组蛋白脱乙酰化酶4 n- 末端和c- 末端片段的原核表达及纯化.pdf

窑712 窑 南方医科大学学报(JSouthMedUniv) 2010;30(4) 组蛋白脱乙酰化酶4N- 末端和C-末端片段的原核表达及纯化 杨 洋袁覃小翠袁刘书虎袁黄威袁王雪敏渊南方医科大学基础医学院神经生物学教研室袁广东 广州 510515冤 摘要院目的在大肠杆菌中分别表达组蛋白脱乙酰化酶4渊HDAC4冤N- 末端渊1-1950bp冤和C-末端渊1708-3255bp冤片段与 谷胱甘肽-S转移酶渊GST冤的融合蛋白袁并进行纯化遥 方法应用PCR 方法扩增HDAC4N- 末端和C-末端片段袁将目 的基因插入GST 融合载体pGEX-6P-1 中袁 重组载体在酶切鉴定和序列测定后袁 在大肠杆菌中经IPTG 诱导表达 GST-HDAC4-N 和GST-HDAC4-C蛋白遥经SDS及Westernblotting鉴定表达产物后袁用谷胱苷肽琼脂糖珠纯化 目的蛋白遥 结果经测序尧酶切鉴定证明成功构建了融合蛋白表达质粒袁表达出的融合蛋白经SDS分析其相对 分子量约为 110500和93080遥Westernblotting 分析证实融合蛋白可以被HDAC4 特异性抗体所识别遥 结论成功构建 了融合表达载体渊pGEX-6P-1/HDAC4-N 和pGEX-6P-1/HDAC4-C冤袁并进行了融合蛋白的诱导表达和鉴定袁可以进一步 用于HDAC4 与其他蛋白质相互作用的研究遥 关键词院组蛋白脱乙酰化酶4曰原核表达曰融合蛋白 中图分类号院R34 文献标识码院A 文章编号院1673-4254(2010)04-0712-04 ProkaryoticexpressionandpurificationofN-terminalandC-terminalfragmentsofhistone deacetylase4 YANGYang,QINXiao-cui,LIUShu-hu,HUANGWei,WANGXue-min DepartmentofNeurobiology,SouthernMedicalUniversity,Guangzhou510515,China Abstract: Objective Toexpre andpurifythefusionproteinsofglutathioneS-transferase (GST)-N-terminalofhistone deacetylase4(HDAC4-N)(1-1952bp)andGST-C-terminalofHDAC4(HDAC4-C)(1708-3255bp)inE.coli.MethodsThe DNAfragment s(HDAC4-NandHDAC4-C) amplifiedbyPCRwereligatedintoGSTfusionvector (pGEX-6P-1) to constructtherecombinantplasmids. AfteridentificationwithrestrictiondigestionandDNAsequencing, therecombinant plasmidsweretransformedinto E.coliBL21andinducedbyIPTGfortheirexpre ion.AfteridentificationbySDSand Westernblotting,thetargetproteinswerepurifiedbyglutathionesepharose4B. ResultsTheresultsofrestrictiondigestionand DNAsequencingconfirmedsucce fulconstructionoftherecombinantplasmids. Therelativemolecularma esofthefusion proteinswereapproximately110500 and 93080a hown bySDS. Westernblottingdemonstratedthat thefusion protein scould be recognized by the specific anti-HDAC4 antibody. Conclusion We have succe full