Hi Eugenio,
It will depend on the expressing system you are using: mamalian
cells, insect cells, yeast.
For enzymatic deglycosylation I suggest you to do first do a small
scale test, using the following enzymes:
PNGase F is extremely efficient.
EndoH (you can try EndoHf which is cheaper and )
Neuraminidase to remove the sialic acid (specially useful for
mammalian cells).
You can check these enzymes at NEB or Prozyme (like Radu mentioned
before. Check also his great paper Chang et al, 2007.).
http://www.neb.com/nebecomm/default.asp
You can also use glycosylation inhibitors like tunicamycin,
kifunensine, swainsonine, etc...
Another approach is site-directed mutagenesis.
And yes, "usually" (this means not always...) it gives you better
crystals which will diffract at higher resolution.
Good luck,
Joao
João M. Dias, Ph. D.
Ollmann Saphire Lab
The Scripps Research Institute
10550 North Torrey Pines Rd. IMM-2
La Jolla, CA 92037 USA
tel (858)784-8925
http://www.scripps.edu/~jmdias/
On Sep 25, 2008, at 1:40 PM, A. Radu Aricescu wrote:
Dear Eugenio,
I believe the paper by Chang VT et al 2007 (PMID: 17355862) might
offer the answers you're after (assuming N-glycosylation is your
main concern). For O-linked sugars, the situation is much more
complex, reflecting their diversity: mucin-type can usually be
dealt with by construct design (eliminate Ser/Thr/Pro-rich
domains), for other types have a look at http://www.prozyme.com/pdf/
gk80110.pdf for example.
Best wishes,
radu
------------------------------------------
A. Radu Aricescu, PhD
University of Oxford
Wellcome Trust Centre for Human Genetics
Division of Structural Biology
Roosevelt Drive, Oxford OX3 7BN
United Kingdom
Phone: +44-1865-287551
Fax: +44-1865-287547
---- Original message ----
Date: Thu, 25 Sep 2008 11:30:24 -0700
From: Eugenio De la Mora <[EMAIL PROTECTED]>
Subject: [ccp4bb] Deglycosylation
To: CCP4BB@JISCMAIL.AC.UK
Dear all,
We have some troubles with the cristallization of glycosylated
proteins and want to try to deglycosate them. We have never done
this so we want to know what enzymes are the best (efficiency;
less protein loss, ... ). And if it gaves better results in
crystallization screens.
Thank you
Eugenio De la Mora
Instituto de Biotecnologia
Universidad NAcional Autonoma de Mexico
