In terms of the expression and characterization of efflux pump proteins, there seem to be several setbacks due to their trans-membrane nature. One method that I know of is the denaturation of the membrane by using Urea, and then attempting to renature the proteins. However this does not seem a suitable method for retaining a sufficient level of the protein activity, thus making further analysis difficult. If possible suggest more suitable methods for the same.
You can always try to re-modify your clone to allow the express of protein towards the membrane fraction of the bacteria; that way you can somewhat obtain the membrane protein in the native-like state and use different mild detergents for refolding of protein without interrupting/denaturing the structural integrity of the protein. Please take a look at our paper for reference.
Another way is cell-free expression that are being used more and more these days. I also attached a reference here for you to take a look
Article Expression, folding, and proton transport activity of human ...
Article Cell-Free Membrane Protein Expression for Solid-State NMR
It may be possible to characterize the activity of the efflux pump using plasma membrane vesicles purified from the overexpressing cells using a fluorescence-based assay, as long as the protein is inserted into the membrane properly. This saves the difficulty of trying to purify it.
However, if the protein is not inserted into the membrane, it will be in a precipitated state. It will then be necessary to dissolve it in a denaturant, such as urea and/or detergent. You can then attempt to reconstitute it into proteoliposomes by adding lipids and detergent, and removing the denaturant and detergent. The transport assay could then be done with the proteoliposomes. This will be much more difficult a project than the purified plasma membrane approach, but it has the advantage of providing the opportunity to purify the protein first.
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