Soluble expression and purification of heparinase I in Escherichia coli using a hexahistidine-tagged small ubiquitin-like modifier as a fusion partner

摘要

ABSTRACT Heparinase has an important application in the preparation of low-molecular-weight heparins and the deheparinization of heparin-treated blood. To increase the soluble expression of heparinase I (HepI) in recombinant Escherichia coli , the hepA gene (coding for HepI) from Flavobacterium heparinum was obtained through chemical synthesis and fused with the gene encoding hexahistidine, small ubiquitin-like modifier (SUMO), a flexible peptide linker (G 4 S) and a bovine enterokinase site (D 4 K). The constructed fusion protein (SUMO?￠????HepI) was expressed in E. coli BL21 (DE3), and then purified with Ni 2+ -chelating affinity chromatography. The fermentation conditions were optimized and the enzymatic properties were also analyzed. As the results showed, the recombinant SUMO?￠????HepI was successfully expressed in E. coli BL21 (DE3) and purified with affinity chromatography. The recombinant protein reached the highest soluble expression when the expression strain was induced by 0.6????mmol/L isopropyl ???2-D-thiogalactoside and grown at 30 ???°C with a shaking speed of 150 r/min for 9 h. The fusion protein could exhibit high enzyme activity without requirements of in vitro refolding and SUMO-tag releasing process, and the optimum enzyme activity was obtained at 30 ???°C, pH 7.0 and 10????mmol/L Ca 2+ in the reaction buffer. This work provided a novel simple approach for the soluble expression of HepI in E. coli , and might establish a foundation for the following production and application of HepI in the industry.