Expression and analysis of the extracellular domain of human glucocorticoid-induced tumor necrosis factor receptor ligand in Escherichia coli.
- Author:
Yanli JIAO
1
;
Fang ZHENG
;
Xiaoxia LI
;
Baoli WANG
;
Shanyi GUO
Author Information
1. Institute of Endocrinology, Tianjin Medical University, Tianjin 300070, China.
- Publication Type:Journal Article
- MeSH:
Base Sequence;
Escherichia coli;
genetics;
metabolism;
Extracellular Space;
metabolism;
Humans;
Molecular Sequence Data;
Recombinant Proteins;
analysis;
biosynthesis;
genetics;
Tumor Necrosis Factors;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2009;25(5):708-713
- CountryChina
- Language:Chinese
-
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 beta-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.
