含RIZ1 PR结构域融合蛋白的原核表达、提纯与功能分析-东南大学学报医学版

含RIZ1 PR结构域融合蛋白的原核表达、提纯与功能分析

单位：1.东南大学基础医学院,病理学与病理生理学系,江苏,南京,210009;加拿大Saskatchewan大学医学院,癌症研究中心,Saskatchewan Saskatoon,S7N 4H4； 2.东南大学基础医学院,病理学与病理生理学系,江苏,南京,210009； 3.加拿大Saskatchewan大学医学院,癌症研究中心,Saskatchewan Sas

关键词：RIZ1蛋白 PR结构域表达载体组蛋白甲基化作用

分类号：Q78, R730.231

出版年·卷·期（页码）：2006·25·第四期（240-243）

摘要：

目的:表达与提纯RIZ1第1个至第200个氨基酸含PR结构域的活性GST融合蛋白质(GST-PR200融合蛋白).方法:设计引物以含人RIZ1 cDNA全序列的pRIZ1 RH质粒为模板克隆获得目的基因片段,测序核对后经EcoRⅠ与XhoⅠ双酶切,以正确阅读框架插入相同酶切的pGEX-6P-3中,经转染与IPTG诱导表达筛选到阳性大肠杆菌克隆并提纯该融合蛋白质.分别采用SDS-PAGE蛋白电泳法、质谱分析法及组蛋白甲基化转移酶(HMT)测定法鉴定其性质与功能.结果:获得的纯化融合蛋白质相对分子质量约为47 000,其CPM值明显高于对照组(P<0.01),且与作用时间呈正相关.结论:所构建的融合蛋白原核表达系统能有效表达活性GST-PR200融合蛋白,可供大量提取作进一步研究.

Objective To express fusion protein of GST and 1-200 residue region containing PR domain of human RIZ1 protein(GSTPR200 fusion protein).Methods pRIZ1 RH plasmid which contains full-length human RIZ1 cDNA was used as a template for PCR.Then for correct reading code frame,the sequence-checked fragment was cleaved with EcoRⅠ/XhoⅠ,melted and linked with pGEX-6P-3 expression victor.Positive clones was identified with IPTG-induced expression and the fusion protein was purified from transformed E.coli cells.Results Both SDS PAGE analysis and mass spectrometry analysis showed that the relative molecular mass of the fusion protein was about 47 000.Histone methyltransferase(HMT) assay indicated that the protein had the HMT activity.Conclusion The prokaryotic expression system of the fusion protein constructed in this study provides the basis of a large scale expression and purification of GST-PR200 fusion protein for further study.

参考文献：

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