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| 丹参二萜合酶基因CPS4的原核表达体系优化及活性蛋白纯化 | |
| 引用本文： | 靳保龙,崔光红,党伯岳,刘世会,漆小泉. 丹参二萜合酶基因CPS4的原核表达体系优化及活性蛋白纯化[J]. 中国实验方剂学杂志, 2014, 20(10): 94-98 |
| 作者姓名： | 靳保龙 崔光红 党伯岳 刘世会 漆小泉 |
| 作者单位： | 贵州大学药学院, 贵阳 550025;中国科学院植物研究所, 北京 100093;中国科学院植物研究所, 北京 100093;中国中医科学院中药资源中心, 北京 100700;贵州大学药学院, 贵阳 550025;贵州大学药学院, 贵阳 550025;中国科学院植物研究所, 北京 100093 |
| 基金项目： | 国家自然科学基金项目（81001604）；中国博士后科学基金（2012T50159）；贵州省中药现代化科技产业研究开发专项项目（黔科合ZY字[2013]3009号） |
| 摘 要： | 目的:优化丹参柯巴基焦磷酸合酶基因家族新基因CPS4的原核表达体系并对重组蛋白进行纯化。方法:对2种原核表达菌株进行比较。对诱导条件,包括异丙基-β-D-硫代半乳糖苷(IPTG)诱导浓度、诱导时宿主菌的密度(A600)和诱导时间进行正交试验。利用HisTrap HP蛋白纯化柱对重组蛋白进行纯化。结果:Escherichia coli Tuner(DE3)表达菌株能诱导产生更多的可溶重组蛋白。最佳诱导表达条件为诱导时宿主菌的A600值0.7,IPTG浓度0.2 mmol·L-1,诱导表达时间10 h。HisTrap HP蛋白纯化柱纯化时梯度洗脱能得到纯度高的重组蛋白。结论:利用此优化体系可得到足量用于后续研究的重组蛋白,为其他二萜类合酶基因的原核表达提供参考。 |
| 关 键 词： | 丹参 原核表达 正交试验 纯化 |
| 收稿时间： | 2013-12-07 |
Heterologous Expression and Purification of Recombinant CPS4 from Salvia miltiorrhiza |
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| JIN Bao-long,CUI Guang-hong,DANG Bo-yue,LIU Shi-hui and QI Xiao-quan. Heterologous Expression and Purification of Recombinant CPS4 from Salvia miltiorrhiza[J]. China Journal of Experimental Traditional Medical Formulae, 2014, 20(10): 94-98 | |
| Authors: | JIN Bao-long CUI Guang-hong DANG Bo-yue LIU Shi-hui QI Xiao-quan |
| Affiliation: | College of Pharmacy, Guizhou University, Guiyang 550025, China;Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China;Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China;National Resource Center of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China;College of Pharmacy, Guizhou University, Guiyang 550025, China;College of Pharmacy, Guizhou University, Guiyang 550025, China;Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China |
| Abstract: | Objective: To find the optimal heterologous expression conditions and purification system for the new gene CPS4 from Salvia miltiorrhiza. Method: Compare two different prokaryotic expression strains and optimizing the induction conditions including A600 values of Escherichia coli, isopropyl-beta-D-galactose and glycosides(IPTG) concentration and induction time with orthogonal experiment. The recombinant protein was purified by HisTrap HP. Result: E. coli Tuner (DE3) induced more soluble recombinant protein than BL21 (DE3). The optimal expression conditions are A600 is 0.7, IPTG concentration is 0.2 mmol·L-1 and induction time for 10 h. The recombinant protein was purified by gradient elution with HisTrap HP. Conclusion: This optimal system can obtain enough protein for further study and provide a reference for the prokaryotic expression of the other diterpene synthase. |
| Keywords: | Salvia miltiorrhiza prokaryotic expression orthogonal experiment purifying |
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