Problems with Protein Expression
fant.1 at gmx.net
Thu Jul 27 03:46:04 EST 2000
> I donot know if this is the right group to place this question ...!!
It is, but you should better have started a new thread (by not
replying, but simply sending a "new message" or however it is called).
> ..does anyone have any experience in solubilizing a protein ..found in
> large amounts..in a Sf-9 cell line Pellet?? Molecular wt is around 60kD
> ..and..is estimated to be expressed at high conc...(150mg/lit) ...The
> protein was harvested from large plates as well as a spinner flask 48 hrs
> post infection.
I have only some experience with proteins in inclusion bodies in
E.coli, but the principle should be the same.
You can try to solubilize the pellet in 6-8 M Guanidinium
hydrochloride; perhaps you can remove membrane particles and stuff
like this before.
Then comes the tricky thing: Now your protein is solubilized, but
denatured, you have to bring it to native, non-denaturing conditions
and hope it refolds with a high yield. This can be achieved by:
- Dialysis against a "native" buffer
- dilution into native buffer.
each time at a low concentration (5-50 mikrograms/ml). Sometimes
addition of arginine increases the yield.
If the second way works, you can try stepwise addition of denatured
protein to the same solution, after the preceeding sample has been
given some time to fold to its native state. This is sensible because
its the folding intermediates that have a low solubility (and force
you to use such small concentrations), but once it's native there's no
problem. Thus you'll get a solution with higher concentration and
won't need to do a subsequent concentration step (Ultrafiltration or
ammonium sulfate precipitation).
Dein Ironie-Modul hat gerade einen schweren Ausnahmefehler erzeugt.
[Boris 'pi' Piwinger in dang]
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