CN-122029206-A - Photoprotein

CN122029206ACN 122029206 ACN122029206 ACN 122029206ACN-122029206-A

Abstract

The present invention provides a luminescent protein capable of emitting high-intensity light in a plurality of wavelength regions. The luminescent protein comprises a fusion construct of bacterial luciferase and a fluorescent protein, wherein the bacterial luciferase comprises LuxA subunit and LuxB subunit, and the fluorescent protein is fused with the bacterial luciferase through LuxA subunit.

Inventors

NAGAI TAKEHARU
Suban Hardy Kusuma
Fu Buman

Assignees

国立大学法人大阪大学

Dates

Publication Date: 20260512
Application Date: 20241010
Priority Date: 20231018

Claims (7)

1. A luminescent protein comprising a fusion construct of a bacterial luciferase and a fluorescent protein, wherein the bacterial luciferase comprises LuxA subunit and LuxB subunit and the fluorescent protein is fused to the LuxA subunit; Wherein, the The LuxA subunit is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 1 or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 1; The LuxB subunit is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO.2 or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO.2, and The fluorescent protein is selected from the group consisting of a cyan fluorescent protein, a green fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, and a red fluorescent protein; Wherein, the The cyan fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 3, or a polypeptide consisting of an amino acid sequence which has 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 3 and can emit fluorescence with a maximum emission wavelength of 450 to 500 nm, or a circular arrangement form of any of the above; The green fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 4, or a polypeptide consisting of an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 4 and capable of emitting fluorescence with a maximum emission wavelength of 500 to 520 nm, or a circular arrangement form of any of the above; The yellow fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 5, and when fused with the LuxA subunit, the fusion protein comprises amino acids 1 to 229 of SEQ ID NO. 5, which are connected with amino acids 2 to 359 of SEQ ID NO.1 through two amino acids E and L; the orange fluorescent protein is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 6, and when fused with LuxA subunit, the resulting fusion protein comprises amino acids 1 to 218 of SEQ ID NO. 6, which are linked with amino acids 2 to 359 of SEQ ID NO.1 through two amino acids E and L, or The red fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 7, or a polypeptide consisting of an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 7 and capable of emitting fluorescence with a maximum emission wavelength of 580 to 620 nm, or a circular arrangement form of any of the above polypeptides.
2. A nucleic acid comprising a nucleotide sequence encoding a fusion construct of a LuxA subunit of bacterial luciferase with a fluorescent protein, wherein, The LuxA subunit is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1 or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO.1, and The fluorescent protein is selected from the group consisting of a cyan fluorescent protein, a green fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, and a red fluorescent protein; Wherein: The cyan fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 3, or a polypeptide consisting of an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 3 and capable of emitting fluorescence with a maximum emission wavelength of 450 to 500 nm, or a circular arrangement form of any of the above; The green fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 4, or a polypeptide consisting of an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 4 and capable of emitting fluorescence with a maximum emission wavelength of 500 to 520 nm, or a circular arrangement form of any of the above; The yellow fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 5, and when fused with the LuxA subunit, the fusion protein comprises amino acids 1 to 229 of SEQ ID NO. 5, which are connected with amino acids 2 to 359 of SEQ ID NO.1 through two amino acids E and L; The orange fluorescent protein is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 6, and when fused with LuxA subunit, the resulting fusion protein comprises amino acids 1 to 218 of SEQ ID NO. 6, which are linked with amino acids 2 to 359 of SEQ ID NO. 1 through two amino acids E and L, or The red fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 7, or a polypeptide consisting of an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 7 and capable of emitting fluorescence with a maximum emission wavelength of 580 to 620 nm, or a circular arrangement form of any of the above polypeptides.
3. The nucleic acid of claim 2, further comprising a nucleotide sequence encoding a LuxB subunit of bacterial luciferase, wherein, The LuxB subunit is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 2 or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 2.
4. An expression vector comprising the nucleic acid of claim 2 integrated therein.
5. A cell comprising a nucleic acid comprising a nucleotide sequence encoding a fusion construct of a LuxA subunit of bacterial luciferase and a fluorescent protein, and a nucleic acid comprising a nucleotide sequence encoding a LuxB subunit of bacterial luciferase; Wherein, the The LuxA subunit is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO.1 or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 1; The LuxB subunit is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 2 or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 2, and The fluorescent protein is selected from the group consisting of a cyan fluorescent protein, a green fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, and a red fluorescent protein; Wherein: The cyan fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 3, or a polypeptide consisting of an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 3 and capable of emitting fluorescence with a maximum emission wavelength of 450 to 500 nm, or a circular arrangement form of any of the above; The green fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 4, or a polypeptide consisting of an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 4 and capable of emitting fluorescence with a maximum emission wavelength of 500 to 520 nm, or a circular arrangement form of any of the above; The yellow fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 5, and when fused with the LuxA subunit, the fusion protein comprises amino acids 1 to 229 of SEQ ID NO. 5, which are connected with amino acids 2 to 359 of SEQ ID NO.1 through two amino acids E and L; The orange fluorescent protein is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 6, and when fused with LuxA subunit, the fusion construct consists of amino acids 1 to 218 of SEQ ID NO. 6 linked with amino acids 2 to 359 of SEQ ID NO. 1 through two amino acids E and L, or The red fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 7, or a polypeptide consisting of an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 7 and capable of emitting fluorescence with a maximum emission wavelength of 580 to 620 nm, or a circular arrangement form of any of the above polypeptides.
6. The cell of claim 5 which is a bacterial, fungal, plant or animal cell.
7. A method of preparing a luminescent protein comprising expressing a nucleic acid comprising a nucleotide sequence encoding a fusion construct of a LuxA subunit of bacterial luciferase and a fluorescent protein, and expressing a nucleic acid comprising a nucleotide sequence encoding a LuxB subunit of bacterial luciferase; Wherein, the The LuxA subunit is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 1 or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 1; The LuxB subunit is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO.2 or a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO.2, and The fluorescent protein is selected from the group consisting of a cyan fluorescent protein, a green fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, and a red fluorescent protein; Wherein: The cyan fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 3, or a polypeptide consisting of an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 3 and capable of emitting fluorescence with a maximum emission wavelength of 450 to 500 nm, or a circular arrangement form of any of the above; The green fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 4, or a polypeptide consisting of an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 4 and capable of emitting fluorescence with a maximum emission wavelength of 500 to 520 nm, or a circular arrangement form of any of the above; The yellow fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 5, and when fused with the LuxA subunit, the fusion protein comprises amino acids 1 to 229 of SEQ ID NO. 5, which are connected with amino acids 2 to 359 of SEQ ID NO.1 through two amino acids E and L; the orange fluorescent protein is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 6, and when fused with LuxA subunit, the resulting fusion protein comprises amino acids 1 to 218 of SEQ ID NO. 6, which are linked with amino acids 2 to 359 of SEQ ID NO.1 through two amino acids E and L, or The red fluorescent protein is a polypeptide consisting of an amino acid sequence shown in SEQ ID NO. 7, or a polypeptide consisting of an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 7 and capable of emitting fluorescence with a maximum emission wavelength of 580 to 620 nm, or a circular arrangement form of any of the above polypeptides.

Description

Photoprotein Technical Field The present invention relates to a photoprotein comprising luciferase derived from a luminescent bacterium. Background Among various types of luminescence phenomena, those involving luminescence of a luminescent substrate by oxidation with a catalytic enzyme called luciferase, which occurs in a living organism, are classified as bioluminescence. Up to thousands of bioluminescent organism species are currently known from which a variety of different luciferases and luminescent substrates have been identified. Such luciferases and luminescent substrates have been used in the fields of genetic engineering and the like, and quantitative analysis of intracellular proteins has been carried out using luminescent signals as indicators as reporters. A variety of eubacteria (i.e., bacteria) have luminescence properties (hereinafter collectively referred to as luminescent bacteria). The light emission phenomenon of light-emitting bacteria is caused by an enzymatic reaction catalyzed by bacterial luciferase (non-patent documents 1 and 2). Specifically, bacterial luciferase catalyzes the oxidation reaction of reduced flavin mononucleotide (FMNH 2) with long chain fatty aldehyde (RCHO) to produce Flavin Mononucleotide (FMN) and corresponding long chain fatty acid (RCOOH). During this reaction, light having a maximum emission wavelength of about 490nm is generated. The key enzyme required for the luminescence of the luminescent bacteria is encoded by a single operon called "luxCDABE" operon (simply lux operon) which is conserved among luminescent bacterial species (non-patent documents 1 and 2). Wherein the luxA and luxB genes encode the alpha subunit (LuxA) and the beta subunit (LuxB) of the heterodimeric bacterial luciferase respectively, and the luxC, luxD and luxE genes encode enzymes associated with synthesis and recycling of fatty aldehyde substrates of the luciferase. These 5 lux genes are co-expressed in non-luminescent bacteria or yeast cells, and autonomous luminescence (self luminescence) can be produced without adding a substrate from an external source (non-patent documents 3 and 4). In mammalian cells or plant cells, FMN oxidoreductase (LuxG) is co-expressed in addition to the 5 lux genes, and FMNH 2 can be produced in a sufficient amount to achieve stable bioluminescence (non-patent documents 5 to 8). Unlike fluorescent protein-based imaging, bioluminescence imaging requires no external excitation light. Therefore, bioluminescence imaging has an advantage in that problems such as phototoxicity, photofading, and background fluorescence of sample sources are not likely to occur. Particularly, the reporter gene based on bacterial luciferase can realize high signal to noise ratio (S/N ratio) and is simple and convenient to operate. In addition, since excitation light is not required, spectral interference caused by the excitation light is not generated, and analysis can be performed in a wider spectral range than fluorescent protein imaging. However, imaging based on bacterial origin luciferases has disadvantages of low brightness and long exposure time, which limits its application (non-patent documents 9 and 10). Non-patent document 11 describes that by fusing LuxB derived from bacteria with a yellow fluorescent protein Venus in a circular arrangement, bioluminescence Resonance Energy Transfer (BRET) can be induced in a cell, and that the luminescence intensity of about 10 times when Lux is used alone can be achieved in a shorter time. Non-patent document 11 also describes that the luminous intensity in cells can be increased after introducing such a fusion construct into plant cells and animal cells. List of references Non-patent literature Non-patent document 1, meighen, E., A., microbiol. Rev. 55, 123-142, 1991) Non-patent document 2 Dunlap, P., adv, biochem, eng, biotechnol, 144, 37-64, 2014 Non-patent document 3: frackman, S.et al, J.Bacteriol., 172, 5767-5773, 1990 Non-patent document 4 Gupta, R.K. et al, FEMS YEAST Res, 4, 305-313, 2003 Non-patent document 5, close, d.m. et al, PLoS One 5, e12441, 2010 Non-patent document 6 Krichevsky, A.et al, PLoS One 5, e15461, 2010 Non-patent document 7, xu, t.et al., PLoS One 9, e96347, 2014 Non-patent document 8: gregor, C.et al, proc. Natl. Acad. Sci., U.S. A., 116, 26491-26496, 2019 Non-patent document 9: close, d.m. et al, j. Biomed. Opt. 16, 047003, 2011 Non-patent document 10 Gregor, C.et al, proc.Natl. Acad. Sci., U.S. A., 115, 962-967, 2018 Non-patent document 11 Kaku, T.et al, nature Scientific Reports, 11, 14994, 2021 Summary of The Invention The object to be achieved As described above, it has been confirmed that a luminescent protein capable of achieving high-intensity self-luminescence in a cell can be constructed by using a lux operon derived from a bacterium and fusing a fluorescent protein Venus with LuxB protein. The system emits light in a yellow-green color having a maximum wavelength of approximately 528 nm. However