Overexpression of the Alanine Carrier Protein Gene from Thermophilic Bacterium PS3 in Escherichia coli.

概要

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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-β-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 3h 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.
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