I have two protein X and Y, X protein isoelectric point is ~5.5, Y is ~9.5. western blot result indicated that they can directly bind each other. I want to purify large amount of this XY complex and go to crystallization.
I purified this two proteins in Buffer (25 mM HEPES pH 6.9, 100 mM NaCl) respectively, after adjusting the concentration to 4 mg/mL each by centrifugal filter, I mixed clear solution 2 mL X and 2 mL Y, but the 4 mL solution became cloudy immediately. Then I filtrated the 4 mL solution by 0.22um filter, the flow through was clear, and I loaded on gel filtration chromatography. But the XY complex peak come out near the void volume position. Is this peak aggregation of XY? I checked this peak by native page, it indicated there was binding.
But if I mix the 2 mL X and 2 mL in low concentration less than 1 mg/mL each, the solution will not become cloudy and there are no binding peak in gel filtration chromatography. I also tried pH 7.9, there was no binding.
How can I purify this XY complex? My supervisor also did not know, so I really need your help. Thank you very much.
Hi there,
So interaction of the proteins ends up in precipitation which means the complex is less soluble than the individual proteins. What I would do in a first move would be to increase the ionic strength of the buffer in order to improve the complex solubility as 0.1 M NaCl is not that much actually (you may try 0.5M and even more). The issue is to know what is the main factor leading to precipitation : is it because of structural remodeling of the proteins induced by the interaction and possibly modifying solvant exposed surface rendering the complex more hydrophobic or is it because the interaction is just masking some hydrophilic patches and as a result increases the hydrophoby of the object...
Hi,
I'm a little confused by your opening statement "western blot result indicated that they can directly bind each other". I'm not sure how you can use WB to assess interaction between two proteins, do you have any other evidence of interaction? Can you do experiments such as SPR or ITC (the latter may also give some hint about possible large conformation change).
If the 2 proteins indeed interact, they are very likely to interact between hydrophilic patches as Dominique suggested: under the conditions you are using one is positively charged, the other negative. If this is the case just increasing salt concentration may not be much help, actually yuo could end up with weaker interaction between the two proteins. Alternatively, you can try adding small concentrations of detergents (well below CMC) or low concentration of ammonium salts (e.g. sulfate).
In case of protein rearrangement the situation is more complex: I would try to obtain small amounts of XnYm and characterize it: Mw, pI, Rh would give you indications about protein structure (before and) after association.
I wonder whether the turbidity you see when mixing X and Y at high concentration is caused by a non-specific aggregation or the formation of a specific very large structure such as a fibril. You could have a look by transmission EM with negative staining.
I agree with Dominique Liger that one thing to try is to adjust the ionic strength. There may be a range of ionic strengths at which the complex of the proteins is in the "right" stoichiometry. I would suggest using a non-chaotropic salt like ammonium sulfate, rather than the mildly chaotropic sodium chloride.
Hi, Dominique, thanks for your analysis of the possible reasons, still now I do not know what is the main factor leading to precipitation, I am afraid the 0.5M NaCl will dissociated the binding, but next week when I repeat this experiment, I will take 50uL each and adjust NaCl concentration to 0.5M, then mix to check there is still precipitation or not.
Hi, Carlo, thanks for your answer, I am sorry I did not state clear. X protein is GST-tagged, Y protein is his-tagged. I did GST-pull down and then go to western blot by anti-his tag. the result shows they can directly bind each other. Today in the group meeting, one people also suggest me to do ITC in some other lab. Thank you, I learned some new knowledge.
Hi, Adam, thank you so much for so many times help. I also read a paper, they checked the peak near the void volume on gel filtration by negative staining EM, but we do not have this equipment. My X protein is positively charged, Y is negative, if I mix this two protein, usually this binding is non-specific or not? sorry I did not understand this charged binding.
Non-specific electrostatic binding is, of course, a possibility. This will be reduced by increasing the ionic strength. (This is the principle of ion exchange chromatography.) However, electrostatic interactions can also play an important role in specific binding interactions, so it is not so simple to make the distinction.
Protein-protein interactions usually occur over a broad surface, with many points of contact and sometimes one or more very important "hotspots." Such hotspots can be detected by mutagenesis experiments that reveal a substantial loss of binding affinity due to a point mutation.
Hi Zixun,
Could you solve that problem of cloudiness of the mixture of two proteins?
- Badges
- Science method