JP2011155839A - Method for detecting or screening interaction between highly compatible proteins - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently detecting or screening an interaction between highly compatible proteins. <P>SOLUTION: The method for detecting the interaction between the proteins with a yeast includes (1) a step for preparing a first expression cassette for expressing the fused protein of a first protein with a G protein γ-subunit cell membrane-nonbinding variant, (2) a step for preparing a second expression cassette for expressing the fused protein of a second protein with a cell membrane-localized signal sequence, (3) a step for preparing a third expression cassette for expressing a third protein in cell membrane, (4) a step for transferring the first, second, and third cassettes obtained in the steps (1), (2) and (3) to a monoploid yeast to prepare a transformed yeast, and (5) a step for detecting pheromone signal transmission in the transformed yeast obtained in the step (4) or screening the transformed yeast for expressing the pheromone signal transmission. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a high affinity protein-protein interaction detection / screening method. In particular, the present invention relates to a protein interaction detection / screening method using a competitive protein.

  A yeast two-hybrid method is known as a protein-protein interaction detection method. In this method, a transcription factor involved in gene expression regulation recognizes a specific base sequence on a gene (DNA) and binds to it (DNA binding domain: BD), and a basic factor essential for gene transcription. It is composed of domains that act to activate gene transcription (transcription activation domain: AD), based on the fact that each domain can be separated. That is, of a pair of proteins (X and Y) for which protein-protein interaction is desired to be detected, one (X) is fused with a DNA binding domain of a transcription factor to form one hybrid protein (X-BD), and the other When (Y) is fused with the transcriptional activation domain of a transcription factor to form another hybrid protein (Y-AD), transcription of the gene is activated only when there is an interaction between XY. . Transcriptional activation of this gene can be detected by expression of the reporter gene by linking the reporter gene downstream of a specific base sequence to which BD binds. Examples of transcription factors used in the yeast two-hybrid method include Gal4, LexA-B42, LexA-VP16 and the like.

  Yeast is a unicellular eukaryote and has been used for food processing for a long time. Yeast is the most studied in eukaryotes, and yeast genetic recombination technology has also been developed. Yeast is common with higher eukaryotes including mammals such as human beings in terms of eukaryotes, and is therefore widely used as an expression host for these proteins in order to analyze higher eukaryotic proteins. . The yeast two-hybrid method is useful as a method for detecting protein-protein interactions in eukaryotes.

  However, the yeast two-hybrid method using transcription factors in the cell nucleus has a problem that it is difficult to apply to proteins that are difficult to translocate into the nucleus and proteins that originally have a transcription activation domain (for example, transcription factors).

  Therefore, in recent years, a modified method of the yeast two-hybrid method has been devised depending on the localization and function of the protein. Non-Patent Documents 1 and 2 describe methods utilizing the Ras signaling pathway of yeast. In this method, the protein-protein interaction is detected using the growth of yeast as an index by replacing the cell membrane binding ability of Ras with the protein-protein interaction. By utilizing the signal amplification effect of the Ras transmission pathway, protein-protein interactions can be detected with high sensitivity. That is, among a pair of proteins (X and Y) for which protein-protein interaction is to be detected, one (X) is immobilized on the cell membrane, and the other (Y) is a human-derived guanine nucleotide exchange factor (hSOS) or cell membrane binding ability. When fused with the deficient Ras constitutively active mutant (mRas) to form a hybrid protein (Y-hSOS or Y-mRas, respectively), hSOS or mRas is only present in the cell membrane when there is an interaction between XY. Localizes and activates the Ras signaling pathway. Activation of this signal transduction pathway can be detected using yeast growth as an indicator.

  However, this method requires the use of a temperature sensitive mutant of yeast (cdc25-2), which places a temperature limit on yeast growth. That is, since it is necessary to construct a protein library at 25 ° C., which is not a sensitive temperature, and to perform detection or screening of protein-protein interactions at a sensitive temperature of 36 ° C., compared to 30 ° C., which is the optimal growth temperature for yeast. There is a problem that the growth rate of yeast is greatly reduced, and it is difficult to rapidly detect or screen for protein-protein interactions.

Non-Patent Document 3 describes a method for detecting a protein-protein interaction using a junction signal (pheromone signal) transmission pathway by G protein. A haploid yeast secretes a pheromone with respect to a mating type haploid yeast as a pair, and when a partner pheromone is sensed, a signal that promotes mating is transmitted into the cell. A receptor that binds to pheromone exists on the cell membrane, and this receptor is coupled to a trimeric protein called G protein in the cell. When the pheromone binds to the receptor, the G protein dissociates into an α subunit and a βγ complex. Since the α subunit and the γ subunit are respectively bound to the cell membrane, the α subunit and the βγ complex are localized on the cell membrane even after dissociation of the G protein. Signals are transmitted downstream by the β subunit of the βγ complex acting on the effector via the cell membrane. On the other hand, in the mutant type (Gγ _cyto ) lacking the cell membrane binding ability of the γ subunit, the β γ complex (Gβ γ _cyto complex) is released into the cytoplasm due to the dissociation of the G protein and cannot act on the effector. , No signal is transmitted downstream. In Non-Patent Document 3, protein-protein interaction is used to localize this Gβγ _cyto complex to the cell membrane. First, to express the protein (A) fused to G.gamma _cyto fusion protein (A-Gγ _cyto). Next, the protein (B) that interacts with the protein (A) is expressed in the cell membrane (B _mem ). When the protein (A) interacts with the protein (B), the Gβγ _cyto complex composed of A-Gγ _cyto is localized in the cell membrane and the junction signaling is restored. Therefore, the interaction between the protein (A) and the protein (B) can be detected by detecting the junction signaling. In yeast subjected to pheromone stimulation, various responses occur through conjugation signal transduction by G protein, and transcription of various genes is activated. Therefore, a reporter gene is incorporated downstream of a pheromone-responsive promoter, and junction signaling is detected as reporter gene expression. In Non-Patent Document 3, Fc portion of human IgG is used as protein (A), Z domain derived from protein A of Staphylococcus aureus is used as protein (B), and as a reporter gene, By using the green fluorescent protein EGFP, the fluorescence intensity emitted by the reporter is analyzed with a flow cytometer to quantitatively evaluate the protein-protein interaction. As a result, a relatively weak interaction with a binding constant of 8.0 × 10 ³ M ⁻¹ has been successfully detected.

  However, in the methods of Non-Patent Documents 1 to 3, it is difficult to detect or screen for interactions between proteins having a certain affinity, and to selectively detect or screen for interactions between proteins with high affinity. .

A. Aronheim et al., "Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway", Cell, 1994, Vol. 78, pp.949-961. Y.C. Broder et al., "The ras recruitment system, a novel approach to the study of protein-protein interactions", Curr. Biol., 1998, Vol. 8, pp. 1121-1124 N. Fukuda et al., “Construction of a novel detection system for protein-protein interactions using yeast G-protein signaling”, FEBS J., 2009, 276, pp.2636-2644.

  An object of the present invention is to provide a method for efficiently detecting or screening an interaction between proteins having high affinity.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors competed with the yeast cytoplasm in a method for detecting protein-protein interaction using a junction signal (pheromone signal) transmission pathway by yeast G protein. The inventors have found that by expressing a protein, a protein having a higher affinity than the competitive protein can be efficiently detected or screened, and the present invention has been completed.

  The present invention provides a method for detecting a protein-protein interaction using yeast, the method comprising (1) a first expression that expresses a fusion protein of a first protein and a G protein γ subunit cell membrane non-binding mutant. A step of preparing a cassette, (2) a step of preparing a second expression cassette expressing a fusion protein of the second protein and a cell membrane localization signal sequence, and (3) a step of expressing the third protein in the cytoplasm. (4) preparing the expression cassette of (3), (4) introducing the first, second and third expression cassettes obtained in the steps (1), (2) and (3), respectively, into a yeast And (5) detecting pheromone signaling in the transformed yeast obtained in the step (4), or screening transformed yeast exhibiting pheromone signaling. Comprising the step of.

  The present invention also provides a further embodiment of a method for detecting protein-protein interaction using yeast, the method expressing (1) a fusion protein of a first protein and a G protein γ subunit cell membrane non-binding mutant. Preparing a first expression cassette, (2) preparing a second expression cassette expressing a fusion protein of the second protein and a cell membrane localization signal sequence, (3) a third protein A step of preparing a third expression cassette to be expressed in the cytoplasm, (4) the first, second and third expression cassettes obtained in the steps (1), (2) and (3), respectively, are haploid yeast (5) the transformed yeast obtained in the step (4) is joined to a haploid yeast that can be joined to the transformed yeast, and a diploid yeast The process of preparing Beauty (6) selecting the diploid yeast obtained in the step (5).

  In one embodiment, the third protein is a competitive protein of the first protein or the second protein.

  According to the present invention, protein-protein interactions with high affinity can be detected or screened efficiently.

It is a schematic diagram which shows the outline of the principle of the protein protein interaction detection method of this invention. Photograph (A) showing growth of diploid yeast produced on diploid selection medium when no competitive protein is used, and 1 mL of yeast suspension adjusted to cell concentration OD ₆₀₀ = 1.0 It is a graph (B) which shows the colony number of the diploid yeast which can produce | generate. In the case of using the case (A) and _{Z WT} using _{Z K35A} as competitor protein (B), shows the expression of green fluorescent protein (GFP) reporter gene in the haploid yeast in the α-factor the presence of 5μM It is a graph. Photograph showing the case of using the Z _K35A as competitor protein in the case of using the (A and C) and Z _WT (B and D), the growth of diploid yeast produced on diploid selection medium (A and B) and graphs (C and D) showing the number of colonies of diploid yeast that can be produced by a 1 mL yeast suspension adjusted to a cell concentration OD ₆₀₀ = 1.0.

An outline of the principle of the protein-protein interaction detection method of the present invention is shown in FIG. The method of the present invention will be described with reference to FIG. For example, protein (A) is a target protein, protein (B) is a binding protein to the target protein, and protein (C) competes with protein (B) in interaction with protein (A). It is a protein. That is, protein (A) and protein (B) interact, and protein (A) and protein (C) interact. In this case, the competitive protein refers to each other's protein in the relationship between the protein (B) and the protein (C). Here, when the binding constant between the proteins (A) and (B) is KA _-B , and the binding constant between the proteins (A) and (C) is KA _-C , KA _-B is more than _KA-C . When small (right figure in FIG. 1), the Gβγ complex is trapped in the cytoplasm and the transmission of the junction signal is inhibited. On the other hand, when K _A-B is larger than K _A-C (the left diagram in FIG. 1), the Gβγ complex can bind to the cell membrane, and thus a conjugation signal is transmitted. For this reason, by detecting the interaction between the protein (A) and the protein (B) by junction signaling, the target protein (A) has higher affinity than the competing protein (C). It becomes possible to detect or screen for the binding protein (B) (with a large binding constant).

  Here, protein-protein interaction refers to the binding or affinity between proteins.

  In the first embodiment, the method for detecting protein-protein interaction using the yeast of the present invention is as follows: (1) a first expression that expresses a fusion protein of the first protein and a G protein γ subunit cell membrane non-binding mutant. A step of preparing a cassette, (2) a step of preparing a second expression cassette expressing a fusion protein of the second protein and a cell membrane localization signal sequence, and (3) a step of expressing the third protein in the cytoplasm. (4) preparing the expression cassette of (3), (4) introducing the first, second and third expression cassettes obtained in the steps (1), (2) and (3), respectively, into a yeast And (5) detecting pheromone signaling in the transformed yeast obtained in the step (4), or transforming yeast exhibiting pheromone signaling Including the grayed to process.

  The transformed yeast obtained in the above method needs to be activated so that the signal transduction ability can be evaluated. The activation method is not particularly limited as long as it activates the upstream side of the G protein. For example, a method of adding a ligand (agonist) to a yeast endogenous G protein-coupled receptor can be mentioned. In the case of a mating type cell, for example, it is encoded by a gene having the base sequence shown in SEQ ID NO: 1 (GenBank accession number: NC001148), for example, an amino acid sequence shown in SEQ ID NO: 2 (GenBank accession number: NP015137) There is a method of adding α-factor which is a 13 amino acid peptide produced from MF (alpha) 1 which is a precursor having γ.

  The method for detecting pheromone signaling or the method for screening transformed yeast exhibiting pheromone signaling is not particularly limited. Preferably, modification is performed so that expression of a reporter gene is induced under the control of a pheromone signal responsive promoter (eg, FUS1, FIG1, AGA1, etc.). Examples of the reporter gene include a fluorescent reporter gene, a growth reporter gene, a chromogenic reporter gene, and a luminescent reporter gene. Examples of the fluorescent reporter gene include green fluorescent protein (GFP), red fluorescent protein (DsRed), and cyan fluorescent protein (CFP). Examples of the growth reporter gene include HIS3, ADE2, URA3, and CAN1. Examples of the chromogenic reporter gene include β-galactosidase (lacZ). Examples of the luminescent reporter gene include luciferase (luc). Cell cycle suppression induced by pheromone signaling may also be used. Yeast whose cell cycle is suppressed by signal is inhibited from growing. Therefore, it is possible to detect signal transduction ability or signal transduction ability by measuring halo formation on solid medium and cell density in liquid medium. Can be screened for.

  The second embodiment of the protein-protein interaction detection method using the yeast of the present invention is as follows: (1) a first expression cassette that expresses a fusion protein of the first protein and a G protein γ subunit cell membrane non-binding mutant. (2) preparing a second expression cassette that expresses a fusion protein of the second protein and a cell membrane localization signal sequence; (3) expressing a third protein in the cytoplasm; (4) introducing the first, second and third expression cassettes obtained in the steps (1), (2) and (3), respectively, into a haploid yeast for transformation A step of preparing yeast, (5) a step of preparing a diploid yeast by joining the transformed yeast obtained in step (4) with a haploid yeast that can be joined with the transformed yeast, and (6) In the step (5) It was comprising the step of selecting the diploid yeast.

  In said 2nd embodiment, the haploid yeast used for transformation needs to have mating ability. Preferred is an a / α mating type a-type or α-type haploid yeast. In any case, it is necessary to have an appropriate selection marker so that diploid yeast produced by conjugation can be selected separately from haploid yeast. Moreover, yeast lacking the gene encoding the endogenous G protein γ subunit is preferred. In the case of using yeast in which the gene encoding the endogenous G protein γ subunit is not deleted, in the step of preparing transformed yeast by introducing the first expression cassette into haploid yeast, It is necessary to replace the endogenous G protein γ subunit and the first expression cassette by replacement. Examples of yeast species include Saccharomyces cerevisiae and Schizosaccharomyces pombe.

  Examples of selection markers include auxotrophic markers and drug resistance markers. Examples of the auxotrophic marker include various amino acid auxotrophic markers and various nucleic acid auxotrophic markers. Examples of amino acid requirement markers include lysine requirement markers, methionine requirement markers, histidine requirement markers, tryptophan requirement markers, leucine requirement markers, and the like. Examples of nucleic acid requirement markers include uracil requirement markers and adenine requirement markers. Examples of drug resistance markers include geneticin (G418) resistance markers, aureobasidin A resistance markers, and the like. The pair of haploid yeasts to be joined must have different selection markers so that the diploid yeast produced by the joining can be selected separately from the haploid yeast.

  Here, a haploid refers to a yeast individual having one set of genomes, and is also referred to as a haploid. In the present invention, the genes of the first protein (A), the second protein (B), and the third protein (C) are introduced into haploid yeast.

  A diploid refers to a yeast individual having two sets of genomes, also referred to as a polyploid. It is generated by joining haploids. In the present invention, protein-protein interactions are detected or screened based on the efficiency of diploid production.

  The G protein used in the present invention refers to a heterotrimeric G protein composed of a complex formed by an α subunit, a β subunit, and a γ subunit, and G protein is an abbreviation for a guanine nucleotide-binding protein. Each of the α subunit and the γ subunit is bound to the cell membrane. When the pheromone binds to the receptor, the G protein dissociates into an α subunit and a βγ complex. Even after dissociation of the G protein, the α subunit and the βγ complex are localized in the cell membrane. Signals are transmitted downstream by the β subunit of the βγ complex acting on the effector via the cell membrane. The origin of the G protein is not particularly limited as long as it is involved in conjugation signaling.

The γ subunit of the G protein used in the present invention is encoded by, for example, a gene having the nucleotide sequence shown in SEQ ID NO: 3 (GenBank accession number: AY557888), for example, an amino acid sequence shown in SEQ ID NO: 4 (GenBank Ste18 which is a protein having an accession number: CAA89613). The γ subunit cell membrane non-binding mutant (Gγ _cyto ) is not particularly limited as long as it lacks cell membrane binding ability. Examples of Gγ _cyto include mutants in which positions 106 to 110 of the amino acid sequence represented by SEQ ID NO: 4 of γ subunit Ste18 are deleted.

In the present invention, the method for fusing the first protein (A) and Gγ _cyto is not particularly limited as long as the function of Gγ _cyto can be exhibited. For example, the N-terminus of protein (A) is linked to position 105 of the amino acid sequence shown in SEQ ID NO: 4 of γ subunit Ste18.

  The cell membrane localization signal sequence used in the present invention is not particularly limited as long as the second protein (B) can be localized on the cell membrane. Preferably, it is a cell membrane localization signal sequence of the γ subunit of G protein. For example, this signal corresponds to positions 102 to 110 of the amino acid sequence shown in SEQ ID NO: 4 of γ subunit Ste18.

  The method for fusing the second protein (B) and the cell membrane localization signal sequence in the present invention is not particularly limited as long as the function of the cell membrane localization signal sequence can be exhibited. For example, a fragment consisting of positions 102 to 110 of the amino acid sequence shown in SEQ ID NO: 4 of γ subunit Ste18 is linked to the C terminus of protein (B).

  The method for expressing the protein (C) in the cytoplasm in the present invention is not particularly limited.

Expression cassettes for expressing protein (A) -Gγ _cyto fusion protein, cell membrane-binding protein (B) and cytoplasmic protein (C) in yeast usually function in yeast in addition to the coding region of these proteins. A gene expression regulatory region such as a promoter and terminator, a selection marker for selecting a transformed yeast introduced with an expression cassette, and the like. Examples of the promoter include PGK1 promoter, TDH3 promoter, and STE18 promoter. Examples of the terminator include PGK1 terminator, TDH3 terminator, and CYC1 terminator. Examples of the selection marker include a leucine-requiring marker, a uracil-requiring marker, and a geneticin (G418) resistance marker.

  Expression cassettes are usually made as plasmids. In addition to the expression cassette, the plasmid has a fragment necessary for integrating the expression cassette into the yeast chromosome by homologous recombination, or an origin of replication necessary for allowing the plasmid to replicate autonomously in yeast. As a fragment necessary for homologous recombination, a fragment of a gene present at a position on the chromosome to be integrated is preferable. Integration of the expression cassette into the yeast chromosome by homologous recombination is performed by methods commonly used by those skilled in the art. Examples of the origin of replication in yeast include 2 μ (multicopy type) and CEN / ARS (single copy type). The autonomous replication of the plasmid in yeast is performed by a method commonly used by those skilled in the art. The plasmid is amplified and prepared in an appropriate host such as Escherichia coli by a method commonly used by those skilled in the art. The host for preparing the plasmid is not particularly limited. Preferably, it is E. coli. The plasmid also usually has a selection marker for selecting a transformed E. coli or the like into which the plasmid has been introduced in order to facilitate preparation of the plasmid. Examples of selection markers include antibiotic resistance such as ampicillin resistance and kanamycin resistance.

  The expression cassette may be introduced into yeast in the form of a plasmid, or only the expression cassette may be introduced into yeast. The expression cassette can be efficiently integrated into the yeast chromosome by homologous recombination. The plasmid can be autonomously replicated in yeast by continuing to culture the transformed yeast under selective conditions corresponding to the selectable marker.

  The transformed yeast introduced with the expression cassette can be obtained under selection conditions corresponding to the selection marker, that is, by culturing in the absence of amino acids or nucleic acids corresponding to the selection marker, or by culturing in the presence of a drug.

  In the second embodiment of the method of the present invention, a transformed yeast introduced with an expression cassette is mated with a corresponding mating type haploid yeast, preferably a wild type haploid yeast. The haploid yeast used here is not particularly limited as long as it has a selection marker and can be joined. For example, if the transformed yeast introduced with the expression cassette is derived from type a, it is joined to the corresponding a / α mating type α type wild yeast. The haploid wild yeast is not particularly limited as long as it has a selection marker that can select only diploid yeast produced by conjugation with transformed yeast, and has a conjugation ability with transformed yeast. The diploid yeast produced by conjugation can be confirmed and selected by using the above selection marker.

  In the present invention, the joining efficiency can be appropriately quantified. For example, it can be quantified by appropriately diluting the joined yeast suspension to form colonies on the solid medium for selection and counting the number of colonies.

  In the present invention, the first protein (A), the second protein (B), and the third protein (C) may be specific proteins or unspecific proteins. An unspecified protein refers to a protein constituting a library. The third protein (C) is characterized in that a competitive protein of the first protein (A) or the second protein (B) is used. Due to this feature, when the specific first protein (A) is used and the second protein (B) constituting the library is used, the third protein that is a competitive protein with respect to the first protein (A) Compared with (C), it becomes possible to detect or screen the second protein (B) having a higher affinity (a large binding constant) from the library.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

(Culture medium)
YPD medium containing 1% (w / v) yeast extract, 2% (w / v) peptone and 2% (w / v) glucose, or 0.67% (w / v) amino acid-free yeast nitrogen base The yeast was cultured in SD medium containing Becton Dickinson and 2% (w / v) glucose. An amino acid and a nucleic acid corresponding to the selection marker were appropriately added to the SD medium. YPD and SD solid media were prepared by adding 2% (w / v) agar to these media.

(yeast)
Yeast Saccharomyces cerevisiae haploid strains BY4741 (methionine-requiring a cell) and BY4742 (lysine-requiring α-cell), and the genotypes of the yeast strain used and the yeast strain produced in the following examples. Table 1 and the proteins expressed in each strain are shown in Table 2. Transformed strain MC-F1 produced by gene recombination using a-type BY4741 as a parent strain expresses an EGFP reporter gene under the control of a pheromone-inducible FIG1 promoter. Transformed strains BFG2Z18-K35A, BFG2Z18-WT and BZFG2118 prepared by genetic recombination with MC-F1 as the parent strain are all proteins (A), and the Fc portion of human IgG is not bound to the cell membrane of G protein γ subunit It is expressed in the cytoplasm as a fusion protein (Gγ _cyto -Fc) with a mutant type (Gγ _cyto ). BFG2Z18-K35A, BFG2Z18-WT and BZFG2118 bind to the cell membrane a Z domain derived from protein A of Staphylococcus aureus having different affinity for the Fc part of human IgG as protein (B). To express. BFG2Z18-K35A expresses a Z domain variant (Z _{K35A, mem} ) with a binding constant of 4.6 × 10 ⁶ M ⁻¹ , and BFG2Z18-WT has a Z domain with a binding constant of 5.9 × 10 ⁷ M ⁻¹ . Wild type (Z _{WT, mem} ) is expressed, and BZFG2118 expresses a Z domain dimer (ZZ _mem ) with a binding constant of 6.8 × 10 ⁸ M ⁻¹ . BFG2118 is a negative control and expresses only Gγ _cyto -Fc.

(Reference example)
(Method for detecting protein-protein interaction using bonding ability as an index)
BFG2Z18-K35A, BFG2Z18-WT, BZFG2118, and BFG2118 yeast strains were seeded in 5 mL of YPD medium at an initial cell concentration of OD ₆₀₀ = 0.1 and co-cultured at 30 ° C. with untreated mating partner BY4742 for 3 hours. did. After culturing, the yeast was collected, washed, and resuspended in distilled water. In order to evaluate the conjugation capacity of each strain, dilution series of OD ₆₀₀ = 1.0, 0.1 and 0.001 of the yeast suspension were prepared, methionine and lysine free, 20 mg / L histidine, Spotted on diploid selection SD solid medium (SD-Met, Lys plate) containing 30 mg / L leucine and 20 mg / L uracil. As a result, BFG2118 did not grow, but the other three strains grew (FIG. 2A).

  In the three grown strains, there is an interaction between the protein (A) and the protein (B), so it seems that the a-type and α-type were joined by pheromone signaling through the G protein. It is. On the other hand, since BFG2118 lacks protein (B), such an interaction does not exist and proliferation was not observed. Thus, the ability of haploid yeast to join the a-type and α-type via pheromone signaling is effective as a method for detecting the presence or absence of the interaction between the protein (A) and the protein (B). Indicated.

Next, in order to quantitatively evaluate the conjugation ability of the three grown strains, 100 to 1000 colonies grow on a plate of 1 mL of yeast suspension adjusted to a bacterial cell concentration OD ₆₀₀ = 1.0. Thus, it diluted suitably and apply | coated to SD-Met and Lys plate. The number of colonies was counted, and the number of colonies of diploid yeast that could be produced by 1 mL of yeast suspension adjusted to the bacterial cell concentration OD ₆₀₀ = 1.0 was determined by multiplying the measured number of colonies by the dilution rate. The result is shown in FIG. 2B. Error bars in FIG. 2B indicate the standard deviation in the results of three independent experiments.

  As can be seen from FIG. 2B, there was a difference in the number of colonies of diploid yeast that could be generated corresponding to the binding constant between protein (A) and protein (B). Thus, the conjugation ability via pheromone signaling between a-type and α-type of haploid yeast is also effective as a method for quantitatively evaluating the interaction between protein (A) and protein (B). It was shown that.

Example 1
(Preparation of transformant that expresses competitive protein (C) in cytoplasm)
In order to examine whether the expression of the competitive protein (C) in the cytoplasm inhibits the restoration of conjugation signaling via the G protein, BFG2Z18-K35A, BFG2Z18-WT and BZFG2118 were genetically recombined to obtain , A transformant expressing the Z domain wild type (Z _WT ) or the Z domain mutant type (Z _K35A ) in the cytoplasm was prepared.

To _{incorporate the} Z domain wild type gene (Z _WT ) and the Z domain mutant gene (Z _K35A ) expressed as competing proteins in the cytoplasm by homologous recombination upstream of the HOP2 gene on the yeast chromosome (P _HOP2 : HOP2 promoter region). The following plasmid was constructed: The Z _WT and _{Z K35A,} from plasmid pUMZ-WT and pUMZ-K35A using primers 1 (SEQ ID NO: 5) and primer 2 (SEQ ID NO: 6) was amplified by PCR, inserted between SalI-BamHI site of plasmid pGK425 Were designated as plasmids pLMZ-WT and pLMZ-K35A, respectively. A DNA fragment used for homologous recombination in the HOP2 promoter region was amplified by PCR from the genomic DNA of MC-F1 using primer 3 (SEQ ID NO: 7) and primer 4 (SEQ ID NO: 8), and plasmids pLMZ-WT and pLMZ -Each of them was inserted between NotI-SacI sites of K35A to obtain plasmids pLMZ-WT-H and pLMZ-K35A-H, respectively.

Next, a DNA fragment containing LEU2-PGK5′-Z-PGK3′- _PHOP2 (PGK5 ′: PGK1 promoter; PGK3 ′: PGK1 terminator) was immediately upstream of _PHOP2 from pLMZ-WT-H and pLMZ-K35A-H. Amplified by PCR using primer 5 (SEQ ID NO: 9) and primer 6 (SEQ ID NO: 10) of 50 bases containing a region homologous to. The amplified DNA fragment was used for transformation of BGF2Z18-K35A, BFG2Z18-WT and BZFG2118. For transformation, the lithium acetate method was used. Transformants were selected on SD medium (SD-Ura, Leu plates) containing 20 mg / L histidine and 30 mg / L methionine without uracil and leucine, and FC1-1, FC2-1, FC3-1. FC1-2, FC2-2 and FC3-2 strains were obtained (Table 1).

(Example 2)
(Method for detecting protein-protein interaction using fluorescent reporter gene as an indicator in a transformant expressing a competing protein (C) in the cytoplasm)
The expression of the fluorescent reporter gene of the transformant obtained in Example 1 was evaluated. BFG2Z18-K35A, BFG2Z18-WT and BZFG2118 were used as examples that do not express competing proteins. As an example of expressing _ZK35A as a competitive protein, FC1-1, FC2-1 and FC3-1 were used. As an example of expressing the _{Z WT} as competitor protein, FC1-2, using FC2-2 and FC3-2. Each yeast strain was inoculated in 5 mL of YPD medium at an initial cell concentration of OD ₆₀₀ = 0.1, then α-factor was added to a final concentration of 5 μM, and the cells were cultured at 30 ° C. for 6 hours. In order to evaluate the fluorescence reporter gene expression of each strain, the yeast was collected after the culture, and the fluorescence intensity of the cells was evaluated using a flow cytometer. The results are shown in FIG. Among the two columns arranged side by side in the graph of FIG. 3, the left (dark color) column shows the result of the yeast strain into which the competitive protein was not introduced, and the right (light color) column shows the result of the yeast strain into which the competitive protein was introduced. . The error bar in FIG. 3 shows the standard deviation in the results of three independent experiments.

As a result, the yeast strains BFG2Z18-K35A, BFG2Z18-WT and BZFG2118 that do not express the competitive protein (C) all showed fluorescence. On the other hand, in the yeast strain that expresses the competitive protein (C) Z _K35A , _FC1-1 expressing the second protein (B) having a small binding constant shows no fluorescence, and the second binding constant has a higher binding constant. Only FC2-1 and FC3-1 expressing protein 2 (B) showed fluorescence (FIG. 3A). Similarly, in the yeast strain expressing a competitive protein _{(C) Z WT,} at all showed no fluorescence is being FC1-2 and FC2-2 express small second protein of the binding constant (B), more binding Only FC3-2 expressing the second protein (B) having a large constant showed fluorescence (FIG. 3B).

(Example 3)
(Method for detecting protein-protein interaction using as an index the conjugation ability in a transformant expressing a competing protein (C) in the cytoplasm)
The mating ability of the transformant obtained in Example 1 was evaluated in the same manner as in the reference example. The results are shown in FIG. Among the two columns arranged side by side in the graph of FIG. 4, the left (dark color) column shows the result of the yeast strain into which the competitive protein was not introduced, and the right (light color) column shows the result of the yeast strain into which the competitive protein was introduced. . The error bar in FIG. 4 shows the standard deviation in the results of three independent experiments.

As a result, FC2-1 and FC3-1 expressing the competitive protein _{ZK35A grew} on the diploid selection medium (FIG. 4A). The conjugation ability of FC2-1 and FC3-1 was almost the same as that of the yeast strain not expressing the competitive protein (C) (FIG. 4C). Similar results were obtained for yeast strains expressing the competitive protein (C) _ZWT . FC1-2 and FC2-2 did not show conjugation ability, but only FC3-2 showed conjugation ability almost equivalent to the yeast strain that does not express the competitive protein (C) (FIGS. 4B and 4D).

Example 4
(Screening of target cells from model library)
It was verified using a model library whether the protein-protein interaction detection method using the conjugation ability as an index in a transformant expressing the competitive protein (C) in the cytoplasm is effective. As a model library, two libraries were prepared in which target cells (FC3-2 or BZFG2118) and control cells (FC2-2 or BFGZ18-WT) were mixed at the initial ratio shown in Table 3. One is made from the yeast strain expressing no conflict protein (C), and a BFG2Z18-WT in excess expressing a small amount of BZFG2118 and _{Z WT} expressing ZZ, the other, a competitive protein (C) It consists yeast strain expressing that includes a FC2-2 of excess expressing a small amount of FC3-2 and _{Z WT} expressing ZZ. These libraries were co-cultured with the mating partner BY4742 at 30 ° C. in 10 mL YPD medium for 3 hours at an initial cell concentration of each haploid set to OD ₆₀₀ = 0.1. After the culture, the yeast was recovered, washed, applied to SD-Met and Lys plates, and cultured at 30 ° C. for 2 days. Ten colonies were picked and grown separately overnight in YPD medium. The genome was extracted, and the coding region of protein (B) was amplified by PCR using primer 7 (SEQ ID NO: 11) and primer 8 (SEQ ID NO: 12). Whether the amplified fragment was derived from ZZ or _ZWT was determined by the length of the fragment. The proportion of colonies from which ZZ fragments were obtained in 10 colonies was defined as the final proportion of target cells. A value obtained by dividing the final ratio of the target cells by the initial ratio was calculated as the screening efficiency and evaluated. The results are shown in Table 3.

  As shown in Table 3, the protein-protein interaction detection method using the competitive protein (C) showed significantly better screening efficiency than the conventional method not using the competitive protein, reaching up to 7000 times.

  These results indicate that the protein-protein interaction detection method using the competitive protein (C) has a higher affinity for the target protein (A) than the original binding protein (B) ( B) is suitable for detecting.

  According to the present invention, protein-protein interactions with high affinity can be detected or screened efficiently.

Claims (3)

A method for detecting protein-protein interaction using yeast,
(1) preparing a first expression cassette that expresses a fusion protein of the first protein and a G protein γ subunit cell membrane non-binding mutant,
(2) preparing a second expression cassette that expresses a fusion protein of the second protein and a cell membrane localization signal sequence;
(3) preparing a third expression cassette that expresses the third protein in the cytoplasm;
(4) a step of preparing transformed yeast by introducing the first, second and third expression cassettes obtained in the steps (1), (2) and (3), respectively, and (5) the A method comprising detecting pheromone signaling in the transformed yeast obtained in step (4) or screening a transformed yeast exhibiting pheromone signaling. A method for detecting protein-protein interaction using yeast,
(1) preparing a first expression cassette that expresses a fusion protein of the first protein and a G protein γ subunit cell membrane non-binding mutant,
(2) preparing a second expression cassette that expresses a fusion protein of the second protein and a cell membrane localization signal sequence;
(3) preparing a third expression cassette that expresses the third protein in the cytoplasm;
(4) a step of preparing transformed yeast by introducing the first, second and third expression cassettes obtained in the steps (1), (2) and (3), respectively, into a haploid yeast;
(5) a step of preparing a diploid yeast by joining the transformed yeast obtained in the step (4) with a haploid yeast that can be joined with the transformed yeast; and (6) the step ( A method comprising the step of selecting the diploid yeast obtained in 5).   The method according to claim 1 or 2, wherein the third protein is a competitive protein of the first protein or the second protein.

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