Prepro-Leaders Lacking N-Linked Glycosylation for Secretory Expression in the YeastSaccharomyces cerevisiae

Synthetic prepro-leaders lacking consensus N-linked glycosylation sites confers secretion competence of correctly folded insulin precursor expressed in the yeast speciesSaccharomyces cerevisiaewith a yield comparable to, or better than the α-factor prepro-leader. In contrast, theS. cerevisiaeα-factor prepro-leader's three N-linked oligosaccharide chains are necessary for the ability to facilitate secretion of the insulin precursor fromS. cerevisiae(T. Kjeldsenet al., 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 of a pro-leader/insulin precursor fusion protein in which the insulin precursor was correctly folded. The unprocessed pro-leader/insulin-precursor fusion protein was purified from culture medium and maturedin vitroto desB30 insulin byAchromobacter lyticuslysyl-specific protease providing an alternative yeast expression system not dependent on the Kex2 endoprotease. The synthetic prepro-leader lacking N-linked glycosylation provides the opportunity for secretory expression in yeast utilizing eitherin vivoKex2 endoprotease maturation of the fusion protein during secretion orin vitromaturation of the purified fusion protein with a suitable enzyme.