Dear David,
Following the discussion I am starting to wonder if I have been doing
something wrong all these years.
I always forgot to purify the complexes, I just mixed the two
macromolecules and did the crystallization experiments.
And I will admit that I did not even worry about getting the right
stoichiometry. It is true that I got
crystals with the wrong stoichiometry:
Stura, E.A., Graille, M., Taussig, M.J., Sutton, B.J. Gore, M.G.,
Silverman, G.J. & Charbonnier, J.-B. (2001) Crystallization of
macromolecular complexes: Stoichiometric variation screening. J. Cryst.
Growth 232: 580-590.
But then ... I thought that as long as I followed the standard rules for
crystal growth, even stoichiometry variation could be part of
standard crystallization methodology.
Since you still got all the details of the interaction from the structure
even with extra molecules, I was not ashamed of my mistake and tried
to publish the structure. The referees did not seem to mind:
Graille, M., Stura, E. A., Taussig, M. J., Corper, A., Sutton, B. J.,
Charbonnier, J.-B. & Silverman, G. J. (2000) Crystal structure of a
Staphylococcus aureus protein A domain complexed with the Fab fragment of
a human IgM antibody: structural basis for recognition of B-cell receptors
and superantigen activity. Proc. Natl. Acad. Sci. USA. 97: 5399-5404.
Enrico.
On Tue, 21 Jan 2014 18:02:48 +0100, Eugene Osipov <e.m.osi...@gmail.com>
wrote:
May be your complex components interact with column? I mean: you have
calibration plot for column, right? Does the molecular mass of proteins
calculated from elution volume equals to mass calculated by another
method
(like SDS-PAGE)?
2014/1/21 Keller, Jacob <kell...@janelia.hhmi.org>
Maybe there is something required for interaction that was in the
buffer
used for the other binding studies, but not in your SEC buffer?
JPK
*From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf
Of *David
Briggs
*Sent:* Tuesday, January 21, 2014 10:52 AM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] Isolation of protein-protein complexes.
Dear all,
sorry for the slightly off topic post,
I have 2 proteins that have been shown to interact, by multiple groups,
and by multiple techniques - namely ELISA, SPR and DPI.
The Kd of the interaction as determined by SPR is on the order of 1 nM.
I would very much like to crystallise this protein-protein complex, and
as
a first step I attempted to purify the complex by mixing the two
proteins
(same protein preps and same buffers as the SPR experiment) and then
running them down a gel filtration column (Superose 6 - predicted size
of
the complex is ~500kDa).
Somewhat irritatingly the two proteins separate beautifully on the
column
into two distinct peaks. There is no trace of complex formation when the
peaks are analysed by SDS-PAGE.
As far as I am aware, two proteins that interact this strongly should
remain associated during gel filtration, and I was wondering if anyone
else
has encountered anything similar in the past, and if they managed to
resolve the problem, how they went about it?
Cheers in advance,
Dave
============================
David C. Briggs PhD
http://about.me/david_briggs
--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab
http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
http://scholar.google.com/citations?hl=en&user=Kvm06WIoPAsC&pagesize=100&sortby=pubdate
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
