Secretory expression and characterization of insulin in Pichia pastoris

The yeasts Pichia pastoris and Saccharomyces cerevisiae have similar overall features regarding the secretory expression of insulin. The S. cerevisiae mating factor α (α‐factor) prepro‐leader facilitated the secretion of an insulin precursor, but not proinsulin expressed in P. pastoris. Synthetic prepro‐leaders developed for the secretory expression of the insulin precursor in S. cerevisiae also facilitated the secretion of the insulin precursor expressed in P. pastoris. In contrast with S. cerevisiae, only insulin precursor and no unprocessed hyperglycosylated α‐factor pro‐leader/insulin precursor fusion protein was secreted from P. pastoris. A spacer peptide in the fusion protein increased the fermentation yield of the insulin precursor in P. pastoris. A synthetic prepro‐leader, but not an α‐factor prepro‐leader lacking N‐glycosylation sites, facilitated the secretion of the insulin precursor in P. pastoris. P. pastoris has a capacity for secretory expression of the insulin precursor that is equal to or better than that of S. cerevisiae. Peptide mapping and MS indicated a structure of the insulin precursor expressed in P. pastoris identical with that of human insulin.