JP5392992B2 - Autophosphorylation receptor agonist, antagonist screening method, genetically modified yeast - Google Patents

Description

  The present invention relates to a method for screening a substance (agonist or antagonist) that binds to the receptor by expressing in yeast the receptor whose autophosphorylation (protein kinase) action is activated when the agonist binds to the receptor. , Genetically modified yeast, kits and combinations.

  Many receptors are present in the cell membrane and have a structure for recognizing external stimuli and transmitting information.

As a receptor, a high-throughput screening system for agonists in which a G protein-coupled receptor (GPCR) is incorporated into yeast is known (Patent Documents 1 and 2).
JP2005-176605 WO2006 / 104254 Kim YS, Bhandari R, Cochran JR, Kuriyan J, Wittrup KD. Proteins. 2006 Mar 1; 62 (4): 1026-35. T Yi, AL Mui, G Krystal, and JN Ihle, Mol Cell Biol. 1993 December; 13 (12): 7577-7586 (Common IL-3 receptor beta chain (IL-5 receptor beta chain) phosphorylation) That occurs depending on the agonist). Pratt JC, Weiss M, Sieff CA, Shoelson SE, Burakoff SJ, Ravichandran KS.J Biol. Chem. 1996 May 24; 271 (21): 12137-40 (the beta chain phosphorylated by JAK2 is linked to the Shc adapter protein. (Indicates that this Giordano V, De Falco G, Chiari R, Quinto I, Pelicci PG, Bartholomew L, Delmastro P, Gadina M, Scala G. J Immunol. 1997 May 1; 158 (9): 4097-4103 (phosphorylated by JAK2 (Shows that gp130 binds to Shc adapter protein.)

  Autophosphorylated receptors such as EGF and IL-5 are important in the basic functions of the living body. For example, gefitinib on which the EGF receptor acts as an anticancer agent is marketed.

  On the other hand, an agonist / antagonist high-throughput screening system for autophosphorylated receptors has not been established.

  An object of the present invention is to provide a high-throughput screening system for agonists / antagonists for autophosphorylated receptors.

  G protein-coupled receptors (GPCRs) originally exist in yeast, and it is predicted that mammalian GPCRs can be expressed in yeast. In fact, Patent Documents 1 and 2 screen for agonists / antagonists for GPCRs using the trimeric G protein of GPCRs originally present in yeast.

  On the other hand, since there is no autophosphorylated receptor and an adapter protein capable of recognizing the phosphorylation site of the receptor in yeast, it cannot be predicted at all whether it actually functions in yeast when expressed separately. It was. Reported that only mammalian EGF receptor was expressed in yeast and that its expression was confirmed with antibodies, and deletion mutants of EGF receptor having only extracellular domain expressed in yeast cells can bind to EGF. (Non-Patent Document 1). However, it has not been confirmed that the receptor actually functions in yeast, and the adapter protein is not co-expressed. Therefore, Non-Patent Document 1 does not disclose any method for screening for agonist / antagonist.

In the present invention, as a means for solving the above-mentioned problems, a screening system using a modified yeast cell expressing a mammalian-derived autophosphorylated receptor is provided.
1. A method for screening an agonist / antagonist, wherein an agonist / antagonist candidate substance of the receptor acts on a yeast co-expressing a mammalian autophosphorylated receptor and an adapter protein capable of recognizing the phosphorylated receptor Detecting a yeast in which the agonist / antagonist candidate substance is bound to the receptor, wherein the autophosphorylated receptor is multimerized by binding of a ligand, and the receptor itself has a kinase action or receptor. A method wherein the action of a tyrosine kinase that constitutively binds to the body phosphorylates the receptor itself and / or a tyrosine kinase that constitutively binds to the receptor.
2. Item 2. The method according to Item 1, wherein the autophosphorylated receptor is multimerized by binding of a ligand, and the receptor itself is phosphorylated by the kinase action of the receptor itself.
3. The autophosphorylated receptor is multimerized by the binding of a ligand, and both the receptor itself and the tyrosine kinase that binds constitutively to the receptor are phosphorylated by the action of the tyrosine kinase that constitutively binds to the receptor. Item 2. The method according to Item 1.
4). A method for screening an agonist / antagonist, wherein an agonist / antagonist candidate substance of the receptor is allowed to act on a yeast expressing a mammalian autophosphorylated receptor, and the agonist / antagonist candidate substance binds to the receptor. Detecting the yeast
Tyrosine that binds to the receptor itself or to the receptor by multimerization of the autophosphorylated receptor by the binding of an agonist and by the kinase action of the receptor itself or the action of tyrosine kinase that constitutively binds to the receptor The kinase is phosphorylated,
The phosphorylation detection is performed by lysing the cell wall of the yeast and allowing the antibody capable of detecting phosphorylation to act on the cell wall lysed yeast.
Method.
5. Item 5. The method according to Item 4, wherein the autophosphorylated receptor is multimerized by the binding of an agonist, and the receptor itself is phosphorylated by the kinase action of the receptor itself.
6). The autophosphorylated receptor is multimerized by the binding of an agonist, and both the receptor itself and the tyrosine kinase that binds constitutively to the receptor are phosphorylated by the action of the tyrosine kinase that constitutively binds to the receptor. Item 5. The method according to Item 4.
7). The mammalian autophosphorylated receptor is
(i) the receptor itself has an autophosphorylation effect, or
(ii) a receptor that shares gp130 or βc, and that tyrosine kinase constitutively binds to gp130 or βc and phosphorylates both tyrosine kinase itself and gp130 or βc by the action of the tyrosine kinase. 5. The method according to 1 or 4.
8). Item 5. The method according to Item 1 or 4, wherein the tyrosine kinase that constitutively binds to a receptor is JAK1, JAK2, or JAK3.
9. Item 5. The receptor according to Item 1 or 4, wherein the tyrosine kinase is constitutively bound and the receptor to which the tyrosine kinase is phosphorylated in an agonist-dependent manner includes an α chain and a β chain (βc) of an interleukin receptor. Method.
10. Item 5. The method according to Item 1 or 4, wherein the receptor to which the tyrosine kinase is constitutively bound and the tyrosine kinase is phosphorylated in an agonist-dependent manner comprises gp130.
11. Mammalian autophosphorylation that undergoes tyrosine kinase and / or receptor phosphorylation by the action of tyrosine kinase that is multimerized by the binding of an agonist and constitutively binds to the receptor of the agonist or the receptor itself A genetically modified yeast obtained by co-expressing a type receptor and an adapter protein capable of recognizing the phosphorylation in yeast.
12 Item 12. The gene set according to Item 11, which has a tyrosine kinase action and co-expresses a mammalian receptor that multimerizes upon binding of an agonist and is autophosphorylated, and an adapter protein that can recognize phosphorylation of the receptor. Replacement yeast.
13. A receptor sharing βc or gp130 that multimerizes upon binding of an agonist and a tyrosine kinase that constitutively binds to the receptor (the tyrosine kinase phosphorylates βc or gp130 and tyrosine kinase itself upon binding of the agonist) Item 12. The genetic recombinant yeast according to Item 11, which co-expresses an adapter protein capable of recognizing phosphorylation of βc, gp130, or tyrosine kinase.
14 Mammalian autophosphorylation that undergoes tyrosine kinase and / or receptor phosphorylation by the action of tyrosine kinase that is multimerized by the binding of an agonist and constitutively binds to the receptor of the agonist or the receptor itself A kit for screening an agonist / antagonist for the receptor comprising a type receptor and an antibody capable of detecting the phosphorylation.
15. Item 15. The kit according to Item 14, further comprising an enzyme that dissolves the cell wall of yeast.
16. Mammalian autophosphorylation that undergoes tyrosine kinase and / or receptor phosphorylation by the action of tyrosine kinase that is multimerized by the binding of an agonist and constitutively binds to the receptor of the agonist or the receptor itself A combination of a type receptor and an antibody capable of detecting the phosphorylation.
17. Mammalian autophosphorylation that undergoes tyrosine kinase and / or receptor phosphorylation by the action of tyrosine kinase that is multimerized by the binding of an agonist and that constitutively binds to the receptor of the agonist or the receptor itself A combination of a type receptor, an enzyme that dissolves the cell wall of yeast, and an antibody capable of detecting the phosphorylation.

As used herein, autophosphorylated receptors include the following two types of receptors:
(a) Sharing βc or gp130, forming a multimer containing a receptor and βc or gp130 by binding an agonist, and activating tyrosine kinase constitutively bound to βc or gp130 to activate βc or gp130 and tyrosine A receptor that phosphorylates kinases.

 (b) A receptor that itself has a tyrosine kinase action, the receptor multimerizes by binding of an agonist, and the multimerized receptor autophosphorylates by a tyrosine kinase action.

  In the case of (a) above, the receptor moiety to which the ligand (agonist / antagonist) can bind is not phosphorylated, but the receptor moiety forms a heteromultimer with βc or gp130, and βc or gp130 and tyrosine kinase In the present specification, such a receptor is also referred to as an autophosphorylated receptor because (which constitutively binds to βc or gp130) is phosphorylated and a heteromultimer (receptor complex) is phosphorylated. That's it.

  “Constitutively binds” means that tyrosine kinase and βc or gp130 bind / associate in yeast even when tyrosine kinase and βc or gp130 are expressed separately (not chimeric protein) in yeast. These may be expressed in yeast as separate proteins or may be expressed as chimeric proteins.

The features of the present invention are as follows:
First, a mammalian-derived receptor that is not present in yeast and an adapter protein that is not present in yeast (preferably derived from mammals) are simultaneously expressed in yeast, thereby allowing agonists for the receptor to be expressed in non-yeast receptor systems. It was confirmed that the signal due to binding was correctly transmitted in yeast.

  Second, a mammal-derived autophosphorylated receptor that does not exist in yeast is expressed in yeast, and autophosphorylation by binding of an agonist to the receptor is confirmed by an antibody. In particular, the present inventor has revealed for the first time that phosphorylation in yeast can be confirmed by lysing the cell wall without disrupting the yeast, and that agonists / antagonists can be reliably screened.

The present invention enables screening for agonists / antagonists by detecting the phosphorylation reaction and multimer formation of autophosphorylated receptors caused by binding of an agonist and a receptor by the following two methods ( (See FIGS. 1, 6, and 7). Here, in the case where the receptor itself is a receptor having a tyrosine kinase action (for example, EGF receptor), phosphorylation is performed on the receptor itself, and the receptor itself has no tyrosine kinase action. Receptors in which tyrosine kinases (e.g., Janus kinases (JAK) such as JAK1, JAK2, JAK3) are constitutively bound to βc or gp130 shared by the body (contained in the receptor complex when the ligand is bound) In this case, both βc or gp130 and tyrosine kinase. In the latter case, phosphorylation recognized by the adapter protein may be either βc / gp130 phosphorylation or phosphorylation of the tyrosine kinase itself, but preferably the adapter protein recognizes tyrosine kinase phosphorylation. .
First method: An adapter protein that specifically binds to a phosphorylated receptor is simultaneously expressed in yeast together with a mammalian (especially human) receptor, and the binding between the adapter protein and the receptor is expressed in reporter gene expression or cells. Method to detect by proliferation. In the present specification, the adapter protein means phosphorylation of βc or gp130 shared by the receptor, phosphorylation of tyrosine kinase constitutively bound to βc or gp130, or phosphorylation of the receptor itself having tyrosine kinase action. It means a protein that recognizes oxidation.

  Examples of adapter proteins include Shc and Grb2, and mammal-derived adapter proteins are preferred.

  Shc / Grb2 may be expressed alone in yeast or may be fused to a GDP / GTP exchange factor (eg, Sos). In the present invention, it is preferable to further co-express a GDP / GTP exchange factor (for example, Sos) in yeast. The origin of the GDP / GTP exchange factor is preferably a mammal (particularly human), but may be other. When the Cdc25 thermosensitive mutant is used as yeast, the GDP / GTP exchange factor (for example, Sos) can replace the function of the Cdc25 thermosensitive mutant originally expressed in yeast under the culture conditions of 37 ° C. Shc / Grb2 binds to phosphorylated βc or phosphorylated gp130, and the GDP / GTP exchange factor (eg, Sos) fused to this Shc / Grb2 as necessary is concentrated near the intracellular membrane Then, since the GDP / GTP exchange factor (for example, Sos) promotes the exchange of the GDP bound to the yeast endogenous Ras1 / 2 to the GTP, the growth of the yeast is promoted. This enables screening of agonist binding to the receptor using yeast growth as an indicator, or activation of a transcription factor via Ras1 / 2 upon binding of a GDP / GTP exchange factor (for example, Sos) to the reporter gene. It can also be detected by assay (see FIGS. 1 and 7).

  In a particularly preferred embodiment of the present invention, an adapter protein Shc or adapter protein Grb2 fused with mammalian-derived Sos (GDP / GTP exchange factor) (Shc-Sos or Grb2-Sos) can be used. Under screening conditions at 37 ° C., mammalian-derived Sos acts as a substitute for yeast endogenous Cdc25H (a GDP / GTP exchange factor, heat sensitive), and carries out signal transduction to proliferate cells. In this screening, signal transduction is as follows: agonist binds to receptor → receptor multimerization → phosphorylation of receptor tyrosine residue → receptor phosphorylation tyrosine residue Shc Or, when Grb2 recognizes and binds, Shc-Sos or Grb2-Sos is concentrated near the yeast cell membrane → Shc-Sos or Grb2-Sos concentrated near the cell membrane acts instead of yeast endogenous Cdc25H The cells proliferate.

Second method: detection by an antibody that specifically binds to a phosphorylated receptor. By allowing the antibody to act after dissolving the yeast cell wall, the antibody moves into the yeast, and phosphorylation of an autophosphorylated receptor present on the surface of the cell membrane can be detected. While not wishing to be bound by theory, it is important that antibodies migrate into yeast when pores are opened in the cell membrane when the cell wall is lysed, or part or the entire structure of the cell membrane is weakened. The present inventor believes that it is easy for antibodies to enter.

  Antagonist screening is done by adding a known agonist to the yeast medium in advance, or using a yeast that can express the agonist on the cell wall, and the expression of the reporter gene and cell growth by this known agonist are ineffective. Can be performed by screening.

  Random peptides can be expressed on the surface of yeast cells by combinatorial engineering of agonist peptide proteins in yeast. Specifically, a random peptide is linked to a signal peptide and an anchor peptide that is bound to the cell wall surface. When the random peptide is secreted from the cell membrane, the anchor peptide is caught on the cell wall, allowing the random peptide to bind to the cell membrane receptor. Examples of the anchor protein include: FLO42 (42 peptides at the C-terminal side of FLO1 protein), C-terminal 320 amino acids of α-agglutinin, C-terminal 42 amino acids of yeast aggregating protein FLO1 (FLO42), C-terminal 102 amino acids (FLO102), C-terminal 146 amino acids (FLO146), C-terminal 318 amino acids (FLO318), ethanolamine phosphate-6 mannose α1-2 mannose α1-6 mannose α1-4 glucosamine α1-6 inositol phospholipid Glycolipid (GPI anchor) as the basic structure.

  Alternatively, a peptide that is a candidate for an agonist is linked to a sugar chain-binding protein domain and expressed in yeast, and the sugar chain part of the domain interacts with or entangles with a sugar chain in the cell wall, so that it remains on the cell surface layer and is a receptor. Can also be combined.

  In the present invention, suitable yeasts for expressing the receptor include S. cerevisiae BY4741, ATCC60715, MT8-1, W303a, IFO10150, YPH499, BJ5464 and the like. Since a strain having a large amount of signal transduction is preferable, it is preferable to select such a yeast strain.

In the present specification, preferable examples of the tyrosine kinase include JAK (JAK1, JAK2, JAK3, particularly JAK2), Lyn, Yes, Fyn, Fes, Hck and the like, and preferably JAK2. Autophosphorylated receptors include those that constitutively bind tyrosine kinases such as JAK (JAK1, JAK2, JAK3, especially JAK2), such as gp130 and βc chains, and EGF receptor (EGFR). The receptor (EGFR) itself has tyrosine kinase activity. In particular:
(i) Receptor sharing gp130
IL-6R, IL-11R, LIFR, CNTFR, OSMR, CT-1R, etc.
(ii) Receptor sharing βc
IL-3R, IL-5R, GM-CSFR, etc.
(iii) Ligand of receptor having autophosphorylation effect
AlkR family including Alk, AxlR family including Axl, DDR family including DDR1, EphR family including EGFR, EphAR family including Eph A1, EphBR family including Eph B1, FGFR family including FGFR1, IR including IFG-1R MetR family including Met, MetR family including MuSK, MuSKR family including MuSK, Neurotrophin R family including TrkA, PDGFR family including PDGFR-α, RETR family including RET, RorR family including Ror1, RykR family including Ryk, Sev SevR family including Tie1, TieR family including Tie1, VEGFR family including VEGFR-1.

  A screening system for a beta chain (βc) covalent receptor and a screening for a gp130 covalent receptor can be performed in the same manner. For example, Non-Patent Documents 2 and 3 show that the beta chain is phosphorylated by JAK2 and the adapter protein Shc binds to the phosphorylation site. Non-Patent Document 4 shows that gp130 is phosphorylated to JAK2 and adapter protein Shc binds thereto. That is, Shc shows the behavior of binding to the phosphorylation site of the intracellular domain of the receptor in the same manner for both beta chain and gp130. Therefore, the detection system using Shc is a beta chain shared type. It is clear that it can be used in screening systems for both receptors (eg IL-5) and gp130 covalent receptors (eg IL-6, LIF, etc.).

  In the present invention, tyrosine kinase such as JAK (for example, JAK2) and βc / gp130 may be expressed separately or may be expressed as a chimeric protein. It is known from the literature that tyrosine kinases such as JAK and βc / gp130 are expressed constitutively even if they are expressed separately (Zhao Y, Wagner F, Frank SJ, Kraft AS. J Biol Chem. 1995). Jun 9; 270 (23): 13814-8.).

The screening method of the present invention can screen for a substance capable of binding to the above receptor. When screening for an agonist, an agonist candidate substance is allowed to act on the yeast, and the presence or absence of binding of the candidate substance to the receptor may be detected. In addition, when detecting an antagonist, an antagonist candidate substance is allowed to act in the presence of an appropriate concentration of an agonist to detect how much the binding of the agonist has decreased. In the present invention,
In the "step of detecting a yeast in which an agonist / antagonist candidate substance is bound to a receptor", an agonist candidate substance is allowed to act on yeast to detect the presence or absence of binding of the candidate substance to the receptor. Both include the case where an antagonist candidate substance is allowed to act in the presence of various concentrations of an agonist to detect how much the agonist binding has been reduced.

  The agonist can be bound to the receptor by adopting a method in which autophosphorylated gp130 or βc is expressed by a reporter protein in the growth of yeast cells or in the intracellular transmission pathway, or detected by an antibody. it can.

  For example, as shown in FIG. 6, the IL-5 receptor is composed of an IL-5 receptor α chain and a β chain (βc). Tyrosine kinase JAK2 is constitutively bound to the β chain, and autophosphorylation of JAK2 is caused by binding of IL-5 and IL-5 receptor α chain.

  Similarly, for a receptor having gp130 instead of βc, autophosphorylation by JAK2 constitutively bound to gp130 occurs when a ligand binds to the receptor.

On the other hand, as shown in FIG. 1, in the EGF receptor and the like, dimerization / multimerization of the receptor occurs due to the binding of the ligand (EGF), thereby inducing the autophosphorylation of the receptor.
1. Detection Method of Agonist / Receptor Reaction Using Antibody Detection of receptor autophosphorylation with an anti-phosphotyrosine kinase (for example, JAK2) specific antibody enables screening for agonists. (Figure 1)
Examples of the phosphorylation site recognized by the anti-phosphorylated antibody include the following positions in JAK1, JAK2, and EGF receptors. However, the amino acid number is a number in which the N-terminal amino acid residue in the human-derived protein is the first.
1. JAK2 phosphorylation sites: Tyr221, Tyr813, Tyr1007 / 1008
2. JAK1 phosphorylation site: Tyr1022 / 1023,
3. EGF receptor phosphorylation sites: Tyr974, Tyr992, Tyr1045, Tyr1068, Tyr1086, Tyr1148, Tyr1173
Antibodies that recognize the above phosphorylation sites are known, and autophosphorylated receptors can be detected by using known antibodies. Detection can be performed based on detection of the presence of a secondary antibody label in the vicinity of the yeast membrane, for example, by causing an antibody that specifically recognizes a phosphorylation site and a labeled secondary antibody that recognizes the antibody to act on yeast. . For receptors having tyrosine kinase activity other than JAK1 and JAK2 and tyrosine kinase activity other than EGF receptor, receptors autophosphorylated by antibodies can be detected in the same manner.

2. A method using signal transduction in yeast.
To detect JAK2 phosphorylation, a method using a human-derived adapter protein and signal transduction to yeast endogenous Ras1 or Ras2 similar to the EGF receptor screening method can also be used (FIG. 7). Specifically, a human-derived Sos protein fused with an adapter protein Shc that specifically binds to an agonist-dependent phosphorylated receptor is concentrated in the vicinity of the cell membrane from the cytoplasm in an agonist-dependent manner, so that GDP-bound yeast endogenous This screening method utilizes the fact that yeast cell division is induced by converting sex Ras1 or Ras2 into a GTP-binding type.

  The screening methods shown in FIGS. 6 and 7 can be applied to receptor groups sharing the β chain, that is, IL-3 receptor, GM-CSF, similarly to IL-5 receptor. Also, an agonist of IL-6 receptor, IL-11 receptor, LIF receptor, CNTF receptor, OSM receptor, CT-1 receptor having a molecular structure similar to that of IL-5 receptor and sharing gp130 / Applicable as an antagonist screening method. Furthermore, it has a molecular structure similar to that of IL-5 receptor, and is also applied to IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, and IL-15 receptor sharing the γ chain. Applicable.

  When screening for an antagonist, a known agonist is added in advance to a yeast medium, and a compound that disables autophosphorylation of JAK1 or JAK2 by this known agonist is screened.

  Screening using cell growth as an indicator by using the yeast endogenous gene promoter, which is located downstream of yeast endogenous Ras protein signaling and whose expression increases with Ras activation, as the reporter gene promoter. In addition, screening using reporter gene expression as an index can be performed. Reporter genes include genes for fluorescent proteins including GFP, coloring of β-galactosidase and luciferase, genes for enzymes for which luminescent substrates have been developed, and auxotrophic marker genes. Yeast endogenous Ras activates PDE2, a yeast endogenous cAMP-dependent phosphodiesterase (Sass P, Field J, Nikawa J, Toda T, Wigler M. Proc Natl Acad Sci U. Sci U.S. 1988). : 9303-7.). In addition, genes whose expression is induced by the expression of PDE2 have been comprehensively investigated (Jones DL, Petty J, Hoyle DC, Hayes A, Ragnai E, Popolo L, Oliver SG, Stateva LI. Physiol Genomics. 200). Dec 16; 16 (1): 107-18.). Therefore, a promoter of a gene whose expression is reported to be induced by PDE2 can be a promoter of a reporter gene in this screening method.

In the present invention, an actual drug candidate compound screening method is exemplified below.
I. When adding low molecular weight compounds or proteins to the culture solution from outside-Place a liquid or agar medium in a 96-well plate and seed the yeast. A compound or protein is added thereto, and the expression of the reporter gene is observed using yeast growth, fluorescence and color development as indicators. At this time, if growth or expression of a reporter gene is detected in yeast, the compound is an agonist.
・ Place liquid culture medium or agar culture medium in 96-well plate and seed yeast. Compound or protein is added to each protein, and autophosphorylation in receptor tyrosine kinases such as EGFR, and autophosphorylation of JAK1 and JAK2 in cytokine receptors such as IL-5R and IL-6R. Detection is by immunostaining using specific phosphorylated antibodies. At this time, if autophosphorylation of receptor tyrosine kinase or autophosphorylation of JAK1 or JAK2 is observed, the compound is an agonist. Immunostaining requires treatment to remove yeast cell walls.
・ Place yeast in a liquid or agar medium containing agonist in a 96-well plate. A compound or protein is added thereto, and the expression of the reporter gene is observed using yeast growth, fluorescence and color development as indicators. At this time, if the yeast does not express the reporter gene, the compound is an antagonist.
・ Place yeast in a liquid or agar medium containing agonist in a 96-well plate. Compound or protein is added to the protein, and autophosphorylation in receptor tyrosine kinases such as EGFR, and autophosphorylation of JAK1 and JAK2 in cytokine receptors such as IL-5R and IL-6R. Detection is performed by immunostaining using a typical phosphorylated antibody. At this time, if autophosphorylation of receptor tyrosine kinase or JAK1 or JAK2 is not observed, the compound is an antagonist. Immunostaining requires treatment to remove yeast cell walls.

II. When a random peptide is incorporated into the yeast itself: A yeast expressing a random peptide is spread on an agar medium, and the expression of the reporter gene is observed using yeast growth, fluorescence, and color development as indicators. At this time, if the yeast expresses a reporter gene, the yeast is a yeast that expresses an agonist peptide. Subsequently, the plasmid sequence can be identified by extracting the plasmid introduced to express the peptide from yeast and conducting sequence analysis.
・ Random peptide expressing yeast is plated on agar, autophosphorylation in receptor tyrosine kinases such as EGFR, and JAK1 and JAK2 autophosphorylation in cytokine receptors such as IL-5R and IL-6R Is detected by immunostaining using a phosphorylated antibody specific for each protein. At this time, if autophosphorylation of a receptor tyrosine kinase or autophosphorylation of JAK1 or JAK2 is observed, the yeast is a yeast that expresses an agonist peptide. Immunostaining requires treatment to remove yeast cell walls. Subsequently, the plasmid sequence can be identified by extracting the plasmid introduced to express the peptide from yeast and conducting sequence analysis.
-Seed the expression of the reporter gene by using yeast that expresses a random peptide in an agar medium containing an agonist and using the growth, fluorescence, and color of the yeast as indicators. At this time, if the yeast does not express the reporter gene, the yeast is a yeast that expresses an antagonist peptide. Subsequently, the plasmid sequence can be identified by extracting the plasmid introduced to express the peptide from yeast and conducting sequence analysis.
・ Place yeast expressing a random peptide on an agar medium containing an agonist, autophosphorylate in receptor tyrosine kinases such as EGFR, and JAK1 and IL-5R and cytokine receptors such as IL-6R. JAK2 autophosphorylation is detected by immunostaining using a phosphorylated antibody specific for each protein. If autophosphorylation of a receptor tyrosine kinase or JAK1 or JAK2 is not observed at this time, the yeast expresses an antagonistic peptide. Immunostaining requires treatment to remove yeast cell walls. Subsequently, the plasmid sequence can be identified by extracting the plasmid introduced to express the peptide from yeast and conducting sequence analysis.

As shown in the Examples of the present invention, by detecting the yeast cell wall and allowing the antibody to act, detection by the antibody can be performed while maintaining the yeast form. The following are illustrated as an advantage of the immunostaining method which does not destroy the form of yeast.
-Needless to say, it is necessary to use an antibody for immunostaining, but it was found that the antibody was non-specifically adsorbed on the yeast cell wall. For this reason, treatment to remove the yeast cell wall was required.
・ When processing to remove the yeast cell wall, it is observed whether EGFR autophosphorylation or autophosphorylation such as JAK1 or JAK2 occurs accurately on the yeast cell membrane depending on the compound, protein, or peptide to be screened. In this case, false positives can be excluded, and screening with higher accuracy becomes possible. Therefore, it is preferable not to break the shape of the yeast.
・ When a low molecular weight compound or protein is added to the culture solution from the outside, the compound-dependent autophosphorylation used is accurately determined by observing whether autophosphorylation is occurring accurately on the surface of the yeast cell. Whether it is occurring on the yeast cell membrane.
・ When a random peptide is incorporated into the yeast itself, the target cell is automatically selected by using a single cell handling device, specifically, a FACS with a built-in cell sorter or a target cell sorting device with a built-in equivalent system. It is possible to isolate with high throughput. At this time, it is necessary to extract the plasmid introduced to express the peptide from the yeast and specify the peptide sequence by sequence analysis, and it is an absolute condition not to destroy the shape of the yeast.
-From the above, it is preferable to detect the fluorescence in the vicinity of the yeast cell membrane in any case where a low molecular weight compound or protein is added to the culture solution from the outside or a random peptide is incorporated into the yeast itself. It is advantageous not to break the shape of the yeast.

EXAMPLES Hereinafter, although this invention is demonstrated in detail according to an Example, it cannot be overemphasized that this invention is not limited to these Examples.
Example 1: Construction of plasmid for reconstituting epidermal growth factor (EGF) signaling in yeast 1-1: Construction of EGF receptor expression plasmid Encoding human-derived EGF receptor (EGFR) which is a single transmembrane receptor Using this gene, pGMH20-MFα-FLAG-EGFR was constructed as follows.

1-1-1: Construction of pGMH20-MFα-FLAG Encoding the prepro sequence by PCR using the gene encoding the prepro sequence (J. Bacteriol., 903-906, 1983) of the yeast secretory protein MFα1 as a template A gene encoding a FLAG tag sequence on the 3 'side of the gene to be amplified and an amplified fragment (a) with restriction enzyme sites added to both ends were obtained. The amplified fragment (a) was digested with a restriction enzyme and inserted into the pGMH20 vector to obtain pGMH20-MFα-FLAG.

1-1-2: Construction of pGMH20-MFα-FLAG-EGFR An amplified fragment in which restriction enzyme sites are added to both ends of a gene encoding mature EGFR protein is obtained by PCR using a plasmid encoding EGFR gene as a template. It was. This amplified fragment was digested with a restriction enzyme and inserted into pGMH20-MFα-FLAG to obtain pGMH20-MFα-FLAG-EGFR.

1-2: Construction of EGF surface layer display plasmid Using a gene encoding human-derived EGF, pYES2-MFα-HA-EGF-FLO42 was constructed as follows.

1-2-1: Construction of pYES2-MFα-FLO42 As shown in 1-1-1, by PCR using the gene encoding the prepro sequence of MFα1 as a template, HA was added to the 3 ′ side of the gene encoding the Prepro sequence. A gene encoding the tag sequence and an amplified fragment (a) with restriction enzyme sites added at both ends were obtained. An amplified fragment in which restriction enzyme sites were added to both ends of the gene encoding the 42 amino acid residues on the C-terminal side of FLO1 was obtained by PCR using a plasmid encoding the yeast agglutinated protein FLO1 gene as a template. Plasmid pYES2 in which these amplified fragments (a) and (b) are digested with restriction enzymes and inserted into a pYES2 vector to express MFα and HA on the N-terminal side of the protein and FLO42 on the C-terminal side. -MFα-HA-FLO42 was obtained.

1-2-2: Construction of pYES2-MFα-HA-EGF-FLO42 Amplified fragment in which restriction enzyme sites are added to both ends of the gene encoding mature EGF protein by PCR using a plasmid encoding EGF gene as a template Got. This amplified fragment was digested with a restriction enzyme and inserted into pYES2-MFα-HA-FLO42 to obtain pYES2-MFα-HA-EGF-FLO42.

1-3: Construction of Sos-Grb2 expression plasmid Using a gene encoding Grb2, which is an adapter protein that binds to the phosphorylation site of EGFR that has been autophosphorylated by EGF stimulation, pSos-Grb2 is as follows. Built.

1-3-1: Construction of pSos-Grb2 A plasmid pSos vector was purchased from Stratagene for fusing and expressing a mammalian-derived guanine-nucleotide exchange protein Sos on the N-terminal side of the protein. An amplified fragment in which restriction enzyme sites were added to both ends of the gene encoding the Grb2 protein was obtained by PCR using a plasmid encoding the Grb2 gene as a template. This amplified fragment was digested with a restriction enzyme and inserted into pSos to obtain pSos-Grb2.

Example 2: Reconstitution of EGF signaling in yeast 2-1: Preparation of yeast transformant Yeast Saccharomyces cerevisiae strain D1BR205a (MATa, ura3, his3, ade2, lys2, trp1, leu2, cdc25-2, Gal ⁺ ) D1BR205α (MATa, ura3, His3, ade2, lys2, trp1, leu2, cdc25-2, Gal ⁺ ) was obtained from Stratagene. Each plasmid pYES2, pYES2-MFα-HA-EGF-FLO42, pGMH20, pGMH20-MFα-FLAG-EGFR, and pSos-Grb2 obtained as described in 1 was introduced into the D1BR205 strain by the lithium acetate method, and yeast transformation was performed. D1BR205 / pYES2 / pGMH20 / pSos, D1BR205 / pYES2 / pGMH20 / pSos-Grb2, D1BR205 / pYES2-MFα-HA-EGF-FLO42 / pGMH20 / pSos-Grb2, D1BR2051-pYES20 / pM20 / pM -Grb2, and that of D1BR205 / pYES2-MFα-HA-EGF-FLO42 / pGMH20-MFα-FLAG-EGFR / pSos-Grb2 Les was obtained on SDΔUra / His / Leu agar medium.

2-2: Culture of yeast transformant On the above-mentioned agar medium, a colony of yeast transformant grown to a diameter of 2 to 3 mm was OD ₆₀₀ = 3 mL of SDΔUra / His / Leu liquid medium at 25 ° C. and 300 rpm. Cultivated from 1.0 to 1.5. The cells are collected and washed, suspended in 500 μL of sterilized water, inoculated into 10 mL of SRGΔUra / His / Leu liquid medium so that the initial OD ₆₀₀ = 0.20, and then OD ₆₀₀ at 25 ° C. and 300 rpm. Cultured to = 1.00.

2-3: Expression of EGF receptor protein in yeast transformant and confirmation of presence or absence of sugar chain modification D1BR205 / pYES2-MFα-HA-EGF-FLO42 / pGMH20-MFα-FLAG-EGFR cultured as in 2-2 / PSos-Grb2 and negative control D1BR205 / pYES2 / pGMH20 / pSos were collected and 200 μL of lysate (50 mM Tris-HCl (pH 7.3), 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 2 mM DTT , 1/10 mL-solution) and crushed with glass beads to obtain a crude yeast extract. Centrifugation for 10 minutes separated the supernatant fraction and the precipitate fraction, and SDS and 2-mercaptoethanol were added to the supernatant fraction to a final concentration of 0.5% and 1%, respectively. The precipitated fraction was resuspended in a solution containing SDS and 2-mercaptoethanol in equal amounts to the supernatant fraction at final concentrations of 0.5% and 1%, respectively, and boiled at 100 ° C. for 15 minutes, then 10 Centrifugation was performed for a minute to collect a supernatant fraction. 200 μL of sample was added with 1 μL of N-Glycosidase and 1 μL of O-Glycosidase, reacted at 37 ° C. for 0 and 30 minutes, sampled for each time, SDS-PAGE and anti-FLAG mouse monoclonal antibody were It used for the Western blot used. The results are shown in FIG. In the yeast transformant, a human-derived EGFR band that was not observed before the deglycosylation treatment was confirmed near the calculated molecular weight after the treatment. This confirmed that EGFR protein expression and sugar chain modification were received in yeast.

2-4: Expression of surface-presented EGF-FLO42 protein in yeast transformant and confirmation of presence or absence of sugar chain modification After performing the same operation as in 2-3, a sample was obtained, then SDS-PAGE and anti-HA mouse monoclonal The sample was subjected to Western blot using an antibody. The results are shown in FIG. In the yeast transformant, an EGF-FLO42 band was confirmed in the insoluble fraction. The insoluble fraction has been reported to contain a cell wall, indicating that the surface display of the EGF-FLO42 protein in yeast was successful.

2-5: Expression of Sos-Grb2 protein in yeast transformant Samples were obtained by performing the same operation as in 2-3, and then subjected to Western blotting using SDS-PAGE and anti-Sos mouse monoclonal antibody. The results are shown in FIG. In the yeast transformant, a band of Sos-Grb2 was confirmed in the soluble fraction. Combined with 2-3 and 2-4, expression of EGF, EGFR, and Sos-Grb2 was confirmed in yeast.

2-6: Confirmation of localization of EGF receptor in yeast transformant D1BR205 / pYES2-MFα-HA-EGF-FLO42 / pGMH20-MFα-FLAG-EGFR / pSos-Grb2 cultured as in 2-2 is recovered Then, after immobilization with 4% paraformaldehyde, it was washed twice with sorbitol buffer (40 mM potassium phosphate (pH 6.5), 0.5 mM MgCl ₂ , 1.2 M sorbitol). The immobilized yeast was resuspended in 500 μL of sorbitol buffer, added with 0.3 mg of Zymolase 20T, reacted at 37 ° C. for 10 minutes, and washed twice with sorbitol buffer. An anti-FLAG mouse monoclonal antibody (clone M2) was added as a primary antibody and washed after reaction for 1 hour. Furthermore, a Cy3-labeled anti-mouse IgG goat polyclonal antibody was added as a secondary antibody, washed after 1 hour of reaction, and observed with a fluorescence microscope. The results are shown in FIG. FIG. 3 confirmed that EGFR was localized in the yeast cell membrane.

2-7: Thermosensitivity experiment of EGF / EGFR / Sos-Grb2 If each protein expressed in the yeast transformant functions as shown in FIG. 1, the D1BR205 strain, which is a thermosensitive yeast, grows at 37 ° C. Expected to be possible. Therefore, D1BR205 / pYES2 / pGMH20 / pSos-Grb2, D1BR205 / pYES2-MFα-HA-EGF-FLO42 / pGMH20 / pSos-Grb2, and D1BR205 / pYES2 / pGMH20-MFα1-pFLAG-EGFR / FLAGS-FLAG-EGFR-PGR-EGFR-pGF-EGFRS -Grb2, and D1BR205 / pYES2-MFα-HA-EGF-FLO42 / pGMH20-MFα-FLAG-EGFR / pSos-Grb2 diluted in stock solution 10, 100, and 1000 times to SRGΔUra / His / Leu agar medium 10 μl each was dropped on the top and cultured at 37 ° C. for 14 days. The results are shown in FIG.

  As a result of the thermosensitivity experiment, growth at 37 ° C. was confirmed only in yeast transformants that expressed all of EGF-FLO42, EGFR, and Sos-Grb2, which were considered necessary for EGF signaling reconstitution in yeast. From this, it was considered that the EGFR received the signal of the surface layer-presenting EGF expressed in yeast, and the signal flowed to Sos-Grb2. In order for Grb2 to bind to EGFR, EGFR needs to be autophosphorylated. From the results of this experiment, it was considered that EGFR autophosphorylation also occurred in yeast as in mammals. Therefore, next, EGFR autophosphorylation was confirmed by immunostaining.

2-7: Confirmation of autophosphorylation of EGFR D1BR205 / pYES2 / pGMH20 / pSos-Grb2, D1BR205 / pYES2 / pGMH20-MFα1-FLAG-EGFR / pSos-Grb2, and D1BR205 / pYES2- cultured as in 2-2 MFα-HA-EGF-FLO42 / pGMH20-MFα-FLAG-EGFR / pSos-Grb2 was recovered and the cell wall was removed as in 2-6. Anti-phosphorylated EGFR rabbit polyclonal antibody was added as a primary antibody and washed after 1 hour of reaction. Further, Alexa488-labeled anti-rabbit IgG goat polyclonal antibody was added as a secondary antibody, washed after 1 hour of reaction, and observed with a fluorescence microscope. The results are shown in FIG. As shown in FIG. 5, autophosphorylation was not observed in D1BR205 / pYES2 / pGMH20-MFα1-FLAG-EGFR / pSos-Grb2, but D1BR205 / pYES2-MFα-HA-EGF-FLO42 / pGMH20-MFα-FLAG- Autophosphorylation was confirmed in EGFR / pSos-Grb2. From this result, it was found that EGFR autophosphorylation occurred in yeast in an EGF-dependent manner. This is the first time that EGF-dependent autophosphorylation of EGFR has been detected in yeast. This indicates that an agonist can be screened from a compound library by detecting the autophosphorylation of a receptor caused by the agonist with an antibody. In addition, if the site coding for the EGF gene contained in pYES2-MFα-HA-EGF-FLO42 is a peptide, high-throughput screening of peptides having affinity for EGFR is possible.

Example 3: Construction of plasmid for reconstitution of IL-5 signaling in yeast 3-1: Construction of IL-5 receptor expression plasmid Human-derived IL-5 receptor α chain which is a single transmembrane cytokine receptor ( Plasmid pME18 Hyg-huIL-5R (J. Immunol., 6924-6932, 1993) encoding the IL-5Rα) gene and plasmid pME18-KH97 encoding the human-derived IL-5 receptor β chain (βc) gene (J. Biol. Chem., 28287-28293, 1996) was used to construct pGMH20-MFα-FLAG-IL5Rα and pGMT20-MFα-HA-βc as follows.

3-1-1: Construction of pGMH20-MFα-FLAG and pGMT20-MFα-HA PCR using a gene encoding a prepro sequence (J. Bacteriol., 903-906, 1983) of yeast secretory protein MFα1 as a template As a result, a gene encoding the FLAG tag sequence on the 3 ′ side of the gene encoding the Prepro sequence and an amplified fragment (a) having restriction enzyme sites added to both ends were obtained. By PCR using the gene as a template, a gene encoding the HA tag sequence on the 3 ′ side of the gene encoding the Prepro sequence and an amplified fragment (b) in which restriction enzyme sites were added to both ends were obtained. The amplified fragment (a) was digested with a restriction enzyme and inserted into a pGMH20 vector digested with a restriction enzyme to obtain pGMH20-MFα-FLAG. The amplified fragment (b) was digested with a restriction enzyme and inserted into a pGMT20 vector digested with a restriction enzyme to obtain pGMT20-MFα-HA.

3-1-2: Construction of pGMH20-MFα-FLAG-IL5R Amplification in which restriction enzyme sites are added to both ends of the gene encoding mature IL-5Rα protein by PCR using plasmid pME18 Hyg-huIL-5R as a template A fragment was obtained. This amplified fragment was digested with a restriction enzyme and inserted into pGMH20-MFα-FLAG digested with a restriction enzyme to obtain pGMH20-MFα-FLAG-IL5Rα.

3-1-3: Construction of pGMT20-MFα-HA-βc An amplified fragment in which restriction enzyme sites were added to both ends of the gene encoding the mature βc protein was obtained by PCR using the plasmid pME18-KH97 as a template. This amplified fragment was digested with a restriction enzyme and inserted into pGMT20-MFα-HA digested with a restriction enzyme to obtain pGMH20-MFα-HA-βc.

3-2: Construction of JAK2 expression plasmid Plasmid pSRα-JAK2 encoding mouse-derived JAK2 gene, which is a protein tyrosine kinase that undergoes autophosphorylation upon stimulation with IL-5 (Genes to Cells, pages 431-441, 1998) PAUR123-V5-JAK2 and pAURGAL1p-V5-JAK2 were constructed as follows.

3-2-1: Construction of pAUR123-V5 and pAURGAL1p-V5 An amplified fragment (a) encoding the GAL1p gene was obtained by PCR using the pYES2 vector encoding the GAL1p gene, which is a galactose-inducible promoter, as a template. An amplified fragment (b) lacking ADH1p was obtained by PCR using the pAUR123 vector encoding the non-inducible promoter ADH1p gene as a template. The amplified fragment (a) was digested with KpnI and inserted into the amplified fragment (b) digested with KpnI to obtain a pAURGAL1p vector. A gene encoding a V5 tag sequence was inserted into the pAUR123 vector and pAURGAL1p vector digested with KpnI and SalI to obtain pAUR123-V5 and pAURGAL1p-V5, respectively.

3-2-2: Construction of pGEM-JAK2 An amplified fragment encoding the JAK2 gene was obtained by PCR using the plasmid pSRα-JAK2 as a template. Adenine was added to the 3 ′ side of the amplified fragment using TARGET Clone (TOYOBO), and this was inserted into the pGEM T-Easy vector to obtain pGEM-JAK2.

3-2-3: Construction of pAUR123-V5-JAK2 and pAURGAL1p-V5-JAK2 By PCR using plasmid pGEM-JAK2 as a template, an amplified fragment with restriction enzyme sites added to both ends of the gene encoding JAK2 protein was obtained. It was. This amplified fragment was digested with SalI and SacI and inserted into pAUR123-V5 vector and pAURGAL1p-V5 vector digested with SalI and SacI to obtain pAUR123-V5-JAK2 and pAURGAL1p-V5-JAK2.

Example 4: Reconstitution of IL-5 signaling in yeast 4-1: Preparation of yeast transformants Yeast Saccharomyces cerevisiae D1BR205a strains (MATaura3 His3ade2 lys2 trp1 leu2 cdc25Gal ⁺ ) and D1BR2r33p2 leu2 cdc25 Gal ⁺ ) was obtained from Stratagene.

  Each of the plasmids pGMH20, pGMH20-MFα-FLAG-IL5Rα, pGMT20, pGMT20-MFα-HA-βc, pAUR123, pAUR123-V5-JAK2, pAURGAL1p, and pAURGAL1p-V5-JAK2 The yeast transformants D1BR205 / pGMH20 / pGMT20 / pAUR123, D1BR205 / pGMH20 / pGMT20 / pAUR123-V5-JAK2, D1BR205 / pGMH20-MFα1-FLAG-IL5Rα / FGMT-ILGR -V5-JAK2, D1BR205 / pGMH20 / pGMT20 / pAURGAL1p, D1BR205 / pGMH20 / pGM Each of T20 / pAURGAL1p-V5-JAK2 and D1BR205 / pGMH20-MFα-FLAG-IL5Rα / pGMT20-MFα-HA-βc / pAURGAL1p-V5-JAK2, 0.5 μg / ml aureobasidin A (TaKaRa) Obtained on SDΔHis / Trp agar medium.

4-2: Culture of yeast transformant 3 mL of SDΔHis / Trp liquid medium containing 0.5 μg / ml aureobasidin A as a colony of yeast transformants grown to a diameter of 2 to 3 mm on the agar medium. Incubated at 25 ° C. and 300 rpm to OD ₆₀₀ = 1.0 to 1.5. The cells were collected and washed, suspended in 500 μL of sterilized water, and inoculated in 10 mL of SRGΔHis / Trp liquid medium containing 0.5 μg / ml of aureobasidin A so that the initial OD _{600 was} 0.20. after, it was grown to _OD 600 = _0.30~2.00 at 300rpm at 25 ℃.

4-3: Expression of IL-5Rα protein in yeast transformant and confirmation of sugar chain modification D1BR205 / pGMH20-MFα1-FLAG-IL5Rα / pGMT20-MFα-HA-βc / pAUR123 cultured as in 4-2 -V5-JAK2 and negative control D1BR205 / pGMH20 / pGMT20 / pAUR123 were collected and 200 μL of lysate (50 mM Tris-HCl (pH 7.3), 150 mM NaCl, 0.1% Triton X-100, 1 mM EGTA, 2 mM DTT, 1/10 mL-solution), and crushed with glass beads to obtain a crude yeast extract. Centrifuge for 10 minutes to separate the supernatant fraction and the precipitate fraction, and add SDS and 2-ME to the supernatant fraction to 0.5% (V / V) and 1% (V / V), respectively. did. The precipitate fraction is resuspended in a lysate containing 0.5% (V / V) and 1% (V / V) of SDS and 2-ME in the same amount as the supernatant fraction, respectively, at 100 ° C. After boiling for 15 minutes, the supernatant fraction was collected by centrifugation for 10 minutes. After adding 50 μL of O-glycosidase and 50 μL of N-glycosidase to 50 μL of sample, react at 37 ° C. for 0, 10, 30, and 60 minutes, and sample for each time to obtain SDS-PAGE and anti-antigen It used for the Western blot which used the FLAG mouse monoclonal antibody (clone M2) (FIG. 8). So far, there has been no successful expression of a single-transmembrane cytokine receptor in yeast. For the first time in this example, the expression of human-derived cytokine receptor IL-5Rα protein in yeast was confirmed. Moreover, the band seen in the position larger than the calculated molecular weight of IL-5Rα protein was shifted to the position near the calculated molecular weight by the deglycosylation modification treatment. From this, it was found that IL-5Rα protein undergoes sugar chain modification in yeast.

4-4: Expression of βc protein in yeast transformant and confirmation of presence or absence of sugar chain modification After performing the same operation as 4-3 to obtain a sample, SDS-PAGE and anti-HA mouse monoclonal antibody (clone 12CA5) (Fig. 9). In the yeast transformant, a human-derived βc band that was not seen before the deglycosylation treatment was confirmed in the vicinity of the calculated molecular weight after the treatment. This confirmed that the yeast was subjected to βc protein expression and sugar chain modification. In 2-3, the expression of IL-5 receptor was confirmed in yeast together with the expression of IL-5Rα.

4-5: Confirmation of localization of IL-5 receptor in yeast transformant D1BR205 / pGMH20-MFα-FLAG-IL5Rα / pGMT20-MFα-HA-βc / pAUR1233-V5-JAK2 cultured as in 4-2 The negative control D1BR205 / pGMH20 / pGMT20 / pAUR123 was recovered, immobilized with 4% paraformaldehyde, and then sorbitol buffer (40 mM potassium phosphate (pH 6.5), 0.5 mM MgCl ₂ , 1.2 M sorbitol). ) Twice. The immobilized yeast was resuspended in 500 μL of sorbitol buffer, 0.3 mg of cell wall-removing enzyme Zymolase 20T was added, reacted at 37 ° C. for 30 minutes, and washed twice with sorbitol buffer. The yeast from which the cell wall had been removed was suspended in sorbitol buffer, anti-FLAG mouse monoclonal antibody (clone M2) and anti-HA rabbit polyclonal antibody were added as primary antibodies, and washed after 1 hour of reaction. Furthermore, Cy3-labeled anti-mouse IgG goat polyclonal antibody and Alexa Fluor 430-labeled anti-rabbit IgG goat polyclonal antibody were added as secondary antibodies, washed for 1 hour, and observed with a fluorescence microscope. The results are shown in FIG.

  In D1BR205 / pGMH20-MFα-FLAG-IL5Rα / pGMT20-MFα-HA-βc / pAUR123-V5-JAK2 from which the cell wall was removed, it was confirmed that IL-5Rα and βc were localized in the cell membrane. From this, it is considered that the human-derived IL-5 receptor was successfully localized on the yeast cell membrane.

4-6: Confirmation of expression of JAK2 protein in yeast transformant D1BR205 / pGMH20 / pGMT20 / pAUR123-V5-JAK2, D1BR205 / pGMH20 / pGMT20 / pAURGAL1p-V5-JAK2, cultured as in 4-2 D1BR205 / pGMH20 / pGMT20 / pAUR123 and D1BR205 / pGMH20 / pGMT20 / pAURGAL1p were collected and 200 μL of lysate (50 mM Tris-HCl (pH 7.3), 150 mM NaCl, 0.1% Triton X-100, 1 mM EGTA , 2 mM DTT, Complete protease inhibitor cocktail 1/10 mL-solution), crushed with glass beads to obtain a crude yeast extract, and then SDS-PAGE And subjected to Western blot using anti-V5 mouse monoclonal antibody (clone V5-10) and anti-phosphorylated JAK2 rabbit poclonal antibody (FIG. 11). The expression of JAK2 is confirmed at OD ₆₀₀ = 2.0 for D1BR205 / pGMH20 / pGMT20 / pAUR123-V5-JAK2, and at OD ₆₀₀ = 0.6 for D1BR205 / pGMH20 / pGMT20 / pAURGAL1p-V5-JAK2. It was found that the expression was high at ₆₀₀ = 2.0.

4-7: Confirmation of IL-5-dependent enhancement of autophosphorylation of JAK2 Whether or not JAK2 autophosphorylation shown in 4-6 occurs in yeast depending on IL-5, which is a human-derived ligand, is described below. It carried out like. D1BR205 / pGMH20-MFα-FLAG-IL5Rα / pGMT20-MFα-HA-βc / pAUR123-V5-JAK2, D1BR205 / pGMH20-MFα-FLAG-IL5Rα / pGMT20-MFα-HA-β cultured as 4-2 pAURGAL1p-V5-JAK2, negative controls D1BR205 / pGMH20 / pGMT20 / pAUR123, and D1BR205 / pGMH20 / pGMT20 / pAURGAL1p were collected and washed to a final concentration of 20 mM HEPES (pH 7.0) and a final concentration of 0.5 μg / ml. Was replaced with 1 mL of SRGΔHis / Trp liquid medium containing Aureobasidin A, and IL-5 was added to a final concentration of 4 nM, followed by reaction for 0 h and 1 h. After the reaction, D1BR205 / pGMH20-MFα-FLAG-IL5Rα / pGMT20-MFα-HA-βc / pAUR123-V5-JAK2, D1BR205 / pGMH20-MFα-FLAG-IL5Rα after immobilizing with 4% paraformaldehyde and removing the cell wall / PGMT20-MFα-HA-βc / pAURGAL1p-V5-JAK2, negative controls D1BR205 / pGMH20 / pGMT20 / pAUR123, and D1BR205 / pGMH20 / pGMT20 / pAURGAL1p are suspended in sorbitol buffer and phosphorylated as a primary J antibody. Polyclonal antibody was added and washed after 1 hour reaction. Further, the secondary antibody Alexa Fluor 488-labeled anti-rabbit IgG goat polyclonal antibody was added, washed after 1 hour of reaction, and observed with a fluorescence microscope and analyzed with a flow cytometer (FCM). As shown in FIG. 12, IL1 in D1BR205 / pGMH20-MFα-FLAG-IL5Rα / pGMT20-MFα-HA-βc / pAUR123-V5-JAK2 of OD ₆₀₀ = 2.0 and negative control D1BR205 / pGMH20 / pGMT20 / pAUR123 IL-5-dependent enhancement of fluorescence intensity was confirmed after 5 hours of addition and reaction culture for 1 hour. From the above results, it was shown that agonist-dependent phosphorylation of JAK2 occurs in yeast.

Screening method using yeast cell signaling The results of Western blotting using SDS-PAGE and anti-FLAG mouse monoclonal antibody are shown. Confirmation of localization of EGF receptor in yeast transformants It shows that growth at 37 ° C. was confirmed only in yeast transformants that expressed all of EGF-FLO42, EGFR, and Sos-Grb2, which were considered necessary for EGF signaling rearrangement in yeast. Confirmation of EGFR autophosphorylation Ligand screening method using anti-phosphorylated JAK2 antibody Screening method using yeast cell signal transduction, IL-5 receptor The results of Western blotting using SDS-PAGE and anti-FLAG mouse monoclonal antibody (clone M2) are shown. The results of Western blotting using SDS-PAGE and anti-HA mouse monoclonal antibody (clone 12CA5) are shown. Confirmation of localization of IL-5 receptor in yeast transformants Confirmation of expression of JAK2 protein in yeast transformants Confirmation of IL-5-dependent autophosphorylation of JAK2

Claims (7)

A method for screening an agonist / antagonist, wherein an agonist / antagonist candidate substance of the receptor is allowed to act on a yeast expressing a human-derived autophosphorylated receptor, and the agonist / antagonist candidate substance binds to the receptor. Detecting the yeast
Tyrosine that binds to the receptor itself or to the receptor by multimerization of the autophosphorylated receptor by the binding of an agonist and by the kinase action of the receptor itself or the action of tyrosine kinase that constitutively binds to the receptor The kinase is phosphorylated,
The detection is performed by lysing the cell wall of the yeast and allowing the antibody capable of detecting phosphorylation to act on the cell wall lysed yeast.
Method. The method according to claim 1 , wherein the autophosphorylated receptor is multimerized by the binding of an agonist, and the receptor itself is phosphorylated by the kinase action of the receptor itself. The autophosphorylated receptor is multimerized by the binding of an agonist, and both the receptor itself and the tyrosine kinase that binds constitutively to the receptor are phosphorylated by the action of the tyrosine kinase that constitutively binds to the receptor. The method according to claim 1 . The mammalian autophosphorylated receptor is
(I) the receptor itself has autophosphorylation, or
(Ii) a receptor that shares gp130 or βc, and the tyrosine kinase is constitutively bound to gp130 or βc, and phosphorylates both tyrosine kinase itself and gp130 or βc by the action of the tyrosine kinase. Item 2. The method according to Item 1 . The method of claim 1 tyrosine kinase constitutively bound to the receptor is JAKl, JAK2 or JAK3. The method according to claim 1, wherein the receptor to which the tyrosine kinase is constitutively bound and the tyrosine kinase is phosphorylated in an agonist-dependent manner includes the α chain and β chain (βc) of the interleukin receptor. . Constitutively bound tyrosine kinase and receptor agonist-dependent manner the tyrosine kinase is phosphorylated comprises gp130, The method of claim 1.

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