Dear all,
are there known fusion partners or amino-acid-subsequences that provoke precipitation of the fusion protein in inclusion bodies?
I want to provoke inclusion body formation, caused solely by the aggregation-prone fusion partner and independent of the second, interchangeable partner. So in fact a reversion of the usual strategy of using fusion partners (such as Maltose-Binding-Protein) to enhance solubility.
thanks a lot in advance,
regards,
peter
Any constraints on the size of the aggregation-prone protein? Beacause I am sure that a lot of the people here can name some good candidates - namely proteins they tried hard to express in E. coli, but would ever end up in inclusion bodies...
I've seen a lot of aggregation with short poly-arginine tagging. Alternatively stick something slightly longer to the N-terminus. TEV protease and human FGF2 will aggregate horrendously when using their native N-termini.
"Leukotriene BLT2 Receptor Monomers Activate the Gi2 GTP-binding Protein More Efficiently than Dimers" J Biol Chem. Feb 26, 2010; 285(9): 6337–6347.
In this paper you can find some info about aggregation- enhancing fusion partners. I used one of them, human α5 integrin for my GPCR expression. It worked well.
Best
Dawid
Thanks for alle the input!
John, could you elaborate on the details of "short arginine tagging"?
I would like to use a fusion domain that is hydrophobic and as small as possible, but at the same time directs the partner to inclusion bodies, ideally with allowing the partner to fold correctly before to have native structures in the aggregates.
Before folks had His6 tags, Arg6 targs were used to make the protein purifiable by cation exchange. E.coli doesn't have any strongly basic proteins, so that was a 1-step procedure. That worked OK, except that these things can cause proteins to misfold and aggregate that would otherwise be stable. Not sure if that counts as a "bona fide" inclusion body, but it might suit your purpose.
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