a problem with deglycosylation is that it is often incomplete.
A small fraction of your
protein sample will still be glycosylated, and the glycosylated contaminant
can seriously inhibit
crystal growth, because it resembles the protein actually built in
the crystal lattice.
Deglycosylating enzymes that we have used with success are the EndoF1
by Hampton Research and
the EndoH by Boehringer. But in all cases, there was need for
a purification step using a lectin column,
which retains the glycosylated enzyme, while the deglycosylated protein
can flow through (Concanavalin A
can be used for this purpose).
Did you check the relative fraction of deglycosylated and glycosylated
protein ? (using silver-stained
SDS-PAGE gels, or better M.S.)
Hope this helps
Filip Van Petegem
Kyung Hyun Kim wrote:
Hi,This is not directly related to CCP4 packages. I have been trying to
purify and crystallize a protein which is highly glycosylated.
Deglycosylation using chemical and enzymatic methods is followed by many
different chromatographic procedures. But, no crystallization luck yet.I wonder how people out there approach for the crystallization of these
glycosylated proteins. Particularly, any new method for deglycosylation
and purification would be highly appreciated. Thanks.K.H.
-- =========================================================== Filip Van Petegem Laboratory for Protein Biochemistry and Protein Engineering Department of Biochemistry Physiology and Microbiology K.L. Ledeganckstraat 35 Tel: (32)-9-264-5127 BP 9000 GENT Fax: (32)-9-264-5338 BELGIUM E-mail: Filip.VanPetegem@.rug.ac.be ===========================================================