JP6146797B2 - HER2 protein detection probe, method for producing HER2 protein detection probe, and method for detecting HER2 protein - Google Patents

Description

  The present invention relates to HER2 protein detection technology, and more particularly to a novel fusion protein, a HER2 protein detection probe using the fusion protein, a method for producing a HER2 protein detection probe, and a method for detecting HER2 protein.

  HER2 is a receptor tyrosine kinase that is involved in the development and maintenance of the heart and nerves, and is also involved in the regulation of cell proliferation and differentiation in other cells, and is a glycoprotein of about 185 kDa formed from 1234 residues. Since HER2 is overexpressed in many cancer cells, it is known as a cancer marker. In order to detect HER2, which is a cancer marker, a molecule in which a labeled compound is bound to an antibody against HER2 is used. In order to bind the labeled compound to the antibody, a chemical bonding method is usually used. On the other hand, the Z domain of protein A is known as a molecule that specifically binds to HER2 (see, for example, Non-Patent Documents 1-3). This is smaller in size than an antibody and can be produced by microorganisms such as Escherichia coli. It is known as Affibody (registered trademark), and a hybrid molecule chemically bonded to a labeled molecule has been produced.

  However, a molecule obtained by chemically binding a labeled molecule and an antibody may decrease the binding force of the antibody or the performance of the labeled molecule. In particular, when the labeling molecule is an enzyme, the performance of the enzyme is inevitably lowered. In addition, when a labeled molecule and an antibody are chemically bound, a chemical reaction for binding and a purification operation to remove an extra single molecule that has not been bound after the chemical reaction are required, which is a labor to prepare. Takes a lot. Furthermore, in order to produce antibodies, it is generally necessary to use higher organisms such as rabbits, mice, and goats, and it is inevitable that they are expensive, and experimental animals must be used.

  On the other hand, there is a technique for site-specific enzyme modification of proteins using transglutaminase (TGase), which is an enzyme having an activity of binding side chains of amino acids lysine (Lys) and glutamine (Gln) ( For example, see Patent Document 1 and Non-Patent Document 4).

JP 2008-54658 A

Karin Nord, Elin Gunneriusson, Jenny Ringdahl, Stefan Stahl, Mathias Uhlen, Pre-Ake Nygren, Binding protein s selected from combinatorial libraries of an alpha-helical bacterial receptor domain, Nat. Biotechnol., 15, 772-777 (1997) Anna Orlova, Mikaela Magnusson, Tove LJEriksson, Martin Nilsson, Barbro Larsson, Ingmarie Hoiden-Guthenberg, Charles Widstrom, Jorgen Carlsson, Vladirmir Tolrmachev, Stefan Stahl, Fredrik Y. Nilsson, Tumor Imaging Using a Picomolar Affinity HERecule Affinity HERec Res 2006; 66: (8) .April 15, 2006 Ilya Lyakhov, Rafal Zielinski, Monika Kuban, Gabriela Kramer-Marek, Robert Fisher, 0leg Chertov, Lakshman Bindu, Jacek capala, HER2- and EGFR-Specific Affiprobes: Novel Recombinant Oprical Probes for Cell Imaging, ChemBioChem 2010, 11, 345-350 M. Kitaoka, Y. Tsuruda, Y Tanaka, M. Goto, M. Mitsumori, K. Hayashi, Y. Hiraishi, K. Miyawaki, S. Noji, N. Kamiya, Transglutaminase-mediated synthesis of a novel DNA- (enzyme ) n probe for highly sensitive DNA detection, Chem. Eur. J., 19, 5387-5392 (2011)

  An object of the present invention is to provide a fusion protein that can detect HER2 protein with good sensitivity and can be applied to production of a HER2 protein detection probe and the like.

  Another object of the present invention is to provide a HER2 protein detection probe capable of detecting HER2 protein with good sensitivity, a method for producing a HER2 protein detection probe, and a method for detecting HER2 protein.

The present invention also comprises a polymer carrier having a plurality of glutamine (Gln) residues and a plurality of fusion proteins having a lysine (Lys) residue bound thereto, wherein the plurality of glutamine (Gln) residues are The polymer carrier having a group is a nucleic acid in which a plurality of nucleoside triphosphate derivatives of the following formula (5) are introduced, and the fusion protein is obtained by fusing a protein A mutant capable of binding to the HER2 protein and a labeling enzyme. It is a HER2 protein detection probe having a tag containing a lysine (Lys) residue.
(In formula (5), X and Y are each independently an ethenylene group, — (C ₂ H ₄ O) _n — or — (C ₃ H ₆ O) _n — (where n = 2, 4). , 8, 12, 24) which may be substituted with an alkylene group having 1 to 48 carbon atoms or an alkenylene group having 2 to 48 carbon atoms, and Z is a dinitrophenyl group or L-3,4-dihydroxy group. An alkyl group having 1 to 48 carbon atoms, an alkoxy group having 1 to 48 carbon atoms, an aryl group having 6 to 48 carbon atoms, an aryloxy group having 6 to 48 carbon atoms, and 7 carbon atoms, which may be substituted with a phenyl group An arylalkyl group having ˜48 or an arylalkyloxy group having 7 to 48 carbon atoms, and Y and Z may be independently substituted at least one with an amino acid other than Lys, and B is a hydrogen atom or hydroxyl to the group It is .m is 0 or 1.)

Further, the present invention uses a transglutaminase (TGase), a polymer carrier having a plurality of glutamine (Gln) residues, combines multiple fusion proteins with lysine (Lys) residue, wherein the plurality of glutamine The polymer carrier having a (Gln) residue is a nucleic acid into which a plurality of nucleoside triphosphate derivatives of the following formula (5) are introduced, and the fusion protein includes a protein A mutant capable of binding to HER2 protein and a labeling enzyme Is a method for producing a HER2 protein detection probe having a tag containing a lysine (Lys) residue.
(In formula (5), X and Y are each independently an ethenylene group, — (C ₂ H ₄ O) _n — or — (C ₃ H ₆ O) _n — (where n = 2, 4). , 8, 12, 24) which may be substituted with an alkylene group having 1 to 48 carbon atoms or an alkenylene group having 2 to 48 carbon atoms, and Z is a dinitrophenyl group or L-3,4-dihydroxy group. An alkyl group having 1 to 48 carbon atoms, an alkoxy group having 1 to 48 carbon atoms, an aryl group having 6 to 48 carbon atoms, an aryloxy group having 6 to 48 carbon atoms, and 7 carbon atoms, which may be substituted with a phenyl group An arylalkyl group having ˜48 or an arylalkyloxy group having 7 to 48 carbon atoms, and Y and Z may be independently substituted at least one with an amino acid other than Lys, and B is a hydrogen atom or hydroxyl to the group It is .m is 0 or 1.)

  Further, the present invention is a method for detecting a HER2 protein, wherein the HER2 protein detection probe is bound to a HER2 protein present in an object, and the bound HER2 protein detection probe is bound by the labeling enzyme. This is a method for detecting HER2 protein to be detected.

  In the present invention, a fusion protein that can detect a HER2 protein with good sensitivity by fusing a protein A mutant capable of binding to the HER2 protein and a labeling enzyme, and is applicable to the production of a HER2 protein detection probe, etc. Can be provided.

  Moreover, in this invention, the HER2 protein detection probe which can detect HER2 protein with favorable sensitivity, the manufacturing method of a HER2 protein detection probe, and the detection method of HER2 protein can be provided by using the fusion protein.

Schematic diagram showing an example of a method for obtaining a (ZZ-AP) _n -DNA probe from a fusion protein (ZZ-AP) and a Z-QG-introduced DNA ((Z-QG) _n -DNA) by a reaction using MTG. It is. It is a figure which shows an example of the synthesis | combining method of Z-QG-dUTP. It is a figure which shows the information of pUCminusMCS-ZZ-AP plasmid. It is a figure which shows the structure of pET22b (+)-ZZ-AP. It is a figure which shows construction | assembly of pET22b (+)-Z-AP. It is a figure which shows construction of pET22b (+)-Z-GS-AP. It is a figure which shows construction | assembly of pET22b (+)-ZZ-GS-AP. It is a figure which shows the result of SDS-PAGE of the fusion protein obtained in the Example. It is a figure which shows the enzyme activity evaluation of various AP in an Example. In an Example, it is a figure which shows the example which detected the HER2 positive cell specifically, after making a fluorescent substance couple | bond with the tag containing a lysine with respect to the obtained fusion protein. It is a figure which shows the result of Western Blot in HER2 specific binding strength evaluation of the fusion protein obtained in the Example. It is a figure which shows the result of the agarose gel electrophoresis of (Z-GS-AP) _n -DNA obtained in the Example. It is a figure which shows the result of the anion exchange chromatogram of (Z-GS-AP) _n -DNA obtained in the Example (310bp). It is a figure which shows the result of the anion exchange chromatogram of (Z-GS-AP) _n -DNA obtained in the Example (960 bp). It is a figure which shows the amount of Z-GS-AP labels of (Z-GS-AP) _n -DNA obtained in the Example (310bp). It is a figure which shows the amount of Z-GS-AP labels of (Z-GS-AP) _n -DNA obtained in the Example (960 bp). It is a figure which shows the result of ELISA regarding the effect of signal amplification by the increase in the enzyme number of (Z-GS-AP) _n -DNA obtained in the Example. It is a diagram illustrating a multivalent effect regarding the ELISA results obtained _{(Z-GS-AP) n} -DNA of _{Z 342} in the embodiment.

  Embodiments of the present invention will be described below. This embodiment is an example for carrying out the present invention, and the present invention is not limited to this embodiment.

The present inventors examined the production of a novel fusion protein and a novel high-sensitivity HER2 protein detection probe using the fusion protein. As an affinity protein, for example, Affibody (registered trademark) selected using a phage display method from a library of Staphylococcal protein A-derived Z domains was focused. Affibody (registered trademark) is characterized by being capable of large-scale expression using a microorganism as a host because it has a very small size of about 6 kDa as a characteristic not found in antibodies and does not require post-translational modification. In 2004, Anti-HER2 Affibody was first reported by Wikman et al., And in 2006, the process of affinity maturation was found by Orlova et al., And a higher affinity Z _{HER2: 342} (Z ₃₄₂ ) was found. It was shown that affinity increases when incorporated.

  The inventors of the present invention studied expression of a fusion protein in which a protein A mutant capable of binding to HER2 protein and a labeling enzyme were fused by a genetic engineering technique, and obtained a novel fusion protein. In addition, as a technique for site-specific enzyme modification, attention was paid to site-specific protein modification ability of transglutaminase (TGase) such as microorganism-derived transglutaminase (MTG). TGase is an enzyme that catalyzes an acyl transfer reaction. For example, TGase includes a γ-carboxyamide group of a specific Gln residue (Q) in a protein, an ε-amino group of a Lys residue (K), and various primary amines. An enzyme that catalyzes a covalent bond. During the expression of the fusion protein, a TGase recognition sequence containing a Lys residue (K) or a Gln residue (Q) that can be recognized by TGase as a tag is added as a tag to obtain a fusion protein to which the tag is attached. On the other hand, a DNA into which at least one, preferably a plurality of Gln residues (Q) or Lys residues (K) that can be recognized by TGase is introduced using a PCR method or the like. These are cross-linked by a TGase catalytic reaction to create a HER2 protein detection probe in which at least one protein A mutant capable of binding to the HER2 protein and preferably a plurality of labeling enzymes are introduced.

For example, as shown in FIG. 1, a fusion protein (Z ₃₄₂ ) obtained by fusing a dimer of Z ₃₄₂ (ZZ) capable of specifically binding to HER2 and an alkaline phosphatase (BAP) derived from Escherichia coli by genetic engineering techniques. ) ₂ were expressed as -bap, this time, by applying the MTG recognition sequence K-tag (MRHKGS) the C-terminal side of _{BAP, (Z 342) 2 -BAP} -CK (ZZ-AP) novel fusion such as Get protein. On the other hand, DNA ((Z-QG) _n -DNA), n = 1 or more) into which at least one, preferably a plurality of Z-QGs, which are MTG recognition substrates, has been prepared is prepared using PCR. These By crosslinking by MTG _catalysis, one 1 BAP at least dimers and (ZZ) of _{Z 342,} preferably to create a plurality introduced HER2 protein detection probes _{((ZZ-AP) n -DNA} ) .

By producing such a complex, for example, by increasing the number of labeled enzymes introduced into one molecule of the probe, it is expected that the detection will be highly sensitive by signal amplification. Further, by introducing a dimer of Z ₃₄₂ as the antigen recognition unit in probe (ZZ), the increase in apparent affinity for antigen by multivalent effect of Z ₃₄₂ is expected. Furthermore, by utilizing the characteristics of DNA, it is possible to impart water solubility by a negative charge, and to strictly design a structure. Moreover, since there are only 13 mutations in the Affibody library, it is expected to become an innovative platform for not only HER2 but also any protein detection technology.

<Fusion protein>
The fusion protein according to the embodiment of the present invention is a fusion protein of an affinity molecule that can bind to the HER2 protein and a labeling enzyme, and is a fusion protein in which a protein A mutant that can bind to the HER2 protein and a labeling enzyme are fused. . The fusion protein according to this embodiment can be produced by a microorganism or the like. Using this fusion protein, the HER2 protein can be specifically detected.

  The fusion protein according to this embodiment preferably has a tag capable of site-specific enzyme modification, such as a tag capable of site-specific enzyme modification by TGase, such as a glutamine (Gln) residue or lysine (Lys). It is more preferred to have a tag with residues). A hybrid molecule of a Z domain, which is a protein A mutant known to specifically bind to the HER2 protein, and a labeling enzyme is prepared as a fusion protein. Thereby, a hybrid of an affinity molecule for HER2 and a labeling enzyme can be produced without using a chemical bonding method.

  Regardless of the chemical binding method, obtaining the fusion protein in which the labeling enzyme and the affinity molecule are fused hardly reduces the performance of the affinity molecule. In addition, since the fusion protein has a tag capable of site-specific enzyme modification, it is possible to label a fluorescent substance or the like without substantially reducing the performance of the affinity molecule. Furthermore, it is possible to improve the detection ability and performance of the HER2 protein by using the tag and linking a plurality of fusion proteins to the polymer carrier.

  A protein A variant capable of binding to the HER2 protein, which is an affinity molecule, is known as Affibody (registered trademark). Affibody (registered trademark) is a protein derived from the B domain in the immunoglobulin binding region of Staphylococcal protein A. The B domain is a 58-residue peptide (approximately 6 kDa) that is relatively short and has no Cys residues, and is formed from three α-helices. The folding speed is one of the fastest proteins reported, and in addition has high water solubility and heat resistance. A recombinant having a mutation introduced into this B domain in order to increase chemical resistance is referred to as a Z domain (Zwt, pI 5.16, Mw = 6640). The Z domain by mutagenesis maintains affinity for the Fc region of the antibody, while lacking weak affinity for the Fab region. It is widely used in the field of biotechnology because of its resistance to the Z domain and its ability to specifically bind to a wide variety of immunoglobulin species, protein A, and derivatives of immunoglobulin binding domains. The beneficial nature of the Z domain is not only a property as an antibody binding species, but also an excellent advantage in a wider variety of applications.

  The labeling enzyme is not particularly limited as long as it has a property capable of performing detection using a color development reaction or the like. For example, alkaline phosphatase (AP), glutathione-S-transferase (GST), luciferase, peroxidase and the like can be mentioned. Of these, alkaline phosphatase or peroxidase is preferable from the viewpoint of high catalytic activity and stability. From the viewpoint that a peptide tag can be easily introduced, a protein that can be produced by genetic engineering is preferred. It is preferable to use bacterial-derived alkaline phosphatase as the labeling enzyme so that the fusion protein can be produced in E. coli.

  As a fusion protein in which a protein A mutant capable of binding to the HER2 protein and a labeling enzyme are fused, two Z domains (Z) are linked in series, and AZ-AP fused with alkaline phosphatase (AP), one Z In consideration of the effect on the expression efficiency, etc., in which Z-AP is fused with alkaline phosphatase in the domain, a molecule in which a linker is introduced (ZZ-linker in order to provide a space between ZZ and AP and between Z and AP. -AP, Z-linker-AP) and the like.

  Examples of the linker include GGGGS linker and GGGSGSGGGGS linker.

  The fusion protein according to the embodiment of the present invention can be used for separation, purification and the like in addition to the specific detection of HER2 protein, the production of a HER2 protein detection probe described later, and the like.

  The fusion protein according to the embodiment of the present invention is a fusion of a protein A mutant capable of binding to the HER2 protein and a labeling enzyme, but the affinity molecule is not limited to the protein A mutant capable of binding to the HER2 protein. A single chain antibody, a camel antibody, or the like may be used.

<HER2 protein detection probe and production method thereof>
The HER2 protein detection probe according to the present embodiment has a HER2 protein having a tag capable of site-specific enzyme modification containing a lysine (Lys) residue on a polymer carrier having at least one glutamine (Gln) residue. At least one fusion protein in which a protein A mutant capable of binding to a marker enzyme and a labeling enzyme are fused. Alternatively, the HER2 protein detection probe according to the present embodiment has a tag capable of site-specific enzyme modification containing a glutamine (Gln) residue on a polymer carrier having at least one lysine (Lys) residue. At least one fusion protein obtained by fusing a protein A variant capable of binding to the HER2 protein and a labeling enzyme is bound to the HER2 protein.

  Moreover, the method for producing a HER2 protein detection probe according to the present embodiment uses a transglutaminase (TGase) and a site containing a lysine (Lys) residue on a polymer carrier having at least one glutamine (Gln) residue. This is a method of binding at least one fusion protein having a tag enzyme and a protein A variant capable of binding to HER2 protein having a tag capable of specific enzyme modification. Alternatively, in the method for producing a HER2 protein detection probe according to this embodiment, a site containing a glutamine (Gln) residue on a polymer carrier having at least one lysine (Lys) residue using transglutaminase (TGase). This is a method of binding at least one fusion protein having a tag enzyme and a protein A variant capable of binding to HER2 protein having a tag capable of specific enzyme modification.

  As a technique for covalently introducing the fusion protein into a polymer carrier, attention was paid to the site-specific protein modification ability of transglutaminase (TGase) such as microorganism-derived transglutaminase (MTG). TGase is an enzyme that catalyzes an acyl transfer reaction. For example, TGase includes a γ-carboxyamide group of a specific Gln residue (Q) in a protein, an ε-amino group of a Lys residue (K), and various primary amines. An enzyme that catalyzes a covalent bond. Using this TGase, a HER2 protein detection probe in which the fusion protein is introduced into a polymer carrier is created. Since MTG and the like have high substrate recognition properties, the reaction site of the polymer carrier and the reaction tag portion of the fusion protein can be selectively cross-linked, and can be introduced without substantially reducing the activity of the labeling enzyme.

Specifically, for example, as shown in FIG. 1, by genetic engineering techniques, fusing dimer of Z ₃₄₂ with the ability to specifically bind to HER2 (ZZ) and E. coli alkaline phosphatase (BAP) The fusion protein (Z ₃₄₂ ) ₂ -BAP was expressed, and at that time, the MTG recognition sequence K-tag (MRHKGS) was added to the C-terminal side of BAP, and (Z ₃₄₂ ) ₂ -BAP-CK (ZZ-AP A novel fusion protein such as On the other hand, for example, as shown in FIG. 2, Z-QG is a nucleotide derivative in which Z-QG having Gln (MTG recognition Gln) recognized by TGase is bound to deoxyuridine triphosphate (dUTP). -Synthesize dUTP. DNA using Z-QG-dUTP and at least one, preferably a plurality of Z-QGs, which are MTG recognition substrates, using the PCR method ((Z-QG) _n -DNA), n = 1 or more) To prepare. These By crosslinking by MTG _catalysis, one 1 BAP at least dimers and (ZZ) of _{Z 342,} preferably to create a plurality introduced HER2 protein detection probes _{((ZZ-AP) n -DNA} ) .

In FIG. 1, the Gln residue in (Z-QG) _n -DNA and the Lys residue in the fusion protein may be reversed. That is, when expressed as a fusion protein (Z ₃₄₂ ) ₂ -BAP, a tag containing an MTG recognition Gln residue is added to the C-terminal side of BAP to obtain a fusion protein. On the other hand, DNA into which at least one, preferably a plurality of MTG recognition Lys residues are introduced is prepared. These By crosslinking by MTG _catalysis, one 1 BAP at least dimers and (ZZ) of _{Z 342,} preferably to create a plurality introduced HER2 protein detection probes _{((ZZ-AP) n -DNA} ) .

  In this way, it is possible to suppress the influence on the recognition site of the HER2 protein and the active site of the labeling enzyme, and to introduce the fusion protein containing the labeling moiety into the polymer carrier in a site-specific manner.

  Examples of the polymer carrier include nucleic acids such as DNA, PNA and RNA, and synthetic polymers such as acrylic acid polymer, methacrylic acid polymer and polyacrylamide. There is no particular limitation on the sequence and length of the nucleic acid. When the polymer carrier is DNA, it is possible to impart water solubility due to negative charges and to strictly design the structure of the structure using the characteristics of DNA.

  When the polymer carrier is a nucleic acid, a TGase-recognized Gln residue or Lys residue using a nucleoside triphosphate derivative having a glutamine (Gln) residue or lysine (Lys) residue that can be recognized by TGase such as MTG A DNA into which at least one, preferably a plurality of DNAs are introduced may be prepared. Examples of the nucleoside triphosphate derivative having a glutamine (Gln) residue or a lysine (Lys) residue include a uridine triphosphate (uridine triphosphate: UTP) derivative having a glutamine (Gln) residue or a lysine (Lys) residue. , Adenosine triphosphate (ATP) derivative, guanosine triphosphate (GTP) derivative, cytidine triphosphate (CTP) derivative, deoxyuridine triphosphate (deoxyuridine TP) derivative A deoxyadenosine triphosphate (dATP) derivative, Oxy guanosine triphosphate (deoxyguanosine triphosphate: dGTP) derivatives, deoxycytidine triphosphate (deoxycytidine triphosphate: dCTP) such derivatives. In the nucleoside triphosphate derivative according to the present embodiment, the glutamine (Gln) residue or lysine (Lys) residue is bound to, for example, a uracil, adenine, guanine, or cytosine moiety directly or through a substituent. .

  These nucleoside triphosphate derivatives can be obtained from UTP, ATP, GTP, CTP, dUTP, dATP, dGTP, dCTP or their various derivatives.

  These nucleoside triphosphate derivatives also include uridine, uridine monophosphate (UMP) and diphosphate (UDP), adenosine, adenosine monophosphate (AMP) and diphosphate (ADP), guanosine, guanosine. Monophosphate (GMP) and diphosphate (GDP), cytidine, cytidine monophosphate (CMP) and diphosphate (CDP), deoxyuridine, deoxyuridine monophosphate (dUMP) and diphosphate ( dUDP), deoxyadenosine, deoxyadenosine monophosphate (dAMP) and diphosphate (dADP), deoxyguanosine, deoxyguanosine monophosphate (dGMP) and diphosphate (dGDP), deoxycytidine, deoxycytidine mono Phosphate (dCMP) and diphosphate (dCDP) and their It may be obtained from the seed derivatives.

  For example, phosphorylation of uridine, adenosine, guanosine, cytidine, deoxyuridine, deoxyadenosine, deoxyguanosine, deoxycytidine phosphorylase, etc. (for example, Journal of Biotechnology, 85 (9), p397-399 (2007), Journal of Bioscience and Bioengineering, 87 (6), p. 732-738 (1999), etc.) or phosphorylation with phosphorus oxychloride in the presence of a proton sponge (for example, Tetrahedron Letters, 29 (36), p. 4525- 4528 (1988) and the like) and the triphosphates can be obtained.

The nucleoside triphosphate derivative according to this embodiment is, for example, a uridine triphosphate derivative represented by the following formula (1) and having a Gln residue or a Lys residue that can be recognized by TGase.

(In formula (1), A represents a substituent having a glutamine (Gln) residue or a lysine (Lys) residue, and B represents a hydrogen atom or a hydroxyl group.)

The nucleoside triphosphate derivative according to the present embodiment is, for example, an adenosine triphosphate derivative represented by the following formula (2) and having a Gln residue or a Lys residue that can be recognized by TGase.

(In Formula (2), at least one of A ₁ and A ₂ represents a substituent having a glutamine (Gln) residue or a lysine (Lys) residue, the rest represents a hydrogen atom, and B represents a hydrogen atom or a hydroxyl group) Represents a group.)

The nucleoside triphosphate derivative according to this embodiment is, for example, a cytidine triphosphate derivative represented by the following formula (3) and having a Gln residue or a Lys residue that can be recognized by TGase.

(In Formula (3), A represents a substituent having a glutamine (Gln) residue or a lysine (Lys) residue, and B represents a hydrogen atom or a hydroxyl group.)

The nucleoside triphosphate derivative according to this embodiment is, for example, a guanosine triphosphate derivative represented by the following formula (4) and having a Gln residue or a Lys residue that can be recognized by TGase.

(In Formula (4), A represents a substituent having a glutamine (Gln) residue or a lysine (Lys) residue, and B represents a hydrogen atom or a hydroxyl group.)

  The substituent having a glutamine (Gln) residue or lysine (Lys) residue represented by A is not particularly limited, and for example, a linear chain having a glutamine (Gln) residue or a lysine (Lys) residue. And substituents including a branched or cyclic saturated or unsaturated alkyl group, aminoalkyl group, aryl group, or heteroaryl group, and may be determined in consideration of easiness of synthesis.

The nucleoside triphosphate derivative according to the present embodiment is preferably, for example, a TGase substrate-modified nucleotide derivative having a Gln residue or a Lys residue that is represented by the following formula (5) and can be recognized by TGase.

(In Formula (5), X and Y each independently represent a divalent linking group, Z represents a substituent, B represents a hydrogen atom or a hydroxyl group, and m is 0 or 1. )

The divalent linking groups represented by X and Y are each independently an alkylene group having 1 to 48 carbon atoms such as a methylene group, an ethylene group, a propylene group, or a butylene group, an ethenylene group, a propenylene group, or a butenylene group. C2-C48 alkenylene group etc. are mentioned. Among these, X and Y are each independently preferably an alkylene group having 1 to 48 carbon atoms, an alkenylene group having 2 to 48 carbon atoms, or an alkoxy group having 1 to 48 carbon atoms, and X is an ethenylene group, More preferably, Y is a methylene group. X and Y are further an ethenylene group, an oxyalkylene group, such as — (C ₂ H ₄ O) _n — or — (C ₃ H ₆ O) _n — (n is a repeating number, and n = 2, 4, 8, 12 , 24) may be substituted with a group or the like. The Y, for _{example, - (C 2 H 4 O} ) n -C 2 H 4 - and the like.

  Examples of the substituent represented by Z include an alkyl group having 1 to 48 carbon atoms such as a methyl group, an ethyl group and a propyl group, an alkoxy group having 1 to 48 carbon atoms such as a methoxy group, an ethoxy group and a propoxy group, and a phenyl group. , An aryl group having 6 to 48 carbon atoms such as a naphthyl group, an aryloxy group having 6 to 48 carbon atoms such as a phenyloxy group, an arylalkyl group having 7 to 48 carbon atoms such as a benzyl group, and a carbon number such as a benzyloxy group Examples include 7 to 48 arylalkyloxy groups. Among these, Z is an alkyl group having 1 to 48 carbon atoms, an alkoxy group having 1 to 48 carbon atoms, an aryl group having 6 to 48 carbon atoms, an aryloxy group having 6 to 48 carbon atoms, and a 7 to 48 carbon atom. It is preferably an arylalkyl group or an arylalkyloxy group having 7 to 48 carbon atoms, and Z is more preferably a benzyloxy group. Z may be further substituted with a dinitrophenyl group, an L-3,4-dihydroxyphenyl group, or the like. In combination with the substitution represented by Y described above, at least one of Y and Z may be independently substituted with an amino acid other than Lys.

  By appropriately selecting X and Y, the structure of the linker site for linking Z-QG and UTP can be optimized. For example, by introducing a flexible linker site, access to enzymes and the like can be improved. In addition, by appropriately selecting Y and Z, the substrate peptide sequence can be optimized, and for example, the affinity of an enzyme or the like can be improved.

  When microorganism-derived TGase (MTG) is used, the Gln residue that can be recognized by MTG is preferably present as benzyloxycarbonyl-L-glutamylglycine (Z-QG). Z-QG is preferable because it has a smaller molecular size than digoxigenin (DIG) or the like. In the nucleoside triphosphate derivative represented by the formula (5), a nucleotide derivative in which X is an ethenylene group, Y is a methylene group, Z is a benzyloxy group, B is a hydrogen atom, and m = 0, Z-QG is added to dUTP. The coupled nucleotide derivative Z-QG-dUTP. In addition, it is preferable to select a nucleoside triphosphate derivative that does not coexist with a Gln residue recognizable by TGase and a Lys residue or a primary amine. In the case of coexistence, TGase may cause self-crosslinking, which may adversely affect the yield of the target protein-nucleic acid complex.

  Moreover, as a good substrate of microorganism-derived TGase, LLQG (SEQ ID NO: 1), LAQG (SEQ ID NO: 2), LGQG (SEQ ID NO: 3), PLAQSH (SEQ ID NO: 4), FERQHMDS (SEQ ID NO: 5), Alternatively, a peptide consisting of the amino acid sequence of TEQKLISEEDL (SEQ ID NO: 6), or an amino acid of GLGQGGG (SEQ ID NO: 7), GGGQGGG (SEQ ID NO: 8), GVGQGGGG (SEQ ID NO: 9), or GGLQGGGG (SEQ ID NO: 10) Peptides consisting of sequences are known. Further, as a good substrate for TGase derived from guinea pig river, a peptide consisting of an amino acid sequence of benzyloxycarbonyl-L-glutamylphenylalanine (Z-QF) or EAQQIVM (SEQ ID NO: 11), or GGGQLGGG (SEQ ID NO: 12) , GGGQVGG (SEQ ID NO: 13), GGGQRGG (SEQ ID NO: 14), GQQQLG (SEQ ID NO: 15), PNPQLPF (SEQ ID NO: 16) or PKPQQFM (SEQ ID NO: 17) is known. Yes. A Gln residue that can be recognized by TGase may exist as such a peptide depending on the type of TGase used.

  In addition, since the N-terminal amino group can be a substrate for TGase in the substrate peptide whose N-terminus is glycine (G), a by-product due to self-crosslinking can occur. Therefore, for a substrate peptide whose N-terminus is glycine (G), it can be protected from becoming a substrate for TGase by replacing the hydrogen of the N-terminal amino group with an appropriate group, and desired ligation can be performed. You should be able to do it. In the present specification, “N-terminal protection” is used in this sense except in special cases. It is known that the reactivity varies depending on the N-terminal protection means. Specifically, with regard to mammalian-derived TGase, protection by G-terminal Q-acetylation of GQQQLG (ie, Ac-GQQQLG) and N-terminal amino acid as DOPA (L-3,4-dihydroxyphenylalanine) (ie, DOPA-GQQQLG) It is known that the reactivity is improved. Examples of such protection can also be used in this embodiment.

  A method for preparing Z-QG-dUTP is shown in FIG. This is an example of a method for preparing the nucleoside triphosphate derivative according to the present embodiment, and is not limited thereto.

  First, an N-hydroxysuccinimide (NHS) group or the like is introduced into benzyloxycarbonyl-L-glutamylglycine (Z-QG) and activated (NHS-ized Z-QG). And Z-QG-dUTP can be obtained by condensing dUTP which has the substituent which aminated the ends, such as aminoallyl UTP, and NHS-ized Z-QG.

  In addition to the above-described method for active esterification of the C-terminal carboxyl group, as a method for introducing a peptide having a Gln residue that can be recognized by TGase into dUTP, a functional group highly reactive with an amino group is added to the peptide. There is a way to introduce. For example, if an aldehyded, acylazidated, sulfonyl chlorided, epoxidized, isocyanated, or isothiocyanated substrate peptide can be prepared, it can be reacted with aminated dUTP to have a Gln residue that TGase can recognize. dUTP can be prepared. However, these reactive functional groups need to be introduced into a portion of the substrate peptide that does not affect TGase recognition. Therefore, as described above, the method of activating the C-terminal carboxyl group separated from the Gln residue is one of the most excellent for this purpose.

  Z-QG-dUTP can be purified by high performance liquid chromatography (HPLC), gel filtration chromatography (GPC), or the like. Further, Z-QG-dUTP can be identified by MALDI TOF-MS, NMR, IR, and the like. Moreover, confirmation and a yield of a product can be calculated | required by HPLC.

  In the HER2 protein detection probe, the ratio n between the polymer carrier and the fusion protein is not particularly limited as long as it is 1 or more, and can be appropriately adjusted. However, n is preferably 2 or more, and the larger n is, The detection sensitivity is preferably increased. However, if n is too large, the efficiency of coupling with HER2 may be reduced.

In addition, for example, a plurality of HER2 protein detection probes having different labeling enzymes can be prepared by the following method.
(1) Change the origin of TGase.
(2) Change the substrate specificity of TGase.

  In the method (1), for example, UTP modified with a different substrate peptide may be prepared according to the type of TGase used.

  In the method (2), for example, an amino acid mutation may be introduced into TGase by protein engineering to change the substrate specificity. For example, MTG is prepared in E. coli (see, for example, Christian K. Marx, Thomas C. Hertel and Markus Pietzsch, Enzyme and Microbiology Technology, Volume 40, Issue 6, 15p50, 15 May 2007, 50 May 15p. pro-transglutaminase from Streptomyces mobaraensis in Escherichia coli "), and MTG with improved thermostability can be obtained by making a mutant library (see, for example, Christian K. Marx, Thomas P, and Thomas P., Thomas P. , Journal of Biotechnology, Volume 136, Issues 3-4,10 September 2008, p.156-162, "Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants" reference).

  In the present specification, “binding by TGase” means that, except for a special case, the resulting linking part is formed by forming an ε (γ-glutamyl) lysine bond between a Lys residue and a Gln residue. It is configured.

  In the present embodiment, the Lys residue that can be recognized by TGase may be a primary amine. In the present specification, the Lys residue is described as an example, but the description also applies to a primary amine except in special cases.

  In contrast to TGase, the substrate serving as the Lys residue donor is considered to have fewer structural restrictions than the substrate serving as the Gln residue donor. Therefore, the labeling enzyme to be modified may originally have a Lys residue that can be recognized by TGase, or a tag that includes a Lys residue that can be recognized by TGase may be added to the enzyme.

  The Lys residue (K) that can be recognized by TGase is a peptide having the amino acid sequence of MKHKGS (SEQ ID NO: 18), MRHKGS (SEQ ID NO: 23), MRRKGS (SEQ ID NO: 24), MHRKGS (SEQ ID NO: 25). May exist as Such tagging with a peptide containing a Lys residue that can be recognized by TGase can be used for the purpose of linking a labeling enzyme to a desired site of the protein, for example, the C-terminus or the N-terminus. Examples of other peptides containing Lys residues recognizable by TGase or amino acid sequences thereof include modified S-peptide (GSGMKETAAARFERAHMGSGS (SEQ ID NO: 19)), MGGSTKHKIPGGGS (SEQ ID NO: 20), N-terminal glycine (N -Terminal GGG, N-terminal GGGGG (SEQ ID NO: 21)), MKHKGGGSGGGSGS (SEQ ID NO: 22) in which the linker site between the N-terminal MKHKGS and the target protein is extended.

  A labeling enzyme to which a peptide containing a Lys residue capable of recognizing TGase at the C-terminus or N-terminus can be prepared as a recombinant protein using genetic engineering techniques. Purification of the recombinant protein in which a TGase substrate peptide tag is introduced at the C-terminus or N-terminus is performed by purifying the peptide tag for purification added to the N-terminus or C-terminus, respectively (for example, (His) 6-tag (hexahistidine tag) (To avoid a decrease in TGase reactivity, it may be designed so that the purification peptide tag is inserted at a terminal different from the terminal where the substrate peptide tag is inserted.) The amino acid sequence can be confirmed by confirming the gene sequence of the plasmid vector encoding the protein with a DNA sequencer, or the substrate peptide introduced at the N-terminus can be directly identified by N-terminus analysis. Confirmation of protein purification can be performed by SDS-PAGE or the like.

  When using a fusion protein or HER2 protein detection probe under conditions of relatively high temperature (for example, 70 ° C. or higher), there is a concern about loss of activity if an enzyme derived from thermophilic bacteria is used. In that case, the enzyme is preferably an alkaline phosphatase (PfuAP) derived from the hyperthermophilic bacterium Pyrococcus furiosus.

  Enzymes derived from hyperthermophilic bacteria are preferred because they are generally known to exhibit high stability against organic solvents and heat (for example, H. Atomi, Current Opinion in Chemical Biology, 9, p. 166). -173 (2005)), and is also preferable in that a large-scale preparation using E. coli as a host can be performed relatively easily. When preparing a thermostable enzyme using Escherichia coli as a host, the cell disruption solution is subjected to high-temperature treatment (for example, incubation at 80 ° C. for 30 minutes) to precipitate most of the E. coli-derived concomitant proteins and facilitate crude purification. It can be carried out.

  Since hyperthermophilic bacteria are microorganisms that can grow in extreme environments where general organisms hardly grow, proteins derived from hyperthermophilic bacteria have very high heat resistance. Furthermore, not only the resistance to heat, but generally the resistance to denaturing agents, organic solvents, pH, etc. is extremely higher than that of enzymes derived from room temperature bacteria, so the use of PfuAP does not cause enzyme inactivation. It is thought that detection can be performed.

  Further, in the fusion protein and HER2 protein detection probe, the enzyme may be an enzyme that is stable against organic solvents and heat. Such highly stable enzymes are screened from nature (for example, Chemistry and Industry, vol. 61 (No. 6), p. 571-575 (2008), Taku Uchiyama, Kentaro Miyazaki, Bioscience and Industry, vol.66 (No.5), p.234-239 (2008), Noriyuki Michihisa, Bioscience and Industry, vol.66 (No.12), p.667-670 (2008)) and protein engineering Techniques for improving stability by techniques (for example, Hiroyasu Kanno, BIO INDUSTRY, vol. 25 (No. 7), p. 16-23 (2008), Kentaro Miyazaki, BIO INDUSTRY, vol. 25 (No. 7), p. .52-58 (2008)). By these techniques, even an enzyme derived from a thermophilic bacterium can be converted into an enzyme having resistance to organic solvents and heat resistance.

  Various types of transglutaminase (TGase) can be used. Currently, TGases are known to be derived from mammals (guinea pigs, humans), invertebrates (insects, horseshoe crabs, sea urchins), plants, fungi, and protists (slime molds). Eight types of isozymes have been found. A preferred example of TGase that can be used in this embodiment is microorganism-derived microorganism-derived transglutaminase (MTG) in terms of stability, ease of handling, and bulk production.

When MTG is used in the present embodiment, a ligation reaction between a fusion protein containing a labeling enzyme having a Lys residue and (Z-QG) _n -DNA is predicted to occur at the MTG active center based on the expected catalytic reaction of MTG. Formation of an acyl-enzyme complex by nucleophilic substitution of a cysteine (Cys) residue with (Z-QG) _n -DNA to Gln, followed by the labeling compound to the acyl-enzyme complex with Lys It is expected to proceed in two stages: MTG elimination by nucleophilic substitution reaction.

In a preferred aspect of this embodiment, the molar concentration ratio of the fusion protein having a Lys residue recognizable by TGase to (Z-QG) _n -DNA having a Gln residue recognizable by TGase is preferably 2 It is above, More preferably, it is 5 or more. In the present specification, the term “concentration ratio” refers to a ratio based on molar concentration unless otherwise specified.

  When performing a ligation reaction using MTG as TGase, in addition to adjusting the molar concentration ratio to an appropriate range as described above, pH 5.5 to 8.0, temperature 4 to 50 ° C. (for example, , Preferably at room temperature (for example, 18 ° C. to 22 ° C.). In this way, a sufficiently high reaction rate can be achieved within 12 hours, preferably within 6 hours, more preferably within 3 hours.

<Method for detecting HER2 protein>
The HER2 protein detection method according to the present embodiment is a method in which the HER2 protein detection probe is bound to the HER2 protein present in the target, and the bound HER2 protein detection probe is detected by a labeling enzyme. .

  The HER2 protein detection method according to the present embodiment can be used for the purpose of qualitative, quantitative, identification, staining, localization investigation, etc. of the HER2 protein.

  It is also possible to make the target other than the HER2 protein by changing the affinity molecule. Examples of affinity molecules for EGFR include ZEGFR: 1907, and affinity molecules for TNF include ZTNFα: 2 and ZTNFα: 185.

  Hereinafter, although an example and a comparative example are given and the present invention is explained more concretely in detail, the present invention is not limited to the following examples.

As protein A mutant-BAP fusion proteins, Z-AP- (His) ₆ -CK-tag and ZZ-AP- (His) ₆ -CK-tag are candidates, and the influence on the expression efficiency is considered. GGGSGSGGGGS was introduced as the linker length introduced between the A mutant and the AP. And when GGGSGSGGGGS linker is introduced, it shall be expressed as Z-GS-AP- (His) ₆ -CK-tag, ZZ-GS-AP- (His) ₆ -CK-tag, and thereafter, The notation of-(His) ₆ -CK-tag is omitted and expressed as ZZ-AP, Z-GS-AP, and ZZ-GS-AP.

<Construction of ZZ-AP expression vector>
[Transformation of ZZ-AP gene sequence to Escherichia coli JM109 for genetic recombination]
“pUCminusMCS-ZZ-AP (5129 bp)” in which the gene of pel B- (Z342) ₂ -BAP-His-CRK (ZZ-AP) (1905 bp) was encoded in the pUCminusMCS plasmid was transformed into Escherichia coli for recombination (Escherichia coil, JM109). Strain) and stored as a glycerol stock at −80 ° C. (FIG. 3).

[Construction of ZZ-AP expression vector by gene recombination]
The resulting glycerol stock of E. coli strain JM109 transformed with pUCminusMCS-ZZ-AP was cultured overnight and then extracted with a plasmid. The obtained pUCminusMCS-ZZ-AP was treated with two types of restriction enzymes, NdeI and HindIII. This was separated by agarose gel electrophoresis, and only the ZZ-AP gene product was purified by gel cutting. Next, pET22b (+) (5493 bp) was similarly treated with Nde I and Hind III, and then dephosphorylated using CIAP. Thereafter, the amplified ZZ-AP gene product was inserted by ligase to obtain “pET22b (+)-ZZ-AP (7278 bp)” (FIG. 4, SEQ ID NO: 26). The prepared pET22b (+)-ZZ-AP was transformed into E. coli strain JM109 and stored at -80 ° C as a glycerol stock.

<Construction of Z-AP expression vector>
[Construction of Z-AP expression vector by gene recombination using inverse PCR method]
In order to remove the second Z ₃₄₂ sequence from the N-terminal side by inverse PCR from pET22b (+)-ZZ-AP (7278 bp). The primer on the side is complementary to the GGGS (linker) + BAP sequence, Rev. The primer on the side was designed to be complementary to the C-terminal side + G (linker) of the _Z342 sequence (Table 1).

  A glycerol stock of Escherichia coli JM109 strain transformed with pET22b (+)-ZZ-AP was cultured overnight and then extracted with a plasmid. From the obtained pET22b (+)-ZZ-AP, inverse PCR of pET22b (+)-ZZ-AP was performed using primer Z-AP For and primer Z-AP Rev (Table 1), and pET22b (+ ) -Z-AP PCR product was obtained. Then, after digesting Original pET22b (+)-ZZ-AP with an enzyme, self-ligation of pET22b (+)-Z-AP was performed using Lyation High kit, and “pET22b (+)-Z-AP (7062 bp) "was prepared (FIG. 5, SEQ ID NO: 27). The prepared pET22b (+)-Z-AP was transformed into E. coli strain JM109 and stored at −80 ° C. as a glycerol stock.

<Construction of Z-GS-AP expression vector>
[Construction of Z-GS-AP expression vector by gene recombination using inverse PCR method]
For p.22b (+)-ZZ-AP (7278 bp), the second Z ₃₄₂ sequence from the N-terminal side was removed by inverse PCR, and an additional GS linker was introduced. The primer on the side is designed to be complementary to the S insert + GGGGS (linker) + BAP sequence, and Rev. Side Primers were designed first _{Z 342} C-terminal + GGGS (linker) sequence + G insertion portion complementary to become so (Table 2).

  From pET22b (+)-ZZ-AP, using primer Z-GS-AP For and primer Z-GS-AP Rev, inverse PCR of pET22b (+)-ZZ-AP was performed, and pET22b (+)-Z -A PCR product of GS-AP was obtained. Then, after digesting the original pET22b (+)-ZZ-AP with an enzyme, the PCR product of pET22b (+)-Z-AP was self-ligated using Lyation High kit, and “pET22b (+)-Z -GS-AP (7080 bp) "was prepared (FIG. 6, SEQ ID NO: 28). The prepared pET22b (+)-Z-GS-AP was transformed into Escherichia coli JM109 strain and stored at −80 ° C. as a glycerol stock.

<Construction of ZZ-GS-AP expression vector>
[Construction of ZZ-GS-AP expression vector by gene recombination using inverse PCR method]
For additional GS linker can be introduced from pET22b (+)-ZZ-AP (7278 bp) by inverse PCR. The primer on the side is designed to be complementary to the SGS insert + GGGGS (linker) + BAP sequence, and Rev. The primer on the side was designed to be complementary to the C-terminal side + GGG insertion part of the second _Z342 sequence (Table 3).

  Using pET22b (+)-ZZ-AP, inverse PCR of pET22b (+)-ZZ-AP is performed using primer ZZ-GS-AP For and primer ZZ-GS-AP Rev, and pET22b (+)-ZZ -A PCR product of GS-AP was obtained. Then, after digesting the original pET22b (+)-ZZ-AP with an enzyme, the PCR product of pET22b (+)-ZZ-AP was self-ligated using Lyation High kit, and “pET22b (+)-ZZ” was obtained. -GS-AP (7296 bp) "was prepared (FIG. 7, SEQ ID NO: 29). The prepared pET22b (+)-ZZ-GS-AP was transformed into E. coli strain JM109 and stored at −80 ° C. as a glycerol stock.

  PCR in each stage was performed using a thermal cycler under the composition and reaction conditions shown in Tables 4 and 5. Various restriction enzyme treatments, phosphorylation, dephosphorylation, and ligation were performed using the buffer solution, reaction temperature, and reaction time recommended in the TaKaRa catalog. Tables 6 and 7 show typical reaction conditions for each. In addition, when transforming various plasmid vectors into E. coli for recombination JM109 and E. coli for expression (Escherichia coli, BL21), 0.30 μL to 0.60 μL of various plasmid vectors are added to each competent cell. The heat treatment was performed by a thermal cycler according to the temperature program shown in Table 8.

<(1) Expression of ZZ-AP>
[Expression of ZZ-AP]
ZZ-AP was expressed using Escherichia coli BL21 strain. First, a plasmid encoding the ZZ-AP gene was transformed into E. coli BL21 strain to obtain a glycerol stock. The transformant was cultured in a large amount in 1 L of LB medium containing Anpicillin (100 mg / L). After about 2 hours, when OD ₆₀₀ = 0.6, IPTG was adjusted to a final concentration of 0.1 mM. ZZ-AP was expressed by culturing at 27 ° C. overnight (SEQ ID NO: 30).

[Recovering and washing bacterial cells]
The obtained bacterial cells were collected by centrifugation and washed twice with sucrose buffer (50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 20% (w / w) sucrose). Then, it was resuspended using Wash buffer (sucrose buffer (pH 7.4), 5 mM MgCl ₂ ). Then, after freezing in liquid nitrogen, it was stored in a deep freezer for a day.

[Ultrasonic disruption of E. coli]
The frozen E. coli solution was thawed and then crushed by sonication. After centrifugation, the resulting cell-free extract was filtered using a 0.45 μm filter and a 0.22 μm filter.

[Protein purification using Ni-NTA column]
Purification was performed with HisTrap HP column (5 mL volume) using His-tag. First, a protein solution was applied to a column equilibrated with Ni-NTA-equilibrated buffer (20 mM Tris-HCl (pH 7.4), 500 mM NaCl, 35 mM imidazole), washed again with equilibrated buffer, Ni-NTA- The target recombinant protein was eluted using an elution buffer (20 mM Tris-HCl (pH 7.4), 500 mM NaCl, 500 mM imidazole). The purified protein solution was subjected to buffer exchange with 10 mM Tris-HCl (pH 8.0) using a PD-10 column.

  In Ni-NTA column purification, it was considered that the degree of purification was insufficient, and therefore purification using a further column was examined. Since the pI of AP is 4.5, it is considered that the AP is usually negatively charged at neutral pH. Therefore, purification was performed using HiTrap DEAE column.

[Protein purification using weak anion exchange column]
The protein solution was applied to a column equilibrated with DEAE-equilibrated buffer (20 mM Tris-HCl (pH 8.0)). At this time, the eluted solution was left unfixed on the DEAE column. After washing with the equilibration buffer again, gradient elution (NaCl concentration: 0M → 0.75M) using DEAE-elution buffer (20 mM Tris-HCl (pH 8.0), 1M NaCl) and slowly changing the NaCl concentration. The target protein was eluted. The protein solution after purification was concentrated by ultrafiltration (30 kDa cut off), and then buffer exchanged to 10 mM Tris-HCl (pH 8.0) using a PD-10 column. Confirmation of expression and purification was performed using SDS-PAGE.

[Concentration calculation by BCA assay]
Proteins have the effect of reducing Cu (II) to Cu (I) under alkaline conditions. Utilizing this, the protein concentration was calculated from the absorption wavelength of 562 nm derived from the complex of bicinchonic acid and Cu (I). Thereafter, unless otherwise stated, the protein concentration is measured using BCA assay.

<(2) Expression of Z-GS-AP and ZZ-GS-AP and evaluation of enzyme activity>
For BAP, a recombinant of Z-GS-AP and ZZ-GS-AP in which one or two _Z342 were inserted by inserting a GS linker (SEQ ID NO: 31, 32), ammonium sulfate fraction, Ni -Purification by NTA column and cation exchange column was performed.

[Expression of Z-GS-AP and ZZ-GS-AP]
Z-GS-AP and ZZ-GS-AP use a transformed glycerol stock of Escherichia coli BL21, and this transformant was cultured in a large amount in 1 L of LB medium containing Anpicillin (100 mg / L). It was. After about 2 hours, when OD ₆₀₀ = 0.6, IPTG was added to a final concentration of 0.1 mM, and cultured overnight at 15 ° C. to express the target protein.

[Recovering and washing bacterial cells]
The obtained bacterial cells were collected by centrifugation and washed twice with TBS. Thereafter, the suspension was resuspended using Wash buffer (1 × TBS (pH 7.4), 5 mM MgCl ₂ , 3 tablets / 30 mL of protease inhibitor cocktail mini). Then, after freezing in liquid nitrogen, it was stored in a deep freezer for a day.

[Ultrasonic disruption of E. coli]
The frozen E. coli solution was thawed and then crushed by sonication. After centrifugation, the resulting cell-free extract was filtered using a 0.45 μm filter and a 0.22 μm filter.

[Rough fractionation of target protein using ammonium sulfate precipitation]
A crude fractionation of the expressed recombinant protein was performed using ammonium sulfate precipitation. First, powdered ammonium sulfate was added little by little so that it might become 40% saturation. After standing for 30 minutes, centrifugation was performed to obtain a supernatant containing the target protein. Similarly, ammonium sulfate was saturated to 60%, the target protein was precipitated, and the concentration operation was performed. This precipitate was suspended with 1 × TBS, and then buffer exchange was performed with 1 × TBS using a PD-10 column.

[Protein purification using Ni-NTA column]
Purification was performed with HisTrap HP column (1 mL volume) using His-tag. First, a protein solution was applied to a column equilibrated with Ni-NTA-equilibrated buffer (20 mM Tris-HCl (pH 7.4), 500 mM NaCl, 35 mM imidazole), washed again with equilibrated buffer, Ni-NTA- The target recombinant protein was eluted using an elution buffer (20 mM Tris-HCl (pH 7.4), 500 mM NaCl, 500 mM imidazole). The protein solution after purification was buffer-exchanged to 50 mM HEPES buffer (pH 7.5) using a PD MidiTrap ™ G-25 column.

[Protein purification using cation exchange column]
Purification was performed by HiTrap SP column (GE Healthcare) using the positive charge of the protein derived from Z. First, the protein solution is passed through a column equilibrated with SP-equilibrated buffer (50 mM HEPES buffer (pH 7.5)), washed again with equilibrated buffer, and then SP-eluted buffer (50 mM HEPES buffer (pH 8.0)). ) 1M NaCl) was used to elute the target protein by gradient elution with a gradual change in NaCl concentration. The purified protein solution was subjected to buffer exchange with 10 mM Tris-HCl (pH 8.0) using a PD MidiTrap ™ G-25 column, and concentrated using a 30 kDa cut off ultrafiltration column.

  The purity of the prepared protein was evaluated by SDS-PAGE. The results are shown in FIG. Although the presence of partially cleaved contaminating proteins was suggested for ZZ-GS-AP, fusion proteins Z-GS-AP and ZZ-GS-AP, which are the desired recombinant proteins, were obtained.

  Moreover, 10 μL of various 1 μM BAP solutions were added to 1 mL of the substrate solution (1 M Tris-HCl (pH 8.0), 1 mM p-Nitro Phenyl phosphate), and after sufficient stirring, changes over time in absorption at 410 nm were measured. . Using the obtained slope as the initial activity, the enzyme activities of various BAPs were compared. The results of measuring the enzyme activities of Z-GS-AP and ZZ-GS-AP are shown in FIG. As can be seen from FIG. 9, even if one Z is introduced into AP by genetic engineering (Z-GS-AP), the enzyme activity is not affected, and the alkaline phosphatase activity is not reduced. When two of these were introduced (ZZ-GS-AP), the enzyme activity slightly decreased. This is probably because the specific activity was measured to be low because impurities were included as can be seen from the electrophoresis results.

<(3) Evaluation of HER2 binding ability by cell-free fluorescence staining of FITC-labeled Z-GS-AP and ZZ-GS-AP>
As a technique for labeling fluorescent groups to Z-GS-AP and ZZ-GS-AP, K introduced in Z-GS-AP and ZZ-GS-AP so as not to affect the function of Z342. A technique for site-specific cross-linking of a fluorescent group using microorganism-derived transglutaminase (MTG) was selected using -tag, and FITC-SSYALQMR in which a fluorescent group was introduced into the selected substrate sequence was used. The prepared FITC-labeled Z-GS-AP and ZZ-GS-AP had FITC label amounts of 2.2 and 3.2, respectively, with respect to the AP dimer. In the immunofluorescence staining experiment of cells, SK-BR-3 cell line (corresponding to HER2 expression level 3+) was used as positive cells, and HeLa cell line (corresponding to HER2 expression level 0) was used as negative cells. 300 μL of FITC-labeled Z-GS-AP and ZZ-GS-AP solutions prepared to 83.3 nM were added dropwise to HeLa cells and SK-BR-3 cells, and allowed to stand at 37 ° C. for 1 hour. . After fixing with formalin and nuclear staining with DAPI, immunofluorescence staining for HER2 was observed using a fluorescence microscope. The results are shown in FIG. Green fluorescence was observed only for HER2-positive SK-BR-3 cells, and it was found that HER2 can be specifically stained for both Z-GS-AP and ZZ-GS-AP.

<(4) Evaluation of HER2-specific binding force by Western Blot of cell-free extracts of Z-GS-AP and ZZ-GS-AP>
Whether Z-GS-AP and ZZ-GS-AP were specifically bound to HER2, and the HER2 detection ability of both were evaluated by Western Blot method. The cell-free extracts of SK-BR-3 cells and HeLa cells were electrophoresed using Blue-Native PAGE (BN-PAGE), which can fractionate proteins in their native state. After transferring the protein from the gel after BN-PAGE to the PVDF membrane by semi-dry blotting, immunoreaction with Z-GS-AP and ZZ-GS-AP prepared to 2.09 nM was performed. In order to suppress endogenous AP activity, staining was performed using a 0.02 mM ECF substrate solution added with 2 mM Levamisole. The results are shown in FIG. Although the experimental conditions were different, a band around 90 kDa that was considered to be HER2 confirmed in an experiment using a commercially available Biotin-Affibody was also confirmed in Z-GS-AP and ZZ-GS-AP. Since no non-specific band was confirmed, it was found that both had specific affinity for HER2. Moreover, the results suggesting that Z-GS-AP has higher detection sensitivity than ZZ-GS-AP were obtained, and in consideration of the degree of purification and specific activity, Z-GS was used as a labeling enzyme in the subsequent experiments. -AP was selected.

<Preparation of (5) (Z-GS-AP) _n -DNA>
[Synthesis and purification of Z-QG-dUTP]
First, in 4 mL of N, N-dimethylformamide (DMF), 100 mM N, N′-diisopropylcarbodiimide (DIC), 100 mM N-hydroxysuccinimide (NHS), and 50 mM Z-QG were obtained at room temperature (the day of preparation was 18 NHS-ized Z-QG (50 mM) was prepared by reacting at about -22 ° C for 20 hours. Meanwhile, 16 μL of 10 mM Tris-HCl (pH 7.5) solution (manufactured by Ambion) containing 50 mM 5- (3-aminoallyl) -dUTP (hereinafter abbreviated as “aminoallyl-dUTP”, manufactured by SIGMA) and 200 mM borate buffer (pH 8) .8) 40 μL and 16 μL of sterilized water were mixed to prepare 80 μL of a 10 mM aminoallyl-dUTP solution. To this solution, 80 μL of the NHS-modified Z-QG solution prepared above was added and reacted at 25 ° C. overnight. After completion of the reaction, the sample was diluted 10-fold with Milli-Q, and HPLC (manufactured by JASCO, high-performance liquid chromatograph pump: TRI ROTAR-V type, variable loop injector: VL-613 type, UV-visible spectroscopic detector : UVIDEC-100-IV type) under the conditions shown in Table 9. The product was identified by MALDI TOF-MS (manufactured by BRUKER DALTONICS, autoflex III). At this time, 3-hydroxypicolinic acid (3-HPA) was used as a matrix.

  After synthesizing Z-QG-dUTP, reverse phase HPLC was performed. As a result, a new peak appeared on the hydrophobic side as compared with aminoallyl-dUTP, and this peak is Z-QG-dUTP. It was speculated. Therefore, when a peak with a retention time of 19.1 minutes was collected and subjected to MALDI TOF-MS analysis, a peak of 841.46 was confirmed, and a result that closely matched the theoretical molecular weight of 842.13 was obtained. The synthesis of -QG-dUTP was shown.

[Preparation of (Z-GS-AP) _n -DNA]
Regarding the dTTP among the four nucleotides constituting the DNA, the “dTTP + Z-QG-dUTP” concentration is kept constant (0.4 mM), and the Z-QG-dUTP concentration ratio is changed to 0, 10, 40, 80% to perform PCR. As a result, (X% Z-QG) _n -DNA (X = 0, 10, 40, 80) in which Z-QG groups were introduced at different ratios was prepared. Using FITC-modified primer, 310 bp (SEQ ID NO: 33) and 960 bp (SEQ ID NO: 34) scaffold DNAs, which were fluorescently labeled at the 5 ′ end, were prepared. As a result of confirming the production of DNA by agarose gel electrophoresis, only 960 bp of (80% Z-QG) _n -DNA was not obtained. This is thought to be due to the increase in the proportion of modified nucleotides that are not the original substrate, resulting in a sequence site that cannot continue the extension reaction.

About 3 μM of Z-GS-AP was added to 20 mM Tris-HCl buffer (pH 8.0) with respect to various prepared (Z-QG) _n -DNA (10 ng / μL, 310 bp: 52.2 nM, 960 bp: 16.9 nM). ) And 0.01 U / mL of MTG (manufactured by Ajinomoto Co., Inc.), and a crosslinking reaction was carried out at 37 ° C. for 1 hour. About 3 μM of Z-GS-AP is set to be equal to the molar concentration of Z-QG groups that are theoretically incorporated into (40% Z-QG) _n -DNA. The progress of the reaction was evaluated by agarose gel electrophoresis. The results are shown in FIG. From this result, it was confirmed that the crosslinking reaction proceeded only in the presence of MTG and the band was shifted to the high molecular weight side, and (Z-GS-AP) _n -DNA could be prepared.

<(6) Purification using various (Z-GS-AP) _n -DNA anion exchange columns and evaluation of the amount of Z-GS-AP label>
(Z-QG) _n -DNA, Z-GS-AP, and MTG have different pI and different surface charges. Using this property, purification was performed using HiTrap Q HP, which is an anion exchange column. Q-elution buffer (20 mM Tris-HCl (pH 7.5), 1M NaCl) was used, and the target (Z-GS-AP) _n -DNA was eluted by applying a salt concentration gradient. As a reference experiment, only Z-GS-AP was applied to the column, and the peak position of Z-GS-AP was confirmed. The chromatograms obtained in the experiment are shown in FIGS. As the Z-QG introduction amount increased, the peak near the retention time of 1100 seconds derived from Z-GS-AP decreased, and it was confirmed that a new peak appeared. In addition, since the new peak moves to the Z-GS-AP side as the amount of Z-QG introduced increases, the amount of Z-GS-AP label increases, and elution occurs by relieving the strong negative charge of DNA. It seems that time has been accelerated.

In order to quantitatively evaluate the number of Z-GS-AP labels on DNA, with respect to (Z-GS-AP) _n -DNA obtained after purification, the fluorescence intensity by FITC modified at the 5 ′ end, and Z-GS From the enzyme activity derived from -AP, the amount of Z-GS-AP label was quantified. The results are shown in FIGS. It was found that the amount of Z-GS-AP label increased, and a maximum of about 40 molecules of Z-GS-AP was labeled at a DNA chain length of 310 bp and a maximum of about 77 molecules at a DNA chain length of 960 bp.

<(7) (Z-GS-AP) _n -DNA signal amplification (DNA equimolar) and examination of multivalent effect (Z-GS-AP equimolar)>
Here, signal amplification by (a) increasing the number enzyme in the probe, was aimed at increasing the apparent affinity by multivalent effect of (b) Z _342. Therefore, it was evaluated by ELISA using HER2-Fc Chimera protein whether the prepared (Z-GS-AP) _n -DNA was a probe having the two functions described above. In order to examine the effect of (a), 100 ng of the HER2 protein was immobilized, and the ELISA was performed with a DNA molar concentration of 0.258 nM. Moreover, in order to examine the effect of (b), a dilution series of HER2 protein 100 ng to 0.025 ng was immobilized, and the Z-GS-AP molar concentration was unified to 1.0 nM, and ELISA was performed. From the result of FIG. 17, it was possible to obtain a result that the signal was amplified in accordance with the amount of Z-QG introduction and the increase in the DNA chain length with respect to the signal amplification effect of (a). Further, FIG. 18 shows only the result of 310 bp (Z-GS-AP) _n -DNA, but it can be seen that the immune reaction proceeds even in a low concentration region with respect to the multivalent effect of (b). -The detection sensitivity was improved about 10 times compared with AP alone. Therefore, the results of both experiments showed the usefulness of the probe of this example.

  As described above, using a new molecular probe that presents a plurality of fusion proteins having a protein A mutant capable of binding to HER2 protein and a labeling enzyme as a basic unit (Z-GS-AP) on DNA, Z-GS is used. -The target antigen could be detected with detection sensitivity about 10 times higher than that of AP alone.

Claims (3)

A plurality of fusion proteins having a lysine (Lys) residue are bound to a polymer carrier having a plurality of glutamine (Gln) residues,
The polymer carrier having a plurality of glutamine (Gln) residues is a nucleic acid into which a plurality of nucleoside triphosphate derivatives of the following formula (5) are introduced,
The HER2 protein detection probe, wherein the fusion protein is a protein A mutant capable of binding to the HER2 protein and a labeling enzyme, and has a tag containing a lysine (Lys) residue.
(In formula (5), X and Y are each independently an ethenylene group, — (C ₂ H ₄ O) _n — or — (C ₃ H ₆ O) _n — (where n = 2, 4). , 8, 12, 24) which may be substituted with an alkylene group having 1 to 48 carbon atoms or an alkenylene group having 2 to 48 carbon atoms, and Z is a dinitrophenyl group or L-3,4-dihydroxy group. An alkyl group having 1 to 48 carbon atoms, an alkoxy group having 1 to 48 carbon atoms, an aryl group having 6 to 48 carbon atoms, an aryloxy group having 6 to 48 carbon atoms, and 7 carbon atoms, which may be substituted with a phenyl group An arylalkyl group having ˜48 or an arylalkyloxy group having 7 to 48 carbon atoms, and Y and Z may be independently substituted at least one with an amino acid other than Lys, and B is a hydrogen atom or hydroxyl to the group It is .m is 0 or 1.) Using transglutaminase (TGase), a plurality of fusion proteins having lysine (Lys) residues are bound to a polymer carrier having a plurality of glutamine (Gln) residues,
The polymer carrier having a plurality of glutamine (Gln) residues is a nucleic acid into which a plurality of nucleoside triphosphate derivatives of the following formula (5) are introduced,
The method for producing a HER2 protein detection probe, wherein the fusion protein is obtained by fusing a protein A mutant capable of binding to the HER2 protein and a labeling enzyme, and has a tag containing a lysine (Lys) residue. .
(In formula (5), X and Y are each independently an ethenylene group, — (C ₂ H ₄ O) _n — or — (C ₃ H ₆ O) _n — (where n = 2, 4). , 8, 12, 24) which may be substituted with an alkylene group having 1 to 48 carbon atoms or an alkenylene group having 2 to 48 carbon atoms, and Z is a dinitrophenyl group or L-3,4-dihydroxy group. An alkyl group having 1 to 48 carbon atoms, an alkoxy group having 1 to 48 carbon atoms, an aryl group having 6 to 48 carbon atoms, an aryloxy group having 6 to 48 carbon atoms, and 7 carbon atoms, which may be substituted with a phenyl group An arylalkyl group having ˜48 or an arylalkyloxy group having 7 to 48 carbon atoms, and Y and Z may be independently substituted at least one with an amino acid other than Lys, and B is a hydrogen atom or hydroxyl to the group It is .m is 0 or 1.) A method for detecting a HER2 protein, comprising:
The HER2 protein detection probe according to claim 1 is bound to the HER2 protein present in an object, and the bound HER2 protein detection probe is detected by the labeling enzyme. Detection method.