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。
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 A_600 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 A_600 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.
出处
《中国实验方剂学杂志》
CAS
北大核心
2014年第10期94-98,共5页
Chinese Journal of Experimental Traditional Medical Formulae
基金
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)
关键词
丹参
原核表达
正交试验
纯化
Salvia miltiorrhiza
prokaryotic expression
orthogonal experiment
purifying