Dear Pascal and Matthias, I am sorry for the delay of reply, thanks very much for your suggestions on the glycosylation protein. Now I am trying to do a stable cell line with CHO lec 3.8.2.1 cells, this cell line could express protein with shorter glycans. I hope several weeks later I could get some better result. I will also try to use the Glycosylation deficient cell lines.
I am still working on it, thanks again for your valuable advice. Best regards, Wei From: Matthias Zebisch [mailto:matthias.zebi...@bbz.uni-leipzig.de] Sent: 2011年5月13日 18:15 To: Wei Li Subject: Re: [ccp4bb] highly glycosylated protein Try to get hold of GntI deficient HEK293S cells (not commercially available). Expression takes two weeks but you can achieve comparable yields to HEK293T. These cells yield very homogenous bands on SDS PAGE. However, check also for O glycosylation prediction. As you appear to be from Braunschweig, ask Prof. Sträter in Leipzig. He can send you these cells. Good luck, Matthias From: Pascal Egea [mailto:pas...@msg.ucsf.edu] Sent: 2011年5月13日 18:01 To: Wei Li Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] highly glycosylated protein Hi Wei, Glycosylation usually stabilize proteins although it is a source of structural heterogeneity for us crystallographers.Since you are expressing in HEK293 cells, there is a strain of cells that is deficient for glycosylation (it was designed by Gobind Khorana at the MIT I believe). You may want to try this. This is particularly useful when you express membrane proteins, it avoids hyperglycosylation. You may want to try a lightly glycosylated version of your protein and see if it behaves correctly, The other extreme solution is to identify all occupied sequons in your protein and eventually inactivate them by mutagenesis to have a completely deglycosylated protein. This solution is probably not the best since glycosylation usually stabilize proteins and may be essential to their biological function and activity. So it is to be considered with a lot of caution. Hope this helps. -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 356 Boyer Hall office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu<mailto:pe...@mednet.ucla.edu> ________________________________ Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124 Braunschweig | www.helmholtz-hzi.de Vorsitzende des Aufsichtsrates: MinDir’in Bärbel Brumme-Bothe, Bundesministerium für Bildung und Forschung Stellvertreter: MinDirig Heiko Gevers, Niedersächsisches Ministerium für Wissenschaft und Kultur Geschäftsführung: Prof. Dr. Dirk Heinz; Ulf Richter, MBA Gesellschaft mit beschränkter Haftung (GmbH) Sitz der Gesellschaft: Braunschweig Handelsregister: Amtsgericht Braunschweig, HRB 477
