Chinese Journal of Biotechnology 2013;29(7):998-1005

Expression and purification of hPARP1 by baculovirus system.

Haiyan ZHOU 1 ; Jun MA ; Xueli YANG ; Xiaohai GONG ; Qiuping LI ; Jian JIN

Affiliations

+expand

Country

China

Language

Chinese

Abstract

PARP1 is an important part of DNA repair machinery. In recent years, PARP1 as novel anti-cancer therapeutic target has been broadly explored. In this study, we expressed hPARP1 enzyme in the baculovirus system and tested its activity. We inserted hPARP1 gene into the pFastBac1, a baculovirus transfer vector and then transformed it into DH10Bac containing a shuttle vector of Bacmid. After co-transfecting the recombinant plasmid into Sf9 insect cells, the expressed hPARP1 was purified by 3-aminobezamide affinity chromatography. The expression of hPARPI was visualized by SDS-PAGE and Western blotting; the activity of expressed and purified hPARP1 was confirmed by the reaction of consumption of NAD+ by hPARP1 in vitro. After the purification by 3-aminobezamide affinity column, 3.2 mg protein was obtained and its specific activity was 1.988 nmol/(min x microg).