Expression And Characterization Of Heparan Sulfate3-O Sulfotransferase V In Escherichia Coli

Heparan sulfate3-O-sulfotransferase isoform V （3-OST-V） is a key enzyme in the process ofenzymatic synthesizing low molecular weight heparin. As a bifunctional enzyme,3-OST-V generatesboth an antithrombin-binding site to exhibit anticoagulant activity and a binding site for herpessimplex virus I glycoprotein D to serve as an entry receptor for herpes simplex virus. In this paper,the enzyme was expressed in Escherichia coli heterologously, then its expression conditions wereoptimized, and its characterization was also studied preliminary. The recombinant strain E.coliBL21/pET15b/3-OST-V was Successfully connstructed, which carried the target gene3-OST-V, thehost strain E. coli BL21（DE3） and the expression vector pET-15b. After induced the recombinantbacteria by IPTG, we got the target protein. And the activity of crude enzyme is0.11U/mg,indicating the successful expression of exogenous protein-heparan sulfate3-OST-V.In order to investigate the optimal conditions of producing3-OST-V by recombinant bacteriaBL21/pET15b/3-OST-V, the combination Single Factor and orthogonality methods were designedand employed for the fermentation conditions. The optimal conditions of cultivation were as follows:100mL culture in500mL shake flask, the OD600was1.3, the final concentration of IPTG was0.2mmol/L, induced temperature was20℃, and the cultivated time was6h. Under the optimalcultivation conditions the activity of3-OST-V reached42.17U/mL.3-OST-V was purified by Ni+affinity chromatography, and identified by SDS-PAGE. Themolecular weight estimated by SDS-PAGE was31kDa. And the specific activity of3-OST-V is0.58U/mg, which is5.27-fold than crude enzyme.On this basis, a preliminary study on the enzymatic characterization of purified3-OST-V. Theresults showed that the optimal temperature was35℃, temperature stability was good when thetemperature below40℃. And the optimal pH was7.0. The enzyme was stable in the range from pH7.0to pH9.0. A final concentration of1mmol/L K⁺, Ca²⁺, Ba²⁺can activate the enzyme.Considering the characterization of AST-IV and3-OST-V, The reaction system of determining3-OST-V activity were as follows: pH7.0, reaction temperature30℃, Ca²⁺and Ba²⁺with a finalconcentration of1mmol/L.In this study,3-OST-V which was a key enzyme in the biosynthetic of heparin was expressedheterologous, then its inducible conditions were optimized, the expressed product was purified andits characterization were studied preliminary. It laid a certain foundation for large-scale synthesis3-OST-V, provided the necessary enzyme for large-scale synthesis artificial heparan sulfate, and alsoprovided the reaction system of synthetic heparan sulfate.