JP2011067190A - Single molecular format probe and utilization of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a single molecular format probe having a high emitted light brightness and easily designable. <P>SOLUTION: This fused protein used as a probe for detecting a target material includes a primary binding protein having a binding part with which the target material binds, a secondary binding protein recognizing that the target material binds with the primary binding protein and an active type enzyme positioning between the primary protein and secondary protein or its precursor, and in the case that the secondary protein recognizes that the target material binds with the primary protein, the protein increases the enzyme activity. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a fusion protein for detecting a target substance in a test sample and use thereof, and more specifically to a fusion protein used as a single-molecule probe for detecting a target substance in a test sample and use thereof. is there.

  Examples of biological phenomena based on protein deformation, binding between protein molecules, and the like include a nuclear receptor (NR) that becomes an activated state through a structural change due to hormone stimulation and becomes a dimer. Then, the dimerized nuclear receptor moves into the nucleus and binds to a nuclear receptor response element (NRE) in the nucleus to cause transcriptional activity (Non-patent Document 1).

  In addition, cells respond to growth hormone (growth factor) stimulation from the outside of the cell via a specific receptor (growth factor receptor) and transmit signals inside the cell by mediating a series of protein phosphorylation. (Non-Patent Document 2). Furthermore, the cell has a system for monitoring changes in the cholesterol concentration inside the cell. As an example, the cell has a protein that regulates cholesterol concentration (SREBP-2: Sterol-response element binding protein-2). When the intracellular cholesterol concentration falls below a certain level, the cell senses it and SREBP-2 is divided. A part of the fragmented SREBP-2 fragment dimerizes and translocates into the nucleus, and binds to a sterol-response element (SRE) in the nucleus, thereby a series of cholesterol regulatory factors. Is expressed (Non-patent Document 3).

  Such intracellular molecular phenomenon is a mechanism for a living body to quickly respond to external environmental changes while maintaining homeostasis of life activity. The proteins responsible for these signal transmissions play an extremely important role in allowing cells to react quickly to and cope with external stimuli.

  Several methods for measuring intracellular molecular phenomena have been disclosed. For example, techniques for measuring protein-protein interactions, changes in protein tertiary structure, protein phosphorylation, and the like are known. FRET (fluorescence resonance energy transfer) and BRET (bioluminescence resonance energy transfer) methods are widely used as methods for detecting protein-protein interactions (Non-patent Documents 4 and 5).

In addition, the present inventors have previously proposed monomolecular and bimolecular bioluminescent probes (Non-patent Documents 6 and 7 and Patent Documents 1 to 3). These bioluminescent probes are bioluminescent visualization probes in a form in which a luminescent enzyme is divided into two and one or two proteins are inserted between them. As an example of the single-molecule type bioluminescent probe, a luminescent enzyme derived from Gaussia is divided into two, and a calcium-recognizing protein calmodulin (CaM) is inserted between them. When this monomolecular bioluminescent probe is introduced into a eukaryotic cell, a change in bioluminescence depending on the Ca ²⁺ concentration can be observed (Non-patent Document 8 and Patent Document 3).

  Furthermore, the present inventors have previously proposed a bioluminescent probe using a circularly permuted luminescent enzyme (Non-patent Document 9). In this bioluminescent probe, circular permutation is performed by linking the original N-terminus and C-terminus of the luminescent enzyme, while cutting the enzyme active site of the enzyme to create an artificial N-terminus and C-terminus. Yes. Then, the protein is linked to each of these artificially prepared ends. By designing the molecule in this way, the enzyme active site of the luminescent enzyme is divided into two, and the divided enzyme active sites are respectively arranged outward from the center of the molecule. Therefore, the collision probability between enzyme active sites can be remarkably suppressed. As a result, the signal-noise ratio (S / N ratio) can be significantly improved.

  In addition, the present inventors have previously proposed a bioluminescence imaging probe that uses androgen receptor (AR) nuclear translocation as an index (Non-patent Document 1). In this probe, while the luminescent enzyme is divided into two, the protein (DnaE) that causes the protein joining reaction is also divided into two. Then, each of the two divided fragments is linked to the AR and normally localized in the cytoplasm, and the remaining fragments are localized in the nucleus. AR moves into the nucleus in a steroid hormone-dependent manner, and the fragment linked to AR interacts with the remaining fragment localized in the nucleus. The protein-protein interaction is detected by measuring the recovered bioluminescence intensity.

  As another method for measuring intracellular molecular phenomena, Patent Document 4 describes a method for measuring the amount of luminescence that is reduced by external stimulation using a probe in which one protein is inserted into firefly luciferase.

International Publication No. 2008/084869 Pamphlet JP 2009-34059 A (published February 19, 2009) US Patent Application Publication No. 2009/0176239 (published July 9, 2009) International Publication No. 2005/038029 Pamphlet (April 28, 2005)

Kim, S. B .; Ozawa, T .; Watanabe, S .; Umezawa, Y. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11542-11547. Kaihara, A .; Umezawa, Y. Chemistry, An Asian Journal 2008, 3, 38-45. Kim, S. B .; Takao, R .; Ozawa, T .; Umezawa, Y. Analytical Chemistry 2005, 77, 6928-6934. Awais, M .; Sato, M .; Lee, X. F .; Umezawa, Y. Angew. Chem. Int. Ed. 2006, 45, 2707-2712. Hoshino, H .; Nakajima, Y .; Ohmiya, Y. Nat.Methods 2007, 4, 637-639. Kim, S. B .; Otani, Y .; Umezawa, Y .; Tao, H. Anal. Chem. 2007, 79, 4820-4826. Kim, S. B .; Awais, M .; Sato, M .; Umezawa, Y .; Tao, H. Anal. Chem. 2007, 79, 1874-1880. Kim, S. B .; Sato, M .; Tao, H. Anal. Chem. 2009, 81, 67-74. Kim, S. B .; Sato, M .; Tao, H. Bioconjugate Chemistry 2008, 19, 2480-2486. Hodges, Y. K .; Tung, L .; Yan, X. D .; Graham, J. D .; Horwitz, K. B .; Horwitz, L. D. Circulation 2000, 101, 1792-1798. Loening, A. M .; Wu, A. M .; Gambhir, S.S. Nat.Methods 2007, 4, 641-643.

  The above-described detection method using a luminescent probe containing a luminescent enzyme has a high background fluorescence due to autofluorescence, an external light source is required in FRET for detecting a protein-protein interaction, and a fluorescence measurement method. Therefore, problems such as the need for a large fluorescent microscope, the limited number of cells that can be analyzed, and qualitative rather than quantitative results can be solved.

  Therefore, further improvement in detection accuracy by such a luminescent probe is required, and in particular, a luminescent probe with higher stability is required in order to obtain sufficient detection accuracy. In addition, it is required to be easy to design in order to reduce the development cost of the light emitting probe and to use it widely.

  Therefore, an object of the present invention is to provide a novel probe that has high emission intensity and is easy to design.

  The present inventors have intensively studied to solve the above problems, and have completed the present invention by considering the following points.

  First, it was found that the full length of the luminescent enzyme was used as it was, considering that the enzyme activity inherent to the luminescent enzyme was not violated. By using the change in enzyme activity due to structural deformation of the luminescent enzyme as an index, it is possible to measure at high luminescence intensity, simplifying the structure of the luminescent probe and making trial and error in design. It was found that it can be easily avoided.

  In order to solve the above problems, a fusion protein according to the present invention is a fusion protein for detecting a target substance, and has a primary binding protein having a binding site to which the target substance binds, and the target substance to the primary binding protein. A secondary binding protein that recognizes binding, and an active enzyme or a precursor thereof positioned between the primary binding protein and the secondary binding protein, and the target is bound to the primary binding protein. When the secondary binding protein recognizes that a substance is bound, the enzyme activity is increased. In the fusion protein according to the present invention, the enzyme preferably has a substantially spherical shape.

  In the fusion protein according to the present invention, the enzyme is a luminescent enzyme, and increases the amount of luminescence when the secondary binding protein recognizes that the target substance is bound to the primary binding protein. It is preferable. Moreover, the fusion protein according to the present invention preferably further comprises a fluorescent protein located between the primary binding protein and the secondary binding protein. In the fusion protein according to the present invention, the luminescent enzyme is preferably selected from the group consisting of Renilla luciferase, Gaussia luciferase, click beetle luciferase, and firefly luciferase.

  Furthermore, in the fusion protein according to the present invention, the primary binding protein is preferably selected from the group consisting of a nuclear receptor, a cytokine receptor, a protein kinase, a second messenger recognition protein, and a transcription factor. In the fusion protein according to the present invention, the primary binding protein is a ligand binding domain of an estrogen receptor, the secondary binding protein is an SH2 domain of an Src protein, and the luminescent enzyme is a Renilla luciferase. preferable.

  In addition, the fusion protein according to the present invention further includes a first linker that links the primary binding protein and the enzyme, and a second linker that links the secondary binding protein and the enzyme. Each of the second linkers preferably has an amino acid sequence of 5 amino acids or less. Furthermore, in the fusion protein according to the present invention, it is preferable that the first linker is composed of an amino acid sequence represented by Gly-Ser, and the second linker is composed of an amino acid sequence represented by Gly-Thr.

  Furthermore, the fusion protein according to the present invention comprises a fusion protein consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 and 13 to 14; or any one of SEQ ID NOs: 1 to 3 and 13 to 14 When the secondary binding protein recognizes that the target substance has bound to the primary binding protein consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence shown in FIG. It is characterized by being a fusion protein that increases

  The polynucleotide according to the present invention is a polynucleotide encoding any one of the above fusion proteins. The polynucleotide according to the present invention is a polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 4 to 6 and 15 to 16; the base sequence represented by any of SEQ ID NOs: 4 to 6 and 15 to 16 When the secondary binding protein recognizes that the target substance has bound to the primary binding protein, the enzyme activity is increased. A polynucleotide comprising a base sequence encoding a fusion protein; hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in any of SEQ ID NOs: 4-6 and 15-16 , A fusion protein that increases enzyme activity when the secondary binding protein recognizes that the target substance has bound to the primary binding protein A polynucleotide comprising the encoding base sequence; or at least 66% identical to the base sequence shown in any of SEQ ID NOs: 4-6 and 15-16, and secondary binding of the target substance bound to the primary binding protein It is characterized by being a polynucleotide comprising a base sequence encoding a fusion protein that increases the enzyme activity when the protein is recognized.

  The vector according to the present invention is characterized by including any of the polynucleotides described above. The transformant according to the present invention is characterized by containing any one of the above polynucleotides or the above vector.

  A method for detecting a target substance according to the present invention is a method for detecting a target substance in a test sample, comprising the step of bringing the test sample into contact with any one of the above fusion proteins. . A kit for detecting a target substance according to the present invention is characterized by comprising any one of the above fusion proteins.

  The method for producing a probe according to the present invention is characterized by including a step of transforming a cell using any one of the above polynucleotides or the above vector. A probe production kit according to the present invention is characterized by comprising any of the polynucleotides or vectors described above.

  According to the fusion protein of the present invention, by using an active enzyme or a precursor thereof, the design is easy, the luminescence intensity is high, and the visual detection of various protein-protein interactions is performed with higher accuracy. be able to.

It is a figure which shows typically the ligand recognition mechanism using an example of the light emission probe which concerns on this invention. It is a figure which shows the crystal structure of Renilla luciferase (RLuc8). It is a figure which shows the crystal structure of a firefly luciferase (FLuc). It is a figure which shows the interaction between each amino acid in the enzyme active site of RLuc8, and a substrate, The amino acid near from the enzyme terminal is enclosed with a broken line, and is shown. It is a schematic diagram which shows the ligand recognition mechanism using the luminescent probe which concerns on this invention. It is a figure which shows an example of the DNA construct structure for producing the luminescent probe which concerns on this invention. It is a graph which shows the emission spectrum in the transformant which expressed the luminescent probe (ERS) which inserted RLuc8. It is a graph which shows the ratio of the emitted light intensity in the transformant which each expressed ERS, EGS (luminescent probe which inserted GLuc), ECS (luminescent probe which inserted CBLuc), and EFS (luminescent probe which inserted FLuc). It is a figure which shows the chemical structure of the various substrate of a luminescent enzyme, and the site | part which hits an important side chain is enclosed with a broken line, and is shown. It is a graph which shows the emission spectrum in the presence of various substrates. It is a graph which shows the ratio of the peak intensity of the emission spectrum in the presence of various substrates. It is a graph which shows the emission spectrum in the transformant which expresses RLuc8, and the transformant which expresses ERS. It is a figure which shows the ligand response curve in the transformant which expresses ERS. Expresses ERS, EmRS (luminescent probe in which phosphorylation site of ER LBD is mutated), ER + RS (probe in which RLuc8 and ER LBD are linked (ER) and probe in which RLuc8 and SH2 domain are linked (RS)) It is a graph which shows the relative luminescence intensity in a transformant. It is a figure which shows the DNA construct structure for expressing ER or RS. It is a figure which shows the structure of ER and RS typically. It is a figure which shows the chemical structure of various ligands. It is a graph which shows the emitted light intensity in response to the various ligand in the transformant which expresses ERS. It is a figure which shows an example of the structure for producing the light emission probe (EERS) based on this invention. It is a graph which shows the emission spectrum in the transformant which expressed the luminescence probe (pEERS) which inserted EYFP and RLuc8. It is a figure which shows typically the ligand recognition mechanism of the luminescent probe (EERS) which is the luminescent probe which concerns on this invention, and raise | generates the luminescence resonance energy transfer in the inside.

[1. Fusion protein and polynucleotide)
The fusion protein according to the present invention is a fusion protein that is used as a single-molecule bioluminescent probe and detects a target substance, and includes a primary binding protein having a binding site to which the target substance binds, and the primary binding protein. A secondary binding protein that recognizes that a target substance has bound, and an active enzyme or a precursor thereof positioned between the primary binding protein and the secondary binding protein, the primary binding protein It is a fusion protein that increases the enzyme activity when the secondary binding protein recognizes that the target substance has bound.

  As described later, a typical example of the active enzyme or precursor thereof contained in the fusion protein according to the present invention is an active luminescent enzyme or precursor thereof. Therefore, hereinafter, as an example, the enzyme in the fusion protein according to the present invention will be described as a luminescent enzyme, and the fusion protein according to the present invention may be referred to as a luminescent probe, but the present invention is not limited to this, Even an enzyme can be explained similarly.

  In the present specification, “luminescent probe” is used interchangeably with “bioluminescent probe” or “probe” to cause various molecular phenomena caused by a target substance specific to a target in a living cell or living body. It is a probe that can be visualized and imaged as bioluminescence. In addition, the “single molecule luminescent probe” is a probe including all the components used for the above-described visualization imaging in a single fusion molecule. For example, a fusion protein containing (i) a primary binding protein, (ii) an active enzyme or a precursor thereof, and (iii) a secondary binding protein as basic components may be included.

  The fusion protein of the present invention may be in any order as long as an active enzyme or a precursor thereof is inserted between the primary binding protein and the secondary binding protein, but the primary binding protein, the active enzyme or a precursor thereof. It is preferable that it is a form that is linearly linked in the order of the body and the secondary binding protein, and is linearly linked in the order of the primary binding protein, the active enzyme or its precursor, and the secondary binding protein from the amino terminus. More preferably, it is in the form.

  In addition, the fusion protein of the present invention is preferably one that increases the amount of luminescence when the enzyme is a luminescent enzyme and the secondary binding protein recognizes that the target substance is bound to the primary binding protein. Furthermore, the fusion protein of the present invention preferably further comprises a fluorescent protein located between the primary binding protein and the secondary binding protein. With this configuration, emission resonance energy transfer can be caused.

  Here, the “primary binding protein” is intended to be a protein that binds the target substance to the binding site of the target substance, and is sometimes referred to as “target substance binding protein”. As will be described later, when the target substance to be detected according to the present invention is referred to as a ligand, the target substance binding protein may be referred to as a “ligand binding protein”.

  The primary binding protein can be, for example, a protein that can change the steric structure by binding to a target substance and bind to a molecular recognition site of a secondary binding protein described later. As such a primary binding protein, for example, a nuclear receptor (NR) having a hormone, chemical substance or signal transduction protein as a ligand, a cytokine receptor, or various protein kinases are used. The primary binding protein is appropriately selected depending on the target substance of interest.

  The “target substance” in the present specification is a substance to be detected by the luminescent probe according to the present invention, and can specifically bind to a specific protein on a living cell membrane or in a living cell to change its function. It means a substance, sometimes called “ligand”. For example, steroids or synthetic chemicals are used for nuclear receptors, cytokines are used for cytokine receptors, and stimulants containing insulin are used for receptors on cell membranes such as insulin receptors. Sure.

  The ligand to be detected by the present invention is not particularly limited as long as it binds to a ligand-binding protein, and may be an extracellular ligand that is taken into the cell from outside the cell. It may be an intracellular ligand that is produced. For example, it can be an agonist or antagonist for a receptor protein (eg, nuclear receptor, G protein-coupled receptor, etc.). In addition, signaling proteins such as cytokines, chemokines, and insulin that specifically bind to molecules involved in intracellular signal transduction, intracellular second messengers, lipid second messengers, phosphorylated amino acid residues, G protein-coupled receptor ligands Etc.

For example, when an intracellular second messenger, a lipid second messenger, or the like is targeted as a ligand, the binding protein (second messenger recognition protein) of each second messenger can be used as the ligand binding protein. The second messenger is intended to be another type of intracellular signaling substance that is newly generated in the cell by binding extracellular signaling substances such as hormones and neurotransmitters to receptors present on the cell membrane. . Examples of the second messenger include cGMP, cAMP, PIP, PIP2, PIP3, inositol triphosphate (IP3), IP4, Ca ²⁺ , diacylglycerol, arachidonic acid and the like. For example, for the second messenger Ca ²⁺ , calmodulin (CaM) can be used as a ligand binding protein.

  For example, when a ligand specific to a nuclear receptor is used as a ligand, a known binding domain (LBD) of the nuclear receptor can be used as a ligand binding protein. . Furthermore, when targeting phosphorylated amino acid residues or G protein-coupled receptor ligands, phosphorylated amino acid binding domains or G protein-coupled receptors can be used as ligand-binding proteins, respectively. As the nuclear receptor ligand binding domain, the estrogen receptor (ER), glucocorticoid receptor (GR), androgen receptor (AR) or progesterone receptor (PR) ligand binding domain having estrogen as a ligand is preferable. Used.

  For example, the LBD of the estrogen receptor is based on the sequence information of the full-length human estrogen receptor (GenBank / P03372), and the LBD region (amino acid numbers 305 to 550) prepared by genetic engineering or by PCR synthesis is used. can do. In addition, for example, the androgen receptor LBD is based on the sequence information of the full-length human androgen receptor (GenBank / AF162704), and its LBD region (amino acid numbers 672 to 910) prepared by genetic engineering or PCR synthesis is used. can do. In addition, for example, the LBD of the glucocorticoid receptor is prepared by genetic engineering or PCR synthesis of the LBD region (amino acid numbers 527 to 777) based on the sequence information of the full-length human glucocorticoid receptor (GenBank / 1201277A). Can be used. Similarly, for the LBD of progesterone receptor, the LBD region (amino acid numbers 677-933) prepared by genetic engineering or PCR synthesis based on the sequence information of the full-length human progesterone receptor (GenBank / P06401) Can be used.

  In the present specification, the “secondary binding protein” is intended to be a protein having a molecular recognition site that recognizes that a ligand is bound to the above-described ligand binding protein, and may be referred to as “recognition protein”. The recognition protein includes, for example, a protein that binds to a ligand-binding protein whose steric structure has been changed by binding of the ligand.

  For example, when calmodulin is used as the ligand binding protein, the M13 peptide that is a peptide derived from miosin light chain kinase is preferably used as the recognition protein. In addition to M13, examples of proteins that can bind to calmodulin include CaM-dependent protein kinases such as adenylyl cyclase and calmodulin kinase II. A portion of such a protein can be used in place of M13.

  For example, when a nuclear receptor such as an androgen receptor or estrogen receptor is used as a ligand-binding protein, an LXXLL motif (SEQ ID NO: 12) derived from a co-transcription factor (coactivator) as a recognition protein, the N-terminal side of AR (AR NTD) -derived FQNLF motif, FXXXLF motif, WXXLF motif and the like can be used. Preferably, Rip140 (GenBank / NP003480), which is a type of co-transcription factor, or LXXLL motif (about 15 amino acids) of Src-1a (steroid receptor coactivator 1 isoform 1; GenBank / NP003734) is used.

  Furthermore, SH2 domains of various kinase proteins, which are domains capable of recognizing phosphorylated amino acid residues, may be used as recognition proteins. For example, Src protein (proto-oncogene tyrosine-protein kinase Src; GenBank / NP938033), a phosphorylation recognition domain (SH2 domain; amino acid numbers 150 to 248), a growth factor receptor related to cell proliferation, carcinogenesis, etc. For example, the SH2 domain of the binding protein Grb2 (growth factor receptor-binding protein 2) can be used. When a G protein-coupled receptor is used as the ligand binding protein, G protein or the like can be suitably used as the recognition protein.

  The combination of the ligand binding protein and the recognition protein in the present invention may be two proteins that bind in a condition-dependent manner. For example, FRB and BRAD, ER LBD and LXXLL motif, AR LBD and FQNLF motif, CaM and M13, nuclear receptor and its coactivator, phosphorylated protein and its phosphorylated recognition protein, homo or hetero bond between nuclear receptors A pair of two proteins or the like for performing (dimerization) can be used.

  As used herein, “active enzyme or precursor thereof” is intended to be an activated enzyme or precursor thereof capable of binding a substrate to the enzyme active site, and includes the full-length sequence of the enzyme. It is intended to be unwanted. The “full-length sequence of the enzyme” includes at least the region including the enzyme active site as it is without being deleted or divided, and may be any one that retains the original enzyme activity. For example, the deletion of a part of the N-terminal side or C-terminal side that does not affect the enzyme activity even if it is deleted is also included in the “full-length sequence of the enzyme”.

  Here, the precursor of the enzyme is inactive, but is intended to be activated by being subjected to some modification to become an active enzyme, and from the same sequence as the active enzyme. It will be. For example, when using an enzyme that is activated from an inactive state by phosphorylation, it is inserted into a luminescent probe in the precursor state, activated by phosphorylation when detecting a ligand, and used as an active enzyme Can do. In the following, when describing “full-length enzyme” or simply “enzyme” used in the present invention, an active enzyme or a precursor thereof is intended.

  The enzyme used in the present invention has an external molecular tension due to the interaction between the ligand-binding protein and the recognition protein that are connected to the N-terminal side and the C-terminal side, respectively. Can be an enzyme in which the enzyme activity from the luminescent probe changes. A typical example of such an enzyme is a luminescent enzyme (LE).

  In addition, the enzyme used in the present invention may be inserted between a ligand binding protein and a recognition protein in a manner linked to a fluorescent protein. In this case, the enzyme is preferably a luminescent enzyme. In addition, the fluorescent protein is preferably one that can serve as a receptacle for luminescence resonance energy transfer (BRET). That is, the fluorescent protein is preferably one that can absorb the luminescence energy generated by the activity of the luminescent enzyme. This makes it possible to induce emission resonance (cause emission resonance energy transfer).

  The combination of the luminescent enzyme and the fluorescent protein used in the present invention is affected by part of the luminescence resonance energy transfer between the luminescent enzyme and the fluorescent protein by being inserted between the ligand binding protein and the recognition protein. It is desirable that In addition, the combination of a luminescent enzyme and a fluorescent protein allows the ligand-binding protein and the recognition protein to interact with each other, thereby adding molecular tension from the outside and causing their internal structural deformation to occur. It can be a combination of a luminescent enzyme and a fluorescent protein, in which the luminescence resonance energy transfer with the protein is affected, thereby changing the enzyme activity from the luminescent probe.

  The fluorescent protein may be linked to the N-terminal side of the enzyme or may be linked to the C-terminal side of the enzyme.

  In the present specification, the “molecular tension” is a force (tension) applied from the N-terminus and the C-terminus to the above-described enzyme (enzyme molecule) or a combination of an enzyme and a fluorescent protein, preferably an enzyme molecule, or It is a force that causes structural deformation in the combination of an enzyme and a fluorescent protein.

  In the present specification, “structural deformation” refers to an enzyme molecule or a molecule of a combination of an enzyme and a fluorescent protein due to a molecular tension applied from the outside to the combination of the enzyme molecule or the enzyme and the fluorescent protein. Is intended to be a structural change such as twisting, and the structure does not have to be greatly deformed, and the deformation may increase the enzyme activity from the luminescent probe. That's fine.

  The ligand binding protein, the full length of the enzyme or the full length of the enzyme and the fluorescent protein, and the recognition protein, which are each component of the luminescent probe according to the present invention, are each directly or optimally selected so as to form a linear fusion molecule. It is linked via a linker peptide. In the case of linking via a linker peptide, the distance between each component can be appropriately adjusted by changing the type and length of the linker peptide sequence.

  Such a linker peptide is preferably a linking peptide for expressing the luminescent probe according to the present invention as a single-chain fusion protein. The linker peptide may be via a flexible amino acid (for example, glycine (G)) without a side chain. The length of the linker peptide is preferably short, for example, it is preferably a short amino acid sequence of 5 amino acids or less. Thus, a probe with good ligand responsiveness can be designed by minimizing the length of the linker peptide between each element. The first linker for linking the ligand binding protein and the enzyme or the fluorescent protein inserted in combination with the enzyme consists of an amino acid sequence represented by Gly-Ser, and the recognition protein and the enzyme or the enzyme The second linker that links the fluorescent protein that is inserted in combination with an amino acid sequence preferably consists of an amino acid sequence represented by Gly-Thr, whereby a molecule generated by the interaction between the ligand-binding protein and the recognition protein Tension can be efficiently transmitted to the enzyme, and structural deformation can be surely generated in the enzyme.

  Here, a ligand recognition mechanism using an example of the luminescent probe according to the present invention is schematically shown in FIG. As an example of the luminescent probe according to the present invention, as shown in FIG. 1, for example, the full length of a luciferase (Luciferase), which is a luminescent enzyme, is inserted between a ligand binding protein (A) and a recognition protein (B). . The full length may be a part lacking in enzyme activity. When stimulated by the ligand, the ligand binds to the ligand binding protein, and the recognition protein recognizes this binding, whereby the ligand binding protein and the recognition protein interact. The interaction between the ligand binding protein and the recognition protein inevitably causes a certain structural deformation (strain) in the luminescent enzyme present between them, and this deformation increases the enzyme activity from the luminescent probe.

  FIG. 21 schematically shows a ligand recognition mechanism using another example of the luminescent probe according to the present invention. Another example of the luminescent probe according to the present invention is, as shown in FIG. 21, for example, between the ligand binding protein (A) and the recognition protein (B), and the total length of luciferase (RLuc8), which is a luminescent enzyme, is enhanced. Yellow Fluorescent Protein (EYFP) is inserted. EYFP is linked to the N-terminal side of RLuc8. In the absence of the ligand, the ligand-binding protein and the recognition protein do not interact, the bioluminescence resonance energy transfer (BRET) between EYFP and RLuc8 is weak, and the luminescence intensity is weak. On the other hand, when stimulated by a ligand, the ligand binds to the ligand binding protein, and the recognition protein recognizes this binding, whereby the ligand binding protein and the recognition protein interact. The interaction between the ligand binding protein and the recognition protein inevitably causes a certain structural deformation (strain) in the luminescent enzyme and the fluorescent protein existing between them, and the bioluminescence resonance energy between the luminescent enzyme and the fluorescent protein. By affecting the migration, the enzyme activity from the luminescent probe is increased.

  Since the increase in enzyme activity depends on the concentration, nature, etc. of the ligand, the nature, amount, etc. of the ligand can be measured using the increase in enzyme activity, ie, the increase in luminescence intensity as an index. In order to detect the ligand based on the principle as described above, the luminescent probe according to the present invention may be referred to as a “strain probe”.

  In this way, the luminescent enzyme inserted into the luminescent probe is subjected to molecular tension due to the interaction between the proteins linked on both sides of the full-length luminescent enzyme, resulting in structural deformation of the molecule (molecular deformation). As long as it increases the enzyme activity, the type, luminescent properties, molecular weight, etc. are not particularly limited. For example, Renilla luciferase (RLuc8: Renilla luciferase 8), Gaussia luciferase (GLuc: Gaussia luciferase), click beetle luciferase (CBLuc: click beetle luciferase), a type of beetle (insect) luciferase, firefly luciferase (FLluc: firefly) ), RLuc, MpLuc1 (Metridia pacifica luciferase 1), MpLuc2 (Metridia pacifica luciferase 2), MLuc (Metridia longa luciferase), OLuc (Oplophorus), RWluc (Rail-road worm luciferase), etc. Can be used without particular limitation. Among these luminescent enzymes, RLuc8, GLuc, CBLuc, FLuc and the like can be suitably used.

  These luminescent enzymes have known amino acid sequences and gene (DNA) base sequences (for example, FLuc for GenBank / AB062786 etc., CBLuc for GenBank / AY258592.1 etc., GLuc for GenBank / AY015993 etc.), and these sequences DNA can be obtained by a known method based on the information.

  Here, the structures of RLuc8 and FLuc are compared. FIG. 2 is a diagram showing the crystal structure of RLuc8, and FIG. 3 is a diagram showing the crystal structure of firefly luciferase (FLuc). In FIG. 2, N represents the N-terminus, and C represents the C-terminus. The enzyme active site is indicated by an arrow site (a). As shown in FIG. 2, RLuc8 is composed of one unit having a substantially spherical shape and has a rigid molecular structure. On the other hand, as shown in FIG. 3, FLuc is composed of two domains, and the N-terminal domain and the C-terminal domain are connected by a highly flexible hydrophilic amino acid. Because of these structural differences, when an enzyme is inserted alone between a ligand binding protein and a recognition protein, an enzyme having a structure such as RLuc8 is more likely to be a molecule than a two-domain structure such as FLuc. The molecular tension from the end is efficiently transmitted to the enzyme body, and structural deformation is likely to occur, which is preferable.

  Next, the enzyme active site of RLuc8 will be considered. FIG. 4 is a view showing an interaction (binding) between each amino acid and a substrate in the enzyme active site of RLuc8 (Non-patent Document 11). In FIG. 4, amino acids surrounded by a broken line are those close to the end of the enzyme. Among the amino acids constituting the enzyme active site of RLuc8, H285, F286, F261, and the like are close to the C-terminal (Q311), and thus are particularly easily affected by external molecular tension applied to the C-terminal. Therefore, RLuc8 has a structure in which the enzyme activity is easily changed by deformation because external molecular tension is efficiently transmitted to the enzyme active site.

  As described above, the enzyme used in the luminescent probe according to the present invention is composed of one substantially spherical domain such as RLuc8 when the enzyme is inserted between the ligand binding protein and the recognition protein alone. In addition, it is preferable that the enzyme active site is close to the end and is easily deformed and the enzyme activity is easily changed by the deformation.

  In the present specification, “the enzyme has a substantially spherical shape” means that the three-dimensional structure of the enzyme molecule as a whole has a substantially spherical shape. Therefore, for example, even if an enzyme molecule is composed of two domains, if the enzyme molecule as a whole has a substantially spherical shape, the enzyme has a substantially spherical shape. The “substantially spherical shape” is a shape having a relatively small difference from a perfect spherical shape, and is a shape in which the distance from the center of the enzyme molecule to an arbitrary point on the surface of the enzyme molecule is substantially constant. I just need it.

  In addition, when a luminescent enzyme and a fluorescent protein are combined and inserted into a luminescent probe, the luminescent enzyme described above can be used without any particular limitation. The fluorescent protein combined with the luminescent enzyme is preferably one that can efficiently absorb the luminescent energy generated by the activity of the luminescent enzyme. For example, as for fluorescent protein, it is desirable that all or part of the absorption spectrum thereof overlaps with all or part of the emission spectrum of the luminescent enzyme. Examples of the fluorescent protein include Venus, mOrange, Kaede, Dronpa and the like in addition to the above-described EYFP.

The luminescent probe according to the present invention uses a ligand-binding domain (LBD) of an estrogen (female hormone) receptor (ER) as a ligand-binding protein, and phosphorylation of kinase Src as a recognition protein. A luminescent probe (ERS) using a recognition domain (Src SH2) and RLuc8 as the full length of the luminescent enzyme is preferable. ER LBD responds to ligands such as estrogens such as 17β-estradiol (E ₂ ) and estrogen antagonists (OHT; 4-hydroxytamoxifen). This luminescent probe can be used to detect a ligand that phosphorylates ER LBD.

  Thus, by selecting the optimal luminescent enzyme, the molecular tension due to the structural change of the luminescent probe is efficiently transmitted to the luminescent enzyme, and the structure of the luminescent probe can be further simplified. By configuring the luminescent probe in this way, the amount of luminescence due to the interaction between the ligand-binding protein and the recognition protein is remarkably maintained while maintaining a much higher luminescence intensity from the luminescent probe compared to the conventional enzyme-splitting type. Can be increased. For example, as shown in the examples described later, when RLuc8 is used as the luminescent enzyme of the luminescent probe, the amount of luminescence compared to the case where they do not interact due to the interaction between the ligand binding protein and the recognition protein. Can be increased to 650% (see fcp in FIG. 10). As described above, the luminescent probe according to the present invention is preferably one that causes enzyme deformation due to molecular tension and accompanying increase in enzyme activity.

  FIG. 5 shows a schematic diagram showing a ligand recognition mechanism using the thus configured luminescent probe. As shown in FIG. 5, ER LBD undergoes a structural change (activation) in response to stimulation with a ligand such as OHT. As a result of this structural change, when Y537 in ER LBD is phosphorylated, the SH2 domain of Src recognizes this phosphorylation site, and ER LBD and SH2 domain are bound by intramolecular interaction. Binding between the ER LBD-SH2 domains causes structural deformation of RLuc8 and increases the enzymatic activity of the luminescent probe. This increase in enzyme activity depends on the ligand concentration and the like. Therefore, the amount of ligand can be measured using the change in emission intensity from the luminescent probe as an index.

  For example, in order to detect a ligand for the androgen receptor, AR LBD can be used as a ligand-binding protein and an LXXLL motif can be used as a recognition protein.

  In the luminescent probe according to the present invention, when the recognition protein in the same molecule recognizes that the ligand is bound to the ligand binding protein, the enzyme activity of the luminescent probe is caused by structural deformation of the enzyme, That is, the amount of luminescence increases more than the amount of luminescence from the luminescent probe in the state before the ligand binds to the ligand binding protein. That is, the amount of light emitted from the luminescent probe is different before and after the ligand is bound, and the change in the amount of light emitted may be caused by an increase in the quantum yield of the enzyme due to deformation, Examples include an improvement in turn-over rate (improved stability) of the luminescent probe due to enzyme deformation. However, it goes without saying that in the detection of a ligand by the luminescent probe according to the present invention, an increase in the luminescence amount from the luminescence probe may be used as an index regardless of the cause of the change in the luminescence amount from the luminescence probe.

  The luminescent probe according to the present invention may be obtained by linking each component polypeptide after obtaining the polypeptide of each component by a chemical synthesis method or the like. It can also be obtained by transforming a cell using a polynucleotide linked to the DNA, that is, a vector into which a chimeric DNA has been inserted, and expressing it in the transformed cell.

  Here, the chimeric DNA is DNA in which several DNA fragments having different origins are artificially linked in a linear form, and can express a fusion protein comprising several polypeptides as constituent elements. It is.

  In this specification, “live cells” include not only cells in living organisms (prokaryotic cells, yeast cells, insect cells, mammalian cells including humans, etc.), but also cultured cells that maintain their original functions. (Prokaryotic cells and eukaryotic cells), cells transplanted and infected in living organisms, and particularly cells of laboratory animals such as mice. It is also a concept that includes the individual organism itself.

  In the present specification, the fusion protein may be one in which several proteins or polypeptides having different origins are artificially linked.

  In the present specification, “polypeptide” is used interchangeably with “peptide” or “protein”. The polypeptides according to the invention may also be chemically synthesized or isolated from natural sources. The term “isolated” polypeptide or protein is intended to be a polypeptide or protein that has been removed from its natural environment. For example, recombinant polypeptides and proteins expressed and produced in host cells can be isolated in the same manner as natural or recombinant polypeptides and proteins that have been substantially purified by any suitable technique. It is thought that.

  In the luminescent probe according to the present invention, each constituent polypeptide includes natural purified products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells, higher plant cells, insects). Cells, and products produced by recombinant techniques from mammalian cells).

  Further, the luminescent probe and the polypeptide of each component according to the present invention may contain an additional peptide. Examples of the additional peptide include epitope-tagged peptides such as His tag (SEQ ID NO: 7), Myc tag (SEQ ID NO: 9), and Flag tag (SEQ ID NO: 8). The fusion protein according to the present invention can be modified into a preferred form and expressed recombinantly. For example, the fusion protein according to the present invention has stability in host cells, purification, operations following purification, storage, etc. by adding a specific amino acid, particularly a charged amino acid region, to the N-terminus or C-terminus. Sustainability etc. can be improved.

  In one embodiment, the luminescent probe according to the present invention is preferably a fusion protein consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 3 and 13 to 14 or a variant thereof. Here, the mutant is a fusion protein that increases the amount of luminescence when the recognition protein recognizes that the ligand is bound to the ligand binding protein, and is in any one of SEQ ID NOs: 1-3 and 13-14. In the amino acid sequence shown, it is preferably a fusion protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added.

  As used herein with respect to a polypeptide or protein, the term “variant” refers to a polypeptide or protein in which at least one or more of the original amino acids are point mutations, insertions, inversions, repeats, deletions, type substitutions. Thus, a polypeptide or protein that increases the amount of luminescence when the recognition protein recognizes that the ligand has bound to the ligand binding protein is intended.

  Such variants include deletions, insertions, inversions, repeats, type substitutions (eg, replacement of a hydrophilic residue with another residue, but usually a strong hydrophilic residue with a strong hydrophobic residue. And a mutant containing a point mutation or the like. The amino acid substitution includes, for example, a case where a neutral amino acid in a polypeptide is substituted with another neutral amino acid.

  It is well known in the art that some amino acids in the amino acid sequence of a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide. Furthermore, it is also well known that there are variants that not only artificially modify, but also do not significantly change the structure or function of the protein.

  One skilled in the art can readily mutate one or several amino acids in the amino acid sequence of a polypeptide using well-known techniques. For example, according to a known point mutation introduction method, an arbitrary base of a polynucleotide encoding a polypeptide can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide. Furthermore, if the method described in this specification is used, it can be easily determined whether the produced mutant has a desired activity.

  The mutant is preferably a mutant having a mutation such as a conservative or non-conservative amino acid substitution, deletion, or addition. The mutation possessed by the mutant is preferably silent substitution, addition and deletion, and particularly preferably conservative substitution. These do not change the luminescent activity of the luminescent probe according to the present invention.

  Typical conservative substitutions are substitutions of one amino acid for another in the aliphatic amino acids Ala, Val, Leu, and Ile; exchange of hydroxyl residues Ser and Thr, acidic residue Asp And Glu exchange, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe and Tyr.

  In another embodiment, the variant of the luminescent probe according to the present invention is a fusion protein that increases the amount of luminescence when the recognition protein recognizes that the ligand is bound to the ligand-binding protein, comprising SEQ ID NO: 4 to It is preferable that one or several base sequences of base sequences shown in any of 6 and 15 to 16 are encoded by a polynucleotide comprising a base sequence deleted, substituted or added.

  In another embodiment, the variant of the luminescent probe according to the present invention is a fusion protein that increases the amount of luminescence when the recognition protein recognizes that the ligand is bound to the ligand-binding protein, comprising SEQ ID NO: 4 to It is preferably encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence shown in any of 6 and 15-16.

  Hybridization is described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed. , Cold Spring Harbor Laboratory (1989). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous polynucleotide can be obtained. The appropriate hybridization temperature varies depending on the base sequence and the length of the base sequence. For example, when a DNA fragment consisting of 18 bases encoding 6 amino acids is used as a probe, a temperature of 50 ° C. or lower is preferable.

  As used herein, the term “stringent (hybridization) conditions” refers to hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM phosphorous. After overnight incubation at 42 ° C. in sodium acid (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA, It is intended to wash the filter in 1 × SSC. Depending on the polynucleotide that hybridizes to a “portion” of the polynucleotide, at least about 15 nucleotides (nt) of the reference polynucleotide, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably about Polynucleotides (either DNA or RNA) that hybridize to polynucleotides longer than 30 nt are contemplated.

  In another embodiment, the variant of the luminescent probe according to the present invention is a fusion protein that increases the amount of luminescence when the recognition protein recognizes that the ligand is bound to the ligand-binding protein, and is SEQ ID NO: At least 66% identical to the nucleotide sequence shown in any of 4-6 and 15-16, more preferably at least 80% identical, even more preferably at least 85%, 90%, 92%, 95%, 96%, 97% , 98% or 99% are preferably encoded by a polynucleotide consisting of a base sequence.

  If 66% or more of the DNA base sequence is the same, a silent mutation can be considered as a basis for producing a fusion protein having exactly the same activity. According to the gene memorization form, for example, the gene encoding the amino acid valine may be any of GUU, GUC, GUA, and GUG. Therefore, since the same fusion protein probe can be produced even if the maximum of 33% of the gene bases are different, it has a reasonable meaning in determining whether 66% of the numbers have the same base sequence.

  Many proteins whose functions have been elucidated have natural mutants or isoforms. Even in the ER given in this example, ERα and ERβ exist in nature, and it is known to have the same sensing ability for female hormones (Non-patent Document 10). However, it should be considered that the homology between the amino acids is only 30% (N-terminal region; NTD) and 53% (ligand binding region; LBD).

  Furthermore, in glucocorticoid (GR), which is the same nuclear receptor as ER used in this example, there are isoforms such as GRαA, GRβA, GRα2, GRβ2, GRAα, GRAβ, GR-P, GR-βB, It must be taken into account that each functions in vivo as the same GRs (Swiss-Prot P04150).

  For example, the target base sequence that is “a polynucleotide comprising a base sequence at least 66% identical to the reference (QUERY) base sequence of the polynucleotide encoding the fusion protein according to the present invention” is 100 nucleotides (base) of the reference base sequence. ) Is a sequence encoding a protein having the same phenotype (amino acid sequence) as the reference base sequence, although it may contain up to 33 mismatches. A polynucleotide comprising a base sequence that is at least 66% identical to the reference base sequence may have, for example, up to 33% of the bases in the reference sequence deleted or substituted with another base. In addition, a number of bases corresponding to up to 33% of all bases in the reference base sequence may be inserted into the reference base sequence. Bases that do not match the reference base sequence in the target base sequence are dispersed individually or in one or more adjacent groups in the 5 ′ or 3 ′ end position of the target base sequence, or in other base sequences. It may be anywhere in the target base sequence.

  The present invention also provides a polynucleotide encoding the luminescent probe according to the present invention. The polynucleotide according to the present invention is a polynucleotide capable of expressing the luminescent probe according to the present invention inside or outside a living cell. The polynucleotide according to the present invention is prepared by, for example, linearly linking cDNA encoding each of the ligand binding protein, the full length of the luminescent enzyme, and the fusion protein in which the recognition protein is linearly arranged. Chimera DNA.

  As used herein, the term “polynucleotide” is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. As used herein, the term “base sequence” is used interchangeably with “nucleic acid sequence” or “nucleotide sequence” and refers to deoxyribonucleotides (abbreviated A, G, C, and T) or ribonucleic acid. Shown as a sequence of nucleotides (C, A, G and U). The “polynucleotide comprising the base sequence shown in SEQ ID NO: 4 or a fragment thereof” means a polynucleotide containing the sequence shown by each deoxynucleotide A, G, C and / or T of SEQ ID NO: 4 or a fragment thereof Is intended.

  The polynucleotide according to the present invention may exist in the form of RNA (for example, mRNA) or in the form of DNA (for example, cDNA or genomic DNA). DNA can be double-stranded or single-stranded. Single stranded DNA or RNA can be the coding strand (also known as the sense strand) or it can be the non-coding strand (also known as the antisense strand).

  In one embodiment, the polynucleotide according to the present invention is preferably a polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 4 to 6 and 15 to 16, or a variant thereof.

  In one embodiment, the variant of the polynucleotide according to the present invention encodes a fusion protein that increases the amount of luminescence when the recognition protein recognizes that the ligand has bound to the ligand binding protein, and the following polynucleotide: Preferably, the polynucleotide is a polynucleotide comprising a nucleotide sequence in which one or several nucleotide sequences of the nucleotide sequences represented by any of SEQ ID NOs: 4 to 6 and 15 to 16 are deleted, substituted or added A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by any of SEQ ID NOs: 4-6 and 15-16; SEQ ID NOs: 4-6 and 15-16 Is at least 66% identical, more preferably at least 80% identical, At least 85% preferred, 90%, 92%, 95%, 96%, 97%, 98%, or a polynucleotide consisting of a nucleotide sequence 99% identical.

  The polynucleotide according to the present invention may include a sequence such as an untranslated region (UTR) sequence or a vector sequence (including an expression vector sequence).

  Although it does not specifically limit as a supply source for acquiring the polynucleotide based on this invention, For example, it is preferable that it is a biological material (for example, various organs, such as a human or a mouse | mouth). As used herein, the term “biological material” intends a biological sample (a tissue sample or a cell sample obtained from an organism).

  The polynucleotide according to the present invention is, for example, subcloned into an expression vector or the like to prepare a vector (plasmid) for expressing a luminescent probe and introduce it into a cell, whereby the polynucleotide is encoded. A luminescent probe can be expressed in the cell. The polynucleotide according to the present invention may be one in which a promoter sequence for expressing a luminescent probe in a cell is incorporated upstream of a region encoding a fusion protein.

  The fusion protein or polynucleotide according to the present invention can be used as a luminescent probe for visualization imaging of molecules inside and outside cells, as further described herein. More specifically, the fusion protein according to the present invention is configured as a single-chain fusion polypeptide, and when a ligand binds to the ligand-binding protein, its three-dimensional structure changes, and the ligand-binding protein is recognized by the recognition protein. . As a result, when the ligand binding protein interacts with the recognition protein, the full-length luminescent enzyme located between the ligand binding protein and the recognition protein, or the linked full-length luminescent enzyme and fluorescent protein, is deformed. Increase light emission. Thus, according to the fusion protein of the present invention, it is possible to visualize and detect the movement of molecules, signal transmission and the like inside and outside the cell based on the change in the amount of luminescence.

  Therefore, by using the fusion protein according to the present invention, it is possible to detect the physiological activity, concentration, etc. of the target-specific ligand with high accuracy. For example, screening of in vivo risk factors such as carcinogens, pharmaceutical field Useful for screening new drug candidate substances, quantitative evaluation of pharmacological effects of anticancer drugs (development of new drugs), determination of environmental pollution / toxic substances, diagnostic kits, measurement of differentiation / physiological effects in new all-purpose cells (iPS), etc. is there.

  That is, an object of the present invention is to provide a luminescent probe and a polynucleotide encoding the same, and resides in a fusion protein preparation method, a polynucleotide preparation method and the like specifically described in the present specification. is not. Therefore, it should be noted that luminescent probes obtained by methods other than the above methods and polynucleotides encoding the same also belong to the technical scope of the present invention.

[2. vector〕
The present invention also provides a vector for expressing the fusion protein according to the present invention in an organism or a cell. When the vector according to the present invention is used, the polynucleotide encoding the fusion protein according to the present invention can be introduced into an organism or cell, and the fusion protein according to the present invention can be expressed in the organism or cell.

  The vector according to the present invention is characterized by containing the above-described polynucleotide according to the present invention. The vector according to the present invention is not particularly limited as long as it contains the polynucleotide according to the present invention. Examples thereof include a recombinant expression vector into which the polynucleotide cDNA according to the present invention is inserted. A method for producing a recombinant expression vector includes, but is not limited to, a method using a plasmid, phage, cosmid or the like.

  The specific type of the expression vector is not particularly limited, and a vector that can be expressed in the host used for expression can be appropriately selected. For example, a known eukaryotic or prokaryotic cell vector can be used. In addition, the fusion protein according to the present invention can be selectively expressed in a specific organ by using a known vector that specifically resides in a certain organ.

  In addition, the vector according to the present invention may incorporate a known tissue-specific promoter sequence in order to control the expression of the polynucleotide according to the present invention (for example, expression in a specific tissue in an individual organism). In addition, a known specific stimulus-specific promoter sequence may be incorporated in order to control the expression of the polynucleotide according to the present invention depending on the presence or absence of a specific ligand, stimulus or the like.

  Moreover, the vector according to the present invention may be one in which the polynucleotide according to the present invention is connected to a retroviral vector such as pMX. By introducing this into a packaging cell PLATE having a high-titer virus-producing ability, it is possible to produce a retrovirus that infects animals. By infecting each organ of the animal with this virus, real-time imaging of the second messenger characteristics in each organ can be performed.

  That is, the vector according to the present invention appropriately selects a promoter sequence in order to reliably express the fusion protein encoded by the polynucleotide according to the present invention, and combines this and the polynucleotide according to the present invention into various plasmids and the like. It may be incorporated.

  The vector according to the present invention can be introduced into a host by a known method such as a microinjection method, an electroporation method, or a transfection method using a lipid reagent (for example, TransIT, manufactured by Miras). The host is not particularly limited, and various conventionally known cells, living organisms, etc. can be suitably used.

  In addition, it is not limited to the case where the target fusion protein according to the present invention is always synthesized, and synthesis may be induced by adding IPTG. In addition, the vector may include a sequence to which a tag sequence such as a histidine tag, HA tag (SEQ ID NO: 10), Myc tag, Flag or the like is added to the N-terminus or C-terminus of the protein to be synthesized.

  In addition, the vector according to the present invention is not intended to express the fusion protein, for example, by connecting a control sequence capable of controlling the expression upstream of the sequence encoding the fusion protein, thereby using the presence or absence of expression of the fusion protein as an index. It can be used for further bioanalysis.

  The vector according to the present invention preferably contains at least one selectable marker. Such markers include, for eukaryotic cell cultures, dihydrofolate reductase or drug resistance genes such as neomycin, zeocin, geneticin, blasticidin S, hygromycin B and the like. Examples of culture in E. coli and other bacteria include drug resistance genes such as kanamycin, zeocin, actinomycin D, cefotaxime, streptomycin, carbenicillin, puromycin, tetracycline, and ampicillin. By using the above selectable marker, it can be confirmed whether or not the polynucleotide according to the present invention has been introduced into the host, and whether or not it is reliably expressed in the host.

  When the vector according to the present invention is used, the fusion protein according to the present invention can be expressed in the organism or cell by introduction into the organism or cell. In addition, the fusion protein can be extracted from cells into which the vector according to the present invention has been introduced and purified or unpurified (non-cell system) and used as a sensor component of a diagnostic kit. For example, by measuring steroid hormones or chemical substances as an example of various physiological substances in a biological sample, the physiological activity of each ligand can be rapidly measured.

  Furthermore, if the vector according to the present invention is used in a cell-free protein synthesis system, the fusion protein according to the present invention can be synthesized.

  The vector according to the present invention only needs to contain at least a polynucleotide encoding the fusion protein according to the present invention. That is, it should be noted that vectors other than the expression vector are also included in the technical scope of the present invention.

[3. Transformant)
The present invention also provides a transformant capable of expressing the fusion protein according to the present invention. The transformant according to the present invention includes the polynucleotide according to the present invention and a vector containing the polynucleotide. In the present specification, the “transformant” is intended to include not only a cell, tissue or organ but also an individual organism. The transformant according to the present invention is not particularly limited as long as it contains the polynucleotide according to the present invention and a vector containing the polynucleotide.

  The transformant according to the present invention can be obtained by introducing the polynucleotide according to the present invention or a recombinant vector containing the polynucleotide into an organism or cell so that the fusion protein according to the present invention can be expressed. . The host organism and cells into which the recombinant vector is introduced are not particularly limited. For example, eukaryotic cultured cells such as COS-7, MCF-7, HeLa, and CHO, and somatic cell-derived new all-purpose cells (iPS). Prokaryotic cells, plant cells, higher animals and the like can be used.

  A method for introducing a polynucleotide according to the present invention or a vector containing the polynucleotide into a host, that is, a transformation method is not particularly limited, and conventional methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, etc. A known method can be suitably used. Further, the transformant according to the present invention may be a transient transformant that is transiently expressed in a state where the polynucleotide according to the present invention is not integrated into the host genome. It may be a stable transformant in which such a polynucleotide is integrated into the host genome and continues to be stably expressed.

  Since the fusion protein according to the present invention can be expressed by using the transformant according to the present invention, the transformant according to the present invention is stimulated with a ligand, and the change in the amount of luminescence from the transformant is measured. By doing so, the nature, concentration, activity level, etc. of the ligand can be evaluated.

[4. Target substance detection method and kit]
The present invention further provides a target substance detection method and kit for detecting a target substance in a test sample. When the target substance is referred to as a ligand, the target substance detection method and kit are referred to as a ligand detection method and kit. The target substance detection method according to the present invention includes a step of allowing a test sample and the fusion protein according to the present invention to coexist. In this method, the enzyme substrate contained in the fusion protein to be contacted with the test sample may be appropriately contacted. Thereby, the target substance in the test sample is detected using the light emission amount from the fusion protein according to the present invention as an index. In the present specification, the “test sample” may be the ligand itself or a biological sample containing the ligand. The test sample can also be an intracellular substance that is secondarily produced in cells in which the fusion protein according to the present invention is expressed.

  In the above step, when the amount of luminescence from the fusion protein according to the present invention increases in the presence and absence of the test sample, the ligand that binds to the ligand binding protein of the fusion protein according to the present invention in the test sample Can be detected. According to the ligand detection method of the present invention, it is possible to analyze the nature of the ligand, the degree of ligand activity, the concentration of the ligand, etc., as shown in the examples described later.

  According to the target substance detection method of the present invention, an unknown antagonist with respect to a known agonist can also be detected. For example, a test sample containing a candidate substance is brought into contact with the fusion protein according to the present invention, and then a known agonist is brought into contact therewith, and the amount of luminescence from the fusion protein according to the present invention is measured. If the candidate substance in the test sample has an antagonistic action, since the agonist binding site of the primary binding protein is occupied by the candidate substance, the subsequently introduced agonist cannot bind to the primary binding protein.

  At this time, if a primary binding protein whose steric structure does not increase depending on the binding of the antagonist is used, the amount of luminescence from the fusion protein does not increase due to the binding of the antagonist. Low. On the other hand, if a primary binding protein whose steric structure does not change depending on the binding of an agonist is used, an unknown agonist can also be detected using a known antagonist.

  In the target substance detection method according to the present invention, typically, a living cell transformed with an expression vector containing a polynucleotide encoding the fusion protein according to the present invention is contacted with a test sample to stimulate. The increase in the amount of luminescence from the fusion protein according to the present invention before and after stimulation is measured. The method for bringing the fusion protein according to the present invention into contact with the test sample is not particularly limited. For example, the test sample is added to the culture medium of the living cells in which the fusion protein according to the present invention is expressed, and the test sample is subjected to endocytosis. May be brought into contact with cells. When the test sample is an intracellular substance, the fusion protein according to the present invention may be contacted by expressing it in the cell. In addition, even in the absence of living cells, for example, if the culture solution containing the fusion protein according to the present invention secreted into the medium or the purified fusion protein according to the present invention is used, the substrates of the respective luminescent enzymes exist simultaneously. In-vitro measurement is also possible.

In the target substance detection method according to the present invention, when a luminescent probe expressed in a living cell is used, the following method can be applied, but is not limited to this method.
(I) A plasmid having a polynucleotide encoding the luminescent probe according to the present invention is introduced into a living cell on a 24-well plate and further cultured for 16 hours.
(Ii) Stimulate the cells with the test sample for 20 minutes.
(Iii) In the presence of the substrate, the change in the amount of luminescence in the cells is measured with a luminometer.

In the target substance detection method according to the present invention, when a non-cell system experiment (in vitro) using the luminescent probe according to the present invention is performed, the following method can be applied, but is not limited to this method.
(I) A luminescent probe purified by a known method is hung on the end of a cruciform paper piece having a diameter of 1.2 cm and dried.
(Ii) A substrate solution containing a stimulating substance (ligand) is instilled on the other end of the cross-shaped paper piece, and a change in the amount of luminescence is immediately measured with a luminescence scanner (for example, RAS-3000; FujiFilm).

  A target substance detection kit for detecting a target substance according to the present invention includes the fusion protein according to the present invention. The kit may further include a substrate for a luminescent enzyme contained in the fusion protein according to the present invention.

  Here, the fusion protein according to the present invention provided in the kit can be purified after being expressed in large quantities in eukaryotic cells and prokaryotic cells. For example, when mammalian cells are used, a large amount of fusion that can be used for analysis without a purification step by connecting a suitable signal sequence known in the art (for example, MGVKVLFALICIAVAEA) and secreting it in the medium in large quantities. A protein-containing culture supernatant is obtained. Further, by attaching a purification tag (for example, His Tag), the fusion protein can be easily purified in large quantities.

  Similarly to the kit provided with the fusion protein thus obtained, a kit provided with a transformant expressing the fusion protein according to the present invention can be used for detection of a target substance in a test sample. In addition, for example, a kit combining a paper strip having a purified luminescent probe and a substrate of a luminescent enzyme is similar to a kit having a transformant expressing a luminescent probe, and external stimuli and secondary phenomena caused thereby. It is possible to make a kit configuration capable of qualitative quantitative analysis of the second messenger. Similarly, a kit comprising a polynucleotide according to the present invention and a cell transformed with the polynucleotide, and a kit comprising a vector according to the present invention and a cell transformed with the vector, are similarly tested samples. It can be used for the detection of target substances in it.

[5. Probe production method and kit]
The present invention further provides a probe production method and kit according to the present invention. The method for producing a probe according to the present invention is characterized by including a step of transforming cells using the polynucleotide according to the present invention or the vector according to the present invention. In this method, the probe according to the present invention is produced by transforming a cell using the polynucleotide according to the present invention or the vector according to the present invention and expressing the probe according to the present invention in the cell. The probe expressed in the cell can be recovered from the cell and purified by a known method.

  Specifically, after transforming and culturing cells using the polynucleotide or vector according to the present invention, conventional methods (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, etc.) from the culture etc. , Ion exchange chromatography, etc.), the probe according to the present invention can be recovered and purified. Further, when a purification tag is added to a polynucleotide expressed in a cell, it can be recovered more easily.

  The probe production kit according to the present invention comprises the polynucleotide according to the present invention or the vector according to the present invention. The kit may further include cells transformed with the polynucleotide according to the present invention or the vector according to the present invention. Furthermore, you may provide the substrate of the enzyme contained in the fusion protein which concerns on this invention. The probe production kit according to the present invention may include a polynucleotide encoding the fusion protein according to the present invention.

  The present invention provides a fusion protein used as a novel single-molecule probe that uses the full length of an enzyme in order to overcome the problems in the conventional methods. For example, a novel bioluminescence visualization means using a luminescent enzyme as an enzyme is provided. The technique of the present invention also provides a basic principle using a bioluminescent enzyme.

  The present invention uses a unique bioanalytical method that uses the twisting phenomenon of molecules related to enzymes as an index. Since the present invention uses the full length of the enzyme, high-luminance bioluminescence imaging is possible when a luminescent enzyme is used as the enzyme. In addition, the fusion protein according to the present invention is highly stable because degradation, misfolding and the like hardly occur. In addition, since one intramolecular protein interaction is used as an index, it can be applied to biological analysis in a non-cellular system. Therefore, the fusion protein according to the present invention provides a highly accurate and highly stable analysis means and provides a simple technique that can be applied to both cell and non-cell systems. The analysis principle in the present invention can be widely applied to screening of new drug candidate substances, evaluation of toxic substances and environmental pollutants.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  Note that the terms in this specification are basically based on IUPAC-IUB Commission on Biochemical Nomenclature, or based on the meaning of terms commonly used in this field. Various techniques used for carrying out the invention can be easily and surely implemented by those skilled in the art based on known literatures and the like, except for the techniques that clearly indicate the source. For example, genetic engineering and molecular biology techniques are described in J. Sambrook, EF Fritsch & T. Maniatis, "Molecular Cloning: A Laboratory Manual (2nd edition)", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995); Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995; Japan Biochemical Society, “Sequel Biochemistry Experiment Course 1, Genetic Research Method II”, Tokyo Chemical Doujin (1986); Japan Biochemical Society, “Shinsei Chemistry” Laboratory Lecture 2, Nucleic Acid III (Recombinant DNA Technology), Tokyo Kagaku Dojin (1992); R. Wu ed., "Methods in Enzymology", Vol. 68 (Recombinant DNA), Academic Press, New York (1980); R. Wu et al. Ed., "Methods in Enzymology", Vol. 100 (Recombinant DNA, Part B) & 101 (Recombinant DNA, Part C), Academic Press, New York (1983); R. Wu et al. ed., "Methods in Enzymolog y ", Vol. 153 (Recombinant DNA, Part D), 154 (Recombinant DNA, Part E) & 155 (Recombinant DNA, Part F), Academic Press, New York (1987) It can be carried out by the methods described in the literatures cited in the literature, methods substantially the same as these, or modified methods thereof. Various proteins, peptides, or DNAs encoding them used in the present invention can be obtained from existing databases (URL: http://www.ncbi.nlm.nih.gov/ etc.).

[Example 1: Design of strain probe inserted with luminescent enzyme]
First, with respect to cDNAs encoding RLuc8 (1-234 amino acids), GLuc (17-185 amino acids), CBLuc (1-515 amino acids), and FLuc (1-550 amino acids), ER LBD is located at the N-terminal side. The Src SH2 domain cDNA was ligated to the C-terminal side thereof to produce a chimeric DNA construct in which these cDNAs were linearly linked (SEQ ID NOs: 4 to 6, 15). The constructed construct was subcloned into pcDNA 3.1 (+) (Invitrogen).

  FIG. 6 is a diagram showing an example of a plasmid structure for producing a strain probe. FIG. 6 shows a DNA construct configuration of only a plasmid (pERS) for expressing a strain probe (ERS probe) inserted with RLuc8, but a plasmid (EGS probe) for expressing a strain probe (EGS probe) inserted with GLuc. pEGS), a plasmid (pECS) that expresses a strain probe (ECS probe) inserted with CBLuc, or a plasmid (pEFS) that expresses a strain probe (EFS probe) inserted with FLuc was constructed in the same manner.

These plasmids were then transformed into COS-7 cells (ATCC). Specifically, COS-7 cells were added to a 24-well plate containing Dulbecco's modified Eagle medium (DMEM; manufactured by Sigma) supplemented with 10% steroid-free fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S). The cells were cultured in a cell incubator (manufactured by Sanyo) at 37 ° C. in the presence of 5% CO ₂ . TransOS-LT1 (manufactured by Miras) was used as an introduction reagent, and CERS-7 cells were transiently transformed with pERS, pEGS, pECS and pEFS (0.2 μg per well). The transformed cells were incubated in the presence of 5% CO ₂ at 16 hours.

  Using the obtained transformant, the luminescence intensity in the presence and absence of 4-hydroxy tamoxifen (OHT) used as a ligand was observed.

First, COS-7 cells into which the prepared pERS, pEGS, pECS or pEFS had been introduced were stimulated for 20 minutes in the presence or absence of OHT ^10-6 M (final concentration), and then coelenterazine or D-luciferin as a substrate. The emission intensity was measured using (D-luciferin).

  Measurement of luminescence intensity was performed as follows. For COS-7 cells into which pERS had been introduced, the stimulated cells were lysed in 200 μL of cell lysis buffer containing the substrate, and the entire lysate was transferred to a quartz cell (45 × 12.5 × 7.5 mm). Using a spectrophotometer (FP-750; Jasco), light emission in the range of 400 nm to 700 nm was measured (n = 2). Moreover, the luminescence intensity was measured using a luminometer (Minilamat LB9506) for COS-7 cells into which pERS, pEGS, pECS or pEFS had been introduced. The results are shown in FIGS.

  FIG. 7 is a graph showing an emission spectrum of a transformant introduced with pERS. As shown in FIG. 7, in the ERS probe inserted with RLuc8, it was shown that the luminescence intensity greatly increased depending on the OHT stimulation. Further, the emission spectrum showed the maximum value in the vicinity of 475 nm.

  FIG. 8 is a graph showing the ratio of the luminescence intensity when the background (-OHT) is 1 for the transformants expressing each strain probe. As shown in FIG. 8, it was shown that the emission intensity increased depending on the OHT stimulus in each strain probe. Thus, from the result that the emission intensity from the strain probe is increased by OHT stimulation, the turnover rate of the strain probe itself is improved or the quantum yield of the strain probe is improved by receiving an external stimulus. It was suggested that

  In addition, as shown in FIG. 8, in the ERS probe inserted with RLuc8, the luminescence intensity increased particularly compared with the strain probe inserted with other luminescent enzymes. From this result, it was suggested that the change in the amount of luminescence of the strain probe due to external stimulation strongly depends on the molecular structure of the inserted luminescent enzyme. That is, it is suggested that whether or not the inserted luminescent enzyme has a molecular structure in which the structure of the enzyme active site is easily deformed by external molecular tension exerts an influence on the change in the amount of luminescence of the strain probe. It was. As described above, since RLuc8 has a substantially spherical shape, molecular tension from the outside is efficiently transmitted to the enzyme active site, and the structure is easily deformed (strain is easily applied). Therefore, it can be said that the result of the increase in the luminescence intensity by the external stimulus was larger than that of other luminescent enzymes. In addition, GLuc has a very small molecular weight (18-185 amino acids; 18 kD) compared to other luminescent enzymes, so it is difficult for external molecular tension to be transmitted to the enzyme body, and the structure of its active site is not easily deformed. Compared to RLuc8, it can be said that the amount of increase in emission intensity due to external stimulation was small.

  Moreover, the luminescence intensity from the transformant expressing the ERS probe was very high. It can be said that this is because the ERS probe includes the entire length of RLuc8.

  Thus, it was shown that the interaction between various proteins can be measured with high sensitivity and high luminescence intensity by using the strain probe according to the present invention.

[Example 2: Substrate selectivity of strain probe]
Next, the substrate selectivity of the luminescent enzyme RLuc8 in the ERS probe was examined. After COS-7 cells were sufficiently cultured in a 12-well plate, pERS was introduced into COS-7 cells using a lipid reagent and further cultured for 16 hours. Thereafter, the cells were stimulated with a solvent or OHT10 ⁻⁶ M (final concentration) for 20 minutes, and the luminescence intensity in the presence or absence (background) of various substrates was determined in the same manner as in Example 1 using a spectrophotometer ( FP-750; Jasco). As substrates, coelenterazine (coel), coelenterazine cp (cp), coelenterazine hcp (hcp), coelenterazine fcp (fcp), coelenterazine ip (ip), and coelenterazine 400A (400A) shown in FIG. 9 were used. In FIG. 9, a portion corresponding to an important side chain is shown surrounded by a broken line. The results are shown in FIGS.

  FIG. 10 is a graph showing emission spectra of COS7 cells into which pERS has been introduced in the presence of various substrates. FIG. 11 is a graph showing the ratio of the peak intensity of the emission spectrum of COS7 cells introduced with pERS in the presence of various substrates, with a background of 1. As shown in FIGS. 10 and 11, the luminescence intensity increased in an OHT-dependent manner regardless of which substrate was used, but the rate of increase varied depending on the type of substrate. The emission intensity was highest when coelenterazine was used (not shown). On the other hand, the signal ratio to the background was the highest when coelenterazine fcp was used.

  From these results, it was suggested that the substrate can be properly used according to the purpose of the experiment. In addition, it was suggested that by comparing the side chains of each coelenterazine, important side chains in the substrate of the strain probe of the present invention can be identified.

  Further, the emission intensity when RLuc8 was used alone or when ERS was used was measured using the same method. Only cDNA encoding the full length of RLuc8 was subcloned into pcDNA 3.1 (+) (Invitrogen) to construct a plasmid, and RLuc8 was expressed alone by transforming the plasmid into COS-7 cells. A transformant was obtained. The results are shown in FIG.

  FIG. 12 is a graph showing an emission spectrum of a transformant expressing RLuc8 alone or a transformant expressing ERS. As shown in FIG. 12, in the transformant expressing RLuc8 alone in the coexistence of coelenterazine fcp, no change in OHT-dependent emission spectrum was observed, but in the transformant expressing ERS, A dependent emission spectrum change was observed.

[Example 3: Ligand dependence, detection limit and ligand selectivity of ERS]
First, the ligand dependence and detection limit of ERS were examined. After introducing (transfecting) pERS into COS-7 cells cultured in a 12-well plate, the cells were further cultured for 16 hours. The cells were then stimulated with various concentrations of OHT or testosterone (T) for 20 minutes and then lysate was made by introducing lysate reagent for 15 minutes. About the produced lysate, the change of luminescence intensity was measured using a luminometer (Minilamat LB9506) in the presence of coelenterazine as a substrate. The results are shown in FIG.

FIG. 13 is a diagram showing a ligand response curve in a transformant expressing ERS. As shown in FIG. 13, the luminescence intensity in the transformant expressing ERS began to increase by OHT stimulation at a concentration of about 10 ⁻¹⁰ , and reached the maximum value in the vicinity of the concentration of 10 ⁻⁷ . On the other hand, no response was observed in response to testosterone stimulation due to an increase in luminescence intensity. This showed that ERS specifically responded to OHT.

  Next, the effect of phosphorylation of ER LBD on ERS and the effect of ERS single-molecule structure change on the change in luminescence intensity were examined. A plasmid expressing the EmRS probe, in which a mutation was introduced into Y537, which is the phosphorylation site of ER LBD, was constructed, and a transformant in which this was introduced into COS-7 cells was obtained. Further, as shown in FIG. 15, a plasmid (pER) having a DNA construct structure for expressing a probe (ER probe) in which only ER LBD is connected to the N-terminal side of RLuc8, and only the SH2 domain on the C-terminal side of RLuc8 Plasmids (pRS) having a DNA construct structure for expressing a probe (RS probe) connected to each other were constructed. And the transformant (ER + RS) which introduce | transduced the constructed pER and pRS simultaneously into the COS-7 cell was obtained.

  The resulting transformant was transformed into OHT or 0. Stimulation was performed with 1% DMSO (control), and the luminescence intensity was measured and compared in the same manner as described above. The results are shown in FIG. FIG. 14 is a graph showing relative luminescence intensity when transforms expressing ERS, EmRS, and ER and RS, respectively, were set to 1.

  As shown in FIG. 14, the transformant expressing ERS exhibited a luminescence intensity about 3 times that of the control with respect to the OHT stimulation. On the other hand, in the transformant expressing EmRS, the increase in luminescence intensity against OHT stimulation was about 1.7 times that in the control. This suggests that in EmRS in which the phosphorylation site is mutated, the interaction between the ER LBD and the SH2 domain due to the external stimulus is unlikely to occur, and the change in the amount of luminescence depending on the external stimulus is difficult to be seen.

  Further, in the transformant (ER + RS) co-expressing ER and RS, no increase in luminescence intensity was observed with respect to OHT stimulation. Here, the structure of ER and RS is typically shown in FIG. Compared with the ERS shown in FIG. 5, in the transformant (ER + RS) co-expressing ER and RS shown in FIG. 16, even though the SH2 domain interacts with ER LBD by OHT stimulation, RLuc8 No external molecular tension is applied. Therefore, since RLuc8 was not deformed, that is, twisted, the enzyme active site was not deformed, and the luminescence intensity did not increase.

Next, ligand selectivity in ERS was examined. As shown in FIG. 17, OHT, E ₂ (17β-estradiol), and Genistein, a partial agonist, as well as dehydrotestosterone (DHT), testosterone (T), and cortisol (cortisol). ) Was used as the ligand. After stimulating a transformant expressing ERS with each ligand at a concentration of 10 ⁻⁶ M, the luminescence intensity was measured by the same method as above, and the luminescence when a solvent was used instead of the ligand (vehicle) Compared with strength. The results are shown in FIG.

FIG. 18 is a graph showing the luminescence intensity of the transformant expressing ERS by various ligand responses. Transformants that expressed ERS showed increased approximately equal luminous intensity relative OHT and E _2. Further, with respect to genistein, although lower than OHT and E _2, showed an increase in emission intensity. On the other hand, DHT, testosterone, and cortisol did not show an increase in luminescence intensity.

  From this result, it was shown that ERS has a specific ligand sensitivity to the female hormone receptor. It was also shown that ERS can respond with high sensitivity to ligands.

[Example 4: Design of strain probe inserted with a combination of a luminescent enzyme and a fluorescent protein]
First, cDNA encoding the fluorescent protein EYFP was ligated to the N-terminal side of cDNA encoding RLuc8 (1-234 amino acids). The ER LBD cDNA is further ligated to the N-terminal side of the ligated cDNA and the Src SH2 domain cDNA is ligated to the C-terminal side to produce a chimeric DNA construct in which this cDNA is linearly linked. (SEQ ID NO: 16). The constructed construct was subcloned into pcDNA 3.1 (+) (Invitrogen).

  FIG. 19 is a diagram showing an example of a plasmid structure for preparing a strain probe. FIG. 19 shows a DNA construct structure of a plasmid (pEERS) for expressing a strain probe (EERS probe) in which RLuc8 is linked to the C-terminal side of EYFP.

  Next, this plasmid was transformed into COS-7 cells (ATCC) using the same method as shown in Example 1, and the resulting transformant was used in the presence or absence of OHT. The emission intensity in each of the following was measured. The results are shown in FIG.

  FIG. 20 is a graph showing an emission spectrum of a transformant introduced with pEERS. As shown in FIG. 20, it was shown that the luminescence intensity increased depending on the OHT stimulation in the EERS probe into which EYFP and RLuc8 were inserted.

  From this result, the following was shown. As shown in FIG. 21, in the absence of ligand (OHT), the ligand binding protein (ER LBD) and the recognition protein (Src SH2) do not interact, and the bioluminescence resonance energy transfer between EYFP and RLuc8 The emission intensity was weak because it was weak. On the other hand, when stimulated by a ligand, the ligand binds to the ligand binding protein, and the recognition protein recognizes this binding, whereby the ligand binding protein and the recognition protein interact. The interaction between the ligand binding protein and the recognition protein inevitably causes a certain structural deformation (strain) in the luminescent enzyme (RLuc8) and the fluorescent protein (EYFP) existing between them, and the interaction between the luminescent enzyme and the fluorescent protein. As the bioluminescence resonance energy transfer between them was affected, the enzyme activity from the luminescent probe, that is, the total amount of luminescence increased. That is, it was shown that the EERS probe causes luminescence resonance energy transfer inside.

  Therefore, it is shown that the luminescent probe according to the present invention can change the ligand-dependent enzyme activity even when two protein molecules including the enzyme are inserted between the ligand-binding protein and the recognition protein. It was done.

  Since the fusion protein according to the present invention has high luminescence intensity and is easy to design, it can be applied to a wide range of fields such as bio industry, pharmaceutical industry, food industry and the like. In particular, it can be suitably used in the diagnostic medical field, nanobio field, pharmacological action evaluation field, environmental analysis field, and the like.

Claims (21)

A fusion protein for detecting a target substance,
A primary binding protein having a binding site to which the target substance binds;
A secondary binding protein that recognizes that the target substance is bound to the primary binding protein;
An active enzyme or a precursor thereof located between the primary binding protein and the secondary binding protein;
A fusion protein, which increases enzyme activity when the secondary binding protein recognizes that the target substance is bound to the primary binding protein.   The fusion protein according to claim 1, wherein the enzyme has a substantially spherical shape. The enzyme is a luminescent enzyme;
The fusion protein according to claim 1 or 2, wherein when the secondary binding protein recognizes that the target substance has bound to the primary binding protein, the amount of luminescence is increased.   The fusion protein according to claim 3, further comprising a fluorescent protein located between the primary binding protein and the secondary binding protein.   The fusion protein according to claim 3 or 4, wherein the luminescent enzyme is selected from the group consisting of Renilla luciferase, Gaussia luciferase, Crick beetle luciferase, and Firefly luciferase.   The primary binding protein is selected from the group consisting of a nuclear receptor, a cytokine receptor, a protein kinase, a second messenger recognition protein, and a transcription factor. Fusion protein.   5. The primary binding protein is a ligand binding domain of an estrogen receptor, the secondary binding protein is an SH2 domain of an Src protein, and the luminescent enzyme is a Renilla luciferase. Fusion protein. A first linker that links the primary binding protein and the enzyme; and a second linker that links the secondary binding protein and the enzyme;
The fusion protein according to any one of claims 1 to 7, wherein each of the first linker and the second linker has an amino acid sequence of 5 amino acids or less.   The fusion protein according to claim 8, wherein the first linker is composed of an amino acid sequence represented by Gly-Ser, and the second linker is composed of an amino acid sequence represented by Gly-Thr. A fusion protein comprising the amino acid sequence shown in any one of SEQ ID NOs: 1-3 and 13-14; or one or several amino acid sequences shown in any one of SEQ ID NOs: 1-3, 13-14 A fusion protein comprising an amino acid sequence in which the amino acid is deleted, substituted or added, and the enzyme activity increases when the secondary binding protein recognizes that the target substance has bound to the primary binding protein.   A polynucleotide encoding the fusion protein according to any one of claims 1 to 10. A polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 4-6 and 15-16;
It consists of a base sequence in which one or several base sequences shown in any of SEQ ID NOs: 4 to 6 and 15 to 16 are deleted, substituted or added, and the target substance is bound to the primary binding protein. A polynucleotide consisting of a base sequence encoding a fusion protein that increases enzyme activity when a secondary binding protein recognizes
Hybridization under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence shown in any one of SEQ ID NOs: 4-6 and 15-16 indicates that the target substance has bound to the primary binding protein. A polynucleotide comprising a base sequence encoding a fusion protein that increases enzyme activity when the next binding protein is recognized; or at least 66% of the base sequence shown in any of SEQ ID NOs: 4-6 and 15-16 A polynucleotide comprising a base sequence that encodes a fusion protein that is the same and encodes a fusion protein that increases enzyme activity when a secondary binding protein recognizes that a target substance has bound to the primary binding protein.   A vector comprising the polynucleotide according to claim 11 or 12.   A transformant comprising the polynucleotide according to claim 11 or 12.   A transformant comprising the vector according to claim 13. A method for detecting a target substance in a test sample, comprising:
A method comprising contacting the test sample with the fusion protein according to any one of claims 1 to 10.   A kit for detecting a target substance in a test sample, comprising the fusion protein according to any one of claims 1 to 10.   A method for producing a probe, comprising a step of transforming a cell using the polynucleotide according to claim 11 or 12.   A probe production kit comprising the polynucleotide according to claim 11.   A method for producing a probe comprising the step of transforming a cell using the vector according to claim 13.   A probe production kit comprising the vector according to claim 13.