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High‐yield secretion of recombinant gelatins by Pichia pastoris
Author(s) -
Werten Marc W. T.,
van den Bosch Tanja J.,
Wind Richèle D.,
Mooibroek Hans,
de Wolf Frits A.
Publication year - 1999
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(199908)15:11<1087::aid-yea436>3.0.co;2-f
Subject(s) - pichia pastoris , heterologous , biology , proteolysis , recombinant dna , protease , yeast , biochemistry , secretion , saccharomyces cerevisiae , aspergillus oryzae , mutagenesis , gelatin , fermentation , enzyme , mutation , gene
Recombinant non‐hydroxylated gelatins based on mouse type I and rat type III collagen sequences were secreted from the methylotrophic yeast Pichia pastoris , using the Saccharomyces cerevisiae α ‐mating factor prepro signal. Proteolytic degradation could be minimized to a large extent by performing fermentations at pH 3·0 and by adding casamino acids to the medium, even though gelatin is extremely susceptible to proteolysis due to its open, unfolded structure. Proteolytic cleavage at specific mono‐arginylic sites, by a putative Kex2‐like protease, could be successfully abolished by site‐directed mutagenesis of these sites. Production levels as high as 14·8 g/l clarified both were obtained, using multicopy tranformants. To our knowledge, this represents the highest level of heterologous protein secretion reported to date for P. pastoris . Copyright © 1999 John Wiley & Sons, Ltd.