Ligand-controllable degron (Shield-1) for tunable protein stability - (Jan/22/2014 )
I was wondering if anyone here has done any work utilizing Shield-1 technology (ligand-controllable degron for rapidly regulation levels of exogenous protein). It's quite a fascinating system (no Shield-1 ligand --> degron-fused protein is degraded vs. add Shield-1 ligand --> degron-fused protein is stabilized). We have had some success so far, where we can transfect cells with our degron-tagged constructs and see a wonderful time-dependent stabilization upon the addition of the Shield-1 ligand.
However, we noticed that baseline levels (in cells transfected with our degron-construct, but not treated with Shield-1) is not as low as I had originally hoped (although we see a clear increase in protein levels upon the addition of Shield-1). Obviously there will be variability based upon cell type being used and the protein being studied, but does anyone know if this is a common problem? If so, has anyone heard of any suggestions or any possible workaround solutions for reducing the protein level (or is this just a caveat of the system in general)?
I have a very similar result, low expression in the absence of Shield-1. What I also found is that the DD tag does impair function of the protein (we had problems with dominant negative kinases not working when fused to DD domain for example). Sorry, I don't know any way around this, maybe titrate your protein?