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
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 ;)
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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