Graduate School of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Protein Pept Lett. 2021;28(10):1180-1190. doi: 10.2174/0929866528666210805120715.
Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level.
In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium.
First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression).
Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture.
The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.
自动诱导是一种无需添加诱导剂即可使用乳糖操纵子控制的大肠杆菌表达系统生产重组蛋白的便捷方法。自动诱导可能会在使用混合培养底物制备的复杂培养基中无意中发生。培养底物的差异有时会导致诱导水平的变化。
在这项研究中，我们研究了使用葡萄糖和乳糖作为复杂培养基自动诱导增强剂的可行性。
首先，通过定量测定 T7 lac 启动子控制下的重组 GFPuv 表达来评估自动诱导水平。使用绵羊血管紧张素原（tac 启动子表达）和嗜热栖热菌锰过氧化氢酶（T7 lac 启动子表达）检查含添加剂培养基的有效性。
用酶蛋白消化物 Polypepton 观察到自动诱导 GFPuv 表达，但用另一种消化物 tryptone 则没有。无论蛋白质消化物的类型如何，在 Terrific Broth 培养基中添加葡萄糖（终浓度为 2.9 g/L）和乳糖（终浓度为 7.6 g/L）均可成功获得与市售自动诱导培养基相似的诱导水平。两种重组蛋白的产量均为每升培养物毫克级纯化蛋白。
本研究中所示的培养基组成对于实现基于大肠杆菌的重组蛋白生产的可靠自动诱导将具有实际意义。
