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KMID : 0578319970070030367
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Molecules and Cells
1997 Volume.7 No. 3 p.367 ~ p.373
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Cloning and Expression of a Gene Encoding Moraxalla sp. CK-1 Autolysin
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Ohn, tak Bum
Kim, Chul Ho/Min, Byung Re/Choi, Yong Keel
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Abstract
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A gene of Moraxella sp. CK-1 encoding cell wall lytic activity has been clones and expressed in E. coli. A genomic library of Moraxella sp. CK-1 was constructed in the multi-functional phagemid vector of pT7T3 19u, with partial Sau3A digests of Moraxella sp. CK-1 genomic DNA inserted at the BamHⅠ restriction site. Screening of about 5,000 transformants for cell lysis activity in LB agar plates containing Micrococcus lutes cells gave one positive clone harboring the 3.7 kb insert (pMXA282). Restriction mapping and deletion analysis of the recombinant plasmid carrying a 3.7 kb insert suggested that the autolysin gene was located within a 1.1 kb BamHⅠ-PstⅠ fragment. Analysis of extracts of E. coli clone harboring recombinant plasmids on renaturing SDS-polyacrylamide gels containing heat-killed Micrococcus luteus cells showed a clear zone around a polypeptide of about 32 kDa. Lytic activity against Micrococcus luteus cell walls by the cloned autolysin was maximun at pH 9.0. Even in conditions of over pH 10.0, this cloned autolysin showed a vigrous lytic activity. Southern blot analysis suggested the existence of other homologous regions in Moraxella sp. CK-1 genome.
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