Institute of Chemistry , University of Graz , Graz 8010 , Austria.
Biochemistry. 2019 Jun 25;58(25):2799-2803. doi: 10.1021/acs.biochem.9b00403. Epub 2019 Jun 14.
Isotopic labeling of recombinant proteins is crucial for studying proteins by liquid state NMR spectroscopy. Nowadays, conventional E. coli-based expression systems like BL21 (DE3) are typically used to express recombinant proteins. Still, the production of isotopically labeled proteins is often costly and time-consuming, and yields are not sufficient enough for structural studies. Here, we present Vibrio natriegens (Vmax) as an alternative expression system in M9 minimal medium. Due to our optimized M9 minimal medium and conditions and the early time point of induction, we obtained a 2- to 4-fold higher protein yield for two test proteins, FKBP and EYFP, compared to E. coli BL21 (DE3). Production of proteins in V. natriegens in minimal medium is not only more cost-effective and convenient but also less time-consuming than in E. coli. Comparing N HSQC spectra of FKBP and EYFP expressed in Vmax and BL21 (DE3) revealed correct folding during expression.
对重组蛋白进行同位素标记对于通过液相 NMR 光谱法研究蛋白质至关重要。如今，通常使用传统的基于大肠杆菌的表达系统（如 BL21（DE3））来表达重组蛋白。然而，同位素标记蛋白的生产通常成本高昂且耗时，产量不足以满足结构研究的需求。在这里，我们提出了一种替代的表达系统，即使用 V. natriegens（Vmax）在 M9 基础培养基中表达重组蛋白。由于我们优化了 M9 基础培养基和条件，并在早期诱导，与大肠杆菌 BL21（DE3）相比，两种测试蛋白 FKBP 和 EYFP 的蛋白产量提高了 2 到 4 倍。在 V. natriegens 中在基础培养基中生产蛋白不仅更具成本效益和方便，而且比在大肠杆菌中更省时。比较在 Vmax 和 BL21（DE3）中表达的 FKBP 和 EYFP 的 N HSQC 谱表明，在表达过程中正确折叠。
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