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COLIPHAGE T4 ENDONUCLEASE II A RECOMBINASE DISGUISED AS RESTRICTION
The overall goal of this project is to understand precisely how EndoII interacts with its target, understand its short-term and long-term biological roles ands how what appears to have been a recombinational enzyme has evolved towards restriction functions. This requires good access to pure enzyme, not a trivial task for an enzyme that degades its own DNA template. Either of the following two projects, starting with the wild-type clone we have, will likely yield functional enzyme for such studies:
(i)Mutagenesis of the cloned gene through error-prone PCR to generate mutants producing enzymes functional at low but not at high temperatures. Mapping of the ensuing mutations will tell what parts of the protein are essential for catalysis, and expression at high temperatures will yield large quantitites of enzyme the function of which will be studied at lower temperatures
(ii) Purification of the wildtype enzyme from phage-infected cells. A mutant protein that we have purified will be used to prepare antibodies (polyclonal or monoclonal). These antibodies will then be used to pull out EndoII from lysates through affinity chromatography.
With either approach, the functional enzyme will be used for in vitro studies of how it interacts with its target.
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