Hello,
I am trying to express and purify recombinant nuclear pore proteins in E. coli cells. All the published protocols I have found use 8M urea in the lysis buffer and subsequently during purification using Ni-NTA column. Is there any other role of urea in the buffer, besides the solubilization of nuclear pore proteins?
Thank you.
Yes! It's been >50 years since I took biochemistry, but 8M Urea ensures the proteins have not denatured and remain active. 8M Urea has a pH near 7 and 2 electrons that are available (from the -C=O). Many other solvents/solutions are harsh and do not provide the environment that proteins enjoy or require.
8 M urea does not dissolve cells, it denatures proteins, which ends up dissolving them if they were insoluble.
Bruce Neagle I believe that there is a misunderstanding of the terms "denatured" and "active" in this context. 8 M urea will most likely unfold, i.e. denature, most proteins efficiently so there will be no activity of your protein! There are some exceptions with extremely stable proteins that do not easily get unfolded (e.g. streptavidin still binds biotin, thus the structure is stable unless you heat it up).
The 8M urea does not only dissolve the nucelar pore proteins but all of the proteins so there is less unspecific binding to Ni-NTA (in native conditions, some E coli proteins are bound to Ni-NTA).
As others mentioned, 8 M urea will denature (unfold) nearly all proteins; this is particularly good at solubilizing recombinantly-expressed proteins that aggregate and form inclusion bodies. However, because the protein of interest has been unfolded, there is limited capacity for any biochemical characterization of it. It is possible to re-fold some proteins after urea solubilization, but it will always be difficult to say if the resulting re-folded protein is characteristically identical to its native form.
Given that your protein of interest is a membrane protein, use of non-denaturing detergents may be a good route to explore; these could also impact protein characteristics / function but the protein would be closer to its native form.
- Badges
- Science topic