WO2000042198A1

WO2000042198A1 - An e. coli protein tyrosine kinase and its uses

Info

Publication number: WO2000042198A1
Application number: PCT/IL2000/000023
Authority: WO
Inventors: Ilan Rosenshine; Ofir Ilan
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem
Priority date: 1999-01-12
Filing date: 2000-01-11
Publication date: 2000-07-20
Also published as: AU1999300A

Abstract

An isolated protein tyrosine kinase having an amino acid sequence substantially homologous to the amino acid sequence of Etk protein of E. coli, particularly proteins encoded by DNA SEQ ID NO:1 and/or a functional equivalent or derivative thereof. Expression vectors comprising the encoding DNA and transformed host cells are also encompassed. The proteins can be tagged with detectable and/or functional moieties. The proteins can be used for tyrosine phosphorylating target proteins. The invention also relates to agents that can selectively bind and/or inhibit proteins that are substantially homologous to E. coli Etk, and to methods of screening for such agents. The agents can be used as active therapeutic agents in the treatment and/or prevention of bacterial infections.

Description

A E . COLI PROTEIN TYROSINE KINASE AND ITS USES

Field of the Invention

This invention is directed to a protein tyrosine kinase having an amino acid sequence substantially homologous to the amino acid sequence of Etk protein of E. coli., and/or a functional equivalent or derivative thereof and their different uses.

Background of the Invention

Tyrosine protein phosphorylation and its biological significance has been extensively characterized in eukaryotes [a few recent reviews include: Schlaepfer, D.D. and Hunter, T., Trends Cell Biol 8: 151-7 (1998); Tzahar, E. and Yarden, Y„ et al, Biochim Biophys Acta 1377:M25-37 (1998); Williams, J.C., el al, Trends Biochem Sci 23: 179-84 (1998); Hubbard, S.R., et al, J Biol Chem 273: 11987-90 (1998)]. In contrast only a few reports describe protein tyrosine phosphorylation in prokaryotes [Atkinson, M., et al, J Bacteriol 174:4356-60 (1992); Kelly- Wintenberg, K., et al., J Bacteriol 172:5135-9 (1990); South, S.L., et al, Mol Microbiol 12:903-10 (1994); Frasch, S.C. and Dworkin, M., J Bacteriol 178:4084-8 (1996)], and one report describes a prokaryotic protein tyrosine kinase, the Ptk protein of Acinetobacter johnsonii [Grangeasse, C, et al, Gene 204:259-65 (1997)]. Except Ptk, none of the bacterial tyrosine phosphorylated proteins or the corresponding kinases have been identified. Moreover, the biological significance of these protein tyrosine phosphorylation events remained elusive. Thus, protein tyrosine phosphorylation in prokaryotes is regarded as rare and is poorly defined [Zhang, C.C., Mol Microbiol 20:9-15 (1996); Cozzone, A.J., Biochimie 80:43-8 (1998)].

Exopolysaccharides (EPS) are important virulence factors of many animal and plant pathogens. The role of the EPS in forming a capsule that protects the pathogen from phagocytosis is well documented [Ofek, I., et al., Infect Immun 61 :4208-16 (1993)]. However EPS may contribute to virulence in other ways. In Erwinia amylovora and Pseudomonas solannaceanim the EPS appears to be required for initial attachment of these pathogens to the host plant tissue [Bugert, P. and Geider, K., Mol Microbiol 15:917-33 (1995); Cook, D. and Sequeira, L., J Bacteriol 173: 1654-62 (1991)], a step which may be needed for efficient delivery of virulence factors via the type III secretion systems. In Pseudomonas aeruginosa the loose EPS aggravates the lung infections in cystic fibrosis patients. In the plant symbiont Rhizubium meliloti, the succinoglycan EPS play a more specific role in interaction with the host plant. The succinoglycan is sloughed off into the surroundings and mediates signaling to the host cell which is essential for the formation of fully matured nodules [Leigh, J.A. and Walker, C.G., Trends Genet 10:63-67 (1994)].

The inventors have earlier reported that enteropathogenic E. coli (EPEC) possesses a tyrosine-phosphorylated protein Ep85 [Rosenshine, I., et al, EMBO J 11 :3551-60 (1992)]. The present invention is based on the isolation and identification p85, now renamed as Etk, and the characterization of its coding gene.

Summary of the Invention

The present invention relates to an isolated protein tyrosine kinase having an amino acid sequence substantially homologous to the amino acid sequence of Etk protein of E. coli.

The invention further relates to protein tyrosine kinase having an amino acid sequence substantially homologous to the amino acid sequence of Etk protein of E. coli encoded by a DNA sequence substantially as shown in SEQ ID NO: l or a functional equivalent or derivative thereof.

The invention relates to a recombinant protein tyrosine kinase having an amino acid sequence substantially homologous to the amino acid sequence of Etk protein of E. coli.

In a further aspect, the invention relates to a recombinant tagged protein, as herein defined, having protein tyrosine kinase activity, comprising an amino acid sequence substantially homologous to the amino acid sequence of E. coli Etk protein and tag moiety.

In addition, the invention relates to an expression vector comprising a DNA sequence encoding protein which is a protein tyrosine kinase having an amino acid sequence substantially homologous to the amino acid sequence of Etk protein from E. coli. In preferred embodiments said DNA has the nucleotide sequence substantially as shown in SEQ ID NO: l . The expression vectors of the invention may further comprise operably linked regulatory elements, which may be, for example, promotor/operator elements, ribosome binding sites, repressors, initiators and other expression control elements. A preferred expression vector is the plasmid pOI194.

The invention also relates to a host transformed with an expression vector of the invention. The hosts can be a unicellular organism or a mammalian cell in culture.

The invention additionally relates to a method of producing a protein having protein tyrosine kinase activity and having the amino acid sequence substantially homologous to the amino acid sequence of Etk protein of E. coli comprising providing a host cell, transforming the host cell with an expression vector comprising a DNA sequence encoding a protein having the amino acid sequence substantially homologous to the amino acid sequence of Etk protein of E. coli, culturing the transformed cell; harvesting the cultured cells so obtained and isolating the protein from the harvested cells by suitable liquid chromatography methods. The DNA sequence encoding the protein having protein tyrosine kinase activity is preferably the DNA sequence substantially as shown in SEQ ID NO: l or a functional equivalent or derivative thereof. The invention also relates to the recombinant protein produced by the method.

A particular aspect of the invention relates to a method of producing a recombinant tagged protein, as defined herein, having protein tyrosine kinase activity and having an amino acid sequence substantially homologous to the amino acid sequence of Etk protein of E. coli, comprising the steps of providing a host cell, transforming the host cell with an expression vector comprising a DNA sequence encoding a protein having the amino acid sequence substantially homologous to the amino acid sequence of Etk protein ofE. coli, preferably SEQ ID NO: l, and further comprising operably linked nucleotide sequence encoding a tag moiety as herein defined, culturing the transformed cells so obtained and harvesting the cultured cells, and isolating the recombinant tagged protein from the harvested cells. The invention relates also to the tagged proteins obtained by this method.

In a further aspect the invention relates to a method for the purification of a protein or a tagged protein according to the invention by loading a membrane extract of a transformed host according to the invention on a suitable affinity chromatography column, washing off any non-bound material and separating bound material by elution with a suitable eluant.

Still further, the invention relates to an agent that can selectively bind to a protein, which protein is substantially homologous to E. coli Etk and to a functional equivalent or derivative thereof. Such agent can be a low molecular weight compound.

The invention also relates to agents that are capable of inhibiting the protein tyrosine kinase activity of a protein or tagged protein according to the invention.

The invention further relates to a method of screening a sample for the presence of an agent that selectively binds to a protein substantially homologous to E. coli Etk or to a functional equivalent or derivative thereof, comprising depositing a protein having an amino acid sequence substantially homologous to the amino acid sequence of Etk protein of E. coli or a functional equivalent or derivative thereof on a suitable solid phase carrier, incubating the sample with the deposited protein, washing off any non-bound sample material, separating bound material from said deposited protein; and identifying the material thus obtained.

In addition, the invention relates to an automated method of screening a sample for the presence of an agent that is capable of inhibiting the protein tyrosine kinase activity of a protein substantially homologous to E. coli Etk or of a functional equivalent or derivative thereof, comprising coating a multiter plate with a suitable protein tyrosine kinase substrate, adding the sample, adding a solution containing the protein and a phosphate donor and incubating the plate for a suitable period of time, washing off any non-bound material and monitoring the level of tyrosine phosphorylation.

Still further, the invention relates to a method of screening a sample for the presence of an agent that selectively binds to a nucleic acid substantially homologous to E. coli etk gene and to a functional equivalent or derivative thereof or its mRNA transcript, comprising depositing the nucleic acid or functional equivalent or derivative thereof on a suitable solid phase carrier, incubating the sample with the deposited nucleic acid, washing off any non-bound sample material, separating bound material from said deposited protein and identifying the material thus obtained. A suitable substrate is, for example, poly-Glu:Tyr.

A particular aspect of the invention relates to an agent that is capable of inhibiting the protein tyrosine kinase activity of a protein substantially homologous to E. coli Etk or of a functional equivalent or derivative thereof, which is a therapeutic agent that can be used in the treatment and/or prevention of bacterial infection.

The invention also relates to an agent that is capable of binding to a DNA sequence substantially homologous to the etk gene of E. coli, substantially as shown in SEQ ID NO: 1, which agent is also a therapeutic agent for the treatment and/or prevention of bacterial infections. Such agents can be, for example, nucleic acids or peptide nucleic acids (PNAs).

The invention relates also to a method of labeling a tyrosine residue of a target protein with a phosphate group, in which the labeling is catalysed by a protein substantially homologous to E. coli Etk. This labeling method comprises mixing a target protein with a protein substantially homologous to E. coli Etk and with a phosphate donor, for example ATP, in suitable buffer for a suitable period of time; and isolating the phosphorylated target protein thus obtained. The protein substantially homologous to E. coli Etk may be covalently or non-covalently attached to a solid phase carrier. The target protein can be the protein that is substantially homologous to E. coli Etk itself, resulting in autophosphorylation.

A further aspect of the invention is a pharmaceutical composition for the treatment and/or prevention of an infection caused by a bacterial pathogen expressing the etk gene, comprising at least one agent according to the invention.

A kit for labeling a tyrosine residue of a target protein with phosphate comprising at least a protein having an amino acid sequence substantially homologous to E. coli Etk or a functional equivalent or derivative thereof and a phosphate donor is also encompassed.

Brief Description of the Drawings

Figure 1 Amino acid sequence of Etk

Figure 1 the amino acid sequence of Etk of E. coli. (SEQ ID NO: l).

Figures 2A to 2D Ep85/Etk catalyses auto-tyrosine phosphorylation Figure 2A: E. coli K12 MCI 061 that does not normally express Etk (Fig. 5), was transformed with pOI194. The culture was grown to OD 0.6 and IPTG was added to a final concentration of 0.1 mM. The culture was sampled at 0, 5, 10, 20, and 30 min after IPTG was added, bacterial proteins were extracted and used for immunoblot analysis with anti-Etk antibody (anti-Etk) and anti-phosphotyrosine antibody (anti-P-Tyr). Tyrosine phosphorylated Etk (indicated by arrowhead) was detected even before the addition of IPTG, representing the normal basal level of Ptαc activity. IPTG induction increased expression of phosphorylated Etk, and phosphorylation of an additional protein, larger than Etk. that was not detected with the anti-Etk antibody (indicated by arrow).

Figure 2B: the purified Etk was used for auto-phosphorylation assay with [γ-32p]ATP as a phosphate donor. The reaction was stopped at 0, 15, 30, 45, 60, 75, 90, and 120 seconds after starting the reaction and aliquots were analysed by SDS-PAGE and autoradiography (panel 1), and by immunoblot analysis with anti-Etk to verify an equal amount of Etk in the different aliquots. Rapid auto-phosphorylation of Etk was detected.

Figure 2C: the purified tyrosine phosphorylated Etk was treated with YopH, a specific tyrosine protein phosphatase. The reaction was stopped 30, 60 and 90 seconds after adding YopH and aliquots were subjected for immunoblot analysis with anti-Etk to verify equal amount of Etk and with anti-phosphotyrosine antibody to determine the tyrosine phosphorylation levels. Rapid tyrosine dephosphorylation of Etk was detected.

Figure 2D: the purified Etk was labeled with [γ-32p]ATP was hydrolysed and phosphoamino acids were analyzed by cellulose TLC. The position of phosphoamino acid markers (P-Ser designating phosphoserine, P-Thr designating phosphothreonine and P-Tyr designating phosphotyrosine) as detected by nin ydrin staining of the TLC plate is shown in lane 1. The corresponding autoradiogram of the TLC plate is shown in lane 2. All the labeling was associated with phosphotyrosine but not with phosphothreonine nor phosphoserine. O designates Origin.

Figures 3 A and 3B Etk phosphorylates poly Glu:Tyr

Figure 3A: Poly Glu:Tyr at concentrations of 0.008 to 2 mg/ml was used as an exogenic substrate for Etk. The reaction was carried out for 30 min at 24°C. PTK (cpm) designates phosphotyrosine kinase activity in cpm.

Figure 3B: poly Glu:Tyr at concentration of 1 mg/ml was used as the Etk substrate.

The reaction was carried out for the indicated time at 30 C. 1 pmol of ATP corresponds to about 400 cpm. IP (cpm) designates incorporated phosphate in CPM. T designates time in minutes. Figure 4 Localization of Etk to the inner-membrane

EPEC culture, grown to the mid-logarithmic growth phase was fractionated into three fractions containing i) cytoplasmic and periplasmic soluble proteins (S-fraction), ii) outer-membrane proteins (O-fraction), and ii) inner-membrane proteins (I-fraction). The amount of proteins in each fraction was normalized, and proteins were used for immunoblot analysis with different antibodies as indicated beneath each panel. Anti-phosphotyrosine and anti-Etk antibody detected most of the Etk in the I-fraction and only a small amount of it in the S-fraction. Anti-DnaK, anti-FtsH and anti-intimin antibody were used as controls to determine the quality of the fractionation. Anti-DnaK and anti-FtsH antibody reacted as expected with soluble or inner-membrane proteins of the same size as DnaK and FtsH. Intimin is an outer membrane protein and subjected to N-terminal processing upon secretion. As expected, the anti-intimin antibody reacted with processed intimin in the O-fraction and with the pre-processed intimin and a small amount of processed intimin in the I-fraction. Molecular weight markers are shown at the left hand side.

Figure 5 Expression of Etk during EPEC growth

EPEC growth in LB broth, 37 C, with shaking was monitored by measuring the OD600 of the culture. This culture was sampled at 30 min intervals and proteins were extracted. Equal protein concentrations were used for immunoblot analysis with anti- phosphotyrosine and anti-Etk antibody. The time values in the X axis of the growth curve correspond also to the immunoblot beneath the axis. Molecular weight markers are shown at the left-hand side. Insert: demonstration that the anti-Etk recognized Etk epitopes other than phosphotyrosine. This control was needed since anti-Etk antibody were generated by injecting rabbits with phosphorylated Etk. Proteins were extracted from mid-logarithmic EPEC cultures, and were used for immunoblot analysis with anti-phosphotyrosine antibody (PT66, Sigma) (lane 1); PT66 supplemented with 10 mM phenyl phosphate (lane 2); nonspecific rabbit antiserum (pre-bleed of anti-Etk) (lane 3); anti-Etk antibody (lane 4), and anti-Etk antibody supplemented with 50 mM phenyl phosphate (lane 5). Molecular weight markers are shown at the left-hand side. The phenyl phosphate, which is identical to the side chain of the tyrosine phosphorylated residue, fully out-competed the labeling with PT66 but not with anti-Etk antibody. This indicates that anti-Etk reacted with Etk-epitopes other than the tyrosine phosphorylated residues. T designates time in minutes. OD designates optical density.

Figures 6A and 6B Expression of Etk by different E. coli strains Proteins were extracted from cultures of different E. coli strains grown to OD600⁼L0- Etk expression and its phosphorylation state were analyzed by immonoblotting with anti-Etk and anti- phosphotyrosine antibody. In addition, DNA was extracted from the different strains and the existence of the elk gene was examined by PCR with specific etk primers.

Figure 6 A, six clinical isolates of EPEC, including E815/71, 0142:H6 (lane 1), E2348/69, 0127:H6 (lane 2), 2430/78, Ol l lab:NM (lane 3), 1092-80, 0127:NM ( lane 4),2087-77, 055.Η6 (lane 5), and 0659-79, 0119:H6 (lane 6), and three non-pathogenic laboratory E. coli strains including MCI 061 (lane 7), K91 (lane 8), and HB 101 (lane 9) were tested. The specific etk primers amplified etk in all the strains but it was expressed only in the EPEC isolates.

Figure 6B, different E. coli virotype were compared including EIEC E5273/0,

028ac:H" (lane 1), EAEC 17-2, 03:H2 (lane 2), ETEC H10407, 078:H1 1 (lane 3) and EPEC E2348/69 (lane 4). As before, the PCR analysis indicates that all strains possess etk but only EPEC and ETEC express it.

Figures 7A and 7B Expression of Etk homologues by K. pneumonia and E. amylovora

Figure 7A: Proteins were extracted from EPEC 2348/69 (lane 1), K. pneumonia K2 KPA1 (lane 2), both grown on LB, and from E. amylovora Eal/14 (lane 4) or the isogenic AmsA mutant Ea7/74-A56 (lane 3), both grown on LB supplemented with 0.2% sorbitol. The extracted proteins were used for immunoblot analysis with anti-Etk supplemented with 10 mM phenyl phosphate and with anti- phosphotyrosine antibody. The anti-Etk antibody reacted with proteins of similar size to Orf6 and AmsA in the K. pneumonia and E. amylovora extracts (arrows). These proteins also reacted with the anti-phosphotyrosine antibody (arrows). In contrast, neither of these antibodies reacted with corresponding protein in the E. amylovora amsA mutant. This indicates that like Etk, Orf6 and AmsA are tyrosine phosphorylated and probably are PTKs.

Figure 2B: A recombinant AmsA was expressed in E. coli K12 XL1 Blue/pfdC4Z-α»ιsΑ (lane 1), protein were extracted and analyzed with anti-Etk and anti-phosphotyrosine antibody. As negative control we used XL1 Blue (lanes 2) and as positive control we used XL1 Blue transformed with pOI194 that express Etk (lane 3). AmsA reacted specifically with anti-Etk and anti-phosphotyrosine.

Detailed Description of the Invention

As mentioned above, the inventors have previously shown that enteropathogenic E. coli (EPEC) possesses a tyrosine-phosphorylated protein Ep85 [Rosenshine, 1., et al, ibid.], which was revealed by use of anti-phosphotyrosine antibody for immunoblot analysis of crude extract of enteropathogenic E. coli. One of the proteins in the extract in the size of 85 kD (Ep85) reacted with the antibody. This could indicate that Ep85 is either a substrate to some kinase unknown activity, or it incidentally cross-reacted with the antibody or that the protein was modified in some other way to cross-react with the antibody. Upon further and extensive investigation, described in detail in this application, the inventors interestingly found that this protein, now designated Etk, is a protein tyrosine kinase and that it undergoes self-phosphorylation. The present invention is based on these findings, the purification of Ep85, now renamed as Etk, and the characterization of Etk and its coding gene.

Many gram-negative and gram-positive pathogenic bacteria produce high molecular size exopolysaccharides (EPS). These polysaccharides either form capsule, or sloughed off into the surroundings and are essential in formation of bacterial biofilm. Many pathogenic bacterial mutants that can not produce EPS are also non-virulent to animals or plants. These mutants can not establish colonization and are effectively cleared by the host. While the role of capsule in protection against phagocytosis and resistance to complement is well-documented [Finlay B.B. and Falkow S. Microbiol Mol Biol Rev, 61 : 136-69 (1997)], EPSs may contribute to virulence in other ways. In Erwinia amylovora, Ralstonia solannacearum, and enteropathogenic E. coli (EPEC) the EPS is requires for initial attachment of these pathogens to the host tissue ]Cook and Sequeira, ibid.; Bugert and Geider, 1995, ibid.]. This initial attachment is needed for efficient delivery of virulence factors into the host cells, via the type III secretion systems of these pathogens. In cystic fibrosis (CF) patients, Pseudomonas aeruginosa cells secret loose EPS to form a biofilm in the host's lungs. This biofilm is essential for establishing infection that is lethal to infected individuals with CF. In addition, bacteria embedded in biofilms are highly resistant to antibiotic treatments. Hence, formation of biofilm that is mediated by EPS is essential for the virulence of many pathogens [Costerton JW el al., Science 284(5418): 1318-22 (1999)]. In the plant symbiont Rhizobium meliloti, the succinoglycan EPSs play a specific role in interaction with the host plant. The succinoglycan is released into the surroundings and mediates signaling to the host cell, which is essential for the formation of fully matured nodules [Leigh and Walker, ibid.]. Similar signaling role for EPS is yet to be defined in the case of bacterial virulence.

Antimicrobial resistance is emerging as an important public health problem in both hospitals and the community [Cohen, Science 257: 1050-1055 (1992)]. Untreatable infections with many pathogens including Klebsiella pneomonia, Pseudomonas aeruginosa and Staphylococcus aureus are being recognized more frequently. As bacterial pathogens become increasingly resistant, the lack of new alternative antimicrobial agents is causing serious problems. Namely, the existence of bacterial strains that remain susceptible to only a single clinically available antibiotic: vancomycin. Furthermore, the emergence of vancomycin resistance in enterococci has been described [Travis, (1994)] as the "physician's worst nightmare". This resistance is spread to staphylococci and pneumococci that cause common diseases such as pneumonia and many ear infections, which are then beyond the reach of effective therapy. The recent report of vancomycin-resistant Staphylococcus aureus in Japan [Hiramatsu, K., et al, J. Am. Med. Assoc. 275:300-304 (1997)], emphasizes even more the great need for new antibacterial drugs that are not subjected to current resistance mechanism. EPS is en essential virulence factor of all of these pathogens and developing new drugs that will inhibit EPS production may represent a new approach for antibiotic development.

All the currently used antibiotics interfere with basic functions that are specific to the bacteria but not the host. These include drugs that inhibit translation, transcription, replication, cell wall biosynthesis and others basic bacterial functions. The end result of treating bacteria with either of these drugs is similar; growth inhibition and/or bacterial death. This, of course, imposes a strong selection pressure for the emergence of drug-resistant strains due to endogenous mutations or acquisition of resistance genes. Consequently, scientists in research institutes and pharmaceutical companies are engaged in an on-going effort to develop new and potent derivatives of known antibiotics and search for new types of antibiotic agents acting on a completely new molecular targets.

The latter approach includes developing drugs which will not kill or inhibit bacterial growth and hence will not impose a selective pressure that leads to resistant mutants. Instead, these drugs aim to inactivate the virulence mechanism of the pathogens, leading to self-limiting infection without progression to stages of systemic diseases. Inactivation of the virulence mechanism will not pose a direct strong selection force for development of drug resistance strains. Furthermore, it is expected that the treated pathogen will be an easy "prey" to the host immune system. Infection without progression to disease stages, which will occur in the presence of such drugs, is analogous, in some way, to vaccination with attenuated pathogen. Therefore, an added bonus of using this type of drugs, might be development of immunity and vaccination against some pathogens. An example for such drug might be inhibitors of EPS production. EPS inhibitors, which inhibit capsule and biofilm production, are also expected to increase the sensitivity of the infecting bacteria to other, more conventional, antibiotics. Etk is a member of a newly defined family of prokaryotic PTKs. This inner-membrane protein catalyses auto-tyrosine phosphorylation and phosphorylation of an exogenic synthetic substrate. Etk is expressed by several pathogenic E. coli strains including EPEC EHEC and ETEC but not by non-pathogenic E. coli strains. Etk, and Etk-like proteins, play a role in EPS production by many pathogens. Few examples are AmsA of the plant-pathogen Erwinia amylovora and Orf6 of the cps operon of the human-pathogen Klebsiella pneumonia, CapB of Staphylococcus aureus and CpsC of Streptococcus agalactiae [Paulsen I.T., et al., Microbiology 143:2685-99 (1997)].

Production of EPS consists of two major steps. First the bacteria polymerized the sugar residues in the cytoplasm and than it secret the polymers to the bacterial surface. It was hypothesis that Etk and Etk-like proteins, together with a protein tyrosine phosphatase (PTP) and an outer membrane lipoprotein (OMLP), make up the EPS secretion apparatus [Whitfield C. and Roberts I.S., Mol Microbiol 31 : 1307-19 (1999)]. This hypothesis was recently confirmed by the inventors in the EPEC system. EPEC was constructed with knockout in the etk gene. Two methods were used to compare the etk mutant to the wild type strain. The first method is determination of the bacteria density on percol gradient. It was expected that the capsule production decrease the bacterial density. The second method is labeling the bacteria with anti-capsule antibody and immunofluorescent microscopy analysis. Both assays indicated that the elk mutant is deficient in EPS production.

Etk and Etk-like kinases look very different from eukaryotic protein kinases when analyzed using different computer software. Thus, it may be possible to identify an inhibitor that will be specific for the Etk-type kinases but not to eukaryotic kinases. Such an inhibitor may inhibit production or secretion of EPS without effecting the infected human. Such compounds may be used to treat bacterial infection with or without other synergistic antibiotics.

Automated format imposes certain limitations that affect design of an assay in practice. Procedures that are used routinely in bench work including centrifugation are often extremely difficult to automate. Also, the more steps required for an assay, the more difficult to apply to automated HTS. The ideal assay is one that can be performed in a single well with no other manipulation other than injection of samples to be tested. The present invention will enable establishing such an assay for measuring Etk activity.

Recombinant etk clones have now been generated, containing 6xHis tags at their N-terminal. This allows one-step purification procedure of these proteins using suitable affinity columns. The purified proteins show good activity towards polyGlu:Tyr co-polymer. This protein can be used for HTS screening using several alternative methods. The first to coat a multiter plate with poly Glu:Tyr and to react it with soluble 6xHis-Etk in 10 Mm Tris-HCl pH7.2, 10 mM MgC12 and 20 μM ATP as a phosphate donor. Then the enzyme is washed and the level of tyrosine phosphorylation of the solid-phase associated polyGlu:Tyr will be monitored by ELISA assay with anti-phosphotyrosine antibody. Compounds that inhibit tyrosine phosphorylation of polyGlu:Tyr are potential Etk inhibitors. This approach will allow a sensitive, easy and non-radioactive kinase assay. This assay together with measuring the Etk amount with the anti-Etk antibody will allow determine the specific activity of Etk. An alternative approach is to use the protein for screening of phage display libraries to isolate peptides that specifically interact with Etk. This peptide may be used as Etk inhibitor as well.

One may use Etk and a phosphate donor such as ATP to tyrosine phosphorylate proteins in vitro. Tyrosine phosphorylation will tag the treated protein. If the ATP contained radioactive phosphate isotope at the γ position, the reaction will yield radioactive labeling of the protein of interest. In addition, the phosphorylated protein will be detectable also with anti-phosphotyrosine antibody. Labeled proteins may be used for variety of applications, including detection of protein-protein, protein-DNA and protein-RNA interactions. Thus, the present invention relates to an isolated protein tyrosine kinase (Etk) having an amino acid sequence substantially homologous to the amino acid sequence of E. coli Etk and to functional equivalents and derivatives thereof.

More particularly, the invention relates to a protein having an amino acid sequence substantially homologous to the amino acid sequence of E. coli Etk encoded by a DNA sequence substantially as shown in SEQ ID NO: l, or a functional equivalent or derivative thereof.

The term "functional equivalent" as used herein is intended to cover minor variations in the nucleic acid sequence encoding the protein, which, due to degeneracy in the genetic code, do not result in a sequence encoding a different polypeptide.

The term "homologous" as used herein means modified protein that does not show any significant reduction in catalytic activity, compared to wild type Etk. Such modifications can be insertions, deletions or substitutions of one or more amino acid residues (also referred to as "equivalent/s" or "derivative/s").

In specific embodiments, the invention relates to an insertion mutant protein that carries a tag. The term "tag" as used herein is to be taken to mean a functional segment inserted within the frame of the coding region. This segment is capable of being recognized as is, by a certain chemical moiety that may be deposited or form part of a solid phase. An example of such tag is the 6xHis tag, as discussed above and demonstrated in the following Examples. Alternatively, a tag moiety may be modified by attaching another functional group to a certain residue located therein. An example of such tag moiety is the biotin acceptor tag. The tag binds specifically to an immobile phase packed in an affinity column, that can be used for the purification of a desired mutant protein in one step (a protein carries a tag will be hereafter referred to as "tagged protein").

In a further aspect, the invention relates to an expression vector comprising a nucleic acid sequence encoding a protein having an amino acid sequence substantially homologous to the amino acid sequence of E. coli Etk. Preferred expression vectors according to the invention are vectors comprising the DNA sequence substantially as shown in SEQ ID NO: l . The expression vectors of the invention may further comprise operably linked expression control elements.

The invention further relates to hosts transformed with an expression vector of the invention. Specific host cells are unicellular organisms, such a bacterial cells, or mammalian cells in culture. The transformed hosts according to the invention are capable of expressing the protein encoded by the heterologous DNA with which they were transformed.

The invention also relates to a method for the purification of the proteins of the invention, such as various chromatographic techniques that are used in the art. Some typical examples are ion exchange chromatography and size exclusion chromatography.

In one specific embodiment, the invention relates to an affinity chromatography procedure for the purification of tagged Etk, as shown in the following Examples. This is a single stage method for obtaining a highly purified protein in one step. In this method a membranous extract of the cultured expression host is loaded on the affinity column, and non-bound material is washed off. Then, the bound material is eluted, collected and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) analysis.

In another embodiment, the invention relates to an agent that is capable of binding and/or inhibiting Etk or its equivalents. Such preferred agent will be low molecular weight compounds.

In addition, the invention relates to a method of screening for a low molecular weight compound, capable of binding and/or inhibiting Etk and its equivalents. In general, this method is based on the same principle of affinity chromatography. Herein, the purified Etk protein or its equivalent is anchored to the solid phase, and a sample containing a combinatorial synthetic library or phage display library, or any other sample of interest is loaded onto the column. A compound that can bind to the Etk, will bind to the solid phase, while others will remain non-bound and will flow through a solid phase column or can be washed off. Bound material can then be eluted, to be identified by methods such as high performance liquid chromatography (HPLC), mass spectrometry (MS) and any other suitable chemical analytical methods.

Further, the invention relates to an automated method of screening for an inhibitor of Etk. By way of example, for such purpose, potential candidate materials can be placed in separate wells in a multiter plate coated with an artificial Etk substrate, such as polyGlu.Tyr. Then, a phosphate donor, e.g. ATP, and Etk or its equivalent are added. The plate is incubated for a suitable period of time, any non-bound material is washed off, and the level of tyrosine phosphorylation of the substrate is monitored by ELISA with anti-phosphotyrosine. Any material capable of inhibiting phosphorylation will be identified by the mentioned and other suitable analytical methods.

With particular consideration of the of inhibition of bacterial EPS production discussed above, in a further embodiment, the invention relates to a therapeutic agent capable of inhibiting Etk activity, for the prevention and/or treatment of bacterial infections.

In another embodiment of the invention, it relates to a low molecular weight agent, capable of binding to the etk gene, as well as to etk mRNA transcript. It is expected that such agents will block Etk expression leading to an inhibition of bacterial EPS production. Particular examples of said agents are antisense DNA. antisense RNA or peptide nucleic acids (PNA).

In addition, the invention relates to a method of screening for said agent/s In general, this method is based on the principal of affinity chromatography. Herein, a nucleic acid equivalent to etk gene DNA or mRNA is anchored to the column, while a combinatorial synthetic library or phage display library is loaded on the column. The agents can also be endogenous substances. While an agent of interest will bind to the column, others will flow through. The bound agent can then be eluted and identified.

In a special embodiment, the invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of any agent capable of reducing Etk activity. Such agent can be one that binds directly to the Etk protein and inhibit it. Alternatively, such agent can bind to etk gene or RNA transcript while blocking the expression of Etk protein.

The "pharmaceutically effective amount" for purposes herein is that determined by such considerations as are known in the art. The amount must be sufficient to cause any significant change in any physiological or immunological parameter when administered to a sick human or animal.

The pharmaceutical compositions of the invention can be prepared in dosage unit forms and may be prepared by any of the methods well known in the art of pharmacy. In addition, the pharmaceutical compositions of the invention may further comprise pharmaceutically acceptable additives such as pharmaceutically acceptable carriers, excipients or stabilizers, and optionally other therapeutic constituents. Naturally, the pharmaceutically acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed.

The magnitude of therapeutic dose of the composition of the invention will be of course vary with the group of patients (age, sex, etc.), the nature of the condition to be treated and with the route administration and will be determined by the attending physician.

Further, the invention relates to a method for protein phosphorylation, in which Etk or an equivalent thereof may be co-expressed with target protein. In such method, both etk and a target gene can be cloned into expression vectors, which can later be used for transformation of a host. During the biosynthesis of the target protein, it will be phosphorylated by Etk. Alternatively, target protein can be labeled in vitro by incubation with purified Etk in reaction buffer (preferably 10 Mm Tris-HCl pH 7.2, 20 μM preferably ATP, 10 mM MgCl₂), followed by isolation of the phosphorylated target protein. For the in vitro labeling, the Etk can be immobilized on a solid phase.

Further, the invention relates to a kit for labeling of tyrosine residues on a target protein. Such kit will contain any of the following: Etk or equivalent protein, phosphate donor and reaction buffer (preferably 10 Mm Tris-HCl pH 7.2, 20 μM phosphate donor (preferably ATP), 10 mM MgCl₂).

The invention will be described in more detail on hand of the following Examples, which are illustrative only and do not in any sense limit the invention, which is only defined by the appended claims.

Examples Bacterial strains

All pathogenic E. coli strains including EPEC, ETEC, EHEC, EAEC, EIEC strains were obtained from M. Donnenberg (The University of Maryland). The E. coli K12 strains that were used are common laboratory strains available from different suppliers. K. pneumonia K2 strain KPA1 [Ofek et al. ibid.] was obtained from I. Ofek (Tel-Aviv University). All E. coli and K. pneumonia strains were grown in LB agar or LB broth at 37 C. The appropriate antibiotics were added when needed. The E. amylovora strains that were used and their growth conditions were described [Bugert and Geider (1995) ibid.].

Immunoblot analysis

Samples were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and transferred to nitrocellulose membrane (AB-S 83 Schleicher & Schuell Inc.) using a NovaBlot electrophoretic transfer unit (LKB) according to the manufacturer's recommendations [Rosenshine et al, ibid.]. Monoclonal anti- phosphotyrosine (PT66 Sigma) and polyclonal rabbit anti-Etk, anti-DnaK, anti-FtsH and anti-intimin antibody, were diluted in TBS (150 mM NaCl, 20 mM Tris-HCl pH 7.5) containing 1% BSA (Sigma). Binding of secondary anti-mouse IgG or anti-rabbit IgG (Sigma) alkaline phosphatase conjugated antibody was detected using the NBT/BCIP (Promega).

Purification and identification of Etk

Etk was purified from an extract of EPEC culture at the mid-logarithmic growth phase by affinity chromatography with agarose-anti-phosphotyrosine antibody (PT66, Sigma). The eluted protein was resolved by 8% SDS-PAGE, transferred to a PVDF membrane and subjected to N-terminal sequencing using standard procedures.

Recombinant DNA techniques

To clone etk the Ep85 encoding gene, two primers: 5'-ATAAGCTTGCCACTTTCAGTTTTACTCTTTCTCG and 5'-ATGGATCCTATGAATACGCCACCAGGCAG were designed based on a sequence of the yccC/elk gene of f. coli K12 MG1655 [Blattner et al. (1997) ibid.]. The primers were used to amplify a DNA fragment encoding Ep85 using standard PCR protocols and EPEC E2348/69 chromosomal DNA as a template. The amplified product was digested with BamHl and Hmdlll and ligated into the corresponding sites in pQE31 (Quigene). This generated a plasmid pEP19 expressing, via Ptαc, a short His tag fused to the N terminal of full length Ep85. Overexpression of Ep85 was toxic to the expressing bacteria. To avoid this toxicity and to enable better regulation of the expression of Ep85, a Sail fragment containing the lacIQ gene was cloned into the Xhol site of pEP19 to generate pOI194. To clone amsA. a DNA fragment encoding genomic amsA of E. amylovora was PCR amplified with the primers 5'-CGCTGCCCAGAAATGGG and 5'-GCCATTCATCGTCGGCG, cloned into pGEM-T (Promega), digested with Sphl/and Sail, and subcloned into plasmid pfdC4Z' under control of the /αc-promoter to give pfdC4Z-amsA. Purification of recombinant proteins

To prepare Ep85/Etk, E.coli XL1 Blue containing pOI194 was grown in LB to mid-logarithmic growth phase and expression of Ep85 was induced by adding IPTG to a final concentration of 0.1 mM. After 2 h expression, Ep85 was extracted and purified under native conditions using Talon metal-affinity chromatography according to the protocols recommended by the manufacturers (Clontech). The purified Ep85 was used for different assays and to raise anti-Etk antibody in rabbits.

Bacterial cell fractionation

EPEC culture (200 ml) was grown to density of OD 1.0 in LB, 37°C. The culture was harvested, washed, resuspended in 1 ml cold sonication buffer ( 10 mM Tris-HCl pH 7.5, 0.4 mM Vθ4Na3, 0.1 mg/ml phenylmethylsulfonyl fluoride (PMSF), and 10 μg/ml leupeptin), and sonicated. Cell envelopes and unbroken bacteria were removed by centrifuging twice (5,000xg for 5 min). The cleared supernatant containing cytosolic and periplasmic soluble proteins and inner and outer membrane was removed to fresh tubes and further centrifuged for 1 h at

50,000xg, 4°C) to pellet the membranes. The supernatant containing soluble proteins was removed (S-fraction), and the membrane pellet was washed with sonication buffer and resuspended in 0.1 ml of Sarkosyl buffer (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 0.4 mM VO4Na3, 0.1 mg/ml PMSF, 10 μg/ml leupeptin, and 0.5% N-lauroylsarcosine (Sigma)). Under these condition the inner membrane is dissolved but not the outer membrane [Nikaido, H., Methods Enzymol 235:225-34 (1994)]. The outer membranes were precipitated by centrifugation (50,000xg, 1 h) and the supernatant containing the solubilized inner membrane proteins was collected (1-fraction). The outer membrane pellet was washed in Sarkosyl buffer, precipitated again by centrifugation as before, and dissolved in 0.1 ml of SDS loading buffer (O-fraction). Appropriate amounts of 5X SDS loading buffer was added to the different fraction before application to SDS-PAGE. Dephosphorylation with YopH

The purified tyrosine phosphorylated Etk was treated with 5 U of the specific tyrosine protein phosphatase YopH, according to the manufacturer's recommendations (BioLabs). The reaction was stopped at 30, 60 and 90 seconds after adding YopH by removing 20 μl aliquots of the reaction mixture into tubes containing lvμl of 100 mM Na3NO4 . 5 μl of each aliquot was subjected for immunoblot analysis.

Protein kinase assays

Auto-phosphorylation was performed by adding 5 μl of purified Etk (about 1 μg Etk/μl) into 190 μl of reaction mix (150 mM NaCl, 10 mM MgCl2, 10 mM Tris-HCl, pH 7.4). The reaction was started by adding 5 μl of ATP (final concentration of 25 μM ATP, and 10 μCi [γ-32p]ATP). The reaction was stopped by removing 20 μl aliquots at different time points into tubes containing 5 μl 500 mM ETDA. 5 μl of each aliquot was used for analysis by SDS-PAGE. Phosphorylation of poly Glu:Tyr (4: 1, Sigma) was carried out in 40 μl containing

10 mM MgCl2, 20 mM Tris-HCl pH 7.5, 20 μM ATP, 1 μCi [γ-³²P]ATP, 10 μl of suspended Talon beads with bound Etk (about lvμg Etk μl), and poly Glu:Tyr at the indicated concentration. The reaction was stopped by short centrifugation and application of 25 μl of the Etk-free supernatant onto Whatman 3 MM paper which was than washed three times with 10% trichloracetic acid, once with ethanol, dried, and used to count radioactivity. In all experiments we included control with no enzyme (Etk) and control without exogenic substrate (poly Glu:Tyr).

Phosphoamino acid analysis

This analysis was earned out as described by Duclos et al [Methods Enzymol

201 : 10-21 (1991)]. Briefly, the purified Etk was labeled in vitro with [γ-³²P]ATP as described above and hydrolysed in 200 μl of 6N HC1 for 2 h at 110°C. The hydrolysate was dried in a Speed- Vac concentrator and resuspended in 20 μl of water containing 1 mg/ml of each of the phosphoamino acid markers; P-Ser, P-Thr, and P-Tyr (Sigma). Two μl of the hydrolysate were analyzed by ascending thin-layer chromatography (TLC cellulose, Merck Inc.) using a solvent containing mix of isobutyric acid and 0.5 M NH4OH (5:3, v/v). The position of phosphoamino acid markers (P-Ser, P-Thr, and P-Tyr) was detected by ninhydrin staining of the TLC plate (0.25% ninhidrin in acetone). The plate was then exposed to X-ray film to locate the position of the 32p labeled amino acids.

Results

Identification of tyrosine phosphorylated protein in EPEC and its coding gene

While studying aspects of the virulence of EPEC, it was noticed that this pathogen possesses a protein, Ep85, that cross-reacted with several monoclonal anti-phosphotyrosine antibodies, including PY20, 4G10, and PT66 [Rosenshine et al, ibid., and data not shown]. Ep85 was purified , and the amino acid sequence of its N terminal was determined to be MTTKNMNTPPGSTQENE. This sequence matched perfectly with the N-terminal sequence of an E. coli putative protein YccC encoded by an ORF at the appA 3' region of the E. coli K12 MG1655 genome [Blattner, F.R., et al, Science 277: 1453-74 (1997)]. This putative protein is 81.2 kDa in size, similar to the apparent molecular size of Ep85, as determined by SDS-PAGE [Rosenshine et al, ibid.]. A DNA fragment from EPEC E2348/69 that encodes for Ep85 was amplified and cloned under the control of tac promoter, with a His-tag added in the process at its N-terminal. The nucleotide sequence of the Ep85-encoding gene from EPEC (DDBJ/EMBL/GenBank Accession Number AJ238695), was found to be 97.6% identical to the nucleotide sequence of the corresponding E coli K12 gene. It encodes a protein, 80.5 kDa in size, that differs by 3 residues from the corresponding ORF in E. coli K12 (L92, G169 and G216 in the EPEC protein instead of Q92, E169, and E216 respectively in the K12 protein). Both proteins contain the nucleotide binding motif AXXXXGKT (Fig. 1A).

Ep85 catalyzes auto-tyrosine phosphorylation

As shown by many others, tyrosine phosphorylated proteins in E. coli K12, could not be detected using anti-phosphotyrosine antibodies (data not shown and Fig. 5). In contrast, recombinant Ep85 expressed in E. coli K12 strains was detected with anti-Ep85 antibody that we generated, and with anti-phosphotyrosine antibody (Fig 2A). This indicates that in vivo Ep85 becomes tyrosine phosphorylated in E. coli K12. To demonstrate that Ep85 catalyses auto-tyrosine phosphorylation, the recombinant Ep85 was expressed in E. coli K12, affinity purified, and used in a protein kinase (PK) assay with [γ-32p]ATP as a phosphate donor. The purified Ep85 exhibits a rapid autophosphorylation activity, suggesting that it is an ATP-dependent PK (Fig 2B). Accordingly, the purified phosphorylated Ep85 was rapidly dephosphorylated by the specific tyrosine protein phosphatase YopH (Fig. 2C). In addition, phosphoamino acid analysis indicated that the only phosphorylated residues in Ep85 were tyrosines (Fig 2D). Thus, Ep85 was renamed Etk (for E. coli protein tyrosine kinase) and its encoding gene was renamed etk.

Etk catalyses tyrosine phosphorylation of exogenic substrate The pattern of the proteins that were recognized by anti-Ep85 and anti-phosphotyrosine antibody in crude extract oϊ E. coli K12 expressing Etk was not identical. Some of these differentially recognized proteins were smaller than Etk and may represent degradation products of Etk. However, one of these proteins was larger than Etk and may represent an authentic phosphorylation substrate (Fig. 2A). The ability of Etk to phosphorylate in vitro the exogenic substrate poly Glu:Tyr copolymer was further investigated. Affinity purified Etk was incubated with

[γ_32p]ATP as a phosphate donor and poly Glu:Tyr as phosphate acceptor and phosphorylation of poly Glu:Tyr was monitored. Rapid tyrosine phosphoiylation of poly Glu:Tyr was detected (Fig. 3). Taken together, these results indicate the Etk carries out tyrosine phosphorylation of exogenic substrate.

Etk is an inner membrane protein

Cell fractionation and immunoblot analysis ere then used to determine in which cellular compartment Etk resides. These experiments indicate that most of the Etk was associated with the inner membrane and only small fraction of it was soluble (Fig. 4). In concordance with its membrane localization, prediction done using MOMENT program, based on the average hydrophobicity of a sliding window of size 21, identified three potential transmembrane helices in Etk: from F33 to T53, from A425 to A445, and from Y641 to G661 (Fig. 1A). The membrane localization of Etk may suggest that it is involved in signaling or in transport processes.

Differential expression and phosphorylation of Etk during EPEC growth

The inventors then determined the expression pattern of Etk and its tyrosine phosphorylation levels during EPEC growth. While the level of Etk decline upon entering the stationary phase, the levels of the phosphorylated form of the protein remained constant (Fig.5). This may suggest that only an unphosphorylated sub-population of Etk was targeted for degradation. In addition, at different stages of EPEC growth, changes in the mobility of Etk on SDS-PAGE were consistently observed and the Etk bands exhibit irregular M-shape (Fig. 5). This suggests that Etk may be further modified at late growth phases. This mobility shift and the an irregular band-shape were never observed when recombinant Etk was expressed in E. coli K12 (Fig 2A,B and data not shown). These results indicate that Etk expression and its post-translational modifications are dynamic processes during EPEC growth.

Etk is expressed by a subset of pathogenic strains ofE. coli

The presence of etk and its expression were examined in six clinical isolates of EPEC and several E. coli K12 strains (Fig. 6A). In all the strains, the specific etk primers amplified one fragment identical in size to that of elk. This PCR analysis indicated that all the EPEC and E. coli K12 strains possess the etk gene. In contrast, only the EPEC strains express the Etk protein (Fig. 6A). The same analysis was performed with several diarrheagenic virotypes of E. coli including enterotoxigenic E. coli (ETEC), enterohemorragic E. coli (EHEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC). Again, the etk gene was detected in all of the examined E. coli strains but was expressed only by EPEC, ETEC (Fig. 6B), and EHEC (data not shown). The molecular basis for the differential expression of etk between different E. coli strains has yet to be determined. Expression of Etk by only a subset of pathogenic strains of E. coli suggests that Etk may play a part in virulence mechanisms. Etk is a member in a protein family

Etk is homologous to several bacterial proteins that are involved in the production of extracellular polysaccharides (EPS). The Etk-homologs include Wzc, an Etk-like protein in E. coli K12 that is involved in the production of colanic acid capsule [Stevenson, G., et al, J Bacteriol 178:4885-93 (1996)]; Orf6 of Klebsiella pneumonia which is required for the formation of K2 capsule [Arakawa, Y., et al., J Bacteriol 177: 1788-96 (1995)]; AmsA of E. amylovora which is required for amylovoran production [Bugert, P. and Geider, K., FEBS Lett 400:252-6 (1997)]; EpsB of P. solannacearum which is needed for the production of EPS-I [Cook and Sequeira ibid.] and ExoP of R. meliloti that is needed for production of succinoglycan [Becker, A., et al, Mol Gen Genet 241 :367-379 ( 1993)] (Fig 1 and not shown). The K2 capsule, amylovoran, EPS-I and succinoglycan are required for virulence or interaction of the corresponding pathogens with the respective animal or plant host. Wzc, Orf6, AmsA and EpsB exhibit over 50% identity and 70% similarity to Etk while ExoP is somewhat less similar. Etk is also about 36% identical and 40% similar to Ptk, the only known prokaryotic PTK of A. johnsυnii [Grangeasse et al, ibid.].

E. amylovora and K. pneumonia express tyrosine phosphorylated proteins that cross-react with anti-Etk antibody

To test whether like Etk and Ptk, other Etk-homologs are also PTKs, extracts of E. amylovora and K. pneumonia were analyzed by immunoblot analysis with anti- phosphotyrosine and anti-Etk antibody. In agreement with the prediction, E. amylovora Ε.Α /I1 contained a protein similar in size to AmsA that cross-reacts with anti-Etk and with anti-phosphotyrosine antibody (Fig. 7A). Moreover, this cross-reaction was not detected in the isogenic E. amylovora amsA mutant strain Ea7/74-A56 (Fig. 7A). The same results were obtained with a different E. amylovora isolate, strain 1/79, and its isogenic amsA mutant strain 1/79-D49 (data not shown). K pneumonia K2 KPA1 [Ofek et al, ibid.], also contained a protein, presumably Orf6, that cross-reacted with anti-phosphotyrosine and anti-Etk antibody (Fig. 7A). These results support the suggestion that AmsA and Orf6 are PTKs. Interestingly, like Etk, both AmsA and Orf6 exhibit a higher molecular weight than expected and an M-like band-shape in SDS-PAGE (Fig. 7). These characteristics were never observed in recombinant AmsA expressed in E. coli K12

AmsA ofE. amylovora is a PTK

The amsA gene was cloned under the control of lac promoter in the plasmid pfάC4Z-amsA. This plasmid was introduced into E. amylovora amsA -mutant Ea7/74-A56 and it restored the wild type phenotype including formation of mucoid-colonies, production of amylovoran EPS, sensitivity to the EPS-specific phage Ealh, and generation of ooze on slices of infected immature pears (Table 1). pfdC4Z-amsA introduced into E. coli K12 XL 1 -Blue, induced AmsA expression with IPTG and extracted the bacterial proteins. The extracted proteins were analyzed by immunoblot analysis with anti-phosphotyrosine and anti-Etk antibody. The recombinant AmsA reacted specifically with anti-Etk and with anti-phosphotyrosine antibody (Fig. 7B). These results indicate that the recombinant AmsA catalyses auto-tyrosine phosphorylation in vivo.

Table 1

Complementation of an E. amylovora msyl-mutant

Ea7/74 Ea7/74-A56 Ea7/74-A56 (pfdC4Z'-amsAl)

EPS on MMlgal-agar, colony morphology³ muc nmu muc EPS in MMl gal- medium (μg/ml) 160 15 155

Ooze on pear slices⁰: +

Sensitivity to Ealh : + +

^dAmylovoran-specific E. amylovora phage; ^aMM l, the minimal medium has been described in Bugert and Geider (1995); gal: 1% galactose as carbon source; muc, mucoid; nmu, non-mucoid; nd, not done

Claims

1. An isolated protein which is a protein tyrosine kinase having an amino acid sequence substantially homologous to the amino acid sequence of Etk protein of ii. coli.

2. A protein tyrosine kinase encoded by a DNA sequence substantially as shown in SEQ ID NO: 1 or a functional equivalent or derivative thereof.

3. A recombinant protein according to claim 2.

4. A recombinant tagged protein having protein tyrosine kinase activity comprising an amino acid sequence substantially homologous to the amino acid sequence of E. coli Etk protein and tag moiety.

5. An expression vector comprising a DNA sequence encoding protein which is a protein tyrosine kinase having an amino acid sequence substantially homologous to the amino acid sequence of Etk protein from E. coli.

6. An expression vector according to claim 5 wherein said DNA sequence has the nucleotide sequence substantially as shown in SEQ ID NO: l .

7. An expression vector according to claim 5 or claim 6 further comprising operably linked regulatory elements.

8. An expression vector according to claim 7 comprising operably linked regulated 15-lac promoter/operator element, a ribosome binding site derived from the expression vector pQE31, and a repressor lacl^q gene.

9. Plasmid pOI 194.

10. A host transformed with an expression vector according to any one of claims 5 to 9.

11. A transformed host according to claim 10, being a unicellular organism or a mammalian cell in culture.

12. A method of producing a protein having protein tyrosine kinase activity and having the amino acid sequence substantially homologous to the amino acid sequence of Etk protein of E. coli comprising the steps of:

(a) providing a host cell;

(b) transforming said host cell with an expression vector comprising a DNA sequence encoding a protein having the amino acid sequence substantially homologous to the amino acid sequence of Etk protein of

E. coli;

(c) culturing said transformed cell;

(d) harvesting the cultured cells obtained in step (c); and

(e) isolating the protein from the harvested cells obtained in step (d) by suitable liquid chromatography method.

13. A method of producing a protein according to claim 12 wherein said DNA sequence is substantially as shown in SEQ ID NO: l or a functional equivalent or derivative thereof.

14. A recombinant protein having protein tyrosine kinase activity produced by the method of claim 12 or claim 13.

15. A method of producing a recombinant tagged protein having protein tyrosine kinase activity and having an amino acid sequence substantially homologous to the amino acid sequence of Etk protein of E. coli, comprising the steps of:

(a) providing a host cell;

(b) transforming said host cell with an expression vector comprising a DNA sequence encoding a protein having the amino acid sequence substantially homologous to the amino acid sequence of Etk protein of E. coli and further comprising operably linked nucleotide sequence encoding a tag moiety; (c) culturing said transformed cell;

(d) harvesting the cultured cells obtained in step (c); and

(e) isolating the recombinant tagged protein from the harvested cells obtained in step (d).

16. A recombinant tagged protein produced by the method according to claim 15.

17. A method for the purification of a protein according to claim 16, comprising the steps of:

(a) providing a sample of membrane extract from a host according to claim 10 transformed with an expression vector according to claims 5 to 9;

(b) loading of said sample on a suitable affinity chromatography column;

(c) washing off any non-bound material;

(d) separating the bound material by elution with a suitable eluant.

18. An agent that selectively binds to a protein substantially homologous to E. coli Etk and to a functional equivalent or derivative thereof.

19. An agent according to claim 18 being a low molecular weight compound.

20. An agent according to claim 18 or claim 19, capable of inhibiting the protein tyrosine kinase activity of a protein according to any one of claims 1 to 4, 14 and 16.

21. A method of screening a sample for the presence of an agent that selectively binds to a protein substantially homologous to E. coli Etk or to a functional equivalent or derivative thereof, comprising the steps of: (a) depositing a protein having an amino acid sequence substantially homologous to the amino acid sequence of Etk protein of E. coli or a functional equivalent or derivative thereof on a suitable solid phase carrier;

(b) incubating the said sample with the deposited protein obtained in step

(a);

(c) washing off any non-bound sample material;

(d) separating bound material from said deposited protein; and

(e) identifying the material obtained in step (d).

22. An automated method of screening a sample for the presence of an agent that is capable of inhibiting the protein tyrosine kinase activity of a protein substantially homologous to E. coli Etk or of a functional equivalent or derivative thereof, comprising the steps of:

(a) coating a multiter plate with a suitable protein tyrosine kinase substrate;

(b) adding said sample;

(c) adding a solution containing said protein and a phosphate donor;

(d) incubating the plate for a suitable period of time;

(e) washing off any non-bound material; and

(f) monitoring the level of tyrosine phosphorylation.

23. A method of screening a sample for the presence of an agent that selectively binds to a nucleic acid substantially homologous to E. coli etk gene and to a functional equivalent or derivative thereof or its mRNA transcript, comprising the steps of: (a) depositing said nucleic acid or functional equivalent or derivative thereof on a suitable solid phase carrier;

(b) incubating the said sample with the deposited nucleic acid obtained in step (a);

(c) washing off any non-bound sample material;

(d) separating bound material from said deposited protein; and

(e) identifying the material obtained in step (d).

24. A method according to claim 22 wherein said protein tyrosine kinase substrate is poly-Glu:Tyr.

25. An agent that is capable of inhibiting the protein tyrosine kinase activity of a protein substantially homologous to E. coli Etk or of a functional equivalent or derivative thereof which is a therapeutic agent for the treatment of bacterial infection.

26. An agent that is capable of binding to a DNA sequence substantially homologous to the etk gene of E. coli, substantially as shown in SEQ ID NO: l .

27. An agent according to claim 26, which is a therapeutic agent for the treatment of bacterial infection.

28. An agent according to claim 26 or 27, which is a nucleic acid.

29. An agent according to claim 26 or 27, which is a peptide nucleic acid (PNA).

30. A method of labeling a tyrosine residue of a target protein with a phosphate group, wherein said labeling is catalysed by a protein substantially homologous to E. coli Etk, comprising the steps of: (a) mixing said target protein with a protein substantially homologous to E. coli Etk with a phosphate donor in suitable buffer for a suitable period of time; and

(b) isolating the phosphorylated target protein obtained in step (a).

31. A method according to claim 30, wherein said protein that is substantially homologous to E. coli Etk is covalently or non-covalently attached to a solid phase carrier.

32. A method according to claim 30 or 31, wherein said target protein is Etk or an equivalent according to any one of claims 1 to 4, 14 and 16.

33. A pharmaceutical composition for the treatment of an infection caused by a bacterial pathogen expressing etk gene or a homologous gene, comprising at least one agent according to any of claims 18 to 20 and 25 to 29.

34. A kit for labeling a tyrosine residue of a target protein with phosphate comprising at least a protein having an amino acid sequence substantially homologous to E. coli Etk or a functional equivalent or derivative thereof and phosphate donor.

35. A kit according to claim 34, wherein said target protein is Etk or an equivalent according to any one of claims 1 to 4, 14 and 16.

36. A method according to claims 22, 30, or 34 wherein said phosphate donor is

ATP.