细胞可透过性Cre重组酶表达、纯化及生物活性检测(英文)  

Expression, Purification and Biological Activity of a Cell-permeable Cre Recombinase

作　　者：郭中敏[1] 徐康[2] 岳颖[1] 黄冰[1] 唐欢[3] 马芸[1] 洪迅[1] 陈系古[1] 肖东[1] 

机构地区：[1]中山大学实验动物中心,广州510080 [2]中山大学第一附属医院,广州510080 [3]第三军医大学,重庆400010

出　　处：《生物化学与生物物理进展》2004年第9期784-790,共7页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(3 0 2 71177);广东省自然科学基金 (0 2 190 3);中国博士后基金(第二十九批)部分资助项目~~

摘　　要：Cre/loxP系统由Cre位点特异重组酶和可被Cre特异性识别的loxP位点构成,该系统广泛用于条件性基因敲除和表达,以研究基因功能.为了表达和纯化一种细胞可透过性Cre重组酶(即His6 NLS Cre MTS);经IPTG诱导,在BL2 1(DE3)宿主菌成功表达His6 NLS Cre MTS融合蛋白,通过His BondNi NTA树脂分离并纯化了该蛋白质,随后借助细胞和非细胞的重组系统成功检测了His6 NLS Cre MTS的生物活性.The Cre/lox P site-specific recombinase system, which has two components: Cre recombinase and two 34-bp lox P sites that Cre recognizes, has been widely used in conditional gene knockout/activation to study the structure and functions of gene. In the present study, a cell-permeable fusion protein (His6-NLS-Cre-MTS) containing a 12-amino acid membrane translocation sequence (MTS), a nuclear localization signal (NLS) and an N-terminal His6 affinity tag, was expressed in BL21 strains (e. g., DE3) transformed with pDJHisCre by induction of IPTG. The fusion protein was purified with His-Bond Ni-NTA resin. Its functionality was confirmed in a cell-free recombination assay with a plasmid (e. g., pApoE-SCS-EGFP) containing lox P-flanked gene(s), and in an intracellular recombination system using lox P-flanked STOP cassette-modified BEL-7402 cells, by assaying the expression of enhanced green fluorescent protein (EGFP). This cell-permeable Cre recombinase provides a rapid alternative means of manipulating mammalian gene structure and function in vitro and in vivo. Its advantages and potential uses are discussed.

关 键 词：CRE/LOX P系统 细胞可透过性Cre重组酶 表达 纯化 生物活性检测 细胞和非细胞的重组系统 

分 类 号：R11[医药卫生—公共卫生与预防医学] R51