WO1994000581A1 - Lactobacillus expression system using surface protein gene sequences - Google Patents

Abstract

Highly efficient expression/secretion systems for the recombinant engineering of Lactobacillus, Lactococcus, and Bacillus are disclosed. These systems utilize Lactobacillus coat protein (SP) expression and secretion regulatory elements for the expression of recombinant genes.

Description

LACTOBACI LLUS EXPRESSION SYSTEM

USING SURFACE PROTEIN GENE SEQUENCES

Field of the Invention

The invention is in the area of molecular biology of the Lactobacilli. Specifically, the invention is directed to Lactobacilli surface coat protein (SP) expression and secretion units and their use for the expression of recombinant genes in Gram-positive bacteria, and especially in Lactobacillus.

Background of the Invention

Lactic acid bacteria including genera Lactobacillus, Pediococcus, Leuconostoc and Lactococcus have a central role in food processing and are of substantial economic importance. Of the above genera, Lactobacillus is the most widely applied. The food products where fermentation by lactic acid bacteria is used include cheese varieties, yogurt, kefir, acidophilus milk, dry sausages, fermented vegetables and soured bread. The main role of lactic acid bacteria in food fermentation is the preservation of the food by the production of lactic acid and other metabolites, and the production of a desired effect on the flavor and texture of the product by these bacteria (Trends in Food Biotechnology: 121-127 (1989), N. Hen and L. Kong, eds.; FEMS Microbiol. Rev. 87:3-14 (1990)).

In addition to their role in common food products listed above, health benefits like increased digestibility, antitumor factor, anticarcinogens and hypocholesteremic effect have been reported for lactic acid bacteria and therefore there is a growing interest in the use of these bacteria and their products to improve the nutritional content and/or medical benefits of foodstuffs, as well as in the general probiotic use of lactic acid bacteria (Annals of Medicine 22:37-41 (1990); J. Appl. Bacteriol 66:365-378 (1989)). Lactobacilli are also used in the animal feed industry for preservation of silage and to increase the nutritional value or digestibility of the silage or feed.

Due to the central role of lactic acid bacteria in industrial processes, a large number of potential applications for these bacteria have been proposed. Use of lactic acid bacteria has been proposed to provide a host with improved resistance to phage, to stabilize the process activity, to enhance flavor production, produce antimicrobials and exocellular polysaccharide, to accelerate cheese ripening and to control bitterness in the final food product. Thus the targets for Lactobacillus engineering are either improvements of existing industrial process characteristics and food products, or applications where Lactobacilli are used as new production hosts for food and feed industry related products, especially for those requiring GRAS (Generally Recognized As Safe)-status host systems. Additional applications will no doubt emerge as soon as the molecular characterization of Lactobacilli advances.

Research on molecular genetics in the genus Lactobacillus is not as advanced as it is for other bacterial species. Uses such as those described above are limited by our understanding of the genetic engineering of the Lactobacilli. Application of many of the potential uses described above requires new knowledge of the structure and function of Lactobacillus expression and secretion units and new tools to modify the existing expression characteristics.

For example, protoplast transformation and electroporation of Lactobacilli have just recently been described (Appl. and Environ. Microbiol. 54:2599-2602 (1988); FEMS Microbiol. Letters 44: 173-177 (1987); Molecular Microbiol. 2:637-646 (1988)) and construction of various plasmid cloning vectors is underway (Appl. Environ. Microbiol. 57: 1822-1828 (1991)). So far, only a very few genes have been cloned and characterized from Lactobacillus.

These include genes of the central metabolism, like sugar utilization (J. Bacteriol. 173: 1951-1957 (1991); Appl. and Environ. Microbiol. 57:2764-2766 (1991)), the metabolism of lactate and carboxylic acids (Appl. and Environ. Microbiol. 57:2413-2417 (1991); Gene 78:47-57 (1989)) and synthesis of amino acids (J. Biochem. 107:248-255 (1990)). In addition, characterization of some 16S RNA, tRNA and 5S RNA genes from Lactobacillus has been described (Nucleic Acid Res. 18:3402 (1990); Nucleic Acid Res. 18:3041 (1990); Nucleic Acid Res. 17:4873 (1989); Nucleic Acid Res. 16: 10938 (1988)).

Low level heterologous expression of α-amylase, β-glucanase and endoglucanase genes (utilizing their endogenous expression units) and TEM-β-lactamase (utilizing a lactococcal promoter), has also been reported in Lactobacillus (Appl. and Environ. Microbiol. 55:2130-2137 (1989); Appl. and Environ. Microbiol. 55:2095-2097 (1989); Appl. Microbiol. Biotech. 35:334-338 (1991); Appl. and Environ. Microbiol. 57:341-348 (1991)) but so far there are no reports of the characterization of Lactobacillus expression units or the use of such expression units in expression vectors for homologous or heterologous expression in Lactobacillus.

Similarly there are no reports of the isolation of genes encoding Lactobacillus secretory or exported proteins, characterization of the export signals or the use of these signals for export or secretion of homologous or heterologous gene products. Therefore, advances in the industrial uses of Lactobacillus, where such uses require the expression and secretion of desired gene products, have been hampered.

In order to modify and improve the process characteristics of Lactobacilli or use them as production hosts for enzymes, peptides, antimicrobial agents or amino acids for food and feed industry, it is essential to have well characterized and highly efficient expression and secretion units available. We have chosen as a target protein for the isolation of expression and secretion signals a surface layer protein (coat protein or surface coat protein or "SP") of Lactobacillus , and specifically L. brevis. Surface layers, consisting of one or a few different proteins, have been described for both Gram-positive and Gram-negative bacteria. (Ann. Rev. Microbiol. 37:311-339 (1983)). Protein surface layers have also been detected by electron microscopy from several genera of Lactobacillaceae, e.g. L. brevis, L. buchneri, L.fermenti, L. helveticus and L. casei (Microbiol. Immunol. 24:299-308 (1980); Microbiol. Immunol. 23:941-953 (1979); J. Cell Sci. 7:755-785 (1970); Microbiol. Immunol. 29:927-939 (1985); Japan J. Microbiol. 18:469-476 (1974); J. Electron Microscopy 15: 115-122 (1981)). Although these coat proteins have not yet been characterized at molecular level, the available data suggests that, in a given species, the Lactobacillus coat layer is formed by a single type of protein. The size of these proteins in different species, as determined by SDS-PAGE, also appears to be very similar, about 45-50 kd. Other than the self assembly process, there is no further characterization that would describe the structure of the protein or its gene.

Brief Description of the Figures

Figure 1(A-D) depicts the cloning strategy and the primers used for PCR-amplification of the L. brevis DSM20556 SP gene. Figure 1A: PCR-Fragments produced from DSM20556 DNA using oligonucleotides shown below in D. Figure IB: The physical map of the S-layer region with relevant restriction enzyme sites. The open arrow heads refer to the end points of the DNA-sequence shown in Figure 2B. Figure 1C: Cloning strategy of the 5'- and 3'- regions of the SP gene. Figure 1D: Oligonucleotides used for synthesis of PCR fragments 1 to 6 [SEQ ID Nos. 1-12].

Figure 2(A-B) is the nucleotide sequence the L. brevis SP gene and predicted amino acid sequence. Figure 2A: Upstream region of the SP gene from nucleotide position -320 to -1 [SEQ ID No. 13]. Figure 2B: DNA sequence from the nucleotide + 1 [SEQ ID No. 14] and predicted amino acid sequence [SEQ ID No. 15]. The predicted -10 and -35 regions of the promoters PI and P2 are underlined and the 5'- ends of the transcripts found by primer extension (see Fig. 3(A-B)) are marked with arrow heads. The cleavage site of the signal peptide and the mature protein is between amino acids 30 and 31 (↑). The N-terminal amino acid sequences of the intact S- layer protein and its tryptic peptides are underlined and numbered from (1) to (5) (see Table 1). The deduced transcription-terminator is shown with arrows. RBS refers to the predicted ribosome binding site.

Figure 3(A-B) is an analysis of SP mRNA. Figure 3A: Northern blot analysis of transcripts. Total L. brevis GRL1 RNA denatured with glyoxal and DMSO was run in a 0.8% agarose gel using 10 mM phosphate buffer, pH 6.5, followed by blotting to ZETAPROBE^® membrane and hybridization. The [α-³²P] dCTP labelled PCR1 fragment was used as a probe. The filter was washed in 0.5xSSC, 0.1 % SDS at 50°C. RNA molecular weight markers (Bethesda Research Inc.) were used to determine transcript size. Figure 3B: Analysis of mRNA-transcripts after the 5 '-end mapping experiment in a 6% sequencing gel. Approximately 5μg of total RNA of L. brevis was hybridized to 200 pmol of a 19-mer primer (5'-CTTAGCCATATGAGCCTTA-3', [SEQ ID No. 16] see Fig. 2, position 507-489). After ethanol precipitation, the primer extension of the hybrids was performed in the presence of [α-³²P] dCTP, nonlabelled nucleotides, AMV reverse transcriptase, actinomycin C1 and RNASIN. For determination of the lengths of the extended products, sequencing reactions of PCR2-fragment (see Fig. 1(A)) were performed with the same primer. Summary of the Invention

Recognizing the need for the development of recombinant expression systems for use in Lactobacilli, and cognizant of the large amounts of Lactobacillus surface coat protein (SP) that are present in Lactobacilli hosts, the inventors studied the molecular basis of Lactobacillus SP expression. These studies have culminated the identification of the regulatory elements that control SP gene expression and secretion of SP protein, and in the development of a highly efficient system for the expression and secretion of heterologous recombinant proteins in Lactobacillus. Detailed Description of the Preferred Embodiments I. Purification of Lactobacillus Coat Protein

The main reason of choosing this particular type of protein as a possible molecular target for expression of recombinant proteins in Lactobacillus was its abundant amount in the host cell and its presence on the outside of the host membrane. At the time these studies were started it was unknown whether this abundance was associated with a high level of transcriptional and translational expression, and with a high level of efficiency of export to the outer cell surface. However, because the protein coat surrounding the cell must be resynthesized in large amounts every time the cell divides (approximately 5 × 10⁵ copies of the SP protein are needed to cover the cell), and because the cell divides very rapidly, it was speculated that the promoter(s) of the gene encoding the coat protein must be one of strongest expression units in the cell and the coat protein the most efficiently exported protein of Lactobacillus . Even in Bacillus, which is a commercial exoenzyme producer, coat protein production (although the coat protein is structurally very dissimilar to that of the Lactobacillus coat) represents probably the most efficient export process of the Bacillus cell (Appl. Microbiol. Biotechnol., 31:338-341 (1988)).

Further, the Lactobacillus coat protein is apparently synthesized under a wide variety of culture conditions that support host cell replication, indicating that the surface coat protein promoter must be essentially constitutive and thus devoid of common control repression systems (e.g. catabolite repression, response to nitrogen- or phosphate-limitation or aeration). Thus, use of this promoter provides an advantage when it is desired to increase the expression of genes or operons of, for example, biosynthetic or degradative pathways which are otherwise tightly regulated.

Although there have been no previous reports of "purified" Lactobacillus coat protein (SP), the present invention provides a method to isolate SP, such method providing SP essentially free of natural contaminants and therefore of the requisite degree of purity needed to sequence and clone SP genetic sequences from Lactobacillus sources. As a direct result of the method of the invention for the isolation of SP, and of the sequence information gathered from the protein sequence data, cloned constructs that provide recombinant Lactobacillus SP genetic sequences and their regulatory elements have been identified.

Lactobacillus SP may be isolated from frozen or fresh Lactobacilli bacteria. As lactobacilli appear to express SP under all growth conditions, any culture method that supports growth or viability may be used. Because the coat protein is so abundant, it may be isolated by electrophoretic methods, utilizing gel electrophoresis as the medium in which to fractionate the proteins.

For example, in a preferred method, Lactobacillus brevis strain DSM 20556 (German Collection of Microorganisms, Braunschweig, FRG) may be used and grown in MRS-medium (Difco) at 37°C without shaking to saturation (generally overnight). The bacteria are then collected by centrifugation at, for example, 10,000 g for 5 min at room temperature, washed once with a neutral, mild buffer such as 50 mM Tris-C1, pH 7.5, and recentrifuged. The cell pellet is suspended in a small volume of the wash buffer and then directly dissolved by addition of SDS polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (62.5 mM Tris-HCl, ph. 6.8, 10% glycerol, 2% SDS, 0.72M β-mercaptoethanol, and 0.00125 % bromophenol blue, in distilled water), followed by boiling for 5 min. The pelleted cells from a 1 ml culture are suspended in 100 μl of 50 mM Tris-HCl, pH 7.5, and 100 μl SDS-PAGE sample buffer is added and the sample boiled and analyzed on a 10% SDS- PAGE gel system, preferably a preparative gel of sufficient length to provide for protein separation based upon molecular weight, giving consideration to the amount of protein being applied to the gel. Such considerations, (amount of sample, length of gel and electrophoresis running time) and the adaptation of gel electrophoresis technology to the same, are well understood in the art. For example, cells from a 1 ml culture may be suspended in 200 μl of Laemmli sample buffer and this amount applied to a preparative SDS-PAGE gel (approximately 1 μl/200 μl gives a clear visible band in Coomassie Blue staining).

Protein staining of the gel will reveal a single major band of about 45-50 kDaltons (kd). Specifically, SP of Lactobacillus brevis reveals a major band at about 46 kd. Coat protein may be visualized on a preparative gel by treating the gel with 1 M KCl rather than Coomassie Blue, so as to allow for subsequent excision of the protein band from the preparative gel for sequence determinations, etc. Protein is eluted from the cut out pieces of such preparative gels by mixing the gel pieces with a buffered strong denaturant in the presence of a chelator. For example a buffer providing 6 M guanidine hydrochloride, 0.5 M Tris-HCl, 2 mM EDTA, pH 7.5 and mixing for 10 hours, in an end-over-end mixture will suffice to elute the protein from the gel pieces.

SP purified by direct excision from the gel in the above manner, or in a manner wherein equivalents of the above sequence of steps are utilized, is purified to an extent capable of being sequenced by techniques known in the art or may be used to raise antibodies. Preferably, prior to sequencing, the protein eluted from the gel is dialyzed against a low salt, mildly basic buffer such as 10 mM Tris-HCl, pH 8.5, and freeze dried to concentrate the protein.

II. Construction and Identification of Antibodies to SP

In the following description, reference will be made to various methodologies well-known to those skilled in the art of immunology. Standard reference works setting forth the general principles of immunology include the work of Catty, D. (Antibodies, A Practical Approach, Vol. 1, IRL Press, Washington, DC (1988)); Klein, J. (Immunology: The Science of Cell-Noncell Discrimination, John Wiley & Sons, New York (1982)); Kennett, R., et al. in Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, New York (1980)); Campbell, A. ("Monoclonal Antibody Technology," in: Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13 (Burdon, R., et al. , eds.), Elsevier, Amsterdam (1984)); and Eisen, H.N., in: Microbiology, 3rd Ed. (Davis, B.D., et al. , Harper & Row, Philadelphia (1980)).

An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of a hapten which can be recognized and bound by an antibody. An antigen may have one or more than one epitope. An "antigen" is capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

The term "antibody" (Ab) or "monoclonal antibody" (Mab) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab')₂ fragments) which are capable of binding an antigen. Fab and F(ab')₂ fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al. , J. Nucl. Med. 24:316-325 (1983)).

The antibodies of the present invention are prepared by any of a variety of methods. Preferably, purified Lactobacillus SP, or an antigenic fragment thereof, is administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding SP.

Cells expressing Lactobacillus SP, or an antigenic fragment thereof, or, a mixture of proteins containing Lactobacillus SP or antigenic fragments thereof, can also be administered to an animal in order to induce the production of sera containing polyclonal antibodies, some of which will be capable of binding SP. If desired, such SP antibody may be purified from the other polyclonal antibodies in the preparation by standard protein purification techniques and especially by affinity chromatography with purified coat or fragments thereof.

SP or a fragment of SP may be chemically synthesized and purified by HPLC to render it substantially free of contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of high specific activity.

Monoclonal antibodies can be prepared using hybridoma technology known in the art (Kohler et al., Nature 256:495 (1975); Kohler et al. , Eur. J. Immunol. 6:511 (1976); Kohler et al. , Eur. J. Immunol. 6:292 (1976); Hammerling et al. , in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y. , pp. 563-681 (1981)). In general, such procedures involve immunizing an animal with SP. The splenocytes of such animals are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP₂O), available from the American Type Culture Collection, Rockville, Maryland. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands, J.R., et al. , Gastro-enterology 80:225-232 (1981). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the SP.

Through application of the above-described methods, additional cell lines capable of producing antibodies which recognize epitopes of the Lactobacillus coat protein can be obtained.

Antibodies against both highly conserved and poorly conserved regions of Lactobacillus SP are useful for the identification of the isolated protein as being the coat protein by means of physical analyses such as immunogold electromicroscopy imaging, and also for the identification of clones expressing such protein and studies on the control of biosynthesis and catabolism of coat protein in the native Lactobacillus host and in heterologous hosts. III. Cloning of Lactobacillus SP Genetic Sequences

The process for genetically engineering Lactobacillus SP genetic sequences is facilitated through the isolation and sequencing of pure Lactobacillus SP and by the cloning of sequences capable of encoding such SP. As used herein, the term "genetic sequence" is intended to refer to a nucleic acid molecule such as DNA or RNA, preferably DNA. Genetic sequences which encode Lactobacillus SP may be derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof. The preferred source is genomic DNA as genomic DNA will provide not only the SP encoding sequences but also the transcriptional and translational regulatory elements thereof as such, for example, the 5' promoter region of the SP gene, the 3' transcriptional termination region, the genetic sequences which provide the 5' non-translated region of the SP mRNA (if any) and/or the genetic sequences which provide the 3' non-translated region (if any).

To the extent that a host cell can recognize the transcriptional and/or translational regulatory signals associated with the expression of the mRNA and protein, then such regulatory signals may be retained and employed for transcriptional and translational regulation in a host even if such host is not a member of the Lactobacilli.

Lactobacillus SP genomic DNA can be extracted and purified from any Lactobacillus cell which naturally expresses SP by means well known in the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger et al , eds., Academic Press (1987). Some Lactobacillus species have cell walls that are highly resistant to enzymatic lysis. For example, L. brevis strain DSM 20556 is highly resistant to enzymatic lysis. Thus, for isolation of chromosomal DNA from resistant cells such as L. brevis, a novel method has been developed to facilitate membrane lysis. As described below, this method involves the steps of (1) . growth of the cells to midlogarithmic growth phase (Klett₆₆ 80), (2) collection of the cells by centrifugation, (3) suspension in 3M guanidine hydrochloride and (4) incubation for 20 min at room temperature, (5) collection of the incubated cells by centrifugation, (6) a wash with 0.15 M NaCl-0.1 M EDTA and resuspension in same, (7) the addition of (final concentration) 2.85 mg/ml lysozyme, 430 U/ml mutanolysin and 0.43 mM CaCl₂, and finally, (8) cell lysis by addition of 8.75 ml of 20% SDS and incubation at 65°C for 10 min. Na-perchlorate (2.2 ml of 5M Na-perchlorate per 15.75 ml cell volume after the SDS addition) is then added and the cell lysate extracted with chloroform-isoamylalcohol (24: 1). The water phase is removed after centrifugation and nucleic acids are precipitated by bringing the sample to 66% ethanol. Chromosomal DNA is collected around a glass rod and dissolved in an appropriate amount of 10 mM Tris-HCl - 150 mM NaCl, pH 7.5. At this stage, the chromosomal DNA may be cleaved with restriction enzymes such as HaeIll and fragments of the desired size separated by electrophoresis, such as by agarose gel electrophoresis, using methods known in the art. The restriction fragments may be cloned into various commercial vectors, such as, for example, lambda phage vectors, including lambda gt10, according to the manufacturer's instructions, for form a recombinant gene library.

If mRNA is used as the source of the coat protein encoding sequences, such mRNA is preferably enriched in mRNA coding for SP, either naturally, by isolation from cells which are producing large amounts of SP, or in vitro, by techniques commonly used to enrich mRNA preparations for specific sequences, such as sucrose gradient centrifugation, or both. For cloning into a vector, cDNA is prepared from the mRNA, and such cDNA may be enzymatically cleaved and ligated into appropriate vectors to form a recombinant cDNA library.

A DNA sequence encoding SP, or its gene, or regulatory elements thereof, may be inserted into a DNA vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, T., (Maniatis, T. et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, second edition, 1988) and are well known in the art.

Libraries containing sequences coding for SP be screened and a sequence coding for coat protein identified by any means which specifically selects for a sequence coding for coat protein such as, for example, a) by hybridization with an appropriate nucleic acid probe(s) containing a sequence specific for the DNA of SP, or b) by hybridization-selected translational analysis in which native mRNA which hybridizes to the clone in question is translated in vitro and the translation products are further characterized, or, c) if the cloned genetic sequences are themselves capable of expressing mRNA, by immunoprecipitation of a translated SP product produced by the host containing the clone.

Oligonucleotide probes specific for SP which can be used to identify clones to this protein can be designed from knowledge of the amino acid sequence of SP. As used herein, and by convention, when an amino acid sequence is listed horizontally, unless otherwise stated, the amino terminus is intended to be on the left end and the carboxy terminus is intended to be at the right end.

Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D., In: Molecular Biology of the Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), pp. 356- 357). The peptide fragments are analyzed to identify sequences of amino acids which may be encoded by oligonucleotides having the lowest degree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon.

Although occasionally an amino acid sequence may be encoded by only a single oligonucleotide sequence, frequently the amino acid sequence may be encoded by any of a set of similar oligonucleotides. Importantly, whereas all of the members of this set contain oligonucleotide sequences which are capable of encoding the same peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains the nucleotide sequence that is identical to the exon coding sequence of the gene. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.

Using the genetic code, one or more different oligonucleotides can be identified from the amino acid sequence, each of which would be capable of encoding SP. The probability that a particular oligonucleotide will, in fact, constitute an actual SP encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells. Such "codon usage rules" are disclosed by Lathe, R., et al. , J. Molec. Biol. 183: 1-12 (1985). Using the "codon usage rules" of Lathe, a single oligonucleotide sequence, or a set of oligonucleotide sequences, that contain a theoretical "most probable" nucleotide sequence capable of encoding the SP amino acid sequence (or a fragment thereof) is identified.

The suitable oligonucleotide, or set of oligonucleotides, which are capable of encoding at least a fragment of the SP gene (or sequences that are complementary to such coding sequences) may be synthesized by means well known in the art (see, for example, Synthesis and Application of DNA and RNA, S.A. Narang, ed., 1987, Academic Press, San Diego, CA) and employed as a probe to identify and isolate a cloned SP gene by techniques known in the art. Techniques of nucleic acid hybridization and clone identification are disclosed by Maniatis, T., et al , in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)), and by Hames, B.D. , et al , in: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985)), which references are herein incorporated by reference. Those members of the above-described gene library which are found to be capable of such hybridization are then analyzed to determine the extent and nature of coat protein encoding sequences which they contain.

To facilitate the detection of a desired SP DNA encoding sequence, the above-described DNA probe is labeled with a detectable group. Such detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of nucleic acid hybridization and in general most any label useful in such methods can be applied to the present invention. Particularly useful are radioactive labels, such as ³²P, ³H, ¹⁴C, ³⁵S, ¹²⁵I, or the like. Any radioactive label may be employed which provides for an adequate signal and has a sufficient half-life. If single stranded, the oligonucleotide may be radioactively labelled using kinase reactions. Alternatively, polynucleotides are also useful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, an enzyme or a fluorescent group.

Thus, in summary, the elucidation of the SP amino acid sequence permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide. By constructing an oligonucleotide complementary to this theoretical sequence (or by constructing a set of oligonucleotides complementary to the set of "most probable" oligonucleotides), one obtains a DNA molecule (or set of DNA molecules), capable of functioning as a probe(s) for the identification and isolation of clones containing the SP gene.

In a first attempt to generate a DNA probe for the cloning of the SP gene, oligonucleotides were synthesized according to the N-terminal amino acid sequences as shown in Table 1. Table 1: Peptide-sequences of the L. brevis coat protein

Number of Sequence SEQ the ID peptide No.

(1 ) ala-tyr-his-tyr-thr-tyr-thr-tyr-asn-lys 17

(2 ) ser- ala-asp-tyr-phe-arg-ala-tyr-gly-val-lys 18

(3 ) tyr-arg-gly-tyr-val-tyr-gly-gly-lys 19

(4) thr-thr-asn-arg-gly-ser-val-tyr-tyr-arg-val-val-thr-met 20

(5) ser-tyr-ala-thr-ala-gly-ala-tyr-ser-thr-leu-lys 21

N-terminus of the

intact protein NH-₂-lys-ser-tyr-ala-thr-ala-gly-ala-tyr-ser- 22

Deoxyinosine was applied in unknown positions. The oligos were synthesized in such a way that the oligos corresponding to the N-termini of the isolated peptides were in opposite orientation to the oligo corresponding to the N-terminus of the mature SP. These oligos were then used in PCR to generate fragments of the SP gene. The largest of these fragments (1.2 kb) was colinear with the chromosomal coat gene, as shown by Southern blotting, and was used as a probe to screen the gene libraries. The oligos used to generate the 1.2 kb fragment are shown in Figure ID.

Screening of a Lactobacillus brevis HaeIII-lambda gt10 library with the

1.2 kb radioactive probe fragment initially yielded hybridization positive plaques, but these were all lost during subsequent purification steps, suggesting that the coat gene was highly unstable in E. coli. Similarly, when a plasmid gene bank was generated in Bacillus subtillis using a low copy vector pΗP13 (Mol. Gen. Genet. 209:335-342 (1987)) and isolated restriction fragments, known to contain the intact SP gene, no hybridization positive clones were obtained.

Therefore, the above attempts failed to yield the intact SP gene and a new strategy based on reversed PCR was developed. The cloning strategy and the primers that were used are depicted in Figure 1. The 1.2 kb . PCR fragment described above covers about 80% of the DNA that encodes the mature part of the coat region but it lacks both the 5' and 3' regions of the intact gene. To obtain these unknown fragments, restriction fragments of chromosomal DNA, identified by Southern blotting to contain both the missing 5' and 3' fragment, were ligated to linearized pBR322 vector. SP gene and pBR322 specific primers of opposite orientation were then used as primers to generate the missing upstream and downstream regions by PCR from the ligation mixtures. This approach resulted in overlapping PCR-fragments covering about 2.4 kb from the L. brevis genome and includes the complete SP gene. The PCR fragments were sequenced by the dideoxy-chain termination method (Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)) using the sequenase kit with [α³⁵S]-dATP (Amersham) according to manufacturer's instructions.

By sequence analysis of the complete SP gene (Figure 2 and SEQ ID Nos. 14 and 15); the SP gene is 1395 bp long and encodes a protein of 49159 daltons. Removal of the deduced signal peptide (30 amino acids) results in a polypeptide of 435 amino acid residues with calculated molecular mass of 45 kDa. This is in excellent agreement with the apparent molecular mass of 46 kDa obtained in SDS-PAGE analysis of SP that is released from the intact cells of Lactobacillus brevis.

The full DNA sequence of the SP gene enabled the design of new oligos that result in the synthesis of the complete gene in a single PCR fragment from the Lactobacillus chromosome. The DNA and the deduced protein sequences may be analyzed with standard programs, such as the PC/GENE (Genofit) program.

The above discussed methods are, therefore, capable of identifying genetic sequences which are capable of encoding Lactobacillus SP or fragments of this protein. In order to further characterize such genetic sequences, it is desirable to express SP. Such expression identifies those clones which express proteins possessing characteristics of SP. Such characteristics may include the ability to specifically bind SP antibody, the ability to elicit the production of antibody which are capable of binding to SP, and the ability to provide a biological activity that is possessed by SP to a cell, among others. L. brevis was particularly chosen to exemplify herein because of its

GRAS-status. However, any other Lactobacillus species possessing a coat protein, as revealed by electron microscopy or a simple SDS-PAGE screening system, could have been used as a source of the SP gene, using the methods as described herein.

For example, L. buchneri has a SP very similar in size as the L. brevis protein and the L. brevis coat gene hybridizes with the L. buchneri genome. After characterization of the desired SP, the SP gene may be isolated by combined PCR and reverse PCR as described above. PCR is a preferred method because of the difficulties encountered with the cloning of L. brevis SP using the standard E. coli cloning techniques. The regulatory regions may be characterized by DNA sequence analysis and by determination of the 5' end(s) of the SP gene specific mRNA as exemplified for L. brevis.

IV. Expression of Recombinant Protein under the Control of Coat

Protein Sequences

Regulatory elements of the SP gene have been identified from the genetic sequences cloned above and used for the design of expression and secretion units. As described herein, such SP transcriptional and translation or secretory regulatory sequences may be used for the expression of heterologous or homologous recombinant proteins in Lactobacillus. The SP of the invention contains a signal sequence. This is the first instance wherein a sequence of an exported or secreted protein of genus Lactobacillus has been revealed.

According to the invention, SP expression and secretion units can be used for expression or secretion of a heterologous protein in members of the genera Lactobacillus. Furthermore, according to the invention, the same expression/secretion units can be applied to drive secretion of a heterologous protein also in members of the genera Lactococci and the genera Bacillus. The secretion unit can consist either of a full SP signal sequence (MQSSLKKSLYLGLAALSFAGVAAVSTTASA, SEQ ID No. 23, bases 1-30 of SEQ ID No. 15), which is then followed by the coding sequence of the required gene or its relevant part, or a hybrid signal sequence can be constructed in such a way that the SP signal sequence provides the 5' end of the construct, and it is fused to the 3' end of the signal sequence of the target gene so as to create a hybrid secretion sequence. For example, it is known that a signal peptide consists of three domains: a positively charged N-terminal region (N-region), followed by a hydrophobic region (h-region) and a C-terminal region (C-region) which contains the recognition site for the signal peptidase. Typically, there is a helix breaking residue, Pro or Gly at the end of the h-region (for a review see Gierasch, L.M., "Signal Sequences, " Biochemistry 28:923-930 (1989)).

Accordingly, hybrid secretion signals may be designed that possess one or more domains of the SP signal sequence and one or more domains of the signal sequence of a different protein. For example, a hybrid signal sequence may contain only the N-region of the SP signal sequence, or only the h-region, or only the C-region; alternatively, a hybrid signal sequence may contain the SP signal sequence's N-region and h-region (the C-region being provided by a different signal sequence), or, the SP signal sequence's N-region and C-region (the h-region being provided by a different signal sequence), or, the h-region and C-region of the SP signal sequence (the N-region being provided by that of a different signal sequence).

Each of the hybrid secretory signals of the invention is constructed by recombinant techniques so that the amino acid sequences of the three secretory signal domains, the N-region, the h-region and the C-region, are operably linked to each other, in the order "N-h-C," (the amino terminal end being on the left). The operable linkage is such that the hybrid construct will provide for the secretion of a protein that is operably linked to the carboxy terminus of the C-region domain. Preferably, such construct provides for both the secretion and cleavage of the hybrid secretion signal, such that the mature protein of interest is present in the culture medium. If desired, each of the hybrid secretory sequence's three domains may be heterologous to the other (that is, originate from three different secretory signal sequences), or two of the domains may be derived from the same signal sequence.

In theory, any combination of the secretion signal domains may be created. However, it is advisable to construct the hybrid sequence so that the joint of the two heterologous sequences is at the beginning or at the end of the h-region or within the h-region. Since the N-region affects the conformation of the 5' end of the mRNA and thus the frequency of translation initiation, joints made at the initiation methionine (between the 5' non-translated region of the mRNA and the start of translation) often dramatically lower the yield. Therefore, the N-region and any preceding mRNA (5' non-translating sequence) should be derived from the same gene origin, and preferably an SP gene.

A DNA sequence, and especially a DNA sequence encoding the hybrid secretory signal elements of the invention, such as those exemplified below from Bacillus signal sequences, and a Lactobacillus SP signal sequence, or the desired domains thereof, or a specifically desired combination of sequences from each of these signal sequences, may be chemically synthesized using techniques known in the art (for example see Oligonucleotides and Analogues .

R. Eckstein, ed., IRL Press, 1992), or may be prepared from cloned sources.

Many different secretion signals of Bacillus are known which may be used as a source of domains for a hybrid Lactobacillus secretion signal of the invention. For example, the sequence of a domain of any of the Bacillus secretion signals listed in Table 2 is useful to provide the function of such domains to the hybrid secretion signals of the invention, especially for the h-domain and/or the C-domain. The plus and minus sign over the amino acid in Table 2 indicates whether the amino acid is a positively or a negatively charged amino acid.

According to the invention, the promoter and a portion of the signal sequence of a Lactbacillus SP signal sequence is used as the source of the Ndomain and/or h-domain, the promoter without further modification and the signal sequence being altered as described herein as desired to construct the hybrid signal sequence. However, any promoter capable of functioning in the host cell may be used, and the desired domain sequence of any secretion signal may be used, as long as such sequence provides for the desired secretion function in the host cell.

For example, if the α-amylase secretion signal is used as the source of a domain, the different junctions between the α-amylase signal sequence and that of the Lactobacillus signal sequence that may be constructed may be determined in the following manner. As with all other secretion signals, the

B . amy lo li q u efa ci ens α - a m y l a s e s i g n a l s e q u e n c e

(MIQKRKRTVSFRLVLMCTLLFVSLPITKTSA [SEQ ID. No. :28:] is divided into three domains: the N-region (MIQKRKRTVSFR) [SEQ ID

No.:70:], the h-region (LVLMCTLLFVSL) [SEQ ID No. :71:] and the C-region (PITKTSA) [SEQ ID No. :72:]. In a similar manner, the SP secretion signal of SEQ ID No. 23 may be broken into an N-region, h-region and C-region, such the the N-region is positively charged, the h-region is hydrophobic and the C-region provides for the recognition site of the signal peptidase.

Examples of hybrid secretion signal that may be constructed from these domains include sequences containing:

- the h-region of the α-amylase secretion signal and the N-region and C- region of the Lactobacillus SP signal sequence;

- the C-region of the α-amylase secretion signal and the N-region and h- region of the Lactobacillus SP signal sequence; and

- the h-region and C-region of the α-amylase secretion signal and the N- region of the Lactobacillus SP signal sequence.

The location of the junction may affect the protein yield. It is not necessary that the exact native amino acid sequence of a desired secretion element (N-region, h-region, or C-region) be maintained in the hybrid secretion sequence. Changes in the native amino acid sequence of a domain, especially at a junction, such as may be introduced during cloning with restriction enzymes and ligation techniques will not necessarily destroy, or be detrimental to, the functioning of the hybrid secretion sequence.

In a preferred embodiment, the C-region domain or the helix breaker joint provide a signal peptidase recognition site that is homologous to one expressed in the Lactobacillus host cell. However, this is not necessary if the host signal peptidase will recognize the C-region domain on the hybrid secretion sequence. In a highly preferred embodiment, the helix breaker joint (or C-region) provides a signal peptide recognition site that is homologous to the recombinant protein that is operably linked to the C-region of the hybrid secretory signal sequence, and is recognized by the signal peptidase enzyme in the Lactobacillus host cell (even if heterologous to such host cell).

Concerning the N-region, it is preferred that sequence encoding the ribosome-binding site-start-codon- provided by such region originate from an SP gene with a high capacity for efficient translational expression in the desired host cell.

Secretion vectors based either on the intact or hybrid signal sequences can be used for production of food or feed industry-related extracellular enzymes, peptides and peptide antimicrobial agents.

In addition to the signal sequence, secretion vector constructs may be designed that retain part of the SP gene encoding the mature SP protein. This would result in a mature protein that is a fusion protein that is secreted. The desired protein could be later released from the carrier SP protein part either by enzymatic or chemical cleavage. As is known in the art, with simple deletions of the SP gene encoding the mature SP it is straightforward to estimate an appropriate number of amino acids to use for the "carrier" part to ensure efficient secretion and/or protection of the required gene product. A third possibility is to create a fusion protein of the complete SP protein joined to the amino acid sequence of a desired protein of interest, so as to produce the desired protein of interest on the cell surface as a part of the surface layer. This product could be, for example, an antigenic epitope and Lactobacillus cells coated with this epitope could then be used as inexpensive animal vaccines or diagnostic tools (e.g., in screening of autoimmune diseases). The product of the cell surface could also be chosen to improve the probiotic value of a Lactobacillus host. The correct insertion position, e.g., for the epitope coding region within the coat gene can be found for example i) by analyzing the coat sequence with an antigenic determinant screening program (e.g. prediction of antigenic determinants according to Hopp and Woods in PC/Gene (Genofit)); ii) inserting the required DNA fragment in correct reading frame at the most favorable positions; iii) expressing the hybrid coat gene in a chosen Lactobacillus host; and iv) screening intact bacterial clones with antiserum specific with the expressed epitope. Although epitopes have been inserted earlier in some bacterial or phage proteins (Embo J. 5:3029-3037 (1986)), this is the first time this approach has been possible with bacteria that are fully coated with the target protein.

The native SP signal sequence may be used even without a specific proteolytic site within the signal sequence (to allow removal of the signal sequence) if the biological function of the desired protein or fusion protein is not prevented by the presence of the signal sequence.

To express a desired coding sequence, transcriptional and translational signals recognizable by the host are necessary. The cloned SP coding sequences, and specifically, the transcriptional and secretory regulatory elements thereof, obtained through the methods described above, and preferably in a double-stranded form, may be operably linked to a desired gene coding sequence in an expression vector, and introduced into a Lactobacillus, Lactococcus, or Bacillus host cell to produce recombinant protein under the control of such sequences.

Preferably, the present invention encompasses the expression of a desired protein in Lactobacillus cells, and especially L. brevis. A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains expression control sequences which contain transcriptional regulatory information and such sequences are "operably linked" to the nucleotide sequence which encodes the desired polypeptide.

An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (or sequences) in such a way as to place expression of the sequence under the influence or control of the regulatory sequence. For example a sequence encoding a desired protein and a coat protein promoter region sequence joined to the 5' end of the sequence encoding the desired protein, are said to be operably linked if induction of promoter function results in the transcription of mRNA from the sequences encoding the desired protein and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the coat protein expression and secretion regulatory sequences to direct the expression and secretion of the desired protein, or (3) interfere with the ability of the template encoding the desired protein to be transcribed by the promoter region sequence. Thus, a coat protein promoter region would be operably linked to a DNA sequence encoding a desired protein (or antisense RNA) if the coat protein promoter were capable of effecting transcription of that DNA sequence.

The precise nature of the regulatory regions needed for gene expression may vary between species or cell typps, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating (non-coding) sequences involved with initiation of transcription and translation respectively, such as the -35 and -10 regions known to be necessary for prokaryotic promoters. Especially, such 5' non-transcribing control sequences will include a region which contains a promoter for transcriptional expression of the operably linked gene. Such transcriptional control sequences may also include additional activator or repressor sequences as desired. For example, as shown herein, the SP gene has two separate promotes with roughly equal strength under the test conditions. Thus it is possible to either use both promoters in applications requiring maximum strength or only one of them if the total expression capability has to be lowered, or downgraded. The promoter unit(s) can be used for increasing the efficiency or altering the regulation of metabolic pathways or for intracellular production of enzymes (e.g. , peptidases) or peptides (e.g. milk derived opoidic or taste affecting peptides). The promoter unit can be used either alone, devoid of the coat gene derived ribosome binding site by using fusions made e.g., at the nucleotide, encoding 5' end of the mRNA, or as a transcription/ translation initiation unit where joints are made directly after the initiation methionine or further down the structural gene yielding fusion proteins.

Where the coat protein expression and secretion control sequences signals do not function satisfactorily in a host cell, then sequences functional in the host cell may be substituted as necessary.

The vectors of the invention may further comprise other operably linked regulatory elements such as DNA elements which confer antibiotic resistance on a host cell, and which provide for an origin of replication, or for insertion of a desired sequence into the chromosome of a host cell.

To transform a host cell with the DNA constructs of the invention many vector systems are available depending upon whether it is desired to insert the desired protein's DNA construct into the host cell chromosomal DNA, or to allow it to exist in an extrachromosomal form.

If the sequence encoding the desired protein and an operably linked SP promoter and/or secretory signal are introduced into a recipient cell as a non- replicating DNA (or RNA) molecule, the expression of the desired protein may occur through the transient expression of the introduced sequence. Such a non-replicating DNA (or RNA) molecule may be a linear molecule or, more preferably, a closed covalent circular molecule which is incapable of autonomous replication.

Alternatively, genetically stable transformants may be constructed with vector systems, or transformation systems, whereby DNA encoding the desired protein and the expression and secretion regulatory elements thereof are integrated into the host chromosome. Such integration may occur de novo within the cell or, in a most preferred embodiment be assisted by transformation with a vector which functionally inserts itself into the host chromosome, for example, with transposons or homologous DNA integration which promote integration of DNA sequences in chromosomes.

Cells which have stably integrated the introduced DNA into their chromosomes are selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector in the chromosome, for example the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene can either be directly provided on the same vector as that providing the desired DNA gene sequences to be expressed, or such markers may be introduced into the same cell by co-transfection.

Factors of importance in selecting a particular plasmid or phage vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species, such as E. coli and Lactobacillus.

After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the desired protein, or in the production of a fragment of this protein.

When using the SP transcriptional regulatory elements of the invention, this expression preferably takes place in a continuous manner in the transformed cells.

The expressed protein of interest, or fusion protein, is isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like, according to the biochemical characteristics of the desired protein, for example, by affinity purification with antibodies to a desired protein of interest. Such techniques are well known in the art. Therefore, the Lactobacillus SP encoding sequences, obtained through the methods above, will provide sequences which, by definition, encode the Lactobacillus SP transcriptional, translational, and secretory regulatory elements, and which may then be used to obtain direct the synthesis and secretion of a desired protein in the host cell.

Additionally, the Lactobacillus SP encoding sequences, obtained through the methods above, will provide sequences which, by definition, encode the Lactobacillus SP transcriptional, translational, and secretory regulatory elements, and which may then be used to obtain direct the synthesis of an antisense RNA (an RNA complementary to protein's mRNA) in the host cell. An antisense RNA sequence will be that sequence found on the opposite, complementary strand of the strand transcribing the protein's mRNA. An expression vector may be constructed which contains a DNA sequence operably linked to a promoter wherein such DNA sequence expresses the SP antisense RNA sequence. Transformation with this vector results in a host capable of expression of a antisense RNA against a specific protein, under the control of the coat protein transcriptional expression signals. When expressed, antisense RNA interacts with an endogenous DNA or RNA in a manner which inhibits or represses transcription and/or translation of the endogenous protein gene and/or mRNA in a highly specific manner. For example, if SP antisense RNA is expressed, translation and expression of native SP will be reduced or eliminated. This may be desired when it is desired to express a SP fusion protein or other construct on the host surface in place of the native SP protein, or when it is desired to express a coat protein (or other surface protein) that is heterologous to the host.

The examples below are for illustrative purposes only and are not deemed to limit the scope of the invention. Examples

Example 1

Isolation of the Lactobacillus coat (surface) protein

Lactobacillus brevis strain DSM 20556 was used as a source for the isolation of the coat protein. The strain was obtained from the German Collection of Microorganisms (Braunschweig, FRG). To isolate the coat protein, the L. brevis strain was grown in MRS-medium (Difco) overnight (o/n) without shaking at 37°C. The cells were collected by centrifugation at 10,000 g for 5 minutes at room temperature (RT), and washed once with 50 mM Tris-HCl, pH 7.5 buffer. The cell pellet was dissolved in Laemmli sample buffer (Cells from 1 ml culture were suspended in 100 μl of 50 mM Tris-HCl, pH 7.5 followed by addition of 100 μl of Laemmli sample buffer), boiled for 5 minutes and analyzed in SDS-PAGE (10%). This resulted in a single major band of M_r 46 kd after Coomassie Blue staining. To isolate the protein the same cell sample was applied onto a preparative polyacrylamide gel. After electrophoresis the gel was treated with 1 M KCl on ice to visualize the protein band. The band corresponding to the 46 kd protein was excised from the gel and cut into small pieces. The proteins were eluted from the gel pieces with 6 M guanidine hydrochloride, 0.5 M Tris-HCl, 2 mM EDTA, pH 7.5 by incubating in an end-over mixer at RT for 10 hours. The elute was dialyzed against 10 mM Tris-HCl, pH 8.5 o/n and either freeze dried for protein sequencing or used as such for immunizing rabbits. The resulting antiserum was designated KH1225.

Example 2

Characterization of the coat protein by immunogold EM

To confirm that the isolated 46 kd protein corresponds to the coat protein of L. brevis, the intact cells were analyzed by immunogold electromicroscopy using the antiserum (KH1225) raised against the isolated protein band as follows: the bacterial cells were collected with glass rod from MRS-plates and fixed either with 2.5% glutaraldehyde (Electron Microscopy Sciences, Washington, PA 19034) in 0.1 M phosphate buffer (pH 7.5) or with 3.5 % paraformaldehyde and 0.5 % glutaraldehyde dissolved in 0.1 M phosphate buffer. The fixations were carried out at room temperature for 1 hour. Cells were washed three times with the same buffer. The cells were then dehydrated in a series of ethanol (50, 60, 70 and 90%) washes and treated with 90% ethanol and LR-WHITE (2: 1) for one hour as well as (1: 1) for one hour before the final embedding in LR-WHITE (a hydrophilic acrylic resin of low viscosity; London Resin). The resin was polymerized for 24 hours at 60°C. Thin sections were mounted on nickel grids which were placed on droplets of the following solutions: blocking solution (Tween 20 + 1 % BSA = TBST-B) for 10 minutes, antibody solution (1:200) for two hours, blocking solution 10 minutes, solution of protein A-gold (10 nM, Zymed) diluted 1:200 in TBST-B for 1.5 hours and washed twice in Tween 20 for 10 minutes. Grids were extensively washed with distilled water on a shaker. After drying the sections were stained with uranyl acetate and lead citrate (LKB, Calsberg's system). The 0-serum (zero time, that is, serum before immunization) of the same rabbit was used as a control. All micrographs were taken with a JEOL-1200 EX at 60kV. In the cells treated with KH1225 antiserum all the gold particles can be seen to locate mostly in the most outermost part of the cell wall and only some particles are seen in the cell wall and/or in the cytoplasm. In the control cells, no gold particles are seen either in the cell wall or in the cytoplasm. The post embedding immunoelectron microscopy clearly shows that the isolated 46 kd protein locates on the outermost part of the cell wall of L. brevis DSM 20556 cells. Example 3

Protein sequencing of the coat protein

For N-terminal sequence analysis, the coat protein was rerun in an inversed gradient SDS-PAGE (Proc. Int. Meeting on Electrophoresis, Ed. Radola, B.J., pp. 293-397 (1989)) with 12% separation gel and a 5 % running gel. From the gel the proteins were transferred electrophoretically onto a polyvinylidene diflouride (PVDF) membrane (In Current Res. in Prot. Chem. Ed., Villafranca, J. Academic Press, 1990)) and degraded in a gas/pulsed liquid sequencer (J. Prot. Chem. 7:242-243 (1988)). The N-terminal sequence of the coat protein is shown in Table 1. In addition to the N-terminal sequence, also some of the peptides derived from the coat protein were sequenced. To obtain the peptides, the freeze dried protein from part 1 was suspended in 300 μl distilled water and 500 ng of lysylendopeptidase (WAKO, Dallas, TX, USA) was added. The mixture was incubated at 35 °C for 6 hours and the resulting peptides were separated by reverse phase chromatography on a Vydak 218 TPB5 (0.46 × 15 cm) column connected to a Varian 5000 liquid chromatograph. The peptides were eluted using a linear gradient of acetonitrile (0-60% in 90 min) in 0.15 M trifluoroacetic acid. Five lysylendopeptidase digested peptides were sequenced as described above after application on polybrene (2 mg) pretreated glass filters. The N-terminal sequences of the isolated peptides are shown in Table 1. The peptide 5 was shown to be identical to the N-terminal peptide of the protein without the N-terminal lysine which was most probably removed by the lysylendopeptidase digestion. Example 4

Cloning of the gene encoding the 46 kd coat protein

Since the cells of L. brevis strain DSM 20556 were highly resistant to enzymatic lysis, the following method was developed to isolate the chromosomal DNA. The cells were grown in MRS-medium (200 ml) at 37 °C to midlogarithmic growth phase (Klett₆₆ 80), collected by centrifugation at 8000 g for 5 minutes at RT and suspended in 3 ml of 3 M guanidine hydrochloride solution. After incubation for 20 minutes at RT, the cells were collected by centrifugation, washed once with 0.15 M NaCl-0.1 M EDTA solution and suspended in 7 ml of the same solution. 20 mg of lysozyme, 200 μl of mutanolysin (15,000 U/ml, Sigma) and 3 μl of 1 M CaCl₂ were added and the cells were incubated at 55 °C for 2 hours. After addition of 8.75 ml of 20% SDS, followed by incubation of 10 min at 65 °C, 2.2 ml of 5 M Na-perchlorate was added and the cell lysate was extracted with chloroform-isoamylalcohol (24: 1). The water phase was removed after centrifugation and nucleic acid precipitated by bringing the solution to 66% ethanol. The chromosomal DNA was collected around a glass rod. DNA was dissolved in 10 mM Tris-HCl - 150 mM NaCl, pH 7.5. The chromosomal DNA was partially cleaved with HaeIII and fragments of 4-7 kb size were isolated after agarose gel electrophoresis. These HaeIII-fragments were used to generate a lambda gt10 gene bank using a commercially supplied lambda gt10 system (Promega) according to the manufacturer's instructions.

To generate a DNA probe for screening of the coat gene, oligonucleotides were synthesized according to the N-terminal amino acid sequences. Deoxyinosine was applied in unknown positions. The oligos were synthesized in such a way that the oligos corresponding to the N-termini of the isolated peptides were in opposite orientation to the oligo corresponding to the N-terminus of the mature coat protein. These oligos were then used in PCR to generate fragments of the coat gene. The largest of these fragments (1.2 kb) was shown to be colinear with the chromosomal coat gene by Southern blotting and was used as a probe to screen the gene libraries. The oligos used to generate the 1.2 kb fragment are shown in Figure ID.

Screening of the lambda gt10 library with the 1.2 kb radioactive probe fragment yielded initially hybridization positive plaques, but these were all lost during subsequent purification steps, suggesting that the coat gene was highly unstable in E. coli. Similarly, when a plasmid gene bank was generated in Bacillus subtillis using a low copy vector pHP13 (Mol. Gen. Genet. 209:335-342 (1987)) and isolated restriction fragments, known to contain the intact coat gene, no hybridization positive clones were obtained.

As these standard cloning approaches failed to yield the intact coat gene, a new strategy based on reversed PCR was developed. The 1.2 kb PCR fragment covers about 80% of the DNA that encodes the mature part of the coat region but it lacks both the 5' and 3' regions of the intact gene. To obtain three unknown fragments, restriction fragments of chromosomal DNA, identified by Southern blotting to contain both the missing 5' and 3' fragment, were ligated to linearized pBR322 vectors. Coat gene and pBR322 specific primers of opposite orientation were then used as primers to generate the missing upstream and downstream regions by PCR from the ligation mixtures. The cloning strategy and the primers that were used are depicted in Figure 1. This approach resulted in overlapping PCR-fragments covering about 2.4 kb from the L. brevis genome and includes the complete coat gene. The PCR fragments were sequenced by the dideoxy-chain termination method (Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)) using the SEQUENASE kit (using T7 DNA polymerase) with [α³⁵S]-dATP (Amersham) according to manufacturer's instructions. According to the sequence analysis the SP gene is 1395 bp long and encodes a protein of 48159 daltons (Figure 2). Removal of the deduced signal peptide (30aa) results in a polypeptide of 435 amino acid residues with calculated molecular mass of 45 kDa. This is in excellent agreement with the apparent molecular mass of 46 kDa obtained in SDS- PAGE analysis of the coat protein released from the intact cells of Lactobacillus brevis. The available full DNA sequence of the coat gene enabled us to design new oligos that resulted in the synthesis of the complete gene in a single PCR fragment from the L. brevis chromosome (Figure 1). The DNA and the deduced protein sequences were analyzed with PC/GENE (Genofit) program. When the SWISSPROT, NBRF and GenBank data bases were searched for homologous amino acid and DNA sequences with the FASTA program, no significant protein or DNA sequence homology was found with any of the previously known proteins or genes.

Analysis of the mRNA of the coat gene

Total RNA was isolated from L. brevis essentially as described by Palva et al. DNA 7:135-142 (1988) except that mutanolysin and lysozyme were used in concentrations of 900 U/μl and 20 μg/μl, respectively. RNA gel electrophoresis and Northern blot were as described (DNA 7: 135-142 (1988)) using the 1.2 kb coat gene fragment as a probe. The Northern blot analysis revealed a 1.5 kb mRNA which is in good agreement with the DNA data and indicated that the coat gene is monocistronic. The 5' end(s) of the coat mRNA was determined by primer extension method (Appl. and Environ. Microbiol. 57:333-340 (1991)). The primer extension analysis revealed two separate 5' ends for the SP mRNA (Figure 3) indicating together with the DNA sequence data that there are two separate promoters controlling the coat gene expression. The two promoters are situated about 50 and 150 bp upstream of the ribosome binding site and are underlined in Figure 2. Downstream of the translational stop codon of the coat gene there is a typical rho independent transcription termination signal and a similar signal can be found also 460 bp upstream of the structural gene. Example 5

Construction of Lactobacillus insertion vectors

To test the expression and secretion units of the coat gene, integration vectors were constructed for L. brevis, L. Platarum, and L. casei as follows. The vectors were based on the pUC18 plasmid. Similarly to pUC18, any other convenient plasmid vector, either not able to replicate in Lactobacillus or carrying a replicon that is conditionally non-functional in Lactobacillus (e.g., cold- or thermosensitive), could have been used. A selection marker, functional in Lactobacillus, was then added to the pUC18 vector. In this particular case the erythromycin resistance marker (em^r) from S. aureus plasmid pE194 (J. Bacteriol 137:635-643 (1979)) was used. The em^r-gene was released from pE194 by ClaI/HpaII cleavage, blunted with Klenow polymerase and ligated into the HincII site of the pUC18 multilinker. Chromosomal DNA was isolated from L. brevis, L. plantarum, and L. casei as described in example 4. The isolated DNA was cleaved with SphI and fragments of 2-4 kb size were isolated after agarose gel electrophoresis. These random chromosomal fragments were then inserted into the SphI site of the modified pUC18 vector generating a set of integration vectors (carrying the chromosomal insert) for each of the three hosts. The vectors were tested for functional integration by transforming (by electroporation as described i n Appl. Environ. Microbiol. 57: 1822-1828 (1991)) individual vectors into the Lactobacillus hosts. Vectors that yielded stable em-resistant transformants were selected for further use. Selected integration vectors for L. brevis, L. plantarum and L. casei were designated pKTΗ2062, pKTH2064 and pKTH2067, respectively. Instead of integration vectors it may also be possible to apply plasmids that are able to replicate in Lactobacillus although some of these probably would show structural or segregational stability problems related to the high level of expression of the coat promoter and/or the mode of plasmid replication (single stranded intermediates). Example 6

Expression of TEM-β-lactamase in Lactobacillus, Lactococcus and

Bacillus

TEM-β-lactamase was chosen as a model gene to demonstrate the functionality of the coat gene expression and secretion units in various gram-positive hosts, β-lactamase gene was isolated from pBR322 by PCR using oligos 1 and 2 depicted in Table 3.

Table 3. Oligonucleotide primers used for construction of expression and secretion cassettes from L. brevis SP-gene.

SEQ

ID

Number Sequence Use No.

1 5' CCCCGGATCCGTTTTTGCTCACCCAGAAAC 3' 5'-end of bla 73

2 5' CCCCTCTAGATTACCAATGCTTAATCAGTG 3' 3'-end of bla 74

3 5' CCCCGAATTCGGGACAGGTGCTAGAGAC 3' 5'-end of coat , 75

secr./exp.

4 5' GGGGGGATCCCGTTGAAACGGCAGCAACAC 3' 3'-coat ss, 76

secretion unit

5 5' CCCCGGATCCCTAAACTTGATTGCATAATCTTTC 3' 3'-cost. 77

expression unit

Positions of primer sequences 3, 4 and 5 for expression and secretion unit in coat sequence are -311 → -294, 345 → 326 and 282 → 260,

respectively.

The PCR fragment contained a DNA sequence encoding the full mature β-lactamase and four amino acids from the C-terminus of its signal sequence.

The fragment was flanked by 5' BamHI and 3' XbaI sites. The expression/secretion unit was isolated by PCR using oligos 3 and 4 (Table 3) and the full length coat gene (see Example 4) as a template. The resulting PCR fragment contained the promoter region starting from nucleotide -311 (Figure 2A; this fragment also includes a rho independent transcription termination site upstream of the coat promoters to prevent unspecific mRNAs from affecting the system) followed by the ribosome binding site and 30 codons from the signal sequence of the coat gene. The isolated expression/secretion unit was flanked by 5' EcoRI and 3' BamHI sites. The β-lactamase fragment was ligated to the coat gene expression/secretion unit at the common BamHI site and the ligation mixture was amplified by PCR using oligos 3 and 2 to yield a hybrid β-lactamase gene under the coat gene expression and secretion signals. This hybrid gene, flanked by 5' EcoRI and 3' XbaI sites was then inserted to the Lactobacillus integration vectors pKTH2064 and pKTH2067(Εxample 5) between the respective sites. The ligation mixture was transformed by electroporation in L. plantarum and L. casei hosts. The cells were plated on MRS-em plates (10 μg/ml) and em-resistant transformant colonies were screened for β-lactamase activity by using a chromogenic substrate, nitrocefin (Glaxo) that β-lactamase degrades into a red product (J. Mol. Biol. 126:673-690 (1978)). The transformants were suspended in 250 μl of nitrocefin solution in microtiter wells and L. plantarum and L. casei colonies that turned red were picked up for further characterization, β-lactamase positive Lactobacillus colonies were grown to mid-logarithmic phase in 1 ml of MRS-em-solution, the cells were removed by centrifugation and suspended in equal volume of 50 mM phosphate buffer, pH 7.0. Equal volumes of the supernatants and cell suspensions were added to the nitrocefin solution. The supernatant samples contained about ten times more β-lactamase activity than the cell samples (as judged by the change of absorbance during 2 min. incubation), demonstrating that the expression/ secretion signal derived from the coat gene can be used to express and secrete a required protein in Lactobacillus.

The hybrid coat/β-lactamase gene was also joined as a blunt end fragment to the blunted Clal site of plasmid pVS2 (Appl. Environ. Microbiol 53: 1584-1588 (1987)). The ligation mixture was transformed to B. subtillis Marburg strain IH6064 (Gene 19:82-87 (1982)) by physiological transformation (J. Bacteriol. 134:318-329 (1978)) and to Lactococcus lactis subsp. lactis (J. Bacteriol. 154: 1-9 (1983)) by electroporation (Appl. Environ. Microbiol. 55:3119-3123 (1989)). The selection plates were Luria (Virology 1: 190-206 (1955)) and M 17 glucose (Appl. Environ. Microbiol. 29:807-813 (1975)) supplemented with 5 μg/ml chloramphenicol for Bacillus and for Lactococcus, respectively. The cm-resistant transformants were screened by nitrocefin assay as described above and also in the case positive transformants grown to mid-logarithmic phase in L-broth (Bacillus) or M17-glc-broth (Lactococci) secreted over 90% of the β-lactamase activity to the culture medium. This showed that expression/secretion signals derived from the coat gene can also be used in Bacillus and Lactococcus for expression and secretion of a required product. However, these clones in a multicopy plasmid system turned out to be unstable, probably due to high secretion potential and the inherent instability of the pasmid vector pVS2 (which uses a single stranded replication model)).

Example 7

Expression of the chloramphenicol resistance marker in gram-positive hosts

To demonstrate that the coat promoter can be used for intracellular expression of the required gene in various gram-positive host bacteria, the promoter region of the coat gene was isolated as a PCR fragment starting from nucleotide -311 (Figure 2A) and containing both promoters, the ribosome binding site and the four first codons from the 5' end of the signal sequence. The fragment was flanked by a 5' EcoRI site and a 3' BamHI site. The relevant oligos 3 (5') and 5 (3') are shown on Table 3. A promoterless cloramphenicol acetyltransferase gene (cat) in a promoter probe vector pKTH1750 (Appl. Environ. Microbiol. 57:333-340 (1991)) was applied to test the coat gene promoter function. The coat PCR-fragment was ligated with the EcoRI/BglII cleaved pKTH1750 vector and the ligation mixture was transformed into Bacillus subtillis IH6064 and Lactococcus lactis cells as described above. The transformants were selected on cm-(5 μg/ml)-plates. The intact vector pKTH1750 was used as a transformation control. Transformations with the coat/cat ligation mixture yielded large numbers of both the Bacillus and Lactococcus transformants whereas transformations with intact pKTH1750 control plasmid failed to yield any cm-resistant colonies. This shows that the use of the coat derived expression unit results in functional transcription both in Bacillus and Lactococcus and can thus be used for intracellular expression of the required gene in these hosts. Having now fully described the invention, it will be understood by those with skill in the art that the scope may be performed within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: VIAGEN OY

(ii) TITLE OF INVENTION: Lactobacillus Expression System

(iii) NUMBER OF SEQUENCES: 77

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: To be assigned

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/906,320

(B) FILING DATE: 30-JUN-1992

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 1..32

(D) OTHER INFORMATION: /note= "N=deoxyinosine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO :1:

TAYGCNACNG CNGGNGCNTA YWSNACNYTN AA 32

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both (ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 1..29

(D) OTHER INFORMATION: /note= "N=deoxyinosine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

TTRTTRTANG TRTANGTRTA RTGRTANGC 29

(2) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GTATCACGAG GCCCT 15

(2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

TAGTCAGCTG ACTTCTTTGA AGAAG 25

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

ACAACTGACT TGACTGGTGA 20

(2) INFORMATION FOR SEQ ID NO : 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 GTATCACGAG GCCCT 15

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

CCTGAGTTAT AGTGGCGTG 19

(2) INFORMATION FOR SEQ ID NO : 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

TAGCATCTGC AGCATTAGGG TTAG 24

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

AACGACTACT AAGGCTGATA TGCC 24

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

CGTGTTCTCC TCCAATGAAG C 21

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both (ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

CCTGAGTTAT AGTGGCGTG 19

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

CGTGTTCTCC TCCAATGAAG C 21

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 320 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

GCGCCGACTG GGACAGGTGC TAGAGACGGC GATTAAACGC ACCCAGCCAG TACCAGAAGC 60

ATAATATAGA AGAAAAGGGC AAACAAAAAA GCCGGCGAGA ACATTTCGCC GCGCTTTACC 120

TTAAAGAAGT TAATCGTTTC CGTTCTCCGC GGAAGCGATC ACTTTAAGGT AACTATTTCC 180

AGTTGCCAAA AAGCATCACC CCCTTTCATT TGATGCCTGT AGTATGGACC CATCGGAAGA 240

TAAATGCAAG TAAACTTACT TACCTTAACC TCGCTTTAAA TTTAATCATC AATAATGGTT 300

GTCAATGTTG AGAAATAAGC 320 (2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1763 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 268..1662

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

GATTACAAAG GCTTTAAGCA GGTTAGTGAC GTTTTAGTTA TGTAACAATA ACATTACAGG 60

ACACCCATAA TTGTTTCAAT CCAACGACAA TCAGAGCGTA ATCCTTGTAT CTCCTTAAGG 120

AAATCGCTAT ACTTATCTTC GTAGTTAGGG GATAGCTGAT CGGGTCCGCT AATGTTATGA 180 AATAAAATTC TTAACAAAAG CGCTAACTTC GGTTATACTA TTCTTGCTTG ATAAATTACA 240

TATTTTATGT TTGGAGGAAG AAAGATT ATG CAA TCA AGT TTA AAG AAA TCT 291

Met Gln Ser Ser Leu Lys Lys Ser

1 5

CTT TAC TTG GGC CTT GCC GCA TTG AGC TTT GCT GGT GTT GCT GCC GTT 339 Leu Tyr Leu Gly Leu Ala Ala Leu Ser Phe Ala Gly Val Ala Ala Val

10 15 20

TCA ACG ACT GCT TCA GCT AAG TCA TAC GCT ACT GCA GGT GCC TAT TCA 387 Ser Thr Thr Ala Ser Ala Lys Ser Tyr Ala Thr Ala Gly Ala Tyr Ser

25 30 35 40

ACG TTA AAG ACG GAC GCT GCT ACT CGT AAC GTC GAA GCT ACT GGT ACT 435 Thr Leu Lys Thr Asp Ala Ala Thr Arg Asn Val Glu Ala Thr Gly Thr

45 50 55

AAC GCT TTA TAC ACG AAG CCA GGT ACT GTT AAG GGT GCT AAG GTT GTC 483 Asn Ala Leu Tyr Thr Lys Pro Gly Thr Val Lys Gly Ala Lys Val Val

60 65 70

GCT TCT AAG GCT ACT ATG GCT AAG TTA GCT TCT TCA AAG AAG TCA GCT 531 Ala Ser Lys Ala Thr Met Ala Lys Leu Ala Ser Ser Lys Lys Ser Ala

75 80 85

GAC TAC TTC CGT GCT TAC GGT GTT AAG ACC ACT AAC CGT GGT TCA GTT 579 Asp Tyr Phe Arg Ala Tyr Gly Val Lys Thr Thr Asn Arg Gly Ser Val

90 95 100

TAC TAC CGT GTT GTA ACG ATG GAT GGC AAG TAC CGT GGT TAC GTT TAT 627 Tyr Tyr Arg Val Val Thr Met Asp Gly Lys Tyr Arg Gly Tyr Val Tyr

105 110 115 120

GGT GGC AAG TCT GAC ACT GCC TTT GCT GGT GGT ATC AAG TCT GCT GAA 675 Gly Gly Lys Ser Asp Thr Ala Phe Ala Gly Gly Ile Lys Ser Ala Glu

125 130 135

ACG ACT ACT AAG GCT GAT ATG CCT GCA CGT ACT ACT GGG TTC TAC TTA 723 Thr Thr Thr Lys Ala Asp Met Pro Ala Arg Thr Thr Gly Phe Tyr Leu

140 145 150

ACT GAC ACT TCA AAG AAC ACT CTT TGG ACG GCT CCT AAG TAC ACT CAA 771 Thr Asp Thr Ser Lys Asn Thr Leu Trp Thr Ala Pro Lys Tyr Thr Gln

155 160 165

TAC AAG GCA AGT AAA GTT AGC CTT TAT GGT GTT GCT AAG GAC ACC AAG 819 Tyr Lys Ala Ser Lys Val Ser Leu Tyr Gly Val Ala Lys Asp Thr Lys

170 175 180

TTT ACT GTA GAT CAG GCT GCT ACT AAG ACT CGT GAA GGT TCA TTA TAC 867 Phe Thr Val Asp Gln Ala Ala Thr Lys Thr Arg Glu Gly Ser Leu Tyr

185 190 195 200

TAT CAC GTA ACT GCT ACT AAC GGT AGT GGT ATT AGT GGT TGG ATT TAC 915 Tyr His Val Thr Ala Thr Asn Gly Ser Gly Ile Ser Gly Trp Ile Tyr

205 210 215

GCT GGT AAG GGC TTC AGT ACT ACT GCT ACT GGT ACA CAA GTA CTT GGT 963 Ala Gly Lys Gly Phe Ser Thr Thr Ala Thr Gly Thr Gln Val Leu Gly

220 225 230

GGT CTG TCA ACT GAT AAG TCA GTT ACA GCA ACC AAC GAT AAC AGT GTT 1011 Gly Leu Ser Thr Asp Lys Ser Val Thr Ala Thr Asn Asp Asn Ser Val

235 240 245

AAG ATT GTT TAC CGT ACG ACT GAT GGC ACT CAA GTT GGT TCT AAC ACT 1059 Lys Ile Val Tyr Arg Thr Thr Asp Gly Thr Gln Val Gly Ser Asn Thr

250 255 260

TGG GTA ACT TCA ACT GAT GGT ACA AAG GCA GGT TCT AAG GTA AGC GAT 1107 Trp Val Thr Ser Thr Asp Gly Thr Lys Ala Gly Ser Lys Val Ser Asp

265 270 275 280 AAG GCC GCC GAT CAA ACT GCT CTT GAA GCC TAC ATC AAT GCT AAC AAG 1155 Lys Ala Ala Asp Gln Thr Ala Leu Glu Ala Tyr Ile Asn Ala Asn Lys

285 290 295

CCT AGC GGT TAC ACT GTA ACT AAC CCT AAT GCT GCA GAT GCT ACC TAT 1203 Pro Ser Gly Tyr Thr Val Thr Asn Pro Asn Ala Ala Asp Ala Thr Tyr

300 305 310

GGT AAC ACA GTT TAC GCT ACT GTT TCC CAA GCA GCT ACT TCT AAG GTC 1251 Gly Asn Thr Val Tyr Ala Thr Val Ser Gln Ala Ala Thr Ser Lys Val

315 320 325

GCT TTG AAG GTC TCA GGG ACT CCT GTT ACT ACT GCA TTG ACT ACA GCT 1299 Ala Leu Lys Val Ser Gly Thr Pro Val Thr Thr Ala Leu Thr Thr Ala

330 335 340

GAT GCT AAT GAT AAG GTT GCA GCT AAC GAT ACC ACT GCT AAT GGT AGT 1347 Asp Ala Asn Asp Lys Val Ala Ala Asn Asp Thr Thr Ala Asn Gly Ser

345 350 355 360

TCT GTT GCA GGC TCA ACA GTC TAT GCT GCT GGT ACT AAG TTG GCT CAA 1395 Ser Val Ala Gly Ser Thr Val Tyr Ala Ala Gly Thr Lys Leu Ala Gln

365 370 375

TTA ACA ACT GAC TTG ACT GGT GAA AAG GGT CAA GTT GTC ACA TTA ACT 1443 Leu Thr Thr Asp Leu Thr Gly Glu Lys Gly Gln Val Val Thr Leu Thr

380 385 390

GCC ATC GAT ACT GAT TTG GAA GAC GCT ACG TTC ACT GGA ACT ACG ACT 1491 Ala Ile Asp Thr Asp Leu Glu Asp Ala Thr Phe Thr Gly Thr Thr Thr

395 400 405

TAC TAT TCA GAT CTT GGT AAA GCA TAC CAC TAC ACT TAC ACT TAC AAT 1539 Tyr Tyr Ser Asp Leu Gly Lys Ala Tyr His Tyr Thr Tyr Thr Tyr Asn

410 415 420

AAG GAC AGT GCT GCT TCT TCA AAT GCA AGT ACC CAA TTT GGT TCA AAC 1587 Lys Asp Ser Ala Ala Ser Ser Asn Ala Ser Thr Gln Phe Gly Ser Asn

425 430 435 440

GTC ACT GGT ACT TTA ACT GCT ACC CTT GTT ATG GGT AAG TCT ACT GCT 1635 Val Thr Gly Thr Leu Thr Ala Thr Leu Val Met Gly Lys Ser Thr Ala

445 450 455

ACT GCT AAC GGT ACT ACT TGG TTC AAC TAATAATTAT TATTTAGGTG 1682

Thr Ala Asn Gly Thr Thr Trp Phe Asn

460 465

AGCTTTGTTG ATAAAAAGGT CTTTTCAACG TTTATGTTGG GGAGACCTTT TTATATTGAA 1742

AAAATTAGGC CTTTGTTAGG A 1763

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 465 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Met Gln Ser Ser Leu Lys Lys Ser Leu Tyr Leu Gly Leu Ala Ala Leu

1 5 10 15

Ser Phe Ala Gly Val Ala Ala Val Ser Thr Thr Ala Ser Ala Lys Ser

20 25 30

Tyr Ala Thr Ala Gly Ala Tyr Ser Thr Leu Lys Thr Asp Ala Ala Thr

35 40 45 Arg Asn Val Glu Ala Thr Gly Thr Asn Ala Leu Tyr Thr Lys Pro Gly 50 55 60

Thr Val Lys Gly Ala Lys Val Val Ala Ser Lys Ala Thr Met Ala Lys 65 70 75 80

Leu Ala Ser Ser Lys Lys Ser Ala Asp Tyr Phe Arg Ala Tyr Gly Val

85 90 95

Lys Thr Thr Asn Arg Gly Ser Val Tyr Tyr Arg Val Val Thr Met Asp

100 105 110

Gly Lys Tyr Arg Gly Tyr Val Tyr Gly Gly Lys Ser Asp Thr Ala Phe

115 120 125

Ala Gly Gly Ile Lys Ser Ala Glu Thr Thr Thr Lys Ala Asp Met Pro 130 135 140

Ala Arg Thr Thr Gly Phe Tyr Leu Thr Asp Thr Ser Lys Asn Thr Leu 145 150 155 160

Trp Thr Ala Pro Lys Tyr Thr Gln Tyr Lys Ala Ser Lys Val Ser Leu

165 170 175

Tyr Gly Val Ala Lys Asp Thr Lys Phe Thr Val Asp Gln Ala Ala Thr

180 185 190

Lys Thr Arg Glu Gly Ser Leu Tyr Tyr His Val Thr Ala Thr Asn Gly

195 200 205

Ser Gly Ile Ser Gly Trp Ile Tyr Ala Gly Lys Gly Phe Ser Thr Thr 210 215 220

Ala Thr Gly Thr Gln Val Leu Gly Gly Leu Ser Thr Asp Lys Ser Val 225 230 235 240

Thr Ala Thr Asn Asp Asn Ser Val Lys Ile Val Tyr Arg Thr Thr Asp

245 250 255

Gly Thr Gln Val Gly Ser Asn Thr Trp Val Thr Ser Thr Asp Gly Thr

260 265 270

Lys Ala Gly Ser Lys Val Ser Asp Lys Ala Ala Asp Gln Thr Ala Leu

275 280 285

Glu Ala Tyr Ile Asn Ala Asn Lys Pro Ser Gly Tyr Thr Val Thr Asn 290 295 300

Pro Asn Ala Ala Asp Ala Thr Tyr Gly Asn Thr Val Tyr Ala Thr Val 305 310 315 320

Ser Gln Ala Ala Thr Ser Lys Val Ala Leu Lys Val Ser Gly Thr Pro

325 330 335

Val Thr Thr Ala Leu Thr Thr Ala Asp Ala Asn Asp Lys Val Ala Ala

340 345 350

Asn Asp Thr Thr Ala Asn Gly Ser Ser Val Ala Gly Ser Thr Val Tyr

355 360 365

Ala Ala Gly Thr Lys Leu Ala Gln Leu Thr Thr Asp Leu Thr Gly Glu 370 375 380

Lys Gly Gln Val Val Thr Leu Thr Ala Ile Asp Thr Asp Leu Glu Asp 385 390 395 400

Ala Thr Phe Thr Gly Thr Thr Thr Tyr Tyr Ser Asp Leu Gly Lys Ala

405 410 415

Tyr His Tyr Thr Tyr Thr Tyr Asn Lys Asp Ser Ala Ala Ser Ser Asn

420 425 430

Ala Ser Thr Gln Phe Gly Ser Asn Val Thr Gly Thr Leu Thr Ala Thr 435 440 445

Leu Val Met Gly Lys Ser Thr Ala Thr Ala Asn Gly Thr Thr Trp Phe

450 455 460

Asn

465

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

CTTAGCCATA TGAGCCTTA 19

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Ala Tyr His Tyr Thr Tyr Thr Tyr Asn Lys

1 5 10

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Ser Ala Asp Tyr Phe Arg Ala Tyr Gly Val Lys

1 5 10

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Tyr Arg Gly Tyr Val Tyr Gly Gly Lys

1 5

(2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Thr Thr Asn Arg Gly Ser Val Tyr Tyr Arg Val Val Thr Met

1 5 10

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Ser Tyr Ala Thr Ala Gly Ala Tyr Ser Thr Leu Lys

1 5 10

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

Lys Ser Tyr Ala Thr Ala Gly Ala Tyr Ser

1 5 10

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

Met Gln Ser Ser Leu Lys Lys Ser Leu Tyr Leu Gly Leu Ala Ala Leu 1 5 10 15

Ser Phe Ala Gly Val Ala Ala Val Ser Thr Thr Ala Ser Ala

20 25 30

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide (ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (24^25)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (25^26)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (27^28)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (28^29)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

Met Lys Lys Asn Ile Ile Thr Ser Ile Thr Ser Leu Ala Leu Val Ala 1 5 10 15

Gly Leu Ser Leu Thr Ala Phe Ala Ala Thr Thr Ala Thr Val

20 25 30

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (17^18)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

Met Leu Arg Lys Lys Thr Lys Gln Leu Ile Ser Ser Ile Leu Ile Leu 1 5 10 15

Val Leu Leu Leu Ser Leu Phe Pro Thr Ala Leu Ala Ala Glu Gly

20 25 30

(2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site (B) LOCATION: (32^33)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

Leu Lys Lys Phe Pro Lys Lys Leu Leu Pro Ile Ala Val Leu Ser Ser 1 5 10 15 Ile Ala Phe Ser Ser Leu Ala Ser Gly Ser Val Pro Glu Ala Ser Ala

20 25 30

Gln Glu

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (33^34)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

Met Phe Ala Lys Arg Phe Lys Thr Ser Leu Leu Pro Leu Phe Ala Gly 1 5 10 15

Phe Leu Leu Leu Phe Tyr Phe Val Leu Ala Gly Pro Ala Ala Ala Ser

20 25 30

Ala Glu Thr

35

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (31^32)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

Met Ile Gln Lys Arg Lys Arg Thr Val Ser Phe Arg Leu Val Leu Met 1 5 10 15

Cys Thr Leu Leu Phe Val Ser Leu Pro Ile Thr Lys Thr Ser Ala Val

20 25 30

Asn

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both (ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (29^30)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

Met Lys Gln His Lys Arg Leu Tyr Ala Arg Leu Leu Pro Leu Leu Phe 1 5 10 15

Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn

20 25 30

(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (29^30)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

Met Lys Gln Gln Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe 1 5 10 15

Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn

20 25 30

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-s te

(B) LOCATION: (34^35)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

Met Leu Thr Phe His Arg Ile Ile Arg Lys Gly Trp Met Phe Leu Leu 1 5 10 15

Ala Phe Leu Leu Thr Ala Leu Leu Phe Cys Pro Thr Gly Gln Pro Ala

20 25 30

Lys Ala Ala Ala

35

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide (ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (34^35)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Met Leu Thr Phe His Arg Ile Ile Arg Lys Gly Trp Val Phe Leu Leu 1 5 10 15

Ala Phe Trp Leu Thr Ala Ser Leu Phe Cys Pro Thr Gly Gln Pro Ala

20 25 30

Lys Ala Ala Ala

35

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (33^34)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

Met Lys Lys Lys Thr Leu Ser Leu Phe Val Gly Leu Met Leu Leu Ile 1 5 10 15

Gly Leu Leu Phe Ser Gly Ser Leu Pro Tyr Asn Pro Asn Ala Ala Glu

20 25 30

Ala Ser Ser

35

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (35^36)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

Met Thr Leu Tyr Arg Ser Leu Trp Lys Lys Gly Cys Met Leu Leu Leu 1 5 10 15

Ser Leu Val Leu Ser Leu Thr Ala Phe Ile Gly Ser Pro Ser Asn Thr

20 25 30

Ala Ser Ala Ala Val

35

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (27^28)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putatuve and proposed by us or by the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

Met Lys Gly Lys Lys Trp Thr Ala Leu Ala Leu Thr Leu Pro Leu Ala 1 5 10 15

Ala Ser Leu Ser Thr Gly Val Asp Ala Glu Thr Val His

20 25

(2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (33^34)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

Met Lys Met Arg Thr Gly Lys Lys Gly Phe Leu Ser Ile Leu Leu Ala 1 5 10 15

Phe Leu Leu Val Ile Thr Ser Ile Pro Phe Thr Leu Val Asp Val Glu

20 25 30

Ala His His

35

(2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (30^31)

(D) OTHER INFORMATION: /note= "the cleavage site has not bee determined but is putative and proposed by us or the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

Met Arg Lys Lys Thr Lys Asn Arg Leu Ile Ser Ser Val Leu Ser Thr 1 5 10 15

Val Val Ile Ser Ser Leu Leu Phe Pro Gly Ala Ala Gly Ala Ser Ser

20 25 30 (2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (32^33)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (40^41)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

Met Ile Asn Leu Asn Lys His Thr Ala Phe Lys Lys Thr Ala Lys Phe 1 5 10 15

Phe Leu Gly Leu Ser Leu Leu Leu Ser Val Ile Val Pro Ser Phe Ala

20 25 30

Leu Gln Pro Ala Thr Ala Glu Ala Ala Asp

35 40

(2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (27^28)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

Met Lys Arg Phe Met Lys Leu Thr Ala Val Trp Thr Leu Trp Leu Ser 1 5 10 15

Leu Thr Leu Gly Leu Leu Ser Pro Val His Ala Ala Pro

20 25

(2) INFORMATION FOR SEQ ID NO: 40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (31^32) (D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

Met Phe Gln Met Ala Lys Arg Val Leu Leu Ser Thr Thr Leu Thr Phe 1 5 10 15

Ser Leu Leu Ala Gly Ser Ala Leu Pro Phe Leu Pro Ala Ser Ala Ile

20 25 30

Tyr

(2) INFORMATION FOR SEQ ID NO: 41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (27^28)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

Met Lys Ser Arg Tyr Lys Arg Leu Thr Ser Leu Ala Leu Ser Leu Ser 1 5 10 15

Met Ala Leu Gly Ile Ser Leu Pro Ala Trp Ala Ser Pro

20 25

(2) INFORMATION FOR SEQ ID NO: 42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (27^28)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

Met Lys Asn Met Ser Cys Lys Ser Val Val Ser Val Thr Leu Phe Phe 1 5 10 15

Ser Phe Leu Thr Ile Gly Pro Leu Ala His Ala Gln Asn

20 25

(2) INFORMATION FOR SEQ ID NO: 43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide (ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (29^30)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

Met Lys Arg Ser Ile Ser Ile Phe Ile Thr Cys Leu Leu Ile Thr Leu 1 5 10 15

Leu Thr Met Gly Gly Met Ile Ala Ser Pro Ala Ser Ala Ala Gly

20 25 30

(2) INFORMATION FOR SEQ ID NO: 44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (28^29)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

Met Pro Tyr Leu Lys Arg Val Leu Leu Leu Leu Val Thr Gly Leu Phe 1 5 10 15

Met Ser Leu Phe Ala Val Thr Ala Thr Ala Ser Ala Lys Thr

20 25 30

(2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (28^29)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

Met Pro Tyr Leu Lys Arg Val Leu Leu Leu Leu Val Thr Gly Leu Phe 1 5 10 15

Met Ser Leu Phe Ala Val Thr Ser Thr Ala Ser Ala Gln Thr

20 25 30

(2) INFORMATION FOR SEQ ID NO: 46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (32^33) (D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

Met Lys Lys Lys Gly Leu Lys Lys Thr Phe Phe Val Ile Ala Ser Leu 1 5 10 15

Val Met Gly Phe Thr Leu Tyr Gly Tyr Thr Pro Val Ser Ala Asp Ala

20 25 30

Ala Ser

(2) INFORMATION FOR SEQ ID NO: 47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (32^33)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (30^31)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:

Met Lys Lys Arg Arg Ser Ser Lys Val Ile Leu Ser Leu Ala Ile Val 1 5 10 15

Val Ala Leu Leu Ala Ala Val Glu Pro Asn Ala Ala Leu Ala Ala Ala

20 25 30

Pro Pro

(2) INFORMATION FOR SEQ ID NO: 48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (25^26)

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 48 :

Met Lys Asn Lys Arg Met Leu Lys Ile Gly Ile Cys Val Gly Ile Leu 1 5 10 15

Gly Leu Ser Ile Thr Ser Leu Glu Ala Phe Thr 20 25

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (25^26)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

Met Lys Asn Lys Lys Met Leu Lys Ile Gly Met Cys Val Gly Ile Leu 1 5 10 15

Gly Leu Ser Ile Thr Ser Leu Val Thr Phe Thr

20 25

(2) INFORMATION FOR SEQ ID NO: 50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (29^30)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:

Met Lys Asn Thr Leu Leu Lys Leu Gly Val Cys Val Ser Leu Leu Gly 1 5 10 15 Ile Thr Pro Phe Val Ser Thr Ile Ser Ser Val Gln Ala Glu Arg

20 25 30

(2) INFORMATION FOR SEQ ID NO: 51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (30^31)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:

Met Lys Lys Asn Thr Leu Leu Lys Val Gly Leu Cys Val Gly Leu Leu 1 5 10 15 Gly Thr Ile Gln Phe Val Ser Thr Ile Ser Ser Val Gln Ala Ser Gln 20 25 30

(2) INFORMATION FOR SEQ ID NO: 52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (24^25)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:

Met Lys Lys Arg Leu Ile Gln Val Met Ile Met Phe Thr Leu Leu Leu 1 5 10 15

Thr Met Ala Phe Ser Ala Asp Ala Ala Asp

20 25

(2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (29^30)

(xi ) SEQUENCE DESCRI PTION : SEQ ID NO : 53 :

Met Asn Ile Lys Lys Phe Ala Lys Gln Ala Thr Val Leu Thr Phe Thr

1 5 10 15

Thr Ala Leu Leu Ala Gly Gly Ala Thr Gln Ala Phe Ala Lys Glu

20 25 30

(2) INFORMATION FOR SEQ ID NO : 54 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (26^27)

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 54 :

Met Lys Val Tyr Lys Lys Val Ala Phe Val Met Ala Phe Ile Met Phe

1 5 10 15 Phe Ser Val Leu Pro Thr Ile Ser Met Ser Ser Glu 20 25

(2) INFORMATION FOR SEQ ID NO: 55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (34^35)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

Met Lys Leu Val Pro Arg Phe Arg Lys Gln Trp Phe Ala Tyr Leu Thr 1 5 10 15

Val Leu Cys Leu Ala Leu Ala Ala Ala Val Ser Phe Gly Val Pro Ala

20 25 30

Lys Ala Ala Glu

35

(2) INFORMATION FOR SEQ ID NO: 56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (23^24)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:

Met Lys Lys Val Val Asn Ser Val Leu Ala Ser Ala Leu Ala Leu Thr 1 5 10 15

Val Ala Pro Met Ala Phe Ala Ala Glu

20 25

(2) INFORMATION FOR SEQ ID NO:57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (27^28)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:

Met Gly Leu Gly Lys Lys Leu Ser Val Ala Val Ala Ala Ser Phe Met 1 5 10 15

Ser Leu Thr Ile Ser Leu Pro Gly Val Gln Ala Ala Gln

20 25

(2) INFORMATION FOR SEQ ID NO: 58:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (27^28)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:

Met Gly Leu Gly Lys Lys Leu Ser Ser Ala Val Ala Ala Ser Phe Met 1 5 10 15

Ser Leu Thr Ile Ser Leu Pro Gly Val Gln Ala Ala Glu

20 25

(2) INFORMATION FOR SEQ ID NO: 59:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (27^28)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:

Met Gly Leu Gly Lys Lys Leu Ser Val Arg Val Ala Ala Ser Phe Met 1 5 10 15

Ser Leu Ser Ile Ser Leu Pro Gly Val Gln Ala Ala Glu

20 25

(2) INFORMATION FOR SEQ ID NO: 60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (25^26)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:

Met Asn Lys Arg Ala Met Leu Gly Ala Ile Gly Leu Ala Phe Gly Leu 1 5 10 15 Leu Ala Ala Pro Ile Gly Ala Ser Ala Lys Gly

20 25

(2) INFORMATION FOR SEQ ID NO:61:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (33^34)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (31^32)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:

Met Lys Arg Lys Met Lys Met Lys Leu Val Arg Phe Gly Leu Ala Ala 1 5 10 15

Gly Val Ala Ala Gln Val Phe Phe Leu Pro Tyr Asn Ala Leu Ala Ser

20 25 30

Thr Glu His

35

(2) INFORMATION FOR SEQ ID NO: 62:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (24^25)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:

Met Asn Lys Lys Val Val Leu Ser Val Leu Ser Thr Thr Leu Val Ala 1 5 10 15

Ser Val Ala Ala Ser Ala Phe Ala Ala Pro

20 25

(2) INFORMATION FOR SEQ ID NO:63:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Cleavage-site

(B) LOCATION: (26^27)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:

Met Lys Lys Arg Leu Ser Trp Ile Ser Val Lys Leu Leu Val Leu Val 1 5 10 15

Ser Ala Ala Gly Met Leu Phe Ser Thr Ala Ala Lys

20 25

(2) INFORMATION FOR SEQ ID NO:64:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (27^28)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:

Met Lys Gly Lys Leu Leu Lys Gly Val Leu Ser Leu Gly Val Gly Leu 1 5 10 15

Gly Ala Leu Tyr Ser Gly Thr Ser Ala Gln Ala Glu Ala

20 25

(2) INFORMATION FOR SEQ ID NO: 65:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (29^30)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:

Met Arg Ser Lys Lys Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gln Ala Ala Gly

20 25 30

(2) INFORMATION FOR SEQ ID NO:66:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (30^31) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:

Met Arg Gly Lys Lys Val Trp Ile Ser Leu Leu Phe Ala Leu Ala Leu 1 5 10 15 Ile Phe Thr Met Ala Phe Gly Ser Thr Ser Ser Ala Gln Ala Ala Gly

20 25 30

(2) INFORMATION FOR SEQ ID NO: 67:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (29^30)

(D) OTHER INFORMATION: /note= "the cleavage site has not been determined but is putative and proposed by us or by the authors that have published the sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:

Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met 1 5 10 15

Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln

20 25 30

(2) INFORMATION FOR SEQ ID NO: 68:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (27^28)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:

Met Asn Leu Arg Lys Leu Arg Leu Leu Phe Val Met Cys Ile Gly Leu 1 5 10 15

Thr Leu Ile Leu Thr Ala Val Pro Ala His Ala Arg Thr

20 25

(2) INFORMATION FOR SEQ ID NO:69:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: Cleavage-site

(B) LOCATION: (29^30) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:

Met Ile Thr Leu Phe Arg Lys Pro Phe Val Ala Gly Leu Ala Ile Ser 1 5 10 15

Leu Leu Val Gly Gly Gly Ile Gly Asn Val Ala Ala Ala Gln

20 25 30

(2) INFORMATION FOR SEQ ID NO:70:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH : 12 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:

Met Ile Gln Lys Arg Lys Arg Thr Val Ser Phe Arg

1 5 10

(2) INFORMATION FOR SEQ ID NO:71:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:

Leu Val Leu Met Cys Thr Leu Leu Phe Val Ser Leu

1 5 10

(2) INFORMATION FOR SEQ ID NO:72:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:

Pro Ile Thr Lys Thr Ser Ala

1 5

(2) INFORMATION FOR SEQ ID NO:73:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:

CCCCGGATCC GTTTTTGCTC ACCCAGAAAC 30

(2) INFORMATION FOR SEQ ID NO: 74: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:

CCCCTCTAGA TTACCAATGC TTAATCAGTG 30

(2) INFORMATION FOR SEQ ID NO: 75:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:

CCCCGAATTC GGGACAGGTG CTAGAGAC 28

(2) INFORMATION FOR SEQ ID NO: 76:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:

GGGGGGATCC CGTTGAAACG GCAGCAACAC 30

(2) INFORMATION FOR SEQ ID NO: 77:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:

CCCCGGATCC CTAAACTTGA TTGCATAATC TTTC 34

Claims

What Is Claimed Is:

1. Lactobacillus coat protein, essentially free of natural contaminants.

2. The coat protein of claim 1, wherein said coat protein is from a Lactobacillus selected from the group consisting of L brevis, L. buchneri, L. plantarum, L. casei, L. fermenti, and L. helveticus.

3. The coat protein of claim 2, wherein said coat protein is from a Lactobacillus selected from the group consisting of L brevis, and L. buchneri.

4. The coat protein of claim 3, wherein said coat protein comprises the amino acid sequence of the mature coat protein shown in Figure 2.

5. Isolated DNA encoding a Lactobacillus coat protein.

6. The DNA of claim 5, wherein said coat protein is from a Lactobacillus selected from the group consisting of L brevis, L. plantarum, L. casei, L. buchneri, L. fermenti, and L. helveticus.

7. The DNA of claim 6, wherein said coat protein is from a Lactobacillus selected from the group consisting of L brevis, and L. buchneri.

8. The DNA of claim 5, wherein said coat protein comprises the amino acid sequence shown in Figure 2.

9. The DNA of claim 8, wherein said DNA comprises the DNA sequence shown in Figure 2.

10. A recombinant vector comprising DNA encoding a Lactobacillus coat protein.

11. A recombinant vector comprising at least one Lactobacillus coat protein expression regulatory element operably linked to DNA encoding a desired sequence of interest, said regulatory element being selected from the group consisting of a coat protein promoter, a full-length coat protein secretion signal sequence, and one or more domains of a coat protein secretion signal sequence.

12. The recombinant vector of claim 11, wherein said regulatory element is that of the coat protein gene of a Lactobacillus selected from the group consisting of L brevis, L. plantarum, L. casei, L. buchneri, L. fermenti, and L. helveticus.

13. The recombinant vector of claim 12, wherein said Lactobacillus is selected from the group consisting of L brevis, and L. buchneri.

14. The recombinant vector of claim 11, wherein said coat protein secretion signal amino acid sequence comprises the amino acid sequence shown in Figure 2.

15. The recombinant vector of claim 11 , wherein said the sequence of said regulatory element is that shown in Figure 2.

16. The recombinant vector of claim 11 , wherein said desired protein of interest is expressed as a fusion protein with at least a portion of the amino acid sequence of a Lactobacillus coat protein.

17. The recombinant vector of claim 11 , wherein said domain of said coat protein secretion signal is selected from the group consisting of the N-region, the h-region, and the C-region.

18. A host transformed with the recombinant vector of any of claims 11-17.

19. The host of claim 18, wherein said host is selected from the group consisting of a member of the genera Lactobacillus, Lactococcus and Bacillus.

20. The host of claim 19, wherein said host is selected from the group consisting of L brevis, L. buchneri, L. fermenti, L. helveticus, L. casei, B. subtilis, and Lactococcus lactis subsp. lactis.

21. The host of claim 20, wherein said host is L brevis.

22. A method of producing a desired protein of interest, said method comprising transforming a Lactobacillus host with the recombinant vector of any of claims 11-17, and expression of said desired protein of interest in said host.

23. A method of inhibiting expression of an undesired protein in Lactobacillus, said method comprising transforming a Lactobacillus host with the recombinant vector of any of claims 11-13, and expression of said desired sequence of interest, wherein said desired sequence of interest is an antisense RNA directed against the mRNA encoding said undesired protein.