JP2010145238A - Method of determining antigen-binding site in anti-membrane penetration type protein antibody - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of conveniently and efficiently determining a binding site of an antibody to a membrane penetration type protein. <P>SOLUTION: The method of determining an antigen-binding site of the antibody to the membrane penetration type protein having two or more extramembranous regions includes: the process for expressing a group of variants having different extramembranous region and one or more added amino acid variants by a budding yeast expression system; the process for considering a group of the variants expressed by the budding yeast expression system as the antigen, making the antibody react to the membrane penetration type protein, and implementing an antigen-antibody reaction; and the process for identifying the variants in which the antigen-antibody reaction is not detected, and determining the extramembranous regions in which the variants are varied as a region including the antigen-binding site of the antibody or the antigen-binding site. The method determines the antigen-binding site of the antibody to the membrane penetration type protein. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention mainly relates to a method for determining an antigen binding site of an antibody against a transmembrane protein.

  In determining the antigen-binding site of an anti-protein antibody, the three-dimensional structure of the antigen-antibody complex is determined and directly judged, or an infinite number of partial peptides based on the primary sequence of the antigen are prepared, A method called peptide scan that accumulates and estimates the binding information is generally used (see Non-Patent Document 1).

  In the former, accurate bond patterns can be evaluated, but it is difficult to say that it is at an industrially practical level because it takes cost and time to prepare crystals and labels to be used as samples for X-ray crystal structure analysis and NMR. .

  In the latter peptide scan, in order to make an accurate determination, it is necessary to design a partial peptide based on the primary sequence of the antigen protein including duplication. For this reason, partial peptide synthesis requires a great deal of cost depending on the length of the antigen protein sequence. For example, in an example in which the epitope of an antibody against a rat-derived neurotensin receptor was determined by peptide scanning (see Non-Patent Document 2), nine types of peptides were synthesized and investigated in a region near the C-terminal. However, if an antibody that recognizes the other region is given, it will be necessary to prepare a larger number of synthetic peptides.

  On the other hand, recombinant protein expression and its evaluation method have been reported for the purpose of obtaining a quality sample suitable for crystal structure analysis of membrane protein derived from higher organisms (see Non-Patent Document 3). This technology is characterized by introducing a plasmid for expression into Saccharomyces cerevisiae to express a membrane protein fused with a fluorescent protein, and then estimating the success rate of expression and solubilization using the fluorescence of the specimen as an indicator. . In addition, it has been reported that mutant expression plasmid vectors can be efficiently prepared by utilizing homologous recombination in budding yeast (see Non-Patent Document 4).

However, the use of these techniques is limited to the efficient expression of proteins and the evaluation of the expression level. In the review on the implementation method of the above technique (see Non-Patent Document 5), it is not possible to apply the technique to Western analysis. Rather, there was a statement that it was not recommended.
Gershoni, Biodrugs, 2007, 21 (3), 145-56 Niebauer, J. Recept Signal Transduct Res., 2006, 26, 395-415 Newstead, Proc. Natl. Acad. Sci. USA, 2007, 104 (35), 13936-41 Ito K, Biochem. Biophys. Res. Commun., 2008, 371 (4), 841-5 Drew D, Nat.Protocol, 2008, 3 (5), 784-98

  The main object of the present invention is to provide a method capable of quickly and efficiently determining the binding site of an antibody to a transmembrane protein.

  As a result of intensive studies aimed at solving the above-mentioned problems, the present inventor has produced a group of transmembrane proteins and mutants in which sequences in the extramembrane region have been substituted, and budding yeast. It was found that the antigen-binding site of an anti-transmembrane protein antibody can be easily determined by expressing it in an expression system and analyzing the expressed mutant group by performing an antigen-antibody reaction. As a result, the present invention was completed.

  That is, the present invention relates to the following matters.

Item 1: A method for determining an antigen-binding site of an antibody against a transmembrane protein having two or more extra-membrane regions,
Expressing a group of mutants in which one or several amino acid mutations are added to different extramembrane regions in a budding yeast expression system;
A step of performing an antigen-antibody reaction by causing an antibody against the transmembrane protein to act as a group of mutants expressed in the budding yeast expression system,
Identifying a mutant in which an antigen-antibody reaction is not detected, and determining an extra-membrane region in which the mutation is added in the mutant as an antigen-binding site of the antibody or a region containing an antigen-binding site;
A method for determining an antigen-binding site of an antibody against a transmembrane protein, comprising:

Item 2: The step of expressing a group of the mutants comprises:
Item 2. The method according to Item 1, wherein the method is expressed as a fusion protein in which a fluorescent protein is fused to a mutant.

  Item 3: The method according to Item 1 or 2, wherein the transmembrane protein is a seven-transmembrane receptor.

Item 4: Furthermore, a group of mutants in which one or several different amino acid mutations are added to the same outer membrane region is expressed in a budding yeast expression system,
Using a group of the expressed mutants as an antigen, an antibody against the transmembrane protein is allowed to act to perform an antigen-antibody reaction,
Characterized in that it includes a step of identifying a variant in which an antigen-antibody reaction is not detected, and determining a portion where the mutation is added in the variant as an antigen-binding site of the antibody or a region containing the antigen-binding site. Item 4. The method according to any one of Items 1 to 3.

  Hereinafter, the present invention will be described in more detail.

1. Step of expressing a group of mutants in a budding yeast expression system A mutant in the present invention is one or several amino acid residues in one of the transmembrane domains in a transmembrane protein having two or more transmembrane domains. It is a transmembrane protein mutant that has been mutated.

Amino acid mutations include substitution, deletion, insertion and the like.
The amino acid mutation is not particularly limited as long as it damages or reduces the binding between the target protein and the antibody. Usually, the structure is set so as not to be similar to the structure of the same type of protein after considering the structure of the corresponding site from the same type of protein as the target protein.

  In setting the mutation site, a portion corresponding to the extra-membrane region is estimated from the amino acid sequence or gene sequence of the target protein. The estimation method is not particularly limited, but usually the amino acid sequence of the target protein is aligned using a published sequence alignment algorithm such as Clustal or T-coffee with reference to the amino acid sequence of the protein recognized as the same family, This is done by comparing.

  As the reference protein, a protein that can be predicted with high accuracy by an extra-membrane prediction algorithm is adopted, but it is recommended to adopt a protein whose transmembrane region and extra-membrane region are known by empirical means such as three-dimensional structure analysis. The In the case of a 7-transmembrane protein, it is possible to accurately determine the position of the extramembrane region by comparing the adrenoceptor, adenosine receptor, or rhodopsin whose structure has been elucidated as a reference protein. .

  In the present invention, a group of variants in which one or several amino acid mutations are added to different extra-membrane regions means a group of variants in which one or several amino acid mutations are added to each of the extra-membrane regions of a protein. In other words, it is a group of mutants in which one or several amino acid mutations are added in different transmembrane regions.

  For example, in the group of mutants, the first mutation is a group of 7-transmembrane protein mutants in which one or several amino acid mutations are added only to the first transmembrane region. The second variant in which one or several amino acid mutations are added only to the second extra-membrane region, and the third variant in which one or several amino acid alterations are added only to the third extra-membrane region. A variant in which one or several amino acid mutations are added only to the fourth extra-membrane region, and one or several amino acid alterations are added only to the fifth extra-membrane region. The fifth variant, the sixth variant in which one or several amino acid mutations are added only to the sixth extra-membrane region, and the one or several amino acid alterations are added only to the seventh extra-membrane region And the eighth mutation in which one or several amino acid mutations are added only to the eighth extramembranous region The group consisting of and the like.

  Furthermore, in order to pursue the binding site in more detail, one or a plurality of mutants having mutations in the same extra-membrane region but different mutation sites can be further added to the group.

  The amino acid sequence mutation method can follow a known method and is not particularly limited. For example, in the gene sequence of the target protein, the DNA sequence translated into the amino acid residue to be mutated is translated into another amino acid residue. Designing a mutant gene sequence substituted for the DNA sequence to be prepared, preparing a mutant gene fragment by genetic manipulation, and preparing an expression plasmid vector in which the mutant gene fragment is inserted at a predetermined position of the host cell plasmid Can be performed.

  Mutation may be to mutate only one amino acid residue or to mutate two or more amino acid residues simultaneously by one operation.

  Preferably, the mutant is expressed by a transformant of Saccharomyces cerevisiae into which an expression plasmid vector has been introduced.

  For example, the operation of inserting a DNA sequence translated into the peptide chain constituting the mutant into a yeast plasmid and preparing an expression plasmid vector is a biological mechanism of budding yeast in which homologous recombination occurs frequently. This can be done by using the characteristic features. 3 'end side of the upstream part and 5' end side of the extramembranous region part with respect to the DNA region of the upstream part and the downstream part in the transcriptional direction sandwiching the extramembranous part part to be mutated, A fragment having about 30 base pair duplications on the 3 ′ side of the outer membrane region and the 5 ′ end of the downstream portion is prepared by PCR or the like. At this time, the 5 ′ end side of the upstream fragment and the 3 ′ side of the downstream fragment have the same sequence as the upstream and downstream sequences of the cloning site of the yeast plasmid, respectively. When the extra-membrane region is short or when the gene terminal region is mutated, the DNA fragment to be prepared can be a fragment of 2 or less containing the mutated DNA sequence as an overlapping sequence. When transforming a budding yeast together with a yeast plasmid linearized at the cloning site, homologous recombination occurs in a yeast cell with a given sequence, which is retained as an expression plasmid into which the mutant sequence is inserted. . The transformant can then be subjected to mutant expression.

  More preferably, it can be carried out according to the method described in the Examples.

  However, the expression of the mutant by yeast is not limited to the above method, and a method generally performed in a host system other than yeast can be used. For example, it is possible to adopt a means of once obtaining a plasmid vector for expression using the cassette mutation method or Quikchange method and introducing it into budding yeast again.

  The mutant may be expressed as a fusion protein of a mutant protein obtained by adding a mutation to the target protein and a fluorescent protein.

  For example, when yeast is used as the host cell, a mutant green fluorescent protein (yEGFP) gene optimized for yeast expression is placed downstream of the cloning site in the expression plasmid, and the mutant transmembranes. It can be expressed using a protein designed to be expressed as a fusion protein of type protein and yEGFP.

  By expressing as a fusion protein with a fluorescent protein, the determination of the binding site is simple and highly accurate. In particular, in the case of a heterologous expression system in which the target protein is derived from a different host, the expression level may be extremely reduced or the expression may be suppressed depending on the amino acid sequence of the mutant. In this case, bringing in a mutant preparation having no expression to the subsequent steps contributes to erroneous determination. However, if the fluorescence intensity of the mutant expressed as a fusion protein is measured and the antigen-antibody reaction is detected using a sample whose fluorescence intensity is observed, the above-mentioned misjudgment can be avoided and the expressed mutation It becomes possible to accurately detect the antigen-binding site only for the body.

  A culture of the transformant is collected, and a cell membrane fraction expressing the mutant is extracted.

  Specifically, the cells in the culture are collected by centrifuging the culture, the cells are crushed, the carcasses of the cells are discarded by low-speed centrifugation, and the supernatant is collected to collect the cell membrane fraction. Can be obtained. The obtained membrane fraction can be used for the following antigen-antibody reaction as a membrane specimen as it is. However, if necessary, the membrane fraction may be simply purified by centrifugation or the like. By purifying, the preservability of the specimen using the membrane fraction is improved.

2. Step of detecting antigen-antibody reaction An antigen is reacted with a group of transmembrane proteins and mutants expressed in the budding yeast expression system to detect an antigen-antibody reaction.

  The antibody is not particularly limited as long as it is an anti-transmembrane protein antibody against the target protein, but from the viewpoint of determining a specific binding site, a monoclonal antibody is preferably used in the present invention.

  The method for detecting the antigen-antibody reaction can be performed according to a known method, and is not particularly limited.

For example, a method for detecting by directly or indirectly labeling an antibody, specifically, ELISA method or CLIA method for detecting by enzyme labeling such as horseradish peroxidase or alkaline phosphatase, luminol or GFP (Green Fluorescence Protein) An FIA method for detecting a fluorescent label, an RIA method for detecting a radioisotope label such as ¹²⁵ I, and the like can be used.

  In addition, detection methods such as Western blotting and dot blotting can also be used. For example, in Western blotting, a membrane specimen is developed on a polyacrylamide gel, and then the gel contents are transferred to a flat membrane made of PVDF or nitrocellulose. In dot blotting, membrane specimens are spotted directly onto a flat membrane and adsorbed. A monoclonal antibody is allowed to act on the flat membrane on which the membrane specimen is immobilized, and the binding between the antibody and the protein immobilized on the flat membrane is detected directly or indirectly.

3. Step of determining antigen-binding site of antibody By the above-mentioned step, the presence or absence of an antigen-antibody reaction against a certain monoclonal antibody in a group of transmembrane proteins and mutants is confirmed. In this case, the mutant in which the antigen-antibody reaction is detected does not lose the antigen sequence due to the mutation, while the mutant that has not been detected has the antigen sequence completely deleted or partially missing. Can be considered. Therefore, it is determined that the position of the mutation applied to the mutant not detected is involved in the antigen-binding site of the monoclonal antibody.

  For example, a first mutant in which a mutation is added to the first outer membrane region, a second mutant in which a mutation is added to the second outer membrane region, and a mutation in the third outer membrane region A third variant, a fourth variant in which a mutation is added to the fourth extra-membrane region, a fifth variant in which a mutation is added to the fifth extra-membrane region, A sixth mutant in which a mutation is added to the extramembrane region, a seventh mutant in which a mutation is added to the seventh extramembrane region, and an acid mutation in the eighth extramembrane region A group consisting of the eighth mutant is subjected to an antigen-antibody reaction using a monoclonal antibody, and an antigen-antibody reaction is detected for the Xth mutant (X represents an integer of 1 to 8). If not, it can be determined that the antigen-binding site of the monoclonal antibody is present in the Xth extramembrane region.

  When determining the antigen-binding site of an antibody, specimens or analysis results may be arranged two-dimensionally so that the type of mutant and the type of antibody to be investigated are orthogonal.

  For example, using a group of mutants of type Y (Y is an integer of 2 or more), using ELISA or dot blot, analyze the antigen binding site for antibody of type Z (Z is an integer of 2 or more) In this case, a different variant is immobilized for each row of the plate or slot of the Z column × Y row well, and the antigen-antibody reaction is performed by arranging the antibodies in each column, thereby allowing the epitope of the Z type antibody to be detected. The analysis result can be grasped at a glance.

  Also, in the case of Western blotting, the overlay of the mutants to each lane has regularity, and the results of detecting the antigen-antibody reaction using the Z-type antibodies for the Y-type mutants are shown as Z row × Y The results of analyzing the epitopes of the Z-type antibody can be grasped at a glance by arranging them in a row format and comparing them with each other.

  The present invention provides a method for quickly and efficiently determining a region involved in an antigen-binding site of an antibody against a transmembrane protein.

  In the conventional determination method involving a three-dimensional structure analysis, a protein structure analysis is required. Particularly in the case of higher organisms, preparation of samples and a structure analysis are difficult, but in the present invention, a protein structure analysis is performed. The antigen binding site can be determined without going through.

  In addition, in the conventional determination method using peptides, it is necessary to synthesize a large number of peptides, and there is a possibility that an artificial error may occur due to selection of peptide sequences. Since the analysis is performed as a target, the possibility of the above-described problem is eliminated.

  From the above features, the present invention can greatly reduce the time and cost required to determine the antigen-binding site of a membrane protein, and provides a method capable of quickly and efficiently implementing the structure-determining site of a transmembrane protein. To do.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in more detail, this invention is not limited by these.

  Unless otherwise specified, “%” indicates “mass%”.

The binding region in the M2 receptor of the monoclonal antibody against the mouse-derived anti-human muscarinic acetylcholine receptor M2 subtype (hereinafter also referred to as M2 receptor) was analyzed using Western blot.

1-1. Preparation of budding yeast expression plasmid
In order to determine the extramembranous region in the primary sequence of the M2 receptor, pair-wise alignment was performed with the sequence program T-coffee with reference to the primary sequence of the human β2 adrenergic receptor (β2 receptor). The transmembrane region of the β2 receptor was then read from the crystal structure of the receptor (Rasmussen SGF et al. (2007) Nature 450 383-387) and the corresponding primary sequence of the M2 receptor was C- N-terminal loop region (Nter), intracellular first loop region (il), extracellular first loop region (o1), intracellular second loop region (i2), extracellular second loop region ( o2), the inner third loop region (i3), the outer third loop region (o3), and the C-terminal loop region (Cter) were determined as the extramembrane region.

  For the individual mutants corresponding to these extra-membrane regions, in the primary sequence of other muscarinic acetylcholine receptor subtypes, substitution to the amino acid sequence of the receptor having as little similarity as possible among the sequences corresponding to each extra-membrane region Thus, the sequence of each mutant was set as follows.

  The primary structure of the M2 receptors used in this embodiment is a single letter, represented by EmudidiesutidiesuesudienuesuerueierutiesuPiwaikeitiefuibuibuiefuaibuierubuieijiesueruesuerubuitiaiaijienuaierubuiemubuiesuaikeibuienuarueichierukyutibuienuenuwaiefueruefuesuerueishieidieruaiaijibuiefuesuemuenueruwaitieruwaitibuiaijiwaidaburyuPieruJipibuibuishidierudaburyuerueierudiwaibuibuiesuenueiesubuiemuenuerueruaiaiesuefudiaruwaiefushibuitikeiPierutiwaiPibuikeiarutitikeiemueijiemuemuaieieieidaburyubuieruesuefuaierudaburyueiPieiaieruefudaburyukyuefuaibuijibuiarutibuiidijiishiwaiaikyuefuefuesuenueieibuitiefujitieiaieieiefuwaieruPibuiaiaiemutibuieruwaidaburyueichiaiesuarueiesukeiesuaruaikeikeidikeikeiiPibuieienukyudiPibuiesuPiesuerubuiPiesuaruikeikeibuitiarutiaierueiaieruerueiefuaiaitidaburyueiPiwaienubuiemubuieruaienutiefushieiPishiaiPienutibuidaburyutiaijiwaidaburyuerushiwaiaienuesutiaienuPieishiwaieiLCNATFKKTFKHLLMCHYKNIGATR (SEQ ID NO: 1).

As a group of mutants,
Nter mutant in which 2-DDSTDSSDNSLALTSPYKTFE-22 (SEQ ID NO: 2) is replaced with TDDPLGGHTVWQ (SEQ ID NO: 3),
I1 mutant in which 51-NRHLQTVNN-59 (SEQ ID NO: 4) is replaced with DKQLKTVDD (SEQ ID NO: 5),
86-IGYWPLGPVV-95 (SEQ ID NO: 6) replaced with MNRWALGNLA (SEQ ID NO: 7) o1 mutant,
I2 mutant in which 123-CVTKPLTYPVKRTTKM-139 (SEQ ID NO: 8) is replaced with SITRPLTYRAKRSTKR (SEQ ID NO: 9),
An o2 mutant in which 164-FIVGVRTVEDGECYIQFFSN-183 (SEQ ID NO: 10) is replaced with YFVGKRTVPPGECFIQFLSE (SEQ ID NO: 11),
I3 mutant in which 218-KKDKKEPVANQDPVSPSLVPSREK-241 (SEQ ID NO: 12) is replaced with DKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEH (SEQ ID NO: 13),
O3 mutant in which 272-APCIPN-277 (SEQ ID NO: 14) is replaced with DSCVPK (SEQ ID NO: 15),
A Cter mutant was prepared in which 303-ATFKKTFKHLLMCHYKNIGATR-324 (SEQ ID NO: 16) was replaced with KT (SEQ ID NO: 17).

  In the following, the same operation was performed in parallel on each mutant except those explicitly described.

  The yeast plasmid pRS426GAL1-GFP (Newstead S. et al. (2007) Proc. Natl. Acad. Sci. USA 104 (35) 13936-13941) is a tobacco etch virus protease (TEV) downstream of the cloning site of the pRS426GAL1 plasmid. A base sequence arranged in the order of recognition sequence, yEGFP gene, and His × 8 residue tag was inserted. This was digested with the restriction enzyme SmaI and linearized. Next, using the gene sequence of the M2 receptor as a template, a gene fragment necessary for preparing a mutant expression plasmid was amplified by PCR. The reaction volume was 50 μL, and 0.1 U Phusion High-Fidelity DNA Polymerase (Finnzymes) was used as the polymerase.

  Two primers were prepared for the Nter mutant and the Cter mutant, and four primers were prepared for the remaining mutants. Digested fragments and amplified fragments were confirmed by 1% agarose electrophoresis, and the corresponding bands were excised from the gel and purified.

The host strain was then transformed with a linear plasmid and a DNA fragment containing the membrane protein gene and the mutant sequence. The host strain is budding yeast strain FGY217 (Kota J. et al. (2007) J. Cell Biol. 176 617-628) shaken in 10 mL of YPD medium and collected at 2000 xg when OD ₆₀₀ ≈0.6. And washed several times with sterilized water. Transformation was performed by the lithium acetate method (Gietz RD et al. (1995) Yeast 11 355-360).

  The transformed host strain solution was applied to 2% glucose-containing uracil-deficient minimal medium prepared as a 2% agar plate, and allowed to stand at 30 ° C. for 3 days to form colonies. Single colonies were dispensed into two centrifuge tubes containing 10 mL of 0.1% glucose-containing uracil-deficient medium, one of which was collected after shaking culture at 30 ° C. until the cell concentration was saturated. The collected cells were sheared using glass beads (acid-washed, Sigma), and a plasmid solution was obtained from the obtained cell extract using a plasmid miniprep kit (Qiagen). It was confirmed that the mutation was appropriately performed by analyzing the DNA sequence of the plasmid.

1-2. Expression of transmembrane protein in Saccharomyces cerevisiae expression system One of 10 mL of the above 0.1% glucose-containing uracil-deficient medium was shake-cultured at 30 ° C, and when OD ₆₀₀ ≒ 0.6, final concentration of 2% galactose was added. did. After continuing shaking culture at 30 ° C. for 20 hours, the medium was collected and the cells precipitated at 2000 × g were mixed with 50 mM Tris-HCl (pH 7.6), 5 mM EDTA, 10% glycerol, 1 × protease inhibitor. It was suspended in 700 μL of a buffer solution (YSB) consisting of a cocktail (Complete EDTA-free, Roche).

  Add 200 μL of glass beads (Glass beads, acid-washed, Sigma), shake at high speed for 30 minutes at 4 ° C, shear the cells and centrifuge the extract for 1 minute at 14,000 xg for 1 minute. Removed. The supernatant was passed through a small ultracentrifuge at 100,000 × g for 30 minutes, and the precipitate (expression membrane fraction) was collected and suspended in 1 mL of YSB. 2 μL of the suspension was mixed with 2 μL of a solution consisting of 50 mM Tris-HCl (pH 7.6), 5% glycerol, 5 mM EDTA, 0.02% bromophenol blue, and allowed to stand for 30 minutes. Trisglycine 12% polyacrylamide gel (Invitrogen) ) Was used for electrophoresis at 100 V for 120 minutes to develop the composition. The developed gel was photographed with a fluorescence image analyzer (trade name: LAS-1000, Fuji Film), and a single band derived from the GFP fusion mutant protein was confirmed.

  Further, the expression membrane fraction of the unmutated M2 receptor was electrophoresed in the same manner as described above, and expression was confirmed by each detection method of CBB staining, fluorescence detection, and Western blot. For CBB staining, the gel is immersed in an appropriate amount of staining solution (trade name: Imperial Protein Stain (Thermo Scientific)) for 2 hours, then rinsed thoroughly with pure water, and image analyzer (trade name: LAS-1000, Fuji Film). ) Digitizing mode (exposure time 0.25 seconds).

  In fluorescence detection, the electrophoresed gel was directly photographed in a fluorescence detection mode (exposure time 30 seconds) of an image analyzer (trade name: LAS-1000, Fuji Film).

  In Western blotting, 2 mL of a chemiluminescent reagent (trade name: Immobilon Western Detection Reagents, Millipore) was brought into contact with the PVDF membrane after the secondary antibody reaction, allowed to stand for 5 minutes, and then an image analyzer (trade name: LAS-1000, Fuji). Films) and chemiluminescence detection mode (exposure time 30 seconds). For primary antibody reaction, 5 mL of mAb5 diluted to 200 ng / mL with TBS-T, and for secondary antibody reaction, HRP-conjugated anti-IgG antibody diluted to 20 ng / mL with TBS-T (trade name: goat anti-mouse IgG HRP 5 mL of conjugate (manufactured by Santa cruz) was used.

  In FIG. 1, the schematic diagram of the expression of the unmutated body by a budding yeast and the fusion body of a mutant and yEGFP is shown.

  Moreover, the result of having confirmed the expression about each variant and a non-mutant is shown in FIG. FIG. 2 (a) shows the results of confirming the expression of the M2 receptor non-mutant. 1 shows CBB staining, 2 shows fluorescence detection, and 3 shows the result of detection by Western blot.

  As shown in FIG. 2 (a), a myriad of contaminating proteins were detected by CBB staining, but an unmutated M2 receptor was selectively detected by fluorescence detection. In addition, when antibody detection was performed using a monoclonal antibody, a signal was observed at a position where fluorescence was confirmed.

FIG. 2 (b) shows the results of confirming the expression of a group of unmutated and mutants by fluorescence detection.
From the left, the results of unmutated, Nter mutant, i1 mutant, o1 mutant, i2 mutant, o2 mutant, i3 mutant, o3 mutant, and Cter mutant are shown. FIG. 2B shows the band position with the highest fluorescence intensity extracted and displayed.

  For reference, 10 ng of yEGFP and a fluorescent marker (trade name: Benchmark fluorescent, Invitrogen) were also run simultaneously, and the concentration of the mutant expressed from the fluorescence intensity of the band and the relative molecular weight were estimated from the mobility.

  The mutant suspension was diluted with YSB so that the fluorescence intensity was 20 ng / μL, and then stored frozen at −80 ° C.

1-3. Determination of antigen-antibody reaction and binding site for the expressed protein 1 μL of each of the mutant suspensions prepared in 1-2 above was subjected to electricity at 100 V for 120 minutes using 12% polyacrylamide gel (Tris-Glycine Gel, Invitrogen). After electrophoresis, the gel surface is contacted with a PVDF membrane that has been hydrophilized, and 30 V in a tank-type transfer device (Xcell; Invitrogen) filled with a transfer solution consisting of 0.3% Tris, 1.5% glycine, and 0.1% SDS. Transfer was performed in 90 minutes. Next, the membrane was immersed in TBS-T (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% (v / v) Tween-20) containing 2% skim milk for 1 hour to block, and 5 types of primary antibodies were used. Anti-M2 antibodies mAb1 to 5 for determination and HRP-conjugated anti-IgG antibody (trade name: goat anti-mouse IgG HRP conjugate: manufactured by Santa cruz) as a secondary antibody were allowed to act to carry out an antigen-antibody reaction. A secondary antibody was detected using a chemiluminescent reagent (trade name: Immobilon Western Detection Reagents, manufactured by Millipore), and it was determined that the portion of the mutant corresponding to the band that was not detected was the epitope region. .

  FIG. 3 shows the results of testing the antigen-antibody reaction for a group of M2 receptor unmutated and mutants and 5 types of anti-M2 monoclonal antibodies (mAbs 1 to 5). The horizontal axis represents the unmutated mutant and each mutant, and the vertical axis aligned the results of each antibody.

  As shown in the results of FIG. 3, mAb1, mAb2, mAb3, and mAb4 are determined to be antibodies specific for the N-terminal loop region since no band against the Nter mutant has been detected. On the other hand, mAb5 is determined to be an antibody specific for the intracellular third loop region since no band for the i3 mutant was detected.

For the monoclonal antibody in the anti-M2 receptor antibody-producing hybridoma culture supernatant, the epitope region in the M2 receptor was determined by ELISA detection.

2-1. Immobilization and blocking of mutants Among a group of membrane specimens of M2 receptor mutants prepared in Example 1, Nter mutant, i1 mutant, o1 mutant, i2 mutant, o2 mutant, i3 mutant, For o3 mutant and unmutated mutants, 2 μL of each mutant suspension was diluted to 100 μL with YSB and injected into a highly adsorbable 96-well plate (trade name: Maxisorp, manufactured by Nunc), and kept at 4 ° C. for 2 hours. Each component was fixed to the plate wall by leaving it to stand. Thereafter, the suspension was discarded, and the unadsorbed components were removed by exchanging with 150 μL of a washing solution composed of 20 mM Hepes-NaOH (pH 7.5), 150 mM NaCl, 0.2% decyl maltoside. Subsequently, the plate was replaced with 150 μL of a washing solution supplemented with 1% bovine serum albumin, and the plate was allowed to stand at 4 ° C. for 2 hours, thereby reducing nonspecific antibody adsorption in the following procedure.

2-2. Antigen-antibody reaction and determination of the binding site Anti-M2 receptor antibody-producing hybridoma culture supernatant is diluted approximately 5 times with a washing solution, and 100 μL is injected into each well of the ELISA plate to which the unmutated and mutants are immobilized. The mixture was allowed to stand at 4 ° C. for 2 hours, and a primary antibody reaction was performed with 12 kinds of anti-M2 antibodies mAb 6 to 17 for determination dissolved in the hybridoma culture supernatant.

  Thereafter, the culture supernatant dilution was discarded, and washing with 150 μL of the washing solution was repeated twice. Next, 100 μL each of HRP-conjugated anti-IgG antibody (trade name: goat anti-mouse IgG HRP conjugate: manufactured by Santa cruz) diluted to 20 ng / mL with a washing solution as a secondary antibody was injected and allowed to stand at 4 ° C. for 2 hours. After the reaction, the secondary antibody solution was discarded and washing with 200 μL of the washing solution was repeated three times. The remaining solution was removed, and 100 μL of a chemical coloring reagent (trade name: ABTS solution, manufactured by Roche) was added to confirm color development. Further, after standing at room temperature for 15 minutes, absorbance at 415 nm and 562 nm was measured with a plate reader (trade name: Spectramax M2e, manufactured by Molecular Devices), and the difference was recorded. For the same hybridoma culture supernatant, the difference in absorbance between the mutants was compared to analyze the epitope.

  FIG. 4 (a) shows the result of the color development reaction of the plate 30 minutes after the reaction. FIG. 4 (b) shows the difference in absorbance at 415 nm and 562 nm after 15 minutes of reaction. The mutants are arranged in the row direction and the hybridoma culture supernatants are arranged in the column direction. NC is a membrane specimen that does not express the mutant and was used to observe basal level color development.

  Based on the results shown in FIG. 4, the color development of the plate is observed for each column, and in each well in the same column where the color intensity is not significantly observed, the mutation site applied to the corresponding mutant Was determined to be a region having an antibody binding sequence. The color development can be confirmed visually as shown in (a), but if it is not clear, as shown in (b), the difference in absorbance at 415 nm and 562 nm is measured, and the intensity is compared by quantification. It was.

  As a result, in the column using mAb7, mAb9, mAb11-mAb17 as the primary antibody, the color development of the well to which the i3 mutant was fixed was significantly reduced, so that mAb7, mAb9, mAb11-mAb17 The antibody was determined to be specific for the third loop region. In addition, in the column using mAb10, since color development was not observed in the wells to which the N-terminal mutant was fixed, mAb10 was determined to be an antibody specific for the N-terminal loop region.

  As indicated above, the state of the monoclonal antibody does not need to have been purified, and can be applied even to the culture supernatant in the process of hybridoma clone establishment, and the antigen-binding region can be determined. I understood.

The epitope region in the AA2a receptor of the monoclonal antibody (mAb18) against the mouse-derived anti-human adenosine receptor A2a subtype (hereinafter also referred to as AA2a receptor) was analyzed by Western blot as follows.

3-1. Preparation of budding yeast expression plasmid
In order to determine the extramembranous region in the primary sequence of the AA2a receptor, pair-wise alignment was performed using the sequence program T-coffee with reference to the primary sequence of the human β2 adrenergic receptor (β2 receptor). The transmembrane region of the β2 receptor was then read from the crystal structure of the receptor (Rasmussen SGF et al. (2007) Nature 450 383-387) and the amino acid primary sequence of the corresponding M2 receptor was C- Eight regions of Nter, i1, o1, i2, o2, i3, o3, and Cter were determined as the outer membrane region toward the end.

  Substitutions made to individual mutants corresponding to these extramembrane regions have as much similarity as possible among the sequences corresponding to each extramembranous region in the primary sequence of other adenosine receptor subtypes (A1, A2b, A3). The sequence of each mutant was set as follows, considering substitution to the amino acid sequence of the non-receptor.

  The primary structure of AA2a Receptor Using in this embodiment is a single letter, represented by EmuPiaiemujiesuesubuiwaiaitibuiierueiaieibuierueiaierujienubuierubuishidaburyueibuidaburyueruenuesuenuerukyuenubuitienuwaiefubuibuiesuerueieieidiaieibuijibuierueiaiPiefueiaitiaiesutijiefushieieishieichijishieruefuaieishiefubuierubuierutikyuesuesuaiefuesueruerueiaieiaidiaruwaiaieiaiaruaiPieruaruwaienujierubuitijitiarueikeijiaiaieiaishidaburyubuieruesuefueiaijierutiPiemuerujidaburyuenuenushijikyuPikeiijikeikyueichiesukyujishijiijikyubuieishieruefuidibuibuiPiemuenuwaiemubuiwaiefuenuefuefueishibuierubuiPieruerueruemuerujibuiwaieruaruaiefuerueieiaruarukyuerukeikyuemuiesukyuPierupijiiarueiaruesutierukyukeiibuieichieieikeiesuerueiaiaibuijieruefueierushidaburyueruPierueichiaiaienushiefutiefuefushiPidishiesueichieiPierudaburyueruemuwaierueiaibuieruesueichitienuesubuibuienuPiefuaiwaieiwaiaruIREFRQTFRKIIRSHVLRQQEPFKA (SEQ ID NO: 18).

As a group of mutants,
Nter mutant in which 3-IMGSSV-8 (SEQ ID NO: 19) is replaced with MPPSISAFQAA (SEQ ID NO: 20),
An i1 mutant in which 32-WLNSNLQNVTNY-43 (SEQ ID NO: 21) is replaced with KVNQALRDSTFC (SEQ ID NO: 22);
O1 mutant in which 70-FCAACHG-76 (SEQ ID NO: 23) is replaced with ITIHFYS (SEQ ID NO: 24),
An i2 mutant in which 104-IAIRIPLRYNGLVTGT-119 (SEQ ID NO: 25) is replaced with LRVKLTVRYKRVTTHR (SEQ ID NO: 26);
O2 mutant in which 146-CGQPKEGKQHSQGCGEGQVACLFEDVVP-173 (SEQ ID NO: 27) is replaced with LSAVERAWAANGSMGEPVIKCEFEKVIS (SEQ ID NO: 28),
205-RRQLKQMESQPLPGERARSTLQKEVH-230 (SEQ ID NO: 29) is replaced with KRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHK (SEQ ID NO: 30),
An o3 mutant in which 261-DCSHA-265 (SEQ ID NO: 31) is replaced with SCHK (SEQ ID NO: 32);
A Cter mutant was prepared in which 297-QTFRKIIRSHVLRQQEPFKA-316 (SEQ ID NO: 33) was replaced with VT (SEQ ID NO: 34).

  Transformation of budding yeast strain FGY217 by lithium acetate method using yeast plasmid pRS426GAL1-GFP linearized with restriction enzyme SmaI and gene sequence of AA2a receptor or its mutant amplified by PCR method did. The transformed host strain solution was applied to 2% glucose-containing uracil-deficient minimal medium prepared as a 2% agar plate and allowed to stand at 30 ° C. for 3 days to form colonies. Single colonies were dispensed into two centrifuge tubes containing 10 mL of 0.1% glucose-containing uracil-deficient medium, one of which was collected after shaking culture at 30 ° C. until the cell concentration was saturated. The collected cells are sheared using glass beads (trade name: Glass beads, acid-washed, manufactured by Sigma), and a plasmid solution is obtained from the obtained cell extract using a plasmid miniprep kit (Qiagen). did. It was confirmed that the mutation was appropriately performed by analyzing the DNA sequence of the plasmid.

3-2. Expression of transmembrane protein in Saccharomyces cerevisiae expression system In the above 1-2, “AA2a receptor” is used instead of “M2 receptor”, and AA2a receptor mutants are substituted for a group of M2 receptor mutants. Except for using one group, protein was expressed in the same manner.

3-3. Determination of antigen-antibody reaction and binding site for expressed protein In 1-3 above, “AA2a receptor” is used instead of “M2 receptor”, and AA2a receptor mutation is substituted for a group of M2 receptor mutants. The same operation was performed except that a group of bodies was used, and the antigen-antibody reaction and the binding site were determined.

  The upper part of FIG. 5 shows the result of confirming the expression results of the AA2a receptor non-mutant and a group of the mutants by fluorescence detection.

  Further, the lower part of FIG. 5 shows the results of detecting the antigen-antibody reaction against the AA2 receptor non-mutant and a group of the mutants by Western blot.

  As shown in FIG. 5, although the expression was confirmed in the lane where the i3 mutant was developed, no luminescence based on the antigen-antibody reaction was observed. Therefore, it was judged that mAb18 recognizes the sequence 205-RRQLKQMESQPLPGERARSTLQKEVH-230 (SEQ ID NO: 35) located in the cell inner third loop.

3-4. Narrowing down the epitope region using a group of intracellular third loop variants Analyzes the more detailed binding sites of anti-AA2a antibody mAb18 determined to recognize the intracellular third loop in 3-3 above Therefore, a group of 5 mutants with different mutations in the same third loop inside the cell was prepared, and expression confirmation and antigen-antibody reaction detection were performed.

As a group of mutants of AA2a receptor intracellular third loop,
An i3-1 mutant in which 198-LRIFLAARRQ-207 (SEQ ID NO: 36) is replaced with LEVFYLIRKQ (SEQ ID NO: 37);
203-AARRQLKQME-212 (SEQ ID NO: 38) is replaced with IIRNKLSLNL (SEQ ID NO: 39) i3-2 mutant,
I3-3 mutant in which 208-LKQMESQPLP-217 (SEQ ID NO: 40) is replaced with LNKKVSAS (SEQ ID NO: 41),
I3-4 mutant in which 213-SQPLPGERAR-222 (SEQ ID NO: 42) is replaced with SNSKETG (SEQ ID NO: 43),
An i3-5 mutant in which 218-GERARSTLQK-227 (SEQ ID NO: 44) was replaced with SGDPQKYYGK (SEQ ID NO: 45) was prepared.

  For the construction of expression plasmids, transformation, transmembrane protein expression, antigen-antibody reaction and binding site determination, use "AA2a receptor" instead of "M2 receptor", and a group of M2 receptor variants Instead of using a group of AA2a receptor intracellular third loop region mutants, the same procedure as in 1-1 to 1-3 was performed.

  The upper part of FIG. 6 shows the results of confirming the expression results of a group of AA2a receptor intracellular third loop region mutants by fluorescence detection.

  Moreover, the result of having detected the antigen-antibody reaction with respect to the AA2a receptor intracellular 3rd loop area | region mutant using a chemiluminescence reagent (brand name: Immobilon Western Detection Reagents, the product made by Millipore) is shown in the FIG. 6 lower part.

  As shown in FIG. 6, for the i3-1 mutant and the i3-2 mutant, since luminescence was observed in antibody detection, 205-RRQLKQMESQPLPGERARSTLQKEVH-230 determined by the above 3-3 (SEQ ID NO: 35) Among the regions consisting of 205, the region of 205-RRQLKQME-212 (SEQ ID NO: 46) is determined not to be an epitope.

  On the other hand, in the i3-4 mutant and the i3-5 mutant, no luminescence was observed in antibody detection, and thus the mutation was set in the mutant, ie, 213-SQPLPGERARSTLQK-227 (SEQ ID NO: 47). The region is judged to be included in the epitope.

  Further, in the i3-3 mutant, antibody detection was negative, but fluorescence detection was also negative, so it was judged that no expression product was obtained.

  From the results, the epitope sequence of mAb18 in the AA2a receptor was limited to 213-SQPLPGERARSTLQKEVH-230 (SEQ ID NO: 48).

The schematic diagram of the expression of the unmutated body by a budding yeast and the fusion body of a mutant and yEGFP is shown. Native is unmutated, Nter is Nter mutant, i1 is il mutant, o1 is o1 mutant, i2 is i2 mutant, o2 is o2 mutant, i3 is i3 mutant, o3 is o3 mutant, Cter is The expression schematic diagram of a Cter mutant is shown. * Indicates a loop with a mutation. (a) shows the results of confirming the expression of the M2 receptor non-mutant. 1 shows CBB staining, 2 shows fluorescence detection, and 3 shows the result of detection by Western blot. (B) shows the results of confirming the expression of a group of non-mutated M2 receptors and mutants by fluorescence detection. From the left, the results of unmutated, Nter mutant, i1 mutant, o1 mutant, i2 mutant, o2 mutant, i3 mutant, o3 mutant, and Cter mutant are shown. For the band, the band position with the highest fluorescence intensity was extracted and displayed. Shows the results of testing antigen-antibody reaction by Western blot for a group of M2 receptor unmutated and mutants and 5 anti-M2 monoclonal antibodies (mAbs 1-5) The result of having detected antigen-antibody reaction by ELISA about the monoclonal antibody (mAb6-17) in a hybridoma culture supernatant of a group of M2 receptor non-mutant and a variant, and 12 types of anti-M2 receptor antibodies is shown. (A) shows the result of the color development reaction of the plate 30 minutes after the reaction. (B) shows the difference in absorbance at 415 nm and 562 nm after 15 minutes of reaction. The upper fluorescence detection shows the result of confirming the expression results of a group of AA2a receptor non-mutants and the mutants by fluorescence detection. The lower antibody detection shows the result of detecting an antigen-antibody reaction against a group of AA2 receptor unmutated mutants and a group of the mutants by Western blotting. The upper fluorescence detection shows the result of confirming the expression result of a group of AA2a receptor intracellular third loop region mutants by fluorescence detection. The antibody detection in the lower part shows the result of detecting an antigen-antibody reaction against a group of AA2a receptor intracellular third loop region mutants by Western blotting.

Claims (4)

A method for determining an antigen binding site of an antibody against a transmembrane protein having two or more extra-membrane regions,
Expressing a group of mutants in which one or several amino acid mutations are added to different extramembrane regions in a budding yeast expression system;
A step of performing an antigen-antibody reaction by causing an antibody against the transmembrane protein to act as a group of mutants expressed in the budding yeast expression system,
Identifying a mutant in which an antigen-antibody reaction is not detected, and determining an extra-membrane region in which the mutation is added in the mutant as an antigen-binding site of the antibody or a region containing an antigen-binding site;
A method for determining an antigen-binding site of an antibody against a transmembrane protein, comprising: Expressing the group of mutants,
The method according to claim 1, wherein the method is expressed as a fusion protein in which a fluorescent protein is fused to a mutant. The method according to claim 1 or 2, wherein the transmembrane protein is a seven-transmembrane receptor. In addition, a group of mutants in which one or several different amino acid mutations are added to the same outer membrane region are expressed in a budding yeast expression system,
Using a group of the expressed mutants as an antigen, an antibody against the transmembrane protein is allowed to act to perform an antigen-antibody reaction,
Characterized in that it includes a step of identifying a variant in which an antigen-antibody reaction is not detected, and determining a portion where the mutation is added in the variant as an antigen-binding site of the antibody or a region containing the antigen-binding site. The method according to claim 1.

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