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.
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).
