Overexpression of the alanine carrier protein gene from thermophilic bacterium PS3 in Escherichia coli. 1999

M Kanamori, and H Kamata, and H Yagisawa, and H Hirata
Laboratory of Biological Signaling, Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo, 678-1297, Japan.

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.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D002352 Carrier Proteins Proteins that bind or transport specific substances in the blood, within the cell, or across cell membranes. Binding Proteins,Carrier Protein,Transport Protein,Transport Proteins,Binding Protein,Protein, Carrier,Proteins, Carrier
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005798 Genes, Bacterial The functional hereditary units of BACTERIA. Bacterial Gene,Bacterial Genes,Gene, Bacterial
D006088 Gram-Negative Aerobic Bacteria A large group of aerobic bacteria which show up as pink (negative) when treated by the gram-staining method. This is because the cell walls of gram-negative bacteria are low in peptidoglycan and thus have low affinity for violet stain and high affinity for the pink dye safranine. Achromatiaceae,Achromatium,Achromobacteriaceae,Gram Negative Aerobic Bacteria
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001426 Bacterial Proteins Proteins found in any species of bacterium. Bacterial Gene Products,Bacterial Gene Proteins,Gene Products, Bacterial,Bacterial Gene Product,Bacterial Gene Protein,Bacterial Protein,Gene Product, Bacterial,Gene Protein, Bacterial,Gene Proteins, Bacterial,Protein, Bacterial,Proteins, Bacterial
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D015964 Gene Expression Regulation, Bacterial Any of the processes by which cytoplasmic or intercellular factors influence the differential control of gene action in bacteria. Bacterial Gene Expression Regulation,Regulation of Gene Expression, Bacterial,Regulation, Gene Expression, Bacterial

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