SYNTHETIC LEADERS WITH POTENTIAL BIP BINDING MEDIATE HIGH-YIELD SECRETION OF CORRECTLY FOLDED INSULIN PRECURSORS FROM SACCHAROMYCES-CEREVISIAE

Secretion leaders are essential for expression of many heterologous pr oteins including insulin in yeast. The function of secretion leaders a nd their interaction with the secretory pathway is not clear. To deter mine what constitutes functional pre-pro-leader sequences in Saccharom yces cerevisiae, synthetic leader sequences for secretion of the insul in precursor were developed by a combination of rational design and st epwise systematic optimization, The synthetic leaders efficiently faci litate secretion of the insulin precursor from S. cerevisiae when comp ared with the a-factor leader, leading to a high yield of correctly fo lded insulin precursor in the culture supernatant. The synthetic leade rs feature two potential N-linked glycosylation sites which are effici ently glycosylated during secretion. Pulse-chase analysis indicates th at the synthetic leaders/insulin precursor fusion protein have a prolo nged residence in the endoplasmic reticulum compared to the alpha-fact or leader/insulin precursor fusion protein. The longer transition time in the endoplasmic reticulum mediated by the synthetic leaders might provide additional time for correct folding of the insulin precursor a nd account for the increased fermentation yield. (C) 1997 Academic Pre ss.