My protein is DNA binding protein, with the DNA binding domain having a pI of 9.3, while a tail of about 18 acidic amino acids is attached on the C-term end, making effective pI 5.2. I can achieve about 95% purity. The protein is in Tris 30mM, NaCl 200mM and Glycerol 10%, but is still forming soluble aggregates. Can anyone please suggest something.
Hi there,
Gel filtration should be OK. You have to consider the MW of the species you want to separate and use the suitable resin. But it won't stop the process of aggregation to occur in the purified fraction (if this process is spontaneous). An other idea is to optimize the buffer composition and pH in order to minimize the spontaneous aggregation.
It seems the protein is not happy in the buffer/formulation it is in. Screening the chemical space is highly recommended. Finding the right formulation will also impact the yield you end up with after reprepping and using the found hit in terms of buffer/formulation. That is what I would recommend. It is actually tackling the problem at the beginning and not using gel filtration as even after filtrating aggregation might re-occur.
To answer to Thomas,
It all depends on the velocity of aggregates formation compared to the time scale of the things you intend to do with your protein. I wouldn't be so definitive about using gel filtration...
A first goes is that you are stabilizing ionic aggregates. These are probably heterogeneous. A quick test is looking for the effect of ionic strength. Higher ionic strength should decrease the strength of salt bridges. This can be easily tested by 90° light scattering, turbidity, or dynamic light scatter. It would be likely that size exclusion chromatography will give you a smear as will any centrifugation technique. If possible you can also try lopping off the N- and or C- terminus to see if that improves solubility.
@Thomas Wohlfarth.. Thank u so much..
@Dominique Liger.. Its forming aggregates of various sizes which are inseparable, even with superdex200. It was done even at 1M NaCl concentration
@Ross Thomson.. i intend to crystallize protein and use the same for DNA binding assays.. Though the protein is active and binds to DNA, it would be difficult to find conclusive results with this kind of protein.. and also these aggregates are even soluble upto 16mg/ml, however as soon as i add precipitant for crystallization trials.. boom!! everything aggreagates.. trials done even at 3mg/ml
@Adam Zlotnick.. thank u so much.. DLS gives a wide no of particles sizes.. pointing to random aggregation points.. protein is kept at 1M NaCl throughout the purification.. No help :-/
If you are trying to prevent aggregation, I agree that you should work to optimize the chemical space with regard to pH and ionic strength. Perhaps try lowering the ionic strength. If you just want to get rid of the aggregates prior to experiments, you can do so by gel filtration or ultracentrifugation.
I used to have a similar problem with an IMAC-purified SSB I was investigating. I was obtaining what I thought were soluble high MW aggregates of my protein as assessed by size exclusion, followed by SDS-PAGE of the peak fractions. All of the peaks appeared to be highly pure recombinant protein. However, UV spectrophotometry of these samples revealed that the high MW species were contaminated with nucleic acid (presumably non-specific nucleoprotein complexes). I repeated extractions in the presence of DNase I and RNase A, and followed with IMAC and size exclusion to remove the nucleases. This reduced the amount of high MW aggregate significantly.
I am not sure if you're experiencing similar complications, but I though I would share. I think it would be worth running some diagnostics on your aggregate fractions.
Jon E Ramsey :
In fact, i am also purifying a SSB. After asking this question, i changed my buffer to HEPES and added KCl along with NaCl. Though, the type of poly disperse particles have reduced, its still not completely gone. This time a 65kDa impurity came, probably chaperone. I am still trying to optimize it. So you may also try changing the chemical space of your protein. Thank you so much for your input and do share your experience for the same.
Hi Amandeep, you could try making your buffer with 5-10 mM EDTA. Since divalent cations, Ca++, Mg++, etc (even in trace amounts), can bind tightly to acidic amino acids and cause precipitation
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