Firefly Luciferase Enzyme Fragment Complementation for Imaging in Cells and Living Animals

Stanford University, Palo Alto, California, United States
Analytical Chemistry (Impact Factor: 5.64). 04/2005; 77(5):1295-302. DOI: 10.1021/ac0484777
Source: PubMed


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.

16 Reads
  • Source
    • "Since the pioneering work of Richards [3] and Anfinsen [175] with the ribonuclease fragments, complementation has gained importance in the development of applications to the in vivo study of protein folding, protein-protein interactions, aggregation, stability and cellular localization [176] [177] [178] [179] [180] [181] [182] [183]. Furthermore, this strategy is one method to evaluate whether the precise packing of a region -that includes numerous tertiary contacts-is critical to the consolidation of the fold of a given protein. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Thioredoxin (TRX) catalyzes redox reactions via the reversible oxidation of the conserved active center CGPC and it is involved in multiple biological processes, some of them linked to redox activity while others not. TRX is a globular, thermodynamically stable and monomeric alpha/beta protein with a structure characterized by a central beta-sheet surrounded by alpha-helices. In this review we discuss central aspects of folding, dynamics and function of Escherichia coli TRX (EcTRX), pointing to the characterization of the full-length protein and of relevant fragments. In addition, we focus on the critical role that the C-terminal alpha-helical element plays in a late event in the consolidation of the overall EcTRX fold. Furthermore, we address the characterization of internal molecular motions by NMR and molecular dynamics simulation techniques. Finally, we review important aspects of the relationship among structure, dynamics and enzymatic function of this key redox protein.
    Protein and Peptide Letters 07/2015; 22(9). DOI:10.2174/0929866522666150707114309 · 1.07 Impact Factor
  • Source
    • "This strategy depends on division of a monomeric reporter luciferase into two separate inactive fragments whose reconstitution regenerates the enzyme activity. The interaction between proteins brings the luciferase split reporter fragments close enough to enable their reassemblage and subsequently the recovery of its native structure and activity (Binkowski et al., 2009; Paulmurugan and Gambhir, 2005). Moreover, split luciferase is an efficient tool to assay the biochemical metabolites, where a domain is inserted into an internally fragmented luciferase producing ligand binding that causes change in the photon emitted signals (Ataei et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: In this study, we used spilt luciferase complementation assay strategy in order to further elucidate the main role of WD-40 repeats of Apaf-1 molecules in apoptosome formation. In the presence of ATP and cytochrome c, Apaf-1 monomers oligomerize and provide a platform for activation of procaspase-9 and subsequently procaspase-3/7. For a detailed biochemical and structural investigation of Apaf-1 function and apoptosome formation, several studies have been made in recent years. However, many questions related to in vivo evaluation of this phenomenon have been persisting to answer. Some of the most important of these questions are related to WD-40 repeats at the carboxy terminus of Apaf-1 and its function in apoptosome complex formation and caspase activation. When truncated Apaf-1 molecules conjugated with luciferase fragments place in close proximity, light signal emits and real time evaluation of protein-protein interactions becomes possible. Here, we observed, for the first time, the autoassembly of truncated Apaf-1 molecules disappeared after several hours without any caspase-3/7 activation. However, we observed that, truncated Apaf-1 molecules can activate caspase-3/7 upon the induction of apoptosis via doxorubicin. Moreover, oscillation in luciferase activity upon complementation was revealed which implicates the dynamism of apoptosome formation. Copyright © 2015. Published by Elsevier B.V.
    European journal of pharmacology 04/2015; 760. DOI:10.1016/j.ejphar.2015.04.008 · 2.53 Impact Factor
  • Source
    • "Another is how to fully restore the enzyme function after fragment complementation. A detailed knowledge of protein structure, function and mechanism would be of great help in reporter gene engineering 80. For example, the crystal structure of luciferase shows two essentially independent folding domains, the N-terminal domain consisting of residues 1-436 and a C-terminal domain consisting of residues 440-550, connected by a disordered flexible region 81. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Molecular imaging is a newly emerged multiple disciplinary field that aims to visualize, characterize and quantitatively measure biological processes at cellular and molecular levels in humans and other living systems. A reporter gene is a piece of DNA encoding reporter protein, which presents as a readily measurable phenotype that can be distinguished easily from the background of endogenous protein. After being transferred into cells of organ systems (transgenes), the reporter gene can be utilized to visualize transcriptional and posttranscriptional regulation of gene expression, protein-protein interactions, or trafficking of proteins or cells in living subjects. Herein, we review previous classification of reporter genes and regroup the reporter gene based imaging as basic, inducible and activatable, based on the regulation of reporter gene transcription and post-translational modification of reporter proteins. We then focus on activatable reporters, in which the signal can be activated at the posttranslational level for visualizing protein-protein interactions, protein phosphorylation or tertiary structure changes. The applications of several types of activatable reporters will also be summarized. We conclude that activatable reporter imaging can benefit both basic biomedical research and drug development.
    Theranostics 04/2012; 2(4):413-23. DOI:10.7150/thno.3940 · 8.02 Impact Factor
Show more