Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein

Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility1,2,3,4 or may not work in living cells5,6,7. Green fluorescent protein (GFP) fusions can misfold8 or exhibit altered processing9. Fluorogenic biarsenical FLaSH or ReASH10 substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization10. An ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates. One way to accomplish this might be with a split GFP11, but the GFP fragments reported thus far are large and fold poorly11,12, require chemical ligation13 or fused interacting partners to force their association11,12,13,14, or require coexpression or co-refolding to produce detectable folded and fluorescent GFP11,12. We have engineered soluble, self-associating fragments of GFP that can be used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. The split GFP system is simple and does not change fusion protein solubility.