title: [Expression and purification of hPARP1 by baculovirus system].
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).
authors.affiliation: School of Pharmaceutics, Jiangnan University, Wuxi 214122, Jiangsu, China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Zhou
authors.forename: Jun
authors.initials: J
authors.lastname: Ma
authors.forename: Xueli
authors.initials: X
authors.lastname: Yang
authors.forename: Xiaohai
authors.initials: X
authors.lastname: Gong
authors.forename: Qiuping
authors.initials: Q
authors.lastname: Li
authors.forename: Jian
authors.initials: J
authors.lastname: Jin
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000096:biosynthesis*; D011994:Recombinant Proteins; D061987:Sf9 Cells; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases