Hi Bernhard,
I guess you knew all these and is really asking for people's experience,
but please excuse me to start from the theory: N-glycans in eukaryotes
are known to be involved in glycoprotein folding in the ER. They allow
the nascent protein to get into the calnexin/calreticulin cycle in
00
>From: CCP4 bulletin board (on behalf of Savvas
Savvides )
>Subject: Re: [ccp4bb] Glycoprotein expression question
>To: CCP4BB@JISCMAIL.AC.UK
>
>Dear Bernhard
>Our campaigns over the years aiming to produce mammalian cytokines and the
ectodomains of cytokine receptors via euk
Dear Bernhard
Our campaigns over the years aiming to produce mammalian cytokines and the
ectodomains of cytokine receptors via eukaryotic expression systems (mainly in
several HEK293 flavors) for structural biology, have taught us that the
N-linked glycosylation issue remains a very empirical ex
Good morning, Bernhard,
I do not think that you had pure luck (though serendipity helps a lot). You
said that the PNGaseF treated protein was indeed stable, that was already a
good hint.
In my little experience with N- and O-glycoproteins, with not a high percentage
of sugar content, I saw a d
I think it completely depends on the protein: in some proteins, they are
required for folding; in some (eg Fcs), they are not required for folding but
for function); in some, some of them are required and others not; in some they
are required _during_ folding, but afterwards they can be removed
We had experience with a relatively small glycoprotein - when glycosylation
sites were deleted, solubility went drastically down - we could not express
soluble any more. Back to eukaryotic expression system which worked.
So may be you were really lucky.
Jan
On Tue, Apr 11, 2017 at 10:34 PM, Ber
Hi Fellows,
a humble question for our glyco-expressionists:
I have mutated out the Asns of the N-glycoslation consensus sites for Asp
(Asp simply because the PNGaseF treated protein stays stable so I thought
that might be a good guess)
and indeed the unglycosilated mutant expresses wel
