基因表达_____PKB_Akt_；克隆,分子；

PKB/Akt 调节结构域的克隆，表达及初步纯化

http://www.100md.com 《第四军医大学学报》 2003年第6期

基因表达,,PKB,Akt,；克隆,分子；,基因表达,；纯化,PKB,Akt调节结构域的克隆，表达及初步纯化,0引言,1材料和方法,2结果,3讨论,【参考文献】

     (第四军医大学基础部生物化学与分子生物学教研室，陕西西安 710033)

    Cloning，expression and purification of human RD(regulation domain of PKB/Akt) gene in E. coli

    WANG LiFeng , ZHANG Jian , LI Ying，SU Jin , YAO LiBo , LIU XinPing

    Department of Biochemistry & Molecular Biology, Fourth Military Medical University, Xi’an 710033, China

    【Abstract】 AIM: To clone the coding sequences of human RD (regulation domain of PKB/Akt), to express the corresponding protein and to get the purified protein. METHODS： The RD cDNA (regulation domain of PKB/Akt) fragment was amplified using PCR in fetal hepatocytes. The PCR product was then ligated into pMD18T vector. After sequence analysis, it was subcloned into pRSETA which can express the target protein as a (His)6tagged fusion. The bacterial strain BL21 (DE3) was used as the host cells to induce the expression of the fusion protein by IPTG. Cell pellets were lysed by sonication to get the supernatant. RESULTS: RD coding region was cloned into pRSETA and the sequence was confirmed by comparison with the published one. Recombinant pRSETARD was successfully constructed. RD/6×His fusion protein was correctly expressed and the purification was easily achieved on a Ni2NTA affinity column by virtue of the (His)6 tag on the protein. CONCLUSION： Human RD of akt1 gene has been successfully cloned and RD/6×His recombinant plasmid constructed. RD/6×His fusion protein has been correctly expressed in E.coli and the soluble fusion protein purified by Ni2NTA agarose beads. ......

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