enzymes immobilized on columns?
svetlov at oncology.wisc.edu
Tue Apr 14 10:35:23 EST 1998
In article <mwcrepeau-1304981439460001 at curt.ucdavis.edu>,
mwcrepeau at ucdavis.edu (Marc Crepeau) wrote:
People are making all kinds of fusion proteins these days. It
> doesn't seem unreasonable to engineer some kind of linker peptide with an
> end that can be covalently bonded to a polymer bead. Has anyone ever heard
> of such a thing? Or is there some obstacle I'm not seeing?
There are technical obstacles - like some enzymes do not derivatize well or
get easily inactivated in the process, and methodological - like is the
need for such system so great as to pay-off the cost of making the column?
There are already some immobilized enzymes on the market - like the trypsin
columns, the problem with making a restriction enzyme column is obvious and
has to do with operational differences between bulk proteolytic
applications and endonucleolytic treatment during cloning - the amount of
the enzyme utilized in a single cloning digest usually ranges between 1 and
10 units. If that's gonna be a single column with 10U of EcoRI one would
have to buy 500 columns instead of 1 tube of say Boehringer enzyme with
5000 U. In other words, columns or resin make it more difficult to dispense
and mix stuff. There are no endonucleases that can not be removed by other
routine techniques, such as PCI-extraction or now numerous kits like
Promega's PCR clean-up that gets rid of BamHI and other heat-resistant
endonucleases. You also would not want to reuse the column, but because of
the high cost will be tempted to... So, like, the endonuclease column would
work but don't see such a big advantage to pay-off the cumbersomeness and
the high cost.
Vladimir Svetlov, Ph. D.
McArdle Lab for Cancer Research
1400 University Ave.
Madison, WI 53706
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