Journal Article The multidrug-resistance transporter MdfA from Escherichia coli: crystallization and X-ray diffraction analysis

Nagarathinam, Kumar  ,  Jaenecke, Frank  ,  Nakada-Nakura, Yoshiko  ,  Hotta, Yunhon  ,  Liu, Kehong  ,  Iwata, So  ,  Stubbs, Milton T.  ,  Nomura, Norimichi  ,  Tanabe, Mikio

73pp.423 - 430 , 2017-7-1 , International Union of Crystallography (IUCr)
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 P6[1]22.

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