GST-His-Heparinase I双标签融合蛋白的原核表达与纯化

以pETl5b-Hep I为模板，通过PCR技术扩增出上游合有6×His标签的HepI基因序列，克隆至表达载体pGEX-4T-1。测序鉴定后，将重组表达质粒pGEX．His．HepI转入E．coliBL21（DE3）感受态细菌，经IPTG诱导表达。表达产物可溶部分用GSTrapFF和HisTrapHP柱两步亲和纯化，所得产物经SDS—PAGE检测，在66kDa和43kDa处显示特异条带，分别与GST．His．HepI和His-HepI融合蛋白预期分子量相符；最终His—HepI融合蛋白的比酶活为86．45IU／mg，纯度高达99％，与仅一步亲和纯化得到的GST．His—Hep I融合蛋白相比，进一步提高了纯化后重组肝素酶的纯度。本研究为制备高纯度的HepI提供了一种方法，对制备高安全性的LMWH和解析HepI晶体结构具有重要意义。

Prokaryotic expression and purification of GST-His-Heparinase I double labelled fusion protein

The DNA sequence of heparinase I with 6 × His tag added in the gene upstream was amplified with PCR from pET15b-Hep I, and then cloned into the expression vector pGEX-4T-1. The reeombined plasmid was transformed into E. coli BL21 （DE3） and induced by IPTG. The fusion protein was purified by GSTrap FF column followed by HisTrap HP column. The SDS-PAGE result of the fusion protein showed specific bands at the size of 66 kDa and 43 kDa which were consistent with theoretical value of GST-His-Hep I and His-Hep I , respectively. The specific activity of His-Hep was measured as 86.45 IU/mg, and its purity was calculated to be 99% which was further improved compared with GST-His- Hep I. This study provided a way for obtaining high purity Hep I and thus had significant potential application for the production of low molecular weight heparin.