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 solubility or may not work in living cells. Green fluorescent protein (GFP) fusions can misfold or exhibit altered processing. Fluorogenic biarsenical FLaSH or ReASH substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization. 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 GFP, but the GFP fragments reported thus far are large and fold poorly, require chemical ligation or fused interacting partners to force their association, or require coexpression or co-refolding to produce detectable folded and fluorescent GFP. 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.
Cabantous
Stéphanie
S
Bioscience Division, MS-M888, Los Alamos National Laboratory, PO Box 1663, Los Alamos, New Mexico 87545, USA.
Terwilliger
Thomas C
TC
Waldo
Geoffrey S
GS
D020816:Amino Acid Motifs; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D041543:Pyrobaculum / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D012995:Solubility; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003545:Cysteine