Secretory expression and characterization of insulin in Pichia pastoris

The yeasts Pichia pastoris and Saccharomyces cerevisiae have similar overal l features regarding the secretory expression of insulin. The S. cerevisiae mating factor alpha (alpha-factor) prepro-leader facilitated the secretion of an insulin precursor, but not proinsulin expressed in P. pastoris. Synt hetic prepro-leaders developed for the secretory expression of the insulin precursor in S. cerevisiae also facilitated the secretion of the insulin pr ecursor expressed in P. pastoris. In contrast with S. cerevisiae, only insu lin precursor and no unprocessed hyperglycosylated alpha-factor pro-leader/ insulin precursor fusion protein was secreted from P. pastoris. A spacer pe ptide in the fusion protein increased the fermentation yield of the insulin precursor in P. pastoris, A synthetic prepro-leader, but not an alpha-fact or prepro-leader lacking N-glycosylation sites, facilitated the secretion o f the insulin precursor in P. pastoris. P. pastoris has a capacity for secr etory expression of the insulin precursor that is equal to or better than t hat of S, cerevisiae, Peptide mapping and MS indicated a structure of the i nsulin precursor expressed in P. pastoris identical with that of human insu lin.