Methods and cells for identifying primary pilus proteins

Abstract

To provide a method for identifying more kinds of primary ciliary proteins, and to provide a cell usable for the method.SOLUTION: The method for identifying primary cilia proteins includes a culture step of culturing cells having primary cilia, which express complexes of Gpr161 and biotinylating enzymes and / or complexes of Arl13B and biotinylating enzymes, and an identification step of identifying biotin-labeled proteins expressed in the cells obtained by the culture step.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method and cells for identifying primary cilium proteins. [Background technology]

[0002] The primary cilium is a structure that protrudes from the cell and is thought to be an organelle that receives extracellular signals. It is believed that the primary cilium contains many receptors, and efforts to identify the receptors present in the primary cilium are currently underway.

[0003] Biotinylation enzymes are widely used in analyzing protein localization. For example, a method has been developed in which a biotinylation enzyme is localized in a specific organelle to comprehensively biotinylate proteins present in the organelle, and then the biotinylated proteins are identified to identify the proteins localized in the organelle (Non-Patent Documents 1 to 3).

[0004] Conventionally, to identify proteins localized in primary cilia, a complex of ascorbate peroxidase (APEX) and nephrocystin 3 (NPHP3), which is used as a primary cilium marker, has been used (Non-Patent Documents 4 and 5). In this technique, NPHP3 functions to localize the complex to primary cilia, and ascorbate peroxidase functions to biotinylate proteins present in the vicinity of the complex. In this technique, proteins localized in primary cilia are identified by identifying biotinylated proteins. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Nat. Biotechnol. 30. 1143-1148. (2012) [Non-patent document 2] Science. 339. 1328-1331. (2013) [Non-patent document 3] Cell. 178. 473-490. (2019) [Non-patent document 4] Dev. Cell. 35. 497-512. (2015) [Non-Patent Document 5] J. Cell Biol. 220. e202007207. (2021) Summary of the Invention [Problem to be solved by the invention]

[0006] However, conventional techniques have only been able to identify a limited number of primary cilium proteins.

[0007] One aspect of the present invention aims to provide a method for identifying a greater variety of primary cilium proteins and cells that can be used in the method. [Means for solving the problem]

[0008] NPHP3 is a protein that is abundantly localized near the base of the primary cilium. In consideration of the above-mentioned problems, the present inventors discovered that by conjugating two types of proteins localized in the primary cilium, Gpr161, which is localized in the primary cilial membrane, and Arl13B, which is localized throughout the primary cilium, including near the membrane, with biotinylated proteins, it is possible to identify more types of proteins contained in the primary cilium, and thus completed the present invention.

[0009] That is, one aspect of the present invention includes the following configuration.

[0010] <1> A method for identifying primary cilia proteins, comprising a culture step of culturing cells that express a complex of Gpr161 and a biotinylating enzyme, and / or a complex of Arl13B and a biotinylating enzyme, and that have primary cilia, and an identification step of identifying biotin-labeled proteins expressed in the cells obtained by the culture step.

[0011] <2> the cell is an immune-related cell; <1> The method described below.

[0012] <3> The biotinylating enzyme is any one selected from the group consisting of ascorbate peroxidase and BirA. <1> or <2> The method described below.

[0013] <4> A cell that expresses a complex of Gpr161 and a biotinylating enzyme and / or a complex of Arl13B and a biotinylating enzyme, and that has primary cilia.

[0014] <5> the cell is an immune-related cell; <4> The cell described in

[0015] <6> The biotinylating enzyme is any one selected from the group consisting of ascorbate peroxidase and BirA. <4> or <5> The cell described in [Effects of the Invention]

[0016] According to one aspect of the present invention, it is possible to provide a method for identifying a greater variety of primary cilium proteins and cells that can be used in the method. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention is described below, but is not limited thereto. The present invention is not limited to the respective configurations described below, and various modifications are possible within the scope of the claims. Furthermore, embodiments or examples obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic literature and patent documents described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, the term "X to Y" representing a numerical range means "greater than or equal to X and less than or equal to Y."

[0018] 1. Method for identifying primary cilium proteins and cells A method for identifying primary cilium proteins according to one embodiment of the present invention includes a culture step of culturing cells that express a complex of Gpr161 and a biotinylating enzyme and / or a complex of Arl13B and a biotinylating enzyme, the cells having primary cilia, and an identification step of identifying biotin-labeled proteins expressed in the cells (e.g., the primary cilia of the cells) obtained by the culture step.

[0019] As used herein, the term "primary ciliary protein" refers to a protein contained in the primary cilium of a cell or a protein expressed in the primary cilium of a cell. More specifically, the term "primary ciliary protein" refers to a protein localized in the primary ciliary membrane and / or throughout the primary cilium.

[0020] Previously, NHPH3, which is known to be localized in primary cilia, was used to identify primary cilia proteins, but the number of primary cilia proteins that could be identified using NHPH3 was limited.

[0021] The present inventors focused on the fact that NHPH3 is more abundant near the membrane at the base of the primary cilium, and proceeded with their research from the unique perspective that by using proteins that are localized differently in the primary cilium than NHPH3, it may be possible to identify more primary ciliary proteins.

[0022] Gpr161 is a G protein-coupled receptor localized in the primary ciliary membrane. On the other hand, Arl13B is a protein localized throughout the primary cilium, including near the primary ciliary membrane. The present inventors have found that by using a complex of Gpr161 or Arl13B with a biotinylating enzyme, many primary ciliary proteins can be biotinylated, thereby enabling the identification of many primary ciliary proteins.

[0023] The use of a complex that combines specific proteins makes it possible to identify a larger number of primary cilium proteins than was previously possible, which is an excellent effect that could not be predicted from conventional techniques.

[0024] (Culture process) A method for identifying primary cilium proteins according to one embodiment of the present invention includes a culture step of culturing cells that express a complex of Gpr161 and a biotinylating enzyme and / or a complex of Arl13B and a biotinylating enzyme and have primary cilia.

[0025] The cells are not particularly limited as long as they have primary cilia, but are preferably immune-related cells. The immune-related cells include immune cells that are primarily responsible for immune responses and immune function-retaining cells that are indirectly involved in immune responses. Immune function-retaining cells have the function of activating immune cells, for example. Examples of immune cells include skin dendritic cells (e.g., Langerhans cells, dermal dendritic cells), lymphocytic immune cells (e.g., T cells, NK cells, B cells), and monocytic immune cells (e.g., conventional dendritic cells, monocytic dendritic cells (e.g., plasmacytoid dendritic cells)). On the other hand, examples of immune function-retaining cells include keratinocytes, fibroblasts, and epithelial cells. Immune-related cells may be immune-related cells collected from a living body or established immune-related cell lines (e.g., HaCaT cells (human epidermal keratinocyte cell line)).

[0026] The biotinylating enzyme is not particularly limited as long as it is an enzyme that can biotinylate a protein present in the vicinity of the biotinylating enzyme and can form a complex with Gpr161 or Arl13B. Examples of biotinylating enzymes include ascorbate peroxidase (APEX) and BirA. Furthermore, BirA also includes biotinylating enzymes obtained by modifying BirA, such as TurboID, BioID, and AirID. Among these, APEX is preferred because the initiation of the enzyme reaction can be controlled by adding hydrogen peroxide.

[0027] The method for culturing the cells is not particularly limited and can be carried out according to a known method. For example, a known culture medium can be used. The cells in the culture step may all be cultured in the same medium, or the cells may be transferred to a different medium as needed. The culture time is not particularly limited and may be, for example, 1 to 100 hours, 1 to 50 hours, or 1 to 24 hours.

[0028] (Identification step) A method for identifying a primary cilium protein according to one embodiment of the present invention includes an identification step of identifying a biotin-labeled protein expressed in the cells obtained by the culture step.

[0029] Because the primary cilium protein to be identified is labeled with biotin, it can be identified using the biotin as an indicator. The method for identifying a primary cilium protein using biotin as an indicator is not limited. For example, the biotin-labeled protein can be specifically recovered from the cells obtained by the culture step using avidin, neutravidin, streptavidin, an anti-biotin antibody, or the like, and the recovered protein can be subjected to Western blotting, ELISA, silver staining, or the like to identify the biotin-labeled protein.

[0030] Alternatively, the biotin-labeled protein can be identified by subjecting the biotin-labeled protein to liquid chromatography, mass spectrometry, immunostaining, or the like.

[0031] (Cell production process) A method for identifying a primary cilium protein according to one embodiment of the present invention may include, prior to the culture step, a cell preparation step of preparing cells that express a complex of Gpr161 and a biotinylating enzyme and / or a complex of Arl13B and a biotinylating enzyme and have primary cilia.

[0032] The cell production process enables the production of cells according to one embodiment of the present invention, which express a complex of Gpr161 and a biotinylating enzyme and / or a complex of Arl13B and a biotinylating enzyme and have primary cilia.

[0033] Methods for producing the cells are not particularly limited, but include, for example, introducing an expression vector for expressing a complex of Gpr161 and a biotinylating enzyme and / or a complex of Arl13B and a biotinylating enzyme into cells having primary cilia (e.g., immune-related cells).

[0034] The expression vector can be prepared using various commonly used expression vectors. Examples of the expression vector that can be used include phage vectors, plasmid vectors, viral vectors, retroviral vectors (e.g., lentiviral vectors), chromosomal vectors, episomal vectors, and virus-derived vectors. The specific type of vector is not particularly limited, and a vector capable of expressing a complex of Gpr161 and a biotinylating enzyme and / or a complex of Arl13B and a biotinylating enzyme in cells can be appropriately selected.

[0035] The expression vector may comprise a polynucleotide encoding a complex of Gpr161 and a biotinylating enzyme, and / or a polynucleotide encoding a complex of Arl13B and a biotinylating enzyme, so as to be capable of expressing these complexes.

[0036] In a polynucleotide encoding a complex of Gpr161 and a biotinylating enzyme, the positional relationship between the polynucleotide encoding Gpr161 and the polynucleotide encoding the biotinylating enzyme is not limited. The polynucleotide encoding Gpr161 may be located at the 5'-end or 3'-end of the polynucleotide encoding the biotinylating enzyme.

[0037] Furthermore, the polynucleotide encoding Gpr161 and the polynucleotide encoding the biotinylating enzyme may be linked directly or via a desired polynucleotide (for example, a linker).

[0038] In a polynucleotide encoding a complex of Arl13B and a biotinylating enzyme, the positional relationship between the polynucleotide encoding Arl13B and the polynucleotide encoding the biotinylating enzyme is not limited. The polynucleotide encoding Arl13B may be located at the 5'-end or 3'-end of the polynucleotide encoding the biotinylating enzyme.

[0039] Furthermore, the polynucleotide encoding Arl13B and the polynucleotide encoding the biotinylating enzyme may be linked directly or via a desired polynucleotide (for example, a linker).

[0040] As described above, ascorbic acid peroxidase (APEX), BirA, and the like can be used as biotinylating enzymes.

[0041] For example, a polynucleotide encoding APEX from Arabidopsis thaliana is registered under Accession No. NC_003070.9, and a polynucleotide encoding BirA from Escherichia coli is registered under Accession No. NC_000913.3. In one embodiment, the polynucleotide encoding APEX and the polynucleotide encoding BirA may be derived from a biological species other than those mentioned above.

[0042] In one embodiment of the present invention, the polynucleotide encoding the biotinylating enzyme can be (i) the above-mentioned polynucleotide, (ii) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the above-mentioned polynucleotide and encodes a polypeptide having biotinylating enzyme activity, or (iii) a polynucleotide consisting of a polynucleotide with 90% or more sequence identity to the above-mentioned polynucleotide and encoding a polypeptide having biotinylating enzyme activity.

[0043] Whether a desired polypeptide has biotinylation enzyme activity can be determined by whether the substrate is biotinylated when the desired polypeptide is mixed with a substrate. More specifically, after expressing the desired polypeptide in a desired cell, the determination can be determined by whether the biotinylated substrate binds to the streptavidin beads when a lysate of the cell is contacted with streptavidin beads.

[0044] The polynucleotide encoding Arl13B is not limited and may be, for example, any of the following (1) to (3): (1) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1; (2) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 and encodes a polypeptide expressed in primary cilia (e.g., a polypeptide expressed in primary cilia similar to Arl13B); (3) A polynucleotide having a sequence identity of 90% or more with a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 and encoding a polypeptide expressed in primary cilia (e.g., a polypeptide expressed in primary cilia similar to Arl13B).

[0045] The nucleotide sequence shown in SEQ ID NO: 1 corresponds to a polynucleotide encoding human Arl13B. A polynucleotide encoding human Arl13B is publicly known and registered under Accession No. NM_001410782. In one embodiment, the polynucleotide encoding Arl13B may be derived from a source other than human.

[0046] Whether a desired polypeptide is a polypeptide expressed in primary cilia (e.g., a polypeptide expressed in primary cilia similar to Arl13B) can be determined, for example, by expressing the desired polypeptide in a cell having primary cilia, fluorescently staining the polypeptide, and observing the localization of the polypeptide.

[0047] The polynucleotide encoding Gpr161 is not limited and may be, for example, any of the following (4) to (6): (4) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; (5) a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 and encodes a polypeptide expressed in primary cilia (e.g., a polypeptide expressed in primary cilia similar to Gpr161); (6) A polynucleotide having a sequence identity of 90% or more to a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2, and encoding a polypeptide expressed in primary cilia (e.g., a polypeptide expressed in primary cilia similar to Gpr161).

[0048] The base sequence shown in SEQ ID NO: 2 corresponds to a polynucleotide encoding human Gpr161. The polynucleotide encoding human Gpr161 is publicly known and is registered under Accession No. NM_001267612. In one embodiment, the polynucleotide encoding Gpr161 may be derived from a source other than human.

[0049] Whether or not a desired polypeptide is a polypeptide expressed in primary cilia (e.g., a polypeptide expressed in primary cilia similar to Gpr161) can be determined, for example, by expressing the desired polypeptide in a cell having primary cilia, fluorescently staining the polypeptide, and observing the localization of the polypeptide.

[0050] As used herein, "stringent conditions" refers to conditions under which a double-stranded polynucleotide specific to a base sequence is formed, but a non-specific double-stranded polynucleotide is not formed. In other words, these conditions can be defined as conditions under which highly homologous nucleic acids hybridize with each other, for example, at a temperature 15°C lower, preferably 10°C lower, and more preferably 5°C lower than the melting temperature (Tm value) of a perfectly matched hybrid.

[0051] One example of such a condition is hybridization for 16 to 24 hours in a buffer solution consisting of 0.25 M Na2HPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1x Denhardt's solution at a temperature of 60 to 68°C, preferably 65°C, and more preferably 68°C, followed by two 15-minute washes in a buffer solution consisting of 20 mM Na2HPO4, pH 7.2, 1% SDS, and 1 mM EDTA at a temperature of 60 to 68°C, preferably 65°C, and more preferably 68°C.

[0052] Another example is a hybridization solution containing 25% formamide, or more stringent conditions, such as 50% formamide, 4x SSC (sodium chloride / sodium citrate), 50 mM Hepes pH 7.0, 10x Denhardt's solution, and 20 μg / mL denatured salmon sperm DNA, which is prehybridized overnight at 42°C. The labeled probe is then added and incubated overnight at 42°C for hybridization. Subsequent washes can be performed with a solution and temperature conditions of approximately 1x SSC, 0.1% SDS, and 37°C. More stringent conditions include 0.5x SSC, 0.1% SDS, and 42°C, while even more stringent conditions include 0.2x SSC, 0.1% SDS, and 65°C. Thus, the more stringent the hybridization wash conditions, the more specific the hybridization. However, the above combinations of SSC, SDS, and temperature conditions are merely examples, and a person skilled in the art would be able to achieve similar stringency by appropriately combining the above or other factors that determine hybridization stringency (e.g., probe concentration, probe length, hybridization reaction time, etc.), as described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001).

[0053] The sequence identity of amino acid sequences and nucleotide sequences can be determined using the BLASTN (nucleic acid level) or BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). These programs are based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). When analyzing nucleotide sequences using BLASTN, parameters are set, for example, as score = 100 and word length = 12. When analyzing amino acid sequences using BLASTX, parameters are set, for example, as score = 50 and word length = 3. Furthermore, when analyzing amino acid sequences using the Gapped BLAST program, the method can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known. Additions or deletions (e.g., gaps) may be allowed to optimally align the nucleotide or amino acid sequences to be compared.

[0054] The sequence identity is preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, and most preferably 100%.

[0055] [2. Kit] A kit for identifying primary cilium proteins according to one embodiment of the present invention may include cells that express a complex of Gpr161 and a biotinylating enzyme and / or a complex of Arl13B and a biotinylating enzyme, and that have primary cilia.

[0056] A kit for identifying primary cilium proteins according to one embodiment of the present invention may include (i) an expression vector for expressing a complex of Gpr161 and a biotinylating enzyme and / or a complex of Arl13B and a biotinylating enzyme, and (ii) cells having primary cilia.

[0057] The kit may be a package containing containers (e.g., bottles, plates, tubes, dishes, etc.) containing specific materials. The kit of the present invention may be in a form in which each material contained therein exists independently, or in a form in which multiple materials are mixed (e.g., in the form of a composition). The kit preferably includes instructions for using each material. [Example]

[0058] (1. Construction of lentiviral vectors) Using genome extracted from human cultured cells as a template, each exon of the Arl13B gene and each exon of the GPR161 gene were amplified by PCR.

[0059] The amplified exons of the Arl13B gene were joined by joint PCR to obtain the full-length cDNA sequence of the Arl13B gene (SEQ ID NO: 1).

[0060] Furthermore, the exons of the amplified GPR161 gene were joined by joint PCR to synthesize the full-length cDNA sequence of the GPR161 gene (SEQ ID NO: 2).

[0061] The full-length cDNA of the APEX gene was amplified by PCR, and then the cDNA of the GFP gene with the termination codon removed was added to the 5' end of the full-length cDNA of the APEX gene to create "GFP-APEX," a polynucleotide encoding a fusion protein of the GFP protein and the APEX protein.

[0062] Furthermore, the GFP-APEX gene was added to the 3' end of the full-length cDNA sequence of the GPR161 gene (with the termination codon removed) and the full-length cDNA sequence of the Arl13B gene (with the termination codon removed) by joint PCR, thereby generating (i) "GPR161-GFP-APEX," a polynucleotide encoding a fusion protein of GPR161, GFP, and APEX proteins, and (ii) "Arl13B-GFP-APEX," a polynucleotide encoding a fusion protein of Arl13B, GFP, and APEX proteins.

[0063] An expression vector (GPR161) was prepared by inserting "GPR161-GFP-APEX" into the restriction enzyme site of a lentiviral vector.

[0064] On the other hand, an expression vector (Arl13B) was prepared by inserting "Arl13B-GFP-APEX" into the restriction enzyme site of a lentiviral vector.

[0065] In addition, a polynucleotide encoding a fusion protein of NPHP3(WT), GFP, and APEX, "NPHP3(WT)-GFP-APEX," or a polynucleotide encoding a fusion protein of NPHP3(G2A), GFP, and APEX, "NPHP3(G2A)-GFP-APEX," was inserted into a plasmid as a template, and the polynucleotide was amplified by PCR. The amplified polynucleotide was inserted into the restriction enzyme site of a lentiviral vector to prepare an expression vector (NPHP3(WT)) and an expression vector (NPHP3(G2A)).

[0066] Each of the constructed expression vectors was amplified in E. coli to obtain lentiviral vectors.

[0067] (2. Purification of lentivirus and establishment of a constitutively expressing HaCaT cell line) Plasmids into which polynucleotides encoding three types of viral components (Delta8.9, VSVG, and pvcRev) were inserted were used in the following tests.

[0068] After culturing the HEK293T cell line in a 6-cm diameter culture dish, (i) a plasmid containing an inserted polynucleotide encoding the three types of viral components described above, and (ii) an expression vector (GPR161), an expression vector (Arl13B), an expression vector (NPHP3(WT)), or an expression vector (NPHP3(G2A)) were introduced into the HEK293T cell line using polyethyleneimine.

[0069] After 12 hours, the culture medium was replaced, and the HEK293T cell line was then cultured for an additional 60 hours. The culture supernatant containing the lentivirus was collected in a 15 ml tube and centrifuged at 500 g at 4°C for 10 minutes.

[0070] After centrifugation, the supernatant was transferred to a new 15 ml tube and centrifuged at 1500 g at 4°C for 4 hours.

[0071] Since the precipitated pellet was assumed to contain virus particles, the supernatant was removed without disturbing the pellet, and the pellet was suspended in 150 μL of sterile PBS, which was used as a concentrated virus solution.

[0072] After culturing HaCaT cells in a 6-cm culture dish, the medium was replaced with 2 mL of DMEM + 10% FBS medium, and then 8 μg / mL polybrene and 150 μL of concentrated virus solution were added to the medium to infect the HaCaT cells with the virus.

[0073] After 48 hours of incubation, the medium was replaced with fresh medium containing 2 μg / mL puromycin, and incubation was continued. After two weeks of incubation, a cell population that was thought to constitutively express APEX fusion proteins was selected. After incubation, the selected cell population was continued to be cultured in medium containing 2 μg / mL puromycin.

[0074] From the selected cell population, single clones expressing APEX fusion proteins were selected by limiting dilution. Specifically, the cell suspension was diluted to 30 cells / 10 mL to prepare a cell suspension, and 100 μL of the cell suspension was seeded into each well of a 96-well plate and cultured to select single clones with puromycin resistance.

[0075] When single clones grew and formed colonies, they were trypsinized and transferred to 24-well plates for culture. As the colonies grew, they were transferred to 6-well plates, 35mm plates, 6cm dishes, and 10cm dishes, and cultured in that order. The cells derived from the single clones obtained by culture were used as constitutively expressing cell lines.

[0076] (3.Immunostaining method) To confirm whether each fusion protein was expressed in the primary cilia of the constitutively expressing cell line, immunostaining was used.

[0077] Constitutively expressing cell lines were cultured on 24-well plates containing 12 mm cover glasses, washed once with PBS, and then fixed with 4% paraformaldehyde suspended in PBS for 15 minutes at room temperature.

[0078] After fixation, the constitutively expressing cell line was washed three times with PBS, and then blocked and permeabilized for 7 hours using a blocking solution (aqueous solution containing 10% FBS, 0.1% Triton-X100, and 0.1% SDS).

[0079] Thereafter, 100 μL of a solution prepared by diluting the primary antibody with PBS was dropped onto the parafilm, and the cover glass was immersed in the solution and allowed to stand overnight at 4°C.

[0080] After standing, the cover glass was washed once with PBS, and then a solution of secondary antibody diluted with PBS was dropped onto the parafilm, and the cover glass was immersed in the solution and left to stand at 4°C for 1 hour while shielded from light.

[0081] After leaving the coverslips, they were washed once with PBS and then mounted using a mounting medium (Product Name: ProLong Glass Antifade Mountant, Invitrogen, Product Number: P36980). The constitutively expressing cell lines on the coverslips were observed using a confocal microscope (Product Name: FV3000, Olympus). The primary and secondary antibodies were as follows:

[0082] Primary antibody Anti-acetylated tubulin mouse monoclonal antibody (clone 6-11B-1) (Sigma, product number #T7451) (1 / 500 dilution): Anti-Pericentrin Rabbit Polyclonal Antibody (Bethyl, Cat. No. A301-348A) (1 / 1000 dilution): secondary antibody Alexa Fluor 594 Donkey Anti-Mouse IgG (Invitrogen, Cat. #A21203) (1 / 1000 dilution): Alexa Fluor 647 donkey anti-rabbit IgG (Invitrogen, product number #A31573) (1 / 500 dilution).

[0083] [Observation results] In a cell line transfected with the expression vector (GPR161), GFP was confirmed to be localized in the primary cilium, which was positive for acetylated tubulin.

[0084] In a cell line transfected with the expression vector (Arl13B), GFP was confirmed to be localized in the primary cilium, which was positive for acetylated tubulin.

[0085] In a cell line expressing NPHP3(WT) constitutively, GFP was confirmed to be localized in the primary cilium, which was positive for acetylated tubulin.

[0086] In a cell line transfected with an expression vector (NPHP3(G2A)), GFP was not localized to the acetylated tubulin-positive primary cilia, but was uniformly expressed in the cytoplasm.

[0087] Therefore, it was demonstrated that the fusion protein of GPR161 and APEX, the fusion protein of Arl13B and APEX, and the fusion protein of NPHP3(WT) and APEX are localized to primary cilia.

[0088] 4. APEX Reaction and Purification of Biotinylated Enzyme The constitutively expressing cell line obtained above was cultured in the presence of 500 μM biotin-phenol (Biotinyl Tyramide, Tokyo Chemical Industry Co., Ltd.) for 30 minutes, after which H2O2 was added to the constitutively expressing cell line to a final concentration of 1 mM. The medium was then immediately removed, and the constitutively expressing cell line was washed three times with a quencher solution (PBS solution containing 10 mM sodium ascorbate and 5 mM Trolox) to stop the biotinylation reaction.

[0089] The constitutively expressing cell line was lysed in lysis RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS) and then allowed to stand on ice for 10 minutes.

[0090] The resulting lysate was centrifuged at 16,000 g for 20 minutes to remove undissolved material, and a lysate was obtained. Protease inhibitors and 50 μL of streptavidin magnetic beads were added to the lysate, and the mixture was rotated at 4°C for 4 hours to allow the biotinylated molecules to bind to the streptavidin magnetic beads.

[0091] The streptavidin magnetic beads were captured using a magnetic rack and the supernatant was removed. The beads were then washed with lysis buffer. Next, the beads were washed with a buffer containing 4 M urea and 10 mM Tris / HCl, followed by a buffer containing 4 M urea, 10 mM Tris / HCl, and 50 μM biotin. Finally, the beads were washed three times with PBS, and the molecules bound to the beads were analyzed by mass spectrometry.

[0092] Alternatively, 2 mM biotin and 20 mM DTT were added to streptavidin magnetic beads to which biotinylated molecules were bound, and then 10 μL of 4x protein loading Laemmli buffer was added to the streptavidin magnetic beads.The streptavidin magnetic beads were then boiled at 95°C for 10 minutes to elute the proteins bound to the streptavidin magnetic beads.

[0093] The solution containing the eluted proteins was collected and used for Western blot or silver staining analysis. The reagent used for Western blot to detect biotinylated proteins was streptavidin (HRP) (Abcam, product number ab7403) (1 / 500 dilution).

[0094] [Mass spectrometry results] Protein mass spectrometry revealed that 30 proteins were detected in cells expressing the complex of NPHP3 and biotinylating enzyme.

[0095] On the other hand, when cells expressing a complex of Gpr161 and biotinylation enzyme were used, 426 types of primary cilium proteins were detected.

[0096] When cells expressing a complex of Arl13B and a biotinylating enzyme were used, 152 primary cilium proteins could be detected.

[0097] Therefore, it was demonstrated that by complexing Gpr161 and / or Arl13B with a biotinylating enzyme, a larger number of proteins than previously possible could be identified. [Industrial Applicability]

[0098] The present invention can be used as a method for identifying primary cilium proteins.

Claims

1. A culturing step of culturing cells expressing a complex of Gpr161 and a biotinylating enzyme and / or a complex of Arl13B and a biotinylating enzyme, the cells having primary cilia; A method for identifying a primary cilium protein, comprising an identification step of identifying a biotin-labeled protein expressed in the cells obtained by the culture step.

2. The method of claim 1 , wherein the cell is an immune-related cell.

3. The method of claim 1, wherein the biotinylating enzyme is any one selected from the group consisting of ascorbate peroxidase and BirA.

4. A cell expressing a complex of Gpr161 and a biotinylating enzyme and / or a complex of Arl13B and a biotinylating enzyme, the cell having primary cilia.

5. The cell of claim 4 , wherein the cell is an immune-related cell.

6. The cell according to claim 4 , wherein the biotinylating enzyme is any one selected from the group consisting of ascorbate peroxidase and BirA.