[Prokaryotic expression vector construction, expression and polyclonal antibody preparation of the fusion protein of glutathione S-transferase and peroxisome proliferator-activated receptor-gamma coactivator-1].
OBJECTIVE
To express the fusion protein of glutathione S-transferase (GST) and peroxisome proliferator-activated receptor-gamma coactivator-1 (PPARgammaC1) in E. coli. and prepare the polyclonal antibody against PPARgammaC1.


METHODS
The coding sequence of PPARgammaC1 gene was amplified by reverse transcriptase-PCR (RT-PCR) from the total RNA of Hep G2 cells and inserted into pGEX-4T-1 vector. The recombinant vector was identified by restriction endonuclease digestion analysis and the fusion protein GST-PPARgammaC1 was expressed in E. coli. via IPTG induction. The expressed fusion protein was purified by glutathione-agarose affinity chromatography and used to immunize the egg-laying hens for preparing the polyclonal antibody against GST-PPARgammaC1.


RESULTS
Restriction endonuclease digestion analysis demonstrated that the PPARgammaC1 gene had been correctly inserted into pGEX-4T-1 vector, and the expressed fusion protein had a relative molecular mass of approximately 39,000 as shown by SDS-PAGE. The polyclonal antibody obtained from the egg yolk immunoglobulins was found to specifically bind to purified PPARgammaC1 in Western blot analysis.


CONCLUSIONS
The successfully prepared polyclonal antibody against PPARgammaC1 peptide provides a useful reagent for PPARgammaC1 detection.
Zhang
Yan
Y
Department of Endocrinology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. xinchristina@163.com
Xue
Yao-ming
YM
Guo
Ai-lin
AL
Zhang
Wen-min
WM
D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D006801:Humans; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D011387:Prokaryotic Cells / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D006360:Heat-Shock Proteins; C486497:PPARGC1A protein, human; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase