Does your protein have multiple exposed Cys residues? I have observed this before with a protein I worked with that had many exposed Cys residues. In my case I could add more DTT and minutes later the gel would be completely dissipated. My hand-wavy explanation was that the protein stays folded but becomes highly crosslinked when concentrated and exposed to air. I did have some small amount of DTT in my buffer to start with, but I assume it became oxidized relatively quickly (as DTT does), leaving my protein thiols to go wild and make S-S bonds between themselves.
Good luck,
Mike
----- Original Message -----
From: "Pascal Egea" <[log in to unmask]>
To: [log in to unmask]
Sent: Monday, April 22, 2013 3:36:24 PM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] gelification of a pure protein
Dear All,
I am presently faced with a peculiar case in the lab. We are expressing a protein in E. coli and we are able to express it as a fusion protein without problems . Fusion cleavage goes well and the final product looks homogenous by size-exclusion chromatography with the expected molecular weight. There are no signs of aggregation. However when we lower the salt concentration by dialysis then the protein forms a gel. transparent , optically clear, with no fluffy material (in the cold room).
Gelification seems to occur when we lower the concentration below 100 mM NaCl. This protein has a fairly high pI (~9.0). Attempts to reverse the process by gentle heating or salt addition have been so far unsuccessful. It is not a thermophilic protein. We have not been able to obtain crystals so far.
Has anyone already observed this kind of behavior and/or have any suggestions?
Many thanks in advance .
--
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab (310)-983-3516
email [log in to unmask]
--
Michael C. Thompson
Graduate Student
Biochemistry & Molecular Biology Division
Department of Chemistry & Biochemistry
University of California, Los Angeles
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