【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。更多还原

【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 A600values 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 A600is 0. 7,IPTG concentration is 0. 2 mmol·L-1and 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.更多还原