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The multidrug‐resistance transporter MdfA from Escherichia coli : crystallization and X‐ray diffraction analysis
Author(s) -
Nagarathinam Kumar,
Jaenecke Frank,
Nakada-Nakura Yoshiko,
Hotta Yunhon,
Liu Kehong,
Iwata So,
Stubbs Milton T.,
Nomura Norimichi,
Tanabe Mikio
Publication year - 2017
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x17008500
Subject(s) - escherichia coli , multiple drug resistance , crystallization , chemistry , crystallography , transmembrane protein , biochemistry , antibiotics , gene , organic chemistry , receptor
The active efflux of antibiotics by multidrug‐resistance (MDR) transporters is a major pathway of drug resistance and complicates the clinical treatment of bacterial infections. MdfA is a member of the major facilitator superfamily (MFS) from Escherichia coli and provides resistance to a wide variety of dissimilar toxic compounds, including neutral, cationic and zwitterionic substances. The 12‐transmembrane‐helix MdfA was expressed as a GFP‐octahistidine fusion protein with a TEV protease cleavage site. Following tag removal, MdfA was purified using two chromatographic steps, complexed with a Fab fragment and further purified using size‐exclusion chromatography. MdfA and MdfA–Fab complexes were subjected to both vapour‐diffusion and lipidic cubic phase (LCP) crystallization techniques. Vapour‐diffusion‐grown crystals were of type II, with poor diffraction behaviour and weak crystal contacts. LCP lipid screening resulted in type I crystals that diffracted to 3.4 Å resolution and belonged to the hexagonal space group P 6 1 22.