Expression and purification of hPARP1 by baculovirus system / 生物工程学报
Chinese Journal of Biotechnology
; (12): 998-1005, 2013.
Article
in Zh
| WPRIM
| ID: wpr-233180
Responsible library:
WPRO
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).
Full text:
1
Index:
WPRIM
Main subject:
Recombinant Proteins
/
Transfection
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Blotting, Western
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Baculoviridae
/
Poly(ADP-ribose) Polymerases
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Electrophoresis, Polyacrylamide Gel
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Sf9 Cells
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Poly (ADP-Ribose) Polymerase-1
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Genetic Vectors
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Genetics
Limits:
Animals
/
Humans
Language:
Zh
Journal:
Chinese Journal of Biotechnology
Year:
2013
Type:
Article