Renaturation of proteins from 8M Urea - references?

Flip Hoedemaeker, CvF RUL/TNO, Leiden, The Netherlands sbtnfh at rulsfb.LeidenUniv.nl
Sun Sep 18 07:40:06 EST 1994


In article <psl-150994183248 at pokey.ahabs.wisc.edu>, psl at ahabs.wisc.edu (Patric Lundberg) writes:
>In article <DRESNICK.94Sep15133059 at m66-080-9.mit.edu>,
>dresnick at athena.mit.edu (David I Resnick) wrote:
>>I then want to get rid of the urea (and pray that it will fold).  Everything 
>>I've tried so far has resulted in precipitation. 
>Dialysis against 7, 6, 4, 2 M urea, then ddH2O?


In fact, there are many references in the literature, with as many answers.
( See for instance the review by Thomas Keifhaber et al in Bio/Technology
vol9, 825-829 (1991)). The problem is that the succes you have depends very
much on the protein your working with. I've had some succes with the protein
i'm working with, by denaturing in 7M Guanidine, then diluting it 25 times in
buffer containing 1.5M Urea at 0C, followed by dialysis. The 1.5M urea was 
found empirically to be close to the aggregation concentration, so that is 
something you'll have to find out for yourself.
Still, about 95% of the protein precipitates when we cross this border, but the
rest is enough to work with. Whatever protein you use, there's allways this
border to cross, and the best thing to do is cross slowly, and at a very low
temperature. Better still is not to have to renature your protein at all, for
instance by using a secretion system. It didn't work for me, but it might
work for you!

Good luck, Flip




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