Method for producing mammalian cells labeled by chromoprotein, and method for screening chromoprotein

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

[0001] The present disclosure relates to methods for producing mammalian cells labeled with chromoproteins and methods for screening for chromoproteins.

[0002] Chromoproteins are proteins that have a high absorbance in the visible light region (Non-Patent Document 1). Chromoproteins are expected to be used as markers that display coloration when expressed in cells, or as acceptors for fluorescence resonance energy transfer (FRET), taking advantage of their large molar extinction coefficients (Non-Patent Document 2).

[0003] International Publication No. 2023 / 182169

[0004] 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).

[0005] The present inventors attempted to express chromoproteins in mammalian cells by genetic techniques, but discovered a new problem: chromoproteins are difficult to express in mammalian cells under temperature conditions generally used for culturing mammalian cells.

[0006] The present disclosure aims to provide a method for producing mammalian cells labeled with a chromoprotein, and a method for screening for a chromoprotein, which comprises expressing a candidate protein for the chromoprotein in a mammalian cell.

[0007] The present inventors have found that when mammalian cells are cultured at a temperature lower than the general culture temperature, it is possible to efficiently express a pigment protein in the mammalian cells.

[0008] The present disclosure relates to, for example, the following: [1] A method for producing mammalian cells labeled with a chromoprotein, comprising culturing mammalian cells into which a nucleic acid expressing a chromoprotein has been introduced at a temperature below 37°C. [2] A method for evaluating mammalian cell labeling with a chromoprotein, comprising culturing mammalian cells into which a nucleic acid expressing a chromoprotein has been introduced at a temperature below 37°C, and evaluating the visible light absorption of the mammalian cells. [3] A method for screening a chromoprotein, comprising culturing mammalian cells into which a nucleic acid expressing a candidate protein has been introduced at a temperature below 37°C, and evaluating the visible light absorption of the mammalian cells. [4] The method according to [3], further comprising concentrating the mammalian cells cultured at a temperature below 37°C before evaluating the visible light absorption, wherein the evaluation of the visible light absorption of the mammalian cells is carried out using the coloration of the concentrated mammalian cells as an index. [5] The chromoprotein has a molar extinction coefficient of 5.0 x 10 at a maximum absorption wavelength in the 400 to 800 nm region. 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 performed at 33°C or less.

[0009] According to one embodiment of the present disclosure, there is provided a method for producing mammalian cells labeled with a chromoprotein, which allows the chromoprotein to be introduced into cells simply, selectively, and efficiently using genetic techniques.

[0010] According to one embodiment of the present disclosure, there is provided a method for evaluating mammalian cell labeling with a chromoprotein, which allows for simple evaluation of mammalian cell labeling with a chromoprotein using visible light absorption by the mammalian cell as an indicator.

[0011] According to one embodiment of the present disclosure, a method for screening chromoproteins is provided. 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 color change of the concentrated mammalian cells as an indicator.

[0012] This is a diagram showing the results of observing cells in PBS(-) with a phase contrast microscope after washing in step 3 of Example 1. This is a digital photograph of cells in PBS(-) after washing in step 3 of Example 1, taken under room light. This is a digital photograph of the concentrated cells obtained in step 4 in step 5 of Example 1, taken under room light. This is a digital photograph of the concentrated cells obtained in step 4 in step 3 of Example 2, taken under room light at 37°C and 5% CO ２ 1 shows the results of observing cells in PBS(-) under a phase contrast microscope after washing for 6 days under the conditions of 35°C and 5% CO ２ This figure shows the results of observing cells in PBS(-) under a phase contrast microscope after washing for 6 days under the conditions of 32.5°C and 5% CO ２ This figure shows the results of observing cells in PBS(-) under a phase contrast microscope after washing, which were cultured for 6 days under the conditions of 30°C and 5% CO ２ 1 shows the results of observing cells in PBS(-) under a phase contrast microscope after washing for 7 days under the conditions of 37°C and 5% CO ２ 1 shows a digital photograph taken under room light of cells in PBS(-) after culturing for 6 days under the conditions of 35°C and 5% CO in step 3 of Example 2 and subsequent washing. ２1 shows a digital photograph taken under room light of cells in PBS(-) after culturing for 6 days under the conditions of 32.5°C and 5% CO in step 3 of Example 2 and subsequent washing. ２ 1 shows a digital photograph taken under room light of cells in PBS(-) after culturing for 6 days under the conditions of 30°C and 5% CO in step 3 of Example 2 and subsequent washing. ２ 12 is a digital photograph taken under room light of cells in PBS(-) cultured for 7 days under the conditions of (1) and (2) and subsequent washing. 13 is a digital photograph taken under room light of the concentrated cells obtained in step 4 in step 5 of Example 2. 14 is an image of the image shown in FIG. 12 with brightness and contrast adjusted for comparison among conditions. 15 is an image obtained by dividing the RGB image shown in FIG. 12 into independent channels of R (Red), G (Green), and B (Blue). 16 is an image obtained by dividing the RGB image shown in FIG. 12 into HSB color space, H (Hue), S (Saturation), and B (Brightness). 17 is a plot of the results of aeCP597 (37°C, 32.5°C, 30°C) shown in FIG. 15 on polar coordinates, with H (Hue) representing the declination and S (Saturation) representing the distance from the pole. This is a digital photograph taken under room light of a cell suspension prepared by suspending the concentrated cells obtained in step 4 in 500 μL of PBS(−) in step 5 of Example 2. For comparison of each condition, this is an image in which the brightness and contrast of the image shown in FIG.

[0013] Hereinafter, embodiments for carrying out the present disclosure will be described, but the present disclosure is not limited to the following embodiments.

[0014] In the present disclosure, when a protein or nucleic acid comprises 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, the 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, 95% or more sequence identity, and in a most preferred embodiment, 100% sequence identity.

[0015] In the present disclosure, when a sequence contained in a certain protein or nucleic acid has a mutation (i.e., the sequence identity is not 100%) with respect to a predetermined amino acid sequence or nucleotide sequence, the mutation may be a mutation selected from substitution, deletion, insertion, and addition at 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 at 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 at 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 at each of 1 residue or 1 to 3 bases.

[0016] A first embodiment of the present disclosure is a method for producing mammalian cells labeled with a chromoprotein, the method comprising culturing mammalian cells, into which a nucleic acid that expresses the chromoprotein has been introduced, at a temperature below 37° C. (culturing step). Hereinafter, this embodiment is also referred to as the "production method of the first embodiment."

[0017] In the present 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.

[0018] 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. In general, 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 properly when cultured under conditions below 37°C.

[0019] 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 as described above. That is, for example, the chromoprotein may be a chromoprotein derived from an organism as 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 a 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 having a sequence identity of 90% or more 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.

[0020] The chromoprotein of one embodiment may have a molar extinction coefficient at the 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 the maximum absorption wavelength in the visible light region that is 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), for example, the molar extinction coefficient at the maximum absorption wavelength in the 400 to 800 nm region may be equal to or greater than a predetermined lower limit. In these cases, the predetermined lower limit is, for example, 3.0 x 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 x 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.

[0021] The maximum absorption wavelength of the chromoprotein in the 400 to 800 nm region of one embodiment 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 of the chromoprotein in the 400 to 800 nm region of one embodiment 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.

[0022] 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 the detection of a signal 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.

[0023] In one embodiment, the chromoprotein may be expressed in a cell 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 a 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. Such a fusion protein allows the chromoprotein to label a site within a cell that corresponds to the intracellular localization of the modified protein, and also allows the signal from the chromoprotein to be used as an indicator for analyzing the intracellular localization of the modified protein.

[0024] The form of the nucleic acid that expresses the chromoprotein may be any form commonly used for genetically expressing a protein in a cell. Such a nucleic acid contains a sequence encoding the chromoprotein. Such a nucleic acid can be appropriately designed 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 synthesis of nucleic acids, or by inserting a nucleic acid containing a sequence encoding the chromoprotein into a backbone purchased from a supplier using restriction enzyme digestion or the like. Such a nucleic acid can also be amplified by a method commonly used by a person skilled in the art, such as introducing it into bacteria such as Escherichia coli and culturing it.

[0025] 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. The 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. The viral vector can introduce a gene into mammalian cells by utilizing the infectivity of the virus, thereby allowing the target protein to be expressed in the 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.

[0026] 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. The mRNA precursor is not particularly limited as long as it can provide an 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.

[0027] 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 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. 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.

[0028] The nucleic acid to be distributed and introduced into daughter cells of the introduced cell may be a vector that is maintained as an episome (an independent DNA molecule that is not integrated into the cellular chromosome) within the cell. Such a vector (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). Examples of such episome-based vectors include the pEBMulti series (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0029] The nucleic acid into which the region encoding the chromoprotein is incorporated into the genomic DNA of the introduced cell may be, for example, an adenovirus vector, an adeno-associated virus vector, or a reverse transcription virus vector, and among these, an adenovirus vector or an adeno-associated virus vector is preferred from the viewpoint of making it easier to maintain the expression of the chromoprotein during the culture step.

[0030] 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 aspect, may be human. If the mammalian cells are derived from a human (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 for evaluating or screening drugs for humans. Furthermore, if the mammalian cells are derived from a human, 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.

[0031] The production 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).

[0032] The culture temperature of the mammalian cells in the culture step is less than 37° C. and is a temperature at which the chromoprotein is expressed in live mammalian cells. 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 temperature may be from 29° C. to 32° C., from 29° C. to 32.5° C., from 29° C. to 33° C., from 29° C. to 34° C., from 29° C. to 35° C., from 29° C. to 36° C., from 29° C. to less than 37° C., from 30° C. to 31° C., from 30° C. to 32° C., from 30° C. to 32° C., from 30° C. to 32.5° C., from 30° C. to 33° C., from 30° C. to 34° C., from 30° C. to 35° C., from 30° C. to 36° C., or from 30° C. to less 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 such a general culture temperature, the chromoprotein can be expressed in the mammalian cells with high efficiency.

[0033] The culture time of the mammalian cells in the culture step may be any time that allows the mammalian cells to express a chromoprotein when cultured at the above-mentioned culture temperature. The culture time of 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 the 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.

[0034] In the culturing step, culture conditions other than the culture temperature and culture time can be those normally used by those skilled in the art, as long as they do not inhibit the expression of chromoproteins in mammalian cells. For example, as the medium in the culturing step, a medium generally used for culturing mammalian cells can be used, for example, a medium obtained by adding serum components such as fetal bovine serum (FBS) to a general-purpose basal medium such as DMEM medium, RPMI medium, Ham's F-12 medium, or Ham's F-12K medium, can be used, and a medium further containing additives generally used in cell culture (for example, antibiotics such as penicillin / streptomycin) can also be used. In addition, the CO ２ The concentration is, for example, 5%.

[0035] In one aspect, the production 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 is commonly used by those skilled in the art when introducing such nucleic acids can be used. Examples of such methods include contacting a nucleic acid with a cell, lipofection, electroporation, microinjection, sonoporation, etc., which can be appropriately selected by those skilled in the art depending on the type of nucleic acid, and for example, the methods described above in the description of the nucleic acid that expresses the chromoprotein of one aspect can be selected. Furthermore, the amount of nucleic acid to be introduced into mammalian cells can be appropriately determined by those 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.

[0036] When 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. The environment in which nucleic acid introduction 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, and the exposure may be, for example, replacing the culture supernatant of adherent mammalian cells with such a medium or suspending mammalian cells in such a medium. The culture time in the introduction step may be any time period sufficient to introduce nucleic acid into 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 temperature of such culture in the introduction step may be any temperature that allows introduction of nucleic acid into living mammalian cells, and may be a temperature normally 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 one example is 37°C.

[0037] When 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 containing a selection marker and then contacted with an antibiotic, mammalian cells into which the nucleic acid has been introduced survive because they have acquired resistance to the antibiotic, while mammalian cells into which the nucleic acid has not been introduced 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 be those 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.

[0038] According to the production method of the first embodiment, mammalian cells labeled with a chromoprotein can be produced. The labeling of mammalian cells 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. Alternatively, the labeling of mammalian cells with the chromoprotein may be evaluated, for example, by lysing the mammalian cells and then measuring the amount of the chromoprotein contained in the lysate by ELISA or Western blotting.

[0039] The mammalian cells labeled with a chromoprotein produced by the production method of the first embodiment can be used to evaluate the cells using, for example, the coloration due to the visible light absorption of the chromoprotein as an index, which allows, for example, evaluation of the condition dependency of the expression of the chromoprotein (for example, environmental dependency or dependency on the concentration of a specific substance).

[0040] The mammalian cells labeled with a chromoprotein produced by the production method of the first embodiment can be used, for example, as cells co-expressing the chromoprotein as a fluorescence resonance energy transfer (FRET) acceptor and another protein acting as a donor, or as cells expressing a fusion protein of the chromoprotein as a FRET acceptor and a donor protein, to evaluate cells using the fluorescence intensity of the donor protein as an indicator. This allows evaluation of the intracellular environment or events detected by a fluorescent probe operating on FRET, such as protein-protein interactions, ion concentrations, membrane potential, or pH changes.

[0041] Mammalian cells labeled with chromoproteins 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 including: 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 refractive index distributions of the cells labeled at the specific regions in the labeling step at the first wavelength and the second wavelength; 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. A chromoprotein is 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, mammalian cells labeled with chromoproteins produced by the manufacturing method of the first embodiment are considered suitable for cell evaluation methods using refractive index as an indicator, such as the method described in Patent Document 1.

[0042] A second embodiment of the present disclosure is a method for evaluating mammalian cell labeling with a chromoprotein, the method comprising culturing mammalian cells, into which a 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 will also be referred to as the "evaluation method of the second embodiment."

[0043] The chromoprotein, nucleic acid that expresses it, and mammalian cells according to the evaluation method of the second embodiment can be the same as those described in the production method of the first embodiment. Furthermore, the culture step according to the evaluation method of the second embodiment can be carried out in the same manner as described in the production method of the first embodiment. Furthermore, the evaluation method of the second embodiment may include an introduction step prior to the culture step, and the introduction step can be carried out in the same manner as the introduction step described in the production method of the first embodiment.

[0044] In the evaluation step, the visible light absorption of the mammalian cells cultured in the culture step is evaluated, thereby making it possible to evaluate the presence or absence of expression of a pigment protein in the mammalian cells and the amount of expression.

[0045] 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 absorption spectrometer and a microwell plate reader. Using such optical instruments, the visible light absorption of mammalian cells can be evaluated, for example, by measuring the visible light absorption of a cell suspension containing suspended mammalian cells or of mammalian cells in an adherent state. In this case, the visible light absorption may be evaluated using absorbance as an index. For example, the absorbance may be evaluated 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 the maximum absorption wavelength in the visible light region of a chromoprotein expressed in mammalian cells. Alternatively, the absorbance at the maximum absorption wavelength or the maximum absorption wavelength in the visible light region of the chromoprotein may be evaluated.

[0046] The visible light absorption of mammalian cells may be evaluated using the color change of the mammalian cells as an indicator. In this case, the color change of mammalian cells is difficult to observe when the cells are in a low spatial density state, such as when they are adhered in a monolayer on a culture dish, but is easily observed when the cells are in a high spatial density state, such as when they 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 change 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 change of the concentrated mammalian cells as an indicator.

[0047] 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 cell concentration method in the concentration step may be any method commonly used by those skilled in the art for cell concentration, 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.

[0048] In the concentration step, mammalian cells are concentrated to a density at which coloration can be evaluated in the subsequent evaluation step. In the concentration step, for example, the mammalian cells may be concentrated 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, the concentrated product contains 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. In the concentration step, for example, the mammalian cells may be concentrated to a thickness of 1.0 x 10 ５ pcs or more, 2.0×10 ５ pcs or more, 5.0×10 ５ pcs or more, 1.0×10 ６ pcs or more, 1.5×10 ６ or more or 2.0 x 10 ６ The cells or more may be collected separately from the liquid component or collected as a precipitate or a collection on a filter containing such a number of cells.

[0049] A third embodiment of the present disclosure is a method for screening for chromoproteins, comprising culturing mammalian cells into which nucleic acids that express a candidate protein have been introduced at a temperature below 37°C (culturing step), and evaluating the visible light absorption of the mammalian cells. Hereinafter, this embodiment will also be referred to as the "screening method of the third embodiment."

[0050] The nucleic acid that expresses a candidate protein in the screening method of the third embodiment can be the same as that described in the production method of the first embodiment, except that a candidate protein is expressed instead of a chromoprotein. The mammalian cells that can be used in the screening method of the third embodiment can be the same as that described in the production method of the first embodiment. The culture step in the screening method of the third embodiment can be performed in the same manner as that described in the production method of the first embodiment, except that a nucleic acid that expresses a candidate protein is introduced into the mammalian cells. The screening method of the third embodiment may also include an introduction step prior to the culture step, and this introduction step can be performed in the same manner as the introduction step described in the production method of the first embodiment, except that a nucleic acid that expresses a candidate protein is introduced.

[0051] The evaluation step in mammalian cells according to 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, and the concentration step 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 an optical instrument such as an absorbance meter, thereby enabling high-throughput screening of chromoproteins.

[0052] The candidate protein in the screening method of the third embodiment is a candidate protein for a chromoprotein. The candidate protein for a chromoprotein may be, for example, a protein obtained from an organism other than a mammal, a protein expressed from a nucleic acid obtained from an organism other than a mammal, or a randomly modified chromoprotein protein. The randomly modified chromoprotein protein 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 a nucleic acid encoding the chromoprotein. Furthermore, when the candidate protein is a randomly modified chromoprotein protein, the screening method of the third embodiment may be carried out, for example, by introducing a nucleic acid that expresses the randomly modified chromoprotein protein 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).

[0053] The screening method of the third embodiment may include a step of adjusting the number of cells expressing a candidate protein to be used for evaluating 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 for evaluating 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.

[0054] 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. That is, the screening method according to 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).

[0055] 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 a method capable of selecting 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 be, for example, a method capable of selecting a protein that satisfies the requirements for absorbance and fluorescence quantum yield of a chromoprotein in the production method according to the first embodiment. ４ Proteins having a fluorescence quantum yield of 1% or less and a denaturation energy of [L / mol·cm] or more may be selected as chromoproteins.

[0056] Furthermore, when the screening method of the third embodiment includes a concentration step, the selection step may include selecting candidate proteins for which coloration has been observed in the evaluation step. In this case, the selection step may include first selecting, from the candidate proteins, candidate proteins for which coloration has been observed in the evaluation step (primary selection), and then further selecting, from the selected candidate proteins, proteins that satisfy the requirements for chromoproteins, as described above as aspects of the chromoproteins involved in the production method of the first embodiment (secondary selection).

[0057] According to the screening method of the third embodiment, chromoproteins can be selected, and thus chromoproteins that have potential for use in applications such as those described in the production method of the first embodiment can be found with high efficiency.

[0058] Furthermore, in one aspect of the screening method of the third embodiment, when the candidate protein is a randomly modified chromoprotein, it is possible to screen for proteins with improved parameters such as optical properties of the chromoprotein. In this way, by repeating a cycle multiple times 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 by the screening method of the third embodiment, it is possible to obtain a chromoprotein with desired properties in terms of molecular evolution.

[0059] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to the following examples.

[0060] In this example, cjBlue, rpulFkz1, gfasCP, asFP595, Rtms5, and aeCP597 were used as chromoproteins. The optical properties of these chromoproteins are described, for example, in Non-Patent Document 1. cjBlue is a chromoprotein derived from Epiactis japonica (a type of sea anemone), and its amino acid sequence is shown in SEQ ID NO: 1. rpulFkz1 is a chromoprotein derived from Rhizostoma pulmo (a type of jellyfish), and its amino acid sequence is shown in SEQ ID NO: 2. gfasCP is a chromoprotein derived from Galaxea fascicularis (a type of coral), and its amino acid sequence is shown in SEQ ID NO: 3. asFP595 is a chromoprotein derived from Anemonia sulcata (a type of sea anemone), and its amino acid sequence is shown in SEQ ID NO: 4. Rtms5 is a chromoprotein 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.

[0061] Example 1: Evaluation of chromoprotein expression when cultured at 30°C Step 1: Construction of chromoprotein expression plasmid DNA encoding each chromoprotein was obtained by outsourcing synthesis to Eurofins Genomics, where the codons in the coding region of the wild-type DNA encoding each chromoprotein were optimized for expression in human cells and two stop codons were 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.

[0062] The DNA encoding the obtained chromoprotein was subcloned into the multicloning site of the pEBMulti-Neo vector (Fujifilm Wako Pure Chemical Industries, Ltd., 057-08131) using the restriction enzymes BamHI and NotI to obtain chromoprotein expression plasmids (nucleic acids that express the chromoproteins) for each chromoprotein. Note that the pEBMulti series contains the EB 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 also replicated and distributed to daughter cells by an episomal replication system. Therefore, by using the pEBMulti series, it is possible to establish a cell line that constitutively expresses a protein encoded by a target gene, even without integrating the target gene into the genome of the host cell.

[0063] <Step 2: Introduction of the chromoprotein expression plasmid into mammalian cells and culturing the introduced cells> CHO-K1 cells, a cultured cell line derived from Chinese hamsters, were seeded onto a 6-well flat-bottom cell culture plate (VIOLAMO). The seeded cells were cultured at 37°C and 5% CO in a medium (also referred to as "culture medium" in Example 1) prepared by adding 10% (v / v) fetal bovine serum to Ham's F-12K medium (Fujifilm Wako Pure Chemical Industries, Ltd.). ２ The cells were cultured for 2 days under the conditions of 0.1 μg / well of a dye protein expression plasmid and a transfection reagent using ScreenFect (registered trademark) A plus (Fujifilm Wako Pure Chemical Industries, Ltd.) according to the protocol provided by the supplier, and the dye protein expression plasmid was transfected into the cells in each well. After transfection, the cells were incubated at 37°C and 5% CO ２ The cells were cultured for one day under the conditions of 1 mg / ml of antibiotic G418 (Fujifilm Wako Pure Chemical Industries, Ltd.) to each well containing the cultured cells, and cells that expressed proteins from the transfected nucleic acid were selected. The selected cells were then incubated at 30°C and 5% CO ２ The cells were cultured for 35 days under the conditions (culture step). During the main culture, the medium was changed at appropriate times, and no subculture operation was performed.

[0064] <Step 3: Evaluation of labeled cells (before cell concentration)> After the culture 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 under a phase contrast microscope (DM IL LED inverted microscope, Leica, 10x magnification). The cells into which each dye protein had been introduced were adhered to the dish and incubated at 30°C and 5% CO ２ It was confirmed that the cells survived without any problems even after culturing for 35 days under the conditions.

[0065] Figure 2 shows a digital photograph of cells in PBS(-) taken under room light after washing. While the results in Figures 1 and 2 show that cells were indeed present on the dish, it was difficult to visually detect the labeling of the cells with the chromoprotein when the cells were adhered flatly.

[0066] <Step 4: Cell Concentration> The cells obtained in step 3 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 obtained at the bottom of the tube was washed with PBS(-) to obtain concentrated cells.

[0067] <Step 5: Evaluation of labeled cells (after cell concentration)> 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, cell coloration was visually observed in five of them, namely, cjBlue, gfasCP, asFP595, Rtms5, and aeCP597, of which four, gfasCP, asFP595, Rtms5, and aeCP597, showed strong coloration, and two, gfasCP and Rtms5, showed even stronger coloration.

[0068] These results demonstrate that chromoproteins can be expressed in mammalian cells by culturing mammalian cells transfected with a chromoprotein expression plasmid at 30° C. Furthermore, these results suggest that mammalian cell labeling with chromoproteins and screening for chromoproteins can be evaluated by evaluating the visible light absorption of the cells after culturing, and that in particular, screening for chromoproteins can be performed easily and with high throughput by concentrating the cells and using visual coloration as an indicator.

[0069] 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.

[0070] <Step 2 in Comparative Example 1> HepG2 cells, a human-derived cultured cell line, were seeded on a 6-well flat-bottom cell culture plate (VIOLAMO). The seeded cells were incubated at 37°C and 5% CO in a medium (also referred to as "culture medium" in Comparative Example 1) prepared by adding 10% (v / v) fetal bovine serum to DMEM medium (Gibco). ２ The cells were cultured for 2 or 3 days under the conditions of

[0045] . 2.5 μg of a dye protein expression plasmid and a transfection reagent were added to each well using ScreenFect (registered trademark) A plus (Fujifilm Wako Pure Chemical Industries, Ltd.) according to the protocol provided by the supplier, and the dye protein expression plasmid was transfected into the cells in each well. After transfection, the cells were incubated at 37°C and 5% CO ２ The cells were cultured for one day under the conditions of 1 mg / ml of antibiotic G418 (Fujifilm Wako Pure Chemical Industries, Ltd.) to each well containing the cultured cells, and cells that expressed proteins from the transfected nucleic acid were selected. The selected cells were then incubated at 37°C and 5% CO ２ The cells were cultured for 20 days (rpulFkz1, gfasCP, asFP595, Rtms5, aeCP597) or 38 days (cjBlue) under the conditions (culture step). In the main culture, the medium was exchanged at appropriate times, and the cells were subcultured once.

[0071] [Example 2: Evaluation of chromoprotein expression when cultured at various temperatures] The coloration of cells when cultured at four conditions, 37°C, 35°C, 32.5°C, and 30°C, was evaluated and compared in the same manner as in Example 1.

[0072] <Step 1: Construction of a chromoprotein expression plasmid> A plasmid prepared in the same manner as in step 1 of Example 1 was used.

[0073] <Step 2: Introduction of the chromoprotein expression plasmid into mammalian cells and culturing the introduced cells> CHO-K1 cells, a cultured cell line derived from Chinese hamsters, were seeded into a 6-well flat-bottom cell culture plate (VIOLAMO). The seeded cells were cultured in a medium (also referred to as "culture medium" in Example 2) prepared by adding 10% (v / v) fetal bovine serum to Ham's F-12K medium (Fujifilm Wako Pure Chemical Industries, Ltd.) at 37°C and 5% CO ２ The cells were cultured for 2 days under the conditions of 0.1 μg / well of a dye protein expression plasmid and a transfection reagent using ScreenFect (registered trademark) A plus (Fujifilm Wako Pure Chemical Industries, Ltd.) according to the protocol provided by the supplier, and the dye protein expression plasmid was transfected into the cells in each well. After transfection, the cells were incubated at 37°C and 5% CO ２ The cells were cultured for one day under the conditions of 1 mg / ml of the antibiotic G418 (Fujifilm Wako Pure Chemical Industries, Ltd.) to select cells that expressed proteins from the transfected nucleic acid. After the addition of G418, the cells were incubated at 37°C and 5% CO. ２ for 6 days at 35°C and 5% CO ２ for 6 days at 32.5°C and 5% CO ２ for 6 days, or at 30°C and 5% CO ２ The cells were cultured for 7 days under the conditions (culture step). During the main culture, the medium was changed at appropriate times, and no subculture operation was performed.

[0074] <Step 3: Evaluation of labeled cells (before cell concentration)> After the culture in step 2, the culture medium was removed from each well, and the cells were washed with PBS(-). After the culture, the cells in PBS(-) were observed under a phase-contrast microscope (DMi1 inverted microscope, Leica, 10x magnification). Figure 4 shows the results of the culture in PBS(-) at 37°C and 5% CO ２ 5 shows the results of observing cells in PBS(-) under a phase contrast microscope after washing, which were cultured for 6 days under the conditions of 35°C and 5% CO ２6 shows the results of observing cells in PBS(-) under a phase contrast microscope after washing for 6 days under the conditions of 32.5°C and 5% CO ２ 7 shows the results of observing cells in PBS(-) under a phase contrast microscope after washing for 6 days under the conditions of 30°C and 5% CO. ２ The cells were cultured for 7 days under the conditions of 1.0 and 2.1, and then washed. The cells were then observed in PBS(-) under a phase-contrast microscope. The length of the scale bar in Figures 4 to 7 is 50 μm. The cells into which each chromoprotein was introduced were adhered to the dish and incubated at 37°C and 5% CO ２ for 6 days at 35°C and 5% CO ２ for 6 days at 32.5°C and 5% CO ２ for 6 days at 30°C and 5% CO ２ It was confirmed that the cells survived even after 7 days of culture under all of the above conditions.

[0075] Figure 8 shows the results of experiments using a 37°C and 5% CO ２ 9 shows digital photographs taken under room light of cells in PBS(-) after culturing for 6 days at 35°C and 5% CO ２ 10 shows digital photographs taken under room light of cells in PBS(-) after culturing for 6 days at 32.5°C and 5% CO ２ 11 shows digital photographs taken under room light of cells in PBS(-) after culturing for 6 days at 30°C and 5% CO ２ These are digital photographs taken under room light of cells in PBS(-) after culturing for 7 days under the conditions of (1) and (2) and subsequent washing. The results of Figures 4 to 11 show that cells are certainly present on the dish, but when the cells are adhered flat, it is difficult to clearly detect and compare the labeling of the cells with the chromoprotein by visual inspection.

[0076] <Step 4: Cell counting and cell concentration> The cells obtained in step 3 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 number of viable cells was counted by trypan blue staining, and the number of viable cells per tube was found to be 2.5 x 10 ５ The cell suspension was separated into a separate 1.5 mL tube to obtain cells. The tube was 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.

[0077] Step 5: Evaluation of Labeled Cells (After Cell Enrichment) Figure 12 shows digital photographs of the enriched cells obtained in step 4 taken under room light. Figure 13 shows images of 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 the declination angle as H (Hue) and the distance from the pole as S (Saturation). 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.

[0078] 12 to 14 show that of the six chromoproteins tested, five, cjBlue, gfasCP, asFP595, Rtms5, and aeCP597, showed stronger color development 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 development 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 color development was observed at a culture temperature of 37°C, whereas clear color development was observed at a culture temperature of 30°C or 32.5°C. These facts are also evident from the results in Figures 15 and 16, and it was semi-quantitatively confirmed that aeCP597 in particular exhibited greater saturation and more intense color development when the culture temperature was 30°C or 32.5°C compared to when the culture temperature was 37°C.

[0079] 17 and 18, it was difficult to compare the color intensity between culture conditions or between chromoproteins in the case of non-concentrated cell suspensions. This suggests that concentrating cells may enable simple, high-throughput screening using visual coloration as an indicator.

Claims

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. A method for evaluating mammalian cell labeling 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; and evaluating the visible light absorption of the mammalian cells.

3. A method for screening a chromoprotein, comprising: 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.

4. The method according to claim 3, further comprising concentrating the mammalian cells cultured at a temperature below 37°C before assessing the visible light absorption, wherein the assessment of the visible light absorption of the mammalian cells is carried out using the coloration of the concentrated mammalian cells as an indicator.

5. The molar extinction coefficient of the maximum absorption wavelength of the chromoprotein in the range of 400 to 800 nm is 5.0 × 10 ４ The method according to any one of claims 1 to 4, wherein the viscosity is [L / mol cm] or more.

6. 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.

7. 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.

8. The method according to any one of claims 1 to 4, wherein the culturing is carried out at 33°C or below.

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