WO2006057391A1

WO2006057391A1 - Method of detecting interaction between proteins

Info

Publication number: WO2006057391A1
Application number: PCT/JP2005/021824
Authority: WO
Inventors: Nobumasa Hino; Kensaku Sakamoto; Shigeyuki Yokoyama
Original assignee: Riken
Priority date: 2004-11-26
Filing date: 2005-11-28
Publication date: 2006-06-01
Also published as: JP2008035702A

Abstract

A method of detecting an interaction between proteins comprising: the first step wherein a prokaryote-origin TyrRS mutant which is specific to an unnatural amino acid capable of serving as a crosslinking agent, a prokaryote-origin suppressor tRNA which is capable of binding to the above-described unnatural amino acid in the presence of the above-described TyrRS mutant, and a desired protein gene having a nonsense mutation at a desired site are expressed in an animal cell so that the above-described unnatural amino acid is incorporated into the nonsense mutation site of the above-described protein and the protein having the unnatural amino acid integrated thereinto is expressed; the second step wherein the above-described animal cell is irradiated with light having a specific wavelength; and the third step wherein the presence or absence of the interaction between the proteins is detected.

Description

Specification

Method for detecting protein-protein interaction

Technical field

The present invention relates to a method for detecting protein-protein interactions in animal cells using a cross-link method.

This application claims priority based on Japanese Patent Application No. 2004-342914 filed on November 26, 2004, the contents of which are incorporated herein by reference.

Background art

[0002] Non-natural amino acid-incorporated proteins (hereinafter referred to as "aromatic amino acids") in which an amino acid residue at a desired position in a protein is substituted with other than 20 amino acids (non-natural amino acids) involved in normal protein synthesis. Protein ”) can be an effective means for protein function analysis

[0003] In recent years, the present inventors have succeeded in enabling the production of aroprotein, which has been developed in E. coli and cell-free protein synthesis systems, in animal cells (Patent Document 1). That is, the present inventors assigned an aminoacyl t which is assigned to a nonsense codon and is specific for an unnatural amino acid.

Developed suppressor tRNAs that are recognized only by mutants of RNA synthase (hereinafter referred to as “aaRS”) but not aaRS in the host animal cells (specific to these suppressor tRNAs and unnatural tyrosine derivatives) Mutated tyrosyl-tRNA synthesis Enzyme and the production of unnatural amino acid-incorporated protein in animal cells by expressing the desired protein gene with nonsense mutation at the desired position in animal cells Successful.

[0004] By the way, as a means for detecting the presence of protein-protein interaction, immunoprecipitation has been conventionally used. However, the immunoprecipitation method has the following problems. (1) When the protein-protein interaction is based on weak binding force, when an antibody is allowed to act on any protein, the protein-protein interaction may break and dissociate. (2) When the interaction between proteins in cells is detected by immunoprecipitation, it is necessary to first extract the proteins from the cells. Proteins are different competencies in the cell Even if the interaction is not present, the interaction may occur for the first time outside the cell by performing the extraction operation. Therefore, immunoprecipitation may interact within the cell and possibly detect it. For this reason, the accuracy in detecting protein-protein interactions using the immunoprecipitation method is not necessarily high.

[0005] In order to solve the problem of using immunoprecipitation to detect protein-protein interactions, there is a cross-linking method. The cross-linking method is a method in which a protein interacting in a cell and a cross-linking agent are covalently bonded by irradiating the cell with ultraviolet rays. Therefore, even if the protein-protein interaction is based on a weak binding force, the protein is bound by a strong or binding force called a covalent bond. Will not dissociate. In addition, since it is covalently bound within the cell, it is possible to detect only the protein that interacts within the cell even after extraction from the cell.

[0006] Schultz et al. Described a tyrosyl-tRNA synthetase (hereinafter referred to as “TyrRS”) of Methanococcus jannaschii that specifically recognizes p-benzoyl-L-fe-lualanin (hereinafter referred to as “pBpa”). ) Mutant was expressed in Escherichia coli together with a supple search rosyl tRNA derived from Methanococcus yanasi. This TyrRS variant aminoacylates only this tRNA, which is not recognized by endogenous aaRS. Thus, pBpa is assigned to the amber codon of Escherichia coli, and pBpa is transferred into the medium and exposed to 365 nm light for 30 minutes, whereby pBpa is covalently bound as a cross-linking agent. “GST” t) A dimer was produced (Non-patent Document 1). In this way, pBpa, a non-natural amino acid, can be incorporated into prokaryotic E. coli, and its protein interaction can be detected by the cross-linking method. The cross-linking method can be used as an alternative to immunoprecipitation. It became possible.

Patent Document 1: International Publication 2004Z039989 Pamphlet

Non-patent literature l: Chin, JW, Martin, AB, King, DS, Wang, L "& Schultz, PG Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli.Proc Natl Acad Sci USA 99, 11020-11024 (2002) Disclosure of the invention

Problems to be solved by the invention

[0007] By the way, if the cross-linking method can be applied to animal cells, for example, the analysis of the signal transduction system such as the interaction of the receptor for epidermal growth factor (hereinafter referred to as "EGF") (hereinafter referred to as "EGFR") can be performed. It becomes possible and its usefulness is high.

However, animal cells are extremely complex in the cells compared to prokaryotes, and problems such as the above (2) appear strongly in existing methods such as immunoprecipitation. Furthermore, the method of Schultz et al. Described in Non-Patent Document 1 is a method used for prokaryotes and cannot be applied to animal cells as it is.

[0008] Accordingly, an object of the present invention is to provide means for detecting an interaction in an animal cell using a cross-link method.

Means for solving the problem

In order to solve the above problems, the present invention provides:

A prokaryotic-derived TyrRS variant specific to a non-natural amino acid that can be a cross-linking agent, a prokaryotic-derived sub-tRNA capable of binding to the non-natural amino acid in the presence of the TyrRS variant, and a desired tRNA A desired protein gene having a nonsense mutation at a position thereof is expressed in an animal cell, and the non-natural amino acid is incorporated at the position of the nonsense mutation of the protein to express a non-natural amino acid-incorporating protein. About

A second step of irradiating the animal cell with light of a specific wavelength; and

The third step of detecting the presence or absence of protein-protein interactions,

It is a detection method of protein-protein interaction characterized by having.

A non-natural amino acid that can serve as a cross-linking agent is preferred.

The non-natural amino acid that can serve as a cross-linking agent is preferably pBpa. Interaction force between proteins It is preferable that the interaction is a tyrosine kinase type receptor.

The protein-protein interaction is preferably an EGFR interaction. The prokaryotic cell from which the tyrosyl tRNA synthetase variant is derived is preferably E. coli.

Prokaryotic force from which suppressor tRNA is derived. Bacillus stearothermophilus.

The invention's effect

[0010] The present invention makes it possible to easily and reliably detect protein-protein interactions in animal cells.

Brief Description of Drawings

[0011] Fig. 1 is a schematic diagram showing the state of aloprotein expression.

FIG. 2 shows the results of site-specific binding of pBpa to Grb2 in CHO cells.

FIG. 3 shows the analysis results of the binding between Grb2 and EGFR by coprecipitation method.

FIG. 4 shows the analysis results of the crosslink between Grb2 and EGFR.

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] First, the first step will be described.

(Non-natural amino acid)

The non-natural amino acid used in the method of the present invention can be a cross-linking agent. Specifically, it is particularly preferably pBpa, which is preferably an amino acid having a benzophenone skeleton. A non-natural amino acid that can be a cross-linking agent is a non-natural amino acid that has physiological activity by itself, and also serves as a target site for a site-specific label of a protein. · It is useful as a material for structural analysis and may be a target for drug discovery.

[0013] (TyrRS mutant)

The TyrRS mutant used in the present invention specifically recognizes a non-natural amino acid that can be a cross-linking agent as an amino acid, and specifically recognizes a suppressor tRNA to be used in combination as a tRNA. Is a prokaryotic-derived TyrRS variant capable of generating a sub-repressor tRNA bound to. Examples of prokaryotes include Escherichia coli and the like, and K12 and B strains are preferable among them.

TyrRS (wild type) derived from prokaryotes such as E. coli is resistant to tyrosine tRNA in animal cells. Similarly, prokaryotic tyrosine tRNAs do not react with TyrRS in animal cells

[0014] SEQ ID NO: 1 shows the amino acid sequence of TyrRS (wild type) of Escherichia coli.

TyrRS mutants used in the present invention include, for example, 3D structure data of other known TyrRS complexes with tyrosyl AMP (for example, Brick et al., J. Mol. Biol., No. 208 ( 1988) 3-D structure data described in p. 83) Forces After referring to the position where tyrosyl AMP is recognized, the unnatural amino acid that can be a cross-linking agent in the sequence of SEQ ID NO: 1 It can be obtained by estimating the position to be recognized, that is, the position where the mutation is to be introduced, and introducing the mutation in a well-known site-specific manner.

In the case of an unnatural amino acid power ¾ Bpa that can serve as a cross-linking agent, TyrRS mutants include at least a tyrosine at position 37, an aspartic acid at position 182 and a phenotype at position 183 in this sequence. Examples include mutants in which specificity for pBpa is conferred by site-specific substitution of luranin and leucine at position 186 with other amino acids.

[0015] More preferably, the tyrosine at position 37 and the aspartic acid at position 182 are replaced with glycine, and the phalanin at position 183 is replaced with tyrosine, or the leucine at position 186 is replaced with alanine or methionine. Can be used. These variations increase the specificity for pBpa.

Next, as a method for producing these mutants, it is preferable to carry out by a known gene manipulation technique. For example, using a primer in which the base sequence encoding the target amino acid position is replaced with the base sequence encoding the amino acid to be modified, the DNA substituted with the base sequence encoding the amino acid to be modified is amplified and amplified. The DNA fragment can be ligated to obtain DNA encoding the full-length aaRS mutant, which can be easily produced by expressing it in a host cell such as E. coli. The primer used in this method is 20 to 70 bases, preferably about 20 to 50 bases. Since this primer has a mismatch of 1 to 3 bases with the original base sequence before modification, it is preferable to use a relatively long one, for example, 20 bases or more.

[0017] (suppressor tRNA)

The suppressor tRNA used in combination with the above TyrRS mutant It is assigned to a nonsense codon that is not a codon assigned to a type of amino acid, and is strongly recognized only by a TyrRS variant specific to the above-mentioned non-natural amino acid and not recognized by the host's normal aaRS (orthogonal tRNA) It must have the requirements and be expressed in animal cells.

Here, as the nonsense codon, it is preferable to use a UAG (amber) codon, such as UAG (amber), UAA (ocal), and UGA (opal).

[0018] In general, the expression of tRNA in eukaryotic cells requires two internal promoters within the tRNA coding sequence, and the consensus sequences are known as Box A and Box B.

The Bacillus stearothermophilus suppressor synthase tRNA is derived from prokaryotes, but has a box B and box A inside it (M. Sprinzl et al., Nucleic Acids Research 17 , 1-172 (1989)), we thought that it could be expressed in animal cells without any modification.

[0019] And, when the suppressor search synthase tRNA derived from Bacillus stearothermophilus was actually cloned into a vector for introduction into animal cells without modification, it was introduced into animal cells. Expression was confirmed. Then, it was confirmed that it exhibited a substituting activity in combination with the TyrRS mutant of E. coli.

That is, a prokaryotic suppressor tRNA having a box A sequence and a box B sequence and capable of retaining suppressor activity must have a suppressor activity in animal cells in combination with the TyrRS mutant derived from E. coli. I found out that I can.

[0020] The suppressor tRNA used in the present invention includes a mycoplasma genus or a staphylococcus genus authenticity that uses only a suppressor tRNA derived from the genus Bacillus that can bind to an unnatural amino acid in the presence of the tyrosyl tRNA synthetase variant. There is also a sub-lesser tRNA derived from bacteria. For the sequence of these tRNAs, see http: 〃 medlib.med.utah.e du / RNAmoas / trnabase / 3;

http://www.stalf.uni-bayreuth.ae/ btc914 / searcn /

These have a suppressor tRNA sequence that functions in prokaryotes, and two internal promoter consensus sequences that are recognized in animal cells. It is a subletter tRNA capable of binding to a non-natural amino acid that can be a cross-linking agent in the presence of yrRS mutant. Here, `` having a suppressor tRNA sequence that functions in prokaryotes '' is a prokaryotic suppressor tRNA that is a nonsense codon (usually an amber codon (UAG)) for functioning as a suppressor tRNA. It means that it retains complementary anticodon and three-dimensional structure (L-shaped structure part). Further, “having two internal promoter sequences recognized by animal cells inside” means that the consensus sequence of Box A and the consensus sequence of Box B are contained inside. In addition, “can bind to a non-natural amino acid that can be a cross-linking agent in the presence of the TyrRS mutant” means that it binds to a non-natural amino acid that can be specifically recognized by the TyrRS mutant and become a cross-linking agent. Suppressor tRNA that can be expressed, a suppressor mutant derived from tyrosine tRNA that normally binds tyrosine.

[0021] Examples of suppressor tRNAs derived from Bacillus, Mycoplasma, or Staphylococcus true bacterial tyrosine tRNAs include batinoles stearothermophilus tyrosin tRNA-derived suppressor tRNA, Bacillus subtilis tyros Suppressor tRNA (http: t medlib.med.utah.edu/RNAmods/trnab ase 8 registration number 0 丫 1540. \ ^ & \ ^ 0 ^ 61 ^ et al. (1984) J. Biol. Chem. 259 , 3694- 3702), tyrosine tRNA-derived suppressor tRNA from Mycoplasma capricolum (http: 〃 medlib.med.utah.edu/RNAmods/trnabase eight registration number DY1 140; Y. Andachi et al., (1987 ) Proc. Natl. Acd. Sci. USA 84, 7398— 7402), suppressor tRNA from tyrosine tRNA of Staphylococus aureus tRNA (http://medlib.med.utah.edu/RNAmods/trnabase/ Registration number DY1480; C.Green, (1993) J.Bac teriol. 175, 5091-5096), and a suppressor tRNA derived from tyrosine tRNA of Bacillus steer mouth thermophilus is preferably used.

[0022] (TyrRS mutant, suppressor tRNA expression in animal cells)

In order to express the TyrRS mutant in animal cells, any known expression system can be used. For example, commercially available pcDNA3.1 (manufactured by Invitrogen), pAGE107 (Cytotechnology, 3, 133 (1990)) PAGE103 [J. Biochem. L01, 1307 (1987)] and the like can be used. In addition, suppressor tRNA can be any known vector for Escherichia coli cloning. Even if one is used, it can be expressed in animal cells. For example, pBR322 (Proc. Natl. Acad. Sci. USA 75, 3737-3741 (1978)) can be used.

[0023] For the TyrRS mutant, a vector capable of inducible expression can be used if necessary, and for example, a tetracycline responsive promoter commercially available from Clontech, Invitrogene, etc. can be used.

Examples of methods for introducing a vector into cells include electroporation (Nucleic, Acids Res. 15, 1311-1326 (1987)), calcium phosphate method (Mol. Cell Biol. 7,2745- 2752 (1987)), Exclusion method (Cell 7,1025-1037 (1994); Lamb, Nature Genetics 5, 22-30 (1993)).

[0024] (Protein for incorporating unnatural amino acid)

The type of protein into which the unnatural amino acid is incorporated in the present invention is not limited, and any protein that can be expressed may be any heterologous recombinant protein. For example, protein types include so-called signal transduction related proteins, receptors, growth factors, cell cycle related factors, transcription factors, translation factors, transport related proteins, secreted proteins, cell skeleton related proteins, enzymes, chaperones or Examples include disease-related proteins including cancer, diabetes or genetic diseases. These proteins fulfill their functions through interaction and can be used in the present invention.

[0025] In the present invention, it is necessary to introduce a nonsense codon (an amber codon when the subletter tRNA is an amber suppressor) at a position where an unnatural amino acid is incorporated. ) Unnatural amino acid can be specifically incorporated into the site.

[0026] As a method for introducing a site-specific mutation into a protein, a well-known method can be used, and is not particularly limited. Gene 152, 271-275 (1995), Methods Enzymol. 100, 468-500 (1983), Nucleic Acids Res. 12,9441-9456 (1984), Proc. Natl. Acad. Sci. USA 82, 4 88-492 (1985), "Cell engineering separate volume" New cell engineering experiment protocol ", Shujunsha, 241— 248 (1993) "or the method using" QuickChange Site-Directed Mutagenesis Kit "(Stratagene), etc.

[0027] Since the present invention can be expressed in animal cells, Escherichia coli and cell-free proteins have been used so far. In the white matter system, unnatural amino acids can be incorporated into proteins that are not expressed, are low in expression, or cannot be post-translationally modified to become active. Such proteins are known to those skilled in the art, for example, the extracellular domain of a tyrosine kinase receptor such as human EGFR (Cell, 110,775-787 (2002)), human Groucho / TLEl protein (Structure 10,751-761 (2002)), rat muscle-specific kinase (Structure 10.1187-1196 (2002)), etc. The ability to synthesize aloproteins is not limited to these.

In the method of the present invention, since the aloprotein is expressed in animal cells, a non-natural amino acid can be incorporated into a glycoprotein bound to a sugar chain. In particular, in the case of a glycoprotein of a type in which the glycosylation pattern in the cell-free protein system is different from the original pattern, the system in the animal cell of the present invention has the target (original) pattern of sugar. It is considered to be an effective means for obtaining an aloprotein with a chain added.

[0028] (Host)

The host animal cell used in the present invention is preferably a mammalian cell in which a gene recombination system has been established. Examples of useful mammalian host cell lines include Chinese, Muster ovary (CHO) and COS cells. More specific examples are monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, J. Gen Virol , 36:59 (1977)); Chinese nomster ovary cells / -DHFR (CHO, Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)); Mouse Sertoli cells (TM4, Biol. 23: 243-251 (1980); human lung cells (W138, ATCC C CL 75); human liver cells (Hep G2, HB 8065); and mouse breast cancer (MMT 060562, ATCC CC L51). Each has established an expression system and selection of an appropriate host cell is within the skill of the artisan.

This can be carried out according to the method described in Molecular Cloning d | iR, Old Spring Harbor Laboratory Press (2001), etc. for column f.

[0029] Next, the state in which the aloprotein is expressed will be described with reference to FIG. The frame in Fig. 1 represents the cell membrane of animal cells (eg CHO cells). Within the cell (inside the frame), prokaryotic suppressor tRNA and TyrRS mutant are expressed in their respective expression systems. Crosslin A non-natural amino acid (eg pBpa) that can serve as a chelating agent is added to the medium (outside the frame). Non-natural amino acids that can serve as a cross-linking agent in the medium are taken into the cell by the action of the cell itself, and bind to the suppressor tRNA by the action of the TyrRS mutant. The unnatural amino acid that can be a cross-linking agent is then carried on the ribosome by the suppressor tRNA and used to translate the nonsense 'codon (here, the UAG codon). In order to produce a protein containing an unnatural amino acid that can serve as a cross-linking agent at a desired position, the codon at the corresponding position of the protein gene is substituted with UAG, and then this gene is expressed in the cell.

[0030] In this way, the target alloprotein in which the non-natural amino acid that can be a cross-linking agent is incorporated at the target position can be expressed in the animal cell.

Specifically, (A) a TyrRS mutant that expresses a TyrRS mutant with increased specificity for a non-natural amino acid that can be a cross-linking agent in animal cells, and (B) the TyrRS mutation described above. An expression vector for expressing a subletter tRNA derived from a Bacillus genus, Mycoplasma genus, or Staphylococcus spp. And (C) an animal cell having a desired protein gene having a nonsense mutation at a desired position, and a medium suitable for the growth of the animal cell (for example, Opti-MEM I (Gibco Incubate under appropriate conditions in a medium supplemented with a non-natural amino acid that can be used as a target cross-linking agent. For example, in the case of CHO cells, incubate at a temperature of about 37 ° C for about 24 hours. The amount of unnatural amino acid added as a cross-linking agent in the medium is about 0.1 to 3 mM, preferably about 0.3 mM.

[0031] Next, the second step will be described.

Aroprotein strength expressed in animal cells by the above When interacting with other proteins in the animal cell, the animal cell is incorporated into the protein by irradiating the animal cell with light of a specific wavelength. A non-natural amino acid that can be a cross-linking agent forms a covalent bond with the other protein. For example, when the cross-linking agent has a benzophenone skeleton, the carbonyl bond in the benzophenone is cleaved by irradiation with light of a specific wavelength, and the carbon atoms constituting the carbonyl bond are covalently bonded to other interacting proteins. . This In other words, by using the cross-linking method, the aloprotein and the other protein form a strong covalent bond, so that both proteins do not dissociate even after cell extraction operation. Can be easily detected. For this reason, in animal cells having a very complex cell structure compared to prokaryotes, it is easy to detect, for example, tyrosine kinase type receptor interactions, more specifically EGFR interaction, etc. that are peculiar to animal cells. be able to.

[0032] The wavelength of light to be irradiated has a force of 300 to 450 nm that varies depending on the cross-linking agent. S It is more preferable that the wavelength be in the preferred ultraviolet region (for example, 300 to 400 nm). When the cross-linking agent has a benzophenone skeleton, the force is preferably 350 to 380 nm. Further, when the cross-linking agent is pBpa, the force is particularly preferably 360 to 370 nm. The irradiation time is not particularly limited, but for example, 30 seconds to 1 hour is preferable. The irradiation is preferably performed, for example, on an ice bath after washing the cells with an appropriate buffer.

[0033] The third step is a step of detecting the presence or absence of protein-protein interaction, and a known detection method such as a Western plot method can be used. For example, the cells can be lysed with a buffer solution, subjected to SDS-PAGE, transferred to a PVDF membrane or the like, and then detected using an appropriate primary antibody or secondary antibody.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

Example 1

[0035] (Detection of interaction between EGFR and Grb 2 in signal transduction mechanism by EGF)

Intracellular signaling involved in proliferation can be triggered by administration of EGF. EGF specifically binds to EGFR located in the cell membrane. EGF-bound receptors activate their tyrosine (autophosphorylation). This state is a so-called excitement state for cells, and signals that lead to cell proliferation are relayed at once. In general, the relay from EGFR to proliferation typically goes through EGF → EGFR → Grb2 → SOS → Ras → MEK → MAPK → cell proliferation, t, and so on.

In this example, the interaction between EGFR and Grb2 was detected.

In the present invention, Grb2 represents a protein and grb2 represents a nucleobase. [0036] (Method)

Material

pBpa is from Bachem AG, anti-FLAG M2 antibody is from Sigma, anti-HA antibody is from Diagnostica, and anti-EGFR antibody is from Santa Cruz Biotechnology. Antibodies against tyrosine were purchased from Cell Signaling Technology. In addition, HRP (Horseradish Peroxidase) -labeled anti-mouse antibody and anti-rabbit Ig G antibody were purchased from Amersham Biosciences as secondary antibodies.

[0037] Site-directed mutagenesis and plasmid construction

The gene of E. coli TyrRS mutant (EcpBpaRS) that specifically recognizes pBpa was constructed by site-specific mutation of wild-type TyrRS derived from Escherichia coli, and amplified by PCR using mutant primers. Based on the binding structure of Grb2 SH2 domain and ligand peptide, select the position (Leul 11) that is close to the ligand binding site and that is not expected to inhibit binding, as the pBpa introduction position. Replaced by amber codon. More specifically, the leucine codon at position 111 of human grb2 was converted to an amber codon using a Quick Change site-directed mutagenesis kit (Stratagene) (grb2 (Ami 11)). HA tag (Y PYDVPDYA) was attached to the C-terminus of wild type E. coli TyrRS and EcpBpaRS. On the other hand, a FLAG sequence (DYKDDDK) was added to the C-terminus of the wild-type grb2 and grb2 (Ami 11) genes. Wild-type and mutant grb2, TyrRS gene, and EGFR gene were each cloned with pcDNA4 / TO vector (Invitrogen) for expression in cells. The plasmid of the suppressor tRNA was prepared by a known method (Patent Document 1).

[0038] Transfusion

T-REX-CHO cells (Invitrogen) that structurally produce tetracycline ribules grow to 90% confluence in a 90mm dish, and pcDNA4 / TO plasmid and suppressor tRNA expression plasmid can be transferred to Gibco Opti—MEMI medium ( Invitrogen Co., Ltd.) was transferred using LipofectAMINE2000 reagent. 4 hours after transfer, 20 mM HEPES-NaOH (pH 7.2) and 1 g / ml tetra The medium was changed to Salem-free Gibco D-MEM / F-12 medium containing cyclin. pBpa was added to the medium at a concentration of ImM. After an additional 12 hours of incubation, the cells were treated with lOOngZml EGF for 5 minutes at 37 ° C.

[0039] Western blot

The Western blot was performed according to the following procedure. Cells are diluted with buffer A (30 mM Tris-HCl buffer (pH 7.4), 10% glycerol, 1% saline X-100, 5 mM EDTA, 0.05% deoxycholate sodium, 100% protease inhibitor cocktail. (Nacalai Tesque)), and SDS-PAGE was performed and transferred to a PVDF membrane. Proteins were probed with antibodies and then detected with ECL plus immunodetection system (Amersham Biosciences). If the membrane is to be detected with two different antibodies, use 62.5 mM Tris-HCl buffer (pH 6.8) containing 2% SDS and 0.7% 2-mercaptoethanol for 30 minutes at 55 ° C. The first antibody was peeled off after treatment with, and the other antibody was allowed to act to detect. The band intensity was measured using an image analyzer LAS-1000 plus (Fuji Photo Film Co.).

[0040] Immunoprecipitation and cross-linking

In immunoprecipitation, cells were washed twice with phosphate-buffered saline (PBS) at pH 7.2 after EGF treatment and dissolved in buffer A containing ImM orthovanadate. The obtained extract is incubated with a affinity gel linked to anti-FLAG M2 antibody (Sigma) at 4 ° C for 2 hours, and the protein trapped on the gel is eluted with FLAG peptide (Sigma). It was. In the optical cross-link, these cells in PBS were placed on a culture dish on ice, and irradiated with 365 nm light from a distance of 2.5 cm using a handy 8W-lamp, UVP (Upland). Immediately after irradiation, the cell lysate was suspended in buffer A and treated with PTP1 B phosphatase (Calbiochem, EMD Bioscience) at 30 ° C for 30 minutes.

[0041] (Result)

Site-specific binding of pBpa to Grb2

Grb2 is an adapter protein that binds to EGFR and mediates extracellular signals to Ras protein. The interaction between the receptor and this Grb2 involves the SH2 region of Grb2, This region binds directly to receptor Y1068, which is phosphorylated upon EGF stimulation. The tertiary structure of the SH2 region of Grb2 forms a complex with the ligand peptide and has been determined by X-ray crystallography and NMR. The amino acid residue substituted by pBpa is a residue that is near the binding ligand but does not inhibit binding. Based on the crystal structure, we selected leucine 111 as the substitution position.

Figure 2 shows the results of site-specific binding of pBpa to Grb2 in CHO cells. Grb2 (pBpalll) was expressed with an anti-FLAG antibody, and TyrRS was expressed with an anti-HA antibody. In Lane 2 where wild type TyrRS (WTTyrRS), suppressor tRNA and grb2 (Ami 11) were introduced, Grb2 (pBpalll) was generated.The expression level was lower than that of wild type Grb2 (Grb2WT) (lane 1). It was a thing. This amber suppression was dependent on the expression of TyrRS and suppressor tRNA (lanes 2 and 3). When EcpBpaRS was expressed in cells instead of wild-type TyrRS, along with the addition of pBpa to the medium, the expression of Grb2 (pBpal 11) was confirmed (lane 7). From lanes 4, 5, and 6, it was confirmed that the expression of Grb2 (pBpalll) depends on the supply of pBpa into the medium in addition to EcpBpaRS and suppressor tRNA. From these findings, it was shown that the pBpa force Grb2 (pBpal l) was introduced at the position of the amber.

(Grb2 binding to EGFR)

Figure 3 shows the analysis results of the binding between Grb2 and EGFR by the coprecipitation method. First, the obtained cell extract, ie, whole cell lysate (WCL) was detected by Western plot (FIG. 3a). The primary antibody was an anti-EGFR antibody. From Fig. 3a, two bands with different molecular weights of 150 kDa and 170 kDa were detected (Fig. 3a, lanes 1 to 4), but the 150 kDa molecular weight band was derived from being overexpressed in CHO cells. Is a by-product. The results of immunoprecipitation of this extract and detection by Western plot are shown in FIGS. 3b, c and d. The co-precipitation of 170kDa EGFR and Grb2 was promoted by EGF treatment, and it was confirmed that Tyr 1068 of EGFR was phosphorylated in the co-precipitated product. On the other hand, a 150 kDa by-product is immunoprecipitated with the FLAG tag and phosphorylated Tyrl068, but is generated independently of EGF treatment (Figure 3b, c, lanes 1 and 2). When Grb2 (pBpal ll) was expressed, co-precipitation of Grb2 (pBpal ll) and EGFR and EGFR Tyrl068 phosphoric acid S was confirmed (FIGS. 3b and c, lanes 3 and 4). As described above, it was confirmed that Grb2 (pBpal 11) can retain the binding ability to phosphorylated Tyrl068 on EGFR.

[0043] (Grb2 (pBpal l 1) and EGFR cross-link)

Fig. 4 shows the results of cross-linking analysis between Grb2 and EGFR. In the cells that expressed Grb2 (pBpal 11), a single band was detected at a position of about 200 kDa (Figure 4a, b, lanes 8 and 9), and in the cells that expressed wild-type Grb2, the band was detected. (Figures 4a, b, lanes 1-5). It was concluded that this band is a cross-linked complex of Grb2 (pBpal l) and EGFR for the following reasons. First, the molecular mass force corresponds to the sum of the masses of GFR (170 kDa) and Grb2 (25 kDa). Second, the detection of this band depends on the light irradiation (irradiation time: 0, 5, 15 and 30 minutes) and the presence of pBpa. Thirdly, this product was detected not only by anti-FLAG antibody but also by anti-EGFR antibody.

The by-product 150 kDa EGFR did not crosslink with Grb2 (pBpal l l). This by-product is not expressed properly on the cell surface, and even if EGF-independent Y1068 is phosphorylated, it appears that it was unable to bind to Grb2. The co-immunoprecipitation of this by-product with Grb 2 is thought to be due to non-physiological binding that occurred during cell extract preparation or antibody incubation. Therefore, this method is useful for specifically detecting natural complexes formed in the cellular environment. The interaction between EGFR and Grb2 is EGF-dependent. That is, EGFR and Grb2 do not form a structure from the beginning, but specific sites of EGFR are phosphorylated upon stimulation of EGF, and Grb2 binds to it. Without EGF stimulation, no cross-linking occurs and the cells

The cross-linking does not occur until there is a series of signal transductions within. This is because the cross-links often reflect the correctness of the cells.

[0044] A pair of suppressor tRNAs and processed aaRS mutants having amino acid specificity were expressed in Escherichia coli cells, yeast, and mammalian cells, and a liver tree of amino acids binding to proteins was expanded. This technique has already been used to photocrosslink in E. coli between GST molecules containing pBpa. The results of this example relate to cellular signal proteins that interact with each other depending on tyrosine phosphorylation, and the in vivo cross-linking associated with genetic code expansion is applied to various protein-protein interactions in mammalian cells. If possible, suggest that you.

Cross-linked protein complexes are stable due to covalent bonds, and dissociation during cell extraction is not possible. This advantage allows separation of complexes formed by instantaneous or weak interactions between proteins that are not detected by coimmunoprecipitation. In addition, covalent bonds are formed in cells exposed to light, eliminating false interactions that occur during cell extraction or during antibody-incubation (eg, a 150 kDa EGFR by-product). These advantages are useful in studying cell signaling pathways.

Protein cross-linking has been used to determine the position of a protein within a macromolecular complex relative to the position of other proteins. The cross-linking shape of the two proteins indicates direct contact between these proteins in the complex without any intervening factors. Site-specific binding of an amino acid that serves as a cross-linking agent can accurately identify that site in a protein near the interacting protein. It has been reported that cross-linking does not occur when pBpa is bound to a certain GST position away from the dimer boundary. According to this example, the position of Grb2 to which pBpa binds was selected based on the crystal structure of Grb2 having a ligand peptide. The cross-link between Grb2 and EGFR confirmed that the residue at position 111 of Grb2 is located at the boundary where it interacts with the receptor.

Industrial applicability

The detection method of the present invention solves the problems of immunoprecipitation and can detect protein-protein interactions in animal cells with high accuracy. For example, it is useful for detecting interactions unique to animal cells, such as interactions in signal transduction systems and interactions with oncogene products.

Claims

The scope of the claims

[1] A prokaryotic-derived tyrosyl tRNA synthetase variant specific to a non-natural amino acid that can be a cross-linking agent, and a prokaryotic that can bind to the non-natural amino acid in the presence of the tyrosyl tRNA synthetase variant A biological suppressor tRNA and a desired protein gene that has undergone a nonsense mutation at a desired position are expressed in animal cells, and the non-natural amino acid is incorporated at the position of the nonsense mutation in the protein so that it is non-natural. The first step of expressing the amino acid-embedded protein,

A method for detecting an interaction between proteins, comprising:

[2] The method for detecting a protein-protein interaction according to [1], wherein the protein has a non-natural amino acid benzophenone skeleton that can be a cross-linking agent.

[3] The method for detecting a protein-protein interaction according to [1], wherein the non-natural amino acid power p-benzoyl L-feralanin which can be a cross-linking agent is used.

4. The method for detecting a protein-protein interaction according to claim 1, wherein the protein-protein interaction is an interaction of a tyrosine kinase type receptor.

5. The method for detecting a protein-protein interaction according to claim 1, wherein the protein-protein interaction is an epidermal growth factor receptor interaction.

6. The method for detecting protein-protein interaction according to claim 1, wherein the prokaryotic organism from which the tyrosyl tRNA synthetase mutant is derived is Escherichia coli.

[7] The method for detecting a protein-protein interaction according to [1], wherein the suppressor tRNA is a prokaryotic force Bacillus stearothermophilus.