Firefly luciferase enzyme fragment complementation for imaging in cells and living animals.
We identified different fragments of the firefly luciferase gene based on the crystal structure of firefly luciferase. These split reporter genes which encode for protein fragments, unlike the fragments currently used for studying protein-protein interactions, can self-complement and provide luciferase enzyme activity in different cell lines in culture and in living mice. The comparison of the fragment complementation associated recovery of firefly luciferase enzyme activity with intact firefly luciferase was estimated for different fragment combinations and ranged from 0.01 to 4% of the full firefly luciferase activity. Using a cooled optical charge-coupled device camera, the analysis of firefly luciferase fragment complementation in transiently transfected subcutaneous 293T cell implants in living mice showed significant detectable enzyme activity upon injecting d-luciferin, especially from the combinations of fragments identified (Nfluc and Cfluc are the N and C fragments of the firefly luciferase gene, respectively): Nfluc (1-475)/Cfluc (245-550), Nfluc (1-475)/Cfluc (265-550), and Nfluc (1-475)/Cfluc (300-550). The Cfluc (265-550) fragment, upon expression with the nuclear localization signal (NLS) peptide of SV40, shows reduced enzyme activity when the cells are cotransfected with the Nfluc (1-475) fragment expressed without NLS. We also proved in this study that the complementing fragments could be efficiently used for screening macromolecule delivery vehicles by delivering TAT-Cfluc (265-550) to cells stably expressing Nfluc (1-475) and recovering signal. These complementing fragments should be useful for many reporter-based assays including intracellular localization of proteins, studying cellular macromolecule delivery vehicles, studying cell-cell fusions, and also developing intracellular phosphorylation sensors based on fragment complementation.
Paulmurugan
Ramasamy
R
Molecular Imaging Program at Stanford (MIPS), Department of Radiology and the Bio-X Program, Stanford University School of Medicine, James H. Clark Center, 318 Campus Drive, East Wing, First Floor, Stanford, California 94305-5427, USA.
Gambhir
Sanjiv S
SS
D041721:3T3-L1 Cells; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D003592:Cytophotometry / Q000379:methods*; D015696:Gene Products, tat / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D019913:Nuclear Localization Signals / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D015696:Gene Products, tat; D019913:Nuclear Localization Signals; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049409:Luciferases, Firefly; D011494:Protein Kinases; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A; D020123:Sirolimus
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