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.

1 . A polynucleotide coding for a fusion protein, the fusion protein comprising:
 (a) a first polypeptide; and   (b) a second polypeptide fused to the C-terminus of the first polypeptide, wherein the first polypeptide is heterologous with respect to the second polypeptide, the second polypeptide has an amino acid sequence that is at least 95% identical to SEQ ID NO:17, and the second polypeptide has cysteine protease activity when induced by inositol hexakisphosphate such that the second polypeptide is cleaved.   
     
     
         2 . The polynucleotide of  claim 1 , wherein the fusion protein further comprises a peptide tag fused at the C-terminus of the second polypeptide. 
     
     
         3 . The polynucleotide of  claim 2 , 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 polynucleotide of  claim 1 , wherein the second polypeptide has an acid sequence that is at least 97% identical to SEQ ID NO:17. 
     
     
         5 . The polynucleotide of  claim 1 , wherein the second polypeptide has an amino acid sequence that is at least 99% identical to SEQ ID NO:17. 
     
     
         6 . The polynucleotide of  claim 1 , wherein the second polypeptide has an amino acid sequence of SEQ ID NO:17. 
     
     
         7 . The polynucleotide of  claim 1 , wherein the second polypeptide is cleaved between amino acids 2 and 3 of the second polypeptide. 
     
     
         8 . The polynucleotide of  claim 1 , wherein the first polypeptide and the second polypeptide are directly fused. 
     
     
         9 . The polynucleotide of  claim 1 , wherein the first polypeptide and the second polypeptide are fused indirectly via a peptide linker. 
     
     
         10 . A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide of  claim 1 . 
     
     
         11 . An expression vector comprising the recombinant polynucleotide according to  claim 10 . 
     
     
         12 . An isolated cell transformed with the expression vector of  claim 11 . 
     
     
         13 . A method for producing a fusion protein, the method comprising:
 (a) culturing a cell under conditions suitable for expression of the fusion protein, wherein the cell is transformed with a recombinant polynucleotide, and the recombinant polynucleotide comprises a promoter sequence operably linked to the polynucleotide of  claim 1 , and   (b) recovering the fusion protein so expressed.   
     
     
         14 . The method of  claim 13 , wherein the fusion protein comprises a peptide tag and recovering the fusion protein comprises contacting the peptide tag with a resin that binds the peptide tag. 
     
     
         15 . The method of  claim 14 , 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. 
     
     
         16 . The method of  claim 13 , further comprising:
 (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 comprising the first polypeptide; and 
 (ii) a cleaved fragment of the fusion protein comprising a portion of the second polypeptide. 
   
     
     
         17 . The method of  claim 16 , wherein the recovered fusion protein and the inositol hexakisphosphate are contacted in a reaction mixture comprising a protease inhibitor. 
     
     
         18 . The method of  claim 16 , further comprising:
 (d) separating the cleaved fragment of the fusion protein comprising the first polypeptide and the cleaved fragment of the fusion protein comprising the portion of the second polypeptide.   
     
     
         19 . A kit comprising one or more reagents for performing the method of  claim 13 . 
     
     
         20 . A fusion protein comprising:
 (a) a first polypeptide; and   (b) a second polypeptide fused to the C-terminus of the first peptide, wherein the first polypeptide is heterologous with respect to the second polypeptide, the second polypeptide has an amino acid sequence that is at least 95% identical to SEQ ID NO:17, and the second polypeptide has cysteine protease activity when induced by inositol hexakisphosphate such that the second polypeptide is cleaved.