蛋白酶体激活因子REGγ的重组表达及其体外功能的初步探讨  

A preliminary study on recombinant expression and function in vitro of proteasome activator REGγ

作　　者：吴敏[1] 聂晶[2] 张令强[1,2] 汪渊[1] 

机构地区：[1]安徽医科大学分子生物学实验室和生物化学与分子生物学教研室,合肥230032 [2]军事医学科学院放射与辐射医学研究所,北京蛋白质组研究中心,蛋白质组学国家重点实验室,北京100850

出　　处：《军事医学科学院院刊》2010年第1期5-7,11,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：国家重大科学研究计划(2006CB910802);国家自然科学基金重点项目(30830029);国家重点实验室自主研究重点课题(SKLP-K200802)项目部分基金资助

摘　　要：目的蛋白酶体激活因子11S调节蛋白复合物γ亚单位(11S regulator complex gamma subunit,REGγ)的重组表达,初步探讨其与骨形成负调控分子酪蛋白激酶-2相互作用蛋白-1(caseinkinase-2interactingprotein-1,CK-IP-1)的相互作用。方法首先以质粒pCMV-Myc-REGγ为模板,通过聚合酶链反应(PCR)扩增出765bp的REGγcDNA片段,然后亚克隆到原核表达载体pGEX-4T-2并转化入大肠杆菌BL21经IPTG诱导表达,表达产物超声破碎后进行SDS-PAGE和Western印迹鉴定,进而通过GSTPull-down实验验证REGγ是否与CKIP-1存在相互作用。结果通过测序证实原核表达载体pGEX-4T-2-REGγ构建成功,进一步表达鉴定发现GST-REGγ蛋白主要以可溶性的形式存在于裂解上清中;GSTPull-down实验表明REGγ与CKIP-1存在明显的相互作用。结论REGγ与CKIP-1在体外存在相互作用,为进一步深入研究REGγ对CKIP-1的调控作用奠定了基础。Objective To study the expression of the fused proteasome activator REGγ（11S regulator complex gamma subunit） using gene recombination technology and to further study the interaction between REGγ and casein kinase-2 interacting protein-1（CKIP-1）in vitro.Methods Firstly,the full length cDNA fragment of REGγ was amplified through PCR using the plasmid pCMV-Myc-REGγ as template and subcloned into the prokaryotic expression vector pGEX-4T-2 before being transformed into E.coli BL21 cells.The protein expression was induced by isopropyl-β-D-thiogalactoside（IPTG）.Secondly,the protein expression was monitored by SDS-PAGE and Western blotting after ultrasonication.Finally,the GST Pull-down assay was performed to investigate the interaction between REGγ and CKIP-1 in vitro.Results The prokaryotic expression construct pGEX-4T-2-REGγ was generated successfully and confirmed by DNA sequencing.Expression analysis showed that the GST-REGγ protein was easily expressed and isolated mainly in the lysate supernatant after sonication and centrifugation.The GST Pull-down assay revealed the strong mutual interaction between REGγ and CKIP-1 in vitro.Conclusion The proteasome activator REGγ could interact with the negative regulator of osteoblastogenesis CKIP-1 in vitro and the current study has shed light on further investigations of their physiological relevance.

关 键 词：重组表达 质粒 蛋白酶体激活因子REGγ 酪蛋白激酶-2相互作用蛋白-1 聚合酶链反应 

分 类 号：Q78[生物学—分子生物学]