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

FEBS Letters
X D LiD N Wang

Abstract

The gamma-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 A 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 microM, respectively. This protein preparation provides ideal starting materials for future biochemical, biophysical and structural studies of the GABA transporter.

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Citations

Mar 13, 2012·Nature·Bryan K Czyzewski, Da-Neng Wang
Mar 16, 2007·Proceedings of the National Academy of Sciences of the United States of America·Matthias Quick, Jonathan A Javitch
Nov 25, 2003·Protein Science : a Publication of the Protein Society·M Joanne LemieuxDa-Neng Wang
Jun 15, 2007·Protein Science : a Publication of the Protein Society·Daniel A P GutmannBernadette Byrne
Oct 10, 2012·Molecular Microbiology·Sara PlanamenteDenis Faure
Nov 3, 2006·Journal of Molecular Biology·Michael A WhiteMark E Dumont
Jul 22, 2004·The Journal of Biological Chemistry·Sandra GendreauChristoph Fahlke
Feb 18, 2020·Methods : a Companion to Methods in Enzymology·Clarisse FaugierAnass Jawhari
Feb 15, 2003·Biochimica Et Biophysica Acta·Da-Neng WangXiao-Dan Li

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