Chapman-Smith A, Turner D L, Cronan J E, Morris T W, Wallace J C
Department of Biochemistry, University of Adelaide, Australia.
Biochem J. 1994 Sep 15;302 ( Pt 3)(Pt 3):881-7. doi: 10.1042/bj3020881.
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.
由大肠杆菌乙酰辅酶A羧化酶生物素羧基载体蛋白C端87个残基组成的蛋白质片段在大肠杆菌中过表达。通过与过量表达大肠杆菌生物素连接酶的质粒共表达，所表达的生物素结构域肽可以被完全生物素化。生物素化程度在体内有限，但在加入ATP和生物素后，在细胞裂解物中可完成生物素化。我们利用生物素连接酶和受体蛋白的共表达，用[3H]生物素在体外标记生物素结构域肽，这极大地促进了纯化方法的开发。通过在缺乏生物素连接酶过表达质粒的菌株中诱导生物素结构域表达来制备该蛋白的脱辅基（未生物素化）形式。脱辅基结构域可以通过离子交换色谱或非变性聚丙烯酰胺凝胶电泳与生物素化蛋白分离，加入纯化的生物素连接酶后可转化为生物素化形式的肽。通过N端序列分析、氨基酸分析和质谱法确认了纯化的生物素结构域肽的身份。该结构域易于大量产生和纯化，用于核磁共振结构分析。
