JP2023043461A - Luminescence method using luminescent protein - Google Patents

Abstract

To provide methods for allowing a substrate different from the original substrate 3-hydroxyhispidine to luminesce using a fungal luciferase.SOLUTION: The present invention relates to a luminescence method comprising contacting a fungal luciferase or a fusion protein having a chemiluminescent protein derived from a fungal luciferase with coelenterazine.SELECTED DRAWING: Figure 2

Description

The present disclosure relates to luminescence methods using luminescent proteins.

Many organisms, such as fireflies, sea fireflies, and luminescent mushrooms, have the ability to emit light chemically. This chemiluminescence reaction is caused by a chemiluminescent protein (luciferase), which is one of light-emitting proteins, and its luminescent substrate (luciferin), and these structures vary widely depending on species. Currently, various luminescent proteins are applied to life science research as reporters and probes. When luciferase is expressed in cells or the like and used as a reporter or probe, it is necessary to add a luminescent substrate to the cells or the like. The combination of luciferase and its luminescent substrate is highly specific.

Firefly luciferin, a luminescent substrate of firefly luciferase, bacterial luciferin, a luminescent substrate of bacterial luciferase, dinoflagellate luciferin, a luminescent substrate of dinoflagellate luciferase, Vargulin, a luminescent substrate of Cypridina luciferase (Vargula/Cypridina luciferase) structure is known.
Also, the structure of Coelenterazine, a luminescent substrate of photoproteins such as Renilla luciferase, Oplophorus luciferase, Gaussia luciferase, aequorin, and obelin, has been determined.
Furthermore, in recent years, it has been found that the luminescent substrate of luciferase in luminescent mushrooms (luminescent fungi) is 3-hydroxyhistidin (Non-Patent Documents 1 to 3, Patent Document 1).

Kotlobay et al., Genetically encodable bioluminescent system from fungi, 12728-12732, PNAS December 11, 2018 vol. 115 no. 50www.pnas.org/cgi/doi/10.1073/pnas.1803615115 Khakhar et al., Building customizable auto-luminescent luciferase-based reporters in plants eLife 2020;9:e52786.https://doi.org/10.7554/eLife.52786 Mitiouchkina et al., Plants with genetically encoded autoluminescence, NatureBiotechnology VOL 38 August 2020 944-946

Japanese Patent Publication No. 2020-505028

The combination of luciferase and its luminescent substrate, luciferin, is highly specific, and no combination of heterologous luciferase and luciferin capable of chemiluminescence has been reported so far. If a plurality of different luminescent substrates can be used for luciferase, it is expected that the range of applications will be broadened because the substrate can be used according to the situation.

Luciferases of fungi such as luminescent mushrooms usually use 3-hydroxyhistidine as a substrate and catalyze the luminescent reaction.
Thus, in one aspect, the disclosure provides a method of using a fungal luciferase to illuminate a luminescent substrate that is different from the substrates described above.

In one aspect, the present disclosure provides a luminescence method comprising contacting a fungal luciferase or a fusion protein having a chemiluminescent protein portion derived from a fungal luciferase with coelenterazine (hereinafter also referred to as a “luminescence method according to the present disclosure”). ) related.

In one aspect, the present disclosure provides a kit comprising a fungal luciferase or a fusion protein having a fungal luciferase-derived chemiluminescent protein portion, a nucleic acid thereof, or an expression cassette thereof, and coelenterazine (hereinafter also referred to as a "kit according to the present disclosure") ) related.

According to the present disclosure, in one aspect, luciferases from fungi, such as luminescent mushrooms, can be used to illuminate luminescent substrates that differ from the native (endogenous) substrate, 3-hydroxyhistpidine.

FIG. 1 is a graph showing the luminescence intensity when coelenterazine was brought into contact with each luciferase. FIG. 2 is a graph of FIG. 1 excluding Nluc data. FIG. 3 is a graph showing the luminescence intensity when firefly luciferin was brought into contact with each luciferase. FIG. 4 is a graph showing the luminescence intensity when each luciferase was brought into contact with 3-hydroxyhistidine.

In the present disclosure, the combination of luciferase and luciferin is believed to have high specificity, and the luciferase of the luminous mushroom uses coelenterazine, which has a structure that is significantly different from that of endogenous luciferin, 3-hydroxyhistidine, as a substrate to cause a luminescence reaction. Based on the knowledge that it can catalyze.

According to the present disclosure, a fungal luciferase, or a fusion protein having a chemiluminescent protein moiety derived from a fungal luciferase, is used to catalyze a luminescence reaction of coelenterazine distinct from its endogenous luciferin, 3-hydroxyhistidine. be able to.
By using a fungal luciferase to make a luciferin (coelenterazine) different from endogenous luciferin emit light, or by newly increasing fungal luciferase as a luciferase capable of making selenrelazine emit light, the technology using a luminescent protein has been developed. Further expansion and promotion can be expected.

[Luminous luminescent protein]
In the luminescence method of the present disclosure, the luminescent protein that emits light is a fungal luciferase or a fusion protein having a chemiluminescent protein portion derived from a fungal luciferase.

A “fungal luciferase” in the present disclosure may, in one or more embodiments, include native (natural) luciferases of fungi, derivatives thereof, recombinants thereof, and functional fragments thereof.
In the present disclosure, "luciferase" means an enzyme capable of oxidizing luciferin, and the oxidation reaction is accompanied by luminescence and release of oxidized luciferin.
In the present disclosure, a "derivative" is, in one or more embodiments, one or more amino acids added at the N-terminus and/or C-terminus of the original protein and/or within the native amino acid sequence. , and/or substituted and/or deleted and/or inserted proteins.
One or more embodiments of derivatives of naturally occurring fungal luciferases include fungal luciferases with improved luminescence properties over the native form, e.g., fungal luciferases with enhanced luminescence intensity over the native form, luminescence quantum yield Fungal luciferases, which are improved over types, and the like.
In the present disclosure, "functional fragment" with respect to luciferase, in one or more embodiments, refers to a fragment that is a part (fragment) of luciferase and has a function capable of catalyzing a luciferase phosphorylation reaction accompanied by luminescence. say. For example, a functional fragment of luciferase A refers to a portion of luciferase A that has the same luciferase activity (chemiluminescent enzyme activity) as luciferase A.
In the present disclosure, the term “fungi” refers to, in one or more embodiments, fungi capable of bioluminescence, and in one or more embodiments, basidiomycetes capable of bioluminescence, including so-called luminescent mushrooms. sell.
As used herein, "fungi" are, in one or more embodiments, muchurooms.

“Fungal luciferase” in the present disclosure, in one or more embodiments, can be referred to as a luciferase that can catalyze the luminescence reaction of 3-hydroxyhistidin.
In the present disclosure, "fungal luciferase" includes, in one or more embodiments, luciferase disclosed in WO2018/139956 (or Patent Document 1), and the content of the document constitutes a part of the present disclosure. Incorporated as

Fungi that naturally have fungal luciferases, in one or more embodiments, include those described in WO2018/139956 (or Patent Document 1).
In addition, in one or more embodiments, fungi naturally having fungal luciferase include those described in Terashima et al., Mushroom Science and Biotechnology, Vol. 24(4) 176-181, 2017, The contents of this document are incorporated by reference as forming part of the present disclosure.
Fungi that naturally have fungal luciferases, in one or more embodiments, include, but are not limited to:
Mycena lux-coeli
Armillaria mellea
Armillaria tabescens
Dictyopanus gloeocystidiatus
Favolaschia pezizaeformis
Mycena chlorophos
Mycena manipularis
Omphalotus guepiniformis
Pleurotus nitidus
Neonothpanus nambi (Shirohikaritake),
Armillaria gallica
Armillaria ostoyae
Mycena citricolor
Omphalotus olearius
Panellus stipticus
Roridomyces phyllostachydis
Armillaria calvescens
Armillaria cepistipes
Armillaria fuscipes
Armillaria gemina
Armillaria nabsnona
Armillaria sinapina
Collybia tuberosa
Dictyopanus foliicolus
Flammulina velutipes
Favolaschia manipularis
Filoboletus hanedae
Filoboletus pallescens
Filoboletus yunnanensis
Gerronema viridilucens
Mycena asterina
Mycena cahaya
Mycena coralliformis
Mycena daisyogunensis
Kobayashi
Mycena deepha
Aravind. & Manim.
Mycena discobasis
Mycena epipterygia
Mycenae fera
Mycena galopus
Mycena gombakensis
Mycena haematopus
Mycena luminans
Mycena inclinata
Mycena kentingensis
Mycena lacrimans
Mycena lazulina
Mycena lucentipes
Mycena lux-coeli
Mycena luxaeterna
Mycena luxarboricola
Mycena luxperpetua
Mycena maculata
Mycena nocticaeum
Mycena Noctilucens
Mycena olivaceo marginata
Mycena polygram
Mycena pruinosoviscida
Mycena pseudostylobates
Mycena pura
Mycena rosea
Mycena sanguinolenta
Mycenae seminar
Mycena silvaelucens
Mycena sinar
Mycena sinar var. tang kaisinar
Mycena singeri
Mycena stylobates
Mycena sublucens
Mycena tintinnabulum
Mycena zephirus
Neonothopanus gardneri
Neonothopanus nambi
Nothopanus eugrammus
Nothopanus noctilucens
Omphalotus flagelliformis
Omphalotus illustens
Omphalotus japonicus
Omphalotus mangensis
Omphalothus nidiformis
Omphalotus olearius
Omphalotus olivascens
Panellus luminescence
Panellus luxfilamentus
Panelus pusillus
Pleurotus decipiens
Roridomyces irritans
Roridomyces pruinosoviscidus
Roridomyces lamprosporus
Roridomyces roridus
Tricholoma margarita
Tricholoma sciodes
Xylaria hypoxylon
For the “fungal luciferase” in the present disclosure, a person skilled in the art can obtain the gene or protein from the above-described fungi by gene cloning or purification using conventional techniques. For gene cloning, for example, gene sequence information described in WO2018/139956 (or Patent Document 1) can be used. Alternatively, gene cloning may be performed by "degenerate PCR" in which degenerate primers designed based on regions of high homology in existing luciferases are used to amplify the gene.

A "chemiluminescent protein moiety derived from a fungal luciferase" in the present disclosure may, in one or more embodiments, be the fungal luciferase itself or a functional fragment of the fungal luciferase.
In the present disclosure, the fused protein/peptide other than the "fungi luciferase-derived chemiluminescent protein portion" in the fusion protein is not particularly limited.

[Lighting process]
A luminescence method according to the present disclosure comprises contacting a fungal luciferase or a fusion protein having a chemiluminescent protein moiety derived from a fungal luciferase with coelenterazine.
The contact method is not particularly limited as long as coelenterazine is brought into contact with the fungal luciferase or the fusion protein in an amount appropriate for luminescence.

In the present disclosure, "coelenterazine" means naturally occurring coelenterazine as well as analogues thereof unless otherwise specified.
Coelenterazine, in one or more embodiments, is disclosed in US Application No. 12/056073 (paragraph [0086]), for example, coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine -cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine (“coelenterazine-hh”), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methylcoelenterazine. In addition, "coelenterazine" may include those disclosed in WO2012/061529 in one or more other embodiments.
The contents of US Application No. 12/056073 (paragraph [0086]) and WO2012/061529 are incorporated as part of this disclosure.

The luminescence method according to the present disclosure can be applied to reporter systems, analysis, and/or observation, etc., in one or more embodiments.

A luminescence method according to the present disclosure, in one or more embodiments, comprises using a fusion protein having a fungal luciferase or a chemiluminescent protein moiety derived from a fungal luciferase as a reporter, and one or more other In embodiments, detecting luminescence to confirm expression of the reporter.

The luminescence method according to the present disclosure, in one or more embodiments, comprises catalyzing a luminescence reaction with a fungal luciferase or a fusion protein having a fungal luciferase-derived chemiluminescent protein moiety for resonance energy transfer, Another embodiment or embodiments includes catalyzing a luminescent reaction as a Forster Resonance Energy Transfer (FRET) donor.

A luminescence method according to the present disclosure, in one or more embodiments, comprises catalyzing a luminescence reaction for viewing by a fungal luciferase or a chemiluminescent protein moiety derived from a fungal luciferase.

A fungal luciferase that catalyzes a luminescence reaction in the luminescence method according to the present disclosure, or a fusion protein having a chemiluminescent protein moiety derived from a fungal luciferase, in one or more embodiments, is expressed locally or globally in vivo. state of expression locally or throughout the cell, state of expression in the cell wall of a plant, state of purification, state of isolation, and the like.

A luminescence method according to the present disclosure, in one or more embodiments, may include detecting a luminescence signal emitted by the luminescence method according to the present disclosure. The detection may be performed visually or using an optical device.

[Method of making the target glow]
The present disclosure provides, in another aspect, a method of illuminating a subject, the subject comprising a fungal luciferase or a fusion protein having a chemiluminescent protein moiety derived from a fungal luciferase, wherein the method of luminescence according to the present disclosure provides It relates to a method comprising emitting light.

In one or more embodiments, the "subject" includes cells, tissues, and organisms that express or are likely to express a fungal luciferase or a fusion protein having a fungal luciferase-derived chemiluminescent protein moiety. be done.
In another one or more embodiments, the "subject" is a non-cell-based composition containing or potentially containing a fungal luciferase or a fusion protein having a chemiluminescent protein moiety derived from a fungal luciferase. Matters, liquids, and/or articles may be included.

[kit]
The disclosure relates, in another aspect, to a kit comprising a fungal luciferase or a fusion protein having a chemiluminescent protein portion derived from a fungal luciferase, its nucleic acid, or its expression cassette, and coelenterazine.

The fusion protein and coelenterazine having a fungal luciferase or fungal luciferase-derived chemiluminescent protein portion in the kits of the present disclosure are the same as described above.

A nucleic acid in a kit of the present disclosure, in one or more embodiments, is a nucleic acid encoding a fungal luciferase or a fusion protein having a chemiluminescent protein portion derived from a fungal luciferase. In the present disclosure, nucleic acids include single- or double-stranded DNAs selected from synthetic DNAs, cDNAs, genomic DNAs and plasmid DNAs, and transcription products of these DNAs.

An expression cassette in a kit of the present disclosure, in one or more embodiments, is an expression cassette comprising a nucleic acid encoding a fungal luciferase or a fusion protein having a chemiluminescent protein portion derived from a fungal luciferase. In the expression cassette, the nucleic acid is operatively linked with expression control sequences depending on the host cell or cell-free expression system into which it is introduced. Expression control sequences include promoters, enhancers, transcription terminators, etc. Others include initiation codons, intronic splicing signals, and stop codons.

The present disclosure further relates to one or more of the following non-limiting embodiments.
[1] A luminescence method comprising contacting a fungal luciferase or a fusion protein having a fungal luciferase-derived chemiluminescent protein portion with coelenterazine.
[2] A method for illuminating an object,
the subject comprises a fungal luciferase or a fusion protein having a chemiluminescent protein portion derived from a fungal luciferase;
A method comprising emitting light by the method according to [1].
[3] A detection method comprising detecting a luminescence signal emitted by the method of [1] or [2].
[4] The method of any one of [1] to [3], wherein the fungal luciferase is a luciferase that emits light using 3-hydroxyhistidine as a substrate.
[5] a kit comprising fungal luciferase or a fusion protein having a fungal luciferase-derived chemiluminescent protein moiety, its nucleic acid, or its expression cassette, and coelenterazine;

EXAMPLES The present disclosure will be described in more detail below with reference to examples, but these are examples and the present disclosure is not limited to these examples.

The following experiments were performed to confirm that fungal luciferase catalyzes the luminescence reaction using coelenterezin as a substrate.

[Luciferase used]
(1) mcLuz: fungal luciferase of Mycena chlorophos. Acquisition method: Gene cloning from Yakotake mushroom based on WO2018/139956 (or Patent Document 1) (nucleotide sequence is SEQ ID NO: 01)
(2) mlLuz: fungal luciferase of Mycena lux-coeli. Acquisition method: Collected in Ukui, Wakayama Prefecture, gene cloning using degenerate PCR (nucleotide sequence is SEQ ID NO: 02)
(3) nnLuz: fungal luciferase of Neonothopanus nambi. Acquisition method: Purchased from Addgene (Plasmid #139695) (4) Nluc: luciferase derived from Oplophorus gracilirostris. How to obtain: NanoLuc (trade name, manufactured by Promega)
(5) Rluc8: luciferase from Renilla reniformis. Acquisition method: Rluc8_S257G, published by previous paper (Saito et al., Nat. Commun., 3, 2012)
(6) Fluc2: luciferase from firefly (Photinus lyralis). How to obtain: luc2 (trade name, manufactured by Promega)

[Luciferin used]
(1) Coelenterazine: Coelenterazine h (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.)
(2) Firefly luciferin: D-luciferin (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.)
(3) 3-hydroxyhistidine (according to the paper (Kaskova et al., Sci. Adv., 3 (4), 2017). However, when synthesizing organic compound 17 in Supplementary Figure 12 of the same document, Sigma-Aldrich Using purchased Mg(OMe) ₂ in MeOH (Catalog No. 335657-500ML), the reaction was carried out at 60° C. for 15 hours.)

[Luminescence measurement method]
The luminescence measurement of luciferase was performed under the following conditions. The results are shown in FIGS. 1-4.
The genes mcLuz, mlLuz, nnLuz, Nluc, Rluc8, and Fluc2 were each cloned into an E. coli expression vector (pRSETb), transformed into E. coli (JM109 (DE3)), and the E. coli into which each gene was introduced was placed in liquid at 23°C for 69 hours. cultured. Nluc, Rluc8 and Fluc2 proteins were extracted by freeze-thaw method and purified by Ni-NTA column. mcLuz, mlLuz, and nnLuz purified proteins from E. coli according to previous papers (Kotlobay et al., PNAS, 115 (50), 2018). A microplate reader (SH-9000, Corona electric) was used for luminescence measurement, 100 μl of 2 nM purified protein was added, and 10 μl of 5 μM coelenterazine h was added. Luminescence was measured with an exposure time of 1 second for coelenterazine h and D-luciferin, and 10 seconds for 3-hydroxyhistidine.

FIG. 1 is a graph showing the luminescence intensity when coelenterazine was brought into contact with each luciferase. Along with Nluc and Rluc8, which are known coelenterazine luciferases, three fungal luciferases (mcLuz, mlLuz, and nnLuz) were also luminescent.
FIG. 2 is a graph of FIG. 1 excluding Nluc data. Fungal luciferases were found to have luminescence intensities about 1/4 to about 1/2 that of Rluc8.

FIG. 3 is a graph showing the luminescence intensity when each luciferase was brought into contact with D-luciferin, which is firefly luciferin. No luminescence other than firefly luciferase Fluc2 was observed.
FIG. 4 is a graph showing the luminescence intensity when each luciferase was brought into contact with 3-hydroxyhistidin, which is the luciferin of fungal luciferase. No luminescence was observed except for the three fungal luciferases (mcLuz, mlLuz, and nnLuz).

As shown in FIGS. 3 and 4, conventionally, the combination of luciferase and luciferin has a very high specificity, but as shown in FIGS. showed luminescence.

Claims (5)

A method of luminescence comprising contacting a fungal luciferase or a fusion protein having a chemiluminescent protein portion derived from a fungal luciferase with coelenterazine. A method of illuminating an object, comprising:
the subject comprises a fungal luciferase or a fusion protein having a chemiluminescent protein portion derived from a fungal luciferase;
A method comprising emitting light according to the method of claim 1 . A detection method comprising detecting a luminescence signal emitted by the method according to claim 1 or 2. 4. The method according to any one of claims 1 to 3, wherein the fungal luciferase is a luciferase that emits light using 3-hydroxyhistidine as a substrate. A kit comprising a fungal luciferase or a fusion protein having a chemiluminescent protein portion derived from a fungal luciferase, its nucleic acid, or its expression cassette, and coelenterazine.

Images