文献类型：期刊文献

中文题名：重组谷胱甘肽-S-转移酶-肝素酶Ⅰ在大肠杆菌中的表达、纯化及其性质

英文题名：Expression,Purification and Characterization of Recombinant GST-haparinaseⅠ from Flavobacterium heparinum in Escherichia coli

作者：张云康[1];高兴[1];汪德健[1];王富军[2];赵健[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237;[2]上海中医药大学,上海201203

年份：2011

卷号：37

期号：4

起止页码：458

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;Scopus;北大核心:【北大核心2008】；CSCD:【CSCD2011_2012】；

基金：上海市重点学科建设项目资助(B505)

语种：中文

中文关键词：肝素黄杆菌;谷胱甘肽-S-转移酶;肝素酶Ⅰ;融合表达;亲和纯化

外文关键词：Flavobacterium heparinum; glutathione-S-transferase（GST）; heparinase I; fusion expression; affinity chromatography

摘要：肝素酶Ⅰ(HeparinaseⅠ,HepⅠ)是一种多糖裂解酶,可特异性裂解硫酸肝素分子中糖苷键以制备低分子质量肝素(LMWH)。由于重组肝素酶Ⅰ在大肠杆菌中表达时极易形成包涵体,将肝素酶Ⅰ基因的N端融合谷胱甘肽-S-转移酶(GST)标签,在大肠杆菌中进行低温表达。结果显示,约90%的GST-HepⅠ融合蛋白以可溶性形式表达,表达的目标蛋白粗酶经过一步亲和纯化,比活为124.7 U/mg,纯化倍数达到366.7倍,酶活回收率为31.0%;结果表明Ca2+对重组GST-HepⅠ有很强的激活作用,GPC-HPLC结果显示重组GST-HepⅠ与天然肝素酶Ⅰ裂解图谱相同。
HeparinaseⅠis an enzyme that specifically cleaves certain sequences of heparan sulfate.Previous reports showed that this enzyme was highly prone to aggregation in inclusion bodies when it was expressed in Escherichia coli.In this paper,the N-terminus of heparinaseⅠgene was fused a glutathione-S-transferase（GST） tag,which expressed the fusion protein in Escherichia coli.The results showed when induced at 15 ℃,approximately 90% of the fusion protein was found to be produced in the soluble.The enzyme has a specific activity of 124.7 U/mg protein by one-step affinity chromatography,the recovery was 31.0%,and a 366.7-fold-purification was achieved.GST-Hep I was well activated by Ca2＋.Results of GPC-HPLC（Gel permeation chromatography high-performance liquid chromatography） analysis of oligosaccharides of heparin degraded by GST-HepⅠ was similar to those of native Hep Ⅰ.