Hi, Everyone,
I performed oligomerization studies on my protein by applying two methods: size exclusion chromatography (SEC) and photo-induced crosslinking of unmodified proteins (PICUP). The protein is human and there are no literature data indicating that posttranslational modifications are required for its activity/function. The protein was obtained in E. coli BL21-CodonPlus(DE3)-RIL cells as a fusion protein with 6xHis-tag at the C terminus.
A chromatogram from SEC showed that my protein is monomeric only, whereas PICUP analysis indicates that it can also form dimers (please see Figure below, lane 7). Both methods were carried out in a phosphate buffer (10mM pH 7.4 for PICUP and 50mM pH 7.6 for SEC).
Is it possible to obtain such results and if yes, how to explain them? Does anyone have an idea why additional bands (smaller than 25kDa) appear in a sample where oligomerization of my protein occurs (lane 7)?
Perhaps a possible explanation for a dimeric state of my protein is that in physiological conditions a monomer-dimer equilibrium is shifted toward monomers, and by using PICUP method I simply managed to “capture” the oligomeric state of my protein. The other possibility is that I observe some artifactual cross-linking, e.g. due to the presence of 6xHis-tag?
Below I give details for my PICUP analysis:
- protein concentration was 25 µM (according to Vollers et al., 2005)
- the ratio of protein: Ru(Bpy)3: APS was 1:2:40 (according to Vollers et al., 2005)
- samples were irradiated with visible light for 7 s,
- and then gradient SDS-PAGE (7.5, 10, 12.5, 15% running gel and 4% stacking gel) was performed to show the distribution of oligomers.
Thank you for your help and remarks.
"SEC showed..."
you mean chromatogram of SEC or SDS-PAGE gel after SEC?
I would suggest you should be sceptical with SDS-PAGE gels because it is not always reliable. SDS-PAGE Gel is a screen of your sample but not always true!
In my opinion, the best suitable method for your stuff is Native MS and also XL-MS
Hi Erman,
I meant a chromatogram of SEC. SDS-PAGE was used to show results from PICUP.
The data is consistent with your idea that at the low protein concentrations (SEC) you have monomers... and at higher concentrations (25uM or ~0.5 mg/ml in PICUP) you get a small amount of dimer. I agree the dimer band looks promising... but you need to be concerned that your crosslinking may be non-specific... just background caused by driving the protein together at a high concentration. Under the conditions shown you have a lot of background smearing and higher-order complexes. I would next vary the amount of crosslinker keeping the protein concentration (and amount of light) constant. You want to find a good crosslinker condition (concentration/light) that gives very little background smear and a good sharp dimer band. Once you find that concentration then try varying the protein concentration to show you can get dimer formation over a range of protein concentrations and the amount of monomer converted to dimer increases as you increase protein concentration. It would also be good to show that your reagents can specifically crosslink a control protein (like GST) from monomer to dimer at the conditions used for your crosslinking. A mixture of you protein and GST should only give homodimers (GST dimers or your protein dimers and not a mixture). Finally, the 6HIS tag is a worry... you could try adding 5 mM EDTA to soak up any free Nickel that might have leached off the column and is now crosslinking the 6HIS tails.... but I am concerned EDTA might interfere with your APS based chemistry, so you should get rid of it by passage through a desalting column (like a Zeba spin 7K thermo fischer). The smaller bands appear to be UV light induced breakdown products of your protein... I don't find them to be a big problem.
Best of luck,
Chris
Hi Christopher,
Thank you for your comprehensive response and valuable tips. I will try to optimize the procedure.
What other control protein could you recomment instead of GST?
Dear Christopher,
you said: "The smaller bands appear to be UV light induced breakdown products of your protein... I don't find them to be a big problem."
The problem is that I used visible light, not UV light, so I am afraid there must be another reason for the presence of these bands smaller than 25kDa. Do you have any idea?
In addition, Do you think that hemoglobin, known to exist as a tetramer, is a good choice for PICUP control?
Thank you for your comments.
the bands smaller than 25 kDa might be oxidized monomers of your protein due to highly oxidizing conditions(?)
oxidized monomer could possibly run faster on a PAGE-gel compared with the reduced monomer because of compact conformation.
Hi there,
SEC results are sensitive to the dynamics of the system you are looking at. If the protein dimer is not stable enough (roughly Kd above µM range) it will dissociate during the SEC run and the result will be : my protein is monomeric on SEC column. If performing crosslinking, the parameter is mainly distance between reacting chemical functions. As it is performed in solution, the equilibrium association dissociation of the protein monomer will allow the chemical crosslinking to occur and then the conclusion will be : my protein is dimeric in solution. These two results are consistent with a low affinity interaction between monomers.
Hi Dominique,
thank you for your valuable comments. It makes perfect sense to me:) However, I will pass my protein through a desalting column to remove Ni2+ ions and then try to optimize PICUP method to get sharper bands, and finally run some experiments with substrates and cofactors for my enzyme, which, based on my kinetic studies, could affect a monomer-dimer equilibrium. Then I will have no doubts about oligomeric properties of my protein. Thank you once again!
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