GST-His-Heparinase Ⅰ双标签融合蛋白的原核表达与纯化  

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

作　　者：刘卫超[1] 程咏梅[1] 邓超[2] 丁建文 宋志新 陈敬华[1] 

机构地区：[1]江南大学药学院,江苏无锡214122 [2]江南大学无锡医学院,江苏无锡214122 [3]常山生化药业(江苏)有限公司,江苏常州213149

出　　处：《工业微生物》2014年第5期51-57,共7页Industrial Microbiology

基　　金：教育部新世纪优秀人才支持计划(No.NCET-10-0435);教育部博士点基金(No.20110093110008)资助

摘　　要：以pET15b-HepⅠ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.coli BL21(DE3)感受态细菌,经IPTG诱导表达。表达产物可溶部分用GSTrap FF和HisTrap HP柱两步亲和纯化,所得产物经SDS-PAGE检测,在66 kDa和43 kDa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86.45 IU/mg,纯度高达99%,与仅一步亲和纯化得到的GST-His-HepⅠ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。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.

关 键 词：肝素酶I GST.His双标签 原核表达 纯化 

分 类 号：Q78[生物学—分子生物学]