Thanks for the help,
Here is some more information.
Protein is expressed in E. Coli, three methionines in the protein. Protein
forms a disulphide linked homo-dimer. It is prepared from the soluble fraction
of E. coli lysis buffer. Protein is ~12kDa.
Protein runs identical to the native on a non reducing SDS gel.
M. Gordon Joyce,
Visiting Fellow,
Structural Immunology Section,
Laboratory of Immunogenetics,
NIH/NIAID,
12441 Parklawn Drive,
Rockville,
MD 20851
Phone: 301 594 0242 Office
301 496 3792 Lab
From: Pascal Egea [mailto:pas...@msg.ucsf.edu]
Sent: Sunday, March 28, 2010 6:17 PM
To: Joyce, Gordon M. (NIH/NIAID) [F]
Subject: Re: [ccp4bb] Se-met protein as soluble aggregate
Hi Gordon,
You may have to give us more details about your protein.
I would first ask you in what host (Ecoli, yeast ...) are you expressing your
protein and how many methionines do you have in your protein (and what size of
protein). Do you prepare it from a soluble cytoplasmic fraction or a
periplasmic fraction for example?
It is not unusual to observe changes in solubility and behaviour for a given
seleno-protein when compared to its unlabeled equivalent. This is why you
usually have to recalibrate your crystallization conditions (or even change
them drastically for the seleno-labelled protein).
Selenium is overall more hydrophobic than sulfur.
Does your seleno-protein run fine on a non-reducing SDS gel when compared to
the native protein?
If oxidation is the problem you can try to add EDTA and methionine in your IEX
and gel filtration buffers.
I would also see if switching the GF step before the IEX step changes things.
Do you need the IEX step?
If agregation is only due to increased hydrophobicity then may be increasing
ionic strength in your GF buffer will solve your problem.
Alternatively use cobalt chelating resin for affinity purification, it is
milder and requires less imidazole at the elution step, just in case imidazole
is too aggressive on your protein. I have noticed this in the past on some of
my samples.
Hope this helps
Pascal
--
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab (310)-983-3516
email pe...@mednet.ucla.edu<mailto:pe...@mednet.ucla.edu>
