Refold protein 8M Urea:
Sanjiv,
The recommendations for rapid dilution and trying 6M GdmCl are great
recommendations. As an alternative, I had a similar problem years ago with a
membrane-associated receptor domain that was mostly found in inclusion
bodies after expression (I assume this is your problem). We tried expressing
at lower temperatures and in the presence of sorbitol, which are common
methods for getting proteins to express in solution, but I found an
alternative method worked. This will work if you have a tag for column
purification (ie his tag). I incubated my pelets in 8M urea for a couple of
hrs at 37C, spun and collected the supernatant, and then refolded the
protein over a Ni column, as I had a his tag protein. To do this, you load
and wash the protein normally, but before eluting, run b-cyclodextrin (i
think 0.1%, you'll have to try a few concentrations) and then a detergent (I
used Triton X-100). Then you can elute. Here's the paper I used as a guide:
Biotechnol Appl Biochem 2006, 43:137-145
Hope that helps!
Geoff
--
Geoffrey K. Feld
Krantz Lab
College of Chemistry
492 Stanley Hall
University of California, Berkeley
Vigilia pretium libertatis
