Expression and analysis of the extracellular domain of human glucocorticoid-induced tumor necrosis factor receptor ligand in Escherichia coli / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 708-713, 2009.
Article
in Chinese
| WPRIM
| ID: wpr-286653
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Recombinant Proteins
/
Molecular Sequence Data
/
Base Sequence
/
Tumor Necrosis Factors
/
Escherichia coli
/
Extracellular Space
/
Genetics
/
Metabolism
Type of study:
Prognostic study
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2009
Type:
Article
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