Abstract
Native proinsulin belongs to the class of the difficult-to-express proteins in
Escherichia coli. Problems mainly arise due to its small size, a high proteolytic decay, and the necessity to form a native disulfide pattern. In the present study, human proinsulin was produced in the periplasm of
E. coli as a fusion to ecotin, which is a small periplasmic protein of 16
kDa encoded by the host, containing one disulfide bond. The fusion protein was secreted to the periplasm and native proinsulin was determined by ELISA. Cultivation parameters were studied in parallel batch mode fermentations using
E. coli BL21(DE3)Gold as a host. After improvement of fed-batch high density fermentation conditions, 153
mg fusion protein corresponding to 51.5
mg native proinsulin was obtained per L. Proteins were extracted from the periplasm by osmotic shock treatment. The fusion protein was purified in one step by ecotin affinity chromatography on immobilized trypsinogen. After thrombin cleavage of the fusion protein, the products were separated by Ni–NTA chromatography. Proinsulin was quantified by ELISA and characterized by mass spectrometry. To evaluate the influence of periplasmic proteases, the amount of ecotin–proinsulin was determined in
E. coli BL21(DE3)Gold and in a periplasmic protease deficient strain,
E. coli SF120.