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 hexadecanedioic acid is attached via gamma-L-glutamyl spacer to the amino group of the side chain of lysine at position B29 in order to obtain the long-lasting insulin Degludec.
Introduction video: Degludec
Video Procedures
1
Preparation of desB30-insulin
2
Preparation of DEGLUDEC synthetic linker composed of hexadecanedioic acid and a gamma-L-glutamyl spacer
3
Modification of DesB30-insulin with activated deprotected Degludec linker
4
Chromatographic purification of Degludec from the reaction mixture