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KMID : 0438619930170020085
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Journal of Oral Biology
1993 Volume.17 No. 2 p.85 ~ p.92
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Preliminary Experiments on Cloning of Collagenase from C. histolyticum
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Han Song
Sam Seifter
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Abstract
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Collagenase from Clostridium histolyticum was purified to homogeneity using several column chrornatographic methods in the presence of protense inhibitors. The purified enzyme migrated as one band in SDS-PAGE with an apparent Mr of 11O,000 and in several test systems, did not exhibit any other proteolytic activity. Automatic Edman drgradation of the protein revealed that the primary structure of the aminoterminal region of the protein was IANTNSEKYDFEYLNGL. The protein was cleaved with CNBr and the resulting CNBr peptides were separated by HPLC. A polyclonal antibody against the enzyme was raised and its titer determined on a slot blot using an ABC detection method. DNA from C. histolyticum was prepared, digested partially with Sau3AI and inserted onto a multiple cloning site of the vector plasmid pBluescript SK(-). Host E. celi strain XLI Blue transformed with the recombinant plasmids was screened with an oligonucleotide probe; several positive clones were thereby revealed.
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