A new signal peptide useful for secretion of heterologous proteins from yeast and its application for synthesis of hirudin.
The BGL2 gene from Saccharomyces cerevisiae encodes a beta-glucanase which is localized to the yeast cell wall. The ability of a 23-amino acid (aa) signal peptide derived from the BGL2 gene to direct a heterologous protein to the secretory pathway of yeast has been compared to that of the MF alpha 1-encoded signal peptide in a series of gene fusions. As a model protein, the leech anticoagulant, recombinant hirudin variant 2-Lys47 (HIR) has been studied. From a multicopy plasmid chimaeric proteins were produced which carry the BGL2 signal peptide (or the artificial BGL2 pre-Val7 variant) (i) in front of the MF alpha 1 pro sequence (or modified versions of MF alpha 1 pro), i.e., a prepro signal, or (ii) joined directly to the heterologous protein. Accumulation of active HIR in yeast culture supernatants was observed when the BGL2 (or the BGL2 pre-Val7) signal peptide were used in combination with either of three versions of the MF alpha 1 pro peptide: the authentic MF alpha 1 pro, a partially deleted MF alpha 1 pro-delta 22-61, or a pro bearing an aa change (MF alpha 1 pro-Gly22). In each case the BGL2 signal peptide (or its variant) has proven equally productive to the corresponding MF alpha 1 peptide. Four times more active HIR was detected in the culture supernatant when either signal peptide was fused directly to the recombinant protein, as compared to a prepro protein version. Correct signal peptide cleavage was obtained when HIR was produced as a BGL2 pre-Val7::fusion protein.
Achstetter
T
T
Transgene S.A., Strasbourg, France.
Nguyen-Juilleret
M
M
Findeli
A
A
Merkamm
M
M
Lemoine
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002872:Chromosome Deletion; D005800:Genes, Fungal; D014644:Genetic Variation; D005822:Genetic Vectors; D006629:Hirudins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D014633:Valine / Q000235:genetics
D006629:Hirudins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014633:Valine