Gong Xiao-wei, Peng Yi, Liu Jing-hua, Li Zhi-jie, Deng Peng, Qin Qing-he, Jiang Yong
Department of Pathophysiology and Key Laboratory of Shock and Microcirculation of PLA, First Military Medical University, Guangzhou 510515, China.
Di Yi Jun Yi Da Xue Xue Bao. 2004 Jun;24(6):628-30, 635.
To construct the expression vector of His-ARPC2 fusion protein and obtain its expression and purification in E. coli.
ARPC2 cDNA codon region was amplified by PCR from human liver cDNA library and cloned into pET-14b vector following the routine procedures. After identification by enzyme digestion, PCR and sequencing, the positive clones were transformed into BL21 (DE3) competent cells, and the expression of His-ARPC2 fusion protein was induced with IPTG and further purified by Ni-NTA affinity chromatography.
The constructed His-ARPC2 fusion protein vector was highly efficiently expressed in E. coli. With Ni-NTA affinity chromatography, a purified His fusion protein with relative molecular mass of approximately 36 000 was obtained.
The expression vector of His-ARPC2 fusion protein is constructed, expressed and purified under non-denaturing conditions, which may significantly facilitate future study of the physiological functions of ARPC2 and characterization of its interaction proteins.
构建His-ARPC2融合蛋白表达载体，并在大肠杆菌中实现其表达与纯化。
采用常规方法，通过PCR从人肝脏cDNA文库中扩增ARPC2 cDNA编码区，并将其克隆至pET-14b载体。经酶切、PCR及测序鉴定后，将阳性克隆转化至BL21(DE3)感受态细胞，用IPTG诱导His-ARPC2融合蛋白表达，再通过Ni-NTA亲和层析进一步纯化。
构建的His-ARPC2融合蛋白载体在大肠杆菌中高效表达。经Ni-NTA亲和层析，获得了相对分子质量约为36 000的纯化His融合蛋白。
成功构建了His-ARPC2融合蛋白表达载体，并在非变性条件下进行了表达与纯化，这可能为今后深入研究ARPC2的生理功能及其相互作用蛋白的特性提供有力帮助。
