1. High-yield secretion of recombinant gelatins by Pichia pastoris.
- Author
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Werten MW, van den Bosch TJ, Wind RD, Mooibroek H, and de Wolf FA
- Subjects
- Amino Acid Sequence, Collagen metabolism, DNA chemistry, DNA Primers chemistry, Electrophoresis, Polyacrylamide Gel, Fermentation, Gelatin analysis, Genetic Vectors chemistry, Hydrogen-Ion Concentration, Molecular Sequence Data, Mutagenesis, Site-Directed, Pichia genetics, Pichia growth & development, Plasmids chemistry, Recombinant Proteins analysis, Recombinant Proteins metabolism, Saccharomyces cerevisiae genetics, Sequence Analysis, Subtilisins chemistry, Transformation, Genetic, Gelatin metabolism, Pichia metabolism, Proprotein Convertases, Recombinant Proteins biosynthesis, Saccharomyces cerevisiae Proteins
- Abstract
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 alpha-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.)
- Published
- 1999
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