人肿瘤坏死因子受体的配体胞外段的原核表达及分析

Expression and analysis of the extracellular domain of human glucocorticoid-induced tumor necrosis factor receptor ligand in Escherichia coli

摘要:

新近报道糖皮质激素诱导的肿瘤坏死因子受体的配体(GITRL)具有抑制前体破骨细胞的作用, 故命名为Osteostat, 为深入研究其功能和机制, 本研究原核表达人GITRL胞外段并进行活性分析。利用限制性内切酶Eco31I获得大肠杆菌偏嗜性GITRL的胞外段cDNA序列, 构建了基于pQE-30Xa的原核表达载体, 并在M15[pREP4]菌株中经IPTG诱导表达带有His融合标签的GITRL重组蛋白, 主要以包涵体形式存在。经体外包涵体变性、复性及纯化后, 采用SDS-PAGE和Western blotting进行分析和鉴定。同时建立了报告基因技术检测GITRL重组蛋白生物活性的方法, 简单便捷、灵敏而且周期短, 利用此方法分析了重组GITRL胞外段表达蛋白的生物活性。

Abstract:

GITRL (Glucocorticoid-induced tumor necrosis factor receptor ligand) has been recently identified as a novel inhibitor of osteoclastogenesis and hence called Osteostat. In this study, we expressed recombinant extracellular domain of GITRL protein in Escherichia coli and analyzed its bioactivity. Using an Eco31I enzyme-based restriction and ligation method, we obtained an E. coli-preferred DNA sequence coding for the extracellular domain of human GITRL. The DNA was cloned into expression vector pQE-30Xa that encodes a fusion tag of 6xHis before the insert. The resultant recombinant expression vector pQE/GITRL was subsequently transformed into E. coli strain M15[pREP4]. After induction with Isopropyl b-D-Thiogalactoside (IPTG), the cells produced the fusion protein mainly in the form of inclusion bodies as identified by SDS-PAGE. The recombinant protein was purified by affinity chromatography through Ni-NTA column and recognized by anti-His polyclonal antibody using Western blotting analysis. Moreover, we established a simple, efficient and sensitive reporter gene-based method to detect the activity of the recombinant protein. The results showed that the target protein was biologically active.