Dear Wei, If I understood correctly your description, the protein aggregates when produced whith complex N-linked glycosylation, and behaves better when the glycosylation is immature (i.e. following kifunensine treatment I suppose ?). May I suggest that this is a rather atypical situation?
It is also not clear to me whether the behaviour you describe follows deglycosylation attempts or not? Unless your protein is meant to be an ER lumen resident, mature glycosylation should help stabilize it. I would suggest against spending time and effort in making a stable CHO LecR cell line, at this stage at least. It will give you no better protein that kifunensine-trated HEK293 cells. Transient expression in the HEK 293S GnTI-, as suggested by others, will be a lot faster (a week will probably give you enough material to understand your protein, which is what you still need to do now, play with deglycosylation conditions etc) and produce a more homogeneous sample than CHOs (which are leaky, see Chang et al 2007, PMID: 17355862, for details). Best wishes, radu ------------------------------------------ A. Radu Aricescu, PhD University Research Lecturer MRC Career Development Award Fellow University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive, Oxford OX3 7BN United Kingdom Phone: +44-1865-287564 Fax: +44-1865-287547 ---- Original message ---- >Date: Tue, 17 May 2011 18:35:50 +0200 >From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Wei Li ><wei...@helmholtz-hzi.de>) >Subject: [ccp4bb] FW: [ccp4bb] highly glycosylated protein >To: CCP4BB@JISCMAIL.AC.UK > > > > 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
