Enhanced soluble expression and improved catalytic efficiency of heparinase II by fusion of GST tag to the backside of the active pocket

Abstract

Although heparinase II has been widely used in the structural analysis of heparin and HS, it suffers from low activity and inefficient expression and purification. In this study, heparinase II from Mariniphaga anaerophila (MaHepII) was cloned, and the GST tag was fused to the backside of the enzyme's active pocket. The fusion protein GST-MaHepII was efficiently expressed in the recombinant Escherichia coli BL21. Afterward, Ni-NTA affinity chromatography was used to purify GST-MaHepII, with an enzymatic activity recovery yield of 70.3 % and a specific activity of 7.5 U mg⁻¹ toward heparin. When HS was used as the substrate, the enzyme exhibited a specific activity of 11.1 U mg⁻¹. Additionally, the influence of the GST tag on the enzymatic properties of MaHepII was evaluated. After removal of the GST tag by proteolytic cleavage, MaHepII was purified by GSH affinity chromatography. Characterization of the enzymatic properties indicated that the GST tag improved the catalytic efficiency of MaHepII by 3.1-fold toward heparin and 1.4-fold toward HS. Molecular docking revealed that the GST tag fusion might increase the flexibility of the protein structure and alter the size or shape of the substrate channel. These changes facilitate substrate access to the active site and enhance the catalytic efficiency of MaHepII.