蛋白激酶B的PH结构域可溶性表达与纯化及其二级结构分析  

Soluble Expression and Purification of PKB PH Domain and Analysis of Its Secondary Stucture

作　　者：沈岚[1] 季少平[1] 刘新平[1] 王吉村[1] 王立峰[1] 苏金[1] 药立波[1] 

机构地区：[1]第四军医大学生物化学与分子生物学教研室,西安710032

出　　处：《中国生物化学与分子生物学报》2003年第3期383-387,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家杰出青年科学基金 (No.3 982 5 113 );国家重点基础研究发展规划 ( 973 )项目 (No .J19990 75 6)资助~~

摘　　要：The serine/threonine protein kinase B(PKB) is related to cellular survival and regulation. PKB is composed of PH domain, catalytic domain and carboxyl terminal regulator domain. The PH domain of PKB is crucial to the activation of kinase. In order to investigate the function and the structure function relationship of PKB, the cDNA coding fragment of PKB PH domain was amplified from human dental pulp mRNA by RT PCR and cloned into pMD18 T vector to analyze the sequence. The result showed that DNA sequence of cloned human PKB PH domain was consistent with that reported previously. To express PKB PH domain, the cDNA was subcloned into expression vector pRSET A which was then transformed into E.coli BL21(DE3) pLysS, and the strain highly expressing soluble 6His PKB PH domain in minimal medium was obtained. The fusion protein was purified by Ni 2+ NTA agarose beads. The secondary structure of the purified 6His PKB PH domain fusion protein was analysed by circular dichroism. The results indicated that the PH domain was composed of α helix 1 7%,β pleated sheet 80 5% and radom coli 17 8%.The serine/threonine protein kinase B(PKB) is related to cellular survival and regulation. PKB is composed of PH domain, catalytic domain and carboxyl terminal regulator domain. The PH domain of PKB is crucial to the activation of kinase. In order to investigate the function and the structure function relationship of PKB, the cDNA coding fragment of PKB PH domain was amplified from human dental pulp mRNA by RT PCR and cloned into pMD18 T vector to analyze the sequence. The result showed that DNA sequence of cloned human PKB PH domain was consistent with that reported previously. To express PKB PH domain, the cDNA was subcloned into expression vector pRSET A which was then transformed into E.coli BL21(DE3) pLysS, and the strain highly expressing soluble 6His PKB PH domain in minimal medium was obtained. The fusion protein was purified by Ni 2+ NTA agarose beads. The secondary structure of the purified 6His PKB PH domain fusion protein was analysed by circular dichroism. The results indicated that the PH domain was composed of α helix 1 7%,β pleated sheet 80 5% and radom coli 17 8%.

关 键 词：蛋白激酶B PH结构域 可溶性表达 纯化 二级结构 

分 类 号：Q55[生物学—生物化学] Q257