hPARP1酶在杆状病毒/昆虫细胞中的高表达及快速纯化

Expression and purification of hPARP1 by baculovirus system

PARP1是动物细胞内的一种重要的DNA修复酶。近几年PARP1作为新型的抗癌靶点,受到广泛的关注。为了获得高活性的PARP1,首先将hPARP1基因克隆到载体pFastBacTM1中,构建转移载体pFast-hPARP1;然后转化大肠杆菌Escherichia coli DH10Bac感受态细胞中。其次,通过位点特异性转座,将hPARP1基因整合到Bacmid穿梭载体中,构建表达质粒Bacmid-hPARP1。最后,通过脂质体将表达质粒转染Sf9昆虫细胞。Western blotting和酶活测定法对hPARP1的表达和活性进行分析。采用3-氨基苯甲酰胺亲和层析柱对收获的昆虫细胞中表达的hPARP1酶进行纯化。Western blotting结果表明在昆虫细胞中hPARP1酶表达成功。经3-氨基苯甲酰胺亲和层析柱纯化后,Sf9昆虫细胞表达出的hPARP1酶的比活由0.051 nmol/(minμg)提高到了1.988 nmol/(min.μg),而且每100 mL的细胞中能够收获约3.2 mg酶。实验结果为PARP1大规模生产和应用提供了可参考利用的技术。

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 pFastBac^ TM 1,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 hPARP1 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 μg）.