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

Heterologous Expression and Purification of Recombinant CPS4 from Salvia miltiorrhiza

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.