Protein preps become a jelly

From: aidong
Date: 30 August 2011 16:31
Dear Buddies,

Sorry for bothering you with an off-ccp4 question.  We recently are experiencing a very strange phenomena.  A couple of protein preps with reasonably high concentration (10-20mg/ml) become a jelly after storages for overnight or a couple of days at 4C.  All of them have been purified by gel filtration.  Some of these proteins behave like this from very first preps but some of them had been very kind to us previously.  We have googled extensively in CCP4BB and www but it appears this only happens to us.  It would be highly appreciated that you could exchange their experiences or provide your suggestions.

Aidong Han, Ph.D

Department of Biomedical Sciences
School of Life Sciences
Xiamen University
Xiamen, Fujian 361005
China
Web: http://life.xmu.edu.cn/adhanlab/

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From: Laurie Betts
I have had protein crystals (so very high protein concentration) that turn into gummy bear-like objects, where instead of crumbling they are like, well, a gummy bear or a piece of rubber.  I attributed it to oxidation or other chemical ageing processes.  I am sure others will have suggestions for preventing this.

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From: Roger Rowlett

Not necessarily uncommon. To minimize protein-protein interactions that might be causing gelation, you might change the pH of the solution so that is is not close to the pI, use a small amount of chelator to sequester metal ions, and maintain a modest ionic strength. Other additives that might stabilize soluble protein would include glycerol or other polyols, but these may interfere with crystallization. A typical storage buffer for us is 20 mM Tris-Cl, pH 8.0, 10 uM EDTA, 100 mM NaCl. You can add 5-50% glycerol to stabilize protein if desired, and/or omit the NaCl if you protein is tolerant. It is also possible your protein is crosslinking via disulfide bond formation, in which case a modest amount of reducing agent may be necessary, e.g. 10-100 mM beta-ME, 1-10 mM DTT, or a small amount of TCEP. Usually, the less stuff you put in the storage buffer, the easier it is to crystallize the protein, but as always, YMMV.

Cheers,

_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

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From: Patrick Loll
Certainly not unprecedented, or even that unusual (I remember making gels from BSA and IgG solutions during grad school rotations).  Gel formation usually requires crosslinking, so consider whether you might be getting adventitious disulfide bond formation.
Pat

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From: Prince, D Bryan

I had a similar problem with crystals that were obtained from PEG-based precipitants over long periods of time. If I harvested the crystals in about 5 days, they would diffract to ~3 Angstrom. If I let them grow any time past about 7 days, they became PEG-alated and didn't diffract at all. I personally have not had that happen in salt-based precipitant conditions. I would try adding up to 10mM DTT or 5mM TCEP to minimize proteinaceous skin formation, which I believed was crosslinked by the PEG. Attempts to change PEG's did not work and I couldn't find a salt based precipitant condition that would give us crystals where the active site was not occluded by the salt.

BTW, have you purified the prep over SEC followed by IEX or RP columns? Have you dissolved the gel and run mass spec to see if there is any proteolysis going on? If you have an active proteolytic fragment, there may be one or more hydrophobic patches being exposed and this could cause the protein to gum up in order to bury those patches.

Anyway, good luck!

Bryan

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From: Michael Thompson
To add to what Pat has mentioned, I work with a protein that has a number of exposed Cys residues, and it turns into a gel at 4 degrees within a week, even if I store it in buffer with reducing agents. This happens every time I store it at 4 degrees. To test if your "jelly" is due to S-S crosslinking, add a really huge excess of DTT or beta-mercaptoethanol to a small sample and see if it goes back into solution. I usually add 100mM DTT for this test. If it does turn out to be disulfide-related, you can try to add reducing agents with longer half-lives (ie TCEP instead of DTT), but unfortunately you may just have to work with the protein within a couple days of finishing the prep.

Mike

--
Michael C. Thompson

Graduate Student

Biochemistry & Molecular Biology Division

Department of Chemistry & Biochemistry

University of California, Los Angeles

miket@chem.ucla.edu

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From: Hargreaves, David
Hi Aidong,

I've seen this in protein samples that have been snap frozen then thawed on ice. Thawing at room temperature (or in the hand) stopped this state forming. Could temperature be the issue? Maybe try 10C or leave the samples on the bench for a while and see if the state is reversible?

David Hargreaves
Associate Principal Scientist
_____________________________________________________________________
AstraZeneca
DECS, CP&SS
Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF




Gamma delta resolvase catalytic domain stock solutions used to make a nice clear jelly at 4 degrees, but it was perfectly reversible by warming the sample to room T.  In fact, one mutant crystallized in the stock tube after a few trips in and out of the fridge.  The crystals didn't diffract very well (so we never published that), but it was a fun way to grow them.
 Phoebe

=====================================
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago




From: Jacob Keller
Date: 16 September 2011 20:44


I think I know another protein that does this: gelatin! (Well, not the
crystallization part...)

Jacob

Dear Buddies,

I truly appreciate many of you are so kind to spend your time in replying my email with a variety of suggestions regarding this strange problem. Most of you have pointed to oxidation damage.  We tested quickly but high concentrations of DTT or TCEP including EDTA and/or glycerol seem to improve one of three proteins in our hands while other two are still the same.  We are testing different factors, such as pH, salt concentration, detergents, additives and even temperature (Low temp is not good?).  Whether it is intrinsic property of our proteins requires further experiments. I hope to write back as soon as we have clear conclusions.  Many thanks again for sharing your priceless experiences.

Cheers

Aidong