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Secretory expression and characterization of insulin in Pichia pastoris
Author(s) -
Kjeldsen Thomas,
Pettersson Annette Frost,
Hach Morten
Publication year - 1999
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1999.tb01151.x
Subject(s) - pichia pastoris , proinsulin , insulin , saccharomyces cerevisiae , fusion protein , biology , c peptide , biochemistry , yeast , endocrinology , recombinant dna , gene
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.