Expression and purification of hPARP1 by baculovirus system.
- Author:
Haiyan ZHOU
1
;
Jun MA
;
Xueli YANG
;
Xiaohai GONG
;
Qiuping LI
;
Jian JIN
Author Information
1. School of Pharmaceutics, Jiangnan University, Wuxi 214122, Jiangsu, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Baculoviridae;
genetics;
Blotting, Western;
Electrophoresis, Polyacrylamide Gel;
Genetic Vectors;
Humans;
Insecta;
Poly (ADP-Ribose) Polymerase-1;
Poly(ADP-ribose) Polymerases;
biosynthesis;
Recombinant Proteins;
Sf9 Cells;
Transfection
- From:
Chinese Journal of Biotechnology
2013;29(7):998-1005
- CountryChina
- 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).
