Prepro-leaders lacking N-linked glycosylation for secretory expression in the yeast Saccharomyces cerevisiae

Synthetic prepro-leaders lacking consensus N-Linked glycosylation sites con fers secretion competence of correctly folded insulin precursor expressed i n the yeast species Saccharomyces cerevisiae with a yield comparable to, or better than the alpha-factor prepro-leader. In contrast, the S. cerevisiae alpha-factor prepro-leader's three N-linked oligosaccharide chains are nec essary for the ability to facilitate secretion of the insulin precursor fro m S. cerevisiae (T. Kjeldsen ct at, Biotechnol Appl. Biochem. 27, 109-115, 1998). Synthetic prepro-leader lacking both N-glycosylation and the dibasic Kex2 endoprotease processing site also efficiently facilitated secretion o f a pro-leader/insulin precursor fusion protein in which the insulin precur sor was correctly folded. The unprocessed pro-leader/insulin-precursor fusi on protein was purified from culture medium and matured in vitro to desB30 insulin by Achromobacter lyticus lysyl-specific protease providing an alter native yeast expression system not dependent on the Kex2 endoprotease. The synthetic prepro-leader lacking N-linked glycosylation provides the opportu nity for secretory expression in yeast utilizing either in vivo Kex2 endopr otease maturation of the fusion protein during secretion or in vitro matura tion of the purified fusion protein with a suitable enzyme. (C) 1998 Academ ic Press.