The HEK293S/ I-/- strain (Reeves et al PNAS 2002) does not lack glycosylation capacity. It is deficient in a key glucosyl transferase responsible for adding glycans beyond the Man5GlcNac2. This produces N-linked glycosylated proteins with homogeneous glycan trees.
We recently established stable cell lines for inducible expression of a generously glycosylated human protein using the HEK293S/ I-/- strain. This turned out to be a very effective approach for dealing both with low yield transient expressions and the glycosylation issue. Verstraete et al Acta Cryst F67 pp. 325-31 (2011) and Verstraete et al Blood. 2011 PMID: 21389326 Best regards Savvas On 17 May 2011, at 19:09, "Bernhard Rupp (Hofkristallrat a.D.)" <hofkristall...@gmail.com> wrote: > I have limited cautionary experiences with Lec1 CHO expression, although with > a highly and complex glycosilated protein. Subsequent glycan analysis showed > that the glycans are indeed more homogeneous and less branched, but not > exclusively the Man5GlcNac2 type that are reported/expected. There is still > some fraction of higher glycans left, and the crystals are correspondingly > ratty in the first round. On the other hand, successful crystallization has > been reported: > > > > Chen, L., Gorman, J.J., McKimm-Breschkin, J., Lawrence, L.J., > Tulloch, P.A., Smith, B.J., Colman, P.M., and Lawrence, M.C. 2001. The > structure of the fusion glycoprotein of Newcastle disease virus suggests a > novel paradigm for the molecular mechanism of membrane fusion. Structure > 9:255-266. > > > > I’d be interested to hear about the completely glycosylation deficient cell > lines mentioned below – surprised the cells live at all. > > > > BR > > > > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Wei Li > Sent: Tuesday, May 17, 2011 9:36 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] FW: [ccp4bb] highly glycosylated protein > > > > > > 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 > > > > 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 >
