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-   -   [Protein-analysis] Why do disordered regions within proteins decreaseexpression yields? (http://www.molecularstation.com/forum/protein-forum/26700-%5Bprotein-analysis%5D-why-do-disordered-regions-within-proteins-decreaseexpression-yields.html)

S.E. Hart 05-04-2006 01:41 PM

[Protein-analysis] Why do disordered regions within proteins decreaseexpression yields?
 
Hi,

I've been trying to express a protein in the cytosol of E. coli. I'm only
getting very low yields. The protein is predicted to contain a disordered
region of at least 40 amino acids. Would this be enough to explain the low
yields? The disordered region is at the very N-terminus - I truncated the
protein in a loop region after two transmembrane regions. Why do
disordered regions have a negative effect on expression yields - is it
just because they are likely to be targets for proteases or are the other
factors? I've found that adding a tag to the protein greatly increases the
yields. Is that just because the disordered region is no longer at the
N-terminus? I appreciate that tags can have other positive effects e.g. on
the solubility of the protein.

Any help in this matter would be MUCH appreciated.

Thanks,
Sarah


Dr Engelbert Buxbaum 05-08-2006 08:13 AM

[Protein-analysis] Why do disordered regions within proteins decreaseexpression yields?
 
S.E. Hart wrote:


Have you done an SDS-PAGE with the entire lysed bacteria, and is there
no sign of your protein? Often expressed proteins aggregate inside the
bacteria and form "inclusion bodies". If you then use the cytosol as a
source of your protein, yields will, of course, be very poor.

In such a case you isolate the inclusion bodies by centrifugation,
solubilise them in caotropic agents like urea or guanidinium chloride
and then try and re-fold them by slow removal of the caotrope, either by
dialysis or after binding to a column.

Some tags, most notably maltose binding protein (MBP), can reduce the
tendency of expressed protein to form inclusion bodies and keep them in
solution. The molecular mechanism behind this is not well understood,
protein purification is still more art than science.


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