title: Over-expression of Escherichia coli F1F(o)-ATPase subunit a is inhibited by instability of the uncB gene transcript.
abstract: Little is known about the stability of transcripts encoding membrane proteins in strong expression systems and its effect on membrane protein over-production. We have expressed all the genes encoding subunits of the membrane domain F(o) of the ATP synthase in a T7 RNA polymerase-based system. All of them but uncB (subunit a) were expressed separately at very high levels in the bacterial hosts Escherichia coli C41(DE3) and C43(DE3). However, expression of uncB was extremely toxic to the bacteria. Northern blot analysis showed that the level of accumulation of the mRNA from uncB was very low. Deletion of uncB in combination with gene fusion experiments demonstrated that the middle region of the gene, encoding amino acids 92-171, exhibited a dominant toxic phenotype associated with a very poor level of expression. Green fluorescent protein fusions with N- and C-ends of uncB helped to stabilize the mRNA and to obtain high yields of protein.
authors.affiliation: The Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, CB2 2XY, Cambridge, UK.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Arechaga
authors.forename: Bruno
authors.initials: B
authors.lastname: Miroux
authors.forename: Mike J
authors.initials: MJ
authors.lastname: Runswick
authors.forename: John E
authors.initials: JE
authors.lastname: Walker
mesh_terms: D025243:Bacterial Proton-Translocating ATPases / Q000235:genetics*; D015152:Blotting, Northern; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D021122:Protein Subunits / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D029968:Escherichia coli Proteins; D021122:Protein Subunits; D025243:Bacterial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C074428:atpB protein, E coli