Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1995-06-07
2003-04-22
Prouty, Rebecca E. (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S189000, C435S252300, C435S320100
Reexamination Certificate
active
06552179
ABSTRACT:
TECHNICAL FIELD
This invention generally relates to luciferase enzymes that produce luminescence, like that from fireflies. More particularly, the invention concerns mutant luciferases of beetles. The mutant luciferases of the invention are made by genetic engineering, do not occur in nature, and, in each case, include modifications which cause a change in color in the luminescence that is produced. The luciferases of the invention can be used, like their naturally occurring counterparts, to provide luminescent signals in tests or assays for various substances or phenomena.
BACKGROUND OF THE INVENTION
The use of reporter molecules or labels to qualitatively or quantitatively monitor molecular events is well established. They are found in assays for medical diagnosis, for the detection of toxins and other substances in industrial environments, and for basic and applied research in biology, biomedicine, and biochemistry. Such assays include immunoassays, nucleic acid probe hybridization assays, and assays in which a reporter enzyme or other protein is produced by expression under control of a particular promoter. Reporter molecules, or labels in such assay systems, have included radioactive isotopes, fluorescent agents, enzymes and chemiluminescent agents.
Included in the assay system employing chemiluminescence to monitor or measure events of interest are assays which measure the activity of a bioluminescent enzyme, luciferase.
Light-emitting systems have been known and isolated from many luminescent organisms including bacteria, protozoa, coelenterates, molluscs, fish, millipedes, flies, fungi, worms, crustaceans, and beetles, particularly click beetles of genus Pyrophorus and the fireflies of the genera Photinus, Photuris, and Luciola. In many of these organisms, enzymes catalyze monooxygenations and utilize the resulting free energy to excite a molecule to a high energy state. Visible light is emitted when the excited molecule spontaneously returns to the ground state. This emitted light is called “bioluminescence.” Hereinafter it may also be referred to simply as “luminescence.”
The limited occurrence of natural bioluminescence is an advantage of using luciferase enzymes as reporter groups to monitor molecular events. Because natural bioluminescence is so rare, it is unlikely that light production from other biological processes will obscure the activity of a luciferase introduced into a biological system. Therefore, even in a complex environment, light detection will provide a clear indication of luciferase activity.
Luciferases possess additional features which render them particularly useful as reporter molecules for biosensing (using a reporter system to reveal properties of a biological system). Signal transduction in biosensors (sensors which comprise a bilogical component) generally involves a two step process: signal generation through a biological component, and signal transduction and amplification through an electrical component. Signal generation is typically achieved through binding or catalysis. Conversion of these biochemical events into an electrical signal is typically based on electrochemical or caloric detection methods, which are limited by the free energy change of the biochemical reactions. For most reactions this is less than the energy of hydrolysis for two molecules of ATP, or about 70 kJ/mole. However, the luminescence elicited by luciferases carries a much higher energy content. Photons emitted from the reaction catalyzed by firefly luciferase (560 nm) have 214 Kj/einstein. Furthermore, the reaction catalyzed by luciferase is one of the most efficient bioluminescent reactions known, having a quantum yield of nearly 0.9. This enzyme is therefore an extremely efficient transducer of chemical energy.
Since the earliest studies, beetle luciferases, particularly that from the common North American firefly species
Photinus pyralis
, have served as paradigms for understanding of bioluminescence . The fundamental knowledge and applications of luciferase have been based on a single enzyme, called “firefly luciferase,” derived from
Photinus Rvralis
. However, there are roughly 1800 species of luminous beetles worldwide. Thus, the luciferase of Photinus pyralis is a single example of a large and diverse group of beetle luciferases. It is known that all beetle luciferases catalyze a reaction of the same substrate, a polyheterocyclic organic acid, D-(−)-2-(6′-hydroxy-
2′-benzothiazolyl)-A&Dgr;
2
-thiazoline -4-carboxylic acid (hereinafter referred to as “luciferin”, unless otherwise indicated), which is converted to a high energy molecule. It is likely that the catalyzed reaction entails the same mechanism in each case.
The general scheme involved in the mechanism of beetle bioluminescence appears to be one by which the production of light takes place after the oxidative decarboxylation of the luciferin, through interaction of the oxidized luciferin with the enzyme. The color of the light apparently is determined by the spatial organization of the enzyme's amino acids which interact with the oxidized luciferin.
The luciferase-catalyzed reaction which yields bioluminescence (hereinafter referred to simply as “the luciferase-luciferin reaction”) has been described as a two-step process involving luciferin, adenosine triphosphate (ATP), and molecular oxygen. In the initial reaction, the luciferin and ATP react to form luciferyl adenylate with the elimination of inorganic pyro-phosphate, as indicated in the following reaction:
E+LH
2
+ATP→E·LH-AMP+PP
i
where E is the luciferase, LH
2
is luciferin, and PPi is pyrophosphate. The luciferyl adenylate, LH
2
-AMP, remains tightly bound to the catalytic site of luciferase. When this form of the enzyme is exposed to molecular oxygen, the enzyme-bound luciferyl adenylate is oxidized to yield oxyluciferin (L=
0
) in an electronically excited state. The excited oxidized luciferin emits light on returning to the ground state as indicated in the following reaction:
One quantum of light is emitted for each molecule of luciferin oxidized. The electronically excited state of the oxidized luciferin is a characteristic state of the luciferase-luciferin reaction of a beetle luciferase; the color (and, therefore, the energy) of the light emitted upon return of the oxidized luciferin to the ground state is determined by the enzyme, as evidenced by the fact that various species of beetles having the same luciferin emit differently colored light.
Luciferases have been isolated directly from various sources. The cDNAs encoding luciferases of various beetle species have been reported. (See de Wet et al., Molec. Cell. Biol 7, 725-737 (1987); Masuda et al., Gene 77, 265-270 (1989); Wood et al., Science 244, 700-702 (1989)). With the cDNA encoding a beetle luciferase in hand, it is entirely straightforward for the skilled to prepare large amounts of the luciferase by isolation from bacteria (e.g.,
E. coli
), yeast, mammalian cells in culture, or the like, which have been transformed to express the cDNA. Alternatively, the cDNA, under control of an appropriate promoter and other signals for controlling expression, can be used in such a cell to provide luciferase, and ultimately bioluminescence catalyzed thereby, as a signal to indicate activity of the promoter. The activity of the promoter may, in turn, reflect another factor that is sought to be monitored, such as the concentration of a substance that induces or represses the activity of the promoter. Various cell-free systems, that have recently become available to make proteins from nucleic acids encoding them, can also be used to make beetle luciferases.
Further, the availability of cDNAS encoding beetle luciferases and the ability to rapidly screen for CDNAS that encode enzymes which catalyze the luciferase-luciferin reaction (see de Wet et al., supra and Wood et al., supra) also allow the skilled to prepare, and obtain in large amounts, other luciferases that retain activity in catalyzing production of bioluminescence through the l
Gruber Monika G.
Wood Keith V.
Promega Corporation
Prouty Rebecca E.
Schwegman Lundberg Woessner & Kluth P.A.
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