Firefly Luciferase Enzyme Fragment Complementation for Imaging in Cells and Living Animals
ABSTRACT 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.
- SourceAvailable from: Javier Santos
[Show abstract] [Hide abstract]
- "Since the pioneering work of Richards  and Anfinsen  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        . 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. "
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 · 1.74 Impact Factor
[Show abstract] [Hide abstract]
- "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. "
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 · 7.83 Impact Factor
[Show abstract] [Hide abstract]
- "Several methods have been developed to identify, examine and visualize protein interactions and protein complexes in living cells. These methods include the yeast two-hybrid system and its variants optimized for mammalian and plant cells (Ehlert et al., 2006; Fields and Song, 1989; Luo et al., 1997), protein fragment complementation assays (Chen et al., 2008; Johnsson and Varshavsky, 1994; Michnick et al., 2000; Paulmurugan and Gambhir, 2005; Rossi et al., 1997) and fluorescence resonance energy transfer (FRET; Miyawaki et al., 1997; Pollok and Heim, 1999; Fricker et al., 2006; Lalonde et al., 2008). As an alternative approach for the visualization of protein interactions in their natural cellular context, bimolecular fluorescence complementation (BiFC) has been developed (Hu et al., 2002). "
ABSTRACT: The specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such protein-protein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C-terminal and N-terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calcium-mediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.The Plant Journal 11/2008; 56(3):505-16. DOI:10.1111/j.1365-313X.2008.03612.x · 6.82 Impact Factor