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

• Published in: Journal of microbiology and biotechnology pp. 635 - 638
• Main Authors: PARK, MYOUNG JIN, JEI OH YON, 임시규, DEWEY D. Y. RYU, 남두현
• Format: Journal Article
• Language: English
• Published: 한국미생물·생명공학회 01.06.2004
• Subjects: 생물학
• ISSN: 1017-7825

An ATP-binding-cassette (ABC) transporter genein the cephabacin biosynthetic gene cluster of Lysobacterlactamgenus was characterized. The amplified orf10 (cpbJ)gene was subcloned into pET-28a(+) vector and expressed inE. coli BL21(DE3) strain by 0.5 mM IPTG at 30oC. Themembrane fraction of recombinant E. coli cells was separatedby ultracentrifugation, and solubilized using 2.5% octyl-b-Dglucoside.Using the solubilized membrane fraction, the artificialproteoliposomes were reconstituted and analyzed for thebiological activity of CpbJ protein. Upon measuring ATPaseactivity, the proteoliposome made from recombinant E. colimembrane proteins showed slightly higher activity than thatfrom host E. coli membrane proteins. In the measurement ofmembrane transport activity, the reconstituted proteoliposome ofrecombinant E. coli membrane proteins exhibited higher activitywhen both substrates of cephalosporin C and L-Ala-L-Ser wereapplied, compared to the case of cephalosporin C or L-Ala-L-Seronly. It implies that the CpbJ protein is an ABC transportersecreting cephabacin antibiotics synthesized in cytoplasm. KCI Citation Count: 5