Solubilization of inclusion Bodies

[John A. Newitt]

In article <3kkdhh$du4 at threed.uchc.edu>, dliu at panda.uchc.edu (Ding Liu) wrote:

> 
> I need some help about how to complelely solubilze overexpressed
protein. I am using PET system to overexpress a DNA binding protein, after
cell lysis, protein stays in insoluble fraction, and I tried use 8M urea
to solubilize the protein, turns out that more than 250 ml 8M Urea buffer
were needed for about 7 g of wet cell, and I still see quite bit protein
left in pellet.  Is there a better way to do this, since such large volume
is realy not fun to work with ( I am not using His-tag etc. for purifi
> 
First make sure you are completely lysing the bacteria.  If you just want
to solubilize, try 6-7 M guanidine-HCl in 250 mM Tris-HCl pH8.8, 10 mM
EDTA, 100 mM DTT.  Vigorous vortexing or sonication can be used if
needed.  Also, give it a little time to dissolve, say 2-3 hours at 25
degrees (in case there is a lot of disulfide cross-linking, reduction
won't be immediate).  I won't guarantee, however, that  you will ever be
able to re-fold the protein, if that is your intention.

John A. Newitt  <newitt at ncifcrf.gov>
National Institutes of Health
Bethesda, Maryland  USA