CA2237704A1

CA2237704A1 - Cleavage of caulobacter produced recombinant fusion proteins

Info

Publication number: CA2237704A1
Application number: CA 2237704
Authority: CA
Inventors: John Smit
Original assignee: University of British Columbia
Current assignee: Individual
Priority date: 1998-07-14
Filing date: 1998-07-14
Publication date: 2000-01-14
Also published as: JP2002520060A; EP1097227A1; WO2000004170A1; AU4597399A

Abstract

This invention provides a method of cleaving target proteins from Caulobacter S-layer protein under mild acid conditions whereby a fusion protein secreted by the Caulobacter and comprising the target protein and at least a Caulobacter S-layer secretion signal may be cleaved at a 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 envelopes 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 extracelluar 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 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 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. 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 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] ) .
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 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 forming 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.
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 19971 "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. Some commonly used systems are:

1) Glutathione S-transferase (GST) tag, which binds to glutathione-sepharose matrices.
2) Maltose binding protein (MBP) tag, which binds to amylose matrices.
3) Multiple tandem histidine residues (e. g. "His-6") tag, which binds to Nickel-derivatized solid matrices.
4) 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.

_7_ 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 recombinant fusion protein is 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.
SiTi~IP~RY 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 without solubilization of the fusion protein being required. This cleavage may be accomplished when the fusion protein is present as the insoluble aggregate typically formed by Caulobacter S-layer protein.
Cleavage occurs at aspartate-protein dipeptides which may be in the heterologous protein portion of the fusion protein or native to the Caulobacter S-layer portion. The dipeptide may also be placed at a desired location for cleavage by engineering DNA encoding the fusion protein to express the dipeptide at the desired location. Typically, the desired location for cleavage will be at or near the junction of the heterologous (target) protein and the Caulobacter S-layer portion comprising the Caulobacter secretion signal such that a cleavage product will be the -g_ target protein in its entirety and preferably 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. In essence, the S-layer aggregation phenomenon functions like an affinity matrix.
This invention provides a method of cleaving a fusion protein consisting of a first component comprising all or part of a Caulobacter S-layer protein including a Caulobacter C-terminal secretion signal, and a second component comprising a heterologous polypeptide expressed and secreted from Caulobacter wherein the fusion protein comprises at least one aspartate-proline dipeptide, and wherein the method comprises the step of combining said 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. The acid solution may have a pH of from about 1.5 to about 2.5, preferably about 1.65 - 2.35.
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, comprising joining an upstream DNA segment comprising DNA
for a heterologous protein of interest and a downstream DNA
segment for a Caulobacter C-terminal secretion signal which does not encode an aspartate-proline dipeptide, wherein the upstream segment comprises a sequence encoding an _g_ aspartate-proline dipeptide at or near a 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 functions in a manner analogous to a Tag/affinity matrix system except that here, the affinity "tag" is the means of producing the insoluble matrix. In addition, this "matrix"
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 the matrix 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 gene sequence upstream from the secretion signal may also be present and is desirable to contribute to aggregate formation of the secreted protein. The additional Caulobacter sequence may constitute most or all of the remainder of the S-layer protein. Typically, the aggregate forms as loose, gel-like clumps of pure protein that can be readily retrieved and separated from the bacteria by simple filtration.

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.
The creation of fusion proteins is commonly done by preparing the target gene DNA and fusing it in-frame with the C-terminal region of the S-layer gene. There are numerous possible methods; some examples are:
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 Polymerase 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-terminal 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-terminal 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, Incur 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:

1. 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.
2. 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.
3. 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 (see: Appendix 1 and, WO 97/34000) and is suitable for fusions made at the amino acid site.
The methods described above are not the only methods of creating and expressing the fusion recombinant S-layer proteins, nor is it essential 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 would 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 a likely low amount of the corresponding tRNA in Caulobacter. The following is an approximate usage table for C. crescentus.

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 9 s UGC 5 Phe UUC 27.0 Ser UCC 8.5 Try UAC . y .
STOP UAA 0 Cys UGA 1.6 Leu UUA 0.0 Ser CA 1.2 STOP UAG . STOP UGG 7.2 r UCG 7 0.6 Leu UUG 4.4 e .

Leu CUU 4.4 Pro CCU 2.5 His CAU 3.2 Arg CGU 7.6 C 2 Arg CGC 44 Leu CUC 15.7 Pro CCC 15.5 His CA . Arg CGA .
0 Gln CAA 7 3.0 Leu CUA 1.1 Pro CCA . Gln CAG . Arg CGG 12.1 CCG 1 30.2 Leu CUG 72.3 ro .

IIeAUU 2.4 Thr ACU 1.2 Asn AAU 4.1 Ser AGU 0.8 Ile AUC 49.0 Thr ACC 37.3 Asn AAC 23.8 er .
8 s AAA 2.7 Arg AGA 0.4 Ile AUA 0.3 Thr ACA . y 37.9 Arg AGG 1.1 Thr ACG 16 Lys AAG

Met AUG 25.7 .

Val GUU 5.4 Ala GCU 9.5 Asp GAU 11.1 Gly GGU 9.5 5 Gl 8 Val GUC 42.7 Ala GCC 84.1 Asp GAC . y .
GAA 5 Gly GGA 2.3 Gl 20 Val GUA 1.0 Ala GCA 2.2 u . Gly GGG 7.7 GCG 7 Glu GAG 45.4 Val GUG 30.7 Ala .

Large quantities (eg. 12% of total cell protein/3o 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 have lost the ability to produce a native S-layer protein, while retaining a fully functional S-layer protein secretion apparatus. This may be done 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 sizable internal region deleted from the gene (see: Bingle et al 19971 [supra] ; Bingle et al 19972 "Cell Surface Display of a Pseudonomonas aeruaenosa 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 do appropriate modification of 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]).
In the rare case that an entire S-layer gene is 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 either isolate the S-layer gene from a 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 use recombinant fusion S-layer genes produced with C. crescentus S-layer gene and express them 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 take 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 are 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 described above). 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]).
The loose gel like clumps of 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 solubilize one or both of the proteins (for example in 8M urea or 6M guanidine HCL) and separate by chromatography. In this way, only 2 species of protein need to be separated.
Cleavage of Fusion Proteins Conditions for cleavage at aspartate - proline sites are described in Current Protocols in Molecular Bioloay (supp.
28; chapter 16.4) John Wiley & Sons Inc. 1994, and in Landon, M. "Cleavage at Aspartyl - Prolyl Bonds" in Methods in Enzymoloay (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. The 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.
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 1.69. Time and temperature is preferably adjusted 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 12-72 hours. Time or temperature outside of these ranges is permissable 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 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 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 HZS04 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 is 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 BgIII 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 BgIII 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 is shown in Appendix 3.
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 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 5 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.
Lengthy DNA and amino acid sequences referred to in this specification are set out in the following appendixes.
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.

Claims

1. A method of cleaving a fusion protein consisting of a first component comprising all or part of a Caulobacter S-layer protein including a Caulobacter C-terminal secretion signal, and a second component comprising a heterologous polypeptide expressed and secreted from Caulobacter wherein the fusion protein comprises at least one aspartate-proline dipeptide, and wherein the method comprises the step of combining said 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.

2. The method of claim 1 wherein the at least one 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 wherein the acid solution has a pH of from about 1.5 to about 2.5.

4. The method of claim 1 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 comprising the additional step of 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, comprising joining an upstream DNA segment comprising DNA encoding a heterologous protein of interest and a downstream DNA segment for a Caulobacter C-terminal secretion signal which does not encode an aspartate-proline dipeptide, wherein the upstream segment comprises a sequence 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 the steps of expressing a DNA construct prepared as described in claim 7 in Caulobacter and, recovering said fusion protein once secreted by the Caulobacter.