细胞可透过性Cre重组酶表达、纯化及生物活性检测

摘要

Cre/lox P系统由Cre位点特异重组酶和可被Cre特异性识别的lox P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BL21(DE3)宿主菌成功表达His6-NLS-Cre-MTS融合蛋白,通过His-Bond Ni-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.细胞可透过性Cre重组酶提供了一种快捷而高效的在细胞和在体水平进行遗传操作的新工具.%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.