전체메뉴
검색
Article Search

JMB Journal of Microbiolog and Biotechnology

QR Code QR Code

Related articles in JMB

More Related Articles

Article

J. Microbiol. Biotechnol. 2004; 14(3): 635-638

Published online June 28, 2004

Copyright © The Korean Society for Microbiology and Biotechnology.

Biochemical Characterization of an ABC Transporter Gene Involved in Cephabacin Biosynthesis in Lysobacter lactamgenus

Myoung Jin Park , Jei Oh Yon 1, Si Kyu Lim 1, Dewey D. Y. Ryu 2 and Doo Hyun Nam 2*

College of Pharmacy Yeungnam University Gyongsan 712-749 Korea, 1Institute of Biotechnology Yeungnam University Gyongsan 712-749 Korea, 2Biochemical Engineering Program University of California Davis CA 95616 U.S.A

Abstract

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 $30^{\circ}C$. The membrane fraction of recombinant E. coli cells was separated by ultracentrifugation, and solubilized using 2.5% octyl-$\beta$-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. In the measurement of membrane transport activity, the reconstituted proteoliposome of recombinant E. coli membrane proteins exhibited higher activity when both substrates of cephalosporin C and L-Ala-L-Ser were applied, compared to the case of cephalosporin C or L-Ala-L-Ser only. It implies that the CpbJ protein is an ABC transporter secreting cephabacin antibiotics synthesized in cytoplasm.

Keywords: ABCtransporter, cephabacin, cephemantibiotic, Lysobacterlactamgenus, proteoliposome, antibioticbiosynthesis