Monomeric state and ligand binding of recombinant GABA transporter from Escherichia coli

FEBS Letters

Auer, Manfred¹  Li, Xiao-Dan¹  Villa, Anthony¹  Wang, Da-Neng²  Gownley, Colleen¹  Kim, Myong Jin¹  Song, Jinmei¹ 

[1] Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA;Department of Cell Biology, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA

关键词: γ-Aminobutyric acid transporter;    γ-Aminobutyric acid;    Neurotransmitter;    Transporter;    Membrane protein;    Escherichia coli;    BCA;    bicinchoninic acid;    DDM;    dodecylmaltoside;    FC-12;    FOS-Choline 12;    FPLC;    fast-performance liquid chromatography;    GABA;    γ-aminobutyric acid;    GabP;    γ-aminobutyric acid transporter;    HPLC;    high-performance liquid chromatography;    muscimol;    3-hydroxy-5-aminomethylisoxazole;    nipecotic acid;    3-piperidine carboxylic acid;    PAGE;    polyacrylamide gel electrophoresis;    PCR;    polymerase chain reaction;    PMSF;    phenylmethylsulfonyl fluoride;    SDS;    sodium dodecyl sulfate;   

DOI  :  10.1016/S0014-5793(01)02334-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The γ-aminobutyric acid (GABA) transporter from Escherichia coli was homologously overexpressed and purified to homogeneity with a yield of 1.0 mg per liter culture. The purification procedure consists of a cobalt affinity column, proteolytic cleavage of His- and myc-tags, and size-exclusion chromatography. The purified transporter exists as a monomer in FOS-Choline 12 detergent, with a Stokes radius of 45 Å for the protein–detergent complex. In detergent solution the protein binds substrates, as indicated by tryptophan fluorescence quenching. Its dissociation constants (K _d) for GABA, muscimol and nipecotic acid are 13.8, 13.3 and 27.9 μM, respectively. This protein preparation provides ideal starting materials for future biochemical, biophysical and structural studies of the GABA transporter.

【 授权许可】

Unknown