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.