摘要
目的:我们所研创的pRSETa-B7-2-PE40KDEL/BL21pLysE工程菌通常情况下更多的是以包涵体形式为主,而少部分为可溶形式,本研究试图在不改变其结构而仅通过优化各种已知影响原核表达系统的相关因素而获得稳定、高效的几乎全部以可溶性形式表达的重组B7-2-PE40KDEL融合蛋白。方法:针对更换不同的培养基、降低或升高诱导温度、增加或减少IPTG的用量、延长或缩短诱导时间等诸多能够增加可溶性表达量的各种因素进行较为系统的探索。结果:该工程菌在无压力选择条件下传代50代后的表达稳定性约为60%;通过大量对比研究,我们最终确定了pRSETa-B7-2-PE40KDEL/BL21pLysE菌种的最佳培养诱导表达方案:即过夜活化的菌种以2%浓度接种于2YT培养基,22℃培养至D值约为0.4时加入0.1mmol/LIPTG,诱导12h,由此可获得较高水平的、稳定的、几乎全部为可溶性表达的重组B7-2-PE40KDEL融合蛋白,目的蛋白量占菌体总蛋白的≥24%。结论:通过优化各种已知影响原核表达系统的相关因素,获得了稳定、高效的几乎全部以可溶性形式表达的重组B7-2-PE40KDEL融合蛋白的工程菌,为进一步有效分离纯化该蛋白奠定了基础。
Objective：To obtain the high expression level of a new recombinant exotoxin fusion protein BT-2-PE40KDEL in soluble form by optimizing the expression conditions without changing the structure of the expressing E.coli strain. Methods： The factors that may contribute to increase the expression amount in soluble form were explored systematically, such as changing culture medium, lowering or raising the inducing temperature, increasing or decreasing the dosage of IPTG and lengthening or shortening inducing time. Results： Under the condition of no pressure selection, the stable and efficient expression of the engineered bacteria strain after more than 50 passages was about 60%. Based on a series of comparative trials, at last the best induction regime for pRSETa-BT-2-PE40KDEL/BL21 pLysE germ strain was set up, which included firstly diluting the overnight cultured germs to the density of 2% with 2YT culture medium, then culturing at 22℃, adding 0.1 mmol/L IPTG when the D600 reaches 0.4 and culturing further for 12 hours. By this induction method, the aim protein was nearly totally expressed in soluble form and accounted for more than 24% of the total protein. Conclusion：The stable and efficient expression of the recombinant toxin BT-2-PE40KDEL mainly in soluble form was obtained, which lays a foundation, for the purification of B7-2-PE40KDEL.
出处
《军事医学科学院院刊》
CSCD
北大核心
2005年第4期321-324,358,共5页
Bulletin of the Academy of Military Medical Sciences
基金
国家自然科学基金资助课题(30200111)
