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 > 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 <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
