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Fusion protein instability at ER membrane

ER membrane Dimerization FKBP AP20187 Stability

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#1 miST32



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Posted 07 September 2016 - 02:18 PM

Hi all,

I've run into a curious problem with a fusion protein I recently generated.  

Essentially I've got an ER membrane-localized enzyme with functional domains on both sides of the ER membrane.  The cytosolic side has the enzyme domain, and a separate domain is present on the ER lumenal side.  The enzyme is activated by dimerization in the plane of the membrane.

If I express a form that lacks the lumenal domain (retaining only the N-terminal ER signal peptide) - call it "mutant L," it localizes correctly and it looks well-tolerated.  I can drive the expression several fold above endogenous levels to no ill effect.  

So next, I fused a chemically-induced dimerization domain (FKBP variant DmrB)  to the lumenal side, call it "mutant D."  Same signal peptide on the N-terminus as mutant L.

The mutant D construct works biochemically, insofar as I can induce its enzymatic activity by incubating mutant D-expressing cells with the dimerizing compound (AP20187).  However, the expression level of mutant D is dismally low.  I've generated cell lines and cannot get it to express at 1/10 the level of mutant L.  Transient expression also appears to result in very few viable cells expressing high-levels of the protein, as measured by fluorescence microscopy and western blotting.  My guess is that the DmrB fusion destabilizes the protein, renders it toxic, prevents its proper localization, increases turnover, etc.  

This isn't a deal-breaker for the project (low expression is actually good for us provided there are no other problems), but I'm curious why mutant D might be less well-tolerated?  I'm sure there are many potential reasons, but I'd be curious to hear opinions/ideas.  Are there known issues with the DmrB domain in membrane fusions or when localized to different compartments?


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