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 <savvas.savvi...@ugent.be> 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 Nâ>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 <spart...@gmail.com> 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
