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.
Fermentor broth clarification
Insulin Precursor Purification
Transpeptidation of Insulin Precursor
Human insulin ester purification and crystallization
The deprotection reaction
Human insulin purification and lyophilization