Pseudomonas putida由来armXの解析と遺伝子クローニング

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ArmX(hrcA regulon member) is a protein which regulates a role of total control of heat shock chaperone. ArmX was recently discovered, so the specific role of this protein isn't determined in many bacteria. We research Pseudomonas putida KT2440 which produces many kinds of lipase. Lipase is known that it needs the original chaperone to get activity. Many bacteria has the cluster which consists of lipase gene and its chaperone gene, but P.putida KT2440 strain doesn't have its cluster. It suggests that the lipase needs other chaperone in the strain. All chromosomal DNA sequence of the strain has been already determined, and there is ArmX in the strain. The aim of the research was to clone a gene coding for ArmX (armX) from P. putida KT2440 strain, and to ascertain the specific role of this protein, especially whether this protein has chaperone function. In experiment, armX was amplified by PCR (polymerase chain reaction) and then was inserted into pUC118, 119, and the plasmids were transformed into E.coli TOP10. Two plasmids which were obtained from the transformants contained armX, and were naned pARMX1 and 2, respectively. Using three strains which each harbors pARMX1 and 2, the variation of protein expression in these strain was determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). On the other hand, the co-transformant of E.coli, which has armX(pARMX4) and lipase gene(pLIPA1 or 2), was obtained. And lipase activity of the strain was checked to verify that ArmX has chaperone function for lipase. The new heat shock protein and the chaperon activity, however, could not be detected in the E. coli host-vector system.
2013-03-01

Pseudomonas putida由来armXの解析と遺伝子クローニング

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