title: Expression, biotinylation and purification of a biotin-domain peptide from the biotin carboxy carrier protein of Escherichia coli acetyl-CoA carboxylase.
abstract: A protein segment consisting of the C-terminal 87 residues of the biotin carboxy carrier protein from Escherichia coli acetyl-CoA carboxylase was overexpressed in E. coli. The expressed biotin-domain peptide can be fully biotinylated by coexpression with a plasmid that overproduces E. coli biotin ligase. The extent of biotinylation was limited in vivo, but could be taken to completion in cell lysates on addition of ATP and biotin. We used the coexpression of biotin ligase and acceptor protein to label the biotin-domain peptide in vitro with [3H]biotin, which greatly facilitated development of a purification procedure. The apo (unbiotinylated) form of the protein was prepared by induction of biotin-domain expression in a strain lacking the biotin-ligase-overproduction plasmid. The apo domain could be separated from the biotinylated protein by ion-exchange chromatography or non-denaturing PAGE, and was converted into the biotinylated form of the peptide on addition of purified biotin ligase. The identify of the purified biotin-domain peptide was confirmed by N-terminal sequence analysis, amino acid analysis and m.s. The domain was readily produced and purified in sufficient quantities for n.m.r. structural analysis.
authors.affiliation: Department of Biochemistry, University of Adelaide, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Chapman-Smith
authors.forename: D L
authors.initials: DL
authors.lastname: Turner
authors.forename: J E
authors.initials: JE
authors.lastname: Cronan
authors.forename: T W
authors.initials: TW
authors.lastname: Morris
authors.forename: J C
authors.initials: JC
authors.lastname: Wallace
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001710:Biotin / Q000494:pharmacology; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D054889:Fatty Acid Synthase, Type II; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D010446:Peptide Fragments; D001710:Biotin; D000255:Adenosine Triphosphate; D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein