Using Haloarcula marismortui Bacteriorhodopsin as a Fusion Tag for Enhancing and Visible Expression of Integral Membrane Proteins in Escherichia coli

Min Feng Hsu, Tsung Fu Yu, Chia Cheng Chou, Hsu Yuan Fu, Chii Shen Yang, Andrew H.J. Wang

研究成果: 雜誌貢獻文章同行評審

32 引文 斯高帕斯(Scopus)

摘要

Membrane proteins are key targets for pharmacological intervention because of their vital functions. Structural and functional studies of membrane proteins have been severely hampered because of the difficulties in producing sufficient quantities of properly folded and biologically active proteins. Here we generate a high-level expression system of integral membrane proteins in Escherichia coli by using a mutated bacteriorhodopsin (BR) from Haloarcula marismortui (HmBRI/D94N) as a fusion partner. A purification strategy was designed by incorporating a His-tag on the target membrane protein for affinity purification and an appropriate protease cleavage site to generate the final products. The fusion system can be used to detect the intended target membrane proteins during overexpression and purification either with the naked eye or by directly monitoring their characteristic optical absorption. In this study, we applied this approach to produce two functional integral membrane proteins, undecaprenyl pyrophosphate phosphatase and carnitine/butyrobetaine antiporter with significant yield enhancement. This technology could facilitate the development of a high-throughput strategy to screen for conditions that improve the yield of correctly folded target membrane proteins. Other robust BRs can also be incorporated in this system.

原文英語
文章編號e56363
期刊PLoS ONE
8
發行號2
DOIs
出版狀態已發佈 - 2月 15 2013
對外發佈

ASJC Scopus subject areas

  • 生物化學、遺傳與分子生物學 (全部)
  • 農業與生物科學 (全部)
  • 多學科

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