# Patent

## Title
Cysteine protease autoprocessing of fusion proteins

## Abstract

Disclosed are fusion proteins, polynucleotides that encode the disclosed fusion proteins, and methods for expressing and autoprocessing of the disclosed fusion proteins to obtain a target protein. The disclosed fusion proteins include an autoproteolytic cysteine protease fused to a heterologous polypeptide, which may be isolated as the target protein. Preferably, the protease activity of the cysteine protease is inducible. Suitable autoproteolytic cysteine proteases for the fusion proteins include the cysteine protease of the Vibrio cholerae RTX toxin.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a continuation of U.S. application Ser. No. 12/630,603, filed on Dec. 3, 2009, which application was published on Jun. 3, 2010, as US2010/0137563, and claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Number 61/119,489, filed on Dec. 3, 2008, the contents of which are incorporated herein by reference in their entireties.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with U.S. Government Support from the following agency: NIH/NIAID, Grant No. R01AI051490. The U.S. Government has certain rights in the invention.


BACKGROUND
Purification of recombinant proteins from Escherichia coli and other model organisms is an essential component of biochemical and structural biology research. Over the past few decades, numerous strategies have been developed to allow easy purification of recombinant proteins by addition of a fusion protein or protein tag that allows rapid affinity purification. For some proteins, the fusion or tag poses a problem as the additional peptide sequences can interfere with function of the protein in biochemical assays or prevent the protein from forming compact crystals. If this occurs, the tag must often be removed after purification. This process of tag removal can be a cumbersome process.
Recently, a cysteine protease domain (CPD) embedded in a large protein toxin of Vibrio cholerae and other bacterial organisms was discovered that is inactive until induced by addition of the chemical compound inositol hexakisphosphate, also known as inositol-6-phosphate, InsP6, IP6, or phytic acid. Here, it is shown that a target protein can be purified as part of a fusion protein that includes the target protein fused to the CPD and a C-terminal peptide tag of 6-histidine residues. After purification of the fusion protein, the inducer molecule InsP6 is added and the protease and the peptide tag are removed from the fusion protein via autoproteolysis. The remaining portion of the fusion protein includes the target protein and an additional alanine and leucine residue added to the C-terminus of the target protein.
These disclosed strategies for purification of recombinant proteins followed by removal of a peptide tag may be adapted into any available cloning or purification systems. The molecule InsP6 is not produced by bacteria, hence, this strategy may be desirable for any recombinant protein produced in E. coli or other bacterial expression systems. This strategy also is desirable for any recombinant protein produced in plant, fungal, insect, or animal host cell expression systems in which the host cell is modified to block synthesis of InsP6.
SUMMARY
Disclosed are fusion proteins, polynucleotides that encode the disclosed fusion proteins, and methods for expressing and autoprocessing of the disclosed fusion proteins to obtain a target protein. The disclosed fusion proteins include an autoproteolytic cysteine protease fused to a heterologous polypeptide, which may be isolated as the target protein. In some embodiments, the protease activity of the cysteine protease is inducible. Suitable autoproteolytic cysteine proteases for the fusion proteins include the cysteine protease of the Vibrio cholerae RTX toxin. Examples of heterologous polypeptides may include target proteins, including but not limited to, industrial enzymes (process enzymes), enzymes targeted for use in consumer products, or proteins with pharmaceutical activity.
The fusion protein may include: (a) a first polypeptide; and (b) a second polypeptide fused to the C-terminus of the first peptide, where the first polypeptide is heterologous with respect to the second polypeptide and the second polypeptide has an amino acid sequence that includes the cysteine protease domain of the Vibrio cholerae RTX toxin or a cysteine proteases domain from a conserved or related toxin as contemplated herein. In some embodiments, the second polypeptide has an amino acid sequence that is at least 95% identical to any of SEQ ID NOs:1-17 (or at least 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs:1-17), and the second polypeptide has cysteine protease activity when induced by inositol hexakisphosphate such that the second polypeptide is autocleaved (e.g., between amino acids 2 and 3 of the second polypeptide).
The fusion protein further may include a peptide tag fused at the C-terminus of the second polypeptide. Suitable peptide tags may include, but are not limited to a 6×His tag, a hemaglutinin tag, a FLAG tag, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose binding protein tag, a chitin binding protein tag, or another functional sequence of amino acids.
In the fusion protein, the first polypeptide and the second polypeptide may be directly fused. Alternatively, the first polypeptide and second polypeptide may be fused indirectly via a peptide linker (e.g., a linker which optionally is flexible and which optionally has one or more glycine or serine residues or any other compatible amino acid sequence).
Also disclosed are polynucleotides coding for the disclosed fusion proteins. For example, contemplated polynucleotides may include DNA or RNA molecules. The disclosed polynucleotides may be recombinant and may include one or more heterologous polynucleotide sequences fused to the polynucleotide sequence coding for the fusion protein. In some embodiments, contemplated recombinant polynucleotides include a promoter sequence operably linked to a polynucleotide coding for the disclosed fusion protein.
Also disclosed are vectors that include the recombinant polynucleotides. The vectors further may include selectable markers and may be utilized to transform a host cell (e.g., an isolated bacterial, plant, fungal, insect, or animal cell).
Also disclosed are methods for producing the disclosed fusion proteins. The methods may include: (a) culturing or fermenting a cell under conditions suitable for expression of the fusion protein, where the cell is transformed with a recombinant polynucleotide, and the recombinant polynucleotide includes a promoter sequence operably linked to a polynucleotide encoding the fusion protein; and (b) recovering the fusion protein so expressed. The fusion protein further may include a peptide tag as contemplated herein and the method further may include contacting the peptide tag with a molecule or resin that binds the peptide tag (e.g., in order to activate, isolate, separate, or purify the fusion protein). In some embodiments, the method further may include: (c) contacting the recovered fusion protein and inositol hexakisphosphate, thereby inducing cleavage of the fusion protein within the second polypeptide to provide: (i) a cleaved fragment of the fusion protein including the first polypeptide; and (ii) a cleaved fragment of the fusion protein including at least a portion of the second polypeptide. In some embodiments, the recovered fusion protein and the inositol hexakisphosphate are contacted in a reaction mixture comprising a protease inhibitor (e.g., a protease inhibitor that inhibits non-specific protease activity in the reaction mixture). Suitable proteases may include but are not limited to chloromethyl ketones and N-ethylmaleimide. In further embodiments, the method may include: (d) separating the cleaved fragment of the fusion protein that includes the first polypeptide and the cleaved fragment of the fusion protein that includes at least a portion of the second polypeptide. For example, where the fusion protein includes a peptide tag fused at the C-terminus of the second polypeptide, the peptide tag may be contacted with a molecule or resin that binds the peptide tag to remove the cleaved fragment of the fusion protein that includes at least a portion of the second polypeptide.
Also disclosed are kits for preparing and using the fusion proteins contemplated herein. In some embodiments, the kit includes: (1) an expression vector as contemplated herein for expressing the fusion protein; optionally (2) reagents for activating, isolating, separating, or purifying the fusion protein or cleavage products thereof (e.g., resins or columns that bind to the peptide tag present on the fusion protein); and optionally (3) an inducer compound (e.g., inositol hexakisphosphate).



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Model for mechanism of MARTX-Vc-mediated cell rounding.
FIG. 2. HEp-2 cells transfected with CPD fused to EGFP (CPDc) do not have full-length CPD:EGFP as detected by Western blotting with anti-EGFP antibody. Mutagenesis of C3568 (CPDc C-S) or H3519 (CPDc H-A) resulted in proteins at the expected mol. wt.
FIG. 3. Purified recombinant CPD (rCPD) or the C3568S mutant (rCPD C-S) were incubated at 37° C. for 2 hr in the (+)presence or (−)absence of a nuclear-free cell lysate. Arrows indicated full-length (FL) and processed (P) forms of rCPD.
FIG. 4. Demonstrates autoprocessing of rCPD at its N-terminus after addition of 100 μM InsP6.
FIG. 5. Production of recombinant protein as part of a fusion protein and subsequent cleavage at CPD.
FIG. 6. Diagram of overexpression plasmid created to generate a fusion of RhoA to CPD:6×His.
FIG. 7. Clarified lysate (CL) was loaded onto nickel column and Flow through (FT) and wash (W) fractions were collected. Protein was eluted in 250 mM imidazole and Rho was cleaved from CPD:6×His by addition of Inositol 6 phosphate (IP6).



DETAILED DESCRIPTION
The subject matter disclosed herein is described using several definitions and description, as set forth below and throughout the application.
Unless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art. In addition to the definitions of terms provided below, it is to be understood that as used in the specification, embodiments, and in the claims, “a”, “an”, and “the” can mean one or more, depending upon the context in which it is used.
As used herein, “about,” “approximately,” “substantially,” and “significantly” are terms that will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which the term is used or that are not clear in the context of the present disclosure, “about” or “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.
Disclosed herein are fusion proteins that include an autoproteolytic cysteine protease fused to a heterologous polypeptide. As used herein, a “heterologous polypeptide” is a polypeptide that is not naturally cleaved by the autoproteolytic cysteine protease (e.g., a non-Vibrio cholerae RTX polypeptide). Preferably, the enzyme activity of the autoproteolytic cysteine protease is inducible. Suitable autoproteolytic cysteine proteases for the fusion proteins include the cysteine protease domain (CPD) of the Vibrio cholerae RTX toxin or cysteine protease domains from conserved or related toxins (e.g., the conserved toxin in large clostridial glucosylating toxins TcdB, TcdA, TcnA, and TcsL; putative toxins from V. vulnificus, Yersinia sp., Photorhabdus sp., and Xenorhabdus sp.; and a filamentous/hemagglutinin-like protein FhaL from Bordetella sp.). Examples of heterologous polypeptides may include, target proteins, including but not limited to, industrial enzymes (process enzymes), enzymes targeted for use in consumer products, or proteins with pharmaceutical activity. Fusion proteins and methods of making and using fusion proteins are disclosed in U.S. Pat. Nos. 7,378,512; 7,276,355; 7,176,287; and 7,045,677; the contents of which are incorporated herein by reference in their entireties.
Typically, the protease of the disclosed fusion proteins is fused in frame to the C-terminus of the heterologous polypeptide. The heterologous protein and the protease may be directly fused or indirectly fused via a linking peptide. For example, a linking peptide may comprise at least 1, 2, 3, 4, 5, 10, 15, or 20 amino acids. Suitable linkers may comprise any sequence of amino acids. Preferred linkers have neutral structural properties. For example, a linker preferably has a neutral pH and comprises relatively small-sized amino acids (e.g., glycine and serine). A preferred linker may comprise the sequence (GGGGS) (SEQ ID NO:18) or 1, 2, or 3 tandem repeats thereof.
In some embodiments, the enzyme activity of the autoproteolytic cysteine protease of the fusion protein is inducible (e.g., where the proteolytic activity of the protease is induced by contacting the protease with a chemical reagent such as inositol hexakisphosphate in a processing reaction). For example, the fusion protein may be expressed and subsequently the autoproteolytic activity of the protease may be induced in a processing reaction mixture such that the protease cleaves itself. The portion of the fusion protein comprising the heterologous polypeptide may be separated from the other cleaved portion of the fusion protein (i.e., the portion comprising the protease or the majority of the protease). Preferably, the fusion protein further comprises a peptide tag at the C-terminus of the protease, which may be utilized to isolate, separate, or purify the fusion protein or to isolate, separate, or purify the C-terminal portion of the fusion protein. In some embodiments, non-specific protease activity of the fusion protein may be inhibited by including in the processing reaction a protease inhibitor, including but not limited to chloromethyl ketones and N-ethylmaleimide. As contemplated herein, “non-specific protease activity” means cleavage by the autoprotease at a position other than between amino acids 2 and 3 of the autoprotease (or cleavage by another protease in the processing reaction mixture a position other than between amino acids 2 and 3 of the autoprotease). Preferably, the protease inhibitor does not inhibit or does not substantially inhibit specific protease activity of the autoprotease (i.e., cleavage by the autoprotease at the position between amino acids 2 and 3 of the autoprotease). As contemplated herein, a protease inhibitor that does not substantially inhibit the specific protease activity of the autoprotease is a protease inhibitor that does not inhibit the specific protease activity of the autoprotease by more than 50% (preferably that does not inhibit the specific protease activity of the autoprotease by more than 40%, 30%, 20%, or 10%).
Also disclosed are nucleic acid molecules that encode the disclosed fusion proteins. For example, contemplated are nucleic add molecules (e.g., DNA or RNA) which code for a fusion protein in which the fusion protein comprises a first polypeptide fused at its C-terminus to a second polypeptide as disclosed herein.
A preferred nucleic acid molecule is one that encodes the fusion protein contemplated herein in which the encoded second polypeptide of the fusion protein comprises the amino acid sequence of any of SEQ ID NOs:1-17 or the amino acid sequence of a variant, mutant, or derivative thereof with autoproteolytic cysteine protease activity. Variants or derivatives with autoproteolytic cysteine protease activity may include variants or derivative having one or more amino acid substitutions, deletions, additions and/or amino acid insertions, provided that autoproteolytic activity is retained.
The autoproteolytic cysteine protease activity of the disclosed fusion protein or polypeptides can be assayed by methods shown herein or by methods known in the art (e.g., by an in vitro system). For example, a DNA construct encoding a fusion protein or polypeptide may be transcribed into RNA and translated into protein with the aid of an in vitro translation kit. The resulting protein may be labeled by incorporating a detectable amino acid (e.g., a radioactive amino acid). Protease activity may be induced by adding inositol hexakisphosphate to the protein. If a fusion protein or polypeptide exhibits autoproteolysis, the resulting cleavage products can be detected using methods known in the art. For example, the protein can be loaded onto a protein gel (for example SDS-PAGE) and subjected to electrophoresis. The gel may be subsequently stained with suitable dyes or subjected to autoradiography. Alternatively, a Western blot and immunostaining may be performed. Cleavage of the protein can be assessed on the basis of the intensity of the resulting protein bands.
Also contemplated are bacterial, plant, fungal, insect, or animal host cell expression vectors that express the disclosed fusion proteins. Vectors may be used to transform appropriate host cells (e.g., E. coli). The transformed host cell may be cultivated or fermented such that the fusion protein is expressed constitutively or after adding a reagent that induces expression (e.g., via an inducible promoter). The fusion protein may exhibit autoproteolysis after expression. To assess the efficiency of autoproteolytic cleavage, a sample comprising the fusion protein may be taken after the end of the cultivation or induction phase and analyzed by SDS-PAGE or other methods.
Expression vectors as contemplated herein may include control sequences that modulate expression of the fusion protein. Expression control sequences may include constitutive or inducible promoters (e.g., T3, T7, Lac, trp, or phoA), ribosome binding sites, or transcription terminators.
The vectors disclosed herein may be utilized to transform host cells. Suitable host cells include bacterial, plant, fungal, insect, or animal host cell. Suitable bacteria include, but are not limited to: Gram-negative bacteria such as Escherichia species (e.g., E. coli), other Gram-negative bacteria, (e.g., Pseudomonas sp., such as Pseudomonas aeruginosa, or Caulobacter sp., such as Caulobacter crescentus), or Gram-positive bacteria (e.g., Bacillus sp., in particular Bacillus subtilis).
Also disclosed are methods for expressing, preparing, isolating, separating, or purifying fusion protein or polypeptides. In some embodiments, the methods may be utilized to produce the heterologous polypeptide of the fusion protein as disclosed herein. The steps of the methods may include: (i) cultivating or fermenting a transformed host cell (e.g., a bacterial host cell as contemplated herein) which comprises an expression vector (as contemplated herein) which in turn comprises a nucleic acid molecule encoding a fusion protein (as contemplated herein), wherein cultivation occurs under conditions which cause expression of the fusion protein and further autoproteolytic cleavage of the fusion protein; and (ii) isolating, separating, or purifying the cleaved heterologous polypeptide portion of the fusion protein. The transformed bacteria may be cultivated or fermented using methods known in the art in order to express the fusion protein. The cleaved heterologous polypeptide portion of the fusion protein may be isolated, separated, or purified by methods known in the art (see, e.g., M. P. Deutscher, in: Methods in Enzymology: Guide to Protein Purification, Academic Press Inc., (1990), 309-392). An exemplary isolation, separation, or purification method may include one or more of the following steps: a cell disruption step, a clarification step (e.g., via centrifugation or filtration), a chromatographic separation step, a dialysis step, and a precipitation step.
The terms “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand). These phrases also refer to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
The terms “amino acid” and “amino acid sequence” refer to an oligopeptide, peptide, polypeptide, or protein sequence (which terms may be used interchangeably), or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
The amino acid sequences contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, or derivative polypeptide may include conservative amino acid substitutions relative to a reference polypeptide. “Conservative amino acid substitutions” are those substitutions that are predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference protein. Table 1 provides a list of exemplary conservative amino acid substitutions.









TABLE 1






Original




Residue
Conservative Substitution








Ala
Gly, Ser



Arg
His, Lys



Asn
Asp, Gln, His



Asp
Asn, Glu



Cys
Ala, Ser



Gln
Asn, Glu, His



Glu
Asp, Gln, His



Gly
Ala



His
Asn, Arg, Gln, Glu



Ile
Leu, Val



Leu
Ile, Val



Lys
Arg, Gln, Glu



Met
Leu, Ile



Phe
His, Met, Leu, Trp, Tyr



Ser
Cys, Thr



Thr
Ser, Val



Trp
Phe, Tyr



Tyr
His, Phe, Trp



Val
Ile, Leu, Thr









Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.

A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation of a reference polypeptide or a 5′-terminal or 3′-terminal truncation of a reference polynucleotide).
A “fragment” is a portion of an amino acid sequence or a polynucleotide which is identical in sequence to but shorter in length than a reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide.
A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.
“Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences. Homology, sequence similarity, and percentage sequence identity may be determined using methods in the art and described herein.
The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below).
Percent identity may be measured over the length of an entire defined polynucleotide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). Such a pair of nucleic acids may show, for example, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases “percent identity” and “% identity,” as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.
Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
A “variant,” “mutant,” or “derivative” of a particular polypeptide sequence is defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). Such a pair of polypeptides may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides. A “variant” or a “derivative” may have substantially the same functional activity as a reference polypeptide. For example, a variant or derivative of a cysteine protease may have cysteine protease activity (e.g., autoproteolytic cysteine protease activity).
The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides.
“Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1 3, Cold Spring Harbor Press, Plainview N.Y. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
“Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein. The term “substantially isolated or purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.
“Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A “composition comprising a given amino acid sequence” and a “composition comprising a given polynucleotide sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution. The compositions may be stored in any suitable form including, but not limited to, freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. The compositions may be aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, and the like).
The contemplated fusion proteins may include at least a fragment of an amino acid sequence of a proven or putative cysteine protease. (See Sheahan K.L. et al., “Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain,” EMBO J. 2007 May 16;26(10):2552-61, at page 2553, FIG. 1 and supplemental Table 1, the content of which is incorporated herein by reference in its entirety). Proven and putative cysteine protease domains for incorporation into the fusion proteins contemplated herein include, but are not limited to Vibrio-type RTX toxins such as toxins from V. cholerae (VcRtx) (SEQ ID NO:1); V. vulnificus (VvRtx) (SEQ ID NO:2), V. splendidus (VsRtx) (SEQ ID NO:3), Xenorhabdus nematophila (XnRtx) (SEQ ID NO:8), X bovienii (XbRtx) (SEQ ID NO:9), and Photorhabdus luminescens (P1u1344 (SEQ ID NO:4), P1u1341 (SEQ ID NO:5), Plu3217 (SEQ ID NO:6), and Plu3324 (SEQ ID NO:7)); clostridial-type toxins such as Clostridium difficile toxin A (TcdA) (SEQ ID NO:12), toxin B (TcdB) (SEQ ID NO:13), C. sordellii cytotoxin L (TcsL) (SEQ ID NO:15) and C. noveyi alpha toxin (TcnA) (SEQ ID NO:14); putative Yersinia toxins Y. pseudotuberculosis YPTB3219 (YpRtx) (SEQ ID NO:10) and Y. mollaretti Mfp2 (YmMfp2) (SEQ ID NO:11); and four domains arranged in tandem in B. pertussis putative adhesin FhaL (FhaL1-4) (SEQ ID NO:16). Contemplated fusion proteins may include at least a fragment of the proven and putative autoproteases disclosed in Table 2, in particular the listed amino acid fragment.











TABLE 2







Gen Bank






Accession

Amino Acid


Protein
Abbreviation
number
SEQ ID NO:
Fragment







Group 1: Vibrio-type RTX toxins






V. cholerae RTX toxin
VcRtx
gi | 4455065
1
3420-3619


V. vulnificus RTX toxin
VvRtx
gi | 37676690
2
4110-4288


V. splendidus putative RTX toxin
VsRtx
gi | 84386478
3
3751-3975


P. luminescens putative RTX toxins
Plu1341
gi | 37525303
4
2579-2764



Plu1344
gi | 36784731
5
2965-3163



Plu3217
gi | 36786533
6
2425-2620



Plu3324
gi | 37686635
7
2440-2626


Group 2: Putative toxins from Yersinia






Y. pseudotuberculosis putative toxin
YpRtx
gi | 51590811
10
1058-1271


Y. mollaretti putative toxin
YmMfp2
gi | 77962640
11
 1-224


Group 3: Clostridial glucosylating toxins






C. difficile Toxin A
TcdA
gi | 98593
12
535-769


C. difficile Toxin B
TcdB
gi | 761714
13
536-768


C. noveyi alpha toxin
TcnA
gi | 755724
14
532-813


C. sordellii cytotoxin L
TcsL
gi | 1000695
15
526-825


Group 4. Type V secreted adhesin






Bordetella pertussis putative adhesin
cpd1
gi | 33563918
16
2551-2716


FhaL
cpd2
gi | 33563918
16
3079-3119



cdp3
gi | 33563918
16
3375-3971



cpd4
gi | 33563918
16
3397-3562









In some embodiments, the disclosed fusion proteins may include at least a fragment of the amino acid sequence of the MARTX toxin of V. cholerae as the second polypeptide of the fusion protein. (See, e.g., Satchell, K. J., “MARTX, Multifunctional Autoprocessing Repeats-in-Toxin Toxins,” Infection and Immunity, November 2007, p. 5079-5084; Sheahan K. L. et al., “Autoprocessing of the Vibrio cholerase RTX toxin by the cysteine protease domain,” EMBO J. 2007 May 16; 26(10):2552-61; Prochazkova K. et al., “Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin,” J. Biol. Chem. 2008 Aug. 29; 283(35); Prochazkova et al., “Structural and molecular mechanism for autoprocessing of MARTX toxin of Vibrio Cholerae at multiple sites,” J. Biol. Chem. 2009 2009 Sep. 25; 284(39):26557-68 Epub 2009 Jul. 20; the contents of which are incorporated by reference in their entireties). The entire MARTX toxin of V. cholerae is 4545 amino acids. (See Table 2 above where the MARTX toxin is referred to as “VcRtx,” and GenBank Accession No. gi|4455065, the content of which is incorporated herein by reference in its entirety). In some embodiments, the disclosed fusion proteins comprise amino acid sequence 3376-3637 of MARTX (e.g., as the contemplated protease domain or C-terminal second polypeptide of the presently disclosed fusion proteins), or a variant, mutant, or derivative thereof having cysteine protease activity. In other embodiments, the disclosed fusion proteins comprise amino acid sequence 3427-3637 of MARTX (SEQ ID NO:17) (e.g., as the contemplated protease domain or C-terminal second polypeptide of the presently disclosed fusion proteins), or a variant, mutant, or derivative thereof having cysteine protease activity. In other embodiments, the disclosed fusion proteins comprise amino acid sequence 3432-3637 of MARTX (e.g., as the contemplated protease domain or C-terminal second polypeptide of the presently disclosed fusion proteins), or a variant, mutant, or derivative thereof having cysteine protease activity. In even further embodiments, the disclosed fusion proteins comprise amino acid sequence 3441-3637 of MARTX (e.g., as the contemplated protease domain or C-terminal second polypeptide of the presently disclosed fusion proteins), or a variant; mutant, or derivative thereof having cysteine protease activity.
EXAMPLES
The following examples are illustrative and are not intended to limit the disclosed subject matter.
Example 1
Background
Vibrio cholerae MARTX toxin. The life-threatening diarrheal disease cholera is caused by toxigenic strains of the Gram-negative organism Vibrio cholerae. In addition to the well-characterized ADP-ribosylating cholera toxin (CT), V. cholerae secretes a novel cytotoxin that is the founding member of a new family of the RTX (repeats-in-toxin) family (Satchell, 2007). This toxin of V. cholerae contributes to virulence in mice and has been shown to be important for the bacterium to establish prolonged colonization of the small intestine (Olivier et al., 2007b; Olivier et al., 2007a). Hence, it has been proposed that this toxin is important for initiation of disease in cholera patients and is likely an important factor for prolonged colonization in asymptomatic carriers and thereby is important for dissemination of the pathogen.
Beyond its potential importance for pathogenesis, the Multifunctional-Autoprocessing RTX toxin of V. cholerae (MARTX-Vc) is of intrinsic interest due to its novel biochemical properties and mode of action. At 4545 aa and a predicted size of >480 kDa, MARTX-Vc is one of the largest single polypeptide toxins. However, unlike other RTX toxins, MARTX-Vc is not a pore-forming toxin, but rather induces actin depolymerization and cell rounding (Satchell, 2007).
Current knowledge about the process of cell rounding by MARTX-Vc is diagrammed in FIG. 1. Based on the mechanism of translocation of RTX toxin Bordetella pertussis adenylate cyclase (Osickova et al., 1999), it is predicted that MARTX-Vc self-inserts into the eukaryotic cytoplasmic membrane and then transfers activity domains to the cytoplasm. Thus far, at least three activities have been described for this toxin conferred by three discrete activity domains:
1. Cross-linking activity. The toxin has been shown to covalently crosslink actin into oligomers (Fullner and Mekalanos, 2000). This activity is associated with the actin crosslinking domain (ACD) that is shared with a type 6 secretion effector of V. cholerae and with the putative MARTX toxin of Aeromonas hydrophila (MARTX-Ah) (Satchell, 2007; Sheahan et al., 2004). It has been shown that the substrate for crosslinking is free monomeric G-actin and crosslinking occurs dependent upon the hydrolysis of ATP. It has been hypothesized that cell rounding then occurs by depletion of the free G-actin pool leading to an equilibrium shift that drives the depolymerization of assembled actin fibers (Kudryashov et al., 2008; Cordero et al., 2006).
2. RhoGTPase inactivation. It also has been demonstrated that the toxin inactivates RhoGTPases, the master regulators of actin cytoskeletal assembly, by a mechanism that is distinct from all other known Rho-modulating toxins. This activity is associated with a Rho-inactivation domain (RID) that is shared with the MARTX toxin from V. vulnificus (MARTX-Vv) and the putative MARTX toxins from Xenorhabdus sp. (Satchell, 2007; Sheahan and Satchell, 2007).
3. Cysteine protease domain (CPD) autoproteolysis. It also has been shown that the toxin has an autoprocessing activity associated with it cysteine protease domain (CPD). This enzymatic region of the protein cleaves the toxin after binding cytosolic stimulatory factor inositol hexakisphosphate (InsP6), a molecule found exclusively in the eukaryotic cell cytosol. Thus, processing would be induced only after translocation to the eukaryotic cell cytosol to release the ACD and RID to access the substrates (Sheahan et al., 2007).
Identification of the CPD. Transient expression of aa 3376-3637 of the MARTX-Vc toxin in eukaryotic cells resulted in cells that appeared condensed and necrotic. A Western blot of cell lysates with anti-GFP antibody revealed the CPD:EGFP fusion protein expressed from plasmid pCPDc runs on SDS-PAGE at 48 kDa, approx. 9 kDa smaller than predicted (FIG. 2). Since one of only two Cys residues in the entire toxin is located within this region, it was hypothesized that Cys3568 was important for cytotoxicity. Indeed, cytotoxicity was not observed after transfection of a mutant plasmid carrying a C3568S mutation. The fusion protein expressed from pCPDc C-S ran on SDS-PAGE at the expected size of 57 kDa. A His3519A mutant also ran at the predicted mol. wt. (FIG. 2) These results showed this domain is an autoprotease with a His-Cys catalytic dyad in vivo (Sheahan et al., 2007).
Induced autoprocessing of the CPD. In order to demonstrate that the CPD is an autoprocessing cysteine protease, recombinant CPD and recombinant protein carrying the C3568S mutation were purified (rCPD and rCPD C-S, respectively). Both proteins purified from E. coli at the full length and were resolved on SDS-PAGE at the predicted m.w. of 34 kDa demonstrating that rCPD was not cleaved in E. coli or during purification. Cleavage occurred only after addition a nuclear-free eukaryotic cell lysate, which stimulated autoprocessing of rCPD, but not rCPD C-S (FIG. 3) (Sheahan et al., 2007).
Homologous CPDs have been identified in 18 large bacterial proteins including all MARTX toxins, four clostridial glucosylating toxins, 2 putative toxins from Yersinia sp., and an adhesin from Bordetella sp. (Sheahan et al., 2007). Of particular note is Clostridum difficile Toxin B (TcdB). This toxin has previously been demonstrated to undergo processing after translocation to release the glucosyl transferase catalytic domain (Rupnik et al., 2005; Pfeifer et al., 2003). A recent paper showed that autocatalytic processing of TcdB occurs after addition of inositol hexakisphosphate (InsP6), also known as inositol-6-phosphate, IP6, or phytic acid (Reineke et al., 2007). In a subsequent paper, InsP6-induced autoprocessing of both TcdA and TcdB was shown to be due to its cysteine protease domain, the same domain shared with MARTX-Vc (Egerer et al., 2007). Based on the TcdB results, InsP6 was assessed to determine whether it also induces autoprocessing of rCPD. Indeed, InsP6 induces autoprocessing of rCPD and has a binding constant for InsP6 of 0.6 μM. InsP6-induced autoprocessing of rCPD occurred in a time- and concentration-dependent manner (FIG. 4, Prochazkova K., and Satchell K. J., “Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin,” J. Biol. Chem., 2008 Aug. 29; 283(35):23656-64).
Application of CPD to recombinant protein purification. The CPD of V. cholerae is not active in bacteria and is inducible only by adding the inducing compound inositol 6 phosphate in a processing reaction. As such, a recombinant protein fused at the N-terminus of the CPD was created and a peptide tag was added at the C-terminus of the CPD. It was hypothesized that the recombinant protein could be purified via the peptide tag which subsequently could be removed by inducing the CPD, allowing rapid production of native recombinant protein. The concept is diagrammed in FIG. 5 below. The recovered recombinant protein after cleavage includes two additional C-terminal amino acids from the CPD (i.e., an alanine-leucine dipeptide). CPD cleavage of the fusion protein could be induced on a column, after purification of the fusion protein, or during dialysis of the fusion protein. The peptide tag remains associated with the CPD and thus the tagged CPD may be removed from the recombinant protein by affinity chromatography. In some embodiments, protease inhibitors may be included in the processing reaction, including but not limited to chloromethyl ketones or N-ethylmaleimide. For example, protease inhibitors may be utilized to prevent cleavage at a site other than the specific site for CPD cleavage (e.g., at a position other than between amino acids 2 and 3 of the CPD).
In one example, a T7 overexpression plasmid was utilized to fuse the eukaryotic protein RhoA to CPD with a C-terminal 6×His Tag. A map of this vector is diagrammed in FIG. 6. This vector was created from pMCSG7 (Stols et al., 2002).
The vector was transformed to E. coli BL21(DE3) and bacteria were grown and induced with IPTG. RhoA:CPD:6×His fusion protein was purified from 100 ml cell lysate on an AKTA HisTrap column with elution in 250 mM imidazole. Eluted protein was incubated in 1 μM phytic acid (InsP6) from Sigma for 1 hr at 37° C. Result of cleavage is shown in FIG. 7.
REFERENCES
Cordero, C. L., Kudryashov, D. S., Reisler, E. and Satchell, K. J. (2006) The actin cross-linking domain of the Vibrio cholerae RTX toxin directly catalyzes the covalent cross-linking of actin. J Biol. Chem. 281: 32366-32374.
Egerer, M., Giesemann, T., Jank, T., Satchell, K. J. and Aktories, K. (2007) Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on a cysteine protease activity. J Biol Chem epub Jun. 25, 2007.
Fullner, K. J. and Mekalanos, J. J. (2000) In vivo covalent crosslinking of actin by the RTX toxin of Vibrio cholerae. EMBO J. 19: 5315-5323.
Kudryashov, D. S., Cordero, C. L., Reisler, E. and Satchell, K. J. (2008) Characterization of the enzymatic activity of the actin cross-linking domain from the Vibrio cholerae MARTXVc toxin. J Biol. Chem. 283: 445-452.
Olivier, V., Salzman, N. H. and Satchell, K. J. (2007a) Prolonged colonization of mice by Vibrio cholerae El Tor Ol depends on accessory toxins. Infect Immun. 75: 5043-5051.
Olivier, V., Haines, G. K., 3rd, Tan, Y. and Satchell, K. J. (2007b) Hemolysin and the multifunctional autoprocessing RTX toxin are virulence factors during intestinal infection of mice with Vibrio cholerae El Tor Ol strains. Infect Immun. 75: 5035-5042.
Osickova, A., Osicka, R., Maier, E., Benz, R. and Sebo, P. (1999) An amphipathic alpha-helix including glutamates 509 and 516 is crucial for membrane translocation of adenylate cyclase toxin and modulates formation and cation selectivity of its membrane channels. J. Biol. Chem. 274: 37644-37650.
Pfeifer, G., Schirmer, J., Leemhuis, J., Busch, C., Meyer, D. K., Aktories, K. and Barth, H. (2003) Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells. J Biol. Chem. 278: 44535-44541.
Reineke, J., Tenzer, S., Rupnik, M., Koschinski, A., Hasselmayer, O., Schrattenholz, A., et al (2007) Autocatalytic cleavage of Clostridium difficile toxin B. Nature. 446: 415-419.
Rupnik, M., Pabst, S., Rupnik, M., von Eichel-Streiber, C., Urlaub, H. and Soling, H. D. (2005) Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difficile toxin B (TcdB) by host cells. Microbiology. 151: 199-208.
Satchell, K. J. (2007) MARTX: Multifunctional-Autoprocessing RTX Toxins. Infect Immun. 75: 5079-5084.
Sheahan, K. L. and Satchell, K. J. (2007) Inactivation of small Rho GTPases by the multifunctional RTX toxin from Vibrio cholerae. Cell Microbiol. 9: 1324-1335.
Sheahan, K. L., Cordero, C. L. and Satchell, K. J. (2004) Identification of a domain within the multifunctional Vibrio cholerae RTX toxin that covalently cross-links actin. Proc. Natl. Acad. Sci. USA. 101: 9798-9803.
Sheahan, K. L., Cordero, C. L. and Satchell, K. J. (2007) Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain. EMBO J. 26: 2552-2561.
Stols, L., Gu, M., Dieckman, L., Raffen, R., Collart, F. R. and Donnelly, M. I. (2002) A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. Protein Expr Purif. 25: 8-15.
Example 2
Reference is made to the article Prochazkova K., and Satchell K. J., “Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin,” published in the Journal of Biological Chemistry, volume 283(35), pages 23656-64, on Aug. 29, 2008, the content of which was disclosed and incorporated by reference in U.S. Provisional Patent Application No. 61/119,489, filed on Dec. 3, 2008.

## Claims

1. A method for producing a recombinant polypeptide, the method comprising:
(a) culturing a prokaryotic cell transformed with a recombinant polynucleotide, the recombinant polynucleotide encoding a fusion protein comprising:
(i) the recombinant polypeptide; and
(ii) an inducible autoproteolytic cysteine protease fused to the C-terminus of the recombinant polypeptide;

(b) isolating the fusion protein from the cell;
(c) inducing the autoproteolytic cysteine protease of the isolated fusion protein in a reaction mixture by adding a solution consisting_essentially of inositol hexakisphosphate to the reaction mixture to cleave the isolated fusion protein and produce a cleaved fragment comprising the recombinant polypeptide; and
(d) isolating the cleaved fragment comprising the recombinant polypeptide.

2. The method of claim 1, wherein the encoded fusion protein further comprises a peptide tag fused at the C-terminus of the inducible autoproteolytic cysteine protease and the fusion protein is recovered by contacting the fusion protein with a resin that binds the peptide tag.

3. The method of claim 1, wherein the peptide tag is a 6×His tag, a hemaglutinin tag, a FLAG tag, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose binding protein tag, or a chitin binding protein tag.

4. The method of claim 1, wherein the fusion protein comprises a fragment of V. cholerae RTX toxin (SEQ ID NO:1), V. vulnificus RTX toxin (SEQ ID NO:2), V. splendidus putative RTX toxin (SEQ ID NO:3), P. luminescens putative RTX toxin (SEQ ID NO:4-7), Xenorhabdus nematophila (XnRtx) (SEQ ID NO:8), X bovienii (XbRtx) (SEQ ID NO:9), Y. pseudotuberculosis putative toxin (SEQ ID NO:10), Y. mollaretti putative toxin (SEQ ID NO:11), C. difficile Toxin A (SEQ ID NO:12), C. difficile Toxin B (SEQ ID NO:13), C. noveyi alpha toxin (SEQ ID NO:14), C. sordellii cytotoxin L (SEQ ID NO:15), or Bordetella pertussis putative adhesin FhaL (SEQ ID NO:16), and the fragment has inducible autoproteolytic cysteine protease activity.

5. The method of claim 1, wherein the inducible autoproteolytic cysteine protease fused to the C-terminus of the recombinant polypeptide has an amino acid sequence that is at least 95% identical to SEQ ID NO:17, and has cysteine protease activity when induced by inositol hexakisphosphate.

6. The method of claim 5, wherein the inducible autoproteolytic cysteine protease has an amino acid sequence that is at least 97% identical to SEQ ID NO:17.

7. The method of claim 5, wherein the inducible autoproteolytic cysteine protease has an amino acid sequence that is at least 99% identical to SEQ ID NO:17.

8. The method of claim 5, wherein the inducible autoproteolytic cysteine protease has an amino acid sequence of SEQ ID NO:17.

9. The method of claim 8, wherein the inducible autoproteolytic cysteine protease is cleaved between amino acids 2 and 3 of SEQ ID NO:17.

10. The method of claim 1, wherein the recombinant polypeptide and the inducible autoproteolytic cysteine protease are directly fused.

11. The method of claim 1, wherein the recombinant polypeptide and the inducible autoproteolytic cysteine protease are fused indirectly via a peptide linker.

12. A method for producing a recombinant polypeptide, the method comprising:
(a) culturing a prokaryotic cell transformed with a recombinant polynucleotide, the recombinant polynucleotide encoding a fusion protein comprising:
(i) the recombinant polypeptide; and
(ii) an inducible autoproteolytic cysteine protease fused to the C-terminus of the recombinant polypeptide;

(b) isolating the fusion protein from the cell;
(c) inducing the autoproteolytic cysteine protease of the isolated fusion protein in a reaction mixture by adding a solution to the reaction mixture to cleave the isolated fusion protein and produce a cleaved fragment comprising the recombinant polypeptide, wherein the solution consists essentially of hexakisphosphate and a protease inhibitor that inhibits non-specific protease activity and does not inhibit the autoproteolytic cysteine protease; and
(d) isolating the cleaved fragment comprising the recombinant polypeptide.

13. The method of claim 12, wherein the encoded fusion protein further comprises a peptide tag fused at the C-terminus of the inducible autoproteolytic cysteine protease and the fusion protein is recovered by contacting the fusion protein with a resin that binds the peptide tag.

14. The method of claim 12, wherein the fusion protein comprises a fragment of V. cholerae RTX toxin (SEQ ID NO:1), V. vulnificus RTX toxin (SEQ ID NO:2), V. splendidus putative RTX toxin (SEQ ID NO:3), P. luminescens putative RTX toxin (SEQ ID NO:4-7), Xenorhabdus nematophila (XnRtx) (SEQ ID NO:8), X. bovienii (XbRtx) (SEQ ID NO:9), Y. pseudotuberculosis putative toxin (SEQ ID NO:10), Y. mollaretti putative toxin (SEQ ID NO:11), C. difficile Toxin A (SEQ ID NO:12), C. difficile Toxin B (SEQ ID NO:13), C. noveyi alpha toxin (SEQ ID NO:14), C. sordellii cytotoxin L (SEQ ID NO:15), or Bordetella pertussis putative adhesin FhaL (SEQ ID NO:16), and the fragment has inducible autoproteolytic cysteine protease activity.

15. The method of claim 12, wherein the inducible autoproteolytic cysteine protease fused to the C-terminus of the recombinant polypeptide has an amino acid sequence that is at least 95% identical to SEQ ID NO:17, and has cysteine protease activity when induced by inositol hexakisphosphate.

16. The method of claim 15, wherein the inducible autoproteolytic cysteine protease has an amino acid sequence of SEQ ID NO:17.

17. The method of claim 16, wherein the inducible autoproteolytic cysteine protease is cleaved between amino acids 2 and 3 of SEQ ID NO:17.

18. The method of claim 12, wherein the recombinant polypeptide and the inducible autoproteolytic cysteine protease are directly fused.

19. The method of claim 12, wherein the recombinant polypeptide and the inducible autoproteolytic cysteine protease are fused indirectly via a peptide linker.