Abstract
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.
Highlights
Integral membrane proteins constitute an important class of proteins with which are often involved in diverse biological functions, including G-protein coupled receptors (GPCRs), channels, transporters and enzymes
Establishing a highthroughput heterologous expression system is important for membrane protein research
E. coli is the simplest low-cost expression host, and several high-throughput E. coli expression systems for membrane proteins have been published in the literature [6]
Summary
Integral membrane proteins constitute an important class of proteins with which are often involved in diverse biological functions, including G-protein coupled receptors (GPCRs), channels, transporters and enzymes. Membrane proteins account for more than 50% of current drug targets [2,3]. Structural information about these pharmaceutically useful membrane proteins will assist in the design of better drug molecules. Despite the need for identifying membrane protein structures, there are significantly fewer structures available for membrane proteins than for soluble proteins [4]. Some of the major hurdles associated with membrane protein purification include the production of insufficient yields and the inability to obtain diffraction quality crystals
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