Abstract
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.
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Acknowledgements
The authors wish to acknowledge B. Mark and J.-D. Pédelacq for helpful comments and the National Institutes of Health and Laboratory-directed research and development program for generous support.
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The authors are inventors named in a utility patent (Self-assembling fragments of GFP) submitted to the USPTO by Los Alamos National Laboratories on behalf of the University of California.
Supplementary information
Supplementary Fig. 1
Map of pTET-SpecR plasmid. (PDF 32 kb)
Supplementary Fig. 2
Sequential of co-induction of BL21(DE3) containing compatible plasmids expressing GFP 1–10 variants, and sulfite reductase fused to GFP 11 wild type. (PDF 36 kb)
Supplementary Fig. 3
Effect of Urea on the complementation reaction. (PDF 24 kb)
Supplementary Fig. 4
pH dependence of complementation (PDF 88 kb)
Supplementary Table 1
Amino acid sequence of the GFP 11 mutants (PDF 20 kb)
Supplementary Table 2
Comparison of the whole-cell fluorescence of pre-induction and post-induction GFP expression using the pTET-SpecR vector or pPROTET CmR commercial vector (Clontech). (PDF 14 kb)
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Cabantous, S., Terwilliger, T. & Waldo, G. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat Biotechnol 23, 102–107 (2005). https://doi.org/10.1038/nbt1044
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DOI: https://doi.org/10.1038/nbt1044
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