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Role of xylR Gene on the Construction of Xylose-Inducible Expression Vectors Using xylF Promoter of Escherichia coli
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  • Role of xylR Gene on the Construction of Xylose-Inducible Expression Vectors Using xylF Promoter of Escherichia coli
  • Role of xylR Gene on the Construction of Xylose-Inducible Expression Vectors Using xylF Promoter of Escherichia coli
저자명
Kim. Hyun-Ho,So. Jai-Hyun,Shin. Jae-Ho,Rhee. In-Koo
간행물명
Journal of the Korean Society for Applied Biological Chemistry
권/호정보
2010년|53권 6호|pp.790-797 (8 pages)
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한국응용생명화학회
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정기간행물|ENG|
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이 논문은 한국과학기술정보연구원과 논문 연계를 통해 무료로 제공되는 원문입니다.
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For construction of xylose-inducible expression vector, 428 bp of xylF promoter was ligated between AatII and HindIII of pUC18, and named pXF428. To investigate the role of XylR protein encoded by xylR on xylF promoter, 1,988 bp of xylR including its promoter was ligated downstream of multiple cloning site (MCS) in the opposite direction of xylF promoter, and named pXFR428. For the measurement of the expression level, 3,048 bp of lacZ was inserted into MCS downstream of the xylF promoter in plasmids pXF428 and pXFR428 as reporter genes and named pXF428-lacZ and pXFR428-lacZ, respectively. The recombinant plasmids were transformed into Escherichia coli JM109 and DH60, which contains a single point mutation in xylR of E. coli JM109. The constructed plasmids pXF428-lacZ and pXFR428-lacZ inductively produced ${eta}$-galactosidase by addition of xylose. In ${eta}$-galactosidase assays of pXF428-lacZ and pXFR428-lacZ in JMI09, ${eta}$-galactosidase activity of pXFR428-lacZ/JM109 induced with xylose was 2.2-fold higher than that of pXF428-lacZ/JM109 with no induction. In xylR mutant E. coli DH60, ${eta}$-galactosidase activity of pXFR428-lacZ was 7.6 times higher by induction with xylose than that of pXF428-lacZ with no induction. Expression level of lacZ gene in pXF428-lacZ/DH60 was very low, because xylR was defective in both chromosome and plasmid. XylR protein was confirmed to act as an activator on xylF promoter. Inductive production of enzymes in pXF428-lacZ/JM109 and pXFR428-lacZ/JM109 showed over 90% repression by addition of 0.5% glucose in the culture broth. xynA of Streptomyces thermocyaneoviolaceus was successfully expressed in pXF428 and pXFR428 in E. coli BLR(DE3). These results show that both pXF428 and pXFR428 using xylF promoter are sufficient as new expression systems controlled by xylR, and are essential for the gene expression on xylF promoter of the constructed vectors.