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Will 5mM DTT break existing disulfide bonds?

periplasm disulfide bonds purification

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3 replies to this topic

#1 Missle

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Posted 16 January 2014 - 06:07 AM

Hi.  I am isolating/purifying a bacterial recombinant protein that was secreted to the periplasm.  The protein contains 12 cysteines and is predicted to form 6 disulfide bonds.  My concern is that there maybe some free cysteines in the final isolated protein that did not form disulfide bonds and I do not want them to cause aggregation of my sample.  So, I was planning on including 5mM DTT in the isolation/purification/storage buffers to maintain any reduced cysteines and prevent aggregation but I don't want to break any bonds that may already exist.  I thought that 5mM without any heating probably wouldn't be strong enough to break bonds but was hoping one of you might have thoughts/experience on the subject (and have more confidence about yours than I have in mine!)

 

Thanks!



#2 mdfenko

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Posted 16 January 2014 - 12:26 PM

5mM probably won't break existing bonds but we routinely use 2mM to maintain our proteins.


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#3 dimensionsbio

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Posted 16 January 2014 - 06:42 PM

Hi Missle.  I would recommend taking time points (add SDS without extra reducing agent and freeze) during storage, and then running "non-reduced" samples on a gel.  I believe disulfide exchange will occur spontaneously as the protein molecules interact and if so, you can see evidence of multimer formation on the gel. 



#4 Osibisa

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Posted 18 January 2014 - 07:06 PM

I imagine to break most disulfides you will also need some denaturing agent/detergent due to inaccesability within the protein structure. If you want to quench all free thiols and prevent disulfide exchange you can use an agent such as N-ethyl maleimide. If it IS reacting with your protein it may affect its function however.

 

Also consider the properties of your solution. For disulfide exchange the thiolate ion must be formed first, I believe the pKa is normally around 9. I think metal ions, particularly copper catalyses the reaction (can participate in redox rxns).

Using a buffered solution of pH <= 7 with a chelating agent (EDTA) should reduce it to negligable levels.







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