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
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