title: Using Haloarcula marismortui bacteriorhodopsin as a fusion tag for enhancing and visible expression of integral membrane proteins in Escherichia coli.
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.
authors: Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
authors: Min-Feng
authors: MF
authors: Hsu
authors: Tsung-Fu
authors: TF
authors: Yu
authors: Chia-Cheng
authors: CC
authors: Chou
authors: Hsu-Yuan
authors: HY
authors: Fu
authors: Chii-Shen
authors: CS
authors: Yang
authors: Andrew H J
authors: AH
authors: Wang
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D019614:Haloarcula marismortui / Q000235:genetics*; D006639:Histidine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D027701:Organic Cation Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017433:Protein Structure, Secondary; D059748:Proteolysis; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D027701:Organic Cation Transport Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D001436:Bacteriorhodopsins; D011755:Pyrophosphatases; C503879:undecaprenyl pyrophosphate phosphatase