[Expression and analysis of the extracellular domain of human glucocorticoid-induced tumor necrosis factor receptor ligand in Escherichia coli].
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.
Jiao
Yanli
Y
Institute of Endocrinology, Tianjin Medical University, Tianjin 300070, China.
Zheng
Fang
F
Li
Xiaoxia
X
Wang
Baoli
B
Guo
Shanyi
S
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D048069:Tumor Necrosis Factors / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; C478967:TNFSF18 protein, human; D048069:Tumor Necrosis Factors