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 alpha (alpha-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 alpha-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 alpha-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.
Kjeldsen
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Department of Insulin Research and Protein Chemistry, Novo Nordisk A/S, Novo Alle 6B S.90, DK-2880 Bagsvaerd, Denmark.
Pettersson
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AF
Hach
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D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000078790:Insulin Secretion; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011384:Proinsulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D007328:Insulin; D011994:Recombinant Proteins; D011384:Proinsulin