Ngo Duc Tri wrote:
Dear CCP4 experts,
I'm working on a kind of cystein peptidase. It shows a little
degradation after 2 days storing at 4 degree even I tried many kinds
of protease inhibitors, or glycerol and DTT.
So I decided to work fast (1 day) then set xtal. After one week I pick
up some condition and check degradation by SDS-PAGE. It shows heavy
degradation then I think it's impossible to obtain the crystal if I
don't find out how to stop the degradation first.
Do you have any idea about my situation? Is it true that I need to
stop the degradation and how to deal with it?
Thank you for your advices,
My best regards,
TriNgo
If I understand the problem properly, you are attempting to crystallize
a peptidase that autolyzes in solution. If it is not possible to
completely inhibit the protein with an analog or small molecule under
crystallization conditions (and be able to crystallize the complex),
then the most obvious solution to the problem is to prepare an inactive
variant, e.g., with the mutation of the catalytically essential Cys to
Ser. With any luck, the variant protein should still provide valuable
structural and mechanistic context while avoiding pesky autolysis.
Cheers,
--
------------------------------------------------------------------------
Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [EMAIL PROTECTED]
