I have been trying to deglycosylate Chlamydomonas reinhardtii periplasmic
carbonic anhydrase, using Glycopeptidase F (E.C. 32.2.18 or 3.5.1.52) from
SIGMA. So far I haven't been able to get it work. This glycoprotein is a
heterotetramer, with a number of N-asparagine linked oligosaccharides on two
of the subunits.
Essentially, I treat the protein with DTT, then boil with SDS to denature
it. I then incubate in 250 mM sodium phosphate buffer, along with
glycopeptidase, overnite at 37 degree centigrade. Subsequent SDS-PAGE does
not reveal any effect of the enzyme. I'd be happy to supply any further
details as may be required for constructive criticism.
I would appreciate any comments or suggestions that may facilitate
deglycosylation.
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