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

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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

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

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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。

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.

导出引用

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[J]. China Biotechnology, 2010, 30(02): 44-48

参考文献

［1］ Costa A, Onesti S. The MCM complex: (just) a replicative helicase? Biochem Soc Trans, 2008, 36(Pt 1):136140.
［2］ Labib K, Tercero J A，Difley J F．Uninterrupted MCM27 function required for DNA replication fork progression. Science, 2000, 288(5471):16431647.
［3］ Ren B, Yu G, Tseng G C, et al. MCM7 amplification and overexpression are associated with prostate cancer progression. Oncogene, 2006, 25:10901098.
［4］ Fujioka S, Shomori K, Nishihara K, et al. Expression of minichromosome maintenance 7 (MCM7) in small lung adenocarcinomas (pT1): Prognostic implication. Lung Cancer, 2009, 65(2):223229.
［5］ Nishihara K, Shomori K, Fujioka S, et al. Minichromosome maintenance protein 7 in colorectal cancer: implication of prognostic significance. Int J Oncol, 2008, 33(2):245251.
［6］ Nieto M, Finn S, Loda M, et al. Prostate cancer: Refocusing on androgen receptor signaling. Int J Biochem Cell Biol, 2007, 39(9):15621568.
［7］ Golias C H, Iliadis I, Peschos D, et al. Amplification and coregulators of androgen receptor gene in prostate cancer. Ex POncol, 2009, 31(1):38.
［8］ Honeycutt K A, Chen Z, Koster M I, et al. Deregulated minichromosomal maintenance protein MCM7 contributes to oncogene driven tumorigenesis. Oncogen, 2006, 25(29):40274032.
［9］ Koch H B, Zhang R, Verdoodt B, et al. Largescale identification of cMYCassociated proteins using a combined TAP/MudPIT approach. Cell Cycle, 2007, 6(2):205217.
［10］ Buchsbaum S, Morris C, Bochard V, et al. Human INT6 interacts with MCM7 and regulates its stability during S phase of the cell cycle. Oncogene, 2007, 26(35):51325144.
［11］ Tsao C C, Geisen C, Abraham R T. Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling. EMBO J, 2004, 23(23):46604669.
［12］ Tsvetkov L, Stern D F. Interaction of chromatinassociated Plk1 and Mcm7. J Biol Chem, 2005, 280(12):1194311947.
［13］ Ryu M J, Kim B J, Lee J W, et al. Direct interaction between cohesin complex and DNA replication machinery. Biochem Biophys Res Commun, 2006, 341 (3): 770775.
［14］韩昱晨，官忠燕，段红梅，等．酵母双杂交技术筛选肺癌组织中MCM7蛋白结合蛋白，中国肺癌杂志， 2008，11（4）：509512. Han Y C, Guan Z Y, Duan H M, et al. Chineses Journal of Lung Cancer, 2008,11(4): 509512.
［15］ Kraus S, Gioeli D, Vomastek T, et al. Receptor for activated C kinase 1 (RACK1) and Src regulate the tyrosine phosphorylation and function of the androgen receptor, Cancer Res, 2006, 66(22):1104711054.