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Journal of Biochemistry Advance Access originally published online on September 6, 2008
Journal of Biochemistry 2008 144(5):635-642; doi:10.1093/jb/mvn110
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© 2008 The Japanese Biochemical Society

Cell-free Synthesis of the Torque-Generating Membrane Proteins, PomA and PomB, of the Na+-driven Flagellar Motor in Vibrio alginolyticus

Hiroyuki Terashima, Rei Abe-Yoshizumi, Seiji Kojima and Michio Homma*

Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602, Japan

*To whom correspondence should be addressed. Tel: +81-52-789-2991, Fax: +81-52-789-3001, E-mail: g44416a{at}cc.nagoya-u.ac.jp

Received August 18, 2008; Accepted August 29, 2008


  Abstract

Flagellar motor proteins, PomA and PomB, are essential for converting the sodium motive force into rotational energy in the Na+-driven flagella motor of Vibrio alginolyticus. PomA and PomB, which are cytoplasmic membrane proteins, together comprise the stator complex of the motor and form a Na+ channel. We tried to synthesize PomA and PomB by using the cell-free protein synthesis system, PURESYSTEM. We succeeded in doing so in the presence of liposomes, and showed an interaction between them using the pull-down assay. It seems likely that the proteins are inserted into liposomes and assembled spontaneously. The N-terminal region of in vitro synthesized PomB appeared to be lost, but this problem was suppressed by fusing GFP to the N-terminus of PomB or by mutagenesis at Pro-11 or Pro-12. A structural change of the N-terminal region of PomB by these modifications may prevent cleavage during protein synthesis in PURESYSTEM. The mutations did not affect the functioning of the motor. Using this system, biochemical analysis of PomA and PomB can be performed easily and efficiently.

Key Words: bacterial flagellum, in vitro protein synthesis, ion-driven motor, membrane protein, stator

Abbreviations: CHAPS, 3-[(3-cholamidopropyl) dimetylammonio] propanesulfonate; DTT, Dithiothreitol; HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid


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