amino :acids involved in the transglycosylase enzymic function and may be the explanation for the strong evolutionary conservation of this stretch of amiino acids.

EXAN/fIPLE 5.1. Truncated FEcoli PUP 1B A mutant gone encoding the truncated PBP 18 consisting of the.N-terminal 553 amino acids was constructed by PCR amplification using the S'-prinier TG-777 (5'-GA AAA CCA TGG CCG GGA ATG ACC-3') and the 3'primier TG-116 ATG GGA TCC TrA ATC ATT CTG CGG TGA-3').

R t235,1 -36- The 5' end of the primer corresponded to the amino acid 553 in the wild type followed by the stop codon and a site for the restriction enzyme BamHI. A fragment of 1.7 kb was amplified using pBS96 DNA as template.

The PCR amplified fragment was cut with PstI and BamHI and cloned into PstI-BamHI restricted pARC0555 (pARC0555 has the full length ponB gene cloned as NcoI-BamHI fragment into the expression vector pETlld. The NcoI site includes the initiation codon ATG) to obtain pARC0592 (NCIMB 40669; Fig. 21) The expressed protein (SEQ ID NO: 11) was shown to have transglycosylase activity, thus confirming the functional independence of this domain.

The soluble truncated PBP 1B, i.e. PBP 1B with N-terminal 553 amino acids s but lacking the membrane anchoring hydrophobic domain from 65-87, was constructed by replacing the PstI-BamHI fragment of pARC0559 (Fig. 9) with the Pstl-BamHI fragment of pARC0592 to obtain pARC0593 (NCIMB 40670; Fig. 22). The mutant ponB gene encodes the soluble form of PBP 1B and the expressed protein (SEQ ID NOr 12) was found to have transglycosylase activity.

S

5.2. Minimum substrate binding domain of truncated E.coli PBP 1B Detailed computer analysis of the anatomy of the presumptive TG domain (aa 1-553) of PBP 1B indicated that aa 210-368 were probably sufficient for the bindig of the lipid linked substrate and the transglycosylase reaction.

This stretch of amino acids includes the 3 conserved domains Region I, II and III. The mutant gene encoding the truncated protein stretch 210-368 was constructed as follows.

A fragment of approx size 480 bp was amplified from pBS%9 as substrate with the 5'-primer having the sequence TG-154 (5'-CAA TCC ATG GGT GAG CAG CGT CTG TTT were the initiation ATG codon is I R '1235.1 -37immediately followed by the sequence encoding the 210th amino acid of PBP 1B.

The 3'-primer corresponded to the sequence TG-155 CCA GAA TTC CAG TTT TGG GTT ACG-3') were the sequence encoded the amino acid 368 of PBP 1B followed by the nucleotide sequence that provides the restriction site fnr EcoRI, enabling fusion to sequences encoding an enterokinase site and a histidine stretch, which allows rapid purification of the protein on an Ni affinity column (cf. section 6.2 below).

A NcoI-EcoRI fragment was cloned into the plasmid pARC0400 that was restricted with NcoI-EcoRI to obtain the recombinant plasmid pARC0392 (NCIMB 40659; Fig. 23). The recombinant plasmid was transformed into E.coli BL26 (DE3) and a protein of approximately 17 kDa was detected largely in the soluble fraction aItter induction with IPTG.

Along similar lines the minimum substrate binding region of PBP 1A could be predicted to involve the stretch 62-220 in the wild type protein.

Production of this protein as a fusion with a histidine stretch allows high efficiency affinity purification of the expressed product using the Ni 2 column. That the results will be similar to that obtained with truncated PBP 1B can be anticipated.

EXAMPLE 6 6.1. N-terminal fusion of soluble E.coli PBP 1A to glutathione-S-transferase Fusion of the ponAdel23 gene at its 5'-end in frame to sequences coding for glutathione-S-transferase was made as described in the following section.

The vector chosen for the fusion gene construction was pGEX-3X obtained from Pharmacia Biochemicals. In order to fuse the '-initiation ATG of R 1235.1 -38ponAdel23 in frame with the gene encoding glutathione-S-transferase a BarnHI site was introduced using a PCR primer whose sequence included the sequence for the restriction enzyme EcoRI. The 5'-primer used was TC- 115: 51-TCG AGG ATC CCC ATG GCC CTA TAC CGC TAC ATC G-31 EcoRI BamHI The 3'-primer used was TG-106, described in Section 4.2. The PCR amplified DNA Fragment A was digested with Bantfl and PstJ and cloned into the Band-H PsI sites of the standard cloning vector pUC8 to obtain pARC0496. This Fragment A includes the N-terminal 102 amino acids of the PBP lAdel23 protein. A BamHI M10I (site present within the fragment A) 270 bp fragment obtained from Fragment A, a 2.2 kb M10J EcoPJ fragmntn which includes the rest of the portion of the poniA gene obtained :from pARCO49O (pARCO490 has the wild type panzA gene cloned into the too. Xbal BatiHI sites of the low copy vector pWKS29 (Fu Wang et at., 1991) facilitating the 3'-end of the poiiA del 23 gene to be excised as an EcoRI fragment) and a EcoRI Battl-l cleaved pGEX-3X were ligated together and transformed into competent E.coli cells. Individual transformants were screened for recombinant plasmid and the plasmid with the expected structure was designated pARCO499 (NCIMIB 40664; Fig. 24). The encoded fusion product on pARCO499 has the glutathione-S-transferase sequences at its C-termninus linked to PI3P lAdel23 sequences via a Factor Xa cleavage recognition sequence.

Following induction with 1 mivf IPTG, a fusion protein of expected size was found to be induced. The protein bound penicillin and was active in the transglycosylase assay. Following cell lysis by passing the suspension through a French press, the cell free supernatant fraction was prepared as deckiled in Section 1.4. for the purification of P81' lAde23. The supernatant fraction was passod tlirou,3h a (lutathione Sepharose) 1t ti 023s.i -39- (Pharmacia Biochemicals) and the bound GST-PBP lAdel123 was eluted with glutathione. The eluted protein was found to be 80% homogeneous.

Free glutathione was removed by dialysis and the GST-PBP lAdel 23 was cleaved with factor Xa.

PBP lAdel23 thus purified was found to be active in both penicillin binding and the transglycosylase reactions.

6.2. C-termdial fusion of soluble 1,coli PBP 1A to histidine stretch Fu...on of the ponAdel23 gene at its 3'-end in frame to sequences encoding a stretch of 6 histidines was made as described below.

:In the first step the potiAdel23 gene was amplified using pBS98 DNA as template using the 5'-primer TG-115 ('-TCG AGG ATC CCC ATG GGC :i CTA TAG CGC TAG ATC and the 3'-primer 'ITG-121 (5'-GTT AGA MTT CGA ACA ATT CCT GTG-3').

The 3'-primer introduced an EcoRI site at the 3' end of th ponAdt'l23 gene while eliminating the translation stop codon. The PCR amplified modified potiAdel23 gene fragment was digested with I'stI and EcoRi to release a 930 bp 51.end fragment and ligated to PstI-EeoRI digested pBR 329 to obtain the recombinant plasmid pARCO467.

in the next step, a double stranded synthetic oligonuclootide with sequences encoding the six histidines and the DNA sequence coding for aminoacids recogriised as the enterokinase cleavage site was synthiesised and igated to the newly created EcoRt site at the 3'-end of the pollAdel123 gent, on pARCt)467- The synthetic oligonueleotides used were TG412: EcoRlt TTC GAC GAC GAC GAC AAG CAC CAC CAC CAC CAC CAC TGA TAA G-3' ENTEROKINASE fISTIDINES and TG 123 (5'-GAT CCT TAT CAG TGG TGG TGG TGG TGG TGC TTG TCG TCG TCG TCG-3').

The plasmid pARC0467 was linearised with EcoRI and the synthetic double stranded oligonucleotide ligated. Following ligation a PstI Banil (Fragment A) was released from the ligation mixture and cloned into the :Pstl BatnHTf sites of pARCO0558 (Fig. to obtain pARCO400 (NCIMB 40660; Fig. 25). The mutant ponAdel23 fusion gone thus encoded a protein with the PBP IAdel23 sequence fused to the amino acid sequence Asp- Asp-Asp-Asp-Lys fused to His-His-His-His-His-His at its C-termidnus. The .9 Asp-Asp-Asp-Asp-Lys sequence is recognised by the pro tease, en terokinase an caves following the lysine residue, The six histidine residues confer on the protein the ability to bind to the metal nickel.

set* The recombinant plasmid pARCO400 was tra nsformed in E.cvti 13126(1) :cells and induced under culture and temperature conditions identical to those used for the purification of IPBP lAdel23. The cells were Iyedb passing through a French press. The lysate was centrifuged at 10,000 rpin for 10 min. The supernatant obtained after low speed centrifugation was then spun at 200,000 x g for 45 min and the supernatant obtained represented the cytosolic fraction. This fraction contained the protei encoded by the fusion gene and the recombinant fus ion protein was labelled PBP lAdel23PH1. This protein P8UP lAdel23hll1 bound 125 llephradine and was ako active in trngwsls s~.The ,s ouble fraction was passed through a Ni affinity column and bound protein eluted in batches with increasing concentrations of iinidazol et-sentially folloing the procedure deseribed in "The Qia r1Npressionist"' obtained from QLANII Inc. 92.5) iRton Avenue, Chateworth, CA 911311 USA. The miajority of P1131, R'1235 I -41lAdel23EI- eluted with 250 mM imidazole and was approximately homogenous. It was the only cephradine binding protein eluted from the column. Thus the ability of fusion protein to bind to the Ni column can be easily exploited both for efficient purification and immobilisation of tile active protein.

EXAMPLE 7 7.1. Use of cell extracts for enzyme assays and in screening C. .*The crude cell extract made according to Example 6 can be analyzed for the ability to bind penicillin by reacting with 3 1-1ampicillin prepared according to Hackenbeck (1983). To adapt the procedure to large-scale screening, a 96 well microtitre plate is used to contain the reactions and mie assay is performed using a Beckman Biomek robot. Crude cell extract is mixed with 3 1ilampicillin for 15 min at 370C. The proteins in the reactionare are precipitated with TCA and collected onl a glass filter, unbound ampicillin is washed off and filters counted in a scintillation counter. Alternatively, autoradiograph cn be used to assay the degree oif binding of anipicillin.

Based on the above method, a competitive assay can be used to assess thle ability of test compounds to bind to the transpeptidase site of a PBP variant. In this assay, the test compound is exposed to the crude cell extract for 15 min prior to the addition of amipicillin. A positive result is indicated by a reduction in the amount of radioactivity present on the glass filter.

Use of soluble immobilised protein in screeing Protein containing a histidine, peptide which has been purified as demcrbed can be used for scre ning for corn -,ds which inhibit transp-eptidase 42activity or transglycosylase activity. The purified full length or truncated protein is irmmobilised onto agarose gel to which Ni(II) has been coupled.

Aliquots of the beads contaiing immobilised protein are then transferred to the wells of a microtitre plate, tc-st compounds are added to the plate and incubated before unbound test substance is washed free. Compounds which bind to the transpeptidase site of the bifunctional protein can be detected by adding 3 Hlarnpicillin to the reaction vessel and continuing essentially as described above. Alternatively monoclonal antibodies known to bind to the transpeptidase region can be used, Compounds which bind to the transglycosylase site can be assessed in a competitive assay by the use of monoclonal antibodies which bind to the tranisglycosylase region of the protein, EXAMI'L1 8 8.1. Production of mnonoclonal antibodies to PBP IA The protocol for the production of monoclonal antibodies (mAbs) was essentially that described in "Antibodies a laboratory manual" (ed.

H-arlow David Lane, Cold Spring H~arbor, USA). Purified membrane bound PUP IA was used as the inimunogen. Balb-C mice, 6-8 weeks old were inmmunised with 50 jig of purified native IsP 1A in Freunds Complete Adjuvant. A booster injection of 20 pig P131 IA in incomplete Freunds adjuvant was given intraperitonially. Two weeks later thc presonce of serum antibodies was checked by BLISA using PUP 1A as the coated antigen. Mice with circulating antibodies were imnmunised intral.en tonially daily for 4 days with 20 pig of PBP IA in saline and the mice sacrificed for isolating splenocytes for enerating fusions.

The rnyeloma cell line used in fusion experimients, was Mp 210-Ag 14 and these cells were fused with splenoeytes from immunised mnice at an ratio of Fusion was carried out using standard protocols and antibo dy -43production from the clones was monitored by ELISA against PBP 1A when the cells were 90% confluent.

72 high producing clones were expanded to 24 well plates and tle secreted antibody characterised using the following screens: ELISA against m'embrane bound form of PBP IA; ELISA against soluble form of PBP lAdel 23; Dot blot analysis against membrane bound PBP IA to eliminate monoclonals reacting with the detergent solubilised purified PBP 1A protein only due to changes in the configuration during purification; and ELISA against membrane bound form of PBP 113.

Based on these screens, a panel of 5 secreting clones were selected and subcloned twice to ensure monoclonality, Ascites with these hybridomia clones were raised following standard procedures and IgG was purified from these ascites fluids, using Protein G-Sepharoseo affinity chromatography as recommended by the manufacturers of Protein G-SepharoseO (Pharmacia Biochemi cals).

These purified antibodies react specifically with PB13 A in both the membrane bound and the soluble forms in ELISA, Dot blots and in Western. blotting. Clones were obtained by a cloning procedure employing 3 cells well. To ensure the monoclonality these clones were subeloned into 96 well microtitre plates by limiting dilution at 1 cell well. The wells receiving one cell were carefully confirmed under the microscope and allowed to grow with miacrophage feeder layers so as to obtain progeny from a single hybrid cell. Following sub-clornn the secretion, of mAbs to PB13 A was again assayed in liLISA using full length PHI' IA. Finally two clones from each parent lwbridonma were selected and one of thoim was expanded as ascites in pristine primed Balb/c mice. All the five clone!' adapted to grow in peritoneal cavities and produced aseitie inAts.

R .1235-1 -44.- The ascitic mAbs were titrated against purified PBP 1A in ELJSA. All the ascitic mAbs had a litre of 5 x 105 in ELISA and recognised full length protein in western imrnunoblots. The ascitic mAbs were purified by protein-G affinity columns, The immunoglobulin isotype of mAbs was determ~ined by mouse Ig isotype by ELISA using a kit obtained from Sigma chemidcals USA. Four of the monoclonals belonged to IgG1 and one belonged to IgG2a immunoglobulin isotype.

Further characterization of mAbs was done by usig full length membrane bound PBP lA/1B in western blots. In addition the transglycosylase (TG) and transpeptidlase (TP) domain specificity of mAbs was determined by :using various truncated forms of the membrane-bound N-terminal of PBP 1A, N-terminal of PBP 113 and C-terminal of PBP 113 in Western immunoblots. Various full length and truncated membrane bound PBPs were expressed and the prepared membrane fractions were resolved on a SDS-PAGE. The proteins were transferred on to ni trocellulose membranes set and subjected to western blot analysis using polyclonal E~ coli PB13 A antibodies and monoclonal antibodies.

see* Assessment of the penicillin binding inhibitory potential of the mAbs was determined essentially following the protocol described by den Blaauwen et al. (1990). The protein-G affinity purified niAbs was preincubated with PBP IA followed by addition of EkI-1benzyl penicillin or t' 2 lIlcephradine.

Two of the inAbs competitively inhibited binding of the radiolabolled penicillin to P1W IA.

Mnt1onal antibodies specific for the TG doimin of 11131 1A have been obtained by screening the so .vrted antibody of the original hiubridoma clones to react with the prtein representing flt, N-terminal 434 amino acids of P131 IA in, western blots. Antibody from clone TG-2 reaited with the N-terminal truncated 434 amino acid analogue of PBP 1A but also inhibited inhibition) the transglycosylase activity of PBP lA. This indicates that the antibody recognises sequences in the protein which are involved in binding of the substrate; (b catalysing the enzymnic action; or altering conformation of the protein allosterically. In either of the three possibilities, identification of compounds competing for the binding of TG-2 to PBP IA would represent molecules interacting with identical sequences on PBP IA. Thus the competitive binding assay Could be used as a screening assay for the identification of the TG inhiibitory compound.

a....'BRIEF DESCRIPTION OF THE DRAWINGS Fiur I Figure 2 foremtc yrpsatiiypto n of T theanltriona promotegr 2' laetemntr TShemaretin rep rsc ption of the 7tdirn funsaesownaros elpessin ercto pARCO38 sar.hon Nmer e~ o uppcrtwese repre-ssor o (ac zet rigof elcto o) 7coeao Rt 1235-1 -46- Figure 3 Schematic representation of the vector pARCO558 encoding soluble PBP lAdel 23 of Ecoli.

vector sequences nmutant gene encoding PBP lAde!23, kanarnyciin resistance Kmr, lactose.! :prc-ssor (lac 1q) and the origin of replication on,.

Fire 4 Expression of soluble PBP lAdel23. Panel A represents the autoradiograrn of the 125 ,]cephradine binding profile of the uninduced and induced ***cultures of E~coli BL 26 (DE3) harbouring pARCO558. Pantel B represents the Coomassie Brilliant Blue stainting protein profile of the same uninduced and induced cells. Lane uninduced cytosol fraction; uninduced membrane fraction; induced cytosol fraction; induced membrane fraction; molecular weight markers.

Figure SSIDS-PAGE pattern of purified PBP lAdel23. Panel A: Coomnassie blue staining. Panel B: 125 1]cephiradine binding protein profile. Lanes E.coli BL 26(D133)/pARCQ558 cytosolic fraction (200,000g supernatant); :Ammonium sulphate supernatant fraction; 30%1 Anulloniurn sulphate pellet fraction; Cephradine affigel breakthrough fraction; Molecular weight markers; Cephradine affigel eluate.

Figure 6 Transglycosylase activity profile of wvild type PBP IA and Mutant PBP 1 AdeI23 using purified proteins.

represents N, Livity of soluble PBP lAdel23; o) represents activity of membrane bound PBP 1A solubilised with oetyl-p-glucoside. X-axis represents the concentration of thie proteins used in pg. Y-axis represents the quantities of peptidloglycart fornmed.

R 1235-1 -47- Figure 7 Hydropathicity profile of Excoli PBP 1B. The figure represents the expanded hydropathicity profile of (lhe N-terminal 150 amino acids of Ecoli PBP 1B.

Figure 8 Schematic representation of the cloning of the soluble traiisglycosylase diomain of Excoli PBP 1B, vector sequences sequences encoding ponB gene fragments and P-lactamase The NcoI-NruI fragment encoding dhe N-termninal 64 amino acids of PB? lB was cloned into the Ncol-EcoRV sites of pARCO534 to obtain the plasmid This recombinant plasmid harbours the gene encoding amino acid 1 to 480 of PBP 1B with internal deletion of amino acid 65 to 87.

Figure 9 9 Schema tic reprosen ta tion of pARCO5S9 encoding soluble P13? lB.

vector sequences sequences of the mutant ponB gene encoding the soluble form of ~j 20 POBP 10 (solPBP 1B), lactose repressor (lac 1q), kanam-ycin resistance

(K

11 r) and the origin of replication (ori).

Arrows represent direction of transcription of the genes.

Figure Purification of soluble PBP 1B. Panel A: SDS-PAGRi, Coomassie blue staining of the different fractions. Panel B: 12 Ilampicillin binding profile of the same fractions. Lanes and Cytosol)ic fraction of Extr BL 26rW3)/pARC0551) induced colls; Breakthrough fraction of Arnpicillin- Affigel column; M4:NMolecular weight markers; and Fluted fraction from the Ampicillin-Affigol, columin.

Figure 11 R 1235-1 -48- Hydropathicity profile of S.pneumnotdae PBIP IA. The figure shows the expanded profile of the hydropathicity profile of the N-terminal 100 ,nino acids of S.pneumoniae PBP IA.

Figuqre 12 Schematic representation of the plasmid pARC0512 encoding soluble form1 of Spneumioniac PBP IA.

-represents vector sequences represents sequences of the gene encoding soluble PBP 1A of S.pneumoniae (sPBP IA), kanarnycin resistance Kmr and the origin of replication (ori).

Fiue1 *seea Penci.i bidn rfl fslbeSpenta B A ot .CUB ~.:Pnciloli bind inrfl of solnuled St 30cCmor 2U hA; osEoli frcto labelig withula wleight penicillin folodin bytens TheP2 Lanues (o)pands (2)e cosolied fraeione of ces ine at 22 0 for 2 am nd 20A h peivly (3):Ctnosoilics fctin of IA)lls inddate idenica amir 2a;(:cti ratidues Figure Anaid alimen tei o nEved reios oheboringlylasd doiti o ige enolingculat eightaelA:(H~e penicillin bindingprtisThfguecmas prwofi ifena PanP A) (*)ser blt in iate dntic 1 ea anos (cD.rsdu Molecular woight markers; Mlenibrane fraction of E Lt 4 1 JM10 l)t -49cells; Membrane fraction of Excoli 900521 ponB:SpJr cells (This host lacks chromosomal encoded PBP 113); Membrane fraction of Excoli 900521 ponfl:spc./pARCO438 cells; Membrane fraction of Excoli 900521 ponB.-spc/pARCO469; Membrane fraction of Excoli 900521 ponB:spc/pARCO468.

Figitre 16 Schematic representation of plasmid pARCO462 encoding wvild type PSI' 113: vector sequences sequences of the ponB gene, replication origin chioramphenicol acetyl transferaSe (crnr) and portions of the iac Z multiple cloning site.

Figutre 17 Schematic representation of plasinid pARCO463 encoding mutant ponl3 so gene.

.611. -vector sequences sequences of mutant thie ponB gene encodig PSI' 1Bdel8 amino acids, replication origin (ori), chioramphenicol acety! transferase (cmr) and portions of the lac Z multiple cloning site.

Figure IS Schematic representation of plasmid pARCO47O0 encoding mutant polnB gene.

vector sequences sequences of mutant the ponD gene encoding PBP1 113 i

A~

1 2 ~,replication origin (oni), chlorarnphenicol acetyl transferase and portions of the lac Z multiple cloning site.

Schematic representation ef pARC0571 harbouring mutant ponA gene.

R 1235.1 vector sequences sequences of mutant ponA gene (PP IA QQ-AA), kanamycin resistance Kmr origin of replication (06).

Figure 125 JJPenicillin binding protein profile of wild type and mutant Excoli PBP IA. Lane Excoli AMA 1004 ponB:spr/pBS 98 ponA); E.Coli BL21 (DE3) poiiB:spc /pARCO57O ponA); Excoli AMA 1004 del ponA/pARCO57l (QQ-AA ponA); Excoli AMA 1004 del ponA/pBS 98 poniA); Molecular weight markers.

Figure 22 Schema tic representation of plasmidd pARCOS92.

B vector sequences sequences of truncated ponB gene encoding for the N-terminal 553 S amino acids of PBP lB (hinge 1B), kanamyvcin resistance (Kinr) and origin of replication (oni) Figure 22 Schema tic representation of plas mid pARC593.

-~-vector sequences sequences of mutant truncated poOf gene encoding a soluble form of the truncated N-terminal 553 amino acids of PUP lB (;'oluble hinge IB), kanamycin resistance Kxnr and origin of replication (ori).

Figure 23 Schema tic representation of plasmid pARCO396.

vector sequences sequences of mutant gene encoding truncated fragment of PBP 1B protein, representing amino acids 210-368 sequences fused in frame at its 3'-end to sequences encoding a tenterokinase site followed by a R; 1'235 I -51stretch of 6 histiclines, kanamycinl resistance Km' and origin of replication (ori), Figutre 24 Schematic representation of plasn-id pARCO499.

vector sequences sequences Of Mutant pwiAdel23 gene fused at its 5'-end in frame to sequences encoding Glutathilone-S-transferase encoding sequences, P-lactamase ampr and origin of replication (ori).

Figuire **Schematic representation of plasn-id pARC0400.

vector sequences *sequences of mutant poiiAde23 sequences fused in frame at its 3'- 0 15 end to sequencqs encoding a enterokinase site followed by a stretch of 6 histidines, kanarnycin resistance Kin" n rgno elcto (on).

o* 20 RIEFERENCES Balganesh, T.S. and Lacks, S. (1984): Gene 29, 221-230 den Blaauwen, T. et al. (1990): J. Bact. 172, 63-70 Broomne-Smith, J.K. et al. (1985): Hur. J. Biochent 147, 437-446 Covarrubias, L. Bolivar, F. (1982): Gene 17, 79-89 E~delman, A. et al. (1987): Molecular Microbiology 1, 101-0( F~u Wang, R. and Kushner, S.R. (1991): Gene 100, 1951-19 lackenbeck et al. (eds.) The target of penicillin. W. doc Gruvter publications, Berlin/.New York 1983.

Hlamilton, C.A. ot al. (1989): J. Bacteriol. 17 1, 4617-4ok2 Heijenoort, Y. van, et al. (1978): FEI3S Letters 89, 141-144 Rl 1-231,1 -52- Heijenoort, Y. van, et at. (1992): J. Bacteriol. 174, 3549-3557 Ishino, F. et al. (1980): PBiochem. Biophys. Res. Comm. 97, 287- 293 Kunkel, T.A. (1985): Proc. Nail, Acad. Sci. U.S.A. 82, 488-492 Kyte and Doolittle (1982): J. Mol. Biol. 157, 105-132 Lacks, S.A. (1968): Genetics 60, 685-706 Martin, C. et al, (1992): J. Bacteriol, 174, 4517-4523 Miller, J.H. (1972): Experiments in Molecular Genetics. Cold Spring H-arbor Publications.

Nakagawa, J.S. et al. (1984): J. Biol. Chem. 259, 13937-13946 too 0Page, W.f. et al. (1982): J. Bacteriol. 151, 237-242 20 Rojo et.al. (1984): J. Antibiotics. 37, 389-393 Sambrook, Fritsch, E.F. and Maniatis, T. (1989): Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring

NY

255 Saniger, et il. (1977): Proc. Nail. Acad. Sci. U.S.A. 74, 5463-5467 .:..Spratt, 1.5. (1977): Hur. f. Biochern. 72, 341-352" Studier, FEW. etit. (1990): M-ethods in Enzymnology 185, 61-89 Tomb, F. et it. (1991): (;Lne 104, 1-10 Yousif, S.Y. et al. (1985): J. Gen. Microbiol. 131, 2839-2845 Fn 1235i1 SEQUENCE LISTING GENERAL INFOR1ATION: i) APPLICANT: NAME: ASTRA AKTIESOLAG STREET: Kvarnbergagatan 16 CITY: Sodertalje COUNTRY: Sweden POSTAL CODE (ZIP): S-151 TELEPHONE: +46-a 553 290 00 (H TELEFAX: +46-8 553 288 TELEX: 19237 astra s (ii) TITLE OF INVENTION: Novel Polypeptido (iii) NUMBER OF SEQUENCES: 13 (iv) COMPUTER READABLE PORM- MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPETING SYSTEM; PC-DOSiM3-IOS CD) SOFTARE: PentIn Roloaoe Version 01.25 (EIO) INFOPMATION FOR SEQ ID NO: 1: i) SEQUENCE CHAPACTERI.3STIC,3: LEN3GTH: 2487 base pairs TYPE: nucleic acid (Ct .1.TPAN0Et:M=B: d-luble TOFCLOGY: linear (ii) MOLECULtE TYPE: VNIA (gonomicl (Vi) ORIGJIVAL O3CURE: off$ OZt1*11M: kerichlteha coll B: T00rN: (tB5 alpha (vii) nMEDATE SOVRCE: LIP.ARY: PCR e.oning (B I rARC 0558 ljrss olubl;--, PEP 1A de 1 21 (ix) FEATU%: NAM KEY: 'X2 (B LCC'TiCN: 1..2437 V8,N~r7RE: N yM E~y: e ltido ~CAT:cN '..2484 CA TA:' i'33TA 111A 3 CCA CAA T~ 3 3AA 4,3 1! P'z Pr Afl V. 1~ *4.1 1r. (-Ti A-'.J ijir LAAA 143 A6a At j A4 44-- 5.-t S. nS 4fl S t t t rfl 1 S a Sdc b'jblA t i; Il n 235AI -54- ATGOGTC AAA GCC Met Val Lyn Ala TTG CAT CAA ATC CCA CCG Lou Asp so Gin Ile Pro Pro

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O,

L~

n 1235 1 CTG CCA ACC TAT Leu Pe Thr Tyr OCT CCG CTG Giy Pro Leu 325 0 to 060 *to#

CCT

Pro

AGT

CMA

Gin

CMA

Gin 385

CCG

Pro

OTT

Val

CGC

tArq

CTC

Lou

AAC

46U

CM

Gin A la P?:o -4 din

CAO

Gin

ATG

Me t 0 ly 370

ATC

Ile

GMA

Glu Moet

GCC

Ala

TMC

Tlyr 4S0 O3kr Pro

CIGG

Gly v4o t So- k

CMA

Gln

GMA

Glu 355

CCG

Pro

TGG

Trp Val

GCG

Ala

ACC

Thr 435 Acc Thr

GTG

Val1 Lyca Lou Gdy din U-.0

C

Ala 340 GCe Gly Acc, Thr Val1

MAC

Lou 4 ZO

CAG

Gin

C

Pro

GT

Gly Va I Am ACCG C Thr Ala OTT Ccc Val. Ara CCO CGT Pro Arrj COT CAG Arai Gin 390 TCC GCG Sor Ala 405 GTC GOT Val Gly GCA CTG Ala Lou GCG AT(; Ala Mot Ile sozr TCA CCA S3or Pro 485 CA(4 T&Y2 Gln Sor Aq-p Tyr 110 Va I

ATG

Met Trp

AMA

LysJ 375

OTT

CTG

Lou

GCC

C ly

CGT

Ara

CAT

Acp 455 Arg ec Pro

AAA

Lyn A la C AC 3 pklo CTG CCT CCC OCA Leu Pro Ala Ala 330 CTG GCG GAC GO Lou Ala Asp Gly 345 CCC CGT OCT TAC Aia Ara Pro Tiyr 360 GTG ACC CAT OTT Vai Thr Asp Vat GGC CAT GCA TG Oly At Ala Trp 395 GTG TOG ATO MAT Vat Sr Ile Anm 410 vT GAT 'N'C MAT Pho Asp Pho Ann 425 CAG GTG OCT T2C Gin Val Gly Cor 440 AM WT CI A\Ci Lyn Gly Lou Thr TGC- CAT G A MT Trp Aop Ala Co?: CAG TAT G -T GGT Gin Tyr Ala tGly 490 AAC GT G TO ATG Aon Val V41 Mot GCA. GM AT1 CTO Ala 0,1,u Tyr Lou Sz Thr in o?:Lou Ala At:} jl y c tC

OTC

Vat

TCO

Ser

COT

Ara

CTO

Lou

TG

Trp Pro

CAG

Gin

ANC

Akm Lou 460 Ala VA I 4-j -I Aaa c"; 4:a-

ACC

Thr

ACC

Thr

TCG

CAA

Gi1n

CTG

Lou

CAA

Gin

AC

Scr

ATC

I

44S SC A Ala

CGT

2 Lou AGO CC Sor Ala 335 GTC GCA Vat Ala 350 OAT ACT As~p Thr ACG GOT Thr G ly OC'A CAI Ala Gin MAC OCT Asn Gly 41S MAG T'l 4

P

L~ys Pho 430 A1304 A7.11 8or Met 4 9 C' Ala W? t 5 Mo A !Lf ~4

MAT

Ann

TTGO

Lou

GAG

Gin

CAC

Gin

GTG

Va 1 400

CC'

Ala

MAC

As n Pho 'T1(1 Lou 4:3 Avg

NA

Ac3i 1008 1056 1104 1152 1200 1248 1296 1344 1392 14 4 1488~ I,8 §4 163 2~

TI

R W35.1 TOO GAT ATT CCG GTO ATT TAC GT GAT ACC Cys Asp Ile Pro Val Ile Tyr Oly Asp Thr 595 600 CAG AAA TCG MAC OTC CTG *9 9 9 99 9.

9 9 999 0 9 99., bee 99** 9 .9.9 .9 .9 9* 9.

9 9 9

C

.9 4 9* 9 9 999 9999 9 9 9999 99 9 9* 99 9.99 9 99.9

CA;Z

0114

G?]

Val1 625 0CG Ala c t rr Lou

CCA

Pro

CCO

Arg T G Trp 705 Pho AT'r Ile

OCT

Pro

CAG

Gin ser 785 ATz MAT MAC OAT OTT] Asn Asn 610 TCT OTA Ser Va I MOG ACT LY'S Thr GCA TTC Ala Phe GOC TO Gly Trp 67S GAT ATC Asp Ile 690 T'rO TO Phe Sel- GAT GAT Asp Asp AAA CAI' Ly n Aop GcA SIC Ala Trp 75S pro Lou 770 Thr Gly flu (fl li .6 Asp Val G1~ CCA ATG CC( Pro Met Pr 63 000 000 CA( Gly Ala Glr 64S CTC ATTr MC Lou 110 LYS 660 CAG GOT ACI Gin Oly Thr GG0 0450 AAA~ Gly Gly Lys OCT TAC GOT Gly Ty r Gly 710 CAC COT COT His Arq AvV Ck A Tt TCA Gin le Oor 740 GAC GCT TAT Ant) Ala Tyr ACG C C CA Thr Pro Pro CV, 114A GCT Gln Lou Ala *190 380 'GA ON] .u Asp 3CAG

GAO

Glu

ACT

Gly

ACC

Thr 69 S

CCO.

Pro

MAT

Atm 0GGT Gly ATro Met CCo Pro

AAT

Aon Thr CTG GAG Lou Olu TAO OCA Tyr. Ala GCT 'M~r Ala Lou 665 TOG COT Trp Arg 680 000 ACC Oly Thr cc OTT Cly Val CTC OT Leu Gly TAt CM TIyr Oiu 745 AAA CC byo Ala 760 GOT AT Gdy 110 GOkT GtYC Gly Gly CM CA13 Gln (iln

CAG

Gin

CCG

Pro 650

MAC

OCA

Ala

ACT

Thr

GTO

V41I

CAT

I Iis 730 Oly

OTT

ValI

MAC

Alt 810 GTC OCT ATC Val Ala Ile Gin Lys TOO COO Ser Arg 620 OCA MAT Ala Aon 635 CAC GTC His Val Thr An GGT COT Gly Argj MAC ACT Asn Ser 700 ACC TCG Phr 8or 715 ACA ACG Thr Thw GO; T G, tx Gly Ala CTT GMA tLou Glu GTG3 Thr V41 780 Sor Argj 19S *]er 605

GAG

Oiu

CAC

Gin

ATC

Ile

ATO

Ile

CAT

Asp 68 S

TOO

Sor

GTC

VIA I Ala~ 3GT

!IV

765

AAT

koM

IN'

U.

A s

CAC

GC1 Ala

MAC

Pho 670 Lou

AAA

Lys Trp

TCC

3cr v

GTG

Va I

OA';

F A nVai I CAG i Gin Leu

ACT

GOT

Gly CA(3 Gin

CAT

Asp

ATT

Ile

GGA

Gly 1230 A la Pro GAT110

TAT

liy 1 Lou

MAT

Aon

OG

Va 1 640

CO

Pro

GAG

Giu

COT

Arg Ala co0 Gly 1720 GcO Ala

CAG

Glu r 1824 1872 190 1968 2016 2064 2160 23 a 4 14C#0 2 44 [A t 8l 1 1A~ G* 1M IM14T \,lu A1, Gi;:n GinlenL 8--11 I 1 I Fro az-, X~ f r-l n 3, R )X3 1 (ii) MOLECULE 'YPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Gly Lou Tyr 9.

*r S 09 *r *9*9 99 9* *r 9 *r S 9 Leu Giy Lou Acp ArUJ 5cr Arq Giu Ile 14£ T'yr tie Me t A0p Ala Tyr try 9, l U)r~ Lyo Glu Aop Sex Ala Thr Thr Gln 130 Tyr Pho Ala A0p Giu tiet Lhou Ala Pr) Asp Lou Gin Arg Ala Ile Lou 115 Lou Lou aly Gly Arg ly Aan r3o Tht8 ValI Ilte Ile Phe 5cr Thr 100 Mer Lou 0 ly Ly,- Lou A a Tyr Ala Glu

H.-O

Ala U: r Arg Arg Ala Pro Vat Oin Arg Thr Tyr Thr Pro VaI 'o Aon Alap Gin Tyr' ciu 55 Itio Lou Lou Ile Aops 135 Ala AoV Ala ArqJ 215 11io ItI 7yr kin lei ly 40 Met His Pho Ala Lyo i2?0 Giu ryr Gin Pro Arg Gin Ala Gin Ala 33 N11r Pro 25 Glu Va1 Gly r 105 Glu ile Gly Lou cr Aam kiln Pro Giu fPz I-1 VvL1 Tyr Ile Giu Pro Leu Pro Aop Val Ala Thr is Ile Tyr Arg Ile Phe 1le Pro Va- l Ala So r Ph. Lou Leu Lou 140 Ala Am bou G~i Ala Ann ffan t Ala 125 Tyr Ala Giu Pro Thr Pho At,; v 23tL 440: a Gr Ser Pro Ala ly Gin So r Ile Ala Mo~t Lou.

Art A1,3 Lcvoi Ala Val Thr Ilo Gly 9$ Pro Arg An Gin Ala I 1 1 Mot Ala c; r

A-

Asp Thr Gluu Pho Ala Glu lie Ly'G Va I 160 Vat Oiu 243 .t; 4. 4" R 123$.

-58 Pro Gin Gin Ala 340 Thr Ala Met Lou 9.

9. 9 9.

SS

9 9 9*9O .9 9 09 .9 0* 9 4 ft.. *99* 4* 9 9 9996 9. 9 9.

*9 9 9e*4 Gin Gin 38S Pro Val1 Lieu Asti 465 Oin Gin A la Pro) $4 S Gin VaI al lae Met C ly 370 Ile Glu Met Ala Tyr 460 Pro Gly 530 phe G ly ow Aom~ 61 G iu 355 Pro Trrp ValI Ala Thr 43S Thr Val1 Lou G~ly Thr Phe Gly 1V: 159S C iy Thr Val1 Ac n Lieu 420 Gin Ala Pro Aon o ly Soo 1 Aon Pro Lieu ValI Pro Arg 405 V4 1 Ala Ala I 10 485 Gin Aop Va I 56$ 11ic V11 I 11-3 Arg Gin 390 Ala owy Lou Met 47v' Pro~ S'er Tyr k~ln I :o 630 Trp Liys 375 Ila 1 Gly Asp 455 Pro Lya Ala Pro Glu "A n Ala 360 Val Gly ValI Phe Gin 440 Lys Trp Gin A=n Ala 520 lThr *rrp Ala 600 Al Ala 345 Arg Thr Ser Ac p 425 Val1 Aop 'Tyr Va 1 Giu Giu Arfy Pho LY0 Ao Lou Pro Asp Ala lie, 410 Pho Gly Lou Ala A14 490 Val1 Tyr o Ile P?.o Th Min> Val Trp 395 Tr So r Lou Tyor Lyc

A

Sol Lou Trp Pra~ G1.

Lo.;~ Ac: P r Va.

Ser 36$ Gin Stir Ile 44S Ala Gly lie Arg "alI I Ic A la ser Asp Gly Ser Thr Va 1 350 Asp Thr Ala 430 Lyn Ser So r Ala A10 Pho Ala Thr Gly Gin C ly 41S Phe Pro Aop Lou 49S Mot Ala Asn pto

VL

K i Lieu Gin Gin Val1 400 Alai Asn Phe Liou Trp 400 ArU Asp c31n fo% 3.

Ar Ie 1 j Tr a*'Y vltAL o o L -3AtA n1 12", 1 0000% off 0:6f to* 0 U*000 *606 T'rp Phe $or Oly Tyr Gly Pr'o Gly Val Val Thr Ser Val Trp Ile C: 705 710 7157' Ph* Asp Asp IHiz Ar Argj Aun Lou Gly His Thr Thr Ala Ser Oly Al 725 730 735 Xlo Lys Asp Gin Ile Se3r Gly Ty r OGu OGly Ala Lys Ser AlA GI 740 74S 7SO ProQ Ma ~Trp Acp Ala Tyr NotL Lyo Ala Val Lo~u Glu Gly Val Pro GI 755 76075 Gln Pro Lou Thr Pro Pro Pro Gly Ile Val Thr Va I Ann IleO Azp Ar 770 775 720 $or Thr Oly Gin Lou Ala Ann Gly Gly Man Spr AZ'g Mu Glu Tyr P h 785 790 79S so Ilo Olu aly Thr GIn Pro Thr Gin Gin Ala Val H ia Giu Val Gly Th 805 810 815 Thr Ile Xlo Azp Asn GIV Clu Ala Gin Glu Lou Lou 820 82S INVORMATIO1N FOR SM.) ID NO 3 5QUWCC CHACTERrST.XCr, li=-fl 2472 baso pairs (BE TY'PE nualoic acid STRNCICII$Zi d~ouble TOPOLCOGz li.near r='TE pARC OSU SThiubl. PlP It WA NA1CrP: CO~dt (W FEATURE:.24t Me ALA~ ,"ly A=n Aczp ArU Glu Pro 1t Oy Arcj 1yo Gly L or-- 741A~ 71. A a ,a Pro 7Ai "I y in L c Val 0l r A Arg A r~ j r rlu A-zp, A~r Aar, A,3 Attp A~ A 3 gotfl %rg ly 0 11 1235-1 MAA COT MAG Ly/s Gly Lys so AA GOC AAA CGC COT AA( CCT COT Pro Arg Lys Cly Lys 55 Gly Arg Ly.

GO MAA CGC 000 Gly Lys Arg Gly

C

C.

C.

C S COO S

C

0ee4

CC..

C

g.e.

CC C C C 0 CC

CC

C C

C

CC..

C C C. C

CC

C. C

CCC.

C

CC..

eg C C

CICC

C

CCC.

TCO

CTC

Leu

ACC

Thr

COT

Arg

AAC

Asn

GMA

Glu 145

ACC

Tr

GAT

Aop Lou

TAC

03TA Lou

TAT

Tvty 06 1 14

ATC

ii c

GC'I

Ala

ATC

Ile

CAG

Gin

ACC

So r 130

OA

Gly

ATC

Ile Pro Phe Ala 1210 Cor

CAG

zCor lot ho GAT CMA Asp Gin

CCOGOCA

Ala Ala AGC MAC Ser Lys 100 OTC; TCG ValI Ser 115 ATT GAG tie Glu CAG GTG Gin V41 OTC MAT Val Asfl COT CTG Arq1 Lou 180 "al Pro AIZA GAA Trhr C, 1u ATC G3A 01y Ala '~or Giu o Mot rtj Lou 4

AN~

LY~

CT)

Val

MAC

Asn

AAA~

Ly s

ATO

Met Cc Arg

ATC

Met 165

ATC

Ile Cc

GAC

As5p A~ra Sor 245£ 3 "Al

AT'I

70

~TAT

Tyr

GAO

Glu

ATG-

Met

ATT

Ile

GCO

Ala 150

GAG

Glu

ACC

Thr

ACT

So r

COT

Arg Ala 230 jv,-, 'rhr Ala TJr COT AGC COT AT) Arg Ser Arg le GGC CGA ATO GTC Gly Arg Met Val ATGOT GTG CTC Met Val Lys Lou 10s ACC COT CCT GC Thr Arg Pro Oly 120 COO Cr? CCG TTT Arg Arg Pro Phe 135 COT CTG ACC TTT Arg Leu Thr Phe AAC AAC COT CAG Arnn Asn Arg Gin 170 ATO ATC TCT TrCG No:t Ile Ser Sor laS OT TTC CCO CAT Oly Phe Pro Asp 200 CAT TTT TAC GAG HiL$ Phe Tyr, Glu 21 5 GTG CTG CA MAC Val Lou Ala Aon eTO ACO CMI CAG Lou Thr Gin Gin 250O TAC T93 COT A Tryr Trp Argj Lyia 03T TAO. AGS2 AMh Arg Tyr 5cr Lyn CTCO 03T ekAC A fCC Lou Gly Gin 5Cr PCAT 000 MOG Asp Gly Lys 75 MAT CTT GAG Asfl Leu Ciu CTO GAG 000 Lou Giu Ala GMA TTT ACC Glu Phe Thr 125 CAT TTC CCC Asp Phe Pro 140 OAT 0CC OAT Asp Oly Asp 155 TTC GOT TTC Phe Gly Phe CCA MAC OT Pro Asn Cly TTG OTG Ov.O Lou Leu Val CAT GAT GGA4 His Asp Oly 220 OTG ACCOCC0 Lou Thr Ala 235 mT GOWI AM Lou Val Lys G %C MO G 0A Ala Aun Glu OAP. CTY ATW C Aop Arcg til 1 GyAop kin C

CTC

Val

CCA

Pro

ACC

Thr 110

OTG

Val1

GAC

Asp

CAT

His

TC

Phe

GAG

Glu 190

CAT

ATC

Ile 3GA i1y kAC kia .ou

PUN

Trr

GAC

Asp

CAC

Ols

CAG

Gin

ACT

OTO

Lou

CT

Arg 175

CAG

Gin

ACT

Thr

ACT

Sor Cac Arg

OW(

Lou

TAO

GAG

Clu ATi 3CMA

*ATO

Met

*TAT

Tyr

GCC

Ala

A

Lys

CO

Ala 160 cvlv Lou

COT

Arg

TTO

Lou

CTC

Lou

ACO

Thr 240

TTC

Phe Me t Lou Ar g 240 288 336 384 432 480 S76 624 672 720 7 U,8 816 864 lvivj TAT TIN- TIA-k Lou Tyr Tyr h 3 1 S1 A,,Ptr2 Va klu I R 1235-1 -6 1- CTA AGC CTC CAC to 0 0 So 06 Lou

TCC

Ser Asn

CTC

Lou

CG

Gly 385

CTG

Lou

ATC

Ile Ala Lou

GCG

Ala 465

ATO

Met Lou Ala ec Pro Va I

ATG

Mo t, Iser

ATO

Ile

CTG

Lou

TAT

Tyr~ 370

GTG

Va 1

CAG

Gin

TTC

Phe

GTG

Val1

GMA

Giu 450

ATC

Met CACi Gin

ACG

Thr

GAT

Aop

CAG

9330 a AT A o 1 Leu

TAC

Tyr

GTO

Val 3S5

GAC

Asp

ATC

Ile

GCA

Ala

ACT

Thr

GAA

Clii 43S

ACT

Thr

GTC

Val1

GCO

A14 :Occ A14

GC(;

51!5

AAT

Aou

A.AC

As n 340

CTG

Leu

ATG

me t

TCT

Ser

A

Lys

ACC

Thr 420 acc C ly

GCG

Ala CG3A C ly Arg

IVIA

Lou 500

CCIN

pro

GAT

ASP

U~u Gly

CAG

Gizi 325

CCC

Pro

COT

Arg Lou

CCT

Pro

CTG

Leu 405

TTT

Phe Ile

ATT

Ile

CGT

Arg 485 ser

AVP

GA Act, Nlhr

,FF.

Gln Ala TOO COT Z'rp Arg CTG CTC Lou Lou ACT CCC Sor Ala 375 CAG CCA Gin Pro 390 COC CAT Gly Asp GAC 'rCG Asp Sor CCO GCA Pro Ala G TG GTC Val Va 1 TCT GAG Sor Clu 470 TCq ATT4 Sor Ile CA:44 CCa Gin Pro A14 Lou Ar~j Argj B3 S Arq o r t Al Lou

MAC

Asn Lett

CCA

Pro 345 Va 1 330

AAA

Lys CMA CAG CMA Gin Gin Gin 360 CGT ccc OTO Arg Pro Lou OCC TT'rT ATC Ala Phe Met MAG GTA AAA Lys Val. Lys 410 GTG CCC CAC Va1 Ala Gin 425 CTG MCG AAM ,eu Lys Lys 440 GTC CAC CC Val Asp Arg CCG CAO TT Pro Gin PMo GGT TCC CTT C1y Ber Lou 490 kAA AT~C TAT Lyo Tyr CP CAIG CCG Wrq Gin Pro V~yr Sor Gbu 111G A.At G71, ~ot Aon Val al Thr 111u pt, Va I pto CAG C CC TTA GTC GOT ATO Gly Met CTc GCG Lou A14 CAG ATT Gin Ile COG GTT Gly Val 380 Gin Lou 395 CAT CTC Asp Lou GAC C Asp Ala CAG CGT Gin Arg TTT1v AGT Phe S c)r Ala Gly 475S GCA XA CGT CTO3 Arg Leu AAT r3- A -1n Gly gor 01-y S4.

AC Pro *ahr sC~r

OTC

Va: LeL AT9 Ile 365~

CAG

Gir

OTC,

Val1

TCC

Stir

CCA

Ala

MAG

Lys 445 G4T Gly

TAC~

CCA

pro

AXAT

Ag

ITA

3AAA GOG CG 1 Lys Gly Ala 335 GAG CGA COT Glu Arg Arg 350 CAT CAA GMk Asp Gin Glu CCG CCC GOT Pro Arg Gly CGTr CAG GAO Arg Gin Glu 400 CCC CTG AAG Cly Val Lys 415 CAA AAA GCC Gbu Lys Ala 430 TTO AGC GAT Lou Ser Asp CMA OTT CGT Giu Val Arq Md:' COT G Ao'n Arg Ala 480 GC AC.T TAT Ala Thr Tyr 495 A c TOO AT Thr Trp 11o 510 Val TrV c Val Mot Lou Ao r, Lou Gly 1008 1056 1104 11isZ 1200 1248 1296 1344 14408 133 133 I GA Ctq MAA (3AT !CM C~ Val Pro Lyc Asp ""An L-rou S8, Gi;2_A A70 27 Ala *Hot Lou R 1235.1 TTGs AC 'ITA AG LeQ kmn Lett Thr 595 -62- CCA ATC GMA GTO CCAG GGA TTC GAG ACC AT G CC Pro Ile Giu Val Ala Gint Aln Phe Gin Thr Ile Ala 600 605 .9 9 *9 *6 I 9 9 0 0*99 9 9* 9 99 99 9 9 9 9909 9 90 09 9 99 99 9 9999 @09

S

900* 99 9 a.

9S ft...

4

AGC

Ser

GMA

G iv 625

OTT

Vali

GTA

Val

CAT

H ia

TTT

Phe

GAT

Aop 705

ATT

110

GTT

Va 1

TTT

Pho

GGG

Pro

MAT(

ko 785 CQT G Pmo 4 AAG G Lyn A~ Or Gl 61(

GAI

Cco Pro

CAA

oin Lou

GCG

Ala 69 0

MAC

Asn

TAT

Tyr M3C Pro 1411

:AA,

:eo v' GO~ Ai~

CGO

GCc Alz 0CC MCh Ala

CAS

Gin

ACA

Pro

TG

Cyc 755

TCG

Coer Pho vot r AAC rAsn

A

'Lys

CAG

Gin

GOT

Gly 660

CGG

Ile

CAG

Gin r.3T Arg

GAM

740 Lou Acp

CAA

Pho C 8 II"I' ccq

CTO

Val

C

Ala 645

ACC

Thr

A

Lyrs

GAC

As3p cc Pro

TAT

Tyr 725

GAT

Aop SGy ,lu liy

OCA

Ala Leu 630

GCC

Ala

GOT

Gly

ACA

Thr

GO

Gly

ACC

Thr 710

CTG

Lou Gly

CAG

C

S, e)r 0: "19 0 CAS 3 GinL CrCG Pro 615

TAT

TAT

Tyr

COT

Arg

OGG

Gly

ACC

Ser 695 3CT Ala

~CA

kl il ~75

'CT

~or

ICT~

CA(

CTG

Loiu

CAG

Gin

ACT

Thr 680

ACG

Thr

CTG

Lou

AAC

A s n

GAT

Aop

CGT

Arg 7 60

AGC

Cjer CA-3 "In

GAC

r' TCT GCG0 CTG SSer Ala Lou AGC 'VTC CCG Ser Phe Pro 035 ACA CTA TOG Thr Lou Trp 650 CTVD OGG GCG Lou Cly Ala 665 ACC MAC MT Thr Asn Aon OTO ACC ATO Val. Thr Ile TAT GOT GCC Tlyr Gly Ala 715 CAG AGO CCA Gin Thr Pro 730 AV 0 G CGTG Mot Gly Val 745 ATC T110, COG C Ile Loui Pro ATO GAG Giu Mot Gin CCGC A1 CMA c Pro Gin Gin C 195 AG1G4 GAG OS 3T G

COT

Arg 620 CAG 0CC CMA CG OCT Gin Ala GlU ArC, ACC ATG Thr

AAA~

AAC

As n

ACC

Thr 700

AGC

Set Thr G AC la 1 SIn Met:

TAC

Tyr

GTA

ValI 68S

TOO

Trp Gly

CCG

Pro

TAG

Trp "165 CA G ;in

COG

CG

Gin Cco Pro 670

CAT

Asp

GTC

Val1

OCA

Ala

CTO

Leta

GAG

Acp 750

ACC

Thr cc(; Pro

CAA

CAG

655

AAC

Asn

ACC

Thr 000

ATG

Met

MAT

Asn 735 kAC Ser Ser Ala 640 Val Lou Trp

CGT

Arg

TCO

720

CTT

Lou

AT

AT

(11c TOG GTA ATC C Sor Val Ile Ala 1824 1872 192110 1968 2016 2064 2112 2160 22.08 2 3 04 23 5 24E, 2448 Pro qln Gin TrA ycs Asp set Asp 810 dly Vali GTC OCTA TOG ATC Ala Oly Trp 11*e 815S A-1& M.T TA, socr Amr INFRIA01iTICIZ 1A2?f ID 11C3: 4: 8YPE 3 acc~ rl 128L6a -63 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met 1 Ala Gly Asn Arg Glu Pro Ile Arg Lys Gly Lys Pro Thr Arg Pro Val *r 9 9 .9 9 9* 9.

9* *r 9 9.9 9 *999 9* .9 6* *r 9 9*99 *999 9 *9*9 *9 i 9q 9* 9909

S

Asp Lys Ser Lou Thr Arg As n clu 145 Thr Asp, Lou Lou Tyr Val Lou Ala Tyr 1 305 Lou Tyr Gly so Ile Ala Ile 0in Ser 131) Gly lie Prog Phe Ala 210 Ser Gln Sor Lou 290 Iot P'he )o r Asp Lys Asp Ala Ser Val 115 Ile Gin Va 1 Arg 195 Thr Ile Gly 0 e r ilo 27 S Pro Lou Lys Asp Gly Gin Ala Lys 100 Ser Glu Val Aon Lou 180 Pro Glu ,ly Ala G~u 2 60 Mct lIu Lou Aorp Gin Lys Val Tyr Asp Asp Lys Gly Lys 55 Lys Ile Arg Val Tyr Giy An Glu Met Lys Met Thr Met Ile Arg 135 Arg Ala Arg 150 Met Glu Asn 165 lie Thr Met ArV Ger Gly Asp Arg His 215 Arg Ala Val 230 ter Thr Lou 245 Arq Ser Tyr Asp Ala Arg Va I 7yr Lou~ 29S A 14a Ser; L ou 310 c n tYR 9 Ala ins Sex Tyr 40 ciy Ser Arg Va1 Arg 120 Arg Leu Asn Ile Phe 200 Pho Lou Thr Trp Tyrr 280 Gy Ilr Lou Arg 25 Glu Arg Arg Met Lys 105 Pro Pro Thr Arg Ser 1853 Pro Tyr Ala Gin Arg nor Gou Ty r Lou Arg Asp Lys lie Val 90 Lou Gly Phe Phe Gin 170 Asp lu Asn Gin 250 Lys Lyus sor PLO(2 vtal 330 Arg Giu Pro Asp 75 As n Leu Giu Asp Asp 155 Pho Pro Lou His Lou 13 c Lou Ala Asp ;1y Myk Tyr Glu Arg Gly Leu Clu Phe Phe 140 Gly Gly Asr.

Lou Asp 220 Thr 'r I Va.

Aor.

Avj Ah'T Aij Mi~t Glu Pro Oly Lys Glu Ala Thr 125 Pro Asp Phe A5 4 Ji1a '3r a. C Asp Met Lys ValI Pro Thr 110 Val Asp His Phe Glu 190 Ile A, n Lou (31u

"AP

Asp Pro Arg Trp Asp G'n Gin $or Lou Arg 1" *11 Thr Ser Arg Lou Asp Arg cly Gin Met Ty r Ala Lys Ala 160 Lou Arg Lou Lou Thr Phe Met Lu Ar 3 ZD 3 2,i F1 12361 Ser Ile Tyr Asn Pro Trp Arg Asn Pro Lys Lou Ala eu Glu Arg Arg 340 345 350 Asn Leu Val Leu Arg Leu Leu Gin Gin Gin Gin Lie Ile Asp Gin Glu 355 360 365 Leu Tyr Asp Met Leu Ser Ala Arg Pro Leu Gly Val Gin Pro Arg Gly 370 375 380 Giy Val le Ser Pro Gin Pro Ala Phe Met Gin Leu Val Arg Gin Glu 385 390 395 400 Leu Gin Ala Lys Leu Gly Asp Lys Ila l Lys Asp Lou Ser Gly Val Lys 405 410 415 Ile Phe Thr Thr PFe Asp Ser Val Ala Gin Asp Ala Ala Giu Lys Ala 420 425 430 Ala Val Glu Giy Ile Pro Ala Leu Lys Ls Gin Arg Lys Leu Ser Asp 435 440 445 Leu Giu Thr Ala Ile Val Va l Val Asp Arg Pho Ser Gly Glu Val Arg 450 455 460 Ala Met Val Gly Giy Ser Giu Pro Gin Phe Ala Gly Tyr Asn Arg Ala 0:00 465 470 475 480 Met Gin Ala Arg Arg Ser Ile Giy Ser Lou Ala Lys Pro Ala Thr 7yr .485 490 495 Lou Thr Ala Leu Sor Gin Pro Lys le Tyr Arg Lou Ann Thr Trp Ile 500 505 510 Ala Asp Ala Pro lie Ala Lou Arg Gin Pro Ann Gly Gin Val Trp So 515 520 525 Pro Gin Asn Asp Asp Arg Arg Tyr 3Cr Glu Cr Gly Arq Va1l Mot L.ou 530 535 540 Val Asp Ala Lou Thr Arg 3er Mot Asn Val Pro Thr Val Aun Lou Gly 545 550 555 560 Mot Ala Lou Gly Lou Pro Ala Val Thr Glu Thr Trp Ile Lys Lou Gly 56S 570 57 S Goo* ValI Pro Lyo Asp G6n Lou Hi Pro ValI Pro Ala Mot Lou Lou G'y A'a 580 585 590 Lou Asn Lou Thr Pro Ile Glu Val Ala Gin Ala Phe Gin Thr IIc Ala 595 600 605 Sor G1y Gly An Arl Ala Pro Lou Gor Ala Lou Arq Sor Val Ile A.a 610 615 620 0" u Aop Gly Lyo Val Lou Tyr Gin 8cr Pho tro Oln Ala Giu Arg Ala 625 630 535 640 Val Pre. A I a r, Ala Ala I,,r Lou Thr Lou Trp Thr Not G3h Gin 7A.

645 650 Va A Oi Arq G I y 1,r M7. l Art Oln Lou tGly Ala Lys Ty r Pxr A o n Lt) Iti Lou A 33y LyG T.r l'y Thr Thr kin An kmn Val A~p Thr T: 67 680 rho Ala Ghy A p 1y Sr AI,,r VaI Thlr I lo T Tk a' J1 Ar i j 9 S) S1235.1 Asp Asn Asn Gin Pro Thr Lys Lou Tyr Gly Ala Scr Gly Ala Met Sor 705 110 715 720 Ile Tyr Gin Arg Tyr Leu Ala Asn Gin Thr Pro Thr Pro Lou Asn Leu 725 730 735 Val Pro Pro Clu Asp Ile Ala Asp Met Gly Val Asp Tyr Asp Gly Asn 740 745 750 Phe Val Cys Ser Gly Gly Met Arg Ile Leu Pro Val Trp Thr Sr Asp 755 760 765 Pro Gin 5cr Leu Cys Gin Gin Ser Glu Met Gin Gin Gin Pro Ser Gly 770 775 780 Asn Pro Phe Asp Gin Ser Ser Gin Pro Gin Gin Oln Pro Gin Gin Gin 785 790 795 800 Pro Ala Gin Gin Glu Gin Lys Asp Ser Asp Gly Val Ala Gly Trp ile 805 810 815 Lys Asp Met Phe Gly Ser Asn 820 INFORMATION FOR SEQ ID NO; SEQUENCE CHARACTERISTICS: LENGTH: 2049 base pairs TYPE: nucleic acid STRANDEDNESS: doubl'v TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genoric) (vi) ORIGINAL SOURCE: ORGAtTISM: Streptococcus pneumonia( STHAIN: PM I (vii) I1MEDIATE SOURCE: LIBRARY: PCR cloning (1 CLONE: pARC 0512 Soluble PBP IA del 38 (ix) FEATUVRE: NAMEAFEY: CVS LOCATIN: 1.-2049 (ix, FEATURE: NAME.KEY: mnatp~ptite LOCATION: l..1046 SEQUENC-ue DMe3PIiIMC: SEQ ID NO: ATO) G-T CCT AG- CTA TCO Gk3 A-3',T AA A h CTAT (CA ACA ACT TOT A3T 48 Met Ala Pro Sr Lou sor Glu Sor tLYG Leu Vat Ala Thr Thr Ser Sr 1 5 10 is AAA ATC -,AC t3A.- AT AAA A-T CAA O TC AIT GO-T GAO k ,T TCT i Lys n yr Asp Aon Lys An Gin Lou Ile Ala Atop Lou '33y Sor Iu cGc 3O 03 GTC T A4 3OT AAT 33AT ATT CC"- ACA tCi !Tt 3 OTI" AAl Arg Ar Val Aon Ala Glr. Ala Asn Asp 1i0 Pro Thr Azp Lou VataI 40 4 In 1236- 1 OCA ATC OTT TCT ATC GAA GAC CAT CG TTC Ala Ile Val Ser Ile Glu so 0 *9 9.

0 00 9.

40.0

GAT

Asp

TCC

Ser

TAC

GAA

alu

ATC

Ile

GGA

oly 145 Lou

AAC

Ann

MAC

Ac n

CAG

Gin Lou

GAA

olu Thr G~lu

ACC

Thr

CTC

Leu

TTT

Phe

OCT

Ala TTX3 Lou 130

ATO

Me t

ACT

CAA

Gin Lou

TAT

Tyr 210

~AA

Lys 7'rC yva 1 3G*3 11ly Prp

ATC

Ile

CAA

Gin

TCA

Ser

TG

Trp 115

ACC

Thr

CAG

Gin

TTA

Lett

TAT

Tyr OT1C Val1 195

GAG

Glu

TCA

Ser

A=G

I le

ATG

Me t

GAT

Asp 27r ctA, 0f111

COT

Arg

COT

Gly

ACT

Thr 100

TTA

Lou

TAC

Tyr

ACA

Thr

CCT

Pro

GAG

Asp 180

TTA

Lou

A

Ly-s

GCA

Ala

MPT

An 13AT

MIT

I le

ATC

Ile

GGA

Gly

TCO

5cr Al a

TAT

Tryr

GCA

Ala

CAG

Gin 165 CCe Pro

TCT

Sor

GCA

Ala

AGT

Sor

CAA

Gln 245

GTC

Val

TAC

Tyr Ala

CTG

Lou 70

TCA

Ser

ACT

Thr

ATT

Ile

ATA

Ile

OCT

Ala

ISO

TTA

Lou

TAT

TIyr

GMA

Olu

GTC

V4 1

MAT

Ann 2630 L3TT va

TAC

Tyr

AINT

As p 55

OGA

Gly

OCT

Ala

TCC

So r

CG

Gin

M.T

Asn 135

CMA

Gin Ala

TCA

So r

ATOG

Met

MAT

An 215

TAC

Tyr

GMA

Giu

ACA

Thr

ACA

h r OCT TTC Ala Phe CTC ACT Lou Thr GAC CAG Asp Gin 105 TrrA GM Lou Giu 120 MAG GTC Lys Val MAC TAG Asn Tyr TTG CTo Lou Lou CAT CCA Hisc Pro 185 AA MV Ly n Ann 200 ACA CCA Thr Pro COT OCT Pro Ala

GGA(A

Olu Glu AA? GTh Acn va 1 265 Aop Oiu 280 All" G71

TTG

Lou

CAA

Gln 90

ACT

Thr

CAA

Gin

TAC

Ty r

TAT

Tyr

OCT

Ala 170

GMA

O lu

CMA

OMr.

TAG

TIyr

ACA

Th2r

OAC

TAC

GAT

Aszp Rio Arg Phe

A

LyS

ATG

Met

GT

Oly

OGA

O ly

GCA

Ala occ Oly

ACT

Thr

AVG

Met

CAA

Gi1n ValI

OCA

Ala

TCT

So r 140

A

Lys ATro Met 0CC Ala

TAG

TIYr

CAT

Asp 220

GAT

Asp

TAT

GMA

Glu Ala T T Sor

ACC

Thr 125

AAT

Ac n

GAC

Asp

CCT

Pro

CAM

Gin

ATC

Ile Gly

MAT

Asn

AAC

Ann A la

TAT

T

lyr 4111313

AAC

A-M

TTC GAC CAC Phe Asp His COC AAT CTG Arg Asn LeQ CAG TTG ATT Gin Lou Ile ATT TOT COT Ile Ser Arg AGO G00 ATT Ar'g Gly Ile CMA AGC MAT Gin Ser Asn MAG TTG ACT Lys Lou Thr MAG OCT CAG bya Ala Gin 110 MAG CAA GA Lys Gin Giu 000 MAC TAT Gly Ann Tyr CTC MA" MAT Lou A.cm, Ann 160 CG OCA CGA Gin Ala Pro 175 OAC COG CGA Aop Arg Arg 190 TCT GCT GAA Secr Ala Olu CTA GMA A0OT Lou Oin SOL.

rAC CTC AP0 t1yr Lou Lyn

SACA

1.ou Lou Thr .4MA AAA CAT 'In tLyc His ILA: 01v.

Pro A0p ASP4

A

;wl Lyo Va I 192 240 288 36 384 432 480 52"8 87120 L, 4 1 r Ala Gin Lou Ala C~xAT G TCA -T V- 3' Art? lo GIln 0or A=n Val Oer Pho Mly .F1 1235.1 ATT :AtAC CMA GCA GTA GAA ACA AAC CGC GA( 110 Asn Gin Ala Val GlU Thr Ann Arg AsV 325 33( -67- TGG~ OGA Trp dly TCA ACT AVG AAM Sor 'rhr Met Lyn 335 4 4S** *4 4.

S.

*441 4

CCC

Pro

ACT

Thr

ACC

Thr

CAA

CG.n 385

MAC

As n

ATC

Ile

ACC

Thr

GCT

Ala

ATC

48~S

OTC

Va 1 ATO GCT Ala I] Glu G SclA A c Glu r.

54S

AT

Alz CCl *Prc 370

TAC

MAG

Lys

GAC

Asp

,AA

Tyr 460

CAT

~I is, Sly lot

;AA

lu,

TA

0- U

AT

CACA

e Thr

ACT

~Thr 355

OTT

Val

CC

A14

GTC

Val

TAC

Tyr

TCA

So r 43S

OCT

Ala

AAA

Lys

ACT

Thr

MAA

Lys 9] CTC c Lou I ATT G Ile 'P UrV Phe

TA

j(*lA

GA

Asi 34(

AT(

TI

Tyz

CTO

G ly

CCA

Pro 420

OAC

Ala

.,TC

"GT

krg ~hr 0 0

~CT

A

0 V41

M.C

Ann

CAA

CTC

Leu 405

AG?

A

Lyn Phe GTt' Vat 1 Ala t 485 AM C Asn Ai AA1 c An I

CA'

H i

CA;

Glr 390

MAC

As r

AT'I

AAA

Lyn

GCA

.\la Tho 170 eot .ou

'AC

C

GAT GAG Asp Glu 360 GA? AGO Asp Arg 375 TAT CT CCT CC Tyr Ala Pro Ala

TTG

Leu 345

CCC

Pro

GCC

Gly GAG TAC GOT OTC Glu Tyr Gly Val TAT MAC TAC CC? OGG ACA MAT ~TCG CQA $or ArVt CCC CC Arg Ala CAC TAC Hisn Tyr TAT OCA Tyr Gly 440 MAT OCT Aon Gliy 455 AGT GAT 83or As0,p MAA GMA Lys Glu Ai3T TAT' wi T AM.A C3ly L.

A7e AA~ 11') 1Lv r£35 A0243 21"

MAC

Asn

MAG

Toys

TCA

Ser 425

GCA

Ala

OA

C ly C ly

ACG

Thr G0A A rhr

VA

o Tyr Ann Tyr Pro 365 TAC TTT ;OC MAC Tyr Phe Gly An 380 GTC CCA CCC OTO Val Pro Ala Val 395 ACT TTC CTA MAT Thr Pho Lou An 410 MAT 0CC ATT TCA Asn Ala Ile Sor ACT ACT GMA A.C Sor Ser Oiu Lyn 44S

ACT

Thr

ACT

ser

ACA

Thr 490

ACT

Thr o r

TAC

Tyr

GAM

Glu Ala

CA

Cly tGin

TAT

Tyr 460O

A

Lyn

TAT

rg ph(e S4.,3 TAC GAG TCA Tyr Glu Ser 350 Lys GAC2 Glu

ATO

Mo t

MAT

ktm vaI G l

ATC

I le

GAA

Glu

OCT

Oly

ACT

Sor 430

ATOG

Mot C CA

TTC

P he 510

A

la Th

AC(

Th

AC~

Th2

CTC

Lot 415

AAC

Asrn

OCTI

Ala

ATO

Met

A.:C

Thr 495

TAT

A

Thr jT Atm r Lou C' CTA Lou 400 IGly

*ACA

Thr 'OC T Ala

TAT

T

Aim Ant) Lou Asp) Thr 1008 10S6 1104 1152 12 0 0 12 48 1296 1344 14 1483s 1516 1632k 4<r Y* A> t S

A

Ala Ala Lyn Va' t3 4%.r Ar~ 'e~r Not ~4 £3 C Thr t-t L, ti nfIl o t Gl c hI it] I MAT CCA GAG CAT TGG MAT ATA CCA GAG 0OG CTC TAO AGA MAT GOA GMA 18 24 Aon Pro Giu Asp Trp, Asn Ile Pro Giu Gly Lou Ty'r Arg Asn Gly Giu 595 600 605 TTC GTA 'N'T AMA MT OT OCT COT TCT ACG TO ACC TCA COT OT CCA 1872 Phe Val Phe Lys Aon Gly Ala Arg Ser Thr Trp Ser Ser Pro Ala Pro 610 615 620 CMA CMA CCC CCA TCA ACT GAA ACT TCA ACC TCA TCA TCA OAT ACT TCA 1920 Gln Gin Pro Pro Ser Thr Giu Ser Ser Ser Ser Ser Ser Asp $or Ser 625 030 635 640 ACT TCA CAG TOT AGC TCA ACC ACT CCA AGC ACA MAT MT ACT AC O ACT 1968 Thr Sor Gin Sor Scr Ser Thr Thr Pro Scr Thr Asin Asfl Ser Thr Thr 645 6506F ACC AMT CCT MAC MT MAT ACG CAA CMA TCA MAT ACA ACC OCT GAT CMA 2016 Thr Ash Pro Asn Aon Asn Thr Gln Gin Ser Asn Thr Thr Pro Aol Gin 660 665 670 ':AA MAT CAG MT COT CMA CCA CCA CAN OCA TA 204 9 Gin Asn Gin Aon Pro Gin Pro Ala Gin Pro 675 680 INFORMATION FOR SSQ ID NO- 6- W~ SEQUENCE CHARACTERISTICSt LENCTH:t 682 amino acitis TYPE: amino atid TOPOLOGY: linear (ii, MOLECULE TYPE: protein SEQUENCE UOCRIPTION% SEQ ID NO; 6: Met Ala Pro Ser Lou Sor Giu Ser Lya Lou Val Ala Thr Thr 8or Ser 1 510 1 Lya Ile "!yr Aotj Aon Lys Aon Gin Lou Ile A14 Aop Lou 0.1y 0cr Wiu Arg Arg Val A5n Ala Oln Ala Aon Aop Ile Pro Thr Acp Leu Val Lys 3Ei 40 4 Ala Ie Vial 3Cr Ile Sit: Acp Hih Arg Pho Pho Aop Ific Arg GlIy llle Aop Thr le ArU Ile Lou (Ily Ala Pho Lou Arq Aon Lou G,"n 3c~r Pion Scr Lou 1 In Gly (31y 3cer Ala Lou Thr Gl1n Gin Lou le Lys Lo- 11hr Tyr Phe Ocr Thr 'jot 01hr 0)cor Act Gin Thr I I3ocri A.-ij Lyc- Ala G-;An 105 13 Ile Lo u n~th Ty r ti' c Ly~ Va~ I Tyr 'Met Oot 1vr. k- V Ty 130 .3 Leu 3Cr Lou Pr T a Lou L 0u A l A V, 3,11Aa R 12351 S69-- Pro Tyr Ser fi Pro Gu Ala 185 Ann Gln Tyr Asp 180 Ala Gin Aop Arq Arg 190 44 6 4 09 .4 $006 04 44.4 4 444, Arn Lou Val 195 Gin Tyr Glu 210 Lou Lys Ser 225 Glu Vat 1Il Thr Gly Met Lou Trp Asp 27S Glu Lou Gin 290 ZXo Ala Gin 305 Ilo Aon Gln Pro5 le Thr Thr Ala Thr 355 Thr Pr:o Val Gin Tyr Ala 385 Aor, LyJO Val Xho Asp 7yr I Thr Glu Cor 43S Ala 'Pir Ala 2 Ilo 1 Ly0 t VVa 1 Gl Thr2 Mt i 73 Lou d Alca Ttp Lou I I S Lev Ly.

Ala An Asp 260 11c, Va 1 Lu Al Asp 340 I 1 Lou 31y Pro %op Ia tq Sor Ala Sor Gln 245 Val Tyr Ata, Gl.

Val1 325 Tyr Va I Aon Gin Lou 405 Maa 483 (11 1 VaI As 230 Val TIyr Ann Sor Ma 310 Glu Ala His TLp Gin 390 A c r.

ib Ala 4 0 I Me t Ast Ann 215 clu Thr Thr Thr Thr Pro Asp Asp Arg 4%S zLr., p Lys 200 Thr Pro Olu Asn Asp 280 Ile H is Asn Ala Glu 360 Arg A1a Tyyr ly Alu XAJ~l Ann Pro Ala Glu Val 265 Glu Val Gin Arg Lou 345 Pro Oly Ann Lys 425 Ala GIy Glyy Thrr 11e Thr Asp 220 Tyr Met Asp 235 Thr Gly Tyr 250 Asp Gin Glu Tyr Vat Ala Asp Val Sor 300 Sor 5 er m 315 Asp Trp Gly 330 Glu Tyr Gly Tryr An Tir Tyr Phe Gly Va Pro Ala 395 Thr Pho Lou 410 Acn Ala le Gor Cor GIu Thr Tr r 4 Oer Glu 0 'rhr A 1 a 'lr r 4 90', Gily As n An Ala Tyr 285 An Vall S3or V11 I Pro Val A=n 3O~u r Lycn 44-(3 7iu Ilrx A'y'i C, C, Lev Lou Gln 270 Pro ly jor Thr Ityr 350 Gly I io Glu G~ly Sor ~3 "1 Mc.* 4,a Gli Lot Lo5 Lys rp Lys Pho MYr 33M Glu Thr Thr Th r Lo u 41 c-, A n A l.t

IS~

i Sor i Lys 240 I Thr His Asp Val Gly 320 Lys Sor Aon Lou Lou 4 0 qly Thrr Ala 4 K;L As!>~ Gin Gly Tyr Il Sor Ala Glu 205 fBe tSPr; ~tL;f: If;:: 4 rll a A t RI 1235 1 Oiu Lett Phe Ala Gly Tyr Thr Arg Lyni Tyzr Ser Malt Ala Val Trp Thr 545 550 55S 560 Cly Tyr Sor Asn Arg Lou Thr Pro Lou V41. Oly Asn Gly Leu Thr Val.

570 Ala Ala Lys Val Tyr Arq Ser Met Met Thr Tyr Lou Ser Glu Gly Ser 580 585 590 Asn Pro Glu Anp 'rrp Aoin Ile Pro Giu Gly Lou Tyr Arg An n Gly Glu 595 600 Pho Val Phe Lys Asn Gly Al4 Arg Ser Thr Trp Scr Ser Pro Ala Pro 610 615 620 Gin Gin Pro Pro Ser Thr diii Ser Sor Scr Scr Scr $or As3p Scr Ser 61.5 630 63S 640 Thr 8or Gin 5cr Sor $or Thr Thr Pro Sor Thr Aon Aon Ser Thr Thr 645 650 655 Thr Aan Pro Asn Aon Ann Thr din Gin Ser As n Thr Thr Pro Asp Gin **660 665 670 Glfl Asnf G~n Aon Pro GIn Pro Ala Gin Pro INVOP~hT1ON FOR SSE ID NOt it I: SEQUEN'CE CHARACTERXSTCIM LENGTH: 844 amino 41:ida TYPE: am~ino acid Mt) TOPOLOGY: linear MOLECUL!V PYMn: poptilo (,vil ORIGINAf 3'URCE~: 4* )AN E(hric~iia coli WD WCNE: VARC0438 PBP 15 OQAA (Xi) 3QE.1,C MEOCRPTIC,1: 51EQ ID NO: 7: Mot Ala Gly Aon A,,pV Arg kGlu Pro Ile Gly Arg LyG GW4' Lycs Pro Thr 1 510 i Arq Pro Val Lv 31r, 1111- Val Sor Arg Arj Alg Tyr Ju1 A zp 1 A 3 r A 0 ZA0 41 33 Aop lryr Aop Acp Tyr Aop Aop Ty r G 1u Aop, glu Gh1u P toNotPA 3540 4 r G yoy Gly Lypo Gly L-o "IV ArrJ cy Pr: Arjy~ ~A Trp Lcu 1% e I Loe- u 11y 11 Ala t~v 'Va~ Zr? 7 ,a z ALA :lo0 Tyr (I Va*"jI., k" Aqsk1. "7 t- 2, j tA A'.r t. V 4~j p 'It Lou Ala A 1a Va4 1~ 11y At Va

E

Ft 1236,I -71-- Met Thr Il1 Stor 1's Asn 120 Lou Oiu Pro Asp 115 Gin Mt Val Lys Lett Lou 125 Glu Ala 130 Phe Thr 145 Phe Pre Gly Asp Gly Pho Asn Gly 210 Lou Val 225 Asp Gly Thr Ala Thr Gln Tyr Arg Val Asp His Pho 19S Gin Asp cay Gln Sor Lou 180 Arg Gin Thr Arg 260 Ala Lys 165 Ala Asn 150 Glu Thr G It 135 5e Gly Ile

I

Lou Asp Pro Arg Lou Lou 245 Thr Lou Lou 230 Tyr Val Val Ser Lys Mt Thr Arg Pro Gly Glu Phe 21S Ala SOr Gin Ile Oin Val Arg 200 Val Thr Ile Gly GIu Val Asn 185 140 Arg Arg Asn Lou Ile Thr Mo 6 0 set* 0 10 to I *00*0 Otto 00 6 t**I .4 I* I

I

III.

Pro Glu Gly Ala 26S ser Arg 235 Ala Thr G1' 22 Hi Val Lotu Arg

LOU

Asn Pro Phe Thr Pho 175 Avg Gin 190 Asp 160 Asp Pho Val Lys Ann 275 Lou 'he Lou 5r Sor Glu Arg Ser Tyr Ann Glu 290 Ala Tyr Mt Ala Lou Ile MEt 295 Met Asn Glu Arq 305 Asp Arg Not Ala Zio 3855 Val Lou Lou 1Ahla Ile Lou Giu Lou Aon Pro ValI Lou Voo Ile Vat {iov Ala 4S,, Glu Val Lys 3 C' C Gin Asp Pro A'-g in I le Glu 340 0 ly Avg Gin Avg 4206 G 41- 0 Avg 325 1lu Ala Arg Gin GIy 4003 Glu Li,3

ALA

Tj v 310 Gly Lou Lon Lou Pho Soy no Lou Tyr Va P PLo I't Pro Lou

GO~

Val As hop Ala 4 40 kAU Lou AsP 345 Aoll Lou Lot t~iys Asp Al Val T; 31 Ala 3*' 330 Gin GP Pro Tt A rg, Lo Lou C o 39 Pro G 4O PLor rEio l~ r3

P

P~

Arg 300 Lou Lou Arg U8Z Al a Pr,, 41vt Ala1 t lo 205 e Pho 0 Phe Lou Thr Trp 285 T jr Lou Asn Avg 44c' Tyr Ala Ala 270 Arg Gin Lou 3006 3 i ;a Pro Pv., SSor Asp Glu Ala Lys Lys So r Pho 330 Ila tln l Lou 4 pro Lou His 240 Lou Lou Ala Asp liy '1 L08

'C

re; ~1~3 a~s iz~n Lcots kGlu Thr M 'Ae Val £1 1 4 R 12353 1 ao~r Oly Giu Val Arq Ala Met Val Oly Gly S3or Giu Pro Gin Pho Ala 485 490 495 Oly Tyr Asti Arg Ala Met Gin Ala Arq Arg Ser Ile cGly SorL Leu Ala S0o Ojos 510 Lys Pro Ala Thr Tyr Lou Thr Ala Lou Sor Gin Pro Ly; Ile Tyr Argi 515 520 52-5 Lou A~n Thr Trp Ile Ala Asp A14 Pro Ie Ala Lou Arg Gin Pro Aon 530 535 540 Gly Gin Val Tr or Pro Gin Aan Asp Asp Az'q Arq~ Tyr S3or Giu o r 545 550 555 Gly Arg Val Met Lou Val Aqp A la. Lou Thr Arcj Sor Met Aon Va I Pro 565 570 575i Thr Val Asn Lou Gly Mot Ala Lou Gly Lou Pro Ala Val Thr Giu Thr $as 590 Trp Ile Lys Lou Gly Val Pro Lys Asp Oin Lou His Pro Val Pro Ala 595 600 ot Lou Lou Oly Ala Lou Aan Lou Thr Pro Ile Oiu Val Ala Gin Ala 610 615 620 Phe Oln Thr rie Ala So r Gly Oly Aon Arg Ala Pro Lou So r A14 Lou 625 630 635 640 .Arg S3or V41 ile Ala G1u Aop Gly Lys Val Lou Tyr Gin Sor Pho Pro 646 6S 0 666 OlGn Ala Glu Arg Aid Va I Pro Ala Gin Ala Ala Tyr Lou Thr Lou T r: 660 6605 6110 Thr Not GIn CGiln Val VI 7 Gin Artj Glj Thr G ly Atg (I n Lo j'y A la 6 5 600 3 S Lys Tyr Pro Aon Lou flia Lou AlA Oly Lyo T h v 34y Th T.1tr A-sn ilc:.

C 069009 Aon Val Aop Thr Trp Plio A14 Gly Ile As-p (fly Oor Thr Val Thi- Ilo r itrr Val (31y Aop Atm Gin Pr._ 11hr Lyo Lozi T1ir Gi I c 730S 733 Ser (fly Ala I-o ZQr 110 Wr q 1 r Arq I Lou, Ala kt in nl-t I Ple1 745 C Thr p~lou A-Il Loe u 'Aro pfro Giu Ansp 1o Ala As'p 110" Q1Y A01) %ir Asp C;I Y. Aon R.o Iva cyq30tk" Gly GY 11 'AoC Lou li' "a1Tp Tbr r ATzp Ns in Co r lou Cyon 2on or 'tlf Vc Y n Gikn G;I n ptrj or Jfy A t ~o rh Agv '13In So So I os t CGln Pro Ail e (jIn p Ala ;n in ;L 2.ior t A "at Ai la LZ' ki "!-tp n 1236 j INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICr: LENGTH: 844 amuino acids TYPE: amino acid TOPOLOGY: 'ie~ar (ii) MOLECULE TYPE: popt:do (vi) ORIGINAL SOURCE: ORGANIGM: Eschorichi c01i (vii) IMMEDIATE SOURCE: CLONE: pAR(0468 PBP ID QQLL (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Mo Ala Gly Aon Asp Arg Glu Pro Ile Gly Arg Lys Gly Lyo Pro Thr 1 5 10 is Arg Pro Val Lya Gin Lyr Val Sor Arg Arg ArG Tyr Giu Asp Asp Asp 2s Asp Tyr Asp Aop Tyr Anp Asp Tyr Glu Asp Giu Giu Pro Met Pro Arr 40 Lyt Gly Lyo Gly Lyo Gly Lys Gly Arg Lyo Pro Arg Gly Lya ArG Gly $0 sO Trp Lou Trp Lou Lou Lou Lyo Lou Ala iee Val, h e Ala Val Lou Ile 0 Ala IA e Tyr Gly Val 71r Lou Asp Gin Lyc tic A rt 0or Avg ile Asp 90 31y Lys Val Trp Gin Lou Ala Ala Ala Val Ti r Giy Ag Met Pt VaI Aon 130 130 1410 u Glu pecA p Mct~j Th*rh ItiC 5c Lz Azr GI'i P!4o~ Val L3 tLou L? uP Glu Al) r 'r hpr or, yn R3 Ar I Gln Va I5cr L i% rcL Thr AVn Po cly d;u (3 1 a3 13514 4.

G*Iy h Ph "rr VAt 3n la As, 3og to gLu Mcl oio Ar or tc r1F1w Se 145 tOO 1%(I L Pho pPr AoP 3cr Ly.3 iuGi Gn VaI Arq Ak a lAp Lou (,hr I Pt A f14 1,43 lyt aZ Lon~i Ala nrhtsp tic Val1 Ann ?~soe GJ: As:; A2; AE~t Gn E:h~o 130 135 100;i Oly* trhto Pho Arg Loua A~icp Pro ARt Lu lie Thr ;Ct tie Sor 3cr' hY As 1y Jtu 9P I n ArJ Losu -hor- Val ~ro Ar~ Locr 31y" I-he Pro Ag Lsu Loq Th VaIAo 1 tt i Lulu Aa T h. t- 3u Z04) Atrl MN Me L-u 111: 2~4 A0[, t~ 3cr ir Ocr Mtc ct l Ala Val Lou Ala A n Lou 2Sg rAl Ala u u ls~u i I I R 12351 Arg Ser Val Ile Ala.Glu Asp Gly Lys Val Leu Tyr Gin Ser Phe Pro 645 650 655 Gin Ala Glu Arg Ala Val Pro Ala Gin Ala Ala Tyr Leu Thr Leu Trp 660 665 670 Thr Met Gin Gin Val Val Gin Arg Gly Thr Gly Arg Gin Leu Gly Ala 675 680 685 Lys Tyr Pro Asn Leu His Leu Ala Gly Lys Thr Gly Thr Thr Asn Asn 690 695 700 Asn Val Asp Thr Trp Phe Ala Gly Ile Asp Gly Ser Thr Val Thr lie 705 710 715 720 Thr Trp Val Cly Arg Asp Asn Asn Gin Pro Thr Lys Lou Tyr Gly Ala 725 730 735 Ser Gly Ala Met Ser Ile Tyr Gin Arg Tyr Leu Ala Asn Gin Thr Pro 740 745 750 7 Pro Leu Asn Lou Val Pro Pro Giu A~p Ile Ala Asp Met Gly Val 755 760 765 p Tyr Asp Gly Asn Phe Val Cys Ser Gly Gly Met Arg lie Lou Pro 770 775 780 Val Trp Thr Ser Asp Pro Gin Ser Lou Cys Gin Gin Ser Giu Met Gln 785 790 795 800 Gin Gin Pro Ser Gly Asn Pro Phe Asp Gin Ser 5cr Gin Pro Gin Gln 805 810 815 Gn Pro Gin Gin Gin Pro Ala G'n Gin Giu Gin Lys Asp 5cr Asp Gly 820 825 830 Val Ala Gly Trp Ile Lys Asp Met Phe Gly Ser Asn 835 840 INFORMATION FOR SEQ ID NO: 9: Ci) SEQUENCE CHARACTEXRISTICS: LENGTH: 83t amino acids TYPE: amino acid TOPOLOY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: CA) ORGANISM: Eseherichia cali (vii) IMMEDIATE SOURCE: CLONE: pARC0469 PBP 1B del 8 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Mot Ala Gly Asn Asp Arg Glu Pro Tie Gly Arg Ly- Mly Lys Pro Thr 1 5 h1 iS Arg Pro Val Lys Ln tys Val Sor Arg Arj Arg It.,r glu AoF Asp Asp -0 125 z" Asp "Ayr Asp Asp Tyr Asp Asp Tyr Glu Asp Gu 3.u Pro Mo Pro Ar 3S 40 Lys Gly Lys C,'v Ly5 1 tyn iy Arq Lys Prc Arg y s Arg Gy Ilrr II R 1235-1 -76- Trp Leu Trp Leu Leu Lys L.ea Ala Ile Val Phe Ala Val Leu Ile 9.

.9 9 9.

.9 *r 9 9. 9 99 9.

9.

@9 9 9*9 9 9.

69 9 9 @9 9 Ala Gly Leu Glu Phe 145 Phe Gly G ly Asn Leu 225 Asp Thr Glu Met Glu 305 Leu Asp Asn Lou Met I 385 ser I Lys I lie Lys Glu Ala 130 Thr Pro Asp Phe Gly 210 Va1 Gly Ala Arg Asp 290 Val Ala ;In Pro krg 370 ?r5 eul Tyr Val Pro 115 Thr Va 1 Asp His Phe 195 clu Asp lie Gly Ser 275 Ala Tyr Ser Gin Trp 355 Lou Sor Gin G y Gly Trp 100 Asp Gin Gin Spr Leu 180 Arg Gin Thr Ser Ara 260 TY r Arg Leu Lou Ala I 340 Arg Lou Ala Pro As~ 4 Vai Gln Met Tyr Ala Lys 165 Ala Lou Arg Lou eu 245 Thy Trp T'yr Tyr 325 Lou kon Frq krg Ua I S Tyr Leu Asp Gln Lou Thr Arg Asn 150 Glu Thr Asp Leu Lou Lea 230 Tyr Va I Arg Ser Gin 310 Ty r Lou Pro GIn Pro 39 Te Va Ala Gin 135 Ser Gly Ile Pro Phe 215 Ala Ser Gln Lys Ly s 295 Ser Phe Lys Gin 375 Leu mo r L7,,, Ala Ser 120 Val lie Gin Va1 Arg 200 Val Thr Ile Lou Ala 280 Asp Gly Gly CGy Lou 360 GIn Gly 3 In op~ Ala 105 Lys Ser Glu Va1 Asn 185 Lou Pro Glu Gly Va 1 265 Asn Arg Asp Arg Met 1 345 Ala I Ile Lcu Lou 4 -s Lys 90 Va i Asn Lys Met Arg 170 Met Ile Arg Asp Arg 250 Lys Glu Ile Asnn Pro 330 ;a1 cu Ile aIn 10 e r Ile Tyr G'1v Met Ile 155 Ala Glu Thr Soy Arg 235 Ala Asn Ala Leu Giu 315 Val Lys Glu Asp C Pro I 39?5 Gly Arc Gly Met Thr 140 Arg Arg Asn Met

GIN

220 His Val Tyr Glu 300 Ile 31u ly rg :In 380 rg In1 Ser Arg Val 125 Arg Arg Lou Asn Ile 205 Phe Lou Phe Met 285 Lou Arg Glu Ala Arg 365 Clu Gly C lu tys Arg Met 110 Lys Pro Pro Thy Arg 190 Ser Pro Tyr Ala Lou 270 Ala Tyr Gly Lou Ser 350 Asn Lou Loa Ile 430 Ile Val Leu Gly Phe Phe 175 Gin Ser Asp Glu Asn 255 Ser Lou Met: Phe Ser 335 lie Lou T~yr j'ai 3In 415 Pho Asp Asn Leu Glu Asp 160 Asp Phe Pro Lou His 240 Lou Ser le Asn Pro 320 Lou Trv Asp 430 Ala Thr III I slC C I R 1235-1

-I

-77- .Thr Phe Asp 435 Ser Val Ala Gin 9 9.

*r 9 S

S

S S 9.

S.

I

9**S Gly Ala 465 Gly Arg Leu :Pro Asp 545 Leu Gly Asp Thr Asn 625 Ljys Gln Gly Gly Tie 705 Gin Arg Giu S usr Ile 450 Ile Gly Arg Ser Ile 530 Asp Thr Leu Gin Pro 610 Arg Val Ala Thr Lys 690 Asp Pro Tyr As p ;Iy f7 0 Pro Va1 Ser Ser Gin 515 Ala Arg Arg Pro Leu 59s Ile Ala Leu Ala Gly 675 Thr Gly Thr Lou Tie Gly Ala Val Glu Ile 500 Pro Leu Arg Ser Ala 580 His Glu Pro Tyr Tyr 660 Arg Gly Ser Lys Ala Ala Leu Val Pro 485 Gly Lys Arg Tyr Met 565 Val Pro Val Leu Gln 645 Leu Gir Thr Thr Leu Asri Asp Arg Lys Asp 470 GIn Ser Ile Gln Ser 550 Asn Thr Val Ala cr 630 Ser Thr Leu Thr Val 710 Tyr Gin M3 lbe Lys 455 Arg Phe Leu Tyr Pro 535 Glu Val Glu Pro Gin 615 Ala Phe Leu Gly As n 695 Thr Cly Thr G ly Leu f75 Asp 440 Gln Phe Ala Ala Arg 520 Asn Ser Pro Thr Ala 600 Ala Leu Pro Trp Ala 680 Asn Ile Ala Pro "al 760 Pro Arg Ser Gly Lys 505 Lcu Gly Gly Thr Trp 585 Met Phe Arg Gln Thr 665 Lys Asn Thr So r Thr ~a 1 Asp Val.

Lys Gly Tyr 490 Pro Asn Gln Arg Val 570 Ile Leu Gln Ser Ala 650 Met Tyr Val Trp Gly 730 Pro Trp Ala Ala Giu Lys Leu Giu 475 Asn Ala Thr Val Va 1 555 Asn Lys Leu Thr Va 1 635 Glu Gin Pro Asp Val 715 Ala Leu Asp rhr Ser 460 Val Arg Thr Trp Trp 540 Met Leu Leu Gly te 620 Ile Arg Gin As n Thr 700 Gly Met Asn G.y 73: Ala 445 Asp Arg Ala Tyr Ile 525 Ser Leu Gly Gly Ala 605 Ala Ala Ala Val Leu 685 Trp Arg 5cr Lou Asn e65 Asp Ala Leu Ala Met Leu 510 Ala Pro Va I Met Val 590 Leu Ser Glu Val Val 670 His Phe Asp Ile Val 5 C.

Phe P2:cz Val Glu Met Gin 495 Thr Asp Gin Asp Ala 575 Pro Asn Gly Asp Pro 655 Gin Leu Ala IAsn Tyr 735 Pro

I

Val Glu Thr Val 480 Ala Ala Ala Asn Ala 560 Leu Lys Leu cly Gly 640 Ala Arg Ala Gly ASn 720 G:n p3cr ,or LeU C,*S G~n 7 as Ser 5lu Met Gin GMn Min '791 PPn Ser flly Asn Prc) 19S U -rm o*P19mom*Iw IlP R 1235-1 -78- Asp Gin Ser Ser Gin Pro Gin Gin Gin Pro Gin Gin Gin Pro Ala Gin 805 810 815 Gin Giu Gin Lys Asp Ser Asp Giy Val Ala Gly Trp Ile Lys Asp Met 820 825 830 Phe Gly Ser Asn 835 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 850 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: Escherichia coli (vii) IMMEDIATE SOURCE: CLONE: pARC057i PBP IA QQAA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Lys Phe Vai Lys Tyr Phe Leu Ile Leu Ala Va? Cys Cys Ile Leu 1 5 10 is Leu Gly Ala Gly Ser Tie Tyr Gly Leu Tyr Arg Tyr Ile Giu Pro Gin 25 Leu Pro Asp Val Ala Thr Leu Lys Asp Vil Arg Leu Gin Ile Pro Met 40 Gin Ile Tyr Ser Ala Asp Gly Giu Leu Ile Ala Gin Tyr Gly Glu Lys so 55 Arg Arg Ile Pro Val Thr Lea Asp Gin Ile Pro Pro Giu Met Val Lys 70 75 Ala Phe lie Ala Thr Giu Asp Ser Arg Phe Tyr Giu His His Gly Val *2 85 90? Asp Pro Val Gly tie Phe Arg Ala Ala Ser Val Ala Lou Phe 5cr Gly 100 105 110 His Ala Ser Gin Gly Aia Ser Thr Ile Thr Ala Aia Leu Ala Arq Asn 115 120 125 Phe Phe Leu Ser Pro Giu Arg Thr Leu Met Arg Lys lie*-, Lys Giu Va 1 130 135 140 Phe Lou Ala Ile Arg lie Giu GIn Lou Lou Thr Lys Asp Giu lie Lou 145 150 155 160 Glu Leu Tyr Lou Asn Lys lie Tyr Lou Gly 71r Arg Ala Tyr Gly al1 165 1 0 115 Gly Ala Ala Ala in VI Tyr Phe Gly L*s Thr VaI Acp Gin Leu Thr 180 185 190 Leu Asn Gil.. Met Ala Val le Ala Gly Leu Pro Lyso Ala Pri Ser Thr 195 200 6 A Phe Asn 210 Leu Tyr St.r Met A13 Arg Ala Val zis Arg Ari An *.al

~--~ICQP

R 1235.1 -79- Val Leu Ser Arg Met Leu Asp Giu Gly Tyr Ile Thr Gin Gin Gin Phe 225 230 235 240 Asp Gin Thr Arg Thr Glu Ala Ile Asn Ala Asn Tyr His Ala Pro Glu 245 250 255 Ile Ala Phe Ser Ala Pro Tyr Leu Ser Glu Met Val Arg Gin Giu Met 260 265 270 Tyr Asn Arg Tyr Gly Giu Ser Ala Tyr Giu Asp Gly Tyr Arg Ile Tyr 275 280 285 Thr Thr Ile Thr Arg Lys Val Gin Gin Aia Ala Gin Gin Ala Val Arg 290 295 300 Asn Asn Val Leu Asp Tyr Asp Met Arg His Gly

T

yr Arg Gly Pro Ala 305 310 315 320 Asn Val Leu Trp Lys Val Gly Giu Ser Ala Trp Asp Asn Asn Lys Ile 325 330 335 Thr Asp Thr Leu Lys Ala Leu Pro Thr Tyr Giy Pro Leu Leu Pro Ala 340 345 350 Ala Val Thr Ser Ala Asn Pro Gin Gin Ala Thr Ala Met Leu Ala Asp 355 360 365 Gly Ser Thr Val Ala Leu Sar Met Giu Gly Val Arg Trp Ala Arg Pro 370 375 380 Tyr Arg Ser Asp Thr Gin Gin Gly Pro Thr Pro Arg Lys Val Thr Asp 385 390 395 400 Val Leu Gin Thr Gly Gin Gin Ile Trp VAl Arg Gin Val Gly Asp Ala 405 4i0 415 Trp Trp Leu Ala Gln Val Pro Clu Vat I;sn Ser Ala Lau VaIl Ser Ile 420 425 430 Asn Pro Gin Asn Gly Ala Val Met Ala Leu Val Gly Gly Phe Asp Phe 435 440 445 Asn Gin Sor Lys Phe Asn Arg Ala Thr Gin Ala Lou Akrg cln vcl Gly 450 455 460 Ser Asn Ie Lys Pro Phe Lou Tyr Thr Ala Ala Met Asp Lys Gy Leu 465 470 475 480 Thr Lou Ala Ser Met Lou Asn Asp 7al Pro Ile Ser Arg Trp Asp Ala 485 490 495 Ser Ala Gly Ser Asp Trp Gin Pro Lys Aon Ser Pro Pro Gin Tyr Ala 500 53,5 510 Gly Pro ile Arg Leu Arg Gin Gly Lau Gly Gin Ser Ls Asn Val Val 515 520 52S Met Val Arg Ala Met Arg Ala Met Gly Val As Tyr Ala Ala Glu Iy*r 530 535 Lau Gin Arq Phe Gly Ph' Pro Ala Gin Aon lie Val His Thr Giu Sor 545 6 )SS5 0 Leu Ala Lou Gly Ser A Ma Oar Phe Thr Pro Met 31n a L Ala Arg (sy 565 S" 0 51 6yr Ala Val Met Ala Ann tly (3y LOU Vi AGP Pro Trp Phe rSyi -n _r R 1236-1 Val Ser Lys Ile 595 Giu Asn Asp Gin Ile Phe Giu Ala Lys 605 9O 04*e 4**t 9* 9 9 *9 0* 0@ Lys Gin 625 Ser Ala His Thr G ly 705 As n Thr Thr 0 ly Leu 785 Thr Ser Val Ala Cys 610 Lys Ser Asn Arg Giu Gin Asn Gin Al 660 Val Ile Asn 675 Asn Ile Phe 690 Arg Asp Leu Ser Ser Lys Ser Val Trp 740 Thr Ala Ser 755 Ala Lys Ser 770 GIU Gly Val Val A~n Ile Arg Glu Glu 820 Pro Val Gin 645 Leu Thr Giy Gin Asp 725 Ile C ly Ala Pro Asp 805 Ty r Giu Leu 630 Asn Vai Pro Giu Arg 710 Ala G ly Ala Gin C lu 790 Arg Phe Cys 615 Gliu Val1 Ala Leu Pro 695 Arg Trp Phe Ile Pro 775 Gin Ser Ile Asp Asn Ser Lys Ala 680 Gly Asp Phe Asp Lys 760 Ala Pro Thr Giu Ile 840 Ile As n Val1 Thr 665 Phe Trp Ile Ser Asp 745 Asp Trp Leu Gly Gly 825 Pro Asp Pro 650 G ly Leu Gin Gly G ly 730 His Gin Asp Thr Gin 810 Thr Val1 Val1 635 Met Ala Ile Giy G ly 715 Ty r Arg Ile Ala Pro 795 Leu Gi1nr Ile 620 Giu Pro Gin Ly s Thr 700 Lys G ly Arg Ser Tyr 780 Pro Ala Prcn Ty r Asp Gin Glu Ser 685 G ly Thr Pro Asn G iy 765 Met Pro As n Thr Gly Asp Thr Val Ala Ile 640 Leu Giu Gin 655 Tyr Ala Pro 670 Ala Leu Asn Trp Arg Ala Gly Thr Thr 720 Gly Val Val 735 Leu Giy His 750 Tyr Giu Gly Lys Ala Val Gly Ile V a 8C; Giy Gly Asr.

815 Gin Gin Ala 830 Val His Giu Val Gly Thr Thr Ile Asp Asn C17 Giu Ala Gin Giu 845 Leu 835 Phe 850 INFORMATION FOR SEQ I0 NO: 11: Mi SEQUENCE CHARACTERISTICS: LENGTH: 553 amino acids TYPEt amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: pepttide (vi) ORIGINAL SOUR.CE: ORGANIS3M: Escherichia roli (vii) 1WEDIATE SOURCE: CLONE: PAR'- 05S" truncated PBP R i235-1 -81- (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Met Ala Gly Asn Asp Arg Glu Pro Ii:' Gly Arg Lys Gly Lys Pro Thr is 99 9 9 9.

9. 9 *9*9 !99.

*9 9 9.

99 .9 9 9 9*9 9 9*99 .9 .9 9 *39 9 9 ".9 I 99 9 ;e *9 9* 99 99 9999 9 9999 Arg Asp Lys Trp Ala G Ly Leu Giu Phe 145 P he G ly G ly Asn Leu 225 A s p Thr Val As n Arq 305 Asp Pro Ty r Gly LeU Ile Lys Giu Ala 130 Thr Pro Asp Phe Gly 210 Val1 G ly L*ys *sn Val Asp Lys Trp Tyr Val1 Pro 115 Thr Val Asp H~is Phe 195 Giu As, p Ile C ly As n 275 Ala Lou cGlu Lys Asp Gly Leu 0 ly Trp 100 Asp Gin Gin So r Leu 180 Arg Gin Thr Ser Arg 260 Leu Tyr Gliu T le Gin Tyr Lys Leu Val1 Gin Met Tyr Ala Lys Ala Leu Arg Leu Leu 245 Thr ?he Met Lou Arq 325 Lys Asp Gly Leu 70 Tyr Leu Thr Arg Asn I50 clu Thr Asp Leu Leu 210 Tyr Leu kia ryr 10 by Val1 Asp Lys 55 Lys Lcu Ala Ile Gin 135 Ser Gly Ile Pro Phe 215 Ala Ser uln Ser Leu 29S mo t Phe Ser Tyr 40 Gly Lou Asp Ala Ser Va 1 Ile Gin Val1 Arg 200 Va 1 Thr Ile 31Y Ilbe Arg Arg 25 Glu Asp Arg Lys Ala Ile Gin Lys 90 Ala Vai 105 Lys Asn Ser Lys Giu Y e t Val rg 170 Asn Met Leu Ile Pro Arg Giu Asp Gly Arg 250 Ala Ser 265 Glu Arg Met A p Glu Va I Lou Ala 330 Arg Gil Pro Val1 75 Ile Tyr Giu Met Ile 155 Ala Giu Thr S er Arg 235 Ala Ala Ty r 315 TTyr IGlu Arg Phe Arg Gly Met Thr 140 Arg Arg Az n Met Gly 220 Hi~s Val1 Led Tyr Arg 300 Leu Leu Giu Pro G ly Ala Ser Arg Val1 125 Arg Arg Lou As n Ile 205 Phe Phe Lou Thr Trp 285 Tyr~ G..1.

As~ Me Lys Val Arg Met 110 Lys Pro Pro Thr Arg 190 Ser Pro Tyr 31n ,or )Asp :Pro Arg Ile *Val Lou G ly Phe Phe 175 Gin Ser As p Glu Asn 2655 Gin Lyso Lyo 33s- Asp Arg Gly I le Asp Asn Lou Giu Asp 160 Asp Phe Pro Leu His 2140 Leu Arq Pro Val Glu Glu Lou Ser Lou Asp 340 345 01n 31n Ala Leu C C R 1235-1 -82- Asn Pro Met Val Lys 355 Gly Ala Ser Ile Tyr 360 Trp Arg Asn Pro Lys Leu 365 Ala Ile 385 Val Leu Leu Ala Arg 465 Ser G ly Lys Leu Gly 545 Leu 370 Ile Gln Va 1 Ser Ala 450 Lys Gly Tyr Pro Asn 530 Glu Asp Pro Arg Gly 435 Glu Leu Glu Asn Ala 515 Thr Arg Arg Asn Leu Val Leu Arg Leu Leu Gin Gin Gin Gin Arg Gin 420 Val Lys Ser Val Arg 500 Thr Trp Glu Leu 390 Gly Gly 405 Glu Leu Lys Ile Ala Ala Asp Leu 470 Arg Ala 485 Ala Met Tyr Leu Ile Ala 375 Vai Gin Phe Val 455 Glu Met Gin Thr Asp 535 Asp Ile Ala Thr 440 G lu Thr Va1 Ala Ala 520 Ala Met Ser Lys 425 Thr Gly Ala Gly Arg 505 Leu Pro Leu Pro 410 Leu Phe Ile Ile Gly 490 Arg Ser Ile 380 Ser Ala 395 Gin Pro Gly Asp Asp Ser Pro Ala 460 Val Val 475 Ser Glu Ser Ile Gin Pro Ala Leu 540 Arg Ala Lys Va1 445 Leu Val Pro Gly Lys 525 Arg Pro Leu Phe Met 415 Val Lys 430 Ala Gln Lys Lys Asp Arg Gin Phe 495 Ser Leu 510 lie Tyr Gin Pro Gin Gly 400 Gln Asp Asp Gln Phe 480 Ala Ala Arg Asn a.

a *0 *0 a a. a a a.

*a a a** a. a.

a a.

Gin Val. Trp Ser Pro Gin Asn Asp 550 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 532 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TWPE: peptide (vi) ORIGINAL SOURCE: ORGANISM: Eschorichia coli (vii) IMMEDIATE SOURCE: CLONE pARC O93 truncated soluble PBP 1B (xi) SEQUENCE DESCRTIXON: SEQ ID NO: 12: Met Ala Gly Asn Aip Ara Glu Pro lie Gly Arq Lys 1ly Lts Pro Th-,r 1 5 10 Arg Pro V4'al Lys 31n Lys Val Ser Arq Arg Arg Tyr Giu Aop Aop A'o n0 25 Asp Tyr Aup ASp 7r op Asp Anp Tir Glu Asp lu Glu Pro Met Pro A.

40 4S LY- G ly Lys L 1 o 31Y Lys S *I y Ar Lys Prc Arq -1y Lys Av] 3 c II I-P I R 1235-1 -83-- Ser Ile Asp Gin Lys Ile Arg Ser Arg Ile Asp Gly Lys Val Trp Gln 70 75 Le Ala Ala Ala Val Tyr Gly Arg Met Val Asn Leu Glu Pro Asp Met 90 Thr Ile Ser Lys Asn Giu Met Val Lys Leu Leu Glu Ala Thr Gin Tyr 100 105 110 Arg Gin Val Ser Lys Met Thr Arg Pro Gly Giu Phe Thr Val Gin Ala 115 120 125 Asn Ser Ile Glu Met Ile Arg Arg Pro Phe Asp Phe Pro Asp Ser Lys 130 135 140 Glu (ly Gin Val Arg Ala Arg Leu Thr Phe Asp Gly Asp His Leu Ala 145 150 155 160 Thr Ile Val Asn Met Giu Asn Asn Arg Gin Phe Gly Phe Phe Arg Leu 165 170 175 Asp Pro Arg Lou Ile Thr Met Ile Ser Ser Pro Asn Giy Glu Gin Arg 180 185 190 Leu Phe Val Pro Avg Ser Gly Phe Pro Asp Leu Leu Val Asp Thr Lou 195 200 205 Leu Ala Thr Giu Asp Avg His Phe Tyr Giu His Asp Gly Ile Ser Lou 210 215 220 Tyr Ser lie Gly Avg Ala Val Leu Ala Asn Lou Thr Ala Gly Arg Thy 225 230 235 240 Val Gin Gly Ala Sev Thy Lou Thr Gin Gin Lou Val Lys Asn Lou Phe 245 250 255 Leu Ser Ser GIa Arg Ser Tyr Trp Arg Lys Ala Asn Giu Ala Tyr Met 260 265 270 Ala Lou Ile Met Aop Ala Arg Tyr Soy Lys Asp Arg tIe Leu Giu Lou 275 280 285 Tyr Met Asn Clu Val Tyr Lou Gly Gin Ser Gly Asp Asn Giu Ile Arg *290 295 300 44444 Gly Phe Pro Leu A14 5ev Lou Tyr Tyr Phe Gly Arg Pro Val Gu Glu 305 310 315 320 Lou Ser Lou Asp Gin G3n Ala Lou Lou Val Gly Met Val Lys Gly Ala 325 330 335 Ser Ile Tyr Aon Pro Trp Arg Aon Pro Lys Lou Ala Leu Glu Avg Arg 340 345 350 Ann Lou Val Lou Arg Lou Lou Gin Gin Gin Gin Tie Ile Asp Gin Giu 355 360 365 Lou Tyr Asp Mot Lou Ser Ala Avg Prc Lou Gly VaI Gin Pro Avg gly 370 375 38i Gly Val 1le S-or Pro (11n Pro Ala Pho Mot Gin Lou 7a 1,Avg Gln G u 385 303 3q5 401 Lou GIn Ala Lys Lou ys Ac Lys Va I Lys As Lu oev .al 405 410 415 Ile Pho Thy Thy Pho Ann er 7al Ala Gin Ap Ala Ala Gu Lyo Aa 4425 432 1_1~ Irrll- I11, IL Cba O I _I R'1235-1 -84- Ala Val Glu Gly Ile Pro Ala Leu Lys Lys Gin Arg Lys Leu Ser Asp 435 440 445 Leu Glu Thr Ala Ile Val Val Val Asp Arg Phe Ser Gly Glu Val Arg 450 455 460 Ala Met Val Gly Gly Ser Glu Pro Gin Phe Ala Gly Tyr Asn Arg Ala 465 470 475 480 Met Gin Ala Arg Arg Ser Ile Gly Ser Leu Ala Lys Pro Ala Thr Tyr 485 490 495 Leu Thr Ala Leu Ser Gin Pro Lys Ile Tyr Arg Leu Asn Thr Trp Ile 500 505 510 Ala Asp Ala Pro Ile Ala Leu Arg Gin Pro Asn Gly Gin Val Trp Ser 515 520 525 Pro Gin Asn Asp 530 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 159 amino acids TYPE: amino acid STRANDEDNESS: double TOPOLOCY: linear 44 4, (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCEt ORGANISM: Escherichia coli (vii) IMMEDIATE SOURCE; (BI CLONE: pARC 0392 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Gly Giu Gin Arg Leu Phe Val Pro Arg Ser Gly Phe Pro Asp Lou Leu 1 5 10 is 44 4~ Ole Val Asp Thr Leu Lou Ala Thr Giu Asp Arg His Phe Tyr Giu His Asp 25 Gly Ile Ser Lou ?yr 5cr Ile Gly Arg Ala Vaal Lou Ala Aon Lou Thr 40 Ala Gly Arg Thr Val Gin Gly Ala 8or Thr Lou Thr Gin Gin Lou Val 55 Lys Asn Lou Phe Lou Ser 5cr Glu Arg Ser Tyr Trp Arq Lys Ala Asn 70 75 Giu Ala Tyr Met Ala Lou tIe Mt Asp Ala Arg 14ir ."or Lys Ap Arg 8$ 0 90 Ile Lou Glu Lou Tyr Met Aon G1u Val Tyr Lou Gy J3n 8er 51y Asp 10S l11 Aon Glu lie ArAy £he Pro Lou Ala -or Leui tyr Tyr Phtl y A11< luS 12 Pro Ilal1 Glu GlU Lou 0-)r Leu Asp GIn Gth Ala Lou Lu 7a' G*Y Me t 130 135 l43 ;19 '111 I R'1235-1 Val Lys Gly Ala Ser Ile Tyr Asn Pro Trp Arg Asn Pro Lys Leu 145 150 155 The matter contained in each of the following claims is to be read as part of the general description of the present invention.

S6* 0*

WAHM

Claims (16)

1. A polypeptide which is a water-soluble active derivative of a bacterial bifunctional penicillin binding protein, said penicillin binding protein being bound to the cell membrane when expressed in a bacterial cell and being capable of exhibiting both ftaisglycosylase and transpeptidase activities and said derivative lacking a membrane anchoring sequence but retaining the capability to exhibit one or both of said enzymic activities, wherein the derivative has an amino acid sequence which is identical to, or substantially similar to, SEQ ID NO: 2, 4, 6, 12 or 13 in the Sequence Listintg.

2. A polypeptide according to claim 1, wherein the bacterial cell is an Escherichia coli cell or a Streptococciis pneumonia'w cell. too": 3. A polypeptide comprising a first polypeptide according to claimn 1; .0 15and an additional polypeptide which allows binding to an affinity matrix; there being a cleavage site between said polypeptides.

4. A polypeptide according to claim 3 wherein the additional 20polypeptide is glutathione-S-transferase or a polypeptide substantially similar to glutathione-S- trans ferase. A polypeptide according to claim 3 wherein the additional polypeptide is a polypeptide rich in histidine residues.

6. An isolated and purified DNA molecule which has a nucleotide sequence coding for a polypeptide according to claim 1 or 3. '"87-

7. A DNA molecule according to claim 6, which nucleo tide sequence is identical to, or substantially similar to, SEQ ID NO: 1, 3 or 5 in the Sequence Listing.

8. A repl icable expression vector which carries and is capable of mediating the expression of a DNA molecule according to claim 6.

9. A vector according to claim 8 which is the vector pARC0558 (NCIMB No. 40666), pAf(C0559 (NCJIR No. 40667), pARC0512 (NCITYI3 No. 40665), pARCO593 (NCTMI3 No. 40670), pARC0392 (NCIMB No. 40659), pAR.CO499 (NCIMl3 No. 40664), or 15 pARCO400 (NCllvI No. 40660). Acl aburn vector according to claim 8 or 9. A el aborn A process for production of a polypeptide which is a derivative of penicillin binding protein, comprising growing a cell according to P. claim 10 in or on a culture medium for expression of the polypeptide and optionally recovering the polypeptide.

12. A process for the production of a water soluble polypeptide according 25to claim 1 which comprises culturing Eschericlda coli cells harbouring an expression vector wherei a DNA coding sequence for said poly-peptide is under the control of an isopropyl thiogalactoside (IPTG) inducible promoter, said culturing being carried out in the presence of a sub-optimal concentration of IMTG for induction of the said promoter anid at a temperature in the range of 20 to 24'C. U i C.) mT 01' -88-

13. A method of identifying an antibody capable of binding a bacterial bifunctional penicillin binding protein which includes the step of employing a polypeptide according to claim 1 in an antibody binding assay and selecting antibodies that bind to the polypeptide.

14. A method of assaying for compounds which bind to a penicillin binding protein, said method comprising contacting a polypeptide according to claim 1 or 3 with a compound to be investigated; and (b) detecting whether said compound binds to the penicillin binding pro: in. A method of assaying for compounds which bind to a penicillin binding protein, said method comprising culturing cells according to claim 10; lysing the said cells and isolating the crude cell extract; exposing the said cell extract to potential inhibitors of a penicillin binding protein; introducing an agent, known to bind a penicillin binding protein, to the said cell extract; removing the unbound fraction of said agent; and assaying the presence of said agent remaining in the cell extract.

16. A method of assaying for compounds which bind to a penicillin binding protein, said method comprising exposing a polypeptide according to claim 1 or 3 immobilised on a solid support, to a potential inhibitor of a ?enicillin binding protein; exposing an 25 agent, known to bind a penicillin binding protein, to the immobilised polypeptide; removing the unbound fraction of said agent; and (d) assaying the presence of said agent bound to the immobilised polypeptide. I Cq\M <A v^ L -g -89-

17. A method of assaying for compounds which bind to a penicillin binding protein, said method comprising exposing a polypep tide according to claim 1 or 3 to a potential inhibitor of a penicillin binding protein; exposing the said polypeptide to an agent, known to bind a penicillin binding protein, which agent is im~mobilised on a solid support; and assaying the presence of polypep tide bound to the immobilbsed agent.

18. A method of assaying for compounds which bind to the transglycosylase domain of a penicillin binding protein, said viethod comprising exposing the transglylcosylase domain of a polypeptide according to claim 1 or 3, said polypeptide being immobilised on a solid support, to a potential inhibitor of the transglyc'esylase activity of a penicillin binding protein; exposing an agent, known to bind the transglycosylose domain of a penicillin binding protein, to the imynobilised polypeptide; removing the unbound fraction of said agent; and assaying the presence of said agent bound to the immobilised polypeptide.

19. A method of assaying for compounds which bind to the transglycosylase domnain of a penicilin binding protein, said method comprising exposing the transglycosylase domain of a volypeptide :according to claim 1 or 3 to a potential inhibitor of a penicillin binding protein; exposing the said polypeptide to an agent, known to bind to the trans-glycosylase dornain of a penicillin binding protein, which agent is iinmobtJised on a solid supp ort; and assaying the presence of polypeptide bound to the immobilised agent. 'j A/ o A method according to any one of claims 15 to 19 wherein the agent known to bind a penicillin binding protein is a monoclonal antibody.

21. A method according to any one of claims 15 to 19 wherein the agent known to bind a penicillin binding protein is a labelled antibiotic compound.

22. A method of determining the protein structure of a penicillin binding protein, characterized ir that a polypeptide according to claim 1 is utilized in X-ray crystallography. DATEJ this 13th day of May 1998 ASTRA AKTIEBOLAG, By its Patent Attorneys, E WELL IrTON CO., (Bruce Wellinton) 9 9. 0 9*o R 123561 r ABSTRACT The present invention relates to variants of Penicillin Binding Prot:i'.J (PBP), which proteins are involved in bacterial peptidoglycan biosynthesis. Disclosed are also DNA molecules coding for the said PBP variants, as well as vectors and cells harbouring such DNA molecules. The invention is also related to processes for assaying and designing therapeutically useful compounds which have high affinity to PBP, which processes utilize the said PBP variants. 00 S 00 I