Simple Genetic Selection Protocol for Isolation of Overexpressed Genes That Enhance Accumulation of Membrane-Integrated Human G Protein-Coupled Receptors in Escherichia coli
Simple Genetic Selection Protocol for Isolation of Overexpressed Genes That Enhance Accumulation of Membrane-Integrated Human G Protein-Coupled Receptors in Escherichia coli
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DOI：
10.1128/aem.00963-10
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发表时间：
2010-09-01
影响因子：
4.4
通讯作者：
Georgiou, George
中科院分区：
文献类型：
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作者：
Skretas, Georgios;Georgiou, George
在细菌中高效生产膜蛋白仍然是一个重大挑战。在这项工作中，我们试图确定在大肠杆菌中提高重组膜蛋白产量的过表达基因。我们开发了一种用于细菌膜蛋白生产的遗传选择系统，该系统包括膜蛋白与β-内酰胺酶的融合和在含氨苄西林的培养基上对高产菌株的简便选择。利用该系统筛选了ASKA文库，即编码所有已知大肠杆菌开放阅读框架(ORF)的有序质粒库，并筛选出了几个能够积累大量重组膜蛋白的克隆。值得注意的是，共表达编码未知功能的DNA结合蛋白的基因ybaB，被发现能使多种膜整合的人类G蛋白偶联受体和其他完整的膜蛋白在大肠杆菌中的积累增加10倍。这项研究的结果突出了遗传方法在识别影响膜蛋白生物发生的因素和产生用于膜蛋白生产的工程微生物宿主方面的力量。
The efficient production of membrane proteins in bacteria remains a major challenge. In this work, we sought to identify overexpressed genes that enhance the yields of recombinant membrane proteins in Escherichia coli. We developed a genetic selection system for bacterial membrane protein production, consisting of membrane protein fusions with the enzyme beta-lactamase and facile selection of high-production strains on ampicillin-containing media. This system was used to screen the ASKA library, an ordered library of plasmids encoding all the known E. coli open reading frames (ORFs), and several clones with the ability to accumulate enhanced amounts of recombinant membrane proteins were selected. Notably, coexpression of ybaB, a gene encoding a putative DNA-binding protein of unknown function, was found to enhance the accumulation of a variety of membrane-integrated human G protein-coupled receptors and other integral membrane proteins in E. coli by up to 10-fold. The results of this study highlight the power of genetic approaches for identifying factors that impact membrane protein biogenesis and for generating engineered microbial hosts for membrane protein production.