EXPRESSION, BIOTINYLATION AND PURIFICATION OF A BIOTIN-DOMAIN PEPTIDE FROM THE BIOTIN CARBOXY CARRIER PROTEIN OF ESCHERICHIA-COLI ACETYL-COA CARBOXYLASE
EXPRESSION, BIOTINYLATION AND PURIFICATION OF A BIOTIN-DOMAIN PEPTIDE FROM THE BIOTIN CARBOXY CARRIER PROTEIN OF ESCHERICHIA-COLI ACETYL-COA CARBOXYLASE
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DOI：
10.1042/bj3020881
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发表时间：
1994-09-15
影响因子：
4.1
通讯作者：
WALLACE, JC
中科院分区：
文献类型：
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作者：
CHAPMANSMITH, A;TURNER, DL;WALLACE, JC
利用大肠杆菌乙酰辅酶A羧化酶生物素羧基载体蛋白C端87个残基组成的蛋白片段在大肠杆菌中高效表达，表达的生物素结构域的多肽可以通过与大肠杆菌生物素连接酶表达载体的共表达而完全被生物素化。生物素化的程度在体内是有限的，但在细胞裂解产物中可以在添加ATP和生物素的情况下完成。我们利用生物素连接酶和受体蛋白的共表达，在体外将生物素结构域的多肽与[H-3]生物素进行标记，极大地促进了纯化程序的发展。在缺乏生物素连接酶过量生产质粒的菌株中，通过诱导生物素结构域的表达，制备了apo(非生物素化)形式的蛋白质。通过离子交换层析或非变性PAGE将apo结构域从生物素化蛋白中分离出来，并在纯化的生物素连接酶的作用下转化为生物素化形式的多肽。通过N-末端序列分析、氨基酸分析和MS分析，证实了纯化的生物素结构域多肽的同源性。该结构域易于生产和提纯，足以用于n.m.r。结构分析。
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 [H-3]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 identity 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.