[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.edumailto: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
Re: [ccp4bb] FW: [ccp4bb] highly glycosylated protein
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
Re: [ccp4bb] FW: [ccp4bb] highly glycosylated protein
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
Re: [ccp4bb] FW: [ccp4bb] highly glycosylated protein
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