Expression of fusion polypeptides transported out of the cytoplasm without leader sequences

A host cell comprising:

• a) an expression vector capable of expressing in said host cell a fusion polypeptide, said fusion polypeptide comprising,
• i) a fusion partner, said fusion partner comprising a polypeptide selected from the group consisting of IL-1-.alpha., acidic FGF and basic FGF, int-2, hst/KS3, FGF-5, FGF-6, keratinocyte growth factor (KGF);
• hisactophilin;
• soybean trypsin inhibitor, Vibrio cholerae TcpG, leaderless DsbA and leaderless DsbC;
• ii) a linker peptide comprising a cleavage site;
• and
• iii) mutant insulin-like growth factor binding protein 3 (IGFBP-3), wherein said linker peptide is positioned between said fusion partner and the IGFBP-3 and wherein said fusion partner constitutes the amino terminus of the fusion protein;
• and
• b) a nucleic acid capable of expressing in said host cell a proteolytic enzyme that specifically recognizes said cleavage site.

A fusion polypeptide comprising:

• (a) a fusion partner comprising a polypeptide selected from the group consisting of IL-1-.alpha., acidic FGF and basic FGF, int-2, hst/KS3, FGF-5, FGF-6, keratinocyte growth factor (KGF);
• hisactophilin;
• soybean trypsin inhibitor, Vibrio cholerae TcpG, leaderless DsbA, and leaderless DsbC;
• (b) a linker peptide;
• and
• (c) mutant insulin-like growth factor binding protein 3 (IGFBP-3), wherein said linker peptide is positioned between said fusion partner and the IGFBP-3 and wherein said fusion partner constitutes the amino terminus of the fusion protein.

A method of producing a substantially purified fusion polypeptide encoded by a nucleic acid, wherein said fusion polypeptide comprises,

• (i) a fusion partner comprising a polypeptide selected from the group consisting of IL-1-.alpha., acidic FGF and basic FGF, int-2, hst/KS3, FGF-5, FGF-6, keratinocyte growth factor (KGF);
• hisactophilin;
• soybean trypsin inhibitor, Vibrio cholerae TcpG, leaderless DsbA and leaderless DsbC;
• (ii) a linker peptide;
• and
• (iii) mutant insulin-like growth factor binding protein 3 (IGFBP-3), wherein said linker peptide is positioned between said fusion partner and the IGFBP-3 and wherein said fusion partner constitutes the amino terminus of the fusion polypeptide;
• said method comprising the steps of:
• (a) introducing said nucleic acid encoding said fusion polypeptide into a host cell, thereby producing a transformed host cell;
• (b) culturing said transformed host cell under conditions appropriate for expressing said fusion polypeptide;
• and
• (c) purifying said fusion polypeptide, thereby obtaining a substantially purified fusion polypeptide.

A method of producing a substantially purified polypeptide of interest, said method comprising the steps of:

• (a) introducing into a host cell a nucleic acid encoding a fusion polypeptide, said fusion polypeptide comprising,
• (i) a fusion partner comprising a polypeptide selected from the group consisting of IL-1-.alpha., acidic FGF and basic FGF, int-2, hst/KS3, FGF-5, FGF-6, keratinocyte growth factor (KGF);
• hisactophilin;
• soybean trypsin inhibitor, Vibrio cholerae TcpG, leaderless DsbA and leaderless DsbC;
• (ii) a linker peptide encoding a cleavage site, wherein said linker peptide is positioned between said fusion partner and said polypeptide of interest;
• and
• (iii) mutant insulin-like growth factor binding protein 3 (IGFBP-3), wherein said linker peptide is positioned between said fusion protein and the IGFBP-3 and wherein said fusion partner constitutes the amino terminus of the fusion polypeptide, thereby producing a transformed host cell;
• (b) culturing said transformed host cell under conditions appropriate for expressing said fusion polypeptide, thereby expressing said fusion polypeptide;
• (c) cleaving said fusion polypeptide with a proteolytic enzyme or cleavage agent that recognizes said proteolytic cleavage site, thereby producing said mutant IGFBP-3;
• and
• (d) purifying said mutant IGFBP-3, thereby obtaining a substantially purified mutant IGFBP-3.

A method of producing a substantially purified polypeptide of interest, said method comprising the steps of:

• (a) introducing into a host cell a nucleic acid encoding a fusion polypeptide, said fusion polypeptide comprising,
• (i) a fusion partner comprising a polypeptide selected from the group consisting of IL-1-.alpha., acidic FGF and basic FGF, int-2, hst/KS3, FGF-5, FGF-6, keratinocyte growth factor (KGF);
• hisactophilin;
• soybean trypsin inhibitor, Vibrio cholerae TcpG, leaderless DsbA and leaderless DsbC;
• (ii) a linker peptide encoding a cleavage site, wherein said linker peptide is positioned between said fusion partner and said polypeptide of interest;
• and
• (iii) mutant insulin-like growth factor binding protein 3 (IGFBP-3), wherein said linker peptide is positioned between said fusion partner and the IGFBP-3 and wherein said fusion partner constitutes the amino terminus of the fusion protein;
• thereby producing a transformed host cell;
• (b) culturing said transformed host cell under conditions appropriate for expressing said fusion polypeptide, thereby expressing said fusion polypeptide;
• (c) purifying said fusion polypeptide, thereby producing a substantially purified fusion polypeptide;
• (d) cleaving said substantially purified fusion polypeptide with a proteolytic enzyme or cleavage agent that recognizes said proteolytic cleavage site, thereby producing said mutant IGFBP-3;
• and
• (e) purifying said mutant IGFBP-3, thereby obtaining a substantially purified mutant IGFBP-3.

A method of producing a substantially purified polypeptide of interest comprising the steps of:

• (a) introducing into a host cell a nucleic acid encoding a fusion polypeptide, said fusion polypeptide comprising,
• (i) a fusion partner comprising a polypeptide selected from the group consisting of IL-1-.alpha., acidic FGF and basic FGF, int-2, hst/KS3, FGF-5, FGF-6, keratinocyte growth factor (KGF);
• hisactophilin;
• soybean trypsin inhibitor, Vibrio cholerae TcpG, leaderless DsbA and leaderless DsbC;
• (ii) a linker peptide encoding a cleavage site, wherein said linker peptide is positioned between said fusion partner and said polypeptide of interest;
• and
• (iii) mutant insulin-like growth factor binding protein 3 (IGFBP-3), wherein said linker peptide is positioned between said fusion partner and the IGFBP-3 and wherein said fusion partner constitutes the amino terminus of the fusion polypeptide;
• and further wherein said host cell comprises a nucleic acid capable of expressing in said host cell a proteolytic enzyme that specifically recognizes said cleavage site;
• thereby producing a transformed host cell;
• (b) culturing said transformed host cell under conditions appropriate for expressing said fusion polypeptide and said proteolytic enzyme, thereby expressing said fusion polypeptide, causing the in vivo cleavage of said fusion polypeptide, and producing said mutant IGFBP-3;
• and
• (c) purifying said polypeptide of interest, thereby obtaining a substantially purified mutant IGFBP-3.