Hi all,
I am working on a tricky protein. I surpassed some initial aggregation behaviour by adding 0.3% CHAPS and 1mM TCEP, buffer in which is happy until the removal of the SUMO tag, then it crashed out. When I spin the precipitate down and run the soluble fraction in SEC, my protein runs as a monomer. My question is: since the chaperone seems to be keeping it happy in solution, how far would you trust my soluble prep for activity assays? Do you think it would be correctly folded? Is it possible to have mixed populations of soluble, correctly folded protein and insoluble, misfolded one?
I don't wanna have high hopes on my monomeric protein, but still think it's worth giving it a shot in biochemical assays...
Please clarify which protein you want to work with, the SUMO-tagged protein or the supernatant after cutting off the SUMO tag. I suggest you try them both and compare. Please be aware that some journals may not accept enzymological data from the protein with the SUMO tag still attached.
The supernatant contained my protein, and when comparing before and after spinning in the gel, I had only lost around 50% of my total protein amount. When I loaded my supernatant (containing soluble protein) onto gel filtration, it runs as a monomer. However, when concentrating the relevant fractions for storage, my protein crashed out again in 0.3% CHAPS (suggesting a concentration-dependent aggregation behaviour). When doing gel filtration in the same buffer but with 0.8% CHAPS instead, the protein also runs as a monomer, and when concentrating it did not crash out. How far would you trust the monomeric protein in 0.8% CHAPS? Do you think at this concentration CHAPS in preventing aggregation, or disrupting the protein structure? Personally I have seen people using up to 1.5% CHAPS in their lysis buffer, so dont think it's too high
One way to deal with this is to do assays with several different concentrations of CHAPS. If it has no effect other than increasing solubility, you're fine. Otherwise, you at least know you have a problem. The same applies to the tag: if you can show it to have no effect on enzymatic properties, you are free to use it. In both cases, you evidence must be good enough to convince not only yourself, but also the reviewer of your paper.
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