I am trying to crystallize a protein which has a hydrophobic pocket in it. The problem with the protein is that some of it precipitates while concentrating the protein in amicon concentrator. After centrifugation of concentrated protein, I see white precipitated protein at the bottom. After centrifugation, I get 6mg/ml protein in supernatant. The addition of reducing agents (5mM DTT) before protein concentration and increasing salt concentration in purification buffer has helped to prevent precipitation up to some extent. However, When I use 6mg/ml protein to set up a crystallization plate with different screens, I see immediate white precipitation in 70-80 wells out of 96 wells after the addition of mother liquor. Composition of purification buffer is 50mM Tris, 250mM NaCl, 5% glycerol. How can I increase the solubility of protein so that chances of protein crystal formation will increase?
Reducing the salt concentration will probably help. A change in pH may also be beneficial.
Based on what Adam says, you can set the pH of your protein over an incredibly vast range using volumetric ratios of 1 M of Bis-Tris Propane (pH = 11.2 - 11.5) and 1 M Citric Acid (pH = 1.2 - 1.5). Since this mixture has multiple ionizable protons, it covers and incredibly vast range of pH. Scientists at Hampton Research did the calculations for you.
Multiple droplet ratios in the same well may also be helpful.
1:1, 1:2, 2:1 with respect reservoir to protein solution may be helpful.
Sometimes crystals or precipitants show up in one of those drops and not the other.
Agree with both Adam and Adron. One other thing you can do is to perform a Precrystallisation Test (PCT) which is commercially available to determine whether you POI at that concentration is suitable for crystallisation - this will allow to to either increase or decrease the protein concentration. You've mentioned that you have 70-80% precipitation on a 96-well plate. If heavy precipitation then perhaps, decreasing the concentration from 6 mg/ml to say 4 and 3 mg/ml may not be a bad idea.
You could also try a buffer screening on a 96-well format and do a temperature gradient using a PCR machine to get a read out of how stable your your protein is with time.
If your protein has a ligand perhaps adding it might help stabilise your protein.
@Adron protein crystallography is a matter of trial and error! I have worked on a DNA binding protein and crystals are only obtained when the protein form a complex with the DNA. All attempts made so far with the protein alone produced no crystals!
Bubacarr G Kaira , that is good thing. The crystal of a protein:DNA complex is more interesting than that of free protein. I think I have obtained a crystal of the protein:DNA complex. But the crystal did not want to form.
I like explaining to scientists that crystallography is more of an artform than a science and then enduring the backlash for my lateral thinking.
@Adron Did you try to optimise the protein:DNA crystals from the initial hit..
Things to try:
1) Hanging with bugger dropsize.. Sometimes with sitting drop the crystals tend to stick to the bottom of the Well and thus hard to fish and fall apart easily as the solvent content for DNA can be high
2) Try seeding using your own hair or a seed bead in a hanging drop format
3) Nucleix screen can be commercially purchased as it is a sparse matrix designed to crystallised DNA or DNA in complex with proteins.
4)Or try the Noami screen as it helps to crystallised some proteins by providing a surface to form nuclei on.
5) Gel filtrate DNA:protein complex followed by setting crystals trays..
you can make different site directed mutagenesis in the hydrophobic pocket by change residues from hydropbhic to hydrophilic residues which can increase solubility of your protein but you have to check if these changes can affect the protein stability or not, to check that you can use phyre 2 or swiss model servers to create predicted structures of your protein to know where the hydrophobic pocket excatly in your protein structure then you can choose which residues you can change.
second thing you can do is using solubilty tag such as MBP tag with fixed arm linker (three alanines) which was successfully crystallised many proteins as it can increase the crystal packing of the target protein but before that you have to check your protein size as somtimes large solubility tag like MBP tag can interact with target protein , if your protein has high molecular weight then you can use small solubility tag (e.g. Sumo tag )to avoid any interaction with protein target activity.
You can try to measure the second virial coefficient of your protein solution by sedimentation velocity centrifugation, osmotic pressure, fluorescence correlation spectroscopy, small angle X-ray, neutron or light scattering as function of protein concentration. B2 should be small and negative (2-8 e-4 mol ml g-2, doi:10.1016/S0022-0248(01)01076-4, 10.1107/S0907444994001216, 10.1016/S0006-3495(01)75838-9, 10.1016/S0006-3495(02)75513-6). That way, you set up your crystallisation screen only for a promising region.
Hi Sanketkumar,
As mentioned in the previous comments, you can decrease the protein concentration appropriately. I have a protein that is easy to precipitate during crystal screening but crystallized when I decreased the protein concentration to about 3mg/ml. In addition, one of the disadvantages of adding a solubility tag is that it may affect protein-protein interactions. If you study the structure of the complex, and you need to choose the tag carefully.
Good luck!
Roger Davis , if you do go through the trouble of adding a solubility tag by cloning then you should add a cleavage site between your protein and the tag. Cleaving the tag adds another step to the purification which results in purer protein but you lose protein with every purification step.
Hi Sanketkumar,
I hope you solved the problem with the solubility of your protein.
If not, then there something you can try.
1. Use XtalPred to see how easy your protein would crystallize.
http://ffas.godziklab.org/XtalPred-cgi/xtal.pl
2. Use Expasy ProtParam to calculate pI of your protein
https://web.expasy.org/protparam/
- Many crystallization conditions are for acidic proteins with pI below 7.
If your protein has basic pI (above 7) and crystallization conditions are for pH below 7, you are crossing the isoelectric point. That could be a reason for precipitation.
3. Salt like NaCl may increase solubility, but not always. You can test up to 1M NaCl, we had these kind of cases.
4. Bigger drops, low temperature may slow down the crystallization experiment, and that may reduce the precipitation.
Hope this helps,
George.
- Badges
- Science method