A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli

• Published in: Gene Vol. 169; no. 1; pp. 59 - 64
• Main Authors: Tsao, Kwei-Lan, Debarbieri, Barbara, Michel, Hanspeter, Waugh, David S.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 22.02.1996
• Subjects: affinity chromatography; Amino Acid Sequence; ATP-Binding Cassette Transporters; Base Sequence; Biotin; biotin holoenzyme synthetase; BirA; Carrier Proteins - chemistry; DNA Primers - chemistry; Enzymatic biotinylation; Escherichia coli; Escherichia coli Proteins; expression vector; Genetic Vectors; Ma1E; maltose-binding protein; Maltose-Binding Proteins; Molecular Sequence Data; Monosaccharide Transport Proteins; Periplasmic Binding Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Sulfurtransferases - metabolism; affinity chromatography; A; ORF; LB broth; nt; HRP; bio; Ma1E; bp; rop; UTR; rrnT; bla; kb; LC ESI-MS; biotin holoenzyme synthetase; expression vector; aa; P trc; Raf 55–132; RBS; SDS; ori; BirA; MalE; PAGE; re; IPTG; lacI Q; Enzymatic biotinylation; PCR; maltose-binding protein; LacI
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(95)00762-8

A versatile plasmid vector was designed to direct the synthesis of recombinant proteins in either one of two forms that will be biotinylated in Escherichia coli with high efficiency at a single, unique site. The protein of interest can be produced with a peptide substrate for E. coli biotin holoenzyme synthetase (BirA) joined directly to its N terminus, or alternatively, as a fusion to the C terminus of a maltose-binding protein domain (Ma1E) with the peptide substrate on its N terminus. To maximize the yield of biotinylated protein, the vector is designed to express the substrate in a coupled translation arrangement with the enzyme