Overexpression of the alanine carrier protein gene from thermophilic bacterium PS3 in Escherichia coli.
The alanine transporter (alanine carrier protein, ACP) gene of thermophilic bacterium PS3 was previously cloned and expressed in a functionally active form in Escherichia coli cells. To achieve controlled overproduction of the ACP protein, we designed a plasmid encoding a fusion protein comprising ACP joined to the carboxyl terminus of the maltose binding protein (MBP-ACP). Upon transduction of the plasmid into E. coli RM1 cells defective in alanine/glycine transport, the transport activity was expressed even before induction with 1-thio-beta-D-galacto-pyranoside (IPTG), and increased slightly on induction with IPTG at low concentrations. However, overexpression of the MBP-ACP gene, induced by higher concentrations of IPTG, resulted in death of the host cells. Hence we screened other host cells and found that the MBP-ACP fusion protein was produced in a large quantity in E. coli TB1 cells 3 h after IPTG induction. The MBP-ACP fusion protein was accumulated in cytoplasmic membranes in an amount reaching more than 20% of the total membrane protein. The affinity-purified MBP-ACP exhibited very low transport activity when reconstituted into proteoliposomes.
Laboratory of Biological Signaling, Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo, 678-1297, Japan.
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Kanamori
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Kamata
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Yagisawa
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Hirata
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006088:Gram-Negative Aerobic Bacteria / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C042469:alanine-carrier protein, bacteria