We have constructed several recombinant bacterial membrane proteins starting immediately beyond the membrane insertion sequence in hopes that the passenger domain (attached to streptavidin) will fold properly. The biotinylation is invariably folded in and inaccessible, however, and that is the issue. I am going to try binding the streptavidin in the presence of 4-8M urea this weekend to see if the protein will refold (native) during sequential dilutions of the urea (8-4-2-?). Any suggestions?
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How do you expose the C' biotin on membrane proteins?
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