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 <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 <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 <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 > <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 > <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
