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 cleaved by trypsin in order to remove the N-terminal extension and the spacer tripeptide, therefore generating the product DesB30. Finally, a C14 fatty acid chain is activated and attached to the amino acid B29 in order to obtain the long-lasting insulin Detemir.
Preparation of desB30-insulin
Preparation of activated derivatives of Tetradecanoic (Myristic) acid
Modification of DesB30-insulin with activated Myristic acid
Detemir purification and crystallization