I am working on a thermostable protein (host living temperature at 60). After the purification, I set up some trays using fresh purified protein and got crystals diffract to about 3.5A. Afterwards, I did optimizations with the freezed protein(stored at -80 for 2 weeks and the protein stored in fridge for 2 weeks). I found that both of these proteins cannot produce diffracting crystals but can be crystallized easily as I did using fresh purified protein before. It contains 6 cysteines. I have tried heat shock the freezed protein with 5mM DTT/2-mercaptoethanol but it precipitated after 10mins shock at 60.
If anyone knows the reason or has similar experiences please can you let me know?
Kind regards.
Hi there,
I'm afraid you will have to optimize your crystallisation conditions with freshly purified protein prep as you can't reproduce the diffracting crystals with your protein stocks...
Hi there,
I'm afraid you will have to optimize your crystallisation conditions with freshly purified protein prep as you can't reproduce the diffracting crystals with your protein stocks...
You might need to achieve vitrification conditions to keep stable your protein during freezing; especially if you are working with a large protein or one that has multiple subunits. To accomplish this you have to use liquid nitrogen and small amounts of sample (ul) and an appropriate tube (i.e. Cryotops). Then, you have to keep the tubes always in liquid nitrogen. If you do not have all these setups it maybe more simple to use fresh protein. Good luck!
Ruigang, prepare small aliquots when you freeze your fresh protein. Smaller volumes (I always use 50 microliters) will freeze faster and they will also allow you to thaw just enough protein for setting up a tray, or a few drops, or running an assay, etc... Even proteins that freeze well do not like to be frozen and thawed several times, so avoid freezing large aliquots that contain more protein than you will need for a single tray, assay, etc...
Adding 5-10% glycerol is a tried and tested method for improving the survival of protein during storage at -80oC or in liquid nitrogen. However, the glycerol might interfere with crystallisation and I tend to avoid using it unless a protein won't freeze without it. If you can't reproduce the crystals you obtained from fresh protein with protein stored with glycerol, try to screen again as you might get new hits in the presence of glycerol. Conditions that previously gave you showers of microcrystals or clusters, might give you fewer and larger crystals as glycerol reduces nucleation.
As Domingue and Eduardo mentioned, if you simply can't reproduce the crystals or grow different ones with frozen proteinl, then you will have no choice but to work with fresh protein.
Have you looked at the stability of your protein at room temp.? I've previously worked with a protein from a thermostable organism, which behaved better when I just left it on my bench. Even leaving it at 4 C caused it to precipitate, while it was okay at room temp. for weeks. You could test by leaving a small aliquot from your next batch at room temp. and check for degradation by SDS-PAGE (and if you can crystallize it if it still looks good) after some time (or at several time points).
Thank you for all the replies. The protein has no degradation when I stored it in the fridge for two weeks seeing from SDS PAGE. I will use fresh proteins to do crystallization, maybe store them in fridge or room temperature and use them as soon as possible. Glycerol is also worth to try when freeze the protein I think.
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