Dr E. Buxbaum <EB15 at le.ac.uk> wrote in article <342AF9EC.2575 at le.ac.uk>...
> Achim Recktenwal, Ph.D. wrote:
> >
> > I am looking for methods to delipidate protein solutions, or protein &
> > cell-suspensions after cell-disruption. These methods should be
scalable
> > for use in a large-scale protein purification process.
> >
> > I am having big problems with the fowling of my first
> > chromatography-column by lipids in the applied cell-homogenate.
>>
Whilst ammonium sulfate precipitation is an excellent (and cheap !) initial
purification step, we have found that three-phase partioning (TPP) is even
more effective than AS pptation. Tertiary butanol, which is completely
miscible with water, forms a separate phase upon the addition of sufficient
ammonium sulfate. Any protein present in the aqueous phase may precipitate
out, depending on the concentration of ammonium sulfate, due to hydration
effects (like conventional AS pptation). The protein precipitates in the
interfacial layer (between the t-BuOH and aqueous phase).
The advantages of TPP are that any lipids are automatically extracted into
the butanol layer. Further because t-butanol is kosmotropic it is not as
denaturing as conventional organic solvents. The protein(s) that
precipitate at the interfacial are free of salt and can therefore be
directly applied to an ion-exchange column. A disadvantage of the
procedure is that multi-meric proteins can be denatured. You could take a
look at
Pike and Dennison (1989) Biotech. Bioeng. 33, pp221-228
I hope that this helps !
Che
>
