Studies On The Comprehensive Optimization Of Expression And Purification Of Recombinant Human Insulin

This study comprehensive optimize the fermentation of reconbinant Escherichia.coli expressing (His)₆-Arg-Arg-human-proinsulin(RRhPI),and downstream process of recombinant human insulin was investigated. A defined culture medium, fermentation conditions and induction strategy were developed by means of orthogonal design. It allowed growth of the recombinant E. coli M15 up to 43±0. 17g/L wet weight/L (n=3) in shake-flasks, which produced 14±0. 52 g/L wet inclusion bodies. The expression level accounted for about 30% of total bacteria proteins. The recombinant E. coli M15(pQE40) were fermented in a 2.0 L fermentor to produce proinsulin RRhPI. We have obtained 51.82g wet E. coli and 16.23g wet inclusion bodies from 1 liter culture media, respectively. The inclusion bodies were removed from the bacterial pellet by empolying lysozyme / ultrasound to disintegrate the cells. Chromatography of renaturants inclusion bodies on DEAE-SepharoseFF resulted in the resolution of RRhPLAfter the second renaturation, RRhPI was refolded to natural structure with high efficiency. To find an optimal purification and renaturation strategy, effects of various key variables were examined. Then 79 mg human insulin in highly purified form could be obtained from 1 liter fermentation culture. The final products purified by Superdex 75 showed one band by SDS-PAGE analysis and were identical with the native human insulin by all critetia employed. (SDS-PAGE analysis, HPLC analysis, amino acid composition analysis and bioidentity assay.)...