摘要
目的:建立一种以脂代谢靶点蛋白hPPARγ-LBD重组蛋白进行抗肿瘤药物筛选的方法。方法:采用pReceiver-B01-PPARγ-LBD表达质粒转至E.coliBL_(21)(DE_3)细胞,进行表达与纯化得到可溶性靶点蛋白hPPARγ-LBD重组蛋白,以罗格列酮为hPPARγ-LBD的阳性配体,以GW9662作拮抗剂,采用分子排阻色谱-高效液相色谱法(SEC-HPLC)测定hPPARγ-LBD重组蛋白与配体药物的结合活性。结果:在优化条件(16℃、0.6mmol/LIPTG、诱导20h)下,能可溶性表达hPPARγ-LBD重组蛋白;经镍亲和色谱纯化后,以每升LB培养基计可获得41mg、纯度为95%的hPPARγ-LBD重组蛋白;该重组蛋白与罗格列酮特异性结合率约65%,K_d值为625nmol/L;与德氮吡格(Tetrazanbigen,TNBG)特异性结合率约60%,K_d值为1000nmol/L。结论:本文基于脂代谢靶点蛋白hPPARγ-LBD建立了抗肿瘤药物体外筛选模型的方法,该方法快速、稳定、安全及简便,能应用于抗肿瘤药物的筛选。
Objective：To establish a method of antitumor drug screening model based on recombinant hPPARγ-LBD as a target protein for lipid metabolism. Methods：pReceiver-B01-PPARγ- LBD expression plasmid was constructed and transferred into Escherichia coli BL2（1DE3）competent cells. Growth conditionswere optimized to induce soluble expression of hPPARγ-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 tohPPARγ-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.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2010年第6期805-809,共5页
Journal of Chongqing Medical University
基金
国家自然科学基金资助项目(编号:30371632和30772595)
