WO2026004362A1 - Method for producing mammalian cells labeled by chromoprotein, and method for screening chromoprotein - Google Patents

Abstract

Provided are: a method for producing mammalian cells labeled by a chromoprotein that includes culturing, at less than 37° C, mammalian cells into which a nucleic acid that expresses a chromoprotein has been introduced; and a method for screening a chromoprotein that includes culturing, at less than 37° C, mammalian cells into which a nucleic acid that expresses a candidate protein has been introduced and evaluating the visible light absorption of the mammalian cells.

Description

Method for producing mammalian cells labeled with chromoproteins and method for screening chromoproteins

This disclosure relates to a method for producing mammalian cells labeled with a chromoprotein and a method for screening for chromoproteins.

Chromoproteins are proteins that have high absorbance in the visible light region (Non-Patent Document 1). Taking advantage of their large molar extinction coefficient, chromoproteins are expected to be used as markers that display coloration when expressed in cells, and as acceptors for fluorescence resonance energy transfer (FRET) (Non-Patent Document 2).

International Publication No. 2023/182169

Josefine Liljeruhm et al., “Engineering a palette of eukaryotic chromoproteins for bacterial synthetic”, Journal of Biological Engineering 12:8 (2018). F Hafna Ahmed et al., “Over the rainbow: structural characterization of the chromoproteins gfasPurple, amilCP, spisPink and eforRed”, Acta Crystallogr D Struct Biol. 78(Pt 5):599-612 (2022).

When the inventors attempted to express chromoproteins in mammalian cells using genetic techniques, they discovered a new problem: chromoproteins are difficult to express in mammalian cells under the temperature conditions typically used for culturing mammalian cells.

The present disclosure aims to provide a method for producing mammalian cells labeled with chromoproteins. The present disclosure also aims to provide a method for screening chromoproteins, which includes expressing candidate chromoprotein proteins in mammalian cells.

The inventors have discovered that when mammalian cells are cultured at temperatures lower than the general culture temperature, pigment proteins can be efficiently expressed in mammalian cells.

The present disclosure relates, for example, to the following:
[1] A method for producing mammalian cells labeled with a chromoprotein, comprising culturing mammalian cells into which a nucleic acid that expresses the chromoprotein has been introduced at a temperature below 37°C.
[2] Culturing mammalian cells into which a nucleic acid that expresses a pigment protein has been introduced at a temperature below 37°C, and evaluating the visible light absorption of the mammalian cells;
A method for evaluating mammalian cell labeling with a chromoprotein, comprising:
[3] culturing mammalian cells into which a nucleic acid that expresses a candidate protein has been introduced at a temperature below 37°C; and evaluating the visible light absorption of the mammalian cells;
A method for screening a pigment protein, comprising:
[4] The method further comprises concentrating the mammalian cells cultured at less than 37°C before assessing visible light absorption;
The method according to [3], wherein the visible light absorption of the mammalian cells is evaluated using the coloration of the concentrated mammalian cells as an indicator.
[5] The method according to any one of [1] to [4], wherein the molar extinction coefficient of the maximum absorption wavelength of the chromoprotein in the 400 to 800 nm region is 5.0×10 ⁴ [L/mol·cm] or more.
[6] The method according to any one of [1] to [5], wherein the chromoprotein is a chromoprotein derived from an organism other than a mammal or a modified protein thereof.
[7] The method according to any one of [1] to [6], wherein the nucleic acid that expresses the chromoprotein is a nucleic acid that is distributed to daughter cells of the cell into which it has been introduced, or a nucleic acid in which a region encoding the chromoprotein is incorporated into the genomic DNA of the cell into which it has been introduced.
[8] The method according to any one of [1] to [7], wherein the culturing is carried out at 33°C or lower.

One embodiment of the present disclosure provides a method for producing mammalian cells labeled with a chromoprotein. According to this embodiment, the chromoprotein can be introduced into cells simply, selectively, and efficiently using genetic techniques.

One embodiment of the present disclosure provides a method for evaluating mammalian cell labeling with chromoproteins. This embodiment allows for easy evaluation of mammalian cell labeling with chromoproteins using the visible light absorption of mammalian cells as an indicator.

One embodiment of the present disclosure provides a method for screening for chromoproteins. According to this embodiment, chromoproteins suitable for labeling mammalian cells can be easily screened using the visible light absorption of the mammalian cells as an indicator. Furthermore, if one aspect of this embodiment further includes concentrating the mammalian cells, chromoproteins suitable for mammalian cells can be more easily screened using the coloration of the concentrated mammalian cells as an indicator.

FIG. 1 shows the results of observing cells in PBS(−) under a phase-contrast microscope after the washing procedure in step 3 of Example 1. 1 is a digital photograph taken under room light of cells in PBS(-) after the washing procedure in step 3 of Example 1. 1 is a digital photograph taken under room light of the concentrated cells obtained in step 4 in step 5 of Example 1. FIG. 10 shows the results of observing cells in PBS(-) under a phase-contrast microscope after washing, which were cultured for 6 days under conditions of 37°C and 5% _CO2 in step 3 of Example 2. FIG. 10 shows the results of observing cells in PBS(-) under a phase-contrast microscope after washing, which were cultured for 6 days under conditions of 35°C and 5% _CO2 in step 3 of Example 2. FIG. 10 shows the results of observing cells in PBS(-) under a phase-contrast microscope after washing, which were cultured for 6 days under conditions of 32.5°C and 5% _CO2 in step 3 of Example 2. FIG. 10 shows the results of observing cells in PBS(-) under a phase-contrast microscope after washing, which were cultured for 7 days under conditions of 30°C and 5% _CO2 in step 3 of Example 2. This is a digital photograph taken under room light of cells in PBS(-) after culturing for 6 days at 37°C and 5% _CO2 in step 3 of Example 2 and subsequent washing. This is a digital photograph taken under room light of cells in PBS(-) after culturing for 6 days at 35°C and 5% _CO2 in step 3 of Example 2 and subsequent washing. This is a digital photograph taken under room light of cells in PBS(-) after culturing for 6 days at 32.5°C and 5% _CO2 in step 3 of Example 2 and subsequent washing. This is a digital photograph taken under room light of cells in PBS(-) after culturing for 7 days at 30°C and 5% _CO2 in step 3 of Example 2 and subsequent washing. In step 5 of Example 2, a digital photograph was taken under room light of the concentrated cells obtained in step 4. For comparison of each condition, the brightness and contrast of the image shown in FIG. 12 have been adjusted. This is an image obtained by dividing the RGB image shown in FIG. 12 into independent channels of R (Red), G (Green), and B (Blue). The RGB image in FIG. 12 is divided into HSB color space, H (Hue), S (Saturation), and B (Brightness), and the image is shown. The results for aeCP597 (37°C, 32.5°C, 30°C) shown in Figure 15 are plotted on polar coordinates, with the declination angle being H (Hue) and the distance from the pole being S (Saturation). In step 5 of Example 2, a digital photograph was taken under room light of a cell suspension in which the concentrated cells obtained in step 4 were suspended in 500 μL of PBS(−). For comparison of each condition, the brightness and contrast of the image shown in FIG. 17 have been adjusted.

The following describes embodiments for implementing this disclosure, but the disclosure is not limited to the following embodiments.

In the present disclosure, when a protein or nucleic acid contains an amino acid sequence or a nucleotide sequence that has 90% or more sequence identity with a given amino acid sequence or a nucleotide sequence, that protein or nucleic acid may have 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the given sequence, and in a preferred embodiment, may have 95% or more sequence identity, and in a most preferred embodiment, may have 100% sequence identity.

In the present disclosure, when a sequence contained in a certain protein or nucleic acid has a mutation (i.e., sequence identity is not 100%) with respect to a specified amino acid sequence or nucleotide sequence, the mutation may be a mutation selected from substitution, deletion, insertion, and addition in each of 1 to 20 consecutive or dispersed residues or 1 to 60 bases. In a preferred embodiment, the mutation may be a mutation selected from substitution, deletion, insertion, and addition in each of 1 to 10 consecutive or dispersed residues or 1 to 30 bases. In a more preferred embodiment, the mutation may be a mutation selected from substitution, deletion, insertion, and addition in each of 1 to 3 residues or 1 to 10 bases. In an even more preferred embodiment, the mutation may be a mutation selected from substitution, deletion, insertion, and addition in each of 1 residue or 1 to 3 bases.

A first embodiment of the present disclosure is a method for producing mammalian cells labeled with a chromoprotein, which includes culturing mammalian cells into which nucleic acid that expresses the chromoprotein has been introduced at a temperature below 37°C (culturing step). Hereinafter, this embodiment will also be referred to as the "production method of the first embodiment."

In this disclosure, a chromoprotein is a protein that has a high absorbance in the visible light region (Non-Patent Document 1). Proteins with a high absorbance in the visible light region can label cells by absorbing visible light, and their expression in cells can be evaluated using visible light absorption as an indicator.

In one embodiment, the chromoprotein may not be derived from an animal from which the mammalian cells expressing the chromoprotein are derived. In one embodiment, the chromoprotein may be derived from an organism different from the animal from which the mammalian cells expressing the chromoprotein are derived (i.e., derived from a heterologous organism). In one embodiment, the chromoprotein may be derived from an organism other than a mammal, or from a eukaryote other than a mammal. In one embodiment, the chromoprotein may be derived from an organism other than a vertebrate, or from a eukaryote other than a vertebrate. In one embodiment, the chromoprotein may be derived from an invertebrate or a fungus, or may be derived from an invertebrate or a fungus. Invertebrates may be, for example, cnidarians, echinoderms, mollusks, arthropods, annelids, or gastropods, or may be cnidarians, echinoderms, or mollusks, or may be cnidarians, or may be cnidarians belonging to the class Anthozoa, or may be corals or sea anemones. Fungi may be, for example, basidiomycetes or ascomycetes, or may be basidiomycetes. In one embodiment, the chromoprotein may be derived from a cnidarian or a basidiomycete, or may be derived from a cnidarian or an anthozoan cnidarian. In these cases, the organism, eukaryote, animal, or invertebrate from which the chromoprotein is derived may be, for example, a marine organism. Generally, seawater temperatures are below 37°C. Therefore, if the organism, eukaryote, animal, or invertebrate from which the chromoprotein is derived is a marine organism, the chromoprotein may be more easily expressed, or the expressed protein may be more easily folded, when cultured under conditions below 37°C.

In one embodiment, the chromoprotein may be a natural protein or an artificial protein. A chromoprotein that is an artificial protein may be a modified protein of a naturally occurring chromoprotein, such as a modified protein of a chromoprotein derived from an organism such as those described above. That is, for example, the chromoprotein may be a chromoprotein derived from an organism such as those described above or a modified protein thereof, or may be a chromoprotein derived from an organism other than a mammal or a modified protein thereof, a chromoprotein derived from an invertebrate or fungus or a modified protein thereof, a chromoprotein derived from a cnidarian or a basidiomycete or a modified protein thereof, or a chromoprotein derived from a cnidarian or a modified protein thereof. A modified protein of a naturally occurring chromoprotein may be, for example, a protein that has 90% or more sequence identity with the naturally occurring protein. Such modified proteins can be appropriately designed by those skilled in the art based on the sequence information of the original natural protein.

The chromoprotein of one embodiment may have a molar extinction coefficient at its maximum absorption wavelength that is equal to or greater than a predetermined lower limit, for example, the molar extinction coefficient at the maximum absorption wavelength may be equal to or greater than a predetermined lower limit under a physiological environment such as an intracellular environment (e.g., an environment of pH 6.5 to 8.5). The chromoprotein of one embodiment may have a molar extinction coefficient at its maximum absorption wavelength in the visible light region that is equal to or greater than a predetermined lower limit, for example, the molar extinction coefficient at its maximum absorption wavelength in the 400 to 800 nm region may be equal to or greater than a predetermined lower limit under a physiological environment such as an intracellular environment (e.g., an environment of pH 6.5 to 8.5). In these cases, the predetermined lower limit may be, for example, 3.0×10 ⁴ , 5.0×10 ⁴ , 8.0×10 ⁴ , 1.0×10 ⁵ , 1.2×10 ⁵ , 1.4×10 ⁵ , 1.6×10 ⁵ , 1.8× ^{10 5} , or 2.0×10 ⁵ [L/mol cm]. When the maximum absorption wavelength or the local maximum absorption wavelength is equal to or greater than the lower limit, the signal intensity derived from the absorption of the chromoprotein increases, making the compound suitable for use in cell labeling. The fluorescence quantum yield and phosphorescence quantum yield can be measured using, for example, a spectrophotometer or a plate reader.

The maximum absorption wavelength in the 400 to 800 nm region of one embodiment of the chromoprotein may be, for example, 450 nm or more, 500 nm or more, 530 nm or more, 550 nm or more, 560 nm or more, or 565 nm or more, or may be 750 nm or less, 700 nm or less, 670 nm or less, 650 nm or less, 630 nm or less, or 620 nm or less, and these upper limits can be freely combined. The maximum absorption wavelength in the 400 to 800 nm region of one embodiment of the chromoprotein may be, for example, 450 nm or more and 750 nm or less, 530 nm or more and 670 nm or less, 550 nm or more and 650 nm or less, or 565 nm or more and 620 nm or less.

The chromoprotein of one embodiment may have a fluorescence quantum yield of 30% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.3% or less, 0.1% or less, 0.03% or less, 0.01% or less, or 0.001% or less, and the quantum yield may be a value measured in a physiological environment such as an intracellular environment (e.g., an environment of pH 6.5 to 8.5). The chromoprotein of one embodiment may have a phosphorescence quantum yield of 30% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.3% or less, 0.1% or less, 0.03% or less, 0.01% or less, or 0.001% or less, and the quantum yield may be a value measured in a physiological environment such as an intracellular environment (e.g., an environment of pH 6.5 to 8.5). When the fluorescence quantum yield and/or phosphorescence quantum yield of the chromoprotein of one embodiment is equal to or less than the above upper limit, noise due to fluorescence and/or phosphorescence can be suppressed in detecting signals derived from the protein. Furthermore, when the fluorescence quantum yield and/or phosphorescence quantum yield of the chromoprotein of one embodiment is equal to or less than the above upper limit, photobleaching of the chromoprotein due to fluorescence and/or phosphorescence can be suppressed.

In one embodiment, the chromoprotein may be expressed in cells in the form of a fusion protein containing the protein. Such a fusion protein may be obtained by modifying the N-terminus or C-terminus of the modified protein with the chromoprotein, optionally via a linker, and such a fusion protein can be designed according to methods commonly used by those skilled in the art. Using such a fusion protein, it is possible to label with the chromoprotein a location within the cell that corresponds to the intracellular localization of the modified protein, and it is also possible to analyze the localization of the modified protein within the cell using the signal from the chromoprotein as an indicator.

The form of the nucleic acid that expresses the chromoprotein may be any form commonly used when genetically expressing a protein in a cell. Such a nucleic acid contains a sequence that encodes the chromoprotein. Such a nucleic acid can be designed appropriately by a person skilled in the art depending on the form. Such a nucleic acid can be obtained, for example, by outsourcing to a company that performs custom nucleic acid synthesis, or by inserting a nucleic acid containing a sequence that encodes the chromoprotein into a backbone purchased from a supplier using restriction enzyme treatment or the like. Such a nucleic acid can also be amplified using methods commonly used by a person skilled in the art, such as introducing it into bacteria such as E. coli and culturing it.

In one embodiment, the nucleic acid that expresses a chromoprotein may be a vector that expresses the chromoprotein. The vector that expresses the chromoprotein may be, for example, a viral vector or a plasmid vector. The viral vector may be a viral vector whose genome is DNA or a viral vector whose genome is RNA, and may be a viral vector whose genome is DNA in order to facilitate maintaining expression during the culture process. A viral vector whose genome is DNA may be, for example, an Epstein-Barr virus (Human herpesvirus 4, HHV-4) vector, an adenovirus vector, or an adeno-associated virus (AAV) vector. Viral vectors can introduce genes into mammalian cells via their infectivity, allowing the expression of a target protein in mammalian cells. The plasmid vector may be a DNA-based plasmid vector or an RNA-based plasmid vector, and may be a DNA-based plasmid vector in order to facilitate maintaining expression during the culture process. The plasmid vector can be introduced into cells by, for example, lipofection or electroporation. Such vectors can be appropriately designed by those skilled in the art depending on the amino acid sequence of the chromoprotein to be expressed.

In one embodiment, the nucleic acid that expresses a chromoprotein may be mRNA or a precursor thereof containing a sequence encoding the chromoprotein. Such mRNA or a precursor thereof can be used to express the chromoprotein in mammalian cells. There are no particular limitations on the mRNA precursor, as long as it can provide mRNA containing a sequence encoding the chromoprotein through splicing in mammalian cells. Such mRNA and precursors can be appropriately designed by those skilled in the art depending on the amino acid sequence of the chromoprotein to be expressed. Such mRNA and precursors can be introduced into cells by, for example, lipofection or electroporation.

In a preferred embodiment, the nucleic acid that expresses the chromoprotein may be a nucleic acid that can be used to create a strain that constitutively expresses the chromoprotein. For example, the nucleic acid that expresses the chromoprotein may be a nucleic acid that is distributed to daughter cells of the introduced cell, or a nucleic acid in which a region encoding the chromoprotein is incorporated into the genomic DNA of the introduced cell. When the nucleic acid in a preferred embodiment is such a nucleic acid, the expression of the chromoprotein is easily maintained during the culture process.

The nucleic acid that is distributed and introduced into daughter cells of the introduced cell may be a vector that is maintained within the cell as an episome (an independent DNA molecule that is not integrated into the cell's chromosomes). Such a vector (an episome-based vector) is also distributed and introduced into daughter cells. Such an episome-based vector may be, for example, an EB virus vector, or a plasmid vector containing an EB virus-derived OriP (origin of replication) and EBV nuclear antigen 1 (EBNA1, Epstein-Barr Virus (EBV) Nuclear Antigen 1). An example of such an episome-based vector is the pEBMulti series (Fujifilm Wako Pure Chemical Corporation).

The nucleic acid into which the region encoding the chromoprotein is incorporated into the genomic DNA of the transfected cells may be, for example, an adenovirus vector, an adeno-associated virus vector, or a reverse transcription virus vector. Among these, adenovirus vectors and adeno-associated virus vectors are preferred, as they make it easier to maintain the expression of the chromoprotein during the culture process.

The mammalian cells may be, for example, a cultured cell line or primary cultured cells. The mammalian cells may be derived from any mammalian animal, such as a human, mouse, rat, hamster, dog, or monkey, and in one embodiment, may be human. If the mammalian cells are derived from humans (i.e., the mammalian cells are human cells), the influence of species differences in drug efficacy can be eliminated, for example, when the cells produced in this embodiment are used to evaluate or screen drugs for humans. Furthermore, if the mammalian cells are derived from humans, the cells produced in this embodiment can be used, for example, in basic research to elucidate the functions of human proteins. The mammalian cells in this embodiment are living cells.

The manufacturing method of the first embodiment includes culturing mammalian cells into which nucleic acid that expresses a chromoprotein has been introduced at a temperature below 37°C (culturing step).

The culture temperature of the mammalian cells in the culture step is less than 37°C and is a temperature at which the live mammalian cells express the pigment protein. The culture temperature of the mammalian cells in the culture step may be, for example, 10°C or higher, 20°C or higher, 23°C or higher, 25°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, or 30°C or higher, or may be less than 37°C, 36°C or lower, 35°C or lower, 34°C or lower, 33°C or lower, 32.5°C or lower, 32°C or lower, 31°C or lower, or 30°C or lower, and these upper and lower limits can be freely combined. The culture temperature of mammalian cells in the culture step may be, for example, 10°C or higher and 30°C or lower, 10°C or higher and 31°C or lower, 10°C or higher and 32°C or lower, 10°C or higher and 32.5°C or lower, 10°C or higher and 33°C or lower, 10°C or higher and 34°C or lower, 10°C or higher and 35°C or lower, 10°C or higher and 36°C or lower, 10°C or higher and lower than 37°C, 20°C or higher and 30°C or lower, 20°C or higher and 31°C or lower, 20°C or higher and 32°C or lower, 20°C or higher and 32.5°C or lower, 20°C or higher and 33°C or lower, 20°C or higher and 34°C or lower, 20°C or higher and 35°C or lower, 20°C or higher and 36°C or lower, 20°C or higher and 37°C or lower. or lower than 37°C, 23°C or higher and lower than 30°C, 23°C or higher and lower than 31°C, 23°C or higher and lower than 32°C, 23°C or higher and lower than 32.5°C, 23°C or higher and lower than 33°C, 23°C or higher and lower than 34°C, 23°C or higher and lower than 35°C, 23°C or higher and lower than 36°C, 23°C or higher and lower than 37°C, 25°C or higher and lower than 30°C, 25°C or higher and lower than 31°C, 25°C or higher and lower than 32°C, 25°C or higher and lower than 32.5°C, 25°C or higher and lower than 33°C, 25°C or higher and lower than 34°C, 25°C or higher and lower than 35°C, 25°C or higher and lower than 36°C, 25°C or higher and lower than 37°C, 27°C or higher and lower than 30°C Below, 27°C or higher and 31°C or lower, 27°C or higher and 32°C or lower, 27°C or higher and 32.5°C or lower, 27°C or higher and 33°C or lower, 27°C or higher and 34°C or lower, 27°C or higher and 35°C or lower, 27°C or higher and 36°C or lower, 27°C or higher and less than 37°C, 28°C or higher and 30°C or lower, 28°C or higher and 31°C or lower, 28°C or higher and 32°C or lower, 28°C or higher and 32.5°C or lower, 28°C or higher and 33°C or lower, 28°C or higher and 34°C or lower, 28°C or higher and 35°C or lower, 28°C or higher and 36°C or lower, 28°C or higher and less than 37°C, 29°C or higher and 30°C or lower, 29°C or higher and 31°C or lower, 29°C or higher and 31°C or lower, The culture temperature may be 29°C or higher and 32°C or lower, 29°C or higher and 32.5°C or lower, 29°C or higher and 33°C or lower, 29°C or higher and 34°C or lower, 29°C or higher and 35°C or lower, 29°C or higher and 36°C or lower, 29°C or higher and lower than 37°C, 30°C or higher and 31°C or lower, 30°C or higher and 32°C or lower, 30°C or higher and 32.5°C or lower, 30°C or higher and 33°C or lower, 30°C or higher and 34°C or lower, 30°C or higher and 35°C or lower, 30°C or higher and 36°C or lower, or 30°C or higher and lower than 37°C, and examples thereof include 30°C, 31°C, 32°C, 32.5°C, 33°C, 34°C, 35°C, or 36°C. When the culture temperature of the mammalian cells in the culture step is lower than these general culture temperatures, the chromoprotein can be expressed in the mammalian cells with high efficiency.

The culture time for the mammalian cells in the culture step may be any time that allows the mammalian cells to express the pigment protein when cultured at the above-mentioned culture temperature. The culture time for the mammalian cells in the culture step may be, for example, 3 days or more, 5 days or more, 7 days or more, 10 days or more, 14 days or more, 18 days or more, 22 days or more, 26 days or more, or 30 days or more, or 180 days or less, 90 days or less, 60 days or less, 50 days or less, 45 days or less, 40 days or less, 37 days or less, 35 days or less, 34 days or less, 33 days or less, 32 days or less, 31 days or less, 30 days or less, 20 days or less, 15 days or less, or 10 days or less, and these upper and lower limits can be freely combined. The culture time for mammalian cells in the culture step may be, for example, 3 to 180 days, 5 to 180 days, 3 to 30 days, 3 to 20 days, 3 to 15 days, 3 to 10 days, 5 to 30 days, 5 to 20 days, 5 to 15 days, 5 to 10 days, 10 to 90 days, 14 to 60 days, 18 to 50 days, 22 to 40 days, or 26 to 35 days, and may be, for example, 6, 7, 14, or 30 days.

In the culturing step, culture conditions other than the culture temperature and culture time can be those typically used by those skilled in the art, as long as they do not inhibit the expression of chromoproteins in mammalian cells. For example, the medium used in the culturing step can be a medium commonly used for culturing mammalian cells, such as a general-purpose basal medium such as DMEM medium, RPMI medium, Ham's F-12 medium, or Ham's F-12K medium supplemented with a serum component such as fetal bovine serum (FBS), or a medium further supplemented with additives commonly used in cell culture (e.g., antibiotics such as penicillin/streptomycin). The _CO2 concentration in the ambient environment during the culturing step is, for example, 5%.

In one aspect, the manufacturing method of the first embodiment may include introducing a nucleic acid that expresses a chromoprotein into mammalian cells (introduction step) before the culture step. The introduction method in the introduction step is not particularly limited as long as it is a method that can introduce a nucleic acid that expresses a chromoprotein into living mammalian cells, and any method that a person skilled in the art would normally use when introducing such a nucleic acid can be used. Examples of such methods include contacting the nucleic acid with the cell, lipofection, electroporation, microinjection, and sonoporation, which can be selected appropriately by a person skilled in the art depending on the type of nucleic acid. For example, the method described above in the description of the nucleic acid that expresses the chromoprotein of one aspect may be selected. Furthermore, the amount of nucleic acid to be introduced into the mammalian cells can be set appropriately by a person skilled in the art depending on the type of nucleic acid, as an amount that can cause the mammalian cells to express the chromoprotein in the subsequent culture step.

If the introduction of nucleic acid in the introduction step occurs through a time-dependent process such as contact between a viral vector and cells or lipofection, the introduction step may further include culturing mammalian cells in an environment in which introduction of nucleic acid occurs. An environment in which introduction of nucleic acid occurs refers to an environment in which mammalian cells are exposed to a medium containing a viral vector, or a medium containing nucleic acid and reagents necessary for lipofection to occur, and the exposure may be, for example, replacing the culture supernatant of adherent mammalian cells with such a medium or suspending the mammalian cells in such a medium. The culture time in the introduction step may be any time that allows the introduction of nucleic acid into the mammalian cells, and may be, for example, 1 minute or more, 10 minutes or more, 1 hour or more, 6 hours or more, or 24 hours or more, or 96 hours or less, 72 hours or less, 48 hours or less, or 24 hours or less. The culture temperature in the introduction step may be any temperature that allows introduction of nucleic acid into living mammalian cells, and may be a temperature typically used in cell culture, for example, 20°C or higher, 25°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, 36°C or higher, or 37°C or higher, or 50°C or lower, 45°C or lower, 40°C or lower, 39°C or lower, 38°C or lower, or 37°C or lower, and an example is 37°C.

If a selection marker is incorporated into the nucleic acid introduced in the introduction step, the introduction step may include selecting mammalian cells into which the nucleic acid has been introduced after the nucleic acid has been introduced. Selection of mammalian cells into which the nucleic acid has been introduced can be performed by contacting the cells with an antibiotic, and cells that survive after contact with the antibiotic can be selected as mammalian cells into which the nucleic acid has been introduced. The selection marker contains a resistance gene to a specific antibiotic. Therefore, when mammalian cells are introduced with a nucleic acid into which the selection marker has been incorporated and then contacted with an antibiotic, mammalian cells into which the nucleic acid has been introduced will survive because they have acquired resistance to the antibiotic, while mammalian cells into which the nucleic acid has not been introduced will die because they do not have resistance to the antibiotic. The combination of such selection marker and antibiotic, and the conditions for contacting the antibiotic (temperature and concentration), can follow conditions commonly used by those skilled in the art for selection. Examples of antibiotics used for such selection include neomycin, puromycin, genomycin B1 (G418), and hygromycin B.

The manufacturing method of the first embodiment allows the production of mammalian cells labeled with a chromoprotein. The fact that the mammalian cells have been labeled with the chromoprotein may be evaluated, for example, by using the visible light absorption of the mammalian cells as an indicator, according to a method similar to the evaluation step in the evaluation method of the second embodiment described below. The fact that the mammalian cells have been labeled with the chromoprotein may also be evaluated, for example, by lysing the mammalian cells and then measuring the amount of chromoprotein contained in the lysate using ELISA or Western blotting.

Mammalian cells labeled with chromoproteins produced by the manufacturing method of the first embodiment can be used to evaluate cells, for example, using coloration based on the visible light absorption of the chromoprotein as an indicator. This makes it possible to evaluate, for example, the condition dependency of chromoprotein expression (e.g., environmental dependency or dependency on the concentration of a specific substance).

Mammalian cells labeled with a chromoprotein produced by the manufacturing method of the first embodiment can be used to evaluate cells using the fluorescence intensity of the donor protein as an indicator, for example, as cells co-expressing the chromoprotein as a fluorescence resonance energy transfer (FRET) acceptor and another protein that acts as a donor, or as cells expressing a fusion protein of the chromoprotein as a FRET acceptor and a donor protein. This allows for evaluation of the intracellular environment or events detected by fluorescent probes that operate on FRET, such as protein-protein interactions, ion concentrations, membrane potential, or pH changes.

Chromoprotein-labeled mammalian cells produced by the manufacturing method of the first embodiment can be used, for example, to evaluate the distribution of chromoproteins in cells based on changes in refractive index due to the light absorption of the chromoproteins. Patent Document 1 discloses a cell evaluation method comprising: a labeling step of labeling specific regions of cells with a labeling substance having different refractive indices at a first wavelength and a second wavelength; a refractive index distribution acquisition step of acquiring, at the first wavelength and the second wavelength, the refractive index distribution of the cells whose specific regions have been labeled in the labeling step; and an analysis step of evaluating the distribution of the specific regions in the cells by comparing the refractive index distributions at the first wavelength and the second wavelength. Chromoproteins are cited as an example of the labeling substance. This method evaluates cells by utilizing the difference in refractive index between the two wavelengths of the labeling substance. On the other hand, pigments such as chromoproteins are known to exhibit significant changes in refractive index near their maximum absorption wavelengths. Therefore, chromoprotein-labeled mammalian cells produced by the manufacturing method of the first embodiment are considered suitable for cell evaluation methods that use refractive index as an indicator, such as the method described in Patent Document 1.

A second embodiment of the present disclosure is a method for evaluating mammalian cell labeling with a chromoprotein, which includes culturing mammalian cells into which nucleic acid that expresses a chromoprotein has been introduced at a temperature below 37°C (culturing step), and evaluating the visible light absorption of the mammalian cells (evaluation step). Hereinafter, this embodiment is also referred to as the "evaluation method of the second embodiment."

The chromoprotein, nucleic acid that expresses it, and mammalian cells used in the evaluation method of the second embodiment can be the same as those described in the manufacturing method of the first embodiment. Furthermore, the culture step used in the evaluation method of the second embodiment can be carried out in the same manner as described in the manufacturing method of the first embodiment. Furthermore, the evaluation method of the second embodiment may include an introduction step prior to the culture step, and this introduction step can be carried out in the same manner as the introduction step described in the manufacturing method of the first embodiment.

In the evaluation step, the visible light absorption of the mammalian cells cultured in the culture step is evaluated. This makes it possible to evaluate the presence or absence of pigment protein expression in the mammalian cells and the amount of expression.

The visible light absorption of mammalian cells may be evaluated using, for example, an optical instrument capable of measuring absorbance. Examples of optical instruments capable of measuring absorbance include an absorbance meter and a microwell plate reader. Such optical instruments can evaluate the visible light absorption of mammalian cells, for example, by measuring the visible light absorption of a cell suspension in which mammalian cells are suspended, or of mammalian cells in an adherent state. In this case, the visible light absorption may be evaluated using absorbance as an indicator, for example, by evaluating the absorbance at a wavelength within ±80 nm, ±60 nm, ±50 nm, ±40 nm, ±30 nm, ±20 nm, or ±10 nm of the maximum absorption wavelength or maximum absorption wavelength in the visible light region of the pigment protein expressed in the mammalian cells, or by evaluating the absorbance at the maximum absorption wavelength or maximum absorption wavelength in the visible light region of the pigment protein.

The visible light absorption of mammalian cells may be evaluated using the color of the mammalian cells as an indicator. In this case, the color of the mammalian cells is difficult to observe when the spatial density is low, such as when the cells are adhered in a monolayer on a culture dish, but is easily observed when the spatial density is high, such as when the cells are in a pellet or colony state. The visible light absorption of cells in such a high spatial density state can be evaluated, for example, visually, or by measuring the color tone of a digital photograph of the cells using image analysis software (e.g., ImageJ). That is, in the evaluation method according to one aspect of the second embodiment, the visible light absorption of the mammalian cells may be evaluated using the color of the mammalian cells as an indicator. Furthermore, the evaluation method according to one aspect of the second embodiment may further include concentrating the mammalian cells cultured at less than 37°C (concentration step) before the evaluation step, and the visible light absorption of the mammalian cells may be evaluated using the color of the concentrated mammalian cells as an indicator.

In the concentration step, mammalian cells are concentrated. That is, in the concentration step, the density per unit volume of mammalian cells (cell concentration) is increased. The method for concentrating cells in the concentration step may be any method commonly used by those skilled in the art for concentrating cells, such as centrifugation, filtering, or leaving the cell suspension to stand. When the cell suspension is centrifuged, the concentrated cells are obtained as a precipitate (pellet). When the cell suspension is filtered, the concentrated cells are obtained on the filter. When the cell suspension is left to stand, the concentrated cells are obtained as a precipitate caused by gravity.

In the concentration step, the mammalian cells are concentrated to a density that allows color evaluation in the subsequent evaluation step. In the concentration step, the mammalian cells may be concentrated, for example, so that the volume of the concentrated product is occupied by mammalian cells is 10% or more, 30% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more. In the concentration step, the mammalian cells may be concentrated so that the concentrated product contains multiple layers of mammalian cells or a thickness equivalent thereto, for example, 5 or more, 10 or more, 15 or more, 20 or more, 30 or more, 50 or more, or 100 or more layers of mammalian cells or a thickness equivalent thereto. In the concentration step, the mammalian cells may be concentrated so that the maximum thickness of the concentrated product is 0.5 mm or more, 1 mm or more, 1.5 mm or more, 2 mm or more, 3 mm or more, or 5 mm or more. Furthermore, in the concentration step, for example, 1.0 × ¹⁰ or more, 2.0 × ¹⁰ or more, 5.0 × ¹⁰ or more, 1.0 × ¹⁰ or more, 1.5 × ¹⁰ or more, or 2.0 × ¹⁰ or more cells may be collected in a state separated from the liquid component, or may be collected as a precipitate containing such a number of cells or as a matter collected on a filter.

A third embodiment of the present disclosure is a method for screening for chromoproteins, which includes culturing mammalian cells into which nucleic acid that expresses a candidate protein has been introduced at a temperature below 37°C (culturing step), and evaluating the visible light absorption of the mammalian cells. Hereinafter, this embodiment is also referred to as the "screening method of the third embodiment."

In the screening method of the third embodiment, the nucleic acid that expresses the candidate protein can be the same as that described in the manufacturing method of the first embodiment, except that the candidate protein is expressed instead of the chromoprotein. In the screening method of the third embodiment, the mammalian cells that can be used are the same as those described in the manufacturing method of the first embodiment. Furthermore, the culture step in the screening method of the third embodiment can be carried out in the same manner as that described in the manufacturing method of the first embodiment, except that the nucleic acid that expresses the candidate protein is introduced into the mammalian cells. Furthermore, the screening method of the third embodiment may include an introduction step before the culture step, and this introduction step can be carried out in the same manner as the introduction step described in the manufacturing method of the first embodiment, except that the nucleic acid that expresses the candidate protein is introduced.

The evaluation step in mammalian cells in the screening method of the third embodiment can be carried out in the same manner as described for the evaluation step of the second embodiment. Furthermore, the screening method of the third embodiment may further include a concentration step prior to the evaluation step, which can be carried out in the same manner as described for the evaluation method of the second embodiment. When the screening method of the third embodiment includes a concentration step, the expression of chromoproteins in mammalian cells can be easily evaluated using coloration as an indicator without using optical equipment such as an absorbance meter, thereby enabling high-throughput screening of chromoproteins.

The candidate protein in the screening method of the third embodiment is a candidate chromoprotein. The candidate chromoprotein may be, for example, a protein obtained from an organism other than a mammal, a protein expressed from nucleic acid obtained from an organism other than a mammal, or a randomly modified chromoprotein. The randomly modified chromoprotein can be obtained by biosynthesis in bacteria such as Escherichia coli using, as a template, a nucleic acid in which a random base sequence has been introduced into a portion of the nucleic acid encoding the chromoprotein. Furthermore, when the candidate protein is a randomly modified chromoprotein, the screening method of the third embodiment may be carried out, for example, by introducing nucleic acid that expresses the randomly modified chromoprotein into cells (introduction step), allowing the cells to express the randomly modified protein in a culture step, separating the resulting heterogeneous cell population into individual cells by limiting dilution or the like, growing each cell to form colonies, and then evaluating visible light absorption using the color of the colonies as an indicator (evaluation step).

The screening method of the third embodiment may include a step of adjusting the number of cells expressing a candidate protein to be used in the evaluation of visible light absorption (cell number adjustment step) between the culturing step and the evaluation step, or between the culturing step and the concentration step. In the cell number adjustment step, for example, the number of cells used for evaluation may be adjusted to a fixed number. In this case, a chromoprotein that is likely to be colored in the cells can be screened in the subsequent evaluation step. Furthermore, in the cell number adjustment step, for example, the number of cells used for evaluation may be adjusted so that it is inversely proportional to the molar extinction coefficient in the visible light region of the candidate protein to be expressed. In this case, a chromoprotein that is likely to be expressed in the cells can be screened in the subsequent evaluation step. The cell number adjustment step may include, for example, counting the number of cells contained in a unit volume of cell suspension and separating an appropriate volume of the cell suspension so that the desired number of cells is used in the evaluation of visible light absorption. Counting the number of cells contained in a unit volume of cell suspension may be performed according to conventional methods, such as counting the number of viable cells using trypan blue staining.

In the screening method of the third embodiment, chromoproteins are selected from among candidate proteins based on the evaluation results obtained in the evaluation step. In other words, the screening method of one aspect of the third embodiment may include, after the evaluation step, selecting chromoproteins from among candidate proteins based on the evaluation results of visible light absorption in the evaluation step (selection step).

The selection method in the selection step may be any method capable of selecting a protein that satisfies the requirements for a chromoprotein described in the production method according to the first embodiment, and may be, for example, a method capable of selecting a protein that satisfies at least two, three, four, or five of the requirements described as aspects of a chromoprotein in the production method according to the first embodiment. In a preferred embodiment, the selection method in the selection step may be one that can select a protein that satisfies the requirements for absorbance and fluorescence quantum yield of a chromoprotein described as aspects of a chromoprotein in the production method according to the first embodiment, and may, for example, select as a chromoprotein a protein having a molar extinction coefficient of 5.0 × 10 ⁴ [L/mol·cm] or more and a fluorescence quantum yield of 1% or less.

Furthermore, when the screening method of the third embodiment includes a concentration step, the selection step may include selecting candidate proteins for which coloration is observed in the evaluation step. In this case, the selection step may include first selecting candidate proteins for which coloration is observed in the evaluation step from the candidate proteins (primary selection), and then further selecting from the selected candidate proteins proteins that meet the requirements for chromoproteins, as explained above as aspects of the chromoproteins involved in the production method of the first embodiment (secondary selection).

The screening method of the third embodiment allows for the selection of chromoproteins. This makes it possible to efficiently discover chromoproteins that have potential for applications such as those described in the manufacturing method of the first embodiment.

Furthermore, in one aspect of the screening method of the third embodiment, if the candidate protein is a randomly modified chromoprotein, it is possible to screen for proteins with improved parameters, such as optical properties, for the chromoprotein. By repeating multiple times a cycle consisting of forming a candidate protein population by introducing random modifications into the chromoprotein and selecting a chromoprotein with desired properties from the candidate protein population using the screening method of the third embodiment, it is possible to obtain a chromoprotein with desired properties through molecular evolution.

The present disclosure will be explained in more detail below using examples, but the present disclosure is not limited to the following examples.

In this example, cjBlue, rpulFkz1, gfasCP, asFP595, Rtms5, and aeCP597 were used as chromoproteins. The optical properties of these chromoproteins are described in, for example, Non-Patent Document 1.
CjBlue is a pigment protein derived from Epiactis japonica (a type of sea anemone), and its amino acid sequence is shown in SEQ ID NO:1.
rpulFkz1 is a pigment protein derived from Rhizostoma pulmo (a type of jellyfish), and its amino acid sequence is shown in SEQ ID NO:2.
gfasCP is a pigment protein derived from Galaxea fascicularis (a type of coral), and its amino acid sequence is shown in SEQ ID NO:3.
asFP595 is a pigment protein derived from Anemonia sulcata (a type of sea anemone), and its amino acid sequence is shown in SEQ ID NO:4.
Rtms5 is a pigment protein derived from Montipora efflorescens (a type of coral), and its amino acid sequence is shown in SEQ ID NO:5.
aeCP597 is a chromoprotein derived from Actinia equina (a type of sea anemone), and its amino acid sequence is shown in SEQ ID NO:6.

Example 1: Evaluation of chromoprotein expression when cultured at 30°C
<Step 1: Construction of a chromoprotein expression plasmid>
The DNA encoding the chromoproteins was synthesized by Eurofins Genomics, with the codons in the coding region of the wild-type DNA encoding each chromoprotein optimized for expression in human cells and two stop codons added to the 3' end. The nucleotide sequence of the DNA encoding cjBlue is shown in SEQ ID NO: 7. The nucleotide sequence of the DNA encoding rpulFkz1 is shown in SEQ ID NO: 8. The nucleotide sequence of the DNA encoding gfasCP is shown in SEQ ID NO: 9. The nucleotide sequence of the DNA encoding asFP595 is shown in SEQ ID NO: 10. The nucleotide sequence of the DNA encoding Rtms5 is shown in SEQ ID NO: 11. The nucleotide sequence of the DNA encoding aeCP597 is shown in SEQ ID NO: 12.

Using the restriction enzymes BamHI and NotI, the DNA encoding the obtained chromoproteins was subcloned into the multicloning site of the pEBMulti-Neo vector (Fujifilm Wako Pure Chemical Industries, Ltd., 057-08131) to obtain chromoprotein expression plasmids (nucleic acids that express the chromoproteins) for each chromoprotein. The pEBMulti series contains the Epstein-Barr virus-derived OriP (origin of replication) and EBV nuclear antigen 1 (EBNA1, Epstein-Barr Virus (EBV) Nuclear Antigen 1). Therefore, when the pEBMulti-Neo vector is introduced into mammalian cells, the vector is replicated and distributed to daughter cells via an episomal replication system. Therefore, by using the pEBMulti series, it is possible to establish a cell line that constitutively expresses the protein encoded by a target gene, without having to integrate the target gene into the host cell genome.

<Step 2: Introduction of the chromoprotein expression plasmid into mammalian cells and culturing the introduced cells>
CHO-K1 cells, a Chinese hamster-derived cell line, were seeded into six-well flat-bottom cell culture plates (VIOLAMO). The seeded cells were cultured for two days at 37°C and 5% CO2 in Ham's F- _12K medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10% (v/v) fetal bovine serum (also referred to as "culture medium" in Example 1). 2.5 μg of a chromoprotein expression plasmid and a transfection reagent were added to each well using ScreenFect® A plus (Fujifilm Wako Pure Chemical Industries, Ltd.) according to the protocol provided by the supplier, and the cells in each well were transfected with the chromoprotein expression plasmid. The transfected cells were cultured for one day at 37°C and 5% _CO2 . After culturing, the antibiotic G418 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added at 1 mg/ml to each well containing the cells, and cells expressing proteins from the transfected nucleic acid were selected. The selected cells were cultured at 30°C and 5% _CO2 for 35 days (culture step). During this culture, the medium was changed at appropriate times, and no subculture was performed.

<Step 3: Evaluation of labeled cells (before cell enrichment)>
After culturing in step 2, the culture medium was removed from each well, and the cells were washed with PBS(-). Figure 1 shows the results of observing the cells in PBS(-) after the washing procedure using a phase-contrast microscope (DM IL LED inverted microscope, Leica, 10x magnification). It was confirmed that the cells into which each chromoprotein had been introduced adhered to the dish and survived without any problems even after culturing for 35 days under conditions of 30°C and 5% _CO2 .

Figure 2 shows a digital photograph taken under room light of cells in PBS(-) after washing. The results in Figures 1 and 2 show that while cells were indeed present on the dish, when the cells were adhered flat, it was difficult to visually detect the labeling of the cells with the chromoprotein.

<Step 4: Cell Concentration>
After step 3, the cells were treated with 0.25% trypsin (Gibco) to detach them from the dish. The resulting cell suspension was collected in a 1.5 mL tube. The tube was centrifuged at 3,000 rpm at room temperature for 1 to 3 minutes using a centrifuge (Suprema 21, Tomy Seiko Co., Ltd.). The supernatant was removed, and the pellet at the bottom of the tube was washed with PBS(-) to obtain concentrated cells.

Step 5: Evaluation of labeled cells (after cell enrichment)
Figure 3 shows digital photographs taken under room light of the concentrated cells obtained in step 4. As shown in Figure 3, of the six chromoproteins tested, five, namely cjBlue, gfasCP, asFP595, Rtms5, and aeCP597, visually observed cell coloration, of which four, gfasCP, asFP595, Rtms5, and aeCP597, showed strong coloration, and two, gfasCP and Rtms5, showed even stronger coloration.

These results demonstrate that chromoproteins can be expressed in mammalian cells by culturing them at 30°C after introducing a chromoprotein expression plasmid. Furthermore, these results suggest that by assessing the visible light absorption of the cells after culturing, it is possible to evaluate mammalian cell labeling with chromoproteins and screen for chromoproteins, and that in particular, for screening of chromoproteins, simple, high-throughput screening can be performed by concentrating the cells and using visual coloration as an indicator.

Comparative Example 1: Evaluation of chromoprotein expression when cultured at 37°C
The same procedure was carried out as in Example 1, except that the culture step in step 2 was carried out under the following conditions. As a result, no coloration was visually observed in the evaluation of step 5, regardless of which chromoprotein was used.

<Step 2 in Comparative Example 1>
HepG2 cells, a human cell line, were seeded into six-well flat-bottom cell culture plates (VIOLAMO). The seeded cells were cultured for two or three days at 37°C and 5% CO2 in a medium (also referred to as "culture medium" in Comparative Example 1) containing 10% (v/ _v ) fetal bovine serum in DMEM medium (Gibco). 2.5 μg of a chromoprotein expression plasmid and a transfection reagent were added to each well using ScreenFect® A plus (Fujifilm Wako Pure Chemical Industries, Ltd.) according to the supplier's protocol. The transfected cells were cultured for one day at 37°C and 5% _CO2 . The antibiotic G418 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added at 1 mg/ml to each well containing the cultured cells, and cells expressing proteins from the transfected nucleic acids were selected. The selected cells were cultured at 37°C and 5% _CO2 for 20 days (rpulFkz1, gfasCP, asFP595, Rtms5, aeCP597) or 38 days (cjBlue) (culture step). During this culture, the medium was changed at appropriate times and the cells were subcultured once.

Example 2: Evaluation of chromoprotein expression when cultured at various temperatures
The cells were cultured under four conditions of 37°C, 35°C, 32.5°C and 30°C, and the color development of the cells was evaluated in the same manner as in Example 1 and compared.

<Step 1: Construction of a chromoprotein expression plasmid>
A plasmid prepared in the same manner as in step 1 of Example 1 was used.

<Step 2: Introduction of the chromoprotein expression plasmid into mammalian cells and culturing the introduced cells>
CHO-K1 cells, a Chinese hamster-derived cell line, were seeded into 6-well flat-bottom cell culture plates (VIOLAMO). The seeded cells were cultured for 2 days at 37°C and 5% CO2 in Ham's F- _12K medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10% (v/v) fetal bovine serum (also referred to as "culture medium" in Example 2). 2.5 μg of a chromoprotein expression plasmid and a transfection reagent were added to each well using ScreenFect® A plus (Fujifilm Wako Pure Chemical Industries, Ltd.) according to the protocol provided by the supplier, and the cells in each well were transfected with the chromoprotein expression plasmid. The transfected cells were cultured for 1 day at 37°C and 5% _CO2 . To select cells that expressed protein from the transfected nucleic acid, 1 mg/ml of the antibiotic G418 (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each well containing the cultured cells. After adding G418, the cells were cultured at 37°C and 5% _CO2 for 6 days, 35°C and 5% _CO2 for 6 days, 32.5°C and 5% _CO2 for 6 days, or 30°C and 5% _CO2 for 7 days (culture step). During this culture, the medium was changed at appropriate times and no subculture was performed.

<Step 3: Evaluation of labeled cells (before cell enrichment)>
After culturing in step 2, the culture medium was removed from each well, and the cells were washed with PBS(-). After the culturing, the cells in PBS(-) were observed under a phase-contrast microscope (DMi1 inverted microscope, Leica, 10x magnification). Figure 4 shows the results of observing cells in PBS(-) under a phase-contrast _microscope after washing for cells cultured for 6 days at 37°C and 5% CO _2. Figure 5 shows the results of observing cells in PBS(-) under a phase-contrast microscope after washing for cells cultured for 6 days at 35°C and 5% CO 2. Figure 6 shows the results of observing cells in PBS(-) under a phase-contrast microscope after washing for cells cultured for 6 days at 32.5°C and 5% CO _2. Figure 7 shows the results of observing cells in PBS(-) under a phase-contrast microscope after washing for cells cultured for 7 days at 30°C and 5% CO ₂ . The length of the scale bars in Figures 4 to 7 is 50 μm. The cells into which each chromoprotein was introduced adhered to the dish and were confirmed to survive after culturing for 6 days at 37°C and 5% _CO2 , 6 days at 35°C and 5% _CO2 , 6 days at 32.5°C and 5% _CO2 , and 7 days at 30°C and 5% _CO2 .

Figure 8 is a digital photograph taken under room light of cells cultured for 6 days at 37°C and 5% _CO2 in PBS(-) after subsequent washing. Figure 9 is a digital photograph taken under room light of cells cultured for 6 days at 35°C and 5% _CO2 in PBS(-) after subsequent washing. Figure 10 is a digital photograph taken under room light of cells cultured for 6 days at _32.5 °C and 5% CO2 in PBS(-) after subsequent washing. Figure 11 is a digital photograph taken under room light of cells cultured for 7 days at 30°C and 5% _CO2 in PBS(-) after subsequent washing. The results in Figures 4 to 11 show that, while cells are indeed present on the dish, it is difficult to clearly detect and compare the labeling of the cells with the chromoprotein when the cells are adhered flat.

<Step 4: Counting the number of cells and concentrating the cells>
After step 3, the cells were treated with 0.25% trypsin (Gibco) and detached from the dish. The resulting cell suspension was collected in a 1.5 mL tube. The number of viable cells was counted using trypan blue staining, and the cell suspension was aliquoted into separate 1.5 mL tubes so that the number of viable cells per tube was 2.5 x ¹⁰ cells. The tubes were centrifuged at 3,000 rpm for 3 minutes at room temperature using a centrifuge (Suprema 21, Tomy Seiko Co., Ltd.). The supernatant was removed, and the pellet at the bottom of the tube was washed with PBS(-) to obtain concentrated cells.

Step 5: Evaluation of labeled cells (after cell enrichment)
Figure 12 shows a digital photograph of the concentrated cells obtained in step 4 taken under room light. Figure 13 shows the image shown in Figure 12 with brightness and contrast adjusted for comparison between conditions. Figure 14 shows an image obtained by dividing the RGB image shown in Figure 12 into independent channels of R (Red), G (Green), and B (Blue). Figure 15 shows an image obtained by dividing the RGB image shown in Figure 12 into HSB color space, H (Hue), S (Saturation), and B (Brightness). Figure 16 shows the results of aeCP597 (37°C, 32.5°C, and 30°C) shown in Figure 15, plotted on polar coordinates, with H (Hue) representing the angle of deviation and S (Saturation) representing the distance from the pole. Figure 17 shows digital photographs taken under room light of the cell suspension obtained in step 4, in which the concentrated cells were suspended in 500 μL of PBS(-). Figure 18 shows images in which the brightness and contrast of the images shown in Figure 17 have been adjusted for comparison between the various conditions.

The results in Figures 12 to 14 show that of the six chromoproteins tested, five - cjBlue, gfasCP, asFP595, Rtms5, and aeCP597 - showed stronger coloration as the culture temperature approached a lower temperature of 30°C. In particular, for cjBlue, gfasCP, Rtms5, and aeCP597, a visual difference was observed between the color intensity at a culture temperature of 37°C and that at a culture temperature of 30°C or 32.5°C. Furthermore, particularly for cjBlue and aeCP597, almost no visual coloration was observed at a culture temperature of 37°C, whereas clear coloration was observed at a culture temperature of 30°C or 32.5°C. These findings are also evident from the results shown in Figures 15 and 16, and it was semi-quantitatively confirmed that aeCP597 in particular exhibited greater saturation and stronger color development when the culture temperature was 30°C or 32.5°C compared to when the culture temperature was 37°C.

Furthermore, Figures 17 and 18 show that in unconcentrated cell suspensions, it was difficult to compare the color intensity between culture conditions or between pigment proteins. This suggests that concentrating cells enables simple, high-throughput screening using visual coloration as an indicator.

Claims (8)

A method for producing mammalian cells labeled with a chromoprotein, comprising culturing mammalian cells into which nucleic acid that expresses the chromoprotein has been introduced at a temperature below 37°C. Culturing mammalian cells transfected with a nucleic acid that expresses a chromoprotein at a temperature below 37°C; and evaluating the visible light absorption of the mammalian cells.
A method for evaluating mammalian cell labeling with a chromoprotein, comprising: Cultivating mammalian cells transfected with nucleic acids that express a candidate protein at a temperature below 37°C; and evaluating the visible light absorption of the mammalian cells.
A method for screening a pigment protein, comprising: further comprising concentrating the mammalian cells cultured at less than 37°C prior to assessing visible light absorption;
The method according to claim 3 , wherein the evaluation of visible light absorption by the mammalian cells is carried out using coloration of the concentrated mammalian cells as an indicator. 5. The method according to claim 1, wherein the molar extinction coefficient of the chromoprotein at its maximum absorption wavelength in the 400 to 800 nm region is 5.0×10 ⁴ [L/mol·cm] or more. The method according to any one of claims 1 to 4, wherein the chromoprotein is a chromoprotein derived from an organism other than a mammal, or a modified protein thereof. The method according to any one of claims 1 to 4, wherein the nucleic acid that expresses the chromoprotein is a nucleic acid that is distributed to daughter cells of the cell into which it has been introduced, or a nucleic acid in which a region encoding the chromoprotein is incorporated into the genomic DNA of the cell into which it has been introduced. The method according to any one of claims 1 to 4, wherein the culturing is carried out at 33°C or lower.