摘要
目的构建生产辅酶Q10的大肠杆菌基因工程菌并研究相关酶的表达情况。方法将荚膜红细菌(Rhodobacter capsula-tus B10)中十聚异戊二烯焦磷酸合成酶编码基因ddsA分别克隆在Lac,T7和Trc启动子下游,使其在Escherichia coli JM83中表达。同时利用His-tag的表达系统,对该酶的融合表达和镍柱纯化条件进行了研究。结果产物分析发现该基因在E.coli中表达出有活性的十聚异戊二烯焦磷酸合成酶,并使宿主产生辅酶Q10。同时,28℃培养过夜,该酶以可溶形式存在于上清液中。在150 mmol·L-1咪唑的洗涤条件下,从镍柱上解离,并可以达到电泳纯。结论大肠杆菌可以表达 Rhodobacter capsula-tus B10 的十聚异戊二烯焦磷酸合成酶,并合成辅酶Q10。
Abstract
OBJECTIVE To construct Escherichia coli producing coenzyme Q10 and investigate the decaprenyl diphosphate synthase expression.METHODS The ddsA gene encoding decaprenyl diphosphate synthase from Rhodobacter capsulatus B10 was cloned downstream different promoters(Lac,T7 and Trc) and heterologous expressed in E.coli JM83.The purification of ddsA fusion protein was investigated with His-tag system. RESULTS E.coli was observed to synthesize coenzyme Q10 in vivo.The fusion protein was expressed in supernatant after being cultivating overnight at 28 ℃,and was separated by 150 mmol·L-1 imidazole on Ni-sepharose column.CONCLUSION E.coli can produce coenzyme Q10 by heterologous expressing the decaprenyl diphosphate synthase from Rhodobacter capsulatus B10.
关键词
辅酶Q10 /
十聚异戊二烯焦磷酸合成酶 /
融合表达 /
镍柱纯化
{{custom_keyword}} /
Key words
coenzyme Q10 /
decaprenyl diphosphate synthase /
fusion expression /
Ni-sepharose purification
{{custom_keyword}} /
刘欣毅;张惠展;袁勤生.
荚膜红细菌十聚异戊二烯焦磷酸合成酶异源表达及纯化的研究[J]. 中国药学杂志, 2007, 42(01): 13-16
LIU Xin-yi;ZHNG Hui-zhn;YUN Qin-sheng.
Heterologous Expression and Purification of Decaprenyl Diphosphate Synthase from Rhodobacter capsulatus B10 [J]. Chinese Pharmaceutical Journal, 2007, 42(01): 13-16
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] HANS N，ANDREY V K，KATRIN S，et al. The multiple functions of coenzyme Q [J] .Bioorg Chem，2001，29： 1-13.
[2] HANAN C，NAOMI A，ORLY N，et al. Ineffectiveness of oral coenzyme Q10 supplementationin 3-methylglutaconic aciduria，type 3[J] . Brain Development，1998，20： 33-35.
[3] TAKADA M ，IKENOYA A，YUZURIHA T，et al. Simultaneous determination of reduced and oxidized ubiquinones[J] . Methods Enzymol，1984，105：147-155.
[4] BENTLEY R，MEGANATHAN R. Cellular and Molecular Biology [M] . Washington D C：American Society for Microbiology，1987，512-520.
[5] CRANE F L，BARR R. Determination of ubiquinones [J] . Methods Enzymol，1971，18： 137-165.
[6] SAMBROOK J，RUSSELL D W. Molecular Cloning： A Laboratory Manual(分子克降实验指南·第三版)[M] . Beijing： Scientist Press，2002.
[7] TZAGOLOFF A，DIECKMANN C L. PET genes of Saccharomyces cerevisiae [J] .Microbiol Rev，1990，54： 211-225.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
