I store recombinant protein (produced in E. coli) in 8 M Urea. It's aliquoted and frozen in -70 deg. Recently I started to notice that certain monoclonal antibodies had stopped recognizing it. I suggest this is due to protein aggregation. It's known that the protein I use has a domain responsible for aggregation. I wonder is there any way to prevent this process or to slow it down?
In 8m urea you are denaturing your protein - as a result exposed residues (probably the hydrophobic regions) are likely to want to associate and therefore precipitate.
Proteins can vary dramatically in how hydrophilic /charged a matrix they tolerate. Generally, most proteins like a small amount of methanol in the matrix environment to lessen the dominance of charged forces on them but this is a very loose generalisation.
Basically I am saying don't store this particular protein in 8m urea try a pH buffered saline or experiment with lower Molarity Urea.
One option is produces the induction in presence of ethanol 2% to activate chaperon and improves the correct folding
A bit more detail would be helpful. These mAbs that stopped recognizing this protein, in what type of assay did you use them (i.e. ELISA, WB)? The epitope they recognize, is it conformational or sequential?
Loss of recognition by the mAb could be caused by aggregation, but another possibility is that your protein is being degraded.
As Alejandro suggests, a bit more detail on your storage conditions and detection method would be helpful. For example, some proteins, especially when stored at low concentrations, will be lost over time due to binding to the side of the tube. Also, if you detect by Western blot after fractionating over a denaturing SDS gel under reducing conditions, it is unlikely that your reduced signal over time is due to aggregation (since most aggregates should be disrupted by heating, SDS and BME). Finally, I've worked with proteins where disulfide bond formation leads to aggregation. I suggest adding 2-10 mM DTT (or BME) to your stored sample(s). Also consider adding 10-50% glycerol and/or detergent (as Vladimir suggests) in addition to (or perhaps instead of) 8M urea.
Consider storing at a lower dilution. Use a buffer in which the protein is in its folded state.
Does the aggregation happen only on freeze-thaw? Does it happen even at RT? Is the aggregation confirmed on SEC or SDS?
Dear Ilya
You can add arginine to the buffer to increase the stability of your protein to aggregation. You can see some details in the following link
http://pubs.acs.org/doi/abs/10.1021/bi047528r
Best regards
As Alejandro and Jason suggested it would be helpful if you could provide more info about the protein. Why did you choose this particular storage condition? Is there a reducing agent included? What it the protein concentration? What type of assay are you using? As Jason pointed out if you detect by WB it is unlikely that signal reduction is due to protein loss via aggregation since you most likely denatured all of it. As many people here suggested, glycerol would definitely help but we need to know a little more
Thank you for your answers! It's a great pleasure to read them!
Yes, of course, I should have provided more details about the protein. Here they are.
We're screening hybridomas specific to the protein in ELISA. I produced it in E. coli (60 kDa). As it formed inclusion bodies I decided to dissolve them in 8 M urea. The concentration of purified protein is estimated about 1-1.5 mg/ml. It forms precipitate in PBS during about a week of storage and remains dissolved for a bit longer period of time in bicarbonate buffer.
We noticed that some hybridomas had lost their activity to the protein in indirect ELISA. I checked the earlier hybridoma's supernatants and their activity dropped too. So, I concluded that something wrong had happened with the antigen. As I said protein had a domain responsible for its aggregation. That's why I suspect some epitops have got hidden.
I added PMSF and Pepstatin A to protect the protein from degradation due to residual peptidase activity. Today I checked proteins integrity in SDS-PAGE, it was OK. Working solution is stored at 4 deg C protected from light and the aliquotes for long-term storage are placed in a freezer (-70 deg C).
Yes, it seems that aggregation is happening during freezing. Maybe I shouldn't have done that.
Forming disulfide bounds is probable because it has 16 cystein resides. I didn't think about that to be honest.
16 cysteines! I now feel even more strongly that adding a reducing agent will go a long way toward solving your problem.
Since DTT is a better (stronger) reducing agent than BME, I suggest using that if you have some available. Good luck!
You are most likely dealing with a protein sample that is quite heterogeneous. And as Ray suggested, it is possible that a significant portion of the protein is unfolded/denatured in 8 M urea. And as Jason mentioned, DTT should definitely help.
There are several things you could try but none of them is really an easy fix, I'm afraid. I guess you may need to decide how much time and effort you are willing to invest in solving the problem.
Would you be willing to attempt refolding the protein from urea when you have a fresh prep? There are several refolding protocols available online. It could help you find conditions that keep the protein more happy in solution. Your protein seems to like more basic buffers (bicarbonate). Do you know what is the working oligomeric state of the protein in solution? If you have some protein to spare you could try to run a small sample of it on an analytical gel filtration column (maybe in a bicarbonate buffer) to see if you can distinguish several species (that is aggregate and included, folded forms). It would be great if you could do size exclusion runs with a fresh and an old protein sample to see if the distribution of species has changed (while being careful not to clog the column with precipitating samples:)). Later, assuming that you found good refolding conditions, you could run your protein in that particular buffer on the column again. This would all take a while though....
Good luck!
Thank you, Magdalena, for your advice! It seems we deal with some hybridomas specific to confrontational epitopes. So, your thoughts are handy. And, yes, addition of reducing agent really helps to get higher OD value for some clones.
Arginine hydrochloride and glycerol are both effective aggregation inhibitors. I have had success with arginine in minimizing the aggregation of an aggregation-prone intrinsically disordered protein. I had to use a relatively high concentration of arginine to see an effect (~500 mM to 1M). I have also had some success with 40% glycerol (on the same IDP). pH optimization is also important - select a pH that maximizes charge on your protein. I recommend the following papers if you are interested in reading further on the use of arginine hydrochloride and glycerol to prevent aggregation:
Arakawa, Timasheff et al. Biophysical Chem 2007
Vagenende,Trout et al. Biochemistry 2009
I also recomended arginine and a little concentration of CHAPS around 1 mM
Dear Ilya,
Freezing is precypitating urea, I never keep my urea preps below 20 deg. The longest time I was storing the urea - solubilised proteins on the bench was half a year without degradation. In the 8M urea the proteases do not work, thus you can forget about protease inhibitors.
regards - jan
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