热休克蛋白质DnaK的纯化及其二聚体性质研究  

Purification of DnaK Mutants and the Characteristics of DnaK Dimer

作　　者：刘清岱[1,2] 苏家跃 田雪利 杨营[1] 赵飞[1,2] 盛长忠[1,3] 

机构地区：[1]天津科技大学食品工程与生物技术学院,天津300457 [2]天津天隆种业科技有限公司,天津300457 [3]丹娜(天津)生物科技有限公司,天津300457

出　　处：《天津科技大学学报》2016年第3期11-15,共5页Journal of Tianjin University of Science & Technology

基　　金：国家自然科学基金资助项目(31571029);天津市应用基础与前沿技术研究计划资助项目(15JCYBJC30300;15JCQNJC46300;15JCTPJC55400)

摘　　要：通过定点突变技术构建了两个DnaK蛋白质突变体,研究腺嘌呤核苷三磷酸(ATP)和腺苷二磷酸(ADP)条件对热休克蛋白质DnaK二聚体性质的影响.首先诱导表达DnaK蛋白质的两个突变体DnaK-A303C和DnaK-H541C,并采用硫酸镍亲和层析和阴离子交换层析对重组蛋白质进行纯化.对等量的DnaK蛋白质进行氧化交联处理,然后采用聚丙烯酰胺凝胶电泳(SDS-PAGE)检测ATP、ADP对DnaK二聚体的影响.研究表明DnaK突变体在ADP的存在下以同二聚体的形式存在,但是在ATP条件下能够形成异二聚体.由此为深入认识热休克蛋白质两个亚基之间的协同作用提供了实验依据.Two DnaK mutants were constructed by site-directed mutagenesis to study the effect of ATP or ADP on heat shocked protein.DnaK-A303C and DnaK-H541C were expression through induction.Then the recombined proteinswere purified by using sulfate nickel affinity chromatography and anion exchange chromatography.The same amount of proteins was oxidized and the protein cross-linking was detected by SDS-PAGE.The results show that DnaK mutants formed dimmer in the presence of ADP.However,the heterodimer was found under the condition of ATP.This study provides an experimen-tal basis for understanding the synergism between the two subunits of heat shocked protein.

关 键 词：DNAK ATP 二聚体 突变体 

分 类 号：TS201[轻工技术与工程—食品科学]