Problems with inclusion bodies

Roger Wiegand rcwieg at ccmail.monsanto.com
Fri Oct 1 12:55:37 EST 1993


In article <28hmdn$eb0 at BKFUXG.kfunigraz.ac.at>, Landl at bkfug.kfunigraz.ac.at
(Karina Landl) wrote:

> Hey folks out there!
> My enzyme (squalene epoxidase from yeast) expressed in E.coli is
> enriched in inclusion bodies. In order to maintain the enzymes activity
> I cannot use triton X-100 or Urea to solubilize them. Who knows
> something about alternatives ? 
> All suggestions are very welcome !
> Karina


It would be very unusual (unique?) for the enzyme in RBs to be folded and
active. To recover it you will almost certainly have to solubilize and
refold, and possibly take a huge loss or try to force more soluble
expression. Refolding is, unfortunately, very empirical so your best bet is
to try lots of conditions. Solubilization in urea vs guanidine does not
always yield the same result, so try both. Vary pH, salt, time,
temperature, concentration, and phase of the moon. Lower growth
temperatures are the easiest and most commonly successful method of
increasing soluble expression in coli.

If it really doesn't want to refold and you only need 10s-100s of mgs it's
often faster and easier to use insect cells and make soluble protein. The
tradeoff is that starting with purified RBs often gives 50-90% pure
material as starting material for purification, so a 5% refold yield might
not be so bad.

Good luck!
-- 
Thanks,
Roger

rcwieg at ccmail.monsanto.com



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