Research of drug screening model based on recombinant hPPARγ-LBD as a target protein for lipid metabolism

DUAN Wen; et al

doi:10.13406/j.cnki.cyxb.2010.06.052

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Research of drug screening model based on recombinant hPPARγ-LBD as a target protein for lipid metabolism

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- Research of drug screening model based on recombinant hPPARγ-LBD as a target protein for lipid metabolism
- Journal of Chongqing Medical University Vol. 35, Issue 6, Pages: 805-809(2010)
- 作者机构：
  
  1. 重庆医科大学药学院药物化学教研室
  2. 重庆市生物化学与分子药理学重点实验室
  3. 重庆市药物工程研究中心
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2010.06.052
  CLC： R965.1
- Published：2010，
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[1]段雯,李伟,靳红卫,夏铸,程训官,张昕宇,郑小红,余瑜.基于脂代谢靶点蛋白hPPARγ-LBD的抗肿瘤药物筛选模型的研究[J].重庆医科大学学报,2010,35(06):805-809.

DUAN Wen, et al. Research of drug screening model based on recombinant hPPARγ-LBD as a target protein for lipid metabolism. [J]. Journal of Chongqing Medical University 35(6):805-809(2010)
[1]段雯,李伟,靳红卫,夏铸,程训官,张昕宇,郑小红,余瑜.基于脂代谢靶点蛋白hPPARγ-LBD的抗肿瘤药物筛选模型的研究[J].重庆医科大学学报,2010,35(06):805-809. DOI： 10.13406/j.cnki.cyxb.2010.06.052.

DUAN Wen, et al. Research of drug screening model based on recombinant hPPARγ-LBD as a target protein for lipid metabolism. [J]. Journal of Chongqing Medical University 35(6):805-809(2010) DOI： 10.13406/j.cnki.cyxb.2010.06.052.

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

（DE

）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%

值为625nmol/L;与德氮吡格（Tetrazanbigen

TNBG）特异性结合率约60%

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。

Abstract

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.

关键词

脂代谢靶点蛋白hPPARγ-LBD抗肿瘤药物药物筛选模型罗格列酮德氮吡格

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