Biochemical Characterization of an ABC Transporter Gene Involved in Cephabacin Biosynthesis in Lysobacter lactamgenus
2004
Park, M.J. (Yeungnam University, Gyongsan, Republic of Korea) | Ryu, Dewey D.Y. (University of California, Davis, CA, U.S.A.) | Yon, J.O. (Yeungnam University, Gyongsan, Republic of Korea) | Lim, S.K. (Yeungnam University, Gyongsan, Republic of Korea) | Nam, D.H. (Yeungnam University, Gyongsan, Republic of Korea), E-mail: [email protected]
An ATP-binding-cassette (ABC) transporter gene in the cephabacin biosynthetic gene cluster of Lysobacter lactamgenus was characterized. The amplified orf10 (cpbJ) gene was subcloned into pET-28a(+) vector and expressed in E. coli BL21(DE3) strain by 0.5 mM IPTG at 30℃. The membrane fraction of recombinant E. coli cells was separated by ultracentrifugation, and solubilized using 2.5% octyl-β-D-glucoside. Using the solubilized membrane fraction, the artificial proteoliposomes were reconstituted and analyzed for the biological activity of CpbJ protein. Upon measuring ATPase activity, the proteoliposome made from recombinant E. coli membrane proteins showed slightly higher activity than that from host E. coli membrane proteins.
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