Luciferases, fluorescent proteins, nucleic acids encoding...

Details Luciferases, fluorescent proteins, nucleic acids encoding... Luciferases, fluorescent proteins, nucleic acids encoding...

1999-03-26

2001-05-15

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S006120, C435S008000, C435S069100, C435S183000, C435S252200, C435S320100

Reexamination Certificate

active

06232107

ABSTRACT:

FIELD OF INVENTION
The present invention relates to isolated and purified nucleic acids and encoded proteins from the genera Renilla, Gaussia, Philocarpus and Pleuromamma. More particularly, nucleic acids encoding luciferase and fluorescent proteins from species of these genera are provided.
BACKGROUND OF THE INVENTION
Luminescence is a phenomenon in which energy is specifically channeled to a molecule to produce an excited state. Return to a lower energy state is accompanied by release of a photon (hy). Luminescence includes fluorescence, phosphorescence, chemiluminescence and bioluminescence. Bioluminescence is the process by which living organisms emit light that is visible to other organisms. Luminescence may be represented as follows:
A+B→X*+Y X*→X+hv,
where X* is an electronically excited molecule and hy represents light emission upon return of X* to a lower energy state. Where the luminescence is bioluminescence, creation of the excited state derives from an enzyme catalyzed reaction. The color of the emitted light in a bioluminescent (or chemiluminescent or other luminescent) reaction is characteristic of the excited molecule, and is independent from its source of excitation and temperature.
An essential condition for bioluminescence is the use of molecular oxygen, either bound or free in the presence of a luciferase. Luciferases, are oxygenases, that act on a substrate, luciferin, in the presence of molecular oxygen and transform the substrate to an excited state. Upon return to a lower energy level, energy is released in the form of light [for reviews see, e.g., McElroy et al. (1966) in
Molecular Architecture in Cell Physiology,
Hayashi et al., eds., Prentice-Hall, Inc., Englewood Cliffs, N.J., pp. 63-80; Ward et al., Chapter 7 in
Chemi
-
and Bioluminescence,
Burr, ed., Marcel Dekker, Inc. NY, pp.321-358; Hastings, J. W. in (1995)
Cell Physiology:Source Book,
N. Sperelakis (ed.), Academic Press, pp 665-681;
Luminescence, Narcosis and Life in the Deep Sea,
Johnson, Vantage Press, NY, see, esp. pp. 50-56].
Though rare overall, bioluminescence is more common in marine organisms than in terrestrial organisms. Bioluminescence has developed from as many as thirty evolutionarily distinct origins and, thus, is manifested in a variety of ways so that the biochemical and physiological mechanisms responsible for bioluminescence in different organisms are distinct. Bioluminescent species span many genera and include microscopic organisms, such as bacteria [primarily marine bacteria including Vibrio species], fungi, algae and dinoflagellates, to marine organisms, including arthropods, mollusks, echinoderms, and chordates, and terrestrial organism including annelid worms and insects.
Assays employing bioluminescence
During the past twenty years, high-sensitivity biochemical assays used in research and in medicine have increasingly employed luminescence and fluorescence rather than radioisotopes. This change has been driven partly by the increasing expense of radioisotope disposal and partly by the need to find more rapid and convenient assay methods. More recently, the need to perform biochemical assays in situ in living cells and whole animals has driven researchers toward protein-based luminescence and fluorescence. The uses of firefly luciferase for ATP assays, aequorin and obelin as calcium reporters, Vargula luciferase as a neurophysiological indicator, and the Aequorea green fluorescent protein as a protein tracer and pH indicator show the potential of bioluminescence-based methods in research laboratories.
Bioluminescence is also beginning to directly impact medicine and biotechnology; for example, Aequorea GFP is employed to mark cells in murine model systems and as a reporter in high throughput drug screening. Renilla luciferase is under development for use in diagnostic platforms.
Bioluminescence generating systems
Bioluminescence, as well as other types of chemiluminescence, is used for quantitative determinations of specific substances in biology and medicine. For example, luciferase genes have been cloned and exploited as reporter genes in numerous assays, for many purposes. Since the different luciferase systems have different specific requirements, they may be used to detect and quantify a variety of substances. The majority of commercial bioluminescence applications are based on firefly [
Photinus pyralis
] luciferase. One of the first and still widely used assays involves the use of firefly luciferase to detect the presence of ATP. It is also used to detect and quantify other substrates or co-factors in the reaction. Any reaction that produces or utilizes NAD(H), NADP(H) or long chain aldehyde, either directly or indirectly, can be coupled to the light-emitting reaction of bacterial luciferase.
Another luciferase system that has been used commercially for analytical purposes is the Aequorin system. The purified jellyfish photoprotein, aequorin, is used to detect and quantify intracellular Ca
2+
and its changes under various experimental conditions. The Aequorin photoprotein is relatively small [~20 kDa], nontoxic, and can be injected into cells in quantities adequate to detect calcium over a large concentration range [3×10
−7
to 10
−4
M].
Because of their analytical utility, luciferases and substrates have been studied and well-characterized and are commercially available [eq., firefly luciferase is available from Sigma, St. Louis, Mo., and Boehringer Mannheim Biochemicals, Indianapolis, Ind.; recombinantly produced firefly luciferase and other reagents based on this gene or for use with this protein are available from Promega Corporation, Madison, Wis.; the aequorin photoprotein luciferase from jellyfish and luciferase from Renilla are commercially available from Sealite Sciences, Bogart, Ga.; coelenterazine, the naturally-occurring substrate for these luciferases, is available from Molecular Probes, Eugene, Oreg.]. These luciferases and related reagents are used as reagents for diagnostics, quality control, environmental testing and other such analyses.
Because of the utility of luciferases as reagents in analytical systems and the potential for use in high throughput screening systems, there is a need to identify and isolated a variety of luciferases that have improved or different spectral properties compared to those presently available. For all these reasons, it would be advantageous to have luciferases from a variety of species, such as Gaussia and various Renilla species available.
Fluorescent Proteins
Reporter genes, when co-transfected into recipient cells with a gene of interest, provide a means to detect transfection and other events. Among reporter genes are those that encode fluorescent proteins. The bioluminescence generating systems described herein are among those used as reporter genes. To increase the sensitivity bioluminescence generating systems have been combined with fluorescent compounds and proteins, such as naturally fluorescent phycobiliproteins. Also of interest are the fluorescent proteins that are present in a variety of marine invertebrates, such as the green and blue fluorescent proteins, particularly the green fluorescent protein (GFP) of
Aequorea victoria.
The green fluorescent proteins (GFP) constitute a class of chromoproteins found only among certain bioluminescent coelenterates. These accessory proteins are fluorescent and function as the ultimate bioluminescence emitter in these organisms by accepting energy from enzyme-bound, excited-state oxyluciferin (e.g., see Ward et al. (1979)
J. Biol. Chem.
254:781-788; Ward et al. (1978)
Photochem. Photobiol.
27:389-396; Ward et al. (1982)
Biochemistry
21:4535-4540).
The best characterized GFPs are those isolated from the jellyfish species Aequorea, particularly
Aequorea victoria
(
A. victoria
) and
Aequorea forsk{dot over (a)}lea
(Ward et al. (1982)
Biochemistry
21:4535-4540; Prendergast et al. (1978)
Biochemistry
17:3448-3453). Purified
A. victoria

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