人GST-PTEN融合蛋白的原核表达和纯化  

Expression and Purification of Human GST-PTEN Fusion Protein

作　　者：冯智慧 焦淑贤 李长缨 胡彬 刘晓华 

机构地区：[1]青岛市中心血站青岛市输血医学研究所,山东省青岛市266028

出　　处：《医学分子生物学杂志》2008年第4期296-298,共3页Journal of Medical Molecular Biology

基　　金：山东省医药卫生科技发展计划(No.2005034)~~

摘　　要：目的构建人第10号染色体上磷酸酶和张力蛋白同源缺失的基因(phosphatase and tensin homology deleted on chromosome ten,PTEN)原核表达质粒,使其在原核细胞中高效表达并进行纯化。方法将全长片段插入原核表达载体pGEX-4T-1,构建重组子pGEX-4T-1-PTEN,转化BL-21感受态细胞。经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达PTEN融合蛋白的可诱导性表达,通过SDS-PAGE电泳、Western印迹分析证实蛋白表达的特异性。并用谷胱甘肽-S-转移酶(glutathione-Stransferase,GST)亲和层析对融合蛋白进行纯化。结果成功构建了原核表达载体pGEX-4T-1-PTEN,并将PTEN融合蛋白的成功表达,通过SDS-PAGE电泳、Western印迹分析,证实了蛋白表达的特异性。并对蛋白进行了纯化,获得了GST-PTEN融合蛋白的纯品。结论成功表达、纯化了GST-PTEN融合蛋白,为进一步研究PTEN蛋白的功能及其与其它功能性蛋白的相互作用研究奠定了基础。Objective To construct a prokaryotic expression vector efficiently expressing PTEN and purify PTEN fusion protein. Methods The PTEN gene was inserted into the prokaryotic GST fusion protein expression plasmid pGEX-4T-1, to form pGEX-4T-1-PTEN. The recombinant plasmid was transformed into E. coli BL21. After the PTEN protein was induced with IPTG, its expression was analyzed by SDS-PAGE and western blotting. GST-PTEN fusion protein was purified with Glutathion-Sepharose 4B beads. Results The prokaryotic cell expression vector pGEX-4T-1-PTEN was successfully constructed. Highly expressed and purified GST-PTEN fusion protein was obtained. The specificity of fusion protein was verified by SDS-PAGE and western blotting. Conclusion The preparation of GST-PTEN fusion protein can be used to study the biological function of PTEN and its interaction with other proteins.

关 键 词：磷酸酶和张力蛋白同源缺失的基因 原核表达 融合蛋白 蛋白纯化 

分 类 号：R341[医药卫生—基础医学]