METHOD FOR GENERATING PROTEOLYTIC ENZYMES SPECIFIC AGAINST A SELECTED PEPTIDE SEQUENCE

Abstract: The present invention describes methods for screening or selecting novel proteolytic enzymes. These enzymatic functions can be selected from isolated chromosomal DNA libraries or from pools of mutagenized DNA encoding a proteolytic function. In particular, the selection method of the present invention, a phage gene is chosen that encodes a gene product necessary for the production of a phage. The phage carrying the modified gene is introduced into a host. Also, DNA libraries present within a cloning vector are introduced into host cells. The host cells are grown under conditions where the introduced libraries are expressed in the host cells. The presence of an enzymatic function capable of cleaving the target peptide is identified on the basis of production of infective phage.

Description: METHOD FOR GENERATING PROTEOLYTIC ENZYMES SPECIFIC AGAINST A SELECTED PEPTIDE SEQUENCE I. Field of the Invention The present invention relates to screening and selection methods for generating substrate-specific proteolytic enzymes capable of cleaving a specified target peptide sequence, such as present in casein, hyperallergenic peptides, endotoxins, viral coat proteins, and IgG, and for enhancing the proteolytic activity of known proteases against known-sequence peptide targets. H. References Arber, W., et al., in LAMBDA II. edited by R.W. Hendrix, et al., Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pages 433-466 (1983). Ausubel, F.M. , et al. , in CURRENT PROTOCOLS IN MOLECULAR BIOLOOY. John Wiley and Sons, Inc., Media PA. Bachovchin, W.W., et al., Science 24Q:1041 (1988). Bone, R., et al., Nature 222:191-195 (1989). Bricker, J., et al., P.N.A.S. USA 8^:2681-2685 (1983). Chang, A.C.Y., et al., J. Bacteriol. 1 4: 1141 (1978). Ciccarelli, E., et al., Biochem. Biophys. Res. Comrnun. 161:865 (1989). Craik, C.S., et al., Science 228:291 (1985). Crea, R., U.S. Patent No. 4,888,286, issued December 19, 1989. Crissman, J.W., et al., Virolo-rv 132:445 (1984). Cross, C.E., in BRONCHIAL ASTHMA: PRINCIPLES OF DIAONOSIS AND TREATMENT. Second Edition, M.E. Gershwin, Ed., Publ. Grunc and Stratton (Harcourt Brace Jovanovich), pages 39*47 (1986). Davis, C.G. and Fabian, G.R., U.S. Patent No. 5,258,289 issued November 2, 1993. Davis, N.G., and Model, P., Cell 11:607-614 (1985). Davis, R.W., et al., in A MANUAL POR GENETIC ENGINEERING: ADV.ANCED BACTERIAL GENETICS. Cold Spring Harbor Laboratory, Cold Spring Harbor NY (1980). Dayhoff, M.O., et al., Methods in Enzymology 9 .:524 (1983). Doolittle, R.F., Science 214:149 (1981). Eaton, M.A.W., et al., U.S. Patent No. 4,719,180, issued Jan. 12, 1988. Estell, D.A., et al., Science 222:291 (1985). Fowler, R.G., et al., Mol. Gen. Genet. 122:179-191 (1974). Gargiulo, R.J., et al., U.S. Patent No. 4,336,186, issued June 22, 1982. Gilbert J.V., et al., Infection and Immunity 52:7-17 (1991). Goldman, K., et al., FEBS Letters 190(7ι:319 (1985). Goldsmith, M.E., et al., Biochemistry 1£:2686 (1977). Grundy, F.J., et al.. Infection and Immunity 58:320-331 (1990). Gussin, G.N., et al., in LAMBDA II. edited by R.W. Hendrix, et al., Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pages 93-121 (1983). Hedstrom, L., et al., Science 255:1249-1253 (1992). Helm, B., et al., Nature 221:180 (1988). Helm, B., et al., Proc. Natl. Acad. Sci. 8£:9465 (1989). Ho, S.N., et al., Gene 11:51 (1989). Hubacek, J., et al., J. Mol. Biol. 5Q:111 (1970). Huse, W.D., et al.. Science 246:1275 (1989). Hussain, K., et al., Mol. Microbiol. 1(1):73 (1987). Ideda, R.A., et al., Biochemistry 22:9115 (1993). Ishizaka, T., et al., Immunochemistry 2:687 (1970). Jones, E.W., Genetics 85:23 (1977). Jones, E.W., et al., in ALFRED BENZON SYMPOSIUM, von Wettstein, E., et al., 1&183 Copenhagen, Munksgaard. Kabat, E.A., et al., in SEQUENCES OF PROTEINS OF IMMUNOLOOICAL INTEREST. U.S. Public Health Service, National Institutes of Health, Bethesda, MD (1987). Koomey, M.J., et al., P.N.A.S. USA 72:7881-7885 (1982). Lubke, K and Schroder, E., Annalen der Chimie £22:237 (1966). Maniatis, T., et al., in MOLECULAR CLONING: A LABORATORY MANUAL. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982). Mestecky, J. and McGhee, J.R., Adv. in Immunol. 4 : 153-245 (1987). Miller, J.H., in EXPERIMENTS IN MOLECULAR GENETICS. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1972). Mieschendahl, M., et al., J. Bacteriol. 164,(3): 1366 (1985). Morrison, S., et al., Proc. Natl. Acad. Sci. 81:6851 (1984). Mullis, K., U.S. Patent No. 4,683,202, issued July 28, 1987. Needleman, S.B., et al., J. Mol. Biol. 48:443 (1970). Nelson, F.K., et al., Virology lfl8:338 (1981). Oka, A., et al., Mol. Gen. Genet. 122:151 (1982). O'Shannessy, D.J., et al., Immun. Letters 8:273 (1984). Ovchinnikov, Y.A., et al., Gene 6:235 (1979). Radhakrishnan, R., et al., U.S. Patent No. 4,895,719, issued January 23, 1990. Roberts, T. M., et al., Nature 270:274 (1977). Russell, M., et al., Gene 45:333-338 (1986). Short, J. M., et al.. Nucleic Acids Res. 16:7583 (1988). Skerra, A., et al., Science 242:1038 (1988). Smith, G.P., Virology M.: 156 (1988). Smith, R.E., U.S. Patent No. 3,862,011. Sutcliffe, J.G., et al., Cold Spring Harbor Symp. Quant. Biol. 42:77 (1978). Ullmann, A., Gene 22:27 (1984). von Heijne, G., Mid. Acid Res. 14:4683 (1986). Weisberg, R.A., et al., Virology 25:99 (1979). Wilks, H.M., et al., Science 242:1541 (1988). Woo, S.L.C., Methods in Enzymology 68:389 (1979). Yoshio, T., et al., U.S. Patent No. 4,849,350, issued July 18, 1989. Yamada, M., et al., Proc. Natl. Acad. Sci. USA 22:2827 (1982). m. Background of the Invention Substrate specificities associated with different members of the diverse families of proteolytic enzymes can be attributed in part to different sets of amino acids, within the binding domain, that are utilized by each enzyme family for substrate recognition and catalysis. A rational approach to engineering proteases has been successful for several proteases. A conserved amino acid residue (glycine 166) known from crystallographic data to reside within the binding cleft of subtil isin was changed to one of several different amino acid residues. The resulting enzyme derivatives showed dramatic changes in specificity towards substrates with increasing hydrophobicity and amino acid size (Estell, et al.). Another bacterially encoded serine endopeptidase, α-lytic protease, has also been rationally engineered, changing methionine 192 to an alanine. The resulting alteration within the active site of the enzyme appears to have increased structural flexibility of the enzyme active site. The resulting α-lytic protease derivative has a broader substrate specificity towards larger, more hydrophobic targets (Bone, et al.). Although these rational approaches have met with success in the altering of substrate specificity, not all mutations effecting substrate specificity are associated with the known or predicted binding cleft of a given enzyme. The substrate specificity of the serine protease trypsin was altered to a chymotrypsin-like function by alteration of amino acids within the binding domain as well as residues known to be outside of the binding domain (Hedstrom, et al.). Mutations outside of the binding cleft of an enzyme can have a profound effect on amino acid residue packing, conformation strain and conformational charge distribution of residues within the binding cleft and as a result can have a profound effect on substrate recognition, catalysis and enzyme stability. Several other enzymes have also been rationally modified to new substrate-specificities, including T7 DNA polymerase (Ideda, et al.), lactate dehydrogenase (Wilks, et al.). Finally, natural derivatives of the antibiotic resistance determinant, α-lactamase, have been obtained as a result of positive selective pressures to novel substrate specificities. There is a precedence for both rational approaches to altering the substrate-specificities of many different enzymes through a detailed understanding of the conformation and biochemical properties of an enzyme. Furthermore, random events which translate into unique enzymatic functions can also be generated under conditions where the proper selective pressures are applied for a desired catalytic function or substrate specificity. IV. Summary of the Invention The present invention includes a method of selecting a proteolytic enzyme (protease) effective to cleave a polypeptide having a selected target amino acid sequence. The method includes first identifying a natural protease enzyme. The enzyme is preferably one capable of cleaving a polypeptide at an amino acid sequence that is homologous to, i.e., close in amino acid sequence, but distinct from, the target sequence. There is introduced into a host cell, a phage system containing (i) a helper phage capable of expressing mutants of the natural protease, under suitable expression conditions, and (ii) a phage vector bearing a phage gene encoding a gene product necessary for the production of infectious phage. The latter gene has been modified by introducing the target peptide coding sequence into the gene such that the resulting gene product inhibits production of infectious phage, and cleavage of the target peptide results in an active gene product that allows production of infectious phage. The host cells are cultured under conditions in which protease genes are expressed in the host cells, and the cells are then screened for the production of infectious phage. From the screened cells, the protease genes associated with the infectious phage are isolated, for use in the production of protease enzyme specific against the target sequence. 5 In preferred embodiments, the screening includes detecting the presence of infectious phage by plaque formation and the phage gene encodes a phage coat protein. Also in preferred embodiments, the host cells are Escherichia coli cells, the phage gene is gene m of bacteriophage M13, and the target sequence is introduced into gene m in such a fashion as to inhibit export of the gene m product to the periplasmic space of the host cells. The phage gene may encode a fused protein composed of a phage protein required for plaque formation, under selected growth conditions, and a second protein which inactivates the phage protein when linked to one end of the protein, where the target sequence links the second protein to the phage protein. Here the screening includes detecting phage capable of producing plaques when grown under said selected growth conditions. The phage may be a lambda phage, the phage protein may be the cro protein, and the phage may contain a temperature-conditional mutation in its genomic cro gene which is inactive above a selected temperature, wherein the screening is performed above said selected temperature. In another aspect, the invention includes a method of enhancing the proteolytic activity of a native protease against a known target amino acid sequence. The method includes introducing into host cells, a phage system containing (i) a helper phage capable of expressing mutants of the native protease, under suitable expression conditions, and (ii) a phage vector bearing a phage gene encoding a gene product necessary for the production of infectious phage, where the gene is modified by introducing the target peptide coding sequence into the gene such that the resulting gene product inhibits production of infectious phage, and where cleavage of the target peptide results in an active gene product that allows production of infectious phage. The cells are grown under conditions in which the protease genes are expressed in the host cells, and the cells are screened for production of infectious phage, at a level which is elevated with respect to production of infectious phage in cells expressing the native protease gene. Protease genes associated with elevated levels of phage production are then isolated and used for the production of proteases having enhanced activity against said target sequence. These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings. V. Brief Description of the Drawings Fig. 1 show the construction of a CA1000 phage by introduction of a 1.1 kb fragment of pACYC184 (digested with Nhel/Bst l and blunt-ended) into a parental helper phage R408 containing a deleted packaging signal, and digested with Ban followed by flushing of the ends with T4 polymerase. Fig. 2 illustrates bacteriophage coat protein pm, encoded by geneffi of CA1000, and containing an eighteen amino acid leader peptide which is denoted by the one letter amino acid code. Maturation of p , requiring removal of the pm leader peptide, is the result of cleavage by the host encoded leader peptidase at the site denoted by the solid arrow. Bacteriophage derivatives and the introduced modifications in and around the pm leader peptide are outlined. The DNA polylinker enables in-frame insertion of short oligomers for encoding target peptides of choice. Figure 3 gives the schematic overview of the selection scheme. An overview of the positive selection scheme for the isolation of substrate-specific proteolytic functions expressed in E. coli. Bacteriophage p protein has been modified in the helper phage CA2000 to block host-encoded leader peptidase activity. An oligonucleotide encoding the desired target peptide is introduced into the polylinker sequence encoded within the mature portion of pm and is denoted by the solid triangle (A). In bacteria bearing phagemids which encode the desired proteolytic function capable of cleaving the target peptide, assembly and secretion of infective phage particles is restored. Since bacteriophage CA2000 functions as a helper phage, the phagemid DNA, encoding the desired proteolytic function will be preferentially packaged and easily recovered from the infective phage particles generated. The OmpT protease with a dibasic cleavage specificity, is used as an example protease. Figure 4 shows E. coli JK5000(ΔompT) containing the parental secretion vector pSec2 and pSec2OMP which were assayed for OmpT activity. Periplasmic extracts from E. coli JK5000 containing either of these constructs were assayed for activity using the chromogenic substrate BOC-Arg-Val-Ag-Arg-MCA at increasing time intervals. Relative florescence was measured with an excitation of 380 nm and an emission of 460 nm. Figure 5A illustrates an overlap extension PCR (Ho, et al.) used to construct in-frame N-terminal deletions of pm. Each deletion mutant was designed to contain the first three amino acids of the mature pm to ensure proper recognition and cleavage by the host encoded leader peptidase. The pm protein has been divided into two regions; the N-terminus which is critical for infectivity and the C-terminus which is required for functional phage assembly. The portion of pm from each construct is depicted. Each construct was assayed for the production of infective phage particles by measuring the number of chloramphenicol resistant transducing units. Figure 5B shows the p amino acid sequence flanking each deletion junction for each N-terminal in-frame deletion depicted; and Figure 6 shows two bacteriophage CA2000 derivatives containing target peptides. Oligomers introduced in between Xbάl and Sail code for a single or tandem target sites producing bacteriophage derivatives CA2000(OMP1) and CA2000(OMP2). Underlined amino acids have been modified. Amino acids in bold represent beginning of mature pm. VI. Detailed Description of the Invention A. Preparing the Target Phage The methods of the present invention can be used to generate novel substrate-specific protease enzymes capable of cleaving a defined target peptide sequence. Generally, selection of target peptides from larger protein coding sequences only requires that the target sequence is physically accessible to cleavage. Some desirable characteristics for a target peptide include: i) the presence of some charged amino acids; ii) a general hydrophilic nature; iii) a sequence long enough to allow for the desired specificity. With regard to the length of the sequence, if an enzyme having specificity similar to a serine protease is to be isolated, then the recognition sequence need only be similar to that for elastase, the cleavage site is X- Y-, where X is uncharged and non-aromatic (e.g., Ala, Val, Leu, He, Glu, Ser) and Y is non-specific. However, if more cleavage specificity is required or desired, the number of amino acids composing the target site would be increased. Fig. 1 illustrates the construction of a helper phage forming part of a phage system used in practicing the method, in one embodiment. The helper bacteriophage R408 (Russell, et αl.) was linearized within the (-) strand origin of replication with the restriction endonuclease BαnW. A 1.1 kb fragment containing the chloramphenicol resistance determinant from pACYC184 (Chang, et αl.) was introduced into the BαnU site and the resulting bacteriophage construct was designated CAIOOO (Figure 1). Interruption of the minus strand origin of replication was designed to decrease the levels of bacteriophage replicative form (RF) DNA ensuring stable propagation of subsequent assembly incompetent bacteriophage derivatives produced (Smith). The pm bacteriophage coat protein is required for functional phage assembly (Crissman, et αl.) and absolutely essential for phage infectivity (Nelson, etαl.). Under normal circumstances, prior to phage assembly, pm is targeted to the host inner membrane by an eighteen amino acid leader peptide (Goldsmith, et αl.). Upon insertion into the membrane, the pm leader peptide is cleaved by the host encoded leader peptidase, producing an assembly competent pm. Recognition and cleavage of the leader peptide by host encoded leader peptidase relies on amino acid residues -3, -1 relative to the cleavage site (von Heijne). The leader peptide of the bacteriophage coat protein pm, encoded by genelϋ, was modified in and around the pm leader peptide to inhibit leader peptidase cleavage. A schematic diagram of the pm modifications and the designated phage derivatives is shown in Figure 2. The effect each pm modification had on the generation of infective phage particles, quantitated as CmR transducing units, is outlined in Table I. Measuring infective phage particles as a function of antibiotic transducing units was done according to Smith using E. coli K91. Table I The pm leader peptide for the final bacteriophage derivative CA2000 was modified as follows: -10 (Pro > Leu), -6 (Pro > He), -3 (Ser>Met), -1 (Ser>Phe), + 1 (Ala>Glu). In addition to the leader peptide modifications, a short DNA polylinker (Xbal, BglU and Sail) was inserted downstream of the modified leader peptide. The restriction enzyme polylinker was introduced to permit in-frame insertion of short oligonucleotide linkers encoding amino acid target sequences for the selection of target specific proteolytic functions. Bacteriophage CA2000 contains the following properties: i) has a deleted packaging signal and an interrupted minus strand origin of replication which enables it to function as an efficient helper phage; ii) antibiotic resistance determinant, chloramphenicol, which allows for constant selective pressure of any phage derivatives and a means to quantitate the number of infectious phage particles generated by selecting for antibiotic resistant trans¬ ducing units; iii) modified p to be assembly incompetent; and iv) DNA polylinker present downstream of the modified pm leader peptide for the inframe introduction of DNA encoded target peptides. Trans complementation to bacteriophage CA2000 with a wild-type copy of geneffl was used to verify the inability of CA2000 to produce infective phage particles was the result of the modifications within the p leader peptide. A TcR, pACYC184 derivative containing the wild type geneiπ placed under the control of an inducible trc promoter system was transformed into E. coli SURE (has mutations in a number of DNA recombination and repair pathways to inhibit homologous recombination) containing bacteriophage CA2000. A phage lysate harvested from an uninduced overnight culture of CA2000 with a wild type pm supplied in trans-produced 2 x 10 3 chloramphenicol-resistance transducing units/ml of culture. This result confirms the inability of CA2000 to produce infective phage particles derives from the pm materials. The selection system was designed to find target-specific proteolytic activity. However, this intent could be confounded if cleavage of pm outside of the intended target sequence can result in the production of infective phage particles. A series of successively smaller N-terminal in-frame deletions of p were generated to determine the minimum length pm required for functional phage infectivity (Fig. 5A). 22, 47, and 87 amino acid N-terminal inframe deletions of the mature pm proteins were generated. The resulting CAIOOO bacteriophage derivatives, CAIOOO (Δ22), CAIOOO (Δ47) and CAIOOO (Δ87), were assayed for the ability to produce infective bacteriophage particles. Although each of the p N-terminal deletion mutants constructed were able to produce a low level of phage particles, as determined by the isolation of packaged single-stranded DNA, none of the phage constructs was able to produce any infective phage particles. Although the possibility that functional phage infectivity requires the first 25 amino acid residues of pm could not be ruled out, these data suggest filamentous bacteriophage infectivity requires the complete pm protein (Figure 5B). B. Test of the Selection System To test the selection system, targeted expression of a cleavage-specific protease, encoded on a phagemid vector, to die periplasmic space of E. coli is required. The bacterially encoded protease, OmpT, with a dibasic recognition/cleavage site was cloned into an expression vector (pSec2) to target OmpT expression to die periplasmic space of E. coli. Periplasmic extracts from E. coli UT5600 (ompT, ompP) were isolated and tested with the chromogenic substrate BOC-Arg-Val-Arg-Arg-MCA. The results, depicted in Figure 4, show targeted expression of a cleavage-specific protease to die periplasmic space of E. coli. Bacteriophage CA2000 was digested with Xbal/Sall and ligated to two different sets of oligomers designed to have Xbal/Sall sticky ends. The resulting phage constructs, designated CA2000-OMP1 and CA2000-OMP2, are shown in Figure 6. CA2000-OMP1 contains a single Arg-Arg target site and CA2000-OMP2 contains two tandem dibasic recognition sites punctuated by a threonine (Arg-Arg-Thr-Arg-Arg). The overall sensitivity of die phage-based selection system was tested using the above phage constructs and assaying for the production of infectious phage particles from endogenous OmpT expression in E. coli (Figure 3). E. coli DH5α transformed with bacteriophage CA2000, CA2000-OMP1 or CA2000-OMP2 was assayed for the ability to produce infective phage particles resulting from endogenous OmpT dependent maturation of die pm bacterio¬ phage coat protein. The presence of a single dibasic target, present within CA2000-OMP1, did not appear to result in pm maturation resulting from endogenous OmpT activity. On the other hand, greater than ten-fold increase above background (500 OmR T.UJml) in the production of infectious phage particles was observed when die target phage containing two tandem targets was cultured in a strain bearing endogenous OmpT (Table U). As a control, a strain lacking endogenous OmpT activity does not produce any detectable levels of infectious phage particles for any of die three phage constructs tested (Table II). Phage OmpT+ OmpT- CA2000 <50 <50 CA2000 (OMP1) <50 <50 CA2000 (OMP2) 500 <50 Ultimately, the selection system will utilize enzyme derivatives expressed from multicopy phagemid derivatives. Therefore, a phagemid vector to target high level expression of OmpT to the periplasmic space of E. coli UT5600 was used. Overproduction of OmpT, die result of induction of expression in the phagemid vector, appears to be deleterious to E. coli causing cell lysis. To circumvent this problem, a leaky expression of OmpT in the selection system for the production of infective phage particles was used. The average number of ampicillin or chloramphenicol resistant transducing units isolated from each phagemid/phage combination are shown in Table HI. As expected, diere were no detected packaged infective phage particles produced for die control culture CA2000/pSec2OMP, since die parental phage lacks a dibasic target required for OmpT mediated maturation of pm. On die other hand, the presence of a single OmpT target present in CA2000 (Ompl) cultured with pSec2OMP shows die production of 2500 ampicillin-resistant transducing units and 380 chloramphenicol-resistant transducing units/ml of culture. Furthermore, multiple cultures were also assayed for the production of infective phage particles with CA2000 (Omp2) and pSec20MP. Interestingly, even more infectious phage particles were obtained when die target phage contained tandem dibasic targets. Approximately ten-fold increase was obtained in die production of ampicillin-resistant transducing units wiω CA2000 (Omp2) as die target phage, producing 18,000 ampicillin-transducing units/ml of culture. These results convincingly demonstrate that the model selection scheme generated and tested is functionally viable for the selection and isolation of a specific proteolytic function. Phage/Phagemid ApR T.UJml OmR T.UJml | CA2000/pSec2OMP <50 <50 CA2000 (Ompl)/pSec2OMP 2500 380 1 CA2000 (Omp2)/pSec2OMP 18000 660 | Proteins of interest can be examined for a variety of characteristics by using computer assisted sequence analysis and comparisons. For instance, a sequence can be scanned for likely target sites by searching for antigenic sites (ANTIGEN program, Intelligenetics, Mountain View CA) or doing a standard hydropadiicity analysis (SOAP program, Intelligenetics). Antigenic sites tend to be sites available on the surface of proteins. Further, minimum sequences that will distinguish d e target protein from other proteins can be determined by sequence comparisons (e. g. , using die SCANSIM program, Intelligenetics; based on die method of Needleman et al.). In one general embodiment, the target sequence (for which a protease is to be constructed) is compared widi die amino acid sequences recognized by known natural proteases. From these sequences is identified one which is close, and preferably closest, in sequence to die target sequence. The corresponding protease is then used as die starting-point structure for producing die protease of interest. That is, a gene encoding die known, selected natural protease is mutagenized or otherwise treated to introduce mutations, and die mutation- containing sequences are inserted into die phage which expresses the protease. The target which is selected may be one related to a therapeutic application of die protease. For example, the target sequence may be one present in an endotoxin, or a viral protein, or a bacterial wall protein, or a native blood-born peptide related to an auto-immune condition. Here die protease selected is used in a treatment method, by administering the peptide, e.g., by intravenous administration, to a person in need of such treatment. C. Utility Many medically important human pathogens including die respiratory pathogen Haemophilus influenza, the genital pathogen Neisseria gonorrhoeae, Streptococcus sanguis the causative agent of bacterial endocarditis and implicated in periodontal disease and dental caries, Streptococcus pneumonia involved in bronchitis and pneumonia, and Neisseria meningitides involved in bacterial meningitis each contain a protease able to specifically cleave the hinge region of human IgAl (Gilbert et al., Bricker et al., Koomey et al., Grundy et al.). IgAl is the predominant class of antibodies found in mucous membranes and is widely regarded as die primary line of defense against bacterial infection (Mestecky and McGhee). IgA specific cleavage within die hinge region of die IgA antibody by a bacterially encoded IgA protease results in separation of die Fc domain from the antigen binding Fab region of die molecule. Therefore any IgA mediated response to a human pathogen is effectively blocked by IgA cleavage with these IgA specific proteases. Although not definitively proven, these IgA proteases have been implicated in die ability of these pathogenic organisms to invade human tissues by inhibiting normal IgA function. Related but non pathogenic bacteria do not express IgA specific proteases, further suggesting a role of IgA specific proteases in pathogenesis. At least three different classes of proteases (metallo, serine and thiol) specifically cleave the IgA hinge region and have been isolated and characterized from a diverse set of human pathogens. The presence of IgA hinge specific proteases within these pathogens represented by diverse classes of different proteases further supports die suggestion diat this form of convergent evolution may be a necessary component of bacterial pathogenesis. Since these proteases appear to be important for tissue invasion and circumventing die natural IgA mediated defense system of die host, inhibitors of these proteases may be a potential target for use as a new class of antimicrobial therapeutics especially given die dramatic increase of antibiotic resistance within H. influenza, S. pneumonia and N. gonorrhoeae pathogens. 1. Development of a system to determine die effectiveness of inhibitors developed against any therapeutically relevant protease. Our example will focus on the bacterially encoded IgA hinge specific proteases. Several peptide prolyl boronic acids, known to be potent inhibitors of serine proteases, were tested to determine their effectiveness as specific inhibitors of IgA proteases isolated from several different human pathogens (Bachovchin, et al.). These inhibitors were found to be potent inhibitors of botii die Neisseria gonorrhoeae and Hemophilus influenza IgA proteases in the nanomolar range and, as expected, tiiese same inhibitors had no inhibitory effect against the non serine based IgA protease from Streptococcus sanguis (Bachovchin, et al.). In order to determine die effectiveness these or other types of specific inhibitors have on IgA proteases it would be important to know whether mutant IgA protease derivatives are able to circumvent tiiese inhibitors by mutation and inhibited their potential as therapeutic agents. Furthermore, if IgA protease variants are able to overcome these inhibitors, at what frequency would this occur. In order to answer these questions, we purpose to utilize our selection system to determine whetiier IgA mutants can be selected for which are able to circumvent die use of a candidate inhibitors for use as clinical therapeutics. This technique is envisioned to mimic the natural variability inherent in baαerial populations to determine die efficacy and duration at which a prospective inhibitor may expect to function tiierapeutically in a clinical setting. Proteases generated which are active in die presence of a prospective inhibitor could be used as a pool of protease variants which would be used to screen any future catalytic inhibitors generated. These same mutant proteases able to retain activity in the presence of an inhibitor will also be used to aid in die design and testing of more potent inhibitors. 2. We will use a candidate IgA specific protease and introduce it into our secretion phagemid vector pSec2 such that it contains a PelB leader peptide which targets expression to die periplasmic space of E. coli. The DNA encoding die protease will be mutagenized using random or rational based mutagenesis schemes (including in vivo mutagenesis witii an E. coli mutD strain, chemical mutagenesis witii hydroxylamine or a PCR based amplification designed to increase Taq polymerase infidelity) to generate libraries of IgA protease variants. Once generated, each library of IgA protease variants will be electroporated into a strain of E. coli containing a CA2000 bacteriophage derivative with die IgA protease specific hinge target (Thr-Pro-Pro-Thr- Pro-Ser-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser) introduced using the unique Xbal/Sall sites flanking our assembly defective pm. A library of IgA protease derivatives will mimic possible genetic variants within clinic populations and tell at what frequency mutations to an inhibitor will arise. A protease inhibitor, such as the prolyl boronic acids, known to inactivate d e IgA protease, is added to cells containing a bacteriophage CA2000 derivative and die library of IgA protease variants. Protease derivative which do not interact witii die IgA protease inhibitor and are still able to recognize and cleave their target peptide produce an assembly competent version of pm. As a result, infectious phage particles will be produced and phagemid DNA encoding die IgA protease derivative able to function in die presence of a candidate inhibitor will be preferential¬ ly packaged into d e resulting infective phage particles generated. Phage lysates are prepared from these cells and die DNA encoding these IgA protease variants are analyzed at the DNA and biochemical level. Those IgA protease variants mat are able to function in the presence of a candidate inhibitor, are used to aid in the development of new inhibitor derivatives. A pool of proteases able to function in die presence of an inhibitor is then used in screens to find future catalytic inhibitors able to inactivate a wide variety of protease variants which may arise through natural variation and mutation frequencies naturally occurring in a clinical setting. This assumes mat d e bacterial cell is permeable to inhibitor and die inhibitor is highly specific and does not affect any endogenous bacterial protease(s). 3. Generation of a panel of protease variants which recognize variation of die original substrate. Another approach to develop possible candidate therapeutics is to determine the absolute specificity of known clinical variants of a specific IgA protease type as well as to generate IgA proteases with altered substrate specificity. The natural IgA substrate Crhr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser- Pro-Ser) contains a two fold access of symmetry. Here one would generate a number of variants to the hinge target at individual amino acid positions and use these targets to generate a panel of IgA protease variants able to recognize subtle changes with die target peptide. As described above, these IgA protease variants may be found within natural clinical isolates or they may be selected for from libraries of mutagenized IgA proteases developed as outlined above. The Streptococcus sanguis IgA specific protease recognizes the right half of die hinge region consisting of Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser. Using die S. sanguis as a starting protease, a panel of target variants at different positions widiin this target is generated. For example one can generate variants at different positions widiin target with alternative amino acids, denoted as Xaa. Once generated, tiiese targets are reintroduced back into bacteriophage CA2000. SEQ ID NO: 1 Thr-Pro- Pro-Thr-Pro-Ser-Pro-Ser SEQ ID NO:2 Thr-Xaa-Pro-Thr-Pro-Ser-Pro-Ser SEQ ID NO:3 Thr-Pro- Xaa-Thr-Pro-Ser-Pro-Ser SEQ ID NO:4 Thr-Pro- Pro-Xaa-Pro-Ser-Pro-Ser SEQ ID NO:5 Thr-Pro- Pro-Thr-Xaa-Ser-Pro-Ser SEQ ID NO:6 Thr-Pro- Pro-Thr-Pro-Xaa-Pro-Ser SEQ ID NO:7 Thr-Pro- Pro-Thr-Pro-Ser-Xaa-Ser SEQ ID NO:8 Thr-Pro- Pro-Thr-Pro-Ser-Pro-Xaa After generating different target variants verified not to be recognized and cleaved by wild type IgA protease, natural clinical variants as well as testing libraries of IgA protease variants are tested for die ability to cleave new targets. Bacteriophage CA2000 containing die altered target peptides described above is introduced into E. coli cells containing a library of randomly mutagenized protease derivatives. Following overnight growth, phage lysate is generated and tested for die production of infective phage particles. Any protease variants which have altered substrate specificity are included into die panel of IgA specific protease variants used to test any protease inhibitors. The following examples illustrates the construction of vectors useful in practicing a specific embodiment of the invention, and cloning of a selected protease gene. The examples are in no way is intended to limit the scope of die invention. Example I Selection of Enzvmes Capable of Peptide Sequence Specific Target Cleavage A. The Peptide Vector The peptide vector is derived from CA2000. Useful features of CA2000 are the following: (i) it carries all the genes necessary for M13 phage morphogenesis; (ii) it carries a packaging signal, which interacts witii die phage origin of replication to initiate production of single-stranded DNA; (iii) it carries a disrupted phage origin of replication; (iv) and (iv) it carries a chloramphenicol resistance gene. The combination of an inefficient phage origin of replication and an intact plasmid origin of replication favors propagation of CA2000 in the host bacterium as a plasmid (as RF, replicating form, DNA) rather man as a phage. It can dierefore be maintained without killing die host. Furthermore, possession of a plasmid origin means mat it can replicate independent of die efficient phage-like propagation of die phagemid. By virtue of the chloramphenicol resistance gene, CA2000 can be amplified which in turn increases packaging of phagemid DNA into phage particles. The peptide vector of the present invention is generated as follows. Codons -10, -6, -3 and -1 relative to die signal peptidase cleavage site of gene m are modified: codon -3 from a serine to a phenylalanine and -1 from a serine to a tryptophan (Figure 15). The sequence of gene III is known. The modification of these codons is accomplished by standard procedures (Ausubel, et al.). Each of these substitutions independently prevents signal peptidase recognition (von Heijne). Accordingly, a reversion of two mutations would be required to restore cleavage of the signal peptide. Further, unique Xbal and Bglll, and Sail sites are inserted between positions + 1 and +2 relative to d e signal peptidase cleavage site. The Xbal/Sall restriction sites allow the directional cloning of oligonucleotides encoding target peptides of choice. The addition of foreign sequences to the amino terminus of the mature gene m protein product does not interfere with its ability to generate infectious particles. B. Cloning a Target Peptide into the Peptide Vector The target peptide is selected from die protein that is the target for cleavage. The length of the peptide should be approximately four to twenty amino acids. Two oligonucleotides are synthesized. One oligonucleotide, die sense strand which provides a continuous open reading frame in-frame with die gene III protein, contains, in the 5' to 3' direction, die nucleotides of SEQ ID NO:l followed by die coding sequence for die peptide. The second oligonucleotide, die anti-sense strand, contains, in the 5' to 3' direction, die nucleotides of SEQ ID NO:2 followed by die reverse complement of die peptide coding sequence. The two oligos are annealed in a reaction mixture containing 1.0 picomole of each oligo. One tentii of tiiis reaction, corresponding to 0.1 picomole of die double-stranded oligonucleotide, is ligated witii 1 picomole of die peptide vector RF DNA cut witii Spel and Xho I. The one to ten ratio of insert to vector promotes the cloning of a single insert per vector. Alternatively, die insert oligonucleotide may be dephosphorylated using Calf Alkaline Phosphatase (Maniatis, et al.). An appropriate strain of E. coli (e.g. MV 1184 or MV 1190) is transformed with die ligation mix (Maniatis, etal.). Chloramphenicol-resistant colonies are selected. These colonies are screened by hybridization (Ausubel, et al.) with an oligonucleotide corresponding to SEQ ID NO:l or SEQ ID NO:2 that has been end-labelled with 32 P. Small scale plasmid preparations of double-stranded DNA are made from the chloramphenicol resistant colonies mat test positive by hybridization. The isolated plasmid DNA is then sequenced across the peptide cloning site to ensure that (i) a single copy of the oligonucleotide encoding d e target peptide has been inserted, and (ii) a continuous open- reading frame exists through die target peptide encoding sequence and die gene ffl coding sequences. C. Generation of a Protease Enzyme Library A protease enzyme library is generated in Lambda ZAP vector, available from Stratagene. These M13-based plasmids carry the fd origin of replication and are referred to as phagemids since they have bo phage and plasmid-like properties (Figure 18). A combinatorial library of immunoglobulin genes is generated essentially as described in Example 1. The single fragments containing die light and heavy chain genes are cloned into die phagemid vector 3' adjacent the lacZ promoter thus generating a combinatorial Fab expressing library in an M13 based vector. A phagemid is excised from each vector of the combinatorial library (Figure 18) (Short, et al.; as per Lambda ZAP II manufacturer instructions). D. Electroporation of Phagemid Combinatorial Library The phagemid combinatorial library is introduced into E. coli transformed with the peptide vector by electroporation (Maniatis, et al). Electroporation is much more efficient than standard transformation procedures and allows one to generate libraries of over 10* independent clones. Typically, electroporation is performed witii approximately 80 ml of cells and 4 μg of DNA and using a 5 millisecond pulse of 12.5 kV/cm. The cells are then grown in L brotii containing chloramphenicol (25 μg/ml) overnight at 37°C. 18 E. Harvesting and Propagating Infectious Phage Phage particles are recovered from the overnight incubation by standard procedures (Maniatis, et al.) In brief, the media is centrifuged at 12,000 x g for five minutes. Phage particles are precipitated by adding one quarter volume of 2 M NaCl 20% polyethylene glycol, incubating on ice for 15 minutes, and then centrifuging at 12,000 x g for five minutes at 4°C. Only a minute fraction of die phage particles recovered will be infectious, but most of tiiese will contain phagemid DNA encoding enzymes of die desired specificity. These are recovered by coinfection of E. coli strain MV 1184 with CA2000. Sufficient single stranded phagemid DNA can be prepared from individual plaques for further analysis. Exam le 2 Cloning and Specificity Testing of Enzvmes A. Plasmid Cloning from the LAMBDA ZAP II Vectors Enzymes are identified by one of die methods described above in Examples 2 to 6. The corresponding plaques are plaque purified and re-tested as described above. Upon confirmation of a positive result d e catalytic-containing region of die LAMBDA ZAP II clones are excised and expression plasmids generated as previously described (Short, et al.). The plasmids containing genes encoding enzymes are separately transformed into E. coli. The single clones of the plasmid bearing bacteria are inoculated into 5 ml of L-broth (Maniatis, et al.) for overnight cultures. Three mis of the overnight culture are inoculated into 500 ml of L-broth and grown at 37°C for 4 hours (Huse, et al.). Synthesis of the enzyme is induced by die addition of IPTG to a final concentration of 1 mM. The culture is then incubated at 25°C for 10-12 hours. The cultures are harvested and die cells removed by centrifugation. The remaining media, containing the secreted enzyme is concentrated by ultra- filtration using Amicon filters (Amicon, Danvers, MA). The concentrate is then size- fractionated using a TSK-G4000 column. The enzyme containing fractions are identified by screening die fractions by ELISA assays (Ausubel, et al.) using a goat antibody specific against die C„l domains of die heavy chains used to generate d e combinatorial library (Example 1). B. Specificity Testing Human IgE molecules are isolated by standard procedures (Ishizaka, et al.). IgE is added to a final concentration of 10 μg per ml of the Dulbecco's phosphate buffered saline. This solution is tiien divided into 500 μl aliquots. Serial dilutions of die purified enzymes are prepared and added to die IgE-containing aliquots. The reactions are placed at 37°C and 100 μl samples removed at 0, 10, 30, 60, and 120 minutes. The aliquots are then loaded on an SDS-polyacrylamide gel and electrophoretically separated by SDS-PAGE. The proteins are then transferred to nitrocellulose filters (Ausubel, et al.) and probed with a rabbit anti-human- IgE antibody conjugated to alkaline phosphatase. Specific cleavage of die human IgE molecules by an enzyme in target region I will generate three fragments of the IgE molecule under non-reducing conditions, two 50 kilodalton and a 150 kilodalton fragment. Alternatively, specificity can be tested by cleavage of a labelled target peptide itself and analysis of die cleavage products as described above. After a cleavage site is identified a number of variations of the target peptide sequence, witii amino acid substitutions throughout die target region, can be generated by recombinant manipulation of the target peptide sequence. In tins manner die sequence required for cleavage can be more specifically determined. Although the invention has been described with respect to specific methods of making and using enzymes capable of cleaving target polypeptide sequences, it will be apparent that various changes and modifications may be made without departing from die invention.

Claims: IT IS CLAIMED: 1. A method of generating a protease enzyme capable of cleaving a polypeptide having a selected target amino acid sequence, comprising identifying a natural protease enzyme which is capable of cleaving a polypeptide at an amino acid sequence that is related to, but distinct from, the target sequence, introducing into host cells a phage system containing (i) a helper phage capable of expressing mutants of said natural protease, under suitable expression conditions, and (ii) a phage vector bearing a phage gene encoding a gene product necessary for the production of infectious phage, where said gene is modified by introducing the target peptide coding sequence into the gene such that the resulting gene product inhibits production of infectious phage, and where cleavage of said target peptide results in an active gene product that allows production of infectious phage, growing the host cells under conditions in which die protease genes are expressed in die host cells, screening die host cells for production of infectious phage, isolating the protease genes associated with die infectious phage, and using die isolated gene for die production of protease enzyme specific against die target sequence. 2. The method of claim 1, where said screening includes detecting d e presence of infectious phage by plaque formation. 3. The method of claim 1 , where said phage gene encodes a phage coat protein. 4. The method of claim 3, where said host cells are Escherichia coli cells, said phage gene is gene III of bacteriophage M13, and said target sequence is introduced into gene m in such a fashion as to inhibit export of die gene III product to die periplasmic space of die host cells. 5. The method of claim 1, wherein (i) said phage gene encodes a fused protein composed of a phage protein required for plaque formation, under selected growth conditions, a second protein which inactivates said phage protein when linked to one end of die protein, said target links me second protein to the phage protein, and (ii) said screening includes detecting phage capable of producing plaques when grown undo- said selected growth conditions. 6. The method of claim 4, wherein said phage is a lambda phage, said phage protein is the cro protein, and die phage contains a temperature-conditional mutation in its genomic cro gene which is inactive above a selected temperature, and said screening is performed above said selected temperature. 7. The method of claim 5, wherein said phage protein is die lambda cro protein, and said second protein is die Escherichia coli colicin El immunity protein. 8. A method of enhancing die proteolytic activity of a native protease against a known target amino acid sequence, comprising introducing into host cells a phage system containing (i) a helper phage capable of expressing mutants of said protease, under suitable expression conditions, and øi) a phage vector bearing a phage gene encoding a gene product necessary for d e production of infectious phage, where said gene is modified by introducing die target peptide coding sequence into die gene such that die resulting gene product inhibits production of infectious phage, and where cleavage of said target peptide results in an active gene product mat allows production of infectious phage, growing the host cells under conditions in which the protease genes are expressed in d e host cells, screening die host cells for production of infectious phage, at a level which is elevated with respect to production of infectious phage in cells expressing the native protease gene, isolating die protease genes associated witii elevated levels of phage production, and using die isolated genes for the production of proteases having enhanced activity against said target sequence. 9. The method of claim 8, where said screening includes detecting the presence of infectious phage by plaque formation. 10. The method of claim 8, where said phage gene encodes a phage coat protein. 11. The method of claim 10, where said host cells are Escherichia coli cells, said phage gene is gene III of bacteriophage Ml 3, and said target sequence is introduced into gene in such a fashion as to inhibit export of the gene m product to die periplasmic space of the host cells. 12. The method of claim 8, wherein (i) said phage gene encodes a fused protein composed of a phage protein required for plaque formation, under selected growth conditions, a second protein which inactivates said phage protein when linked to one end of the protein, and said target linking the second protein to the phage protein, and (ii) said screening includes detecting phage capable of producing plaques when grown under said selected growth conditions. 13. The method of claim 12, wherein said phage is a lambda phage, said phage protein is the cro protein, and the phage contains a temperature-conditional mutation in its genomic cro gene which is inactive above a selected temperature, and said screening is performed above said selected temperature. 14. The method of claim 12, wherein said phage protein is the lambda cro protein, and said second protein is die Escherichia coli colicin El immunity protein.

Inventors: DAVIS CLAUDE GEOFFREY, GUAY GORDON G, DAVIS, CLAUDE, GEOFFREY, GUAY, GORDON, G.
Applicants: CATALYTIC ANTIBODIES INC, CATALYTIC ANTIBODIES, INC.
Application year: 1995
Main classification: C12N9
IPC codes: C12N9, C12N15, C12Q1, C12R1