JP2010268793A - Method for detecting intermolecular interaction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method which shows a high protein stability, a high reconstitution efficiency and a high specific activity and enables quick detection of an intermolecular interaction or approach, even in a case of using a minor amount of molecules. <P>SOLUTION: The method for detecting an intermolecular interaction or approach comprises: contacting a first molecule, in which a first variant, that is a variant of a reporter enzyme having a two-stage enzymatic reaction mechanism and the reaction in the first stage being the rate-determining step, is bonded to one of two kinds of molecules to be analyzed, with a second molecule, in which a second variant, that is a variant of the reporter enzyme having a two-stage enzymatic reaction mechanism and the reaction in the second stage being the rate-determining step, is bonded to the other of the two kinds of molecules to be analyzed; and detecting the presence or absence of the progress of the enzymatic reaction catalyzed by the reporter enzyme. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for detecting interaction or proximity between molecules using a mutant of a reporter enzyme having a two-step enzyme reaction mechanism.

  Since many in vivo processes are controlled by protein complexes formed by non-covalent bonds, it is important to elucidate interactions between proteins in vivo. Methods for analyzing protein-protein interactions include (1) yeast two-hybrid method, (2) method of mediating reporter protein cleavage along with protein reconstitution, (3) method using protein splicing, (4) Fluorescence resonance energy transfer (FRET) and (5) PCA method (protein fragment complementation assay) are known. Hereinafter, these analysis methods will be described.

(1) Yeast two-hybrid method Yeast Gal4 is a transcriptional regulatory factor comprising an N-terminal DNA binding domain (DBD) and a C-terminal transcription activation domain (AD). Both domains function autonomously, DBD can bind to DNA alone but transcription does not occur, and AD cannot bind to DNA, but can activate transcription. The yeast two-hybrid method was developed by applying this property. That is, when a protein (bait) in which the target protein is fused to the Gal4 DBD and a protein (prey) in which another protein is fused to the Gal4 AD are introduced into yeast cells, the bait and prey interact in the nucleus. Then, the transcription control complex is reconstituted in the yeast cell, and transcription is activated depending on the Gal4 binding site. By detecting this activity using a reporter gene, the interaction between bait and prey can be easily evaluated. However, the yeast two-hybrid method reflects artificial protein association in the nucleus, and may differ from actual life phenomena, and there are many false positives, real-time measurement is difficult, and only in cells. There is a disadvantage that it cannot be measured.

(2) Method of mediating reporter protein cleavage along with protein reconstitution Ubiquitin is a small protein consisting of 76 amino acids and is involved in the degradation of unwanted proteins via the proteasome. In eukaryotic cells, when ubiquitin binds to a protein, the ubiquitin-binding protein (UBP) rapidly cleaves at the ubiquitin-protein binding site, but at this time, ubiquitin must be folded correctly. . Therefore, ubiquitin is divided into N-terminal (Nub) and C-terminal (Cub), and the target protein is linked to each of C-terminal of Nub and N-terminal of Nub via a linker. Binds the reporter protein. If an interaction occurs between the proteins of interest, the ubiquitins that approach each other cause correct folding, and the reporter protein bound by UBP is cleaved, and this cleavage can be confirmed by electrophoresis or the like. However, since the function of the reporter protein does not change before and after the cleavage, the cleavage needs to be assayed by electrophoresis or the like, which is complicated.

(3) A method using protein splicing This is a phenomenon in which the internal protein (Intein) is accurately cut out from the translation product in the nascent stage of yeast and the two protein fragments (Extein) on both sides (outer) are ligated. It is a method using. In other words, when this Intein is divided into two parts, Extein is bound to each, and Prey and Bait are bound to the other end, when they interact, the two Intein-derived fragments approach and return to their original form By being folded, the splicing activity is restored, and Extein is ligated and cut out. When a reporter protein fragment is used for this extein, the reporter protein is thereby reconstituted, and the interaction between the proteins can be evaluated by the function such as the recovered enzyme activity. However, there are problems that the enzyme activity is actually observed for a long time and the background is high.

(4) Fluorescence resonance energy transfer (FRET)
FRET is a phenomenon in which excitation energy is transferred from a fluorescent molecule to another molecule, and molecular sensors using this are widely used. A molecule that gives energy is called a donor, and a molecule that receives energy is called an acceptor. In order for FRET to occur, it is necessary that there is an overlap between the fluorescence spectrum of the donor and the absorption spectrum of the acceptor, and that the donor and acceptor are close to each other. When FRET occurs, the donor fluorescence is weakened, and if the acceptor is a fluorescent molecule, the fluorescence is observed. Therefore, FRET can be measured by monitoring the fluorescence intensity of the donor and the fluorescence intensity of the acceptor when the donor is excited. As the measurement of FRET, FRET measurement using a fluorescent protein such as GFP (Green fluorescent protein) is widely performed. Since fluorescent proteins can be expressed in cells by being fused with specific proteins, they are an effective means for detecting interactions between proteins that occur in cells. In addition to measuring the distance between molecules, FRET is also used as an indicator to know the concentration and activation state of molecules. FRET imaging makes it possible to visualize various molecular-level events that occur in living cells in real time. However, the abundance ratio of the donor and acceptor must be appropriate, and the fluorescent molecules are also quenched, so that there are problems that the conditions and time that can be measured are limited. Moreover, since there is no signal amplification effect by an enzyme etc., a sensitivity is bad.

(5) PCA method (protein fragment complementation assay)
In the PCA method, one functional protein A (enzyme, transcription factor, etc.) is divided into two fragments A1 and A2, and each is fused with bait and prey to produce fusion proteins A1-P and A2-Q. When the bait and prey are combined, the function of the functional protein A is restored, and the interaction between the bait and prey can be determined by detecting the activity. Examples of functional proteins that can be used include β-lactamase, DHFR, β-galactosidase (β-Gal), GFP, and luciferase.

  The method using luciferase is characterized by high luminous efficiency of the enzyme, and can achieve the highest sensitivity. So far, PCA using luciferase derived from Firefly, Renilla and Gaussia has been reported. Firefly luciferase is composed of two domain structures, and there is a large groove between the domains to form an active site. For PCA, a fragment cleaved between positions 420, 437, 445, and 455 near the domain boundary can be used (Paulmurugan et al., Proc. Natl. Acad. Sci. USA 99, 15608-15613, ( 2002), Paulmurugan et al., Anal. Chem. 77, 1295-1302 (2005)). In Renilla luciferase, PCA using fragments cleaved at positions 91 and 229 has been reported (Paulmurugan et al., Anal. Chem. 75, 1584-1589 (2003), Kaihara et al., Anal. Chem. 75, 4176-4181 (2003)). In Gaussia (marine copepod) luciferase, fragments were used at positions 93 and 94. (Remy et al., Nat. Methods 3, 977-979 (2006))

  In PCA using luciferase, there are problems that a high background signal and a low recovery rate of enzyme activity are present. In addition, due to problems such as protein stability, extracellular PCA of luciferase is mainly performed intracellularly, and there are only examples using extracellular cell lysate (I. Remy and SW Michnick, Nat. Methods, 3, 977-979 (2006), Paulmurugan et al., Cancer Res. 15, 7413-7420 (2005)), and purified protein has no examples. Even with PCA using other enzyme fragments, there are very few systems capable of detecting intermolecular interactions as enzyme activities in a test tube.

Paulmurugan et al., Proc. Natl. Acad. Sci. USA 99, 15608-15613, (2002) Paulmurugan et al., Anal. Chem. 77, 1295-1302 (2005) Paulmurugan et al., Anal. Chem. 75, 1584-1589 (2003) Kaihara et al., Anal. Chem. 75, 4176-4181 (2003) Remy et al., Nat.Methods 3, 977-979 (2006) I. Remy and SW Michnick, Nat. Methods, 3, 977-979 (2006) Paulmurugan et al., Cancer Res. 15, 7413-7420 (2005)

  An object of the present invention is to solve the above-described problems of the prior art. That is, the present invention is an object to be solved by providing a method that is excellent in protein stability, reconstitution efficiency, and specific activity, and that can quickly detect interaction or approach between molecules even with a very small amount of molecules. It was.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have analyzed a mutant of a reporter enzyme having a two-stage enzyme reaction mechanism in which the first-stage reaction is a rate-determining process. The first molecule linked to one of the two types of molecules and a variant of a reporter enzyme having a two-step enzyme reaction mechanism in which the second-step reaction is the rate-limiting process It was found that the interaction or approach between the molecules can be detected by contacting a second molecule linked to the other of the molecules and detecting the progress of the enzymatic reaction catalyzed by the reporter enzyme. It came to be completed.

  That is, according to the present invention, a first variant of a reporter enzyme having a two-stage enzyme reaction mechanism, in which the first-stage reaction is a rate-determining process, is one of the two types of molecules to be analyzed. A linked first molecule and a second variant of a reporter enzyme having a two-stage enzyme reaction mechanism in which the second-stage reaction is the rate-determining process. There is provided a method for detecting the interaction or proximity between molecules, comprising contacting a second molecule linked to the other and detecting the progress of an enzymatic reaction catalyzed by the reporter enzyme.

Preferably, the reporter enzyme is luciferase.
Preferably, the reporter enzyme is firefly luciferase.
Preferably, the variant of the reporter enzyme is a firefly luciferase variant having a substitution at any amino acid residue of H245, K529, and / or K443.

  Preferably, the first mutant of the reporter enzyme in which the first-stage reaction is the rate-determining process is a firefly luciferase mutant having a substitution at the amino acid residue of K529 or K443, and the mutation of the reporter enzyme The second mutant in which the second-stage reaction is the rate-determining process is a firefly luciferase mutant having an amino acid substitution at the H245 residue.

Preferably, a second mutant that is a mutant of the reporter enzyme and the second-stage reaction is a rate-limiting process,
(I) a firefly luciferase mutant having an amino acid substitution at K529 and H245, or (ii) a firefly luciferase mutant having an amino acid substitution at K443 and H245.

Preferably, among the mutants of the reporter enzyme, the amino acid substitutions at H245, K443, and K529 are H245D, K443A, and K529A, respectively.
Preferably, the two types of molecules to be analyzed are proteins or nucleic acids.

  Furthermore, according to the present invention, a first variant in which a mutant of a reporter enzyme having a two-stage enzyme reaction mechanism, in which a first-stage reaction is a rate-determining process, is linked to one of two molecules to be analyzed. And a second variant of a reporter enzyme variant having a two-stage enzyme reaction mechanism in which the second-stage reaction is a rate-limiting process linked to the other of the two molecules to be analyzed. A reagent kit for detecting an interaction or access between molecules is provided by the method according to any one of claims 1 to 5, comprising a combination with molecules.

  According to the present invention, since a full-length luciferase having several amino acid mutations is used, the stability and activity are drastically improved, and a protein using luciferase luminescence in a test tube that has been difficult to put into practice so far. It is possible to measure the interaction between the two. According to the present invention, in addition to the interaction between DNA-binding proteins, two proteins of the same size, ie, antibody variable region VH / VL, two single-chain antibodies, antigen and single-chain antibody, ligand It is possible to detect a wide range of interactions such as the receptor.

  In addition, since the method of the present invention is a detection method using luminescence, detection is possible with a sample of about several nM (100 μl or less) and normal substrate luciferin, ATP, and a luminescence detector. Application to the system is easy. The detection time is only a few seconds per sample. Further, it is possible to omit immobilization and washing of the sample, and measurement in a shorter time becomes possible. Known detection systems based on β-galactosidase mutant Δα / Δω complementarity and methods using energy transfer (FRET) between fluorescently labeled proteins are known as detection systems for homogeneous protein-protein interactions that can be detected in vitro. However, the present invention is superior in detection time and S / B ratio even when compared with them (Ueda et al., J. Immunol. Methods, 279, 209-218, 2003). In addition, when compared with the Enzyme Channeling Assay, which is known as a protein-protein interaction detection system using two full-length enzymes, the Enzyme Channeling Assay is known to take a long time for measurement. Detection in a short time of several seconds is possible.

  Furthermore, the detection system of the present invention can be applied to any kind of molecular interaction detection in principle, and can be applied particularly to simple detection kits for diagnostic markers, agricultural chemicals, environmental pollutants and the like. In particular, the sandwich ELISA method can be substituted. In addition, since the detection system probe (reporter enzyme mutant) used in the present invention is a protein, it can be expressed in cells in the same manner as a probe using a GFP mutant. Image diagnosis based on action detection is possible.

FIG. 1 shows a two-step catalytic reaction (ie, luciferin LH ₂ adenylation reaction (LH ₂ -AMP formation reaction) and oxidative luminescence reaction of intermediate LH ₂ -AMP) performed by firefly and light click luciferase. . FIG. 2 shows an overview of the embodiment. When a fusion protein of zinc finger protein Zif12 and luciferase mutant capable of recognizing a specific double-stranded DNA sequence is prepared, and the two recognition sequences of Zif12 are close to each other, the two fusion proteins are present on the double-stranded DNA. Therefore, the intermediate LH ₂ -AMP released from the mutant 1 is efficiently transferred to the mutant 2 and the luminescence reaction rate is increased. FIG. 3 shows the results of adding a LH ₂ solution to a fusion protein of Zif12 and a luciferase mutant and measuring the luminescence intensity for 1 second. FIG. 4 shows the results of measuring the luminescence intensity of two types of Zif12 luciferase mutant fusion proteins alone or mixed (center) when immobilized DNA was bound. (A) Luminescent activity when mixing N / C domain, (B) Luminous activity when mixing K529A / H245D + K529A, and (C) Luminous activity when mixing K443A / H245D + K443A. FIG. 5 shows the results of measuring the luminescence intensity of the mutant pairs K529A / H245D and K529A by changing the mixing ratio. FIG. 6 shows the luminescence activity when the mutant pair (A) K529A / H245D + K529A and (B) K443A / H245D + K443A are simply mixed and measured in a luminescence reaction solution. FIG. 7 shows the time change of the luminescence activity every 0.1 seconds when the substrate is added to the mutant (single or pair). The effect on the activity by the presence or absence of IR DNA is shown. FIG. 8 shows the luminescence activity (integrated value) for 2 minutes from the addition of the substrate when the thioredoxin recognition antibody was added to the mutant pair. FIG. 9 shows the sequence of FKBP12. FIG. 10 shows the FRB sequence. FIG. 11 shows the time change of the luminescence activity every 0.1 seconds when rapamycin is added to the mutant pair. The solid line indicates the time when rapamycin (50 nM) is added, and the dotted line indicates the time when rapamycin is not added.

Hereinafter, embodiments of the present invention will be described more specifically.
As a highly sensitive protein-protein interaction detection system in the cell, the N-terminal side (N domain) and C-terminal side (C domain) of the luminescent enzyme firefly luciferase are fused with the target protein, allowing the intensity of the interaction to be emitted. The Protein Fragment complementation (PCA) method that can be detected by the method is known. However, PCA with N / C domain in vitro has hardly been reported so far because the fusion protein of fragmented luciferase is unstable and the reconstitution efficiency is low. On the other hand, the luciferase catalyzed reaction proceeds in two steps: luciferin LH ₂ adenylation reaction (LH ₂ -AMP formation reaction) and intermediate LH ₂ -AMP oxidative luminescence reaction (FIG. 1). ing. So far, the intermediate LH ₂ -AMP can be generated, but the second stage reaction is extremely slow, so the 245th histidine of firefly luciferase is used as a mutant that releases a large amount of LH ₂ -AMP out of the enzyme. A mutant (H245D) substituted with aspartic acid has been reported (Branchini et al., Biochemistry, 37, 15311-15319, 1998, Ayabe et al., FEBS Lett. 579, 4389-4394, 2005). As another firefly luciferase variant, K529A, in which 529th lysine is substituted with alanine, hardly undergoes the first stage adenylation reaction, but the second stage LH ₂ -AMP oxidative luminescence reaction. It is known that it can be performed efficiently (Branchini et al., Biochemistry, 44, 1385-1393, 2005). Similarly to H245D, the second stage oxidative luminescence reaction is rate-limiting (Branchini et al., Biochemistry, 44, 1385-1393, 2005), while the crystal structure with the reaction intermediate analog (Nakatsu et al. , Nature 440, 372-376, 2006) have reported a mutant (K443A) in which the 443rd lysine is substituted with alanine, whose results have been questioned. Therefore, in the present invention, an intermolecular two-step reaction in which an intermediate produced from one mutant is consumed by the other mutant and emits light from various combinations of these mutants is efficiently performed. Tried to find a combination.

  Specifically, in the examples of the present specification, Zif12 capable of specifically recognizing the mutant and the double-stranded DNA sequence was applied to specific DNA sequence detection. Specifically, expression of fusion protein expression vector of each mutant Fluc, N-domain (1-437 aa), C-domain (394-550 aa) and Zif12, including wild type and K443A with unclear action point Then, it was expressed in Escherichia coli BL21 (DE3, pLysS), and metal affinity purification using a His tag added to the fusion protein C-terminal was performed. Thereafter, the binding ability of Zinc finger was confirmed by ELISA, and the specific activity of each mutant was confirmed to be 1% or less of the wild type as documented. Subsequently, when the activity was measured using the immobilized DNA with the mutants alone and all combinations of the mutants, most combinations were obtained when IR, which is a sequence that specifically binds two Zif12s, was immobilized. The average activity of the two mutants was detected, and no activity was detected when SS, which is a sequence to which only one Zif12 binds weakly, was immobilized. However, in the previously reported N-domain and C-domain and H245D-C domain fusion protein pairs, a slight but above average activity was detected. Therefore, double mutants K529A / H245D (5H) and K443A / H245D (4H), in which mutations were further introduced into H245D, which has higher luminescence activity than C domain fusion protein, were prepared and increased by lowering background activity. Screening was performed in consideration of obtaining a pair showing a response. As a result, it was found that 5H or 4H and four mutant pairs of K529A or K443A showed significantly higher than average luminescence activity. Moreover, by increasing the luminescence measurement time from 3 minutes to 1 second, it was possible to obtain a higher activity increase rate. When these mutant pairs were simply mixed and reacted in a solution, such an increase in activity was hardly observed, so the increase in activity was caused by the proximity of the two mutants via DNA and Zif12. It was thought to reflect that. That is, an interaction detection system was obtained in which the activity was increased by about 7 times the expected value when two proteins interacted with each other through DNA although they were immobilized on a microplate. The obtained signal intensity was about 1000 times that of the conventional N / C complementary system and 1% or more stronger than the wild type. In addition, due to the combination of mutants with increased activity and the rapid response, this increased activity is not due to domain reorganization that is unlikely to occur outside the cell without a molecular chaperone. The body was probably consumed by the other mutant. In other words, the luminescence intensity was significantly increased by the consumption of a small amount of intermediate released by the mutant having the H245D mutation whose rate of oxidative luminescence reaction is limited by K529A to K443A whose rate of adenylation reaction is limited. It was broken. In addition, by introducing double mutations in H245D at this time, the amount of intermediates produced is reduced, so that intermediates that are easily hydrolyzed at neutral pH are localized in the vicinity of the double mutants, and nearby mutations. It was speculated that the condition that only the body enzyme was activated could be realized.

  The method for detecting the interaction or approach between molecules according to the present invention is a variant of a reporter enzyme having a two-stage enzyme reaction mechanism, in which the first mutant whose rate-limiting process is the first-stage reaction is analyzed. A first molecule linked to one of the two types of molecules and a second variant of a reporter enzyme having a two-stage enzyme reaction mechanism, wherein the second-stage reaction is the rate-limiting process. A second molecule linked to the other of the two types of molecules to be analyzed is brought into contact with each other, and the presence or absence of the progress of an enzyme reaction catalyzed by the reporter enzyme is detected.

  The type of reporter enzyme having a two-step enzyme reaction mechanism is not particularly limited, but preferably an enzyme belonging to the acyl adenylate / thioester-forming enzyme superfamily such as firefly luciferase, light-worm luciferase, acetyl CoA synthase, and gramicidin synthase. Can be mentioned. In the present invention, as long as the generated reaction intermediate is common, the two types of mutants used for detection may be derived from different enzymes.

  Luminescence is the term given to photon emission in the visible spectral range caused by excited emitter molecules. In contrast to fluorescence, the energy of light emission is not supplied externally in the form of short wavelength irradiation. There is a clear distinction between chemiluminescence and bioluminescence. Chemiluminescence is a term given to a chemical reaction that gives an excited molecule, and the excited molecule itself emits light when an electron at an excited level returns to a normal energy level. Bioluminescence is the term used when this reaction is catalyzed by an enzyme. The enzyme involved in this reaction is generally called luciferase.

  Luciferases can also be distinguished from each other based on their origin or their substrate specificity (or reaction mechanism). The most important substrates include coelenterazine and luciferin, and derivatives of these two compounds.

The amino acid sequence of American firefly (Photinus pyralis) luciferase used this time is shown below (SEQ ID NO: 17). In addition, when the corresponding residues in luciferases derived from different species are different, the following description can be read as the corresponding residues in the homologous sequence.
1 MEDAKNIKKG PAPFYPLEDG TAGEQLHKAM KRYALVPGTI AFTDAHIEVN ITYAEYFEMS
61 VRLAEAMKRY GLNTNHRIVV CSENSLQFFM PVLGALFIGV AVAPANDIYN ERELLNSMNI
121 SQPTVVFVSK KGLQKILNVQ KKLPIIQKII IMDSKTDYQG FQSMYTFVTS HLPPGFNEYD
181 FVPESFDRDK TIALIMNSSG STGLPKGVAL PHRTACVRFS HARDPIFGNQ IIPDTAILSV
241 VPFH H GFGMF TTLGYLICGF RVVLMYRFEE ELFLRSLQDY KIQSALLVPT LFSFFAKSTL
301 IDKYDLSNLH EIASGGAPLS KEVGEAVAKR FHLPGIRQGY GLTETTSAIL ITPEGDDKPG
361 AVGKVVPFFE AKVVDLDTGK TLGVNQRGEL CVRGPMIMSG YVNNPEATNA LIDKDGWLHS
421 GDIAYWDEDE HFFIVDRLKS LI K YKGYQVA PAELESILLQ HPNIFDAGVA GLPDDDAGEL
481 PAAVVVLEHG KTMTEKEIVD YVASQVTTAK KLRGGVVFVD EVPKGLTG K L DARKIREILI
541 KAKKGGKSKL
Underline: H245, K443, K529

Firefly luciferase luminescence reaction proceeds in two stages as shown in the figure below. First, the carboxyl group of luciferin (LH2) attacks the phosphate group at the α-position of ATP, and once the LH2-AMP intermediate is generated in the enzyme, pyrophosphate (PPi) is released. Then, the enzyme reacts with this intermediate, AMP, together with the CO _2, to produce oxyluciferin excited state, which is moved to the ground state. At this time, the difference energy is emitted as yellowish green visible light (560 nm). The quantum efficiency of this energy conversion is extremely high, reaching almost 90%. Firefly luciferase has been used to examine the activation of in vitro cultured animals, bacteria, insects, plants, yeast and viral cells. The firefly luciferase assay is extremely sensitive, rapid, convenient and safe (Non-radioisotopic) compared to other reporter gene assays. The luciferase assay is 100-1000 times more sensitive and has a wider measurable range than the CAT assay using radioisotopes.

  Other luciferases include Renilla (Renilla) and Gaussia (marine copepod) luciferases. Renilla (Renilla) luciferase is non-ATP-requiring and catalyzes a luminescence reaction using coelenterazine as a substrate (lower figure). Gaussia (marine copepod) luciferase also does not require ATP and emits light (470 nm) by a catalyst for coelenterazine oxidation. Gaussia luciferase has a signal strength that is 1000 times higher than that of Firefly and Renilla luciferase, and is characterized by extremely high stability.

  Studies on amino acid residues involved in firefly luciferase activity have been conducted so far, and the 245th histidine (Branchini et al., Biochemistry, 37, 15311-15319, 1998, Ayabe et al., FEBS Lett. 579, 4389 -4394, 2005), the importance of the 529th lysine (Branchini et al., Biochemistry, 39, 5433-5440, 2000)) and the 443rd lysine has been revealed. The H245D mutant, in which the 245th histidine is converted to aspartic acid, has been found to produce and release a large amount of the reaction intermediate LH2-AMP, a two-step reaction (the luciferin LH2 adenylation reaction, And oxidative luminescence reaction using the reaction intermediate LH2-AMP), the second stage oxidative luminescence reaction was rate-limiting. Therefore, it can be said that the 245th histidine plays an important role in the oxidative luminescence reaction (Ayabe et al., FEBS Lett. 579, 4389-4394, 2005). In addition, in the K529A mutant in which the 529th lysine was converted to alanine, the adenylation reaction rate was greatly reduced. However, the addition of the reaction intermediate LH2-AMP promptly causes an oxidative luminescence reaction. It became clear that the 529th lysine plays a particularly important role in the adenylation reaction. Similarly, the K443A mutant in which the 443rd lysine was converted to alanine had little effect on the adenylation reaction rate, and the oxidative luminescence reaction rate decreased, suggesting its importance in the oxidative luminescence reaction. (Branchini et al., Biochemistry, 44, 1385-1393, 2005). However, based on the results of recent structural analysis (Nakatsu et al., Nature 440, 372-376, 2006), the evaluation of this result has not yet been determined, and there is room for examination of how K443 is involved in the luminescence reaction. is there. As described above from these experimental results, the mutant of the reporter enzyme used in the present invention is preferably a firefly luciferase mutant having any amino acid substitution of H245D and K529A (and / or K443A). Further, for K529 and K443, other amino acid substitutions are considered to be preferable as well.

  In the present invention, the types of the two types of molecules whose interaction is to be investigated are not particularly limited, but are preferably biomolecules, such as proteins or nucleic acids.

  In the present invention, first, a first variant of a reporter enzyme having a two-stage enzyme reaction mechanism in which a first-stage reaction is a rate-determining process is linked to one of two kinds of molecules to be analyzed. One molecule and a reporter enzyme mutant having a two-stage enzyme reaction mechanism, and a second mutant in which the second-stage reaction is the rate-determining process is linked to the other of the two molecules to be analyzed. Prepare a second molecule.

  When the two molecules to be analyzed are proteins (referred to as the first protein and the second protein), the DNA encoding the first protein and the first of the reporter enzyme according to the usual genetic recombination technique. First, a mutant of a reporter enzyme having a two-stage enzyme reaction mechanism in which the DNA encoding the mutant is linked in an appropriate expression vector, and the first-stage reaction is the rate-limiting process. A first recombinant expression vector capable of expressing a first molecule in which the mutant is linked to one of two types of molecules to be analyzed can be constructed. Similarly, a reporter enzyme having a two-stage enzyme reaction mechanism by incorporating a DNA encoding the second protein and a DNA encoding the second variant of the reporter enzyme in a state of being linked in an appropriate expression vector. A second recombinant expression vector capable of expressing a first molecule in which a second mutant whose reaction at the second stage is a rate-limiting process is linked to the other of the two molecules to be analyzed Can be built.

  Then, the first recombinant expression vector constructed as described above and the second recombinant expression vector are individually transformed into an appropriate host, and the host is cultured to obtain the first molecule. And the second molecule can be expressed and the first and second molecules can be individually recovered from the culture. By using the first molecule and the second molecule collected in this manner and bringing them into contact with each other in vitro, for example, it is possible to detect the progress of the enzymatic reaction catalyzed by the reporter enzyme.

  Alternatively, by transforming the first recombinant expression vector and the second recombinant expression vector into a suitable same host and culturing the host, the first molecule and the second molecule are transformed. It can also be expressed and the presence or absence of progress of an enzyme reaction catalyzed by a reporter enzyme in the host can also be detected.

  Combinations of the above expression vectors and suitable hosts are known to those skilled in the art. As the expression vector, a vector that can replicate autonomously in a host cell or can be integrated into a chromosome and contains a promoter at a position where the target DNA can be transcribed is used. Examples of the host include bacteria (eg, Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc.), yeast (Kluyveromyces, Saccharomyces, Zosaccharomyces genus, Trichosporon genus, Siwaniomyces genus), animal cells (Namarva cells, COS1 cells, COS7 cells, CHO cells, etc.), insect cells (Sf9 cells, Sf21 cells, etc.) and the like. In addition, methods for introducing a recombinant expression vector into a host are also known to those skilled in the art, and depending on the type of host, for example, calcium phosphate method, electroporation method, protoplast method, spheroblast method, lithium acetate method, lipofection The method etc. can be used.

  When the two types of molecules whose interaction is to be investigated are nucleic acids (referred to as the first nucleic acid and the second nucleic acid), the first nucleic acid is a variant of the reporter enzyme, A first mutant whose reaction is a rate-limiting process is linked, and a second mutant that is a mutant of a reporter enzyme and a second-stage reaction is a rate-limiting process is linked to the second nucleic acid. Nucleic acid and reporter enzyme mutants can be ligated using nucleic acid binding proteins, biotin labeling and streptavidin (Sano et al., Science 258, 120-122, 1992), Native chemical There are methods using ligation (Takeda et al., Org. Biomol. Chem. 6, 2187-2194, 2008).

  In the present invention, a first molecule in which the first mutant of the reporter enzyme is linked to one of the two types of molecules to be analyzed, and the second mutant of the reporter enzyme is used in the two types of molecules to be analyzed. The interaction or approach between the molecules is detected by contacting the second molecule linked to the other and detecting the progress of the enzymatic reaction catalyzed by the reporter enzyme. In the present invention, the interaction of two types of molecules can be analyzed as described above. Furthermore, in the present invention, the binding of the two kinds of molecules to be analyzed to the target molecule can be detected by making another target molecule to which the two kinds of molecules to be analyzed bind together. That is, when the progress of the enzyme reaction catalyzed by the reporter enzyme is detected, it indicates that the two target molecules are bound to the target molecule, and the progress of the enzyme reaction catalyzed by the reporter enzyme is detected. If not, it indicates that the two molecules to be analyzed are not bound to the target molecule at the same time. When the two types of molecules to be analyzed are antibodies, an antigen against the antibody can be used as the target molecule. When the two types of molecules to be analyzed are nucleic acids, a nucleic acid having a base sequence complementary to the base sequence of the nucleic acid can be used as the target molecule.

  Furthermore, according to the present invention, a mutant of a reporter enzyme having the above-described two-stage enzyme reaction mechanism, in which the first-stage reaction is a rate-determining process, is linked to one of the two types of molecules to be analyzed. A first molecule and a variant of a reporter enzyme having a two-step enzyme reaction mechanism, in which a second-step reaction is a rate-determining process, is linked to the other of the two types of molecules to be analyzed. By combining the two molecules, a reagent kit for detecting the interaction or proximity between the molecules can be provided.

  The following examples further illustrate the present invention, but the scope of the present invention is not limited to these examples.

Example 1:
In this example, a zinc finger protein that can recognize a specific double-stranded DNA sequence, Zif12 (FIG. 2, Yoshitake et al., Biosens. Bioelectron., 23, 1266-1271, 2008) and a luciferase mutant fusion protein Several were made. When two Zif12 recognition sequences are in close proximity, the two fusion proteins are spatially close together on the double-stranded DNA, so that the intermediate LH ₂ -AMP released from mutant 1 is efficiently mutated. It was delivered to the body 2 and expected to increase the luminescence reaction rate (FIG. 2).

  Firefly luciferase mutants fused with Zif12 include H245D, K529A, and K443A, K529A / H245D (5H), which combines amino acid substitutions of K529A and H245D, and K443A /, which combines amino acid substitutions of K443A and H245D A total of five types of H245D (4H) were selected, and the luminescence activity was measured in the presence of specific DNA. In addition, as a reference experiment for comparison with the conventional PCA method, a fusion protein consisting only of the luciferase N domain (positions 1-437) or C domain (positions 394-550) and Zif12 was prepared. Was measured.

Abbreviations used in the following examples are as follows.
LB: Liquid medium containing 1% bactotryptone, 0.5% yeast extract, 0.5% NaCl
LBA: LB containing 100 μg / ml ampicillin
LBAG: LB containing 100 μg / ml ampicillin and 1% glucose
LBAC: LB containing 100 μg / ml ampicillin and 33 μg / ml chloramphenicol
LBAG plate: LB agar containing 100 μg / ml ampicillin and 1% glucose
LBAC plate: LB agar medium containing 100 μg / ml ampicillin and 33 μg / ml chloramphenicol
SOC: Medium containing 2% bactotryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 20 mM glucose, 10 mM MgCl ₂
IPTG: Isopropyl-β-thiogalactopyranoside
PBS: 10 mM phosphate buffer (pH 7.2) containing 137 mM NaCl and 2.7 mM KCl
PBST: PBS containing 0.1% triton-X100
PBSB: PBS containing 1% BSA
TAE buffer: 40 mM Tris-acetate (pH 8.3) containing 1 mM EDTA
Extraction buffer: 50 mM Na ₂ HPO ₄ -NaH ₂ PO ₄ , 300 mM NaCl (pH 7.0)
TALON eluent: 50 mM Na ₂ HPO ₄ -NaH ₂ PO ₄ , 300 mM NaCl, 250 mM imidazole (pH 7.0)
Protein storage buffer: 60 mM Tris-HCl, 150 mM NaCl, 0.8 M ammonium sulfate, 90 mM KCl, 1 mM MgCl ₂ , 90 μM ZnCl ₂
Buffer for luminescence measurement: 10 mM Tris-HCl, 90 mM KCl, 1 mM MgCl ₂ , 90 μM ZnCl ₂ , 50 mM HEPES, 1 mM DTT, 1% BSA, 2.5% glycerol (pH 7.5)
2x LH2 solution: 200 mM MOPS, 20 mM MgSO ₄ , 600 μM luciferin (LH ₂ ), 20 mM ATP, 2 mg / ml BSA, (pH 7.0)

  In all experiments, water purified with Milli-Q (Millipore Co., Billerica, MA) was used. Hereinafter, it is expressed as milliQ water. Unless otherwise indicated, ordinary reagents used were those of Sigma (St. Louis, MO), Nacalai Tesque (Kyoto), Wako Pure Chemical (Osaka), Kanto Chemical (Tokyo). Oligo DNA was synthesized at Texas Genomics Japan (Tokyo) or Invitrogen (Tokyo).

MJ mini personal thermal cycler (BIO-RAD Laboratories, Inc., Hercules, CA) is used for Polymerase chain reaction (PCR), and CEQ ^TM 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA) is used for DNA sequencing. used.

  Enzymes used in this experiment (AscI, NotI, XhoI, EcoRI, DpnI) are Takara Bio (Otsu), Roche Applied Science (Basel, Switzerland), New England BioLabs (Ipswich, MA), Promega Co. (Madison, WI). ) Was used.

The E. coli strains used in this experiment are as follows.
DH5α: F-, Φ80dlacZΔM15, Δ (lacZYA-argF) U169, deoR, recA1, endA1, hsdR17 (rK-, mK +), phoA, supE44, λ-, thi-1, gyrA96, relA1
BL21 (DE3, pLysS): F -, hsdS B (r B - m B -), gal dcm, lacY1, aphC, gor522 :: Tn10 (Tet r), trxB :: Kan r (DE3) pLysS (Cm r)

The oligo DNA sequences used in this experiment are as follows.
Full-1: 5'-ggcgcgcCTCGAGCTTTCCGCCCTTCTTGGCCT-3 '(SEQ ID NO: 1)
Full-2: 5'-ggcgcgccGCGGCCGCCggtggtggtggtagcATGGAAGACGCCAAAAACATAAAG-3 '(SEQ ID NO: 2)
Ndomain-1: 5'-ggcgcgcCTCGAGGCGGTCAACTATGAAGAAGTG-3 '(SEQ ID NO: 3)
Ndomain-2: 5'-ggcgcgccGCGGCCGCCggtggtggtggtagcATGGAAGACGCCAAAAACATAAAG-3 '(SEQ ID NO: 4) = Full-2
Cdomain-1: 5'-ggcgcgcCTCGAGCTTTCCGCCCTTCTTGGCCT-3 '(SEQ ID NO: 5) = Full-1
Cdomain-2: 5'-ggcgcgccGCGGCCGCCggtggtggtggtagcGGACCTATGATTATGTCCGG-3 '(SEQ ID NO: 6)
M13M3: 5'-gtaaaacgacggccagt-3 '(SEQ ID NO: 7)
M13RV: 5'-caggaaacagctatgac-3 '(SEQ ID NO: 8)
T7term: 5'-tagttattgctcagcggtgg-3 '(SEQ ID NO: 9)
TrxFusBack: 5'-ttcctcgacgctaacctg-3 '(SEQ ID NO: 10)
K529A-r: 5'-ctctctgatttttcttgcgtcgagAGCtccggtaagacctttcgg-3 '(SEQ ID NO: 11)
K529A-f: 5'-ccgaaaggtcttaccggaGCTctcgacgcaagaaaaatcagagag-3 '(SEQ ID NO: 12)
K443A-r: 5'-gccacctgatatcctttgtatGCaattaaagacttcaagcggtc-3 '(SEQ ID NO: 13)
K443A-f: 5'-gaccgcttgaagtctttaattGCatacaaaggatatcaggtggc-3 '(SEQ ID NO: 14)

The sequence of the target DNA is as follows. The sequence takes a hairpin structure in an aqueous solution and forms a partial duplex (FIG. 2). In IR, two target sequences (Zif12 recognition sequences) exist as palindrome arrays, but in SS there is only one target sequence.
IR (inverted repeat DNA):
5'-Biotin-ggaTGGGCGCGCGCCCAgggttttcccTGGGCGCGCCCAtcc-3 '(SEQ ID NO: 15)
SS (single site DNA):
5'-Biotin-ggaTGGGCGtatgctgggttttcccagcataCGCCCAtcc-3 '(SEQ ID NO: 16)
Uppercase letters indicate the Zif12 recognition sequence.

The plasmids used in this experiment are as follows.
pGEX-WT: Contains wild-type firefly luciferase gene (Zako et al., FEBS Lett. 579, 4389-4394, 2005)
pGEX-H245D: Contains the gene for firefly luciferase mutant H245D (Ayabe et al., FEBS Lett. 579, 4389-4394, 2005)
pNEB193: Purchased from New England BioLabs
pET32-ZiF12-EYFP: Yoshitake et al., Biosensors and Bioelectronics, 23, 1266-1271, 2008.
pET32-ZiF12-LucWT: Fusion protein expression vector of wild-type luciferase and Zif12
pET32-ZiF12-LucH245D: Fusion protein expression vector of H245D and Zif12
pET32-ZiF12-LucK443A: K443A and Zif12 fusion protein expression vector
pET32-ZiF12-LucK529A: Fusion protein expression vector of K529A and Zif12
pET32-ZiF12-LucH245D / K443A: Fusion protein expression vector of H245D / K443A and Zif12
pET32-ZiF12-LucH245D / K529A: Fusion protein expression vector of H245D / K529A and Zif12
pET32-ZiF12-LucN: fusion protein expression vector of luciferase N domain and Zif12
pET32-ZiF12-LucC: fusion protein expression vector of luciferase C domain and Zif12

Basic experimental operation
Ligation Ligation reaction is performed by mixing an appropriate amount of vector DNA and insert DNA, mixing this mixture with an equal volume of 2 × Ligation high (TOYOBO. CO., LTD., Osaka), and incubating at 16 ° C for 30 minutes. went.
Transformation transformation was performed by the calcium chloride method. Plasmid (ligation reaction product, etc.) and E. coli are mixed, left on ice for 30 minutes, reacted at 42 ° C for 45 seconds, left on ice for 3 minutes, then added 2 times the amount of SOC medium in E. coli solution, 37 Incubated for 20 minutes at ° C. After culturing, it was applied to an LB plate containing an appropriate antibiotic and cultured overnight at 37 ° C.
Agarose gel electrophoresis
Electrophoresis for DNA fragment confirmation was performed on an agarose gel prepared by dissolving Agarose S (NIPPON GENE CO., LTD., Toyama) in TAE to a final concentration of 1%. The DNA solution was prepared, applied, and electrophoresed so as to indicate x. In addition, when the fragment was cut out and purified, the gel around the band was cut out with a knife and purified using the Wizard SV Gel & PCR Clean-up System (Promega Co., Madison, Wis.).

(1) Outline of plasmid construction Genes of wild-type luciferase or H245D, N domain, and C domain are amplified by PCR and incorporated into pET32-ZiF12, which is a plasmid for expression and has a Zif12 gene, and a fusion protein expression vector with pif32 (pET32 -ZiF12-LucWT, pET32-ZiF12-LucH245D, pET32-ZiF12-LucN, pET32-ZiF12-LucC) were constructed. After that, by converting the 443rd and 529th lysine residues to alanine by quick change method to pET32-ZiF12-LucWT or pET32-ZiF12-LucH245D, Zif12 and K443A and K529A, H245D / K443A and H245D / K529A Fusion protein expression vectors (pET32-ZiF12-LucK443A, pET32-ZiF12-LucK529A, pET32-ZiF12-LucH245D / K443A, pET32-ZiF12-LucH245D / K529A) were constructed.

Wild type luciferase and H245D gene were amplified by PCR as follows.
Reaction solution composition
pGEX-WT or pGEX-H245D 1 μl
Primer Full-1 (100 nM) 0.5 μl
Primer Full-2 (100 nM) 0.5 μl
10x ExTaq buffer (Mg2 + 20 mM) (Takara bio Inc., Otsu) 10 μl
dNTP Mixture (2.5 mM each) (Takara bio Inc.) 8 μl
5 U / μl ExTaq DNA polymerase (Takara bio Inc.) 1 μl
milliQ 79 μl

Reaction cycle 1, 94 ℃ 5 min
2, 94 ℃ 30 sec
3, 60 ℃ 1 min 30 sec
4, 72 ℃ 2 min
(2 to 4 25 cycles)
5, 72 ℃ 10 min
6. Hold at 15 ° C

  The gene encoding luciferase N domain or C domain was amplified by PCR as follows. The PCR reaction was performed under the same conditions as above except that Ndomain-1 and Ndomain-2 were used as primers for N domain, and Cdomain-1 and Cdomain-2 were used for C domain.

  The amplified PCR product was cleaved with AscI and then purified using Wizard SV Gel & PCR Clean-up System (Promega Co). Further, pNEB193 (New England BioLabs) was similarly cut with AscI and subjected to agarose gel electrophoresis, and then a DNA band corresponding to open circular pNEB193 was cut out and purified as described above. The AscI-treated and purified PCR product and the open circular pNEB193 were subjected to a ligation reaction as described above, subjected to DH5α transformation, and cultured on an LBA plate. The obtained colonies were analyzed by colony PCR using primers M13RV and M13M3 followed by agarose gel electrophoresis.

Colony PCR reaction solution primer M13RV (5 nM) 1 μl
Primer M13M3 (5 nM) 1 μl
2x GoTaq (Promega Co.) 5 μl
milliQ 3 μl

Colony PCR reaction cycle 1, 94 ℃ 5 min
2, 94 ℃ 30 sec
3 55 ℃ 30 sec
4, 72 ℃ 1 min
(2 to 4 25 cycles)
5, 72 ℃ 2 min
6. Hold at 15 ° C

  For clones in which DNA bands corresponding to luciferase wild type and H245D gene were obtained by colony PCR, the clones were cultured overnight in 4 ml LBA liquid medium, and Wizard Plus SV Minipreps DNA Purification System (Promega Co. ) Was used to extract the plasmid, after which the sequence was confirmed.

  PNEB193 into which luciferase wild type and H245D, N domain, and C domain genes were inserted was cleaved by NotI and XhoI treatment, and after agarose gel electrophoresis, the DNA fragment encoding luciferase wild type and H245D gene was purified as described above. . On the other hand, pET32-ZiF12 was similarly treated with NotI and XhoI, and the open circular pET32-ZiF12 was purified in the same manner as above, ligated with luciferase wild type and H245D gene fragment, transformed, and cultured on LBA plate, colony PCR was performed. Colony PCR was performed under the same conditions as the above colony PCR except that T7 Term and TrxFusBack were used as primers. The colony PCR product was analyzed by agarose gel electrophoresis after a cleavage reaction with EcoRI present in luciferase wild type and H245D gene. Among them, clones that gave the correct cleavage pattern were inoculated into 4 ml of LBA liquid medium, and plasmids were extracted to obtain pET32-ZiF12-LucWT and pET32-ZiF12-LucH245D. It was confirmed by sequencing that each had a DNA sequence as designed.

  Mutants K443A, K529A, H245D / K443A, and H245D / K529A were obtained by the quick change method using the obtained pET32-ZiF12-LucWT and pET32-ZiF12-LucH245D as templates. Primers and reaction conditions are shown below.

Quick change reaction conditions
pET32-ZiF12-LucWT or pET32-ZiF12-LucH245D 1 μl
K443-f or K529A-f (10μM) 1.5 μl
K443-r or K529A-r (10μM) 1.5 μl
Pfu ULTRA Buffer (Stratagene, La Jolla, CA) 5 μl
dNTP 4 μl
Pfu ULTRA (Stratagene) 1 μl
milliQ 36 μl

Reaction cycle 1, 95 ° C 1 min
2, 95 ℃ 30 s
3, 55 ℃ 1 min
4. 68 ℃ 8.5 min
(2 to 4 18 cycles)
5. Hold at 16 ° C

  The obtained quick change product was added with 1 μl DpnI and allowed to stand at 37 ° C. for 1.5 hours, and then several μl was added to DH5α competent cells for transformation. Plasmid extraction was performed as described above from the obtained colonies to obtain pET32-ZiF12-LucK443A, pET32-ZiF12-LucK529A, pET32-ZiF12-LucH245D / K443A, and pET32-ZiF12-LucH245D / K443A. It was confirmed by sequencing that the obtained plasmid had a DNA sequence as designed.

(2) Protein expression E. coli BL21 (DE3) (pLysS) was transformed with the obtained 6 types of vectors and cultured overnight at 37 ° C on an LBAC plate. The resulting colonies were added to 4 ml of LBAC medium. Inoculated and cultured overnight at 37 ° C. Next, 400 μl of the culture solution was inoculated into 100 ml of LBAC liquid medium, and when OD ₆₀₀ reached about 0.5 at 37 ° C., 1 mM IPTG was added to induce protein expression, and at 16 ° C. Cultured overnight.

(3) Protein purification After culturing, the cells are collected by centrifugation, resuspended in 10 ml extraction buffer, sonicated on ice, centrifuged at 10000 xg, 60 min, 4 ° C, The lysate was recovered.
The protein expressed this time was fused with a histidine tag and purified using TALON affinity resin (Clontech Laboratories, Inc., Mountain View, CA). 10 ml of lysate was added to 100 μl of TALON affinity Resin, and proteins were allowed to bind while gently inversion for 20 min at room temperature. After collecting the resin with a tabletop centrifuge and discarding the supernatant, the washing operation of adding 10 ml of Extraction buffer and suspending was repeated three times, and then the Elution Buffer was added and the protein was eluted several times. went. Each eluate was measured for protein concentration by the Bradford method, and the fraction with high concentration was replaced with a protein storage buffer using a NAP5 column (GE Healthcare UK Ltd., Amersham Place, England). And stored at -80 ° C.

  According to SDS-PAGE of the sample after metal affinity purification, the fusion protein of wild type and other mutants (MW 88.3 kDa), N domain (MW 76.7 kDa), C domain (MW 44.8 kDa) and Zif12 was designed. It was confirmed that it could be prepared with sufficient purity. Therefore, the protein concentration was finally measured by Bradford method (protein assay, Bio-Rad).

(4) Evaluation of specific activity The purified Zif12 fusion protein was diluted with a buffer for luminescence measurement to a final concentration of 10 nM, and 50 μl was dispensed into a Costar white 96-well plate (CorningCostar Inc., Corning, NY). 50 μl of 2 × LH2 solution was added, and the luminescence intensity for 1 second was measured using AB-2350 luminometer PHELIOS (Atto Co., Tokyo). FIG. 3 shows the results of measuring the luminescence intensity at each protein final concentration of 10 nM. As a result, it was confirmed that the luminescence activity of the N domain, the C domain and the mutant was 1% or less of the wild type, and it was found that the background was sufficiently low.

(5) Measurement of DNA binding activity 10 μg / ml of streptavidin diluted with a PBS solution was fixed to a white 96-well plate overnight at 4 ° C., blocked with PBSB for 2 hours, and then washed 3 times with PBST. Subsequently, 50 μl of PBSB containing 10 nM biotinylated target DNA (IR, SS) was dispensed and allowed to stand at room temperature for 1 hour. After washing with PBST three times, 50 μl of purified Zif12-fused wild-type luciferase with various concentrations diluted with a luminescence measurement buffer was dispensed and allowed to stand for 1 hour. After washing 3 times with PBST and dispensing 50 μl of the luminescence measurement buffer, 50 μl of 2 × LH2 solution was added and the luminescence intensity was measured. As a result, when the IR with two adjacent target sequences was immobilized, the luminescence intensity increased in proportion to the added protein concentration, whereas the SS with only one target sequence was immobilized. The signal was weak and significant protein concentration dependency could not be confirmed. Therefore, the IR-specific DNA binding ability of the fusion protein was confirmed.

(6) Luminescence intensity enhancement by mixing two kinds of mutants Select two of Zif12 fusion proteins obtained by immobilizing biotinylated IR DNA in a white 96-well plate as described above, and purifying the expression. Were mixed and dispensed so that the final concentration was 20 nM or a final concentration of 20 nM (total 40 nM), and the luminescence intensity was measured after washing as described above. Luminescence measurement was performed for 1 second from 3 seconds after addition of the substrate unless otherwise specified, and the average value of 3 samples was obtained. As a result, when the N domain and C domain, which are conventional PCA methods, were combined, only 1.3 times the luminescence intensity was observed with respect to the value (average value) expected from each luminescence activity (Fig. 4, Measurement time 3 minutes). On the other hand, among the remaining combinations, when mixed in K529A / H245D (5H) + K529A, 5H + K443A, K443A / H245D (4H) + K529A, and 4H + K443A, significantly higher activity than expected was observed, Their emission intensity reached 7.5 times the expected value with 5H + K529A and 6.9 times with 4H + K443A (FIG. 4). Also, the luminescence intensity obtained in the latter was 1000 times or more that of the PCA method obtained in FIG. 4 and well over 1% when the wild type enzyme was used. Further, when the luminescence intensity was measured by changing the mixing ratio of the mutant pair 5H and K529A having the highest activity ratio, the activity was highest when the mixing ratio was 1: 1 as shown in FIG. Reaction time 3 minutes). From this, the increase in activity is due to the fact that the two mutants were not only concentrated on the plate surface by IR-DNA, but also the two mutants were close together at 1: 1 via Zif12. Was suggested. Furthermore, when the activity was measured by simply mixing 5H and K529A, and 4H and K443A in an activity measurement buffer at a 1: 1 concentration (final concentration 40 nM), only the average luminescence intensity of both was obtained. (FIG. 6). As described above, when these mutant pairs are used, enzyme activity is complemented by binding to the target DNA and approaching each other even under conditions where sufficient activity recovery is not observed by the conventional PCA method, and the luminescence intensity is increased. A significant increase was shown. Contrary to expectations, it was revealed that the K443A mutant performs almost the same function as the K529A mutant in this experimental system.

Example 2:
Although the experiment of Example 1 was to measure the activity of the mutant bound to the immobilized DNA, it was examined whether an activity change due to the addition of the antigen was observed in a homogeneous solution. In order to obtain a higher activity change, the L530R mutation (Fujii et al., Anal. Biochem. 366), which is expected to increase the amount of intermediate production compared to the K443A / H245D mutant, which is thought to produce a lot of intermediate LH ₂ -AMP , 131-136, 2007), and also introduced the mutation E354K, which is expected to improve stability for all mutants. That is, the plasmid prepared in Example 2 is as follows.
pET32-ZiF12-LucK443A / H245D / L530R / E354K: Fusion protein expression vector of LucK443A / H245D / L530R / E354K and Zif12
pET32-ZiF12-LucK529A / E354K: Fusion protein expression vector of LucK529A / E354K and Zif12

(1) Construction of mutant plasmid
Applying the quick change method to pET32-ZiF12-LucK443A / H245D, using primers 1 and 2 to mutate the 354th glutamic acid of Fluc to lysine, and using primers 3 and 4 to mutate the 530th leucine residue to arginine PET32-ZiF12-LucK443A / H245D / L530R / E354K was constructed. Similarly, pET32-ZiF12-LucK529A / E354K was constructed by mutating 354rd glutamic acid of Fluc of pET32-ZiF12-LucK529A to lysine. The experimental procedure for plasmid construction was the same as for pET32-ZiF12-LucH245D and the like.

Primer 1:
5'-TTCTGATTACACCCAAGGGGGATGATAAA-3 '(SEQ ID NO: 18)
Primer 2:
5'-TTTATCATCCCCCTTGGGTGTAATCAGAA-3 '(SEQ ID NO: 19)
Primer 3:
5′-CCGAAAGGTCTTACCGGTAAACGCGACGCAAGAAAAATCA-3 ′ (SEQ ID NO: 20)
Primer 4:
5'-TGATTTTTCTTGCGTCGCGTTTACCGGTAAGACCTTTCGG-3 '(SEQ ID NO: 21)

(2) Protein expression purification The two types of vectors obtained were used in the same manner as pET32-ZiF12-LucH245D.

(3) Luminescence intensity enhancement by adding DNA to two mutants Zif fusion LucK529A / E354K and Zif obtained by expression purification in white Half well 96-well plates (Costar 3693, CorningCostar Inc., Corning, NY) Dilute and mix the fused LucK443A / H245D / L530R / E354K with 100 mM Tricine, 1 mg / ml BSA, pH 8.0 to make 5 nM each, or 5 nM each (total 10 nM). Noted. Each mixture was mixed with non-biotinylated IR DNA at a final concentration of 5 nM, and a sample to which no IR DNA was added was prepared as a control. Add 50 μl each of the substrate solution adjusted to 200 mM MOPS, 20 mM MgSO ₄ , 2 mg / ml BSA, 75 μM LH ₂ , 20 mM ATP, pH 7.0 with an ATTO AB-2350 luminometer pump. The luminescence intensity was measured every second for 40 seconds, and the average value and standard deviation of three samples were obtained. First, each mutant alone was measured. When IR DNA was mixed with Zif-fused LucK529A / E354K, it was about 1.05 times 4 seconds after addition of the substrate compared to when nothing was mixed. The emission intensity of was observed. In addition, when IR was mixed with Zif-fused LucK443A / H245D / L530R / E354K, no increase in luminescence intensity was observed compared to the case where nothing was mixed. On the other hand, when IR was mixed in the mixed solution of Zif-fused LucK529A / E354K and Zif-fused LucK443A / H245D / L530R / E354K, compared to the case where nothing was mixed, about 4 seconds after addition of the substrate. An emission intensity of 1.19 times was observed (Fig. 7). From the above, it was strongly suggested that when this mutant pair was used, the enzyme activity was complemented and the luminescence intensity was remarkably increased by approaching both in a target DNA-dependent manner.

(4) Enhancement of luminescence intensity by adding antibody to two mutants
It was investigated whether the effect on the same activity was recognized by bringing two fusion proteins close to each other in a solution by a method other than the addition of DNA. On a Costar 3693 plate, dilute and mix Zif fusion LucK529A / E354K and Zif fusion LucK443A / H245D obtained by expression purification with 100 mM Tricine, 1 mg / ml BSA, pH 8.0 to 10 nM each. 50 μl aliquots were dispensed. Prepare thioredoxin-recognizing antibody (Trx • Tag ^™ Monoclonal Antibody, Novagen, Cat. No. 71542-3) in 1/1000 in each mixture and those without antibody as a control. Add 50 μl of each solution prepared to pH 7.0 containing MOPS, 20 mM MgSO ₄ , 2 mg / ml BSA, 300 μM LH ₂ , 20 mM ATP, measure the luminescence intensity for 2 minutes immediately after the addition, Average values and standard deviations were obtained. When the thioredoxin-recognizing antibody was mixed, the luminescence intensity was measured approximately 1.18 times as the integrated value for 2 minutes after the addition of the substrate compared to the case where the antibody was not mixed (FIG. 8). The above results strongly suggest that when this mutant pair is used, the thioredoxin part encoded by pET32 in the fusion protein binds to the antibody and is brought close to each other, thereby complementing the enzyme activity and significantly increasing the luminescence intensity. It was done.

Example 3:
In Examples 1 and 2, the DNA-binding domain Zif12 was used as an interaction domain. However, DNA induces not only heterodimers of different mutants but also homodimers of the same mutants. There was a problem. Therefore, FKBP12 and FRB were selected as systems capable of selectively forming heterodimers, and the influence of these rapamycin-dependent dimer formation on luminescence activity was evaluated.
(1) Construction of mutant plasmid fused with FKBP12 and FRB FK506 binding protein 12 kd fragment (FKBP12) known to bind to rapamycin and induce dimer formation, and FKBP-rapamycin binding protein FRB (Choi et al., Science 273, 239-242, 1996) was used as an interaction domain instead of Zif12 to investigate whether interaction-dependent enhancement of luminescence intensity could be obtained in solution. Plasmids encoding artificially synthesized FKBP12 and FRB (Mr Gene GMBH, Regensburg, Germany) (FIG. 9 (SEQ ID NOs: 22 and 23) and FIG. 10 (SEQ ID NOs: 24 and 25)) were cloned into restriction enzymes Sfi I and Not I. And fragments encoding FKBP12 and FRB were isolated. Insert these into pET32-ZiF12-LucK443A / H245D / L530R / E354K and pET32-ZiF12- LucK529Q / E354K, which were treated with the same restriction enzymes to remove Zif12 respectively, and pET-FKBP-LucK443A / H245D / L530R / E354K and pET -FRBP-LucK529Q / E354K was obtained. The experimental procedure for plasmid construction was the same as for pET32-ZiF12-LucH245D.

(2) Protein expression purification The two types of vectors obtained were used in the same manner as pET32-ZiF12-LucH245D.

(3) Increasing luminescence intensity by adding rapamycin to two mutants fused with FKBP12 or FRB The association of FKBP12-fused LucK443A / H245D / L530R / E354K and LucK529Q / E354K by the association of FKBP12 and FRB dependent on rapamycin It was examined whether activity was changed as a result. Dilute and mix FKBP fusion LucK443A / H245D / L530R / E354K and FRB fusion LucK529Q / E354K at 250 nM (total 500 nM) with 100 mM Tricine, 1 mg / ml BSA, pH 8.0. 50 μl was dispensed. Each mixture was mixed with a 50 nM rapamycin solution in 1% final methanol, and a sample was prepared by adding only 1% final methanol as a control. Add 50 μl each of the substrate solution adjusted to 200 mM MOPS, 20 mM MgSO4, 2 mg / ml BSA, 75 μM LH2, 20 mM ATP, pH 7.0 with a luminometer pump, and increase the luminescence intensity for 0.1 second for 10 seconds immediately after the addition. Measurements were made and the average and standard deviation of three samples were determined. When rapamycin is mixed in a mixture of FKBP-fused LucK443A / H245D / L530R / E354K and FRB-fused LucK529Q / E354K, it is approximately 2.46 times 0.5 seconds after the start of the reaction compared to the case where rapamycin is not mixed. Was observed (FIG. 11). From the above, it was strongly suggested that when this mutant pair was used, the enzymatic activity was complemented by the close proximity of FKBP and FRB via rapamycin, and the luminescence intensity was significantly increased. At this time, it is considered that higher responsiveness was obtained by forming only heterodimers of different mutants.

Claims (9)

A first molecule that is a variant of a reporter enzyme having a two-stage enzyme reaction mechanism, in which a first mutant whose first-stage reaction is a rate-determining process is linked to one of the two types of molecules to be analyzed; A second molecule, which is a variant of a reporter enzyme having a two-stage enzyme reaction mechanism, in which a second mutant whose rate-limiting process is a second-stage reaction is linked to the other of the two molecules to be analyzed And detecting the progress or progress of an enzyme reaction catalyzed by the reporter enzyme. The method of claim 1, wherein the reporter enzyme is luciferase.
The method according to claim 1 or 2, wherein the reporter enzyme is firefly luciferase. The method according to any one of claims 1 to 3, wherein the reporter enzyme mutant is a firefly luciferase mutant having a substitution at any amino acid of H245, K529 and / or K443. The first mutant of the reporter enzyme, in which the reaction at the first stage is the rate-limiting process, is a firefly luciferase mutant having an amino acid substitution at K529 or K443, and is a mutant of the reporter enzyme in two stages. The method according to any one of claims 1 to 4, wherein the second mutant in which the eye reaction is a rate-limiting process is a firefly luciferase mutant having an amino acid substitution at H245. A second variant of a reporter enzyme, in which the second stage reaction is the rate-limiting process,
The method according to any one of claims 1 to 5, which is (i) a firefly luciferase mutant having an amino acid substitution at H245 and K529, or (ii) a firefly luciferase mutant having an amino acid substitution at H245 and K443. The method according to any one of claims 1 to 6, wherein the amino acid substitutions in H245, K443 and K529 among the mutants of the reporter enzyme are H245D, K443A and K529A, respectively. The method according to any one of claims 1 to 7, wherein the two types of molecules to be analyzed are proteins or nucleic acids. A first molecule in which a mutant of a reporter enzyme having a two-stage enzyme reaction mechanism, in which the first-stage reaction is a rate-determining process, is linked to one of the two types of molecules to be analyzed; A combination of a second molecule in which a mutant of a reporter enzyme having an enzyme reaction mechanism, in which a second-stage reaction is a rate-determining process, is linked to the other of the two types of molecules to be analyzed; A reagent kit for detecting an interaction or approach between molecules by the method according to claim 1.

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