What question did this study set out to answer?

To optimize the high-yield production and secretion of non-hydroxylated recombinant gelatins in Pichia pastoris while minimizing proteolytic degradation.

August 1, 1999Socio-Environmental Systems ModelingOpen Access

High-yield secretion of recombinant gelatins byPichia pastoris

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Authors

MWMarc W. T. WertenWageningen University & Research TBTanja J. van den Bosch RWRichèle D. WindHAN University of Applied Sciences

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Implication

In vitro bioengineering demonstrates high-level secretion of intact recombinant gelatins in Pichia pastoris, indicating an effective strategy to bypass proteolytic breakdown.

Key Points

To optimize the high-yield production and secretion of non-hydroxylated recombinant gelatins in Pichia pastoris while minimizing proteolytic degradation.
Cloned mouse type I and rat type III collagen sequences using the Saccharomyces cerevisiae alpha-mating factor prepro signal for extracellular secretion.
Optimized fermentation parameters by adjusting the broth to pH 3.0 and supplementing with casamino acids to reduce general proteolysis.
Employed site-directed mutagenesis to mutate mono-arginylic sites targeted by a putative Kex2-like protease and utilized multicopy transformants.
Fermentation at pH 3.0 combined with casamino acid supplementation substantially mitigated non-specific degradation of unfolded gelatin chains.
Site-directed mutagenesis of mono-arginylic cleavage sites successfully prevented targeted breakdown by Kex2-like proteases.
Multicopy yeast transformants achieved production yields of up to 14.8 g/L in clarified broth, establishing a record for heterologous protein secretion in Pichia pastoris.

Cite This Study

Werten et al. (1999) studied this question.

synapsesocial.com/papers/6aa04845e47507b78762878c https://doi.org/10.1002/(sici)1097-0061(199908)15:11<1087::aid-yea436>3.0.co;2-f

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Implication

In vitro bioengineering demonstrates high-level secretion of intact recombinant gelatins in Pichia pastoris, indicating an effective strategy to bypass proteolytic breakdown.

High-yield secretion of recombinant gelatins byPichia pastoris

Discussion

Member takes

Implication

Key Points

Cite This Study

High-yield secretion of recombinant gelatins byPichia pastoris

Discussion

Member takes

Implication

Key Points

Cite This Study

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