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

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 (G4S) and a bovine enterokinase site (D4K). The constructed fusion protein (SUMO–HepI) was expressed in E. coli BL21 (DE3), and then purified with Ni2+-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 β-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 Ca2+ 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.