WO2006057391A1 - Procédé de détection d’interaction entre des protéines - Google Patents

Procédé de détection d’interaction entre des protéines Download PDF

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WO2006057391A1
WO2006057391A1 PCT/JP2005/021824 JP2005021824W WO2006057391A1 WO 2006057391 A1 WO2006057391 A1 WO 2006057391A1 JP 2005021824 W JP2005021824 W JP 2005021824W WO 2006057391 A1 WO2006057391 A1 WO 2006057391A1
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protein
amino acid
interaction
detecting
cross
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Japanese (ja)
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Nobumasa Hino
Kensaku Sakamoto
Shigeyuki Yokoyama
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Riken
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/485—Epidermal growth factor [EGF] (urogastrone)

Definitions

  • the present invention relates to a method for detecting protein-protein interactions in animal cells using a cross-link method.
  • 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
  • aaRS RNA synthase
  • 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.
  • 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.
  • TyrRS tyrosyl-tRNA synthetase of Methanococcus jannaschii that specifically recognizes p-benzoyl-L-fe-lualanin
  • pBpa p-benzoyl-L-fe-lualanin
  • 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
  • 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.
  • EGF epidermal growth factor
  • Non-Patent Document 1 is a method used for prokaryotes and cannot be applied to animal cells as it is.
  • an object of the present invention is to provide means for detecting an interaction in an animal cell using a cross-link method.
  • the present invention provides:
  • 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.
  • 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.
  • 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 present invention makes it possible to easily and reliably detect protein-protein interactions in animal cells.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • UAG (amber) codon such as UAG (amber), UAA (ocal), and UGA (opal).
  • Bacillus stearothermophilus suppressor synthase tRNA is derived from prokaryotes, but has a box B and box A inside it (M. Sblul et al., Nucleic Acids Research 17 , 1-172 (1989)), we thought that it could be expressed in animal cells without any modification.
  • 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.
  • 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;
  • 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.
  • ⁇ 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).
  • “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.
  • “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 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 ⁇ 1540. ⁇ ⁇ & ⁇ ⁇ 0 ⁇ 61 ⁇ et al. (1984) J. Biol. Chem.
  • 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.
  • any known expression system can be used.
  • commercially available pcDNA3.1 manufactured by Invitrogen
  • pAGE107 Cellufactured by Invitrogen
  • PAGE103 J. Biochem. L01, 1307 (1987)] and the like
  • suppressor tRNA can be any known vector for Escherichia coli cloning. Even if one is used, it can be expressed in animal cells.
  • pBR322 Proc. Natl. Acad. Sci. USA 75, 3737-3741 (1978)
  • pBR322 Proc. Natl. Acad. Sci. USA 75, 3737-3741 (1978)
  • 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)).
  • 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.
  • 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.
  • 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.
  • the present invention can be expressed in animal cells, Escherichia coli and cell-free proteins have been used so far.
  • 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.
  • 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.
  • the aloprotein since the aloprotein is expressed in animal cells, a non-natural amino acid can be incorporated into a glycoprotein bound to a sugar chain.
  • 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.
  • the host animal cell used in the present invention is preferably a mammalian cell in which a gene recombination system has been established.
  • 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.
  • 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).
  • the codon at the corresponding position of the protein gene is substituted with UAG, and then this gene is expressed in the cell.
  • 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.
  • (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.
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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).
  • the force is preferably 350 to 380 nm.
  • 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.
  • 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.
  • 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.
  • 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.
  • the relay from EGFR to proliferation typically goes through EGF ⁇ EGFR ⁇ Grb2 ⁇ SOS ⁇ Ras ⁇ MEK ⁇ MAPK ⁇ cell proliferation, t, and so on.
  • Grb2 represents a protein and grb2 represents a nucleobase.
  • pBpa is from Bachem AG
  • anti-FLAG M2 antibody is from Sigma
  • anti-HA antibody is from Diagnostica
  • anti-EGFR antibody is from Santa Cruz Biotechnology.
  • Antibodies against tyrosine were purchased from Cell Signaling Technology.
  • HRP Haseradish Peroxidase
  • HRP Haseradish Peroxidase
  • E. coli TyrRS mutant 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)).
  • a Quick Change site-directed mutagenesis kit Stratagene
  • HA tag (Y PYDVPDYA) was attached to the C-terminus of wild type E. coli TyrRS and EcpBpaRS.
  • 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).
  • T-REX-CHO cells 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.
  • 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.).
  • 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.
  • FIG. 2 shows the results of site-specific binding of pBpa to Grb2 in CHO cells.
  • Grb2 pBpalll
  • TyrRS was expressed with an anti-HA antibody.
  • WTTyrRS wild type TyrRS
  • suppressor tRNA and grb2 Ami 11
  • Grb2 pBpalll
  • FIG. 3 shows the analysis results of the binding between Grb2 and EGFR by the coprecipitation method.
  • the obtained cell extract ie, whole cell lysate (WCL) was detected by Western plot (FIG. 3a).
  • the primary antibody was an anti-EGFR antibody.
  • 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.
  • Fig. 4 shows the results of cross-linking analysis between Grb2 and EGFR.
  • 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.
  • the molecular mass force corresponds to the sum of the masses of GFR (170 kDa) and Grb2 (25 kDa).
  • this band depends on the light irradiation (irradiation time: 0, 5, 15 and 30 minutes) and the presence of pBpa.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L’invention concerne un procédé de détection d’une interaction entre des protéines comprenant : la première phase dans laquelle un mutant TyrRS d’origine procaryote qui est spécifique à un acide aminé non naturel capable de servir d’agent de réticulation, un ARNt suppresseur d’origine procaryote qui est capable de se lier à l’acide aminé non naturel décrit plus haut en présence du mutant TyrRS décrit ci-dessus, et un gène de protéine désiré ayant une mutation non-sens en un site désiré sont exprimés dans une cellule animale de sorte que l’acide aminé non naturel décrit ci-dessus est intégré au site de mutation non-sens de la protéine décrite ci-dessus et la protéine ayant l’acide aminé non naturel intégré dans celle-ci est exprimée ; la deuxième phase dans laquelle la cellule animale décrite ci-dessus est irradiée avec une lumière d’une longueur d’onde spécifique ; et la troisième phase dans laquelle la présence ou l’absence de l’interaction entre les protéines est détectée.
PCT/JP2005/021824 2004-11-26 2005-11-28 Procédé de détection d’interaction entre des protéines WO2006057391A1 (fr)

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JP2004342914A JP2008035702A (ja) 2004-11-26 2004-11-26 蛋白質間相互作用の検出方法
JP2004-342914 2004-11-26

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008245591A (ja) * 2007-03-30 2008-10-16 Institute Of Physical & Chemical Research 光反応性官能基を有する非天然型アミノ酸組み込みタンパク質の合成方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004039989A1 (fr) * 2002-10-31 2004-05-13 Riken Procede permettant l'expression d'une proteine contenant un acide amine non naturel
WO2004094593A2 (fr) * 2003-04-17 2004-11-04 The Scripps Research Institute Extension du code genetique eucaryote

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004039989A1 (fr) * 2002-10-31 2004-05-13 Riken Procede permettant l'expression d'une proteine contenant un acide amine non naturel
WO2004094593A2 (fr) * 2003-04-17 2004-11-04 The Scripps Research Institute Extension du code genetique eucaryote

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CHIN J.W. ET AL.: "Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli", PROC. NATL. ACAD. SCI. USA, vol. 99, no. 17, 20 August 2002 (2002-08-20), pages 11020 - 11024, XP003006832 *
HINO N. ET AL: "Dobutsu Saibonai ni okeru Hi Tennengata Amino-san no Tanpakushitsu heno Bui Tokuiteki Donyu ni yoru in vivo Hikari Cross-linking-ho no Kaihatsu", DAI 27 KAI ANNUAL MEETING OF THE MOLECULAR BIOLOGY SCIENTY OF JAPAN PROGRAM KOEN YOSHISHU (1PB-551), 25 November 2004 (2004-11-25), pages 609, XP003006831 *
HINO N. ET AL: "Jinkoteki na Amino-san o Tanpakushitsu ni Donyusuru koto niyoru Dobutsu Saibonai deno Hikari Cross-linking-ho", EXPERIMENTAL MEDICINE, vol. 23, no. 1, 1 August 2005 (2005-08-01), pages 2047 - 2051, XP003006834 *
HINO N. ET AL: "Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid", NAT. METHODS, no. 3, 2 March 2005 (2005-03-02), pages 201 - 206, XP003006833 *
OGISO H. ET AL.: "Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains", CELL, vol. 110, no. 6, 20 September 2002 (2002-09-20), pages 775 - 787, XP002961395 *
RAHUEL J. ET AL.: "Structural basis for the high affinity of amino-aromatic SH2 phosphopeptide ligands", J. MOL. BIOL., vol. 279, no. 4, 19 June 1998 (1998-06-19), pages 1013 - 1022, XP004466356 *
SAKAMOTO K. ET AL: "Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells", NUCLEIC ACIDS RES., vol. 30, no. 21, 1 November 2002 (2002-11-01), pages 4692 - 4699, XP002976283 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008245591A (ja) * 2007-03-30 2008-10-16 Institute Of Physical & Chemical Research 光反応性官能基を有する非天然型アミノ酸組み込みタンパク質の合成方法

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