Isolation of Vibrio alginolyticus sodium-driven flagellar motor complex composed of PomA and PomB solubilized by sucrose monocaprate.
The polar flagella of Vibrio alginolyticus have sodium-driven motors, and four membrane proteins, PomA, PomB, MotX and MotY, are essential for torque generation of the motor. PomA and PomB are believed to form a sodium-conducting channel. This paper reports the purification of the motor complex by using sucrose monocaprate, a non-ionic detergent, to solubilize the complex. Plasmid pKJ301, which encodes intact PomA, and PomB tagged with a C-terminal hexahistidine that does not interfere with PomB function, was constructed. The membrane fraction of cells transformed with pKJ301 was solubilized with sucrose monocaprate, and the solubilized materials were applied to a Ni-NTA column. The imidazole eluate contained both PomA and PomB, which were further purified by anion-exchange chromatography. Gel-filtration chromatography was used to investigate the apparent molecular size of the complex; the PomA/PomB complex was eluted as approx. 900 kDa and PomB alone was eluted as approx. 260 kDa. These findings suggest that the motor complex may have a larger structure than previously assumed.
Yakushi
Toshiharu
T
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
Kojima
Masaru
M
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
Homma
Michio
M
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001426:Bacterial Proteins; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003652:Decanoic Acids; D005407:Flagella / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012995:Solubility; D013395:Sucrose; D014733:Vibrio / Q000235:genetics / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003652:Decanoic Acids; D020409:Molecular Motor Proteins; C103352:PomA protein, bacteria; C107541:PomB protein, Vibrio; D015222:Sodium Channels; C031071:decanoic acid; D013395:Sucrose; D012964:Sodium