I am facing a precipitation problem in crystallizing the iron(II) containing metallo protein under anaerobic condition. When I prepare the low (100 micromolar) concentrtion of metallo protein, no problem. If I go for higher concentration, it will get precipitated. If you are having any idea to over come this hurdle, please suggest me.
Thank you very much
Dear Ramakrishnan,
In addition to what David and Jiang has posted-and very helpful advice it is- I would also try to crystallize the protein under oils using the microbatch method (see the douglas instruments home page for protocols and ideas). If it is oxidation of the protein causing the precipitation, you might try adding sodium dithionite to preserve the reduced state. Hope this is helpful, good luck!
You could change the volume ratio in the crystallization drop in favour of the protein stock solution, i.e. don't mix 1 part protein stock plus 1 part reservoir, but 2 (3,4?) parts protein stock plus 1 part reservoir. However, in this way other components of the protein stock solution (buffer, salt ...) are added in a higher amount as well.
Excellent suggestions so far. If I may add another, try finding a condition where your protein does not precipitate, and then use this as your drop condition and instead of using drop solution in your well, use NaCl as your well solution at varying concentrations say from 0.1 M to 3 M. The optimal salt concentration will slowly dehydrate your drop, and increase the concentration of your protein slowly and hopefully yield crystals.
When exactly is it precipitating? On the concentrator or afterwards (i.e. immediately or slowly or very wrong versus slightly wrong conditions)? Have you tried not keeping the protein in the fridge? Any fancy stuff during purification with his tags and imidazole and chelators and stuff?
How do you achieve anaerobic conditions? Degassing can influence solubility.
Check the Buffer perhaps you need a buffer that have buffer capacity high----If there is no effect of synergistic anion.
I certainly agree with the suggestions about optimising the buffer for your protein. In general, this is the most straightforward way to make a "problem" protein behave.
However, I would also recommend that you look carefully at your construct: if the protein is only moderately soluble, one reason for this might be that as well as your soluble domain, you have some cloning (etc.) artefacts that are affecting the solubility. It's not always the answer, but it's worth double-checking before you expend too much time on buffer optimisation!
HTH!
The solubility of disulfide bond containing protein can be increased by using a host strain with a more oxidizing cytoplasmic environment. Two strains are commercially available (Novagen):
AD494, which has a mutation in thioredoxin reductase (trxB).
Origami, a double mutant in thioredoxin reductase (trxB) and glutathione reductase (gor).
https://www.researchgate.net/publication/309041540_Important_Factors_Influencing_Protein_Crystallization
Article Important Factors Influencing Protein Crystallization
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