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Have you performed N-terminal sequencing after cleavage to absolutely
confirm the cleavage site - sometimes proteases can do odd things...?
J
Neil Paterson wrote:
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I agree that it looks like N-terminal modification but I'm at a loss
to explain how it is being performed - the protein is expressed with
an additional 13 residues following the glycine and is virtually pure
prior to cleavage of the his-tag. At this stage there is no acetate
present although crystallisation conditions include Na acetate. Unless
something is being added by the protease I don't see how this could
occur and I'm unaware of 3C protease modifying the cleaved protein.
Neil
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Hi Neil,
It may depend a lot on your expression system, buffers throughout the
purification/crystallization, etc.
My guess it that it looks like N-terminal acetylation. If your protein
is not too big you should be able to see this by ESI-MS.
Cheers,
Miguel
En/na Neil Paterson ha escrit:
> Dear all,
> > I'm currently puzzled by some N-terminal density visible in a 1.4
> angstrom map (image
<http://www.chem.gla.ac.uk/%7Eneison/blob.png>). My
> protein has been cleaved with 3C protease, cleavage confirmed by
> SDS-PAGE and failure to rebind a NiNTA column. 3C should cleave
> LEVLFQ/GP yet there appears to be strong density preceding the
glycine. > The density is not a great fit for an additional residue
(although there
> shouldn't be one) and I'm sure my glycine is in the correct place
as the
> proline density is very clear. Has anyone seen anything like this
before
> or have any suggestions as to what it may be (and how to model it)?
> > Thanks,
> Neil
- -- Miguel Ortiz Lombardía Centro de Investigaciones Oncológicas C/
Melchor Fernández Almagro, 3 28029 Madrid, Spain Tel. +34 912 246 900
Fax. +34 912 246 976 e-mail: [EMAIL PROTECTED] -
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