Nice to hear from a fellow Wabash alumnus. First, one cannot
completely discount the idea that the protein is naturally unfolded free in
solution (Daughdrill and Dahlquist, ca. 1997, Nature Structure?). But that
is not the most likely explanation, I think.
Most of my refolding tricks are published or come from published
sources (For example, Thompson, Methods in Enzymology, 185, 1990).
However, I was struck by your description of diluting after refolding. I
would dilute, possibly to as low as 10 ug/mL, certainly to not more than
500 ug/mL. I have usually diluted with high urea buffer, but some
protocols dilute with low urea (or guanidinium chloride) buffer, and then
dialyzed the urea away. Another potential problem is cyanate contamination
of the urea, which can be removed with mixed-bed ion exchange resin. While
guanidinium chloride does not have this contaminant, it has other potential
disadvantages.
Hope this helps.
Chris Halkides
>Dear Colleagues: We have expressed a 13kDa mammalian protein fragment from
>bacteria. It was purified from inclusion bodies using a urea nickle column
>procedure. After dialysis to remove the urea and dilution into "refolding
>buffer", the protein is soluble below pH ~6.5 (pI is 8.9) but is unfolded
>according to the NMR spectrum. What we need are some references or
>unpublished "tricks" that others perhaps use to encourage the protein to
>adopt a nonrandom and hopefully native conformation upon purification. If
>you can offer some hints, we would be pleased to receive them. Thanks Ken
>Hapner, Montana State University, khapner@montana.edu
>
>
>----------------------------------------------------------------------------
>--------
>
>Kenneth D. Hapner
>Department of Chemistry and Biochemistry
>Montana State University
>Bozeman, MT 59717-0310
>khapner@gemini.oscs.montana.edu
Christopher Halkides
Dept. of Chemistry, UNCW
601 S. College Road
Wilmington, NC 28403-3297
(910) 962-7427