Sivashanmugam Arun, Murray Victoria, Cui Chunxian, Zhang Yonghong, Wang Jianjun, Li Qianqian
Department of Biochemistry and Molecular Biology, Wayne State University, Detroit, Michigan 48201, USA.
Protein Sci. 2009 May;18(5):936-48. doi: 10.1002/pro.102.
The gram-negative bacterium Escherichia coli offers a mean for rapid, high yield, and economical production of recombinant proteins. However, high-level production of functional eukaryotic proteins in E. coli may not be a routine matter, sometimes it is quite challenging. Techniques to optimize heterologous protein overproduction in E. coli have been explored for host strain selection, plasmid copy numbers, promoter selection, mRNA stability, and codon usage, significantly enhancing the yields of the foreign eukaryotic proteins. We have been working on optimizations of bacterial expression conditions and media with a focus on achieving very high cell density for high-level production of eukaryotic proteins. Two high-cell-density bacterial expression methods have been explored, including an autoinduction introduced by Studier (Protein Expr Purif 2005;41:207-234) recently and a high-cell-density IPTG-induction method described in this study, to achieve a cell-density OD(600) of 10-20 in the normal laboratory setting using a regular incubator shaker. Several practical protocols have been implemented with these high-cell-density expression methods to ensure a very high yield of recombinant protein production. With our methods and protocols, we routinely obtain 14-25 mg of NMR triple-labeled proteins and 17-34 mg of unlabeled proteins from a 50-mL cell culture for all seven proteins we tested. Such a high protein yield used the same DNA constructs, bacterial strains, and a regular incubator shaker and no fermentor is necessary. More importantly, these methods allow us to consistently obtain such a high yield of recombinant proteins using E. coli expression.
革兰氏阴性细菌大肠杆菌为重组蛋白的快速、高产且经济的生产提供了一种手段。然而，在大肠杆菌中高水平生产功能性真核蛋白并非易事，有时颇具挑战性。为优化大肠杆菌中外源蛋白的过量生产，人们已在宿主菌株选择、质粒拷贝数、启动子选择、mRNA稳定性及密码子使用等方面进行了探索，显著提高了外源真核蛋白的产量。我们一直致力于优化细菌表达条件和培养基，重点是实现非常高的细胞密度以进行真核蛋白的高水平生产。我们探索了两种高细胞密度细菌表达方法，包括最近由斯图迪尔引入的自诱导法（《蛋白质表达与纯化》2005年；41:207 - 234）以及本研究中描述的高细胞密度IPTG诱导法，以便在常规实验室环境中使用普通培养箱摇床实现细胞密度OD(600)达到10 - 20。已用这些高细胞密度表达方法实施了几个实用方案，以确保重组蛋白生产的高产率。通过我们的方法和方案，对于我们测试的所有七种蛋白质，我们通常从50 mL细胞培养物中获得14 - 25 mg的NMR三标记蛋白和17 - 34 mg的未标记蛋白。如此高的蛋白产量使用相同的DNA构建体、细菌菌株，且无需发酵罐，仅用普通培养箱摇床即可。更重要的是，这些方法使我们能够始终如一地利用大肠杆菌表达获得如此高产量的重组蛋白。
