Controlling and quantifying protein concentration in Escherichia coli
Controlling and quantifying protein concentration in Escherichia coli
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DOI：
10.1002/pro.3637
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发表时间：
2019-07-01
期刊：
影响因子：
8
通讯作者：
Pielak, Gary J.
中科院分区：
文献类型：
--
作者：
Speer, Shannon L.;Guseman, Alex J.;Pielak, Gary J.
细胞环境是动态和复杂的，涉及数千种不同的大分子，总浓度为每升数百克。然而，大多数生物化学在稀释缓冲液中进行，其中大分子的浓度小于10 g/L。高浓度的大分子会影响蛋白质的稳定性、功能和蛋白质复合物的形成，但为了充分理解这些现象，我们需要知道细胞中测试蛋白质的浓度。在这里，我们定量浓度的过表达的重组蛋白，蛋白G的B1结构域的变体，在调谐器（DE 3）（TM）大肠杆菌细胞作为诱导剂浓度的函数。我们发现，蛋白质的表达水平是可控的，并且在用0.4 mM异丙基β-d-硫代半乳糖苷诱导后，表达在超过2 mM时饱和。我们讨论的结果，什么可以和不能从细胞内蛋白质NMR研究在E。杆菌
The cellular environment is dynamic and complex, involving thousands of different macromolecules with total concentrations of hundreds of grams per liter. However, most biochemistry is conducted in dilute buffer where the concentration of macromolecules is less than 10 g/L. High concentrations of macromolecules affect protein stability, function, and protein complex formation, but to understand these phenomena fully we need to know the concentration of the test protein in cells. Here, we quantify the concentration of an overexpressed recombinant protein, a variant of the B1 domain of protein G, in Tuner (DE3)(TM) Escherichia coli cells as a function of inducer concentration. We find that the protein expression level is controllable, and expression saturates at over 2 mM upon induction with 0.4 mM isopropyl beta-d-thiogalactoside. We discuss the results in terms of what can and cannot be learned from in-cell protein NMR studies in E. coli.