On Nov 13, 2010, at 11:04 PM, Jerry McCully wrote:
> Dear ALL;
>
> A few weeks ago, I posted my problem with MBP fusion protein.
>
> Thank folks very much for the help. Here is my latest result.
> 1) Shortening the linker region would signifcantly reduce the cleavage of
> target proteins from MBP;
>
> 2) Adding a C-terminal tag would minimize the degradation of target proteins
> and facilitate the purification;
>
> 3) Low temperature induction(18 degree) would significantly reduce the
> soluble aggregates;
>
> However, all the optimization procedures resulted in a ~150KD-200KD oligomer
> of MBP fusion protein after gel-filtration.
> Only a very small portion of MBP fusion protein exsited as monomers even in
> the expression condition with low temperature and 0.1mM IPTG.
>
> Does anyone experience the oligomerization of MBP fusion protein?
>
> Thanks again and have a nice weekend!
>
> Jerry McCully
Dear Jerry,
As Artem already pointed out, this is indeed quite common. For an example and a
possible way to recover whatever amount of monomer you are getting, see
Supplementary Figure 1 of:
http://www.ncbi.nlm.nih.gov/pubmed/19052627?dopt=Abstract
having said that, in this case we managed since our our monomeric species was
still ~20% of the total... if it's significantly less, I'd also suggest that
you consider insect/mammalian cell expression.
Good luck,
Luca
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Luca Jovine, Ph.D.
Group Leader & EMBO Young Investigator
Karolinska Institutet
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