Hi Simon, Although they are a source of heterogeneity for crystallization, glycosylations usually stabilize proteins. There are a couple of things that may be important to consider before you deglycosylate your protein.
Do you know how many natural sequons/glycosylation sites your protein has? And in what system/organism are you expressing your protein? I am saying that because I have the case of a bovine enzyme which has three sites. Inactivation of all sequons from Asn to Asp ( they are standards N-glycosylation sites) and expression in Pichia pastoris results in a fully-deglycosylated protein which is unstable and precipitates. However if one specific sequon is kept intact, the obtained protein is glycosylated by Pichia at this functional site, behaves very well and yields good quality crystals. The electron density maps show the two first N-acetyl glucosamine units N-linked to the Asn residue. Interestingly the protein is pretty homogenously glycosylated by Pichia. I know this may sound a little bizarre but you may get around this problems by keeping some sequons or the sequon active (if there is only one) and try to deal with a protein, "glycosylated-light" as you express it. This depends on what your expression system is. You can try to add stabilizing agents like glycerol, ethylene glycol or some di-sugars like trehalose. I hope this helps, Cheers, Pascal F. Egea, PhD University of California San Francisco Department of Biochemistry and Biophysics On Mon, Mar 16, 2009 at 9:29 AM, Yue Li <simon.yu...@yahoo.com> wrote: > Hi everyone, > > Recently, I obtained a soluble glyco-protein. Unfortunately, after I added > PNGase or Endo Hf to remove the glycans, the deglycosylated protein is > precipitated. Is there any method to avoid this kind of precipitation? > > Thanks, > > Simon > >
