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.