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PROCESS FOR PRODUCTION OF PLASMIDE-VECTOR AND FOR CLONOSATION OF ECAI DNS-METHILASE GENE BY APPLICATION OF THIS, AND PROCESS FOR PREPARATION OF ECAI METHILASE IN THE BIG QUANTITY PRODUCING STRAIN
PROCESS FOR PRODUCTION OF PLASMIDE-VECTOR AND FOR CLONOSATION OF ECAI DNS-METHILASE GENE BY APPLICATION OF THIS, AND PROCESS FOR PREPARATION OF ECAI METHILASE IN THE BIG QUANTITY PRODUCING STRAIN
Plasmid-vectors with EcaI detection sites capable of cloning genes of EcaI-DNS-methylase are prepd. A novel cloning process leads to genes for modified methylene-enzyme (M.RcaI) neither described, nor isolated previously and gives rise to a gp. producing EcaI-methylase on large scale. The insertion of a DNS sequence, contg. four EcaI detection sites (GGTNACC) into a known, vector renders it suitable for the cloning task. DNS is isolated subsequently from Enterobacter-cloacae and is partially digested. The proceeding vector and the partially digested DNS is coupled. Escherichia coli cells are transformed then with the previous recombinant DNS and recombinant plasmids are sepd. from transformed cells. Exhaustive digestion of previously isolated DNS follows, with EcaI restrictive endo-nuclease. The determining feature of this method is the destruction of the overwhelming majority of recombinant plasmids, leaving only those which contained active (intact) genes capable of coding M.EcaI enzymes. Repeated transformations of the Escherichia-coli host follows with DNS, exhaustively digested previously. The structure of cloned genes is established by characterising transforming clones. Coding sequences are isolated from plasmids, having obtd. and characterised them earlier and are transplanted into vector plasmids being constructed by them and ensuring high levels of gene development. Thus good yields of EcaI-methylase enzyme are obtd
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