I express a recombinant protein in E. coli that requires Mg2+ and ATP to remove bound chaperones and activate the enzyme, but also requires TCEP. I currently achieve active protein, but with very low specific activity of which a large fraction exists as soluble aggregates.
We have always added all 3 of these reagents during lysis at the same time. I am wondering if this does not maximize their efficacy. The concentrations we use are 1 mM TCEP, 2 mM Mg2+, and 5 mM ATP. I know that metals can interfere with the action of TCEP, and TCEP may interfere with the action of Mg2+. Does anyone else have any experience working with these reagents together? Would it be more efficient to add these reagents step-wise, e.g. starting without Mg2+ and ATP to give the TCEP a chance to work alone and then adding the other reagents after like 10-15 minutes?
Any help you can provide will be appreciated. BTW, we use B-PER as our lysis buffer base and add these reagents to the BPER.
Thanks,
Nathan
I agree that the Mg concentration is low and that it doesn't make sense to use more ATP than Mg, especially since ATP is expensive and Mg is cheap. I would use at least 8 mM of Mg.
If I were you I would add the Mg & ATP when your protein is bound to an affinity column instead of during cell lysis. This way you probably need much less ATP and the chance of chaperone rebinding is much lower. To do this let buffer with Mg & ATP run into the column and let it incubate for 10-15 minutes before eluting the Mg & ATP and hopefully the chaperone with buffer, before you start the elution of your protein. This way you can also see if the chaperone gets eluted. Also make sure the ATP you use is fresh (you can freeze aliquots so that you don't have to freeze and thaw all at once).
I don't think the addition of 1mM TCEP is a problem, but you could always try DTT instead.
Thanks Josy and Dhiraj for the input!
I am following the protocol as it was optimized and published by a post-doc in our lab over 3 years ago. As far as I recall, the [Mg] and [ATP] were set based on a protocol we found in the literature.
Regarding your suggestions Josy, what you say makes a lot of sense. I am definitely going to try your suggestion of applying the Mg and ATP once the enzyme is on the column, though we do currently conduct our first wash with the same buffer we use for lysis, so the chaperones are being washed off in the presence of Mg and ATP. Nonetheless, if it works, it will at least save us money.
We have DEFINITELY noticed that the freshness of our ATP is a big variable to our success. I have been freezing it in dry in aliquots that contain enough ATP for like 5 preps. I always wait for the ATP to come to room temp before I open the aliquot so that no condensation occurs. Do you think I should be freezing them in single use aliquots instead?
What I was mainly concerned about with Mg and TCEP together is not chelation, but rather if TCEP may reduce the Mg to another oxidation state so that it is no longer divalent.
Thanks!
Cytosolic ATP is about 3 mM, so your 5 mM seems ok (perhaps you could go a little lower to save money). If the chaperone you are worried about is from the Hsc70 family, it is ATP-binding rather than ATP-hydrolysis that opens the protein binding site.
The stability of ATP in solution is pH-dependent and highest around neutral. At -80 °C such solutions should last at least 6 months. Bivalent metal ions increase hydrolysis rate and should be absent, freeze-thaw cycles should be avoided.
If you are worried about the TCEP, use DTT instead, but it does not permanently modify the protein and therefore has to be present in all buffers.
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