title: Evolved Escherichia coli strains for amplified, functional expression of membrane proteins.
abstract: The major barrier to the physical characterization and structure determination of membrane proteins is low protein yield and/or low functionality in recombinant expression. The enteric bacterium Escherichia coli is the most widely employed organism for producing recombinant proteins. Beside several advantages of this expression host, one major drawback is that the protein of interest does not always adopt its native conformation and may end up in large insoluble aggregates. We describe a robust strategy to increase the likelihood of overexpressing membrane proteins in a functional state. The method involves fusion in tandem of green fluorescent protein and the erythromycin resistance protein (23S ribosomal RNA adenine N-6 methyltransferase, ErmC) to the C-terminus of a target membrane protein. The fluorescence of green fluorescent protein is used to report the folding state of the target protein, whereas ErmC is used to select for increased expression. By gradually increasing the erythromycin concentration of the medium and testing different membrane protein targets, we obtained a number of evolved strains of which four (NG2, NG3, NG5 and NG6) were characterized and their genome was fully sequenced. Strikingly, each of the strains carried a mutation in the hns gene, whose product is involved in genome organization and transcriptional silencing. The degree of expression of (membrane) proteins correlates with the severity of the hns mutation, but cells in which hns was deleted showed an intermediate expression performance. We propose that (partial) removal of the transcriptional silencing mechanism changes the levels of proteins essential for the functional overexpression of membrane proteins.
authors.affiliation: Department of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre and Zernike Institute for Advanced Materials, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Nadia
authors.initials: N
authors.lastname: Gul
authors.affiliation: Department of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre and Zernike Institute for Advanced Materials, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Linares
authors.affiliation: Department of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre and Zernike Institute for Advanced Materials, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Franz Y
authors.initials: FY
authors.lastname: Ho
authors.affiliation: Department of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre and Zernike Institute for Advanced Materials, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Electronic address: b.poolman@rug.nl.
authors.forename: Bert
authors.initials: B
authors.lastname: Poolman
mesh_terms: D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics; D036781:Fimbriae Proteins / Q000235:genetics; D005470:Fluorometry; D015870:Gene Expression; D016680:Genome, Bacterial; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D008780:Methyltransferases / Q000096:biosynthesis / Q000235:genetics; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D029968:Escherichia coli Proteins; C529458:FimG protein, E coli; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D036781:Fimbriae Proteins; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase
keywords: GFP; VSP; antibiotic-based selection; evolved strains; folding reporters; functional overexpression; green fluorescent protein; membrane protein production; very short patch