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 <[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 >> 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 <[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 > -- 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 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
