Dr Lester Davids wrote:
>> I am currently trying to get proteins out of inclusion bodies in E.coli
> after an overnight culture growth and am really battling. Anyone out
> there with advice to get prots out of inclusion bodies without
> destroying/denaturing the proteins and rendering them inactive?
>I think what you want is impossible, because proteins in inclusion
bodies aren't active, they are aggregated and in a conformation
(partially) different to the native, functional conformation.
Inclusion bodies are formed when bacteria are overexpressing a protein
to such an extent that the bi-(or perhaps multi-)molecular aggregation
reaction of folding intermediates gets faster than their rearrangement
to the native conformation.
Folding is a unimolecular reaction and thus independent of protein
concentration, while aggregation as a bimolecular reaction is. The
critical concentration of intermediates can be achieved simply by
increasing the protein concentration, i.e. overexpression, or when
aggregation-prone off-pathway folding intermediates are stabilized
relative to on-pathway intermediates, e.g. by mutations or by
increased temperature, or by both ways.
If you're not familiar with my use of the term aggregation: In this
context it means association of partially folded proteins, a process
that does not rely on a specific interaction like the one governing
oligomerization, but is also not completely unspecific, because one
usually finds mainly one protein in aggregates (e.g. IB), even if
there's two with similar high concentration. As it is folding
intermediates that aggregate, there's often some native structure even
in the IB, namely secondary structure, but often the beta-sheet
content is higher.
On the other hand, many proteins can fold to their native conformation
/in vitro/ starting from an unfolded chain under apropriate
conditions.
Thus, there's two ways out of your problem:
Either prevent the IBs from beeing formed at all, by:
- lowering the level of expression (which you probably don't want to
do...)
- lowering temperature (_may_ help, or may not)
- coexpressing chaperones (a technology I am not an expert in, so
somebody else should comment on that)
Or by denaturing and solubilizing your protein in the IBs and trying
to renature it. If you're protein is of at most medium size and
monomeric, I bet you can find conditions that allow refolding in
sufficient yields.
Hope this helps, Frank
--
Frank Fuerst, Institute of Biochemistry of Potsdam University
Im Biotechnologiepark, D-14943 Luckenwalde
Tel.: +49-3371-681334; Fax: +49-3371-681339
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