Optimization And Purification Of Three Recombinant Heparinase

The purpose of the present work is to increase the expression level of soluble protein in E.coli. We choose three heparinases as our target protein to increase the soluble productivity in E.coli.Firstly, the expression vector pET-15b-HepI was transformed into E. coli BL21(DE3)/pOFX-T7-SL1. The effects of different expression conditions were systematically investigated to improve the enzymatic activity of Hepl. The optimal conditions were determined as follows:cultivation at25℃in MBL medium, induction at6h after inoculation with0.25mM IPTG, and postinduction expression for8h. Under these conditions, the volumetric enzymatic activity of Hep Ⅰ reached1.49×105IU/L. The recombinant E.coli was further cultivated in a fermentor. As a result, total activity of HepⅠ reached5.55×105IU/L after20h fermentation. One affinity chromatography step was employed to obtain high-purity (96%) with a recovery ratio of37.58%.Secondly, the expression vector pET-19b-HepⅡ was transformed into E. coli BL21(DE3). The effects of different expression conditions were systematically investigated to improve the enzymatic activity of HepⅡ. The optimal conditions were determined as follows:cultivation at25℃in MBL medium, induction at6h after inoculation with0.5mM IPTG, and postinduction expression for6h. Under these conditions, the volumetric enzymatic activity of Hep Ⅱ reached8.70×103IU/L. The recombinant E.coli was further cultivated in a fermentor. As a result, total activity of HepⅠ reached2.35×104IU/L after18h fermentation. One affinity chromatography step was employed to obtain high-purity (90%) with a recovery ratio of47%.At last, the expression vector pET-19b-HepⅢ was transformed into E. coli BL21(DE3). The effects of different expression conditions were systematically investigated to improve the enzymatic activity of HepⅢ. The optimal conditions were determined as follows:cultivation at20℃in MBL medium, induction at6h after inoculation with0.1mM IPTG, and postinduction expression for6h. Under these conditions, the volumetric enzymatic activity of Hep Ⅲ reached4.02×103IU/L. One affinity chromatography step was employed to obtain high-purity (87%) with a recovery ratio of42%.This study indicated that effective expression of Heparinase by the present system would facilitate the large scale preparation of low molecular weight heparin.