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Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly
Published on: February 9, 2015
[Construction and expression of the fusion vector of His-tagged human ARPC2 gene]
Xiao-wei Gong1, Yi Peng, Jing-hua Liu
1Department of Pathophysiology and Key Laboratory of Shock and Microcirculation of PLA, First Military Medical University, Guangzhou 510515, China.
Objective:
To construct the expression vector of His-ARPC2 fusion protein and obtain its expression and purification in E. coli.
Methods:
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.
Results:
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.
Conclusion:
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.
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