Scalable insect cell expression and purification screening applied to CRL4-DCAF substrate receptors
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Abstract
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co-infection/co-expression
I really like your co-infection strategy: it highlights that many proteins require a more complex physiological environment to be studied. Do you have any data showing what happens if you try to un-bind the DCAF from the complex after purification? I wonder how it behaves if it is alone in solution - maybe the other components of the complex are required for its stability post-purification.
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formation
This is a nice bonus. And maybe is another explanation for why a few constructs didn't co-elute with the DDB1 - because the DCAF proteins weren't well folded.
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shown
it would be interesting to estimate the stoichiometry of each protein via the gel (obviously not the most quantitative, but you could get an estimate). Does the size of the complex off of the SEC suggest a 1:1:1 relationship? That would be a nice check that your are purifying what you think you are.
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High-Five
What is your rationale for doing scale up in Hi5 cells when you did your screening in sf9 cells?
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eluate
This could also be an issue of stoichiometry. If the interaction is strong, but the yield of expressed DDB1 for some reason was low, this could also be the case. It would be interesting to assay the amount of each DCAF bound to DDB1 versus free.
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CRBN.
I'm curious if there was any trend or difference that you observed in co-purification with DDB1 compared to the truncated DDB1.
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unstable
It looks like you are eluting your protein in a solution with very high conductivity. Perhaps eluting at a more physiological condition, like 100-150mM salt + imidazole, might reveal some additional interactions. I'm impressed that you co-retained so many complexes in your elution as is, though - those interactions must be quite strong!
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truncations
That's interesting. Do you have any theories why the truncations did not perform as well?
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bacmid DNA
I'm curious about your bacmid preparation in high throughput. Did this require manual colony picking for recombined colonies? Also, did you shake your 24wp on a large or small orbit?
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