I am working on bacterial protein crystallization (protein pI 9.2). During purification, I usually maintain 200mM KCl salt (20 mM Tris 7.5, 10% glycerol) in final elution buffer and followed by protein crystallization. I have tried crystallization hits with 200mM salt conc. I want to check crystallization hit with higher salt concentration, so please suggest me that what other alternative salt conc. should be tried?
Dear Ajeet,
I do not get your question very clearly. I understand that you have already tried several crystallization cocktails, right? Many of those have salt at very high concentration so why would you like to increase the initial KCl concentration within your protein solution? If you could give us more details of your experiments we will be able to help you better.
I have read somewhere as 300 mM. You may try out this if you are keen to go for higher concentration!!! I do not have detailed idea about the protein crystallization, but certainly can say that sometimes, not only salt concentration but also other factors (like, solvent/buffer conditions) are responsible for the protein crystallization process.
@Dear Jose, I have tried crystallization hit at lower protein conc. nearby 3 mg/ml but whenever I am trying crystallization hit at higher protein conc. nearby 6mg/ml, during crystallization, my protein get precipitated in all PEG ion condition. For maintaining protein solubility, I have to increase salt conc. in my protein buffer.
Japanese group have reported if your protein pI and buffer pH difference is more, in that case we can go for higher salt concentration during crystallization.
please find the reference paper for salt optimization-
Dear Ajeet,
Yes, indeed typically to crystallized using PEG ideally you should be separated from the pI +-2 units while to screen with salt it is convenient to be far away from the pI. Therefore it makes sense to be far from the pI when using PEG based precipitant cocktails + salts. But remember that this is for the precipitant cocktail while you are asking how much salt should be within your protein solution and the answer is as much as you need to have a stable and concentrated protein solution.
What I mean is that it is very important the aggregation state of your protein in solution prior crystallization. You could investigate this with DLS but I do not know if you have this available. Still if you realized that to have a stable solution (no precipitation) you need a particular ionic strength them you should consider keeping this ionic strength constant or higher but never lower. Also, did you tried to remove the glycerol from the solution? Glycerol increased protein solubility, which can be good, but is an extra additive that could be affecting crystallization.
I hope this may help you somehow.
To avoid protein precipitation, one may try to use glycine or proline as additives (it is usually used during protein purification processes). PEG is also used for the same reason...
A systematic approach would be to screen your protein against a range of buffers, salts, glycerol, additives etc. using a thermal shift assay a.k.a Differential Scanning Fluorimetry (DSF). A distinct thermal shift signature is a clear indicator of the factors important for protein stabilization, which in turn increases the chances of protein crystallization.
There is no thumb rule for maintaining the concentration of the additives!!! But, in principle one can try up to 0.1M. So, may be you can try out multiple tests at a time with variable concentrations (For example, 30/50/100)....
There is no simple rule for crystallization, which is a trial and error process.
In general, several crystallization screens are available, from several companies, each with tens of different conditions. We also usually test our proteins by Differential Scanning Fluorimetry, in order to obtain information of the most stabilizing/destabilizing conditions. However, we feel that the obtained DSF data are useful especially to maintain the purified proteins in the conditions that warrant the lowest risk of losing the protein by precipitation.
you want the lowest salt concentration possible to keep your protein soluble and mono-dispersed, ie non-aggregated. a simple test/screen is to incubate in various salt concentrations and do a high speed spin and check for pelleting of insoluble protein. you want the lowest possible salt concentration to get the maximum protein concentrating/xtallization effect in your crystallization drop. Glycerol is also bad for crystallization screening, again, it lowers the concentrating/xtallization effect in your drop. 10% glycerol has a huge molar concentration.
@stevan
how much usually glycerol percentage can be preferred according to your previous experiences?
o% is best, but if you have to have it, you can try lowering it to 0.5% or 1%.
I think the best is as low as possible, no matter salt or glycerol. So it is a minimum buffer for crystallization. Ideally, if the protein is stable enough, 20mM buffer might be good start. Let the crystallization screen to take care of pH, salt, salt concentration, and additives. Salt and glycerol are usually added initially to support stablility and solubility. I think protein concentration should be at least 1mg/ml in most case. Some useful additives might come out from first screen, so could be used with protein for 2nd round screen.
But there isn't strict rule for crystallization. I also use salt and glycerol in some cases. And I saw some people routinely use 100mM or 150mM NaCl, 5% or 10% glycerol for crystallization, and they could get diffractable crystals. With 5% 10% glycerol, it will behave a little different with vapor diffusion methods. For some crystallization conditions, the drops could absorb water from well, so the drop might getting bigger.
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