Hi Radu, Many Thanks for your clarifications, and reference for background materials. I will implement all suggestions in my next expression batch.
Best Wishes, Partha On Mon, Jul 16, 2018 at 1:53 PM, <[email protected]> wrote: > Hi Partha, > > In this case (un-treated HEK293), you cannot use EndoH with already > purified > proteins (as per your reply to Artem's email). The N-glycans will be > fucosylated, hence EndoH-insensitive (see PMID: 23623336 for background > infos). You can still use PNGase. But, as Artem suggested, this often > causes > protein precipitation when it can access the sites on folded proteins. > Apart > from shaving the glycan shield, PNGase causes unwanted mutations at the > N-linked sites: Asn -> Asp, which is where most problems stem from. > > Nevertheless, since your fully glycosylated protein doesn't crystallize, > there's not much else you can do with the current prep.... Except for > example > masking your glycans with a nice lectin such as griffithsin, which would be > very elegant for crystallography. Or indeed trying cryo-EM, where wild-type > N-linked glycans are extremely helpful! Why would anyone crystallize > proteins > these days anyway :-) > > 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 > www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu > > > 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 < > [email protected]> > 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 <[email protected]> > 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 > >> ------------------------------ > >> To unsubscribe from the CCP4BB list, click the following link: > >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > ######################################################################## > To > unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > > > > > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
