I am working with a protein and had to dialyze the solution to remove salt. Even though, during purification and AcTEV digestion, the proten did not aggregate, during dialysis, it obviously aggregated. Down the line, I wanted to look at the structure of the protein, and was wondering, if the aggregation would mean it could easily crystallize?
BTW - I know nothing about crystallizing a protein...
Thanks!
A tendency towards aggregation does not necessarily man the protein will easily crystallize, or that if it does crystallize the crystals will be of diffraction quality. It would probably be better for crystallization if the protein didn't have a tendency to aggregate.
Aggregation of the protein when you remove salts is not a good start for crystallization efforts. You will need to have protein at high concentration (at least 10 mg/ml), with the least salt possible and avoiding aggregates. It sounds you will have to do some optimization even before starting screening fro crystallization conditions.
Dear Kanomi
I agree with Carlo you must used high protein concentration with very small salt.
Good luck
I would try to stay away from aggregation. As stated above, I think it will actually make it more difficult to crystallize. You'll want to find conditions that increase solubility and stability (there are kits for that--see Hampton Research, etc.).
See below for an example:
http://hamptonresearch.com/product_detail.aspx?cid=30&sid=201&pid=620
A direct answer to your question is - Likely not.
A lot of scholarly articles have been published in the past 2 decades in this area (e.g. Wilson group, Lenhoff group and many others) - i.e. trying to predict crystallization vs. aggregation behavior from certain physical properties. Such as using osmotic virial coefficient. Prediction still remains "crystal ball" unfortunately. Here is an example reference to learn more.
Biotechnol Prog. 2011 Jan-Feb;27(1):280-9. doi: 10.1002/btpr.536. Epub 2011 Jan 6.
Interactions and phase behavior of a monoclonal antibody.
Lewus RA1, Darcy PA, Lenhoff AM, Sandler SI.
A more direct approach - if you are seriously looking for crystallizing - would be to consult a protein crystallographer and try out some conditions.
I personally would work on the optimization of the purification and dialysis (pH, salt concentrations) to make sure no precipitation occurs. Crystallographers are very superstitious about precipitation. DLS is a good and fast method to check if protein is aggregated or not even if you don't see visible aggregation. The rule I follow is to bring protein to crystallization in the best conditions I can.
Hello Everyone,
Thank you so much, everybody, for your input. There seems to be a consensus regarding aggregation not being a great characteristic wrt crystallization. I appreciate all of the answers.
Kanomi
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