Dear Paul,
Another alternative is to use your original _expression_ system, but with Asn-Asp mutations of the glycosylation sites; assuming of course the mutant proteins will still be soluble which is straightforward to test.
Also, sometimes the EndoH treatment works well under only slightly denaturating conditions. By screening a gradient of denaturating conditions you may be able to pull out a concentration that allows
deglycosylation while not unfolding your protein.
Cheers
Filip Van Petegem
Filip Van Petegem
On 2/2/06, Paul McEwan <[EMAIL PROTECTED]> wrote:
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I'm currently working with a glycoprotein and am having great difficulty deglycosylating it for crystallisation. I can completely remove the sugars very easily when the protein is denatured (not much good when you're wanting to crystalise it), but seems completely resistant to treatment with endo H or pngase F when done under native conditions. Any hints on getting this to work would be greatly appreciated.
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Dr Paul A. McEwan
Protein Crystallography Group
Centre for Biomolecular Science
Clifton Boulevard
University of Nottingham
Nottingham
NG7 2RD
Tel: 0115 8468009
http://www.nottingham.ac.uk/~pazxtal
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Filip Van Petegem
University of California, San Francisco
Cardiovascular Research Institute
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phone: 415 514 2836
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email: [EMAIL PROTECTED]
