Research of drug screening model based on recombinant hPPAR?-LBD as a target protein for lipid metabolism
- VernacularTitle:基于脂代谢靶点蛋白hPPAR?-LBD的抗肿瘤药物筛选模型的研究
- Author:
Wen DUAN
- Publication Type:Journal Article
- Keywords:
Lipid metabolism;
Target protein;
hPPAR ?-LBD;
Antitumour drugs;
Drugscreening model;
Rosiglitazone;
Tetrazanbigen;
TNBG
- From:
Journal of Chongqing Medical University
2003;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a method of antitumor drug screening model based on recombinant hPPAR?-LBD as a target protein for lipid metabolism. Methods:pReceiver-B01-PPAR?- LBD expression plasmid was constructed and transferred into Escherichia coli BL2(1DE3)competent cells. Growth conditionswere optimized to induce soluble expression of hPPAR?-LBD recombinant protein. Purification was carried out by Nickel affinity chromatography. Ligand binding activity of the recombinant protein was determined by novel size exclusion chromatography and high performance liquid chromatography(SEC-HPLC)method with rosiglitazone as reference ligand and GW9662 as antagonist. Results:Under optimal conditions (16℃,0.6 mmol/L IPTG and induction time 20 h),the soluble recombinant protein was expressed successfully. After one-step purification with Nickel affinity chromatography,41 mg of recombinant protein with more than 95% purity could be obtained from per liter Luria-Bertani(LB)medium. The Kd and percentage ofspecific bindingofrosiglitazone and tetrazanbigen tohPPAR?-LBDare 625 nmol/L,65% and 1 000 nmol/L,60%,respectively.Conclusion:In this study,a fast,stable and simple method could be used successfully to screen antitumor drug.
