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Dear all,
Many thanks to all contributors to this topic.
Please find all the entries sent into the ccp4bb below..... plenty of food for
thought (and many things to try).
All the best,
Paul..
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Sigma sells a kit which includes more exotic deglycosidases, these
sometimes access the glycans with different efficiency and make a
difference on a native protein. If you need to go this route, these
enzymes are much more expensive that the routine PNGaseF and EndoH though.
regards,
Pirkko Heikinheimo
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Have you tried deglycosylating while dialyzing against buffer. We
had what we thought was the same problem as you, but we found out
that endoH is very sensitive to product inhibition. By putting the
deglycosylation mixture into a dialysis bag/chamber, and then putting
that against a reservior of deglycosylation buffer, the cleavage went
to completion in 3-4 hr at RT or overnight in the cold room. Without
dialysis, the deglycosylation never went to completion before the
enzyme died. We also prefer the NEB recombinant endoH which is a
maltose-binding protein endoH fusion. It reasonably priced for
making crystallization grade protein.
Michael Garavito
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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 Steiner
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Perhaps you could try a sequential exo-glycosidase(s) then
endo-glycosidase treatment in order give the endoglycosidase better
access?
-John Newitt
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If this is possible, try to mutate your glycosylation sites.
That's what saved us for one project even though
we had fairly reasonable endo H deglycosylation.
Leemor Joshua-Tor, Ph. D.
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If it is that resistant to cleavage, maybe leaving it on would be a good
thing. I assume you have tried crystallising it intact?
We solved the structure of human sulfatase years ago which was not only
30% by mass glycosylated, but also partially proteolysed. Only this
glycosylated, chewed form crystallised. (We saw hardly any sugar in the
electron density).
Dr Charles S. Bond
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If pngase F doesn't work, the glcNac might not be accesable or have an
extra sugar at the 2 (?) position of the first GlcNac.
- You could try a whole series of endoglucosidases.
endo F1, endo F2, endo F3 etc. which cleave one sugar further. Each with a
different specificity. Vary the use of mild detergents like
beta-octyl-glycoside, chaps,etc. (In my case better without, to avoid
precipitation of deglycosylated protein)
- Cleave for a very long time. (24-72h, when 10 min. was enough for
denatured cases. Add some sodium azide to prevent bacterial growth)
- More desperate experiments involve exoglucosidases (neuraminidase,
galactosidase etc) to trim the size of the sugar a bit.
- Some proteins crystalise better with the sugars on. Once, up to 5 sugar
monomers were visible in the my electron density.
- There is a mutant CHO cell line in which all N-linked sugars are
sensitive to pngase F.
- mutate the NxS/T motives and hope it still folds correctly.
- add lectins and crystalize the complex with the sugars nicely covered.
(well, my contact ran out befor I got this desperate.)
Hans Raaijmakers
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have you considered refolding your protein after deglycosylating it? We
have successfully refolded all kinds of proteins lately (including a
membrane protein) and have been able to fish out the correctly refolded
version from the rest by careful runs on an appropriate gel filtration
column. It may be worth a shot in your case.
Savvas N. Savvides
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following up on John Newitt's suggestion: I am working with
a number of N-glycosylated proteins, which can be partially deglycosylated
using a cocktail of three hexo-lycosidases that strip sequentially:
1. The last Nactylneuraminic acid (i.e. take away any sialic acid from
the
end of the glycan). The enzyme is ABS aka arthrobacter sialidase aka
neuraminidase
2. the galactose to which the sialic acid is attached, using
Beta-galactosidase aka BTG
3. the GlcNAc to which the galactose is attached, using
Beta-NAcetyl-hexosaminidase
This leaves only the core glycan GlcNAc2Man3 intact - which you might
(or
might not) then want to treat with your endoglycosidases - but I would watch
out for EndoF - in my case it makes the protein very insoluble - the Asn goes
to Asp. Endo H is a safer bet.
Pietro Roversi.
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when I was working on glucose oxidase from A. Niger in the early '90s, I
used alpha-mannosidase to strip off most of the sugars and then endo H to
finish the job. It worked to give crystals once in about half a dozen
preps.
Possibly a better route would be to clone the gene for your protein into a
prokaryote, to avoid the problem. Might be quicker and more reliable, too.
Harry Powell
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You can express it in GnTI(i) HEK293S cells.
See
Structure and function in rhodopsin: high-level expression of
rhodopsin with restricted and homogeneous N-glycosylation by a
tetracycline-inducible N-acetylglucosaminyltransferase I-negative
HEK293S stable mammalian cell line.
GnTI(-) HEK293S
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?
cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12370423&query_hl=7&itool
=pubmed_docsum
Anastassis Perrakis
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I have had success with removal of N-linked sugars from natively folded
protein with Endo F1. The protein I was working with was expressed in
Pichia and had 2 N-linked sugars. As it turns out, though, the protein
crystallized without removing the sugars. Good luck.
Tim Fritz
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Lec1 Chinese Hamster ovary cells which were the original GnTI
knocked out cells.
Kelvin Luther
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one more pro-glycosylation argument is made by these joint papers:
nature 433 p. 834 24feb05
and
structure 13 pp.197-211 2005
the long-and-short : fully glycosylated SIV gp120. 3.9 Angstroem. gnarly
crystallography. nature/structure paper_s_.
Bryan Lepore
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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.
Filip Van Petegem
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Depends how you have made the protein, insect cells, mammalian cells as to
which enzymes work better.
You can try inhibitors like tunicamycin, mutating out sites, different Lec
CHO cell strains.
you could also do simpler things like incubate the protein with EndoH or
PnGase in a sonicating water bath, to give it a bit more energy!.
otherwise the best thing is to remove sugars bit by bit until you get a
homogenous sample by IEF gel analysis.
Neuraminidase, Endo F1, F2 , F3 (depending on the source), EndoH, PNgase F
there are kits from sigma.
be careful with Neuraminidase though, have to remove it very well it will
crystallize from minute concentrations!
Trevor Huyton
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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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