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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.

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Enzymatic attachment of biotin to proteins requires the interaction of a distinct domain of the acceptor protein (the "biotin domain") with the enzyme, biotin protein ligase, that catalyzes this essential and rare post-translational modification. Both biotin domains and biotin protein ligases are very strongly conserved throughout biology. This review concerns the protein structures and mechanisms involved in the covalent attachment of biotin to proteins.