Expression and epitope tagging of the membrane anchor subunit (DmsC) of Escherichia coli dimethyl sulfoxide reductase.
Escherichia coli dimethyl sulfoxide reductase is a heterotrimer comprising a catalytic subunit (DmsA), an electron transfer subunit (DmsB) and an integral membrane anchor subunit (DmsC). DmsC is not antigenic and the production of antibodies to this subunit has not been successful. We have tagged DmsC at the C-terminus with a dystrophin-specific amino acid sequence (dysp) to which antibodies are readily available. We were able to use this tagging technique to monitor expression and localization of DmsC in E. coli and non-muscle eukaryotic cells. Growth properties of wild-type E. coli, strain HB101, overexpressing DmsC:dysp suggest that the expression of DmsC is lethal to E. coli. The lethality could be overcome by utilizing an E. coli F0F1 ATPase mutant as the host. Growth conditions of culture density, duration of induction, temperature of incubation after induction and media conditions were investigated to optimize DmsC:dysp accumulation levels. In order to alleviate the problem arising from the toxicity of DmsC, expression in eukaryotic tissue culture was also explored. A plasmid expressing DmsC:dysp was transfected into COS-1 or McA-RH777 cells. The presence of expressed DmsC:dysp was confirmed using specific anti-dysp antibodies and immunofluorescence microscopy analysis revealed that the DmsC:dysp was localized to the endoplasmic reticulum. Expression of DmsC:dysp did not appear to be toxic to the eukaryotic cells. These data suggest methodologies to overcome lethality problems associated with the overexpression of integral membrane proteins like DmsC.
Turner
R J
RJ
Department of Biochemistry, University Alberta, Edmonton, Canada.
Busaan
J L
JL
Lee
J H
JH
Michalak
M
M
Weiner
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018604:Epitope Mapping; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D003580:Cytochromes; C102555:DmsB protein, Rhodobacter sphaeroides; C102556:DmsC protein, E coli; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase