Hi Roberto: You could try an expensive way, using Glycosilation inhibitors. Tunicamycine for example Inhibits N-Glycosilation. I know there are also commercial inhibitors for O-Glycosilation but I can't remember any right now. You add them to the cultures when you produce your protein so that all the proteins produced will not have glycosilation. The only problem is that you need to standardize the optimum concentration that makes all your protein be totally sugar free. However, glycosilation inhibitors tend to be quite toxic, and you can end up killing your bug before you get your protein sugar free and if you need lots to produce enough protein it could be expensive.
I tried it a few years ago with Aspergillus fumigatus and A. nidulas proteins and it worked very well. I wasn't crystallising them then, so I don't know if it would have made anything better, but it did work for what I needed. When you say that endo-H doesn't do the job is it that the protein doesn't loose any sugars or is it that it doesn't run (in gel) according to the theoretical fully sugar free size? What I mean is that its not rare for proteins from things like fungus and other "messy" organisms to have an anomalous behavior in gel. If you follow the de-glycosilation process slowly, you may realize that you have actually eliminated all the glycosilations and that your sugar free protein may be running a bit higher that it should. In the .gif Ive attached in the mail you can see an experiment in which after deglycosilation with Endo-H all glycosilation sites had been eliminated but the protein was still running about 10 kDa bigger that according to sequence. So it could be that you protein is fine. Hope some of this long mail is usefull. Juan Juan Sanchez-Weatherby PhD Student School of Bilogical Sciences University of East Anglia -----Original Message----- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Roberto Steiner Sent: 02 February 2006 13:18 To: Paul McEwan Cc: [EMAIL PROTECTED] Subject: Re: [ccp4bb]: off-topic: deglycosylation *** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** Dear Paul, sometimes NOT removing all sugars might even be a good thing. See for example, the case of quercetin 2,3 dioxygenase (quercetinase) from A.japonicus where sugar chains are involved in the formation of a homodimer. We never managed to remove all sugars and the protein crystallized very well. (Crystals diffracted too, btw) Roberto Quoting Paul McEwan <[EMAIL PROTECTED]>: > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > 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. > > ################################## > 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 > ################################## > > > This message has been checked for viruses but the contents of an attachment > may still contain software viruses, which could damage your computer system: > you are advised to perform your own checks. Email communications with the > University of Nottingham may be monitored as permitted by UK legislation. > > > -- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED]
endoH.gif
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