title: Engineering Escherichia coli for functional expression of membrane proteins.
abstract: A major bottleneck in the characterization of membrane proteins is low yield of functional protein in recombinant expression. Microorganisms are widely used for recombinant protein production, because of ease of cultivation and high protein yield. However, the target proteins do not always obtain their native conformation and may end up in a nonfunctional state, in insoluble aggregates. For screening of functional protein, it is thus important to readily discriminate aggregated, mistargeted protein from globally well-folded, membrane-inserted protein. We developed a robust strategy for expression screening of functional proteins in bacteria, which is based on directed evolution. In this strategy, the C-terminus of the target membrane protein is tagged with two additional protein domains in tandem. The first one is green fluorescent protein (GFP), which functions as a reporter of the global folding state of the fusion protein. The other one is the erythromycin resistance protein (23S ribosomal RNA adenine N-6 methyltransferase, ErmC), which confers a means to select for enhanced expression. By gradually increasing the antibiotic concentration in the medium, we force the cells to evolve in a way that allows more functional target-GFP-ErmC to be expressed. The acquired genomic mutations can be generic or membrane protein specific. This strategy is readily adopted for the expression of any protein and ultimately yields a wealth of genomic data that may provide insight into the factors that limit the production of given classes or types of proteins.
authors: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute & Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands.
authors: Franz Y
authors: FY
authors: Ho
authors: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute & Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands. Electronic address: b.poolman@rug.nl.
authors: Bert
authors: B
authors: Poolman
mesh_terms: D001483:Base Sequence; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology / Q000235:genetics*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics*; D008780:Methyltransferases / Q000032:analysis / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase
keywords: Directed evolution; Folding reporter; Membrane proteins; Protein expression; Strain engineering