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

Citation
Xd. Li et al., Monomeric state and ligand binding of recombinant GABA transporter from Escherichia coli, FEBS LETTER, 494(3), 2001, pp. 165-169
Citations number
27
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
FEBS LETTERS
ISSN journal
0014-5793 → ACNP
Volume
494
Issue
3
Year of publication
2001
Pages
165 - 169
Database
ISI
SICI code
0014-5793(20010413)494:3<165:MSALBO>2.0.ZU;2-8
Abstract
The gamma -aminobutyric acid (GABA) transporter from Escherichia coli was h omologously overexpressed and purified to homogeneity with a yield of 1.0 m g per liter culture. The purification procedure consists of a cobalt affini ty column, proteolytic cleavage of His- and myc-tags, and size-exclusion ch romatography. The purified transporter exists as a monomer in FOS-Choline 1 2 detergent, with a Stokes radius of 45 Angstrom for the protein-detergent complex. In detergent solution the protein binds substrates, as indicated b y tryptophan fluorescence quenching, Its dissociation constants (K-d) for G ABA, muscimol and nipecotic acid are 13.8, 13.3 and 27.9 muM, respectively. This protein preparation provides ideal starting materials for future bioc hemical, biophysical and structural studies of the GABA transporter. (C) 20 01 Published by Elsevier Science B.V. on behalf of the Federation of Europe an Biochemical Societies.