人MCM7 N-端融合蛋白的原核表达、纯化及其与AR蛋白相互作用的研究

中国生物工程杂志

2010, Vol. 30

Issue (02): 44-48

研究报告

人MCM7 N-端融合蛋白的原核表达、纯化及其与AR蛋白相互作用的研究

师以康^1**,余金喜²,顾黎¹

1.山东大学国家糖工程技术研究中心济南 250012
2.山东省枣庄矿业集团中心医院枣庄 277011

Expression, Purification of N-terminus Fusion Human MCM7 and a Study on Its Interaction with AR

1.National Glycoengineering Research Center, Shandong University, Jinan 250012, China
2.Central Hospital of Zaozhuang Coal Mining Group Co. Ltd, Zaozhuang 277011, China

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摘要：

目的：制备人MCM7 N-端的GST融合蛋白（GST-MCM7N），研究其是否和雄激素受体（AR）蛋白存在直接的相互作用。方法：利用RT-PCR获得长度为744 bp的人mcm7 基因N-端cDNA碱基片段，把该片段构建到原核表达载体pGEX-5x-3中，转化大肠杆菌BL-21菌株。经IPTG诱导菌株基因表达产生了GST-MCM7N融合蛋白；使用Glutathione Sepharose 4B球珠分离纯化。利用GST pull-down 技术把纯化的融合蛋白分别和前列腺癌细胞LNCaP细胞裂解液及基因工程获得的AR蛋白孵育，检测MCM7蛋白的 N-端是否和AR蛋白直接相互作用。结果：酶切鉴定及基因测序表明，mcm7 基因cDNA的 N-端片段被构建到表达载体pGEX-5x-3中；SDS-PAGE及Western blotting结果分别显示，本研究获得了GST和GST-MCM7N融合蛋白；GST pull-down 结果证实GST-MCM7N和AR蛋白存在相互作用。结论：MCM7蛋白的N-端和AR蛋白至少在体外可以发生直接的相互作用。

关键词： MCM7; AR; 蛋白相互作用; GST融合蛋白

Abstract:

744 bp of N- terminal of mcm7 gene was amplified by PCR and was cloned into prokaryotic expressing vector pGEX-5x-3 to construct pGEX-5x-3/MCM7N. A batch of E.coli BL-21 were transformed with pGEX-5x-3 and pGEX-5x-3/MCM7N, respectively. GST protein and GST-MCM7N fusion protein were obtained from the recombinant E. coli BL-21 after IPTG induction and were purified with Glutathione Sepharose 4B. The fusion proteins with 54 kDa molecular weight were specifically recognized by both anti-GST antibody and anti-MCM7 antibody in Western blotting analyses. GST pull-down analysis showed that GST-MCM7N fusion proteins interacted directly with androgen receptor (AR) protein after GST-MCM7N incubated with prostate cancer LNCaP cell lysate and His-AR fusion protein, respectively. The result demonstrated that the N- terminal of MCM7 protein can interact directly with AR in vitro.

Key words: MCM7 AR Protein interaction GST fusion protein

收稿日期: 2009-06-12 出版日期: 2010-02-26

基金资助:

山东省优秀中青年科学家科研奖励基金资助项目（2008BS02003）

通讯作者: 师以康 E-mail: shiyikang@yahoo.com

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引用本文:

师以康余金喜顾黎. 人MCM7 N-端融合蛋白的原核表达、纯化及其与AR蛋白相互作用的研究[J]. 中国生物工程杂志, 2010, 30(02): 44-48.

SHI Si-Kang, TU Jin-Chi, GU Li. Expression, Purification of N-terminus Fusion Human MCM7 and a Study on Its Interaction with AR. China Biotechnology, 2010, 30(02): 44-48.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I02/44

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