We have developed technology for secretory expression of human insulin precursor (IP) with alpha factor leader under methanol-inducible promoter from yeast Pichia pastoris. IP is purified and converted enzymatically into insulin ester in the transpeptidation reaction with trypsin in the presence of H-Thr(tBu)-OtBu. Trypsin mediates the cleavage of the N-terminal extension and the spacer tripeptide whilst threonine ester is added to B-29 residue. The insulin ester is further purified and crystallized. Finally, the human insulin is obtained in the deprotection reaction and purified by the reversed-phase chromatography.
Video Procedures
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Insulin upstream
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Sample Manipulation
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Fermentor broth clarification
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Insulin Precursor Purification
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Transpeptidation of Insulin Precursor
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Human insulin ester purification and crystallization
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The deprotection reaction
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Human insulin purification and lyophilization