CLEAVAGE OF CAULOBACTER PRODUCED RECOMBINANT FUSION PROTEINS

Abstract: This invention provides a method for cleaving target proteins from <u>Caulobacter</u> S-layer protein under mild acid conditions. A fusion protein secreted by <u>Caulobacter</u> which includes a target protein and a <u>Caulobacter</u> S-layer secretion signal may be cleaved at an aspartate-proline dipeptide without solubilizing the fusion protein. This method may be carried out while the fusion protein is in an insoluble aggregate which facilitates recovery of the protein. This invention also provides a method of preparing a DNA construct for expression of the fusion protein and a method of preparing the fusion protein.

Description: CLEAVAGE OF CAULOBACTER PRODUCED RECOMBINANT FUSION PROTEINS FIELD OF INVENTION This invention relates to the expression and secretion of recombinant fusion proteins from Caulobacter wherein a heterologous polypeptide is fused with all or part of the surface layer protein (S-layer protein) of the bacterium. BACKGROUND OF THE INVENTION Many bacteria assemble layers composed of repetitive, regularly aligned, proteinaceous sub-units on the outer surface of the cell. These layers are essentially two-dimensional paracrystalline arrays, and being the outer molecular layer of the organism, directly interface with the environment. In Caulobacter, the S-layer protein is synthesized by the cell in large quantities and the S-layer completely envelops the cell and thus appears to be a protective layer. Caulobacter are natural inhabitants of most soil and freshwater environments and may persist in waste water treatment systems and effluents. The bacteria alternate between a stalked cell that is attached to a surface, and an adhesive motile dispersal cell that searches to find a new surface upon which to stick and convert to a stalked cell. The bacteria attach tenaciously to nearly all surfaces and do so without producing the extracellular enzymes or polysaccharide "slimes" that are characteristic of most other surface attached bacteria. Caulobacters have simple requirements for growth. The organism is ubiquitous in the environment and has been isolated from oligotrophic to mesotrophic situations. They are known for their ability to tolerate low nutrient level stresses, for example, low phosphate levels. All of the freshwater Caulobacter that produce an S-layer are similar and have S-layers that are substantially the same under election microscopy. The layers are hexagonally arranged in all cases, with a similar centre-centre dimension (see: Walker, S.G., et al... (1992). "Isolation and Comparison of the Paracrystalline Surface Layer Proteins of Freshwater Caulobacters" J. Bacteriol. 174: 1783-1792). 16S rRNA sequence analysis of several S-layer producing Caulobacter strains show that they group closely (see: Stahl, D.A. et al. (1992) "The Phylogeny of Marine and Freshwater Caulobacters Reflects Their Habitat" J. Bacteriol. 174: 2193-2198). DNA probing of Southern blots using the S-layer gene from C. crescentus CB15 5 identifies a single band that is consistent with the presence of a cognate gene (see: MacRae, J.D. and, J. Smit. (1991) "Characterization of Caulobacters Isolated from Wastewater Treatment Systems" Applied and Environmental Microbiology 57:751- 758). Furthermore, antisera raised against the S-layer protein of CB15 reacts against the S-layer protein of other Caulobacter (see: Walker, S.G. et al. (1992) [supra]). All o S-layer proteins isolated from Caulobacter may be substantially purified using the same methods. All strains appear to have a polysaccharide species which may be required for S-layer attachment (see: Walker, S.G. et al. (1992) [supra]). The S-layers elaborated by freshwater isolates of Caulobacter are visibly indistinguishable from the S-layer produced by Caulobacter strains CB2 and CB15. 5 The S-layer proteins from the latter strains have approximately 100,000 m.w. although sizes of S-layer proteins from other species and strains will vary. The hydrophillic S- layer protein has been characterized both structurally and chemically. It is composed of ring-like structures spaced at 22 nm intervals arranged in a hexagonal manner on the outer membrane. The S-layer is bound to the bacterial surface and may be removed by 0 low pH treatment or by treatment with a calcium chelator such as EDTA. The similarity of S-layer proteins in different strains of Caulobacter permits the use of a cloned S-layer protein gene of one Caulobacter strain for retrieval of the corresponding gene in other Caulobacter strains (see: Walker, S.G. et al. (1992) [supra]; and MacRae, J.D. et al. (1991) [supra]). 5 Expression of a heterologous polypeptide as a fusion product with the S-layer protein of Caulobacter provides advantages not previously seen in systems for production of recombinant fusion proteins using other organisms such as E. coli and Salmonella. All known Caulobacter strains are believed to be harmless and are nearly ubiquitous in aquatic environments. In contrast, many Salmonella and E. coli strains 0 are pathogens. Consequently, expression and secretion of a heterologous polypeptide using Caulobacter as a vehicle has the advantage that the expression system will be stable in a variety of outdoor environments and may not present problems associated with the use of a pathogenic organism. Furthermore, Caulobacter are natural biofilm foirning species and may be adapted for use in fixed biofilm bioreators. The quantity of S-layer protein that is synthesized and is secreted by Caulobacter is high, reaching 12% of the cell protein. There is an existing need to produce pure proteins and peptides in an economical manner and in a manner that minimizes or simplifies the purification steps needed after fermentation. Key commercial areas include the production of recombinant human and animal therapeutic antibiotic and vaccine peptides, industrial enzymes, protein polymers, and antibacterial enzymes for foodstuffs. Many of these commercial applications require low production costs and there are few expression systems available that can meet such cost restraints. In addition, there are numerous research applications where rapid methods to produce and purify proteins are needed to facilitate the discovery stage. This is especially true where there is a desire to express a large number of proteins with unknown function (from a collections of cloned cDNA's, for example) or a large number of variants of a single protein, (for example, resulting from site directed mutagenesis) in a search for variants with improved properties. Generally, proteins must be secreted to be produced at low cost. The primary reason is the much reduced cost of purification of the target protein from cell material. However, even for secreted proteins, simple methods of separating the product from spent culture and cells are important for cost reduction and ease of use. An international patent application published as WO 97/34000 on September 18, 1997 describes the expression and secretion of recombinant proteins from Caulobacter in which the recombinant protein is a fusion of all or part of Caulobacter S-layer protein with a heterologous protein of interest (also see: Bingle, W.H., et al. 1997 1 "Linker Mutagenesis of the Caulobacter us S-layer protein: Toward a Definition of an N- terminal Anchoring Region and a C-terminal Secretion Signal and the Potential for Heterologous Protein Secretion" . J. Bacteriol. 179:601-611). The Caulobacter S-layer secretion apparatus is in the category of "Type 1" secretion usually found in pathogenic bacteria and noted for its ability to secrete a wide variety of proteins including large and hydrophillic proteins. The Caulobacter protein secretion system is particularly useful to secrete recombinant proteins. The Caulobacter S-layer Type 1 secretion pathway requires only a C-terminal secretion signal, typically comprising about 200 amino acids at the end of the protein. The export mechanism is capable of tolerating a wide variety of foreign proteins. Recombinant proteins may be conveniently produced as fusion proteins with the target protein being fused to the C-terminal secretion signal. Depending on the application, it may be desirable to remove the secretion signal following secretion. Not removing the secretion signal may be an approach suitable for many subunit vaccine applications, where the remaining S-layer protein serves as a carrier. A unique and desirable feature of fusion proteins produced by the Caulobacter S-layer protein secretion system is that they form insoluble aggregates in the culture medium. This is apparently a consequence of the S-layer sequences associated with secretion signal and reflects the fact that the protein normally self-assembles into a two dimensional crystalline layer on the bacterium's surface. These aggregates are visible to the naked eye and are readily collected by simple filtration. With simple water wash steps, residual bacterial cells are readily flushed away. It is routinely possible to achieve a protein purity of 90% or better with this simple purification procedure. DESCRIPTION OF THE PRIOR ART Most current protein purification systems for recombinant proteins produced by bacteria rely upon an affinity matrix to achieve separation of the target protein and to concentrate the protein for subsequent steps of purification. To accomplish this, genes for recombinant proteins are commonly constructed so that they contain affinity tags, which are protein sequences that will bind to an affinity matrix. Commonly used systems include the following: (a) glutathione S-transferase (GST) tag. which binds to glutathione-sepharose matrices; (b) maltose binding protein (MBP) tag, which binds to amylose matrices; (c) multiple tandem histidine residues (e.g. "His-6") tag, which binds to nickel-derivatized solid matrices; and (d) protein A tag, which binds to Immunoglobulin IgG-derivatized sepharose or comparable matrices. Prior art techniques were typically developed so that removal of a target protein does not disrupt the tag and matrix association. Instead, enzymes that cleave specific sequences of amino acids are employed. The enzyme cleavage sequence is positioned between the tag and the desired recombinant protein and enzymatic cleavage is effected directly on the matrix with attached fusion protein. If a secretion signal is used, the cleavage site is usually positioned such that the secretion signal is separated from the target recombinant protein during the cleavage step. The matrix is regenerated for re- use only after the target recombinant protein has been purified away from the matrix. Typical enzymes used in these methods are Factor Xa, enterokinase and collagenase. Chemical cleavage is generally not used because the conditions required for cleavage will disrupt the binding of affinity tag and matrix or destroy the matrix. When chemical cleavage is used with recombinant fusion proteins to cleave target protein from a secretion signal and/or affinity tag, solubilization and denaturation processes are generally employed. The expectation is that complete or nearly complete unfolding of the protein is a prerequisite for effective cleavage. Mild-acid cleavage is predicated on the inclusion, by happenstance or design, of the acid-sensitive aspartate-proline dipeptide at a desired site for cleavage. The protein to be cleaved is typically exposed to conditions that solubilize and/or completely denature the protein prior to cleavage. The chaotropic agent guanidine hydrochloride (used at 6-7 M) is commonly employed to denature and solubilize the protein prior to, or at the same time as acid treatment. Alternately, high concentrations of acids that also serve as solubilizing agents (as examples: 70-90% formic acid, acetic acid [10%] pyridine, or relatively high concentrations of HCL (60 mM or more) are employed. Because such conditions would disrupt a tag/affinity matrix association, direct cleavage of an affinity tag from the target protein while a protein remains associated with an affinity matrix is not attempted. General conditions for cleavage at aspartate - proline sites are described in Current Protocols in Molecular Biology (supp. 28; chapter 16.4) John Wiley & Sons Inc. 1994, and in Landon, M. "Cleavage at Aspartyl - Prolyl Bonds" in Methods in Enzymology (1977) 47: 145-149. These references suggest that significant variability of cleavage conditions exist for different proteins and that cleavage might occur in some instances without first denaturing or solubilizing the protein. However, in practice, the latter circumstances are rare and proteins to be subjected to acid cleavage at Asp-Pro dipeptides are usually solubilized to a state where there is no visible turbidity. Such solubilized protein will normally not pellet when centrifuged at 100,000 x g for 1 hour. It is now shown that mild-acid conditions may be used for cleavage of aspartate-proline sites in Caulobacter S-layer fusion proteins without placing the protein in a solubilized state as described above. SUMMARY OF INVENTION This invention is based on the unexpected discovery that recombinant fusion proteins produced by the Caulobacter S-layer protein secretion system can be cleaved under mild-acid conditions and solubilization of the fusion protein is not required. Cleavage may be accomplished while the fusion protein is in the form of an insoluble aggregate typical of the Caulobacter S-layer protein. Cleavage occurs at aspartate- protein dipeptides which may be in a heterologous protein portion of the fusion protein or in a portion that is native to the Caulobacter S-layer portion. The dipeptide may be placed at a desired location for cleavage by engineering DNA encoding the fusion protein to express the dipeptide at the desired location. A preferable location for cleavage may be at or near the junction between a heterologous (target) protein and the Caulobacter S-layer portion comprising the Caulobacter secretion signal, such that a cleavage product will be the target protein in its entirety and substantially free of extraneous amino acids. The current invention makes it possible to cleave a heterologous (target) protein from the S-layer protein portion using only mild-acid conditions, even while the fusion protein is in an aggregated form. These cleavage conditions do not result in significant solubilization of the S-layer protein portion. This invention provides a method of cleaving a fusion protein including a first component which comprises all or part of a Caulobacter S-layer protein including a Caulobacter C-terminal secretion signal, and a second component heterologous to Caulobacter. The fusion protein contains at least one aspartate-proline dipeptide. The method comprises combining the fusion protein with an acid solution of a strength insufficient to solubilize the fusion protein for a time sufficient for cleavage of the fusion protein at the aspartate-proline dipeptide. The acid solution may have a pH of from about 1.5 (eg. 1.5 ± 0.1) to about 2.5 (eg. 2.5 ± 0.1), and preferably from about 1.65 (eg. 1.65 ± 0.05) to about 2.35 (eg. 2.35 ± 0.05). Preferred pH conditions may be achieved using an acid equivalent in the range of about 5 to about 20 mM HCL. The method is typically carried out at a temperature in the range of approximately room temperature to about 50°C. This invention also provides a method of preparing a DNA construct suitable for expression of a fusion protein suitable for use in the method of this invention. The method comprises joining an upstream DNA segment including DNA heterologous to Caulobacter which includes a protein of interest to a downstream DNA segment including DNA for a Caulobacter C-terminal secretion signal which does not encode an aspartate-proline dipeptide. The upstream segment contains DNA encoding an aspartate-proline dipeptide at or near the junction between said upstream and downstream segments . This invention also provides a method of preparing a fusion protein, comprising the steps of expressing a DNA construct as described above in Caulobacter and recovering said fusion protein once secreted by the Caulobacter. Once cleavage is accomplished according to this invention, the S-layer portion comprising the Caulobacter secretion signal may remain as an insoluble aggregate. If the target protein is soluble, the S-layer portion may be easily separated from the target recombinant protein by simple centrifugation or filtration methods. Thus the system of this invention facilitates separation as would a Tag/affinity matrix system except that here, the system is also the means for producing an insoluble matrix. In addition, the insoluble matrix produced by this invention is resistant to the effects of the acid treatment, allowing direct cleavage of the target recombinant protein. In this way, a very inexpensive chemical cleavage method can be employed to economically retrieve recombinant proteins from a bacterial fusion protein. In contrast to the cost of most affinity matrices, there is little expense associated with the use of the S-layer secretion signal as it is simply a part of the fermentation secretion process. DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION Production of Recombinant Fusion Proteins Using the Caulobacter S-layer Secretion System Proteins may be produced using the Caulobacter S-layer Type 1 secretion pathway which requires only the C-terminal secretion signal of the Caulobacter. This signal is the C-terminal portion of the S-layer protein, which typically comprises about 200 amino acids. (See: Bingle, et al. (1997) [supra]; and, WO 97/34000). Additional Caulobacter S-layer DNA upstream from the secretion signal may also be present and may be desirable to encode portions of the S-layer protein which will contribute to aggregate formation of the secreted protein. Such additional Caulobacter DNA may constitute most or all of the remainder of the DNA encoding the S-layer protein. Standard techniques (such as methods described in WO 97/34000) may be used to identify the amount of the C-terminal portion of a particular Caulobacter S-layer protein which functions as the secretion signal. Creation of fusion proteins is commonly done by preparing DNA which codes for the target protein and fusing it in-frame with the C-terminal region of the S-layer gene. There are numerous possible methods, with the following being examples. 1. Oligonucleotide Chemical Synthesis. This involves the design of complementary single strands, complete with desirable restriction endonuclease cut sites at the ends, chemical synthesis of the strands followed by annealing, cloning into a plasmid vector, juxtaposed to an appropriate portion of the C-terminal region of the S- layer gene. 2. Production of the Target Gene DNA by Polymer ase Chain Reaction (PCR) Amplification of a Target Sequence. In this case, appropriate in-frame restriction sites are incorporated into the short oligonucleotides used for amplification of a target sequence, such that the final PCR product can be treated with the appropriate restriction enzymes (to create the restriction site "sticky ends"), followed by cloning into a plasmid vector, juxtaposed to an appropriate portion of the C-terminal region of the S-layer gene. 3. Adapting Restriction Endonuclease Cleavage Sites that are Native to a Target Protein Gene Sequence for Fusion to the DNA Coding for the C-terminal S- layer Secretion Signal to Accomplish In-frame Expression of a Chimeric Protein. This can be accomplished by direct ligation (although it is uncommon that an appropriate match will occur), or the use of adapter sequences or methods involving blunting of a restriction site and subsequent blunt-end ligation to change expression reading frame or join unlike restriction site sticky ends. There will be numerous convenient sites for fusion with the C-teπninal regions of the S-layer that lead to the successful expression, secretion and aggregation of a recombinant fusion protein. Some example positions are at or near the DNA sites corresponding to amino acids 622, 690, 784, 892 and 907 of the C. crescentus S-layer gene (see: Appendix 1 and, WO 97/34000). Other sites of fusion with the S-layer gene may also be employed. Most often a plasmid vector is designed such that the C- terminal gene segment is resident on a plasmid with appropriate restriction sites placed at the N-teπninal junction of the S-layer fragment. Target recombinant protein gene segments are then cloned into those restriction sites. It is typical to prepare initial plasmid constructs that are replicated in E.coli. After a construct is produced, it is typically transferred to a broad host range plasmid which can then be introduced into the appropriate Caulobacter strain by electroporation. Suitable broad host range plasmids can be constructed from (but are not limited to) the IncQ, IncW and IncPl plasmid incompatibility groups. The introduction of the aspartate-proline (Asp-Pro) dipeptide at the appropriate site in the fusion protein can be done in several ways. Some examples are: (a) incorporating a DNA sequence necessary to express the Asp-Pro dipeptide into the oligonucleotides used to prepare the target sequence, either by oligonucleotide synthesis or PCR methods; (b) preparing a DNA segment with appropriate restriction sites at the termini so that an Asp-Pro dipeptide can be introduced (most often at the junction between S- layer and target gene) after a fusion recombinant S-layer gene has been made; and (c) use of a native Asp-Pro dipeptide in either the target DNA or the S-layer segment (for example, an Asp-Pro dipeptide is located at amino acids 692 and 693 of the C. crescentus S-layer gene and is suitable for fusions made at the amino acid site). The methods described above are not the only methods that may be used for creating and expressing fusion recombinant S-layer proteins, nor is it necessary to have the engineered genes resident on a plasmid. For example, the expressed gene may be introduced into the chromosome (using well-known gene insertion or replacement techniques) and still achieve secretion of the recombinant proteins (see WO 97/34000). In some cases it may be desirable to produce recombinant fusion proteins as insertions of heterologous DNA in the middle of the S-layer gene. In such a case, Asp-Pro dipeptide sequences could be engineered at the N and C-termini of the target peptide. All possible codon combinations for Asp-Pro will work but the CCA codon for proline is not preferred due to the likelihood of a low amount of the corresponding tRNA being present in Caulobacter. The following is an approximate usage table for C. crescentus. TABLE 1 Caulobacter crescentus Codon Usage Table [Amino Acid] [Triplet Code] [Frequency Per Thousand] Phe UUU 2.5 Ser UCU 1.2 Try UAU 6.6 Cys UGU 0.6 Phe UUC 27.0 Ser UCC 8.5 Try UAC 9.6 Cys UGC 5.5 Leu UUA 0.0 Ser CA 1.2 STOP UAA 0.8 Cys UGA 1.6 Leu UUG 4.4 Ser UCG 25.7 STOP UAG 0.6 STOP UGG 7.2 Leu CUU 4.4 Pro CCU 2.5 His CAU 3.2 Arg CGU 7.6 Leu CUC 15.7 Pro CCC 15.5 His CAC 12.2 Arg CGC 44.7 Leu CUA 1.1 Pro CCA 0.9 Gln CAA 3.7 Arg CGA 3.0 Leu CUG 72.3 Pro CCG 27.1 Gin CAG 30.2 Arg CGG 12.1 HeAUU 2.4 Thr ACU 1.2 Asn AAU 4.1 Ser AGU 0.8 lie AUC 49.0 Thr ACC 37.3 Asn AAC 23.8 Ser AGC 14.9 lie AUA 0.3 ThrACA 0.8 Lys AAA 2.7 Arg AGA 0.4 Met AUG 25.7 Thr ACG 16.8 Lys AAG 37.9 Arg AGG 1.1 Val GUU 5.4 Ala GCU 9.5 Asp GAU 11.1 Gly GGU 9.5 Val GUC 42.7 Ala GCC 84.1 Asp GAC 48.5 Gly GGC 64.8 Val GUA 1.0 Ala GCA 2.2 Glu GAA 20.5 Gly GGA 2.3 Val GUG 30.7 Ala GCG 36.7 Glu GAG 45.4 Gly GGG 7.7 Large quantities (eg. 12% of total cell protein/3% of input organic carbon) of a wide range of proteins can be produced, with yields in the order of 250 mg/liter of batch culture. Fusion proteins with 35 kDa of target peptide are secreted with little difficulty, although proteins with multiple cysteines may be more difficult to express. Post-expression glycosylation of proteins does not occur, an advantage for most peptide expression applications. Host Expression Strains For secretion of recombinant fusion S-layer proteins, the Caulobacter strain will preferably be one which has lost the ability to produce a native S-layer protein, while retaining a fully functional S-layer protein secretion apparatus. Such strains may be obtained by screening for mutants that have spontaneously become S-layer protein negative; or, by directed genetic manipulation, such as (but not limited to) the insertion of a drug resistance cassette in the middle of the S-layer gene or the substitution of a version of the S-layer gene which has had a sizeable internal region deleted from the gene (see: Bingle et al. 1997 1 [supra]; Bingle et al. 1997 2 "Cell Surface Display of a Pseudonomonas aerugenosa PAK Pilin Peptide with the Paracrystalline Layer of Caulobacter crescentus" Molec. Microbiol. 26:277-288; and, Edwards and Smit (1991) " A Transducing Bacteriophage for Caulobacter us Uses the Paracrystalline Surface Layer Protein as a Receptor" J. Bacteriol. 173: 5568-5572). In the case of a genetic manipulation, a common method for producing such strains is to modify a copy of the S-layer gene while on a plasmid and then to use well known gene replacement methods to substitute the modified gene for the native gene in the Caulobacter chromosome (see: Edwards and Smit (1991) [supra]). If an entire S-layer gene is to be used for production of a recombinant protein (via insertion of a target sequence), strains defective in the production of the lipopolysacharide (LPS) used for S-layer attachment to the bacterial surface can be used. These can be prepared by forcing Caulobacter to grow without exogenous calcium. Under these conditions mutants arise that are uniformly defective in producing a proficient version of the S-layer LPS (see: Walker, S.G. et al. (1994) "Characteristics of Mutants of Caulobacter crescentus Defective in Surface Attachment of the Paracrystaline Layer" J. Bacteriol. 176: 6312-6323). All Caulobacter S-layer producing strains are suitable for this technology. One may isolate the S-layer gene from a particular strain (using homology between Caulobacter S-layers to design probes to detect and clone the S-layer genes) and adapt the C-terminal region for recombinant protein expression, in a manner similar to that done for C. crescentus strains (see: MacRae and Smit (1991) [supra], and Walker, S.G. et al. (1992) [supra]). Alternatively, one may construct recombinant fusion S-layer genes using the C. crescentus S-layer gene and express the recombinant genes in alternate Caulobacter hosts. Freshwater Caulobacter producing S-layers may be readily detected by negative stain transmission electron microscopy techniques. Caulobacter may be isolated using the methods outlined by MacRae and Smit (1991) [supra], which lake advantage of the fact that Caulobacter can tolerate periods of starvation while other soil and water bacteria may not and that they all produce a distinctive stalk structure, visible by light microscopy (using either phase contrast or standard dye staining methods). Once Caulobacter strains are isolated in a typical procedure, colonies may be suspended in 2% ammonium molybdate negative stain and applied to plastic-filmed, carbon-stabilized 300 or 400 mesh copper or nickel grids and examined in a transmission electron microscope at 60 kilovolt accelerating voltage (see: Smit, J. (1986) "Protein Surface Layers of Bacteria", in Outer Membranes as Model Systems, (M. Inouge, ed. J.Wiley & Sons, at p. 343-376). S-layers are seen as two-dimensional geometric patterns most readily on those cells in a colony that have lysed and released their internal contents. Recombinant Protein Purification Secreted proteins are separated and shed into the culture media as a macroscopic precipitate (the "aggregate" referred to herein). The shedding phenomenon is a consequence of the absence of the N-terminal region of the S-layer protein in the expressed recombinant protein, or the loss of the lipopolysaccharide species used for S- layer attachment by the Caulobacter (see: Walker, S.G. et al. (1994) [supra]). Typically, the aggregate forms as loose, gel-like lumps of pure protein that can readily be retrieved and separated from the bacteria by simple filtration. The aggregate may be readily separated from a soluble cleaved target protein by any suitable techniques such as filtration of centrifugation. If the target protein is insoluble once cleaved, it may then be convenient to then solubilize one or both of the proteins (for example in 8M urea or 6M quanidine HCL) and separate by chromatography. In this way, only 2 species of protein need to be separated. Cleavage of Fusion Proteins General procedures for performing mild-acid cleavage are known from in the prior art as described above. In the method of this invention, conditions are adjusted to avoid destruction of the target protein or solubilization of the aggregate containing the S-layer secretion signal. Excess acid or too high a temperature may increase the occurrence over time of random cleavages along the length of the fusion protein, which is to be avoided since such random cleavages may lead to undersized fragmentation of the fusion protein or solubilization of the aggregated S-layer portion. Good yields of target protein with minimum random breaks in the fusion protein may generally be achieved by using from 5-20 mM HCL (or its equivalent while employing another acid). The respective pH of these conditions (unbuffered acid solution) is from about 2.3 to about 1.7. Time and temperature is preferably adjusted by routine monitoring to achieve the desired cleavage while minimizing random breaks. For example, temperature may range from room temperature to about 50° C. Time of treatment may range from about 12 to about 72 hours. Time or temperature outside of these ranges is permissible depending upon the strength of the acid and the accepted yield. Generally, lower yields are obtained with less acid strength, less time or lower temperatures. In the following examples, efficiency of cleavage in the order of 40-80% is achieved using conditions the same as or similar to the following alternatives: - 5 mM HCL at 50° C. for 48-72 hours - 20 mM HCL at 30° C. for 48-72 hours. Conditions in excess of the aforementioned values may be employed in some cases with the possibility of random breaks increasing, particularly with increased acid strength or temperature. In the following examples, significant random cleavage occurred with 50 mM HCL at 50° C. after 48 hours. Any acid may be employed in this invention which is normally used in solutions to which proteins are exposed. Acids which have a deleterious effect on proteins under dilute conditions should be avoided. For example, HCL or an equivalent amount of H 2 SO 4 may be used in this invention but oxidizing acids such as nitric acid may not be suitable. Example 1. Cleavage of artificial silk protein sequences from a secretion signal containing a native aspartate-proline cleavage site. An artificial protein sequence resembling spider silk was constructed by synthesis of partially overlapping and complementing oligomers of DNA, which were then completed to a full duplex DNA with Taql polymerase extension, to create a sequence that coded for 97 amino acids. The resulting DNA sequence and corresponding amino acid sequence are shown in Appendix 2. The DNA sequence shown in Appendix 2 was cloned into a gene carrier sequence residing in a pUC8 plasmid cloning vector. The gene segment carrier had BamHl restriction sites at each end and an internal Bglll site. This combination of restrictions sites allowed the production of multimers of the above sequence, relying on the fact that BamHl sticky ends will ligate into BgHI sticky end, with the loss of both restriction sites. Thus one copy of the silk-like sequence within the gene segment carrier can be put inside a second copy of the same to produce a dimer. Using this principle, an 8X repeat was produced, fused to DNA encoding the S-layer secretion signal corresponding to the C-terminal portion of the C. crescentus S-layer protein from about amino acid 690 onwards (see: Appendix 1). This fusion protein gene was introduced into strain CB2A on a broad host range plasmid vector. The 8x multimer appeared to be unstable, resulting in recombination events that reduced the 8X multimer to a 3x size. The 3 fold repeat of the above 97 amino acid sequence, fused to the S- layer secretion signal was secreted. Protein was collected and subjected to treatment with 5mM HCL for 2 days at 50° C. The result was the liberation of about 80% of soluble silk-like polymer which was readily separated by filtration from the S-layer protein which remained completely aggregated under these conditions. Cleavage occurred at native aspartate-proline dimer in the Caulobacter S-layer signal region (see: Appendix 1, amino acids numbered 692-693). Example 2. Cleavage of the salmonid virus Infectious Pancreatic Necrosis Virus (IPNV) surface glycoprotein candidate vaccine sequence from an S-layer secretion signal containing a native aspartate-proline site. The surface glycoprotein of the IPNV strain is a vaccine candidate. For this example and Example 4, the sequence of the first 257 amino acids of the mature protein and the corresponding DNA sequence as shown in Appendix 3 were used. DNA encoding a segment of the major surface glycoprotein gene of IPNV specifying amino acids 145-257 of the protein was fused to DNA sequence specifying two putative T-cell activating epitopes: MVF (SEQ ID No: l; LSEIKGVIVHRLEGV, derived from Measles Virus protein F) and P2 (SEQ ID No:2; QYIKANSKFIGITEL, derived from tetanus toxoid protein). The T-cell epitopes were positioned on the C- terminal end of the IPNV sequence. This chimeric protein was in turn fused in frame with the C -crescentus S-layer gene at about amino acid 690 position of the gene and introduced into Caulobacter on a broad host range plasmid vector. The resulting secreted protein was collected and treated with 5 mM HCL for 2 days at 50° C. Cleavage occurred at the native aspartate-proline dimer described in Example 1. The result was the liberation of about 75% of soluble vaccine candidate chimeric protein from the S-layer secretion signal which remained aggregated. Example 3. Cleavage of segments of an E. coli type I pilus tip subunit from an S-layer secretion signal containing a native aspartate-proline cleavage site. The FimH gene product is the tip pilus subunit of the E. coli strains involved with urinary tract infections. Two segments, T3 (specifying the first 145 amino acids of the mature peptide) and T7 (specifying the entire 258 amino acids of the mature peptide) were fused to the S-layer secretion signal at about amino acid 690 of the S-layer sequence. The T3 and T7 sequences are shown in Appendix 4. The fusion protein genes were introduced into strain CB2A on a broad host range plasmid vector. In both cases the resulting secreted protein was collected and treated with 5 mM HCL for 2 days at 50° C. In both cases, the result was the liberation of about 50% of soluble vaccine candidate chimeric protein from the S-layer secretion signal which remained aggregated. Cleavage occurred at the native aspartate-proline dimer described in Example 1. Example 4. Cleavage of the salmonid virus IPNV surface glycoprotein candidate vaccine sequence from an S-layer secretion signal containing an introduced aspartate-proline cleavage site. A segment of the major surface glycoprotein gene of IPNV specifying amino acids 1-257 of the protein shown in Appendix 4 was fused to a DNA sequence specifying a peptide containing an aspartate-proline dipeptide (SEQ ID No: 3; SPLGPAGDPEAS) such that the aspartate-proline dipeptide was positioned very near the C-terminus of the chimeric protein. This chimeric protein was in turn fused in frame with the C. crescentus S-layer gene at about amino acid 784 position of the gene and introduced in strain CB2A on a broad host range plasmid vector. The resulting secreted protein was collected and treated with 5 mM HCL for 2 days at 50° C. Cleavage occurred at the introduced aspartate-proline dipeptide. The result was the liberation of about 40% of insoluble vaccine candidate chimeric protein from the S- layer secretion signal which remained aggregated. Longer DNA and amino acid sequences referred to above are set out in the following Appendices which are part of this description. Appendix 1 sets out the complete nucleotide sequence of the C. crescentus S-layer gene (SEQ ID No: 4) with the upstream sequence including the -35 and -10 sites of the promoter region and the Shine Dalgarno sequence. The start codon is at nucleotide 101 and the coding sequence run to and includes nucleotide 3179. The amino acid sequence of the C. crescentus S- layer protein (SEQ ID No: 5) included in Appendix 1 is predicted from the DNA sequence. Appendix 2 sets out the artificial spider silk DNA sequence (SEQ ID No: 6) used in Example 1 and the corresponding amino acid sequence (SEQ ID No. 7). Appendix 3 sets out the DNA sequence (SEQ ID No: 8) and corresponding amino acid sequence (SEQ ID No: 9) of the first 257 amino acids of IPNV as described in Examples 2 and 4. Appendix 4 sets out the T3 protein sequence (SEQ ID No: 10) and the T7 protein sequence (SEQ ID No: 11) as described in Example 3. All publications, patents and patent applications referred to herein are hereby incorporated by reference. While this invention has been described according to particular embodiments and by reference to certain examples, it will be apparent to those of skill in the art that variations and modifications of the invention as described herein fall within the spirit and scope of the attached claims. Appendix 1 GCTATTGTCG ACGTATGACG TTTGCTCTAT AGCCATCGCT GCTCCCATGC GCGCCACTCG 60 GTCGCAGGGG GTGTGGGATT TTTTTTGGGA GACAATCCTC ATGGCCTATA CGACGGCCCA 120 GTTGGTGACT GCGTACACCA ACGCCAACCT CGGCAAGGCG CCTGACGCCG CCACCACGCT 180 GACGCTCGAC GCGTACGCGA CTCAAACCCA GACGGGCGGC CTCTCGGACG CCGCTGCGCT 240 GACCAACACC CTGAAGCTGG TCAACAGCAC GACGGCTGTT GCCATCCAGA CCTACCAGTT 300 CTTCACCGGC GTTGCCCCGT CGGCCGCTGG TCTGGACTTC CTGGTCGACT CGACCACCAA 360 CACCAACGAC CTGAACGACG CGTACTACTC GAAGTTCGCT CAGGAAAACC GCTTCATCAA 420 CTTCTCGATC AACCTGGCCA CGGGCGCCGG CGCCGGCGCG ACGGCTTTCG CCGCCGCCTA 480 CACGGGCGTT TCGTACGCCC AGACGGTCGC CACCGCCTAT GACAAGATCA TCGGCAACGC 540 CGTCGCGACC GCCGCTGGCG TCGACGTCGC GGCCGCCGTG GCTTTCCTGA GCCGCCAGGC 600 CAACATCGAC TACCTGACCG CCTTCGTGCG CGCCAACACG CCGTTCACGG CCGCTGCCGA 660 CATCGATCTG GCCGTCAAGG CCGCCCTGAT CGGCACCATC CTGAACGCCG CCACGGTGTC 720 GGGCATCGGT GGTTACGCGA CCGCCACGGC CGCGATGATC AACGACCTGT CGGACGGCGC 780 CCTGTCGACC GACAACGCGG CTGGCGTGAA CCTGTTCACC GCCTATCCGT CGTCGGGCGT 840 GTCGGGTTCG ACCCTCTCGC TGACCACCGG CACCGACACC CTGACGGGCA CCGCCAACAA 900 CGACACGTTC GTTGCGGGTG AAGTCGCCGG CGCTGCGACC CTGACCGTTG GCGACACCCT 960 GAGCGGCGGT GCTGGCACCG ACGTCCTGAA CTGGGTGCAA GCTGCTGCGG TTACGGCTCT 1020 GCCGACCGGC GTGACGATCT CGGGCATCGA AACGATGAAC GTGACGTCGG GCGCTGCGAT 1080 CACCCTGAAC ACGTCTTCGG GCGTGACGGG TCTGACCGCC CTGAACACCA ACACCAGCGG 1140 CGCGGCTCAA ACCGTCACCG CCGGCGCTGG CCAGAACCTG ACCGCCACGA CCGCCGCTCA 1200 AGCCGCGAAC AACGTCGCCG TCGACGGGCG CGCCAACGTC ACCGTCGCCT CGACGGGCGT 1260 GACCTCGGGC ACGACCACGG TCGGCGCCAA CTCGGCCGCT TCGGGCACCG TGTCGGTGAG 1320 CGTCGCGAAC TCGAGCACGA CCACCACGGG CGCTATCGCC GTGACCGGTG GTACGGCCGT 1380 GACCGTGGCT CAAACGGCCG GCAACGCCGT GAACACCACG TTGACGCAAG CCGACGTGAC 1440 CGTGACCGGT AACTCCAGCA CCACGGCCGT GACGGTCACC CAAACCGCCG CCGCCACCGC 1500 CGGCGCTACG GTCGCCGGTC GCGTCAACGG CGCTGTGACG ATCACCGACT CTGCCGCCGC 1560 CTCGGCCACG ACCGCCGGCA AGATCGCCAC GGTCACCCTG GGCAGCT CG GCGCCGCCAC 1620 GATCGACTCG AGCGCTCTGA CGACCGTCAA CCTGTCGGGC ACGGGCACCT CGCTCGGCAT 1680 Appendix 1 (cont'd) CGGCCGCGGC GCTCTGACCG CCACGCCGAC CGCCAACACC CTGACCCTGA ACGTCAATGG 1740 TCTGACGACG ACCGGCGCGA TCACGGACTC GGAAGCGGCT GCTGACGATG GTTTCACCAC 1800 CATCAACATC GCTGGTTCGA CCGCCTCTTC GACGATCGCC AGCCTGGTGG CCGCCGACGC 1860 GACGACCCTG AACATCTCGG GCGACGCTCG CGTCACGATC ACCTCGCACA CCGCTGCCGC 1920 CCTGACGGGC ATCACGGTGA CCAACAGCGT TGGTGCGACC CTCGGCGCCG AACTGGCGAC 1980 CGGTCTGGTC TTCACGGGCG GCGCTGGCCG TGACTCGATC CTGCTGGGCG CCACGACCAA 2040 GGCGATCGTC ATGGGCGCCG GCGACGACAC CGTCACCGTC AGCTCGGCGA CCCTGGGCGC 2100 TGGTGGTTCG GTCAACGGCG GCGACGGCAC CGACGTTCTG GTGGCCAACG TCAACGGTTC 2160 GTCGTTCAGC GCTGACCCGG CCTTCGGCGG CTTCGAAACC CTCCGCGTCG CTGGCGCGGC 2220 GGCTCAAGGC TCGCACAACG CCAACGGCTT CACGGCTCTG CAACTGGGCG CGACGGCGGG 2280 TGCGACGACC TTCACCAACG TTGCGGTGAA TGTCGGCCTG ACCGTTCTGG CGGCTCCGAC 2340 CGGTACGACG ACCGTGACCC TGGCCAACGC CACGGGCACC TCGGACGTGT TCAACCTGAC 2400 CCTGTCGTCC TCGGCCGCTC TGGCCGCTGG TACGGTTGCG CTGGCTGGCG TCGAGACGGT 2460 GAACATCGCC GCCACCGACA CCAACACGAC CGCTCACGTC GACACGCTGA CGCTGCAAGC 2520 CACCTCGGCC AAGTCGATCG TGGTGACGGG CAACGCCGGT CTGAACCTGA CCAACACCGG 2580 CAACACGGCT GTCACCAGCT TCGACGCCAG CGCCGTCACC GGCACGGCTC CGGCTGTGAC 2640 CTTCGTGTCG GCCAACACCA CGGTGGGTGA AGTCGTCACG ATCCGCGGCG GCGCTGGCGC 2700 CGACTCGCTG ACCGGTTCGG CCACCGCCAA TGACACCATC ATCGGTGGCG CTGGCGCTGA 2760 CACCCTGGTC TACACCGGCG GTACGGACAC CTTCACGGGT GGCACGGGCG CGGATATCTT 2820 CGATATCAAC GCTATCGGCA CCTCGACCGC TTTCGTGACG ATCACCGACG CCGCTGTCGG 2880 CGACAAGCTC GACCTCGTCG GCATCTCGAC GAACGGCGCT ATCGCTGACG GCGCCTTCGG 2940 CGCTGCGGTC ACCCTGGGCG CTGCTGCGAC CCTGGCTCAG TACCTGGACG CTGCTGCTGC 3000 CGGCGACGGC AGCGGCACCT CGGTTGCCAA GTGGTTCCAG TTCGGCGGCG ACACCTATGT 3060 CGTCGTTGAC AGCTCGGCTG GCGCGACCTT CGTCAGCGGC GCTGACGCGG TGATCAAGCT 3120 GACCGGTCTG GTCACGCTGA CCACCTCGGC CTTCGCCACC GAAGTCCTGA CGCTCGCCTA 3180 AGCGAACGTC TGATCCTCGC CTAGGCGAGG ATCGCTAGAC TAAGAGACCC CGTCTTCCGA 3240 AAGGGAGGCG GGGTCTTTCT TATGGGCGCT ACGCGCTGGC CGGCCTTGCC TAGTTCCGGT 3300 Appendix 1 (cont'd) Met Ala Tyr Thr Thr Ala Gin Leu Val Thr Ala Tyr Thr Asn Ala Asn 1 5 10 15 Leu Gly Lys Ala Pro Asp Ala Ala Thr Thr Leu Thr Leu Asp Ala Tyr 20 25 30 Ala Thr Gin Thr Gin Thr Gly Gly Leu Ser Asp Ala Ala Ala Leu Thr 35 40 45 Asn Thr Leu Lys Leu Val Asn Ser Thr Thr Ala Val Ala He Gin Thr 50 55 60 Tyr Gin Phe Phe Thr Gly Val Ala Pro Ser Ala Ala Gly Leu Asp Phe 65 70 75 80 Leu Val Asp Ser Thr Thr Asn Thr Asn Asp Leu Asn Asp Ala Tyr Tyr 85 90 95 Ser Lys Phe Ala Gin Glu Asn Arg Phe He Asn Phe Ser He Asn Leu 100 105 110 Ala Thr Gly Ala Gly Ala Gly Ala Thr Ala Phe Ala Ala Ala Tyr Thr 115 120 125 Gly Val Ser Tyr Ala Gin Thr Val Ala Thr Ala Tyr Asp Lys He He 130 135 140 Gly Asn Ala Val Ala Thr Ala Ala Gly Val Asp Val Ala Ala Ala Val 145 150 155 160 Ala Phe Leu Ser Arg Gin Ala Asn He Asp Tyr Leu Thr Ala Phe Val 165 170 175 Arg Ala Asn Thr Pro Phe Thr Ala Ala Ala Asp He Asp Leu Ala Val 180 185 190 Lys Ala Ala Leu He Gly Thr He Leu Asn Ala Ala Thr Val Ser Gly 195 200 205 He Gly Gly Tyr Ala Thr Ala Thr Ala Ala Met He Asn Asp Leu Ser 210 215 220 Asp Gly Ala Leu Ser Thr Asp Asn Ala Ala Gly Val Asn Leu Phe Thr 225 230 235 240 Ala Tyr Pro Ser Ser Gly Val Ser Gly Ser Thr Leu Ser Leu Thr Thr 245 250 255 Gly Thr Asp Thr Leu Thr Gly Thr Ala Asn Asn Asp Thr Phe Val Ala 260 265 270 Gly Glu Val Ala Gly Ala Ala Thr Leu Thr Val Gly Asp Thr Leu Ser 275 280 285 Gly Gly Ala Gly Thr Asp Val Leu Asn Trp Val Gin Ala Ala Ala Val 290 295 300 Thr Ala Leu Pro Thr Gly Val Thr He Ser Gly He Glu Thr Met Asn 305 310 315 320 Val Thr Ser Gly Ala Ala He Thr Leu Asn Thr Ser Ser Gly Val Thr 325 330 335 Gly Leu Thr Ala Leu Asn Thr Asn Thr Ser Gly Ala Ala Gin Thr Val 340 345 350 Appendix 1 (cont'd) Thr Ala Gly Ala Gly Gin Asn Leu Thr Ala Thr Thr Ala Ala Gin Ala 355 360 365 Ala Asn Asn Val Ala Val Asp Gly Arg Ala Asn Val Thr Val Ala Ser 370 375 380 Thr Gly Val Thr Ser Gly Thr Thr Thr Val Gly Ala Asn Ser Ala Ala 385 390 395 400 Ser Gly Thr Val Ser Val Ser Val Ala Asn Ser Ser Thr Thr Thr Thr 405 410 415 Gly Ala He Ala Val Thr Gly Gly Thr Ala Val Thr Val Ala Gin Thr 420 425 430 Ala Gly Asn Ala Val Asn Thr Thr Leu Thr Gin Ala Asp Val Thr Val 435 440 445 Thr Gly Asn Ser Ser Thr Thr Ala Val Thr Val Thr Gin Thr Ala Ala 450 455 460 Ala Thr Ala Gly Ala Thr Val Ala Gly Arg Val Asn Gly Ala Val Thr 465 470 475 480 He Thr Asp Ser Ala Ala Ala Ser Ala Thr Thr Ala Gly Lys He Ala 485 490 495 Thr Val Thr Leu Gly Ser Phe Gly Ala Ala Thr He Asp Ser Ser Ala 500 505 510 Leu Thr Thr Val Asn Leu Ser Gly Thr Gly Thr Ser Leu Gly He Gly 515 520 525 Arg Gly Ala Leu Thr Ala Thr Pro Thr Ala Asn Thr Leu Thr Leu Asn 530 535 540 Val Asn Gly Leu Thr Thr Thr Gly Ala He Thr Asp Ser Glu Ala Ala 545 550 555 560 Ala Asp Asp Gly Phe Thr Thr He Asn He Ala Gly Ser Thr Ala Ser 565 570 575 Ser Thr He Ala Ser Leu Val Ala Ala Asp Ala Thr Thr Leu Asn He 580 585 590 Ser Gly Asp Ala Arg Val Thr He Thr Ser His Thr Ala Ala Ala Leu 595 600 605 Thr Gly He Thr Val Thr Asn Ser Val Gly Ala Thr Leu Gly Ala Glu 610 615 620 Leu Ala Thr Gly Leu Val Phe Thr Gly Gly Ala Gly Arg Asp Ser He 625 630 635 640 Leu Leu Gly Ala Thr Thr Lys Ala He Val Met Gly Ala Gly Asp Asp 645 650 655 Thr Val Thr Val Ser Ser Ala Thr Leu Gly Ala Gly Gly Ser Val Asn 660 665 670 Gly Gly Asp Gly Thr Asp Val Leu Val Ala Asn Val Asn Gly Ser Ser 675 680 685 Phe Ser Ala Asp Pro Ala Phe Gly Gly Phe Glu Thr Leu Arg Val Ala 690 695 700 Appendix 1 (cont'd) Gly Ala Ala Ala Gin Gly Ser His Asn Ala Asn Gly Phe Thr Ala Leu 705 710 715 720 Gin Leu Gly Ala Thr Ala Gly Ala Thr Thr Phe Thr Asn Val Ala Val 725 730 735 Asn Val Gly Leu Thr Val Leu Ala Ala Pro Thr Gly Thr Thr Thr Val 740 745 750 Thr Leu Ala Asn Ala Thr Gly Thr Ser Asp Val Phe Asn Leu Thr Leu 755 760 765 Ser Ser Ser Ala Ala Leu Ala Ala Gly Thr Val Ala Leu Ala Gly Val 770 775 780 Glu Thr Val Asn He Ala Ala Thr Asp Thr Asn Thr Thr Ala His Val 785 790 795 800 Asp Thr Leu Thr Leu Gin Ala Thr Ser Ala Lys Ser He Val Val Thr 805 810 815 Gly Asn Ala Gly Leu Asn Leu Thr Asn Thr Gly Asn Thr Ala Val Thr 820 825 830 Ser Phe Asp Ala Ser Ala Val Thr Gly Thr Ala Pro Ala Val Thr Phe 835 840 845 Val Ser Ala Asn Thr Thr Val Gly Glu Val Val Thr He Arg Gly Gly 850 855 860 Ala Gly Ala Asp Ser Leu Thr Gly Ser Ala Thr Ala Asn Asp Thr He 865 870 875 880 He Gly Gly Ala Gly Ala Asp Thr Leu Val Tyr Thr Gly Gly Thr Asp 885 890 895 Thr Phe Thr Gly Gly Thr Gly Ala Asp He Phe Asp He Asn Ala He 900 905 910 Gly Thr Ser Thr Ala Phe Val Thr He Thr Asp Ala Ala Val Gly Asp 915 920 925 Lys Leu Asp Leu Val Gly He Ser Thr Asn Gly Ala He Ala Asp Gly 930 935 940 Ala Phe Gly Ala Ala Val Thr Leu Gly Ala Ala Ala Thr Leu Ala Gin 945 950 955 960 Tyr Leu Asp Ala Ala Ala Ala Gly Asp Gly Ser Gly Thr Ser Val Ala 965 970 975 Lys Trp Phe Gin Phe Gly Gly Asp Thr Tyr Val Val Val Asp Ser Ser 980 985 990 Ala Gly Ala Thr Phe Val Ser Gly Ala Asp Ala Val He Lys Leu Thr 995 1000 1005 Gly Leu Val Thr Leu Thr Thr Ser Ala Phe Ala Thr Glu Val Leu Thr 1010 1015 1020 Leu Ala 1025 Appendix 2 GAA TTC AGA TCT CAG GGC GCG GGG CAG GGT GGC TAT GGT GGG CTC GGC TCG CAA GGC GCT E F R S Q G A G Q G G Y G G L G S Q G A GGC CTG GGT GGC CAG GGC GCT GGC GCG GCC GCG GCC GCT GCG GCC GGT GGC G R G G Q G A G A A A A A A A G G GCT GGC CAG GGC GGG CTG GGC TCG CAG GGC GCC GGC CAA GGC GCT GGC GCC GCG GCC GCT A G Q G G L G S Q G A G Q G A G A A A A GCG GCC GGT GGC GCC GGC CAG GGT GGC TAC GGC GGC CTG GGC AGC CAG GGC GCC GGT CGC A A G G A G Q G G Y G G L G S Q G A G R GGC GGT CAG GGC GCC GGT GCC GCG GCC GCT GCG GCC GGT GGC GCT GGG CAA GGC GGC TAC G G Q G A G A A A A A A G G A G Q G G Y GGC GGT CTG GGA TCC G G L G S 1/1 Appendix 3 atg aac aca aac aag gca ace gca act tac ttg aaa tec att atg ctt cca gag act g a Met asn thr asn lys ala thr ala thr tyr leu lys ser ile met leu pro glu thr giy 61/21 cca gca age ate ccg gac gac ata acg gag aga cac ate tta aaa caa gag ace teg tea pro ala ser ile pro asp asp ile thr glu arg his ile leu lys gin glu thr ser ser 121/41 tac aac tta gag gtc tec gaa tea gga agt ggc att ctt gtt tgt ttc cct ggg gca cca tyr asn leu glu val ser glu ser gly ser gly ile leu val cys phe pro gly ala pro 181/61 ggc tea egg ate ggt gca cac tac aga tgg aat grg aac cag acg ggg ctg gag ttc gac gly ser arg ile gly ala his tyr arg trp asn ala asn gin thr gly leu glu phe asp 241/81 cag tgg ctg gag acg teg cag gac ctg aag aaa gcc ttc aac tac ggg agg ctg ate tea gin tφ leu glu thr ser gin asp leu lys lys ala phe asn tyr gly arg leu ile ser 301/101 agg aaa tac gac att caa age tec aca eta ccg gcc ggt etc tat get ctg aac ggg acg arg lys tyr asp ile gin ser ser thr leu pro ala gly leu tyr ala leu asn gly thr 361/121 etc aac get gcc ace ttc gaa ggc agt ctg tct gag gtg gag age ctg ace tac aat age leu asn ala ala thr phe glu gly ser leu ser glu val glu ser leu thr tyr asn ser 421/141 ctg atg tec eta act acg aac ccc cag gac aaa gcc aac aac cag ctg gtg ace aaa gga leu met ser leu thr thr asn pro gin asp lys ala asn asn gin leu val thr lys giy 481/161 gtc ace gtc ctg aat eta cca aca ggg ttc gac aaa cca tac gtc cgc eta gag gac gag val thr val leu asn leu pro thr gly phe asp lys pro tyr val arg leu glu asp glu 541/181 aca ccc cag ggt etc cag tea atg aac ggg gcc agg atg agg tgc aca get gca att gca thr pro gin gly leu gin ser met asn gly ala arg met arg cys thr ala ala ile 601/201 cca egg agg tac gag ate gac etc cca tec caa age eta ccc ccc gtt cct gcg aca g9a pro arg arg tyr glu ile asp leu pro ser gin ser leu pro pro val pro ala thr giy 661/221 ace etc ace act etc tac gag gga aac gcc gac ate gtc age tec aca aca gtg acg gga thr leu thr thr leu tyr glu gly asn ala asp ile val ser ser thr thr val thr giy 721/241 gac ata aac ttc agt ctg gca gaa cga ccc gca aac gag ace agg ttc gac ttc cag ctg asp ile asn phe ser leu ala glu arg pro ala asn glu thr arg phe asp phe gin leu Appendix 4 The T3 protein sequence is: FACKTANGTAIPIGGGSANVYVNLAPWNVGQNLWDLSTQIFCHNDYPETITDYVTLQRGSA SYPFPTTSETPRWYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQ CDVSA The T7 protein sequence is: FACKTANGTAIPIGGGSANVYVNLAPWNVGQNLWDLSTQIFCHNDYPETITDYVTLQRGSA SYPFPTTSETPRWYNSRTDKPWPVALYLTPVSSAGGVAIKAGSLIAVLILRQTNNYNSDDFQ CDVSARDVTVTLPDYRGSVPIPLTVYCAKSQNLGYYLSGTHADAGNSIFTNTASFSPAQGVG GAVGTSAVSLGLTANYARTGGQVTAGNVQSIIGVTFVYQ

Claims: WHAT IS CLAIMED IS: A method of cleaving a fusion protein including a first component which comprises all or part of a Caulobacter S-layer protein including a Caulobacter C-terminal secretion signal, and a second component heterologous to Caulobacter., the fusion protein containing at least one aspartate-proline dipeptide, wherein the method comprises combining the fusion protein with an acid solution of a strength insufficient to solubilize the fusion protein for a time sufficient for cleavage of the fusion protein at said aspartate-proline dipeptide. The method of claim 1 wherein a aspartate-proline dipeptide is situated between the first and second components or adjacent a junction between the first and second components. 3. The method of claim 1 or 2, wherein the acid solution has a pH of from about 1.5 to about 2.5. 4. The method of claim 1 or 2, wherein the acid solution has a pH of about 1.65 to about 2.35. 5. The method of any one of claims 1-4 wherein the method is carried out at a temperature in the range of about 30° C. to about 50° C. 6. The method of any one of claims 1-5, wherein the method further comprises separating products cleaved from the fusion protein. 7. A method of preparing a DNA construct for expression of a fusion protein suitable for use in the method of claim 1, wherein the method comprises joining an upstream DNA segment including DNA heterologous to Caulobacter which encodes a protein of interest, to a downstream DNA segment including DNA for a Caulobacter C- terminal secretion signal, wherein the downstream DNA segment does not encode an aspartate-proline dipeptide, and wherein the upstream segment contains DNA encoding an aspartate-proline dipeptide at or near an end of said upstream segment to be joined to said downstream segment. 8. A method of preparing a fusion protein, comprising: (1) expressing a DNA construct prepared as described in claim 7 in Caulobacter and, (2) recovering said fusion protein secreted by the Caulobacter.

Inventors: SMIT JOHN, SMIT, JOHN
Applicants: SMIT JOHN, UNIV BRITISH COLUMBIA, SMIT, JOHN, THE UNIVERSITY OF BRITISH COLUMBIA
Application year: 1999
Main classification: C12N15
IPC codes: C07K14, C07K19, C12N15, C12P21