Hi Engin,
Thanks for the correction. This saved a lot of fancying of mine for moving to
insect cells :-).
Let me also add a little on the yeast side (no personal empirical evidence
either):
wt yeasts generate hypermannosyl N-glycans, which are tens of to more than a
hundred mannose (or modified Man) residues added to the mannose core, forming a
huge N-glycan. I do not know if these hypermannosyl N-glycans are still
sensitive to endo H, but at least I think some people have already complained a
lot about this hyperglycosylation, due to all kinds of concerns.
To my memory, there was a company that mutated the mannosyltransferases
responsible for the polymerization of mannose in pichia pastoris.
Unfortunately, now I cannot find this information again. I wonder if any
crystallographer here can share their experience with respect to these mutant
pichia strains.
Zhijie
From: Engin Özkan
Sent: Saturday, April 09, 2011 10:44 PM
To: Zhijie Li
Subject: Re: [ccp4bb] off topic: Is deglycosylation necessary for
crystallization?
On 4/8/11 1:43 PM, Zhijie Li wrote:
5. Finally, to my knowledge, proteins produced from insect cells are mainly
high-mannose type (I never experimentally tested this though, just took this
idea from some literature). This means: 1) the N-glycans from insect cells are
likely susceptible to Endo H treatment, which is great; 2) the N-glycans are
not charged; 3) the N-glcans themselves are more or less homogenous as they are
likely to be Man3,4,5 only, and are also smaller than the complex type glycans
made from mammalian cells. This is a bless for crystallization without
deglycosylation.
Insect-made glycoproteins are still Endo H resistant. Commonly forgotten, they
have (heterogeneous) core fucosylation at not only 6 but also 3 position of the
innermost NAG.
Endo H resistance can be remedied by addition of certain mannosidase inhibitors
to your culture. Yeast-made N-glycans are, on the other hand, high-mannose and
theoretically Endo-H sensitive (I have no personal empirical evidence).
Engin
