For David Hoover's question regarding PNGase F deglycosylation:
There is a problem to get native deglycosylated protein using PNGase F, as the
enzyme requires denaturation of glycoproteins to be deglycosylated. This is
understandable if you think the position of PNGase F action, i.e., at the base
between sugar chain and the peptide. Whereas endo-H can work perfectly on n
ative glycoproteins, part of the reason is that it works on GlcNAc-GlcNAc linkage a little away from the base. Unfortunately, endo-H only works on high mannoseor hybrid structures.
If you are dealing with biantennary complex, I would suggest to use glycopeptidase A (from Seikagaku, very expensive), which is more efficient than PNGase F.
What are the reasons that kept you from using other systems? I know Genzyme is
using your approach to make their therapeutic recombinant beta-glucosidase for
Gaucher disease. I'd like to know details of your failed approaches to figure
out additional ways to overcome the problem.
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* Ke-Wei Zhao, Ph.D. Phone: (310)825-8722
* Department of Biological Chemistry Fax: (310)206-5272
* UCLA School of Medicine, CHS 33-257 e-mail: kzhao at biovx1.biology.ucla.edu
* Los Angeles, CA 90095-1737
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