Insulin

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

1

Insulin upstream

2

Sample Manipulation

3

Fermentor broth clarification

4

Insulin Precursor Purification

5

Transpeptidation of Insulin Precursor

6

Human insulin ester purification and crystallization

7

The deprotection reaction

8

Human insulin purification and lyophilization

To access the course please contact:

Natasa Skoko, Head

Biotechnology Development Unit

+39 040 3757352

skoko@icgeb.org

Martina Viviani, Head

Technology Transfer Office

+39 040 3757365

viviani@icgeb.org