I am purifying 1-deoxy-D-xylulose 5-phosphate synthase (DXS) enzyme in E. coli BL21 (DE3) cells. I used one-step nickel chromatography to purify it, but I analysed the sample in the SEC column, and it showed an aggregation peak that eluted in the void column. I followed the protocol of a student who have already graduated. I also analysed her sample, it also contained the aggregation peak but was much smaller than that of mine.
Now, I am confusing what this aggregation is, where it occurs, and how to prevent it.
Media: M9/H2O
Lysis buffer: 20 mM Tris, 250 mM NaCl, 2 mM MgCl2, 0.5 mM TPP, 5mM DTT, pH 8.0 containing Complete protease inhibitors, 250U/mL Benzonase. The cells were lysed by sonication (20s on, 40s off, 70%, 5min) after incubating on ice for 30min, then centrifuged at 19,000g, 40min at 4 °C, the supernatant was filtered and applied to Histrap HP 5ml column.
IMAC Equilibration buffer: 20 mM Tris, 250 mM NaCl, 2 mM MgCl2, 0.5 mM TPP, 5mM DTT pH 8.0. 60mM imidazole as the washing buffer and 200mM imidazole as the elution buffer.
DXS is a dimer, the WM of the monomer is ~68kD.
I analysed the fraction without any treatment after nickel purification, it also showed two peaks as shown in Figure A. I have collected these two peaks, the A260/A280 of the first peak (aggregation) is ~1.0 and that of the second one is ~0.7. Did this mean my protein associated with the nucleus? The SDS-PAGE results are in Figure B, and the Native-gel results are in Figure C. In the native gel, the aggregation couldn't run into it.
I have changed the sonication to cell disruption, but it didn't work. All I could think of was to improve the lysis step, and I was not quite sure if the expression step could affect the aggregation. The Bensonaze I used is >90%, and I'd like to use a higher-purity one. Will this help? Did anyone meet this before, how to improve it? Many thanks for your suggestions.
My advice is not to worry about the aggregation, which is common when proteins are overexpressed. Use preparative gel filtration chromatography to remove the aggregated protein after the IMAC step. If you need more of the DXS protein, you can always scale up or repeat the procedure.
The A260/280 result is characteristic of aggregation. This is because of light scattering by aggregated material, which increases sharply with decreasing wavelength. It is not necessarily a symptom of nucleic acid contamination. (By the way, E. coli don't have a nucleus.)
the SEC shows that you have a good method to separate the two products, so maybe do this ? What is the end goal? To biochemically analyze the proteins function? If so I'd move on to that!
There are many subtle effects that can influence the balance between the two forms, the previous worker got lower levels. I'd ask them EXACTLY what they did if you need to reproduce their result.
If the SEC isolated unaggregated protein re-establishes the mixture then something more complex is happening but you can deal with that when/if it happens. Good luck!
Thanks Edward Michelini (sorry for the typo) and Adam B Shapiro . I am using NMR to study the structure of this enzyme. One of the reasons why I want to use one-step purification is that I will use isotopic media to express it, which is quite expensive. I couldn't reproduce the same results as the previous worker just from the experiment records, and not sure if I can contact her now. Maybe introducing a second step is necessary. The good news is once I get the happy protein, it is stable in the buffer and won't form aggregation. The sample of the previous workers I analysed was obtained from LB media, but I expressed it in M9, maybe the protein was less happy in M9.
Hi, you can inclease the NaCl concentrations in your buffers and also can add urea at high concentration (4-8 M). These can prevent the aggregation. But you may have to follow desalting to get rid of the urea after concentrating it.
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