Luciferases and methods for making and using the same
Inventors
Gambhir, Sanjiv S. • Loening, Andreas M. • Wu, Anna M.
Assignees
University of California San Diego UCSD • University of California Irvine • Leland Stanford Junior University
Publication Number
US-8378086-B2
Publication Date
2013-02-19
Expiration Date
2025-09-06
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Abstract
Briefly described, embodiments of this disclosure include polynucleotides that encode mutant Cnidarian luciferases that exhibit modulated properties as compared to the corresponding wild-type luciferases, and the modulated properties include at least one of: modulated stability; enhanced light output; and modulated emission maximum. Embodiments of the present disclosure also include polypeptides or fragments thereof encoded by the polynucleotides, constructs including the polynucleotide, expression cassettes, cells, methods of producing the polynucleotides and polypeptides, antibodies, transgenic cells and/or animals, kits, and the like.
Core Innovation
The invention relates to polynucleotides encoding mutant Cnidarian luciferases, particularly mutants of Renilla luciferase, which exhibit modulated properties as compared to wild-type luciferases. The modulated properties disclosed include one or more of: modulated stability, enhanced light output, and modulated emission maximum. The invention also encompasses polypeptides or fragments encoded by these polynucleotides, fusion proteins, vectors, expression cassettes, host cells, antibodies, transgenic cells or animals, methods for producing the polynucleotides and polypeptides, and kits comprising these components.
The background identifies that while luciferases are widely used as reporter genes and for molecular imaging, existing versions such as beetle luciferases have suboptimal properties for in vivo applications, including large size and ATP-dependence. Coelenterazine-utilizing luciferases, such as Renilla luciferase, are more compatible due to their independence from ATP and smaller size but suffer from limited stability in vivo and an emission spectrum poorly suited for imaging in biological tissues, where red-shifted photons are preferable.
The invention solves these problems by engineering mutant luciferases exhibiting improved stability under in vivo conditions, enhanced light output, and shifted emission spectra. Specific mutations are described that either increase or decrease stability, enhance kinetics and quantum yield, or red- or blue-shift emission wavelengths. The invention provides detailed examples of mutant luciferases, such as variants with specific amino acid substitutions, their production methods, fusion constructs for targeted applications, and validation data demonstrating their improved functional properties in both in vitro and in vivo contexts.
Claims Coverage
There are three principal inventive features covered by the independent claims.
Isolated polynucleotides encoding mutant Cnidarian luciferases with specific amino acid sequences
The invention claims isolated polynucleotides encoding amino acid sequences of mutant Cnidarian luciferases, wherein the mutant luciferase has an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. These sequences represent specific mutants with altered properties including stability and light output as explicitly described in the patent text.
Isolated polynucleotides encoding fusion proteins comprising mutant Cnidarian luciferases and at least one fusion partner
The claims cover isolated polynucleotides encoding a fusion protein composed of a mutant Cnidarian luciferase (from SEQ ID NO: 9, 10, or 11) and at least one fusion partner. This permits the generation of fusion proteins in which the mutant luciferase is linked in-frame with other protein domains, such as targeting moieties, for varied applications.
Vector, expression cassette, and cell-based systems for expressing mutant luciferases
The claims encompass: - Vectors containing the specified polynucleotides encoding the mutant luciferases. - Expression cassettes with a polynucleotide encoding the mutant luciferase. - Isolated cells containing the expression cassette, either as an extrachromosomal element or integrated into the genome, allowing production of the mutant luciferase within a biological or recombinant system. - Methods of producing the luciferase by growing such a cell, expressing, and isolating the mutant luciferase so that it is substantially free of other proteins. - Kits comprising the polynucleotide for use in research or diagnostic settings.
In summary, the claims cover isolated polynucleotides encoding specified mutant Cnidarian luciferases, fusion proteins comprising these mutants, and the necessary components and methods for expressing, isolating, and using these proteins in research, diagnostic, or other kit-based applications.
Stated Advantages
Mutant luciferases exhibit significantly enhanced stability under in vivo conditions compared to wild-type luciferases.
Luciferase variants provide enhanced light output, improving the sensitivity of bioluminescence-based assays.
Certain mutants display shifted emission maxima, including red-shifted variants, enabling improved imaging capabilities in biological tissues due to better tissue penetration of longer-wavelength photons.
Novel mutants allow for more optimal reporter gene systems and bioluminescent tags appropriate for small animal imaging and biochemical assays.
Mutants such as those with increased lability and light output are advantageous for monitoring dynamic gene expression changes.
The invention enables the generation of fusion proteins that retain both targeting and enzymatic activities with high serum stability.
Luciferase mutants with modulated spectral properties are compatible with new substrates, expanding the versatility for various detection systems.
Documented Applications
Use of mutant luciferases as bioluminescent donor proteins and reporter genes for sensitive detection of cellular events in vitro and in vivo.
Imaging of protein-protein interactions, protein dimerization, protein phosphorylation, caspase detection, and cellular ion exchange.
Use in bioluminescence resonance energy transfer (BRET) applications for studying molecular interactions.
Applications in automated high-throughput screening, drug discovery, and functional genomics using cell-based reporter systems.
Design of fusion proteins for targeted imaging, exemplified by RL8-VEGF reporters for imaging angiogenesis and VEGFR-2 signaling in living subjects.
Development of protease cleavage assays where luciferase activity indicates cleavage events.
Marking or identification of specific cell types in vitro or in vivo using fusion proteins comprising targeting and reporter domains.
Use in transgenic animals as in vivo markers, including in tissue-specific gene expression applications.
Preparation and use of kits containing the mutant luciferases, relevant polynucleotides, and substrates for research and diagnostic use.
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