Hi Savvas,
Thank you for kindly pointing to our review. Bernhard, in my experience your
case is a rare exception rather than the rule, so indeed lucky. Adrian
summarised very nicely the wide impact glycans may have on folding,
trafficking and/or function. To keep things simple, there is no need to mutate
the N-glycosilation sites for structural work (other than perhaps to probe
their impact, but people don't seem to do this normally). PMID: 17355862
describes how to deal with these if the aim is to improve crystal quality. For
cryo-EM, glycans should definitely stay on. Why would one risk solving
meaningless structures?
Best wishes,
Radu
--
Radu Aricescu
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: +44-(0)1223-267049
fax: +44-(0)1223-268305
---- Original message ----
>Date: Wed, 12 Apr 2017 10:24:10 +0200
>From: CCP4 bulletin board <[log in to unmask]> (on behalf of Savvas
Savvides <[log in to unmask]>)
>Subject: Re: [ccp4bb] Glycoprotein expression question
>To: [log in to unmask]
>
>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 exercise. Our protein
targets are typically 15-70 kDa and have 2-6 predicted N-linked glycosylation
sites. For instance, we have seen that elimination of even one such site by
mutagenesis can abrogate protein secretion. So for those cases one may even
make an argument for a glycosylation ‘hotspot'. Also, eliminating all
possible N-linked glycosylation sites at once in the couple of cases tried
has been synonymous with zero protein secretion. Our consensus of the
‘magic combo’ in terms of expression levels and stability is a reduction
of N-linked glycosylation sites by up to 1/3. Such reduced levels of
glycosylation also appear to be amenable for enzymatic glycan trimming in
subsequent stages of sample preparation with good results.
>The article http://dx.doi.org/10.1016/j.sbi.2013.04.003 by Aricescu and Owens
may provide some additional perspectives.
>
>best wishes
>Savvas
>
>
>> On 11 Apr 2017, at 22:34, Bernhard Rupp <[log in to unmask]> wrote:
>>
>> 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 well and gets secreted as
planned.
>>
>> But rumor has it that glycoproteins that are mutated to non-glyc often are
not processed correctly and
>> that we had just dumb luck.
>>
>> May I poll the educated opinion of the erudite here?
>>
>> Cheers, BR
>>
>> ------------------------------------------------------
>> Bernhard Rupp
>> Crystallographiae Vindicis Militum Ordo
>> http://www.hofkristallamt.org/
>> [log in to unmask]
>> +1 925 209 7429
>> +43 767 571 0536
>> ------------------------------------------------------
>> :(){ :|: & };:
>> ------------------------------------------------------
