Oops,
I made a mistake in my previous email, apologies! Griffithsin would also
require high-Man glycans, so kifunensine treatment or GnTI-/- cells.... So
yes, it's either PNGase or cryo-EM...
Best wishes,
Radu
> Hi Savvas,
>
> Many Thanks for your inputs and references. This cell line used is not
> HEK293S *MGAT1-/-. *This particular batch was purified from HEK293
> cell-line stably expressing (created using lenti-methods by a
> former colleague) the protein of interest. In future, I will plan to do
> expression in the presence of Kifunensine, followed by EndoH treatment
> before complexation and crystallization.
>
> Best Wishes,
> Partha
>
>
> On Mon, Jul 16, 2018 at 12:53 PM, Savvas Savvides <[log in to unmask]>
> wrote:
>
>> Dear Partha
>> you do not specify which HEK293 cell line you have used, but if it so
>> happens that it is the very handy HEK293S *MGAT1-/- *cell line
>> (previously known as HEK293S *GnTI-/- ) *which produces
>> N-linked Man5GlcNAc2 glycans you might want to consider using EndoH (e.g.
>> see Verstraete et al. DOI: 10.1038/ncomms14937) or even Jack-bean
>> alpha-mannosidase (e.g. see Bloch et al. https://doi.org/10.1016/j.
>> immuni.2017.12.008).
>> Kifunensine, an inhibitor of N-glycosylation processing, tends to work
>> quite well for protein expression in HEK293T and renders N-linked glycans
>> digestible by EndoH (see Chang et al. DOI 10.1016/j.str.2007.01.011 ; Felix
>> et al. http://dx.doi.org/10.1016/j.str.2015.06.019 ; Elegheert et
>> al. doi:10.1038/nsmb.2367).
>>
>> If resources and protein material allow, you might also want to consider
>> the permutation exercise of subjecting the complex to deglycosylation, or
>> the individual components followed by complex formation/purification, or
>> just one of the two components followed by complex formation/purification,
>> or even one of the two components followed by further deglycosylation of
>> the complex. We are becoming more and more apprehensive of the possible
>> role of glycans in complex formation.
>> And then there is of course the option to apply mutagenesis, e.g. via
>> N—>Q, to eliminate certain N-linked glycans either as a standalone
>> approach
>> or in combination with enzymatic glycan digestions as described above.
>>
>> Best wishes
>> Savvas
>>
>>
>> *---*
>> *Savvas Savvides*
>> VIB Center for Inflammation Research
>> Dept. Biochemistry & Microbiology, Ghent University
>> Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium
>> +32 (0)472 928 519 (mobile) ; +32 9 331 36 60 (office) ; Skype:
>> savvas.savvides_skype
>> http://www.vib.be/en/research/scientists/Pages/Savvas-Savvides-Lab.aspx
>>
>> On 16 Jul 2018, at 20:53, Parthasarathy Sampathkumar <[log in to unmask]>
>> wrote:
>>
>> Dear All,
>>
>> I am in a situation, almost for the first time within my limited
>> experience, that deglycosylation might be necessary to obtain crystal. So,
>> I thought of tapping to vast experience of CCP4BBers, while I am searching
>> literature.
>>
>> I have protein that has been expressed in HEK293 cells, secreted into
>> media, purified over IMAC and SEC columns. Crystallization-screens with its
>> binding partners (they form good complexes based on analytical SEC) have
>> not produced any useful hits (whereas complexes with related proteins
>> worked well). So, I plan to re-try complex formation and \crystallization
>> screen after deglysosylation.
>>
>> My question is: In practice, Does a kit (for example here: https://www.
>> sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en®ion=US)
>> containing Endo F1, F2, F3 be sufficient or should this be tried in
>> combination with PNGase (which requires
>> desaturating conditions)?!!
>>
>> Many Thanks in advance for your suggestions, and reference.
>>
>> Best Wishes,
>> Partha
>>
>> ------------------------------
>>
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>>
>>
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--
Radu Aricescu
MRC Laboratory of Molecular Biology
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