Multidrug transporter MexB of Pseudomonas aeruginosa: overexpression, purification, and initial structural characterization.
Structural and functional characterization of the multidrug transporter, MexB, of Pseudomonas aeruginosa is significantly restricted due to a low yield of approximately 0.1 mg/L of culture from natural sources. To facilitate structural studies of this medically important transporter protein, we developed a large-scale system for expression of the genetically engineered recombinant, MexB, in the Escherichia coli cell. Using the system, the eventual yield of MexB attained was about 10mg/L of culture. The optimized purification protocol in the presence of dodecyl beta-D-maltoside allowed isolation of highly homogeneous MexB. The oligomeric state of the protein in detergent solution has been characterized to verify that the native state of the purified protein has been preserved. The molecular mass of the protein-detergent complex was found to be 380-450kDa. The MexB-dodecyl beta-d-maltoside mass ratio was determined to be 1.8 +/- 0.05. Taking into account the monomeric MexB molecular mass deduced from its amino acid sequence (112.8 kDa), we concluded that the purified MexB exists as the homotrimer in the surfactant solution. Circular dichroism analysis of MexB showed dominance of the alpha-helix structures. High yield, homogeneity, and stability of MexB position it as a good candidate for structural and functional characterization.
Mokhonov
Vladislav
V
Department of Molecular Life Science, Tokai University, School of Medicine, 143 Shimokasuya, Isehara 259-1193, Japan.
Mokhonova
Ekaterina
E
Yoshihara
Eisaku
E
Masui
Ryoji
R
Sakai
Miyo
M
Akama
Hiroyuki
H
Nakae
Taiji
T
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis; D015854:Up-Regulation
D001425:Bacterial Outer Membrane Proteins; D026901:Membrane Transport Proteins; C118597:MexB protein, Pseudomonas aeruginosa; D011994:Recombinant Proteins