protein solubility

Richard P. Grant rpg14 at yahoo.co.uk.invalid
Fri Dec 14 04:38:39 EST 2001


In article <20011213180044.18521.qmail at ww02.jatek.com>,
 shan <hshandilya at yahoo.co.in> wrote:

> . This protein has a
> cellulose binding domain tag. the size of the proein is almost 35kDa. This
> protein has huge expression but it is going in the inclusion body in
> insoluble form.

Try expressing *less* - eg. express at, say 30C or even lower.  Try a 
different tag, or try just the protein alone.  Try dropping the 
concentration of IPTG if you're using that to induce.

> I tried to solubilize it by 6M urea it became solubalised
> but when I dialysed against decrease urea gradient it again became
> insoluble. 

Try changing the urea gradient.  You might also try adding the 
solubilized (i.e. in urea) protein dropwise to a large volume - say 2l - 
of urea-free buffer with rapid mixing.  Sometimes this works better than 
bringing the urea conc. down gently. 

There are things to try, but no one can give you the answer except by 
chance - it's all pretty empirical this sort of thing ;)

r

-- 
Richard P. Grant  0x5F9559B1                         MRC Lab of Mol Biol
rpg 'at' mrc-lmb.cam.ac.uk   http://www2.mrc-lmb.cam.ac.uk/personal/rpg/
                     -- Non cogito ergo goon --




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