WO2017010381A1

WO2017010381A1 - N terminal labeling agent, fluorescent-labeled protein using same, and production method therefor

Info

Publication number: WO2017010381A1
Application number: PCT/JP2016/070058
Authority: WO
Inventors: 貴弘芳坂; 貴嘉渡邉; 亮二阿部; 広行大橋
Original assignee: ウシオ電機株式会社
Priority date: 2015-07-10
Filing date: 2016-07-07
Publication date: 2017-01-19
Also published as: JP2017020896A

Abstract

Provided is an N-terminal labeling agent for a protein, comprising a compound represented by general formula (1) (where F is a fluorescent group, and R is an optionally substituted linker group having a linear soft segment formed from a C5 or higher linear hydrocarbon or a linear soft segment where the main chain carbons have been substituted in part by -O-, -N-, -S-, -CO-, -CONH-, -COO-, or -POO-).

Description

N-terminal labeling agent, fluorescently labeled protein using the same, and method for producing the same

The present invention relates to an N-terminal labeling agent for simply labeling a protein, a fluorescent-labeled protein labeled with such an N-terminal labeling agent, and a method for producing the same, and a fluorescent-labeled antibody used in a fluorescence immunoassay and the like is prepared. Useful as technology.

As an immunological measurement method, a fluorescently labeled antibody is quenched in a state where no antigen is bound (in this specification, it may be referred to as quenching or quenching), whereas when the antigen is bound, the quenching is performed. There is known a “homogeneous fluorescence immunoassay method” using a phenomenon in which is canceled (for example, see Patent Document 1 below). This method is carried out by mixing a fluorescently labeled antibody fragment or a fluorescently labeled single-chain antibody with a test sample solution for examining whether or not an antigen is contained. According to this method, the presence of the antigen is concluded when the fluorescence intensity of the fluorescent dye and the antigen concentration are positively correlated, and the concentration of the antigen can be measured based on the degree of correlation. The main cause of such quenching and its release is that the highly conserved tryptophan residue in the antibody molecule interacts with the fluorescent dye to quench the fluorescent dye and bind to the antigen in an antigen-dependent manner. Is to be resolved.

In the case of using a fluorescently labeled antibody fragment in the measurement, more specifically, two antibody fragments fluorescently labeled with either one of the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide are used. It is done. Here, the antibody light chain variable region may be abbreviated as “VL”, and the antibody heavy chain variable region may be abbreviated as “VH”.

In the case of using a fluorescently labeled single chain antibody for measurement, more specifically, a single chain antibody (abbreviated as “scFv”) formed by binding a fluorescently labeled VL polypeptide and a VH polypeptide. May be used).

The above two types of fluorescently labeled antibodies may be collectively referred to as “Quenchbody (registered trademark, hereinafter the same)” or “Q-body (registered trademark, hereinafter the same)”. In particular, when the two are distinguished, the former (when two antibody fragments are used) is referred to as “VH + VL type Q-body”, and the latter (when a single chain antibody is used) is referred to as “scFv type Q-body”. May be called.

The features of the method described in Patent Document 1 are mainly in the following three points.

The first feature is that no cleaning process is required. Thus, since the measurement is completed simply by mixing a small amount of sample and Q-body and measuring the fluorescence intensity, this method can be said to be an extremely simple measurement technique. The second feature is that tryptophan having a high degree of preservation present in the antibody is used for quenching. That is, since the quenching effect can be obtained even if the type of antibody is changed, it can be said that this technique is excellent in versatility in that it can be applied to detection of various substances using various antibodies. The third feature is that only one antigenic site is required. For this reason, it can be applied to a low molecular compound in principle.

Thus, this method is a simple measurement method that does not require a washing step. For this reason, it is possible to reduce the size of the measurement device based on this method to a portable size, for example, and by using this measurement device, ordinary people who have not received specialized education can make measurements on-site. It is assumed that this is possible.

Further, a Fab antibody (Fragment, antigen binding) consisting of a single molecule heterodimeric protein in which a polypeptide consisting of VL and an antibody light chain constant region and a polypeptide consisting of VH and an antibody heavy chain constant region are linked by a disulfide bond. And a fluorescent labeling complex has been developed in which the same or different fluorescent dyes are labeled on each of VL and VH (Patent Document 2). In the fluorescent labeling complex in which fluorescent dyes of the same color are labeled on each of VL and VH, the quenching effect (H-dimer) between the dyes is added in addition to the quenching due to the interaction between the fluorescent dye and the tryptophan residue, which is high. Detection sensitivity can be obtained (FIG. 7, FIG. 8, etc. of Patent Document 2). In the fluorescent labeling complex in which each of VL and VH is labeled with a different color fluorescent dye, in addition to quenching effect due to interaction between the fluorescent dye and tryptophan residue and quenching effect between the dyes, a quenching effect due to the FRET effect is added. High detection sensitivity can be obtained (FIGS. 9 and 10 in Patent Document 2). Such fluorescently labeled Fab antibody complexes are sometimes collectively referred to as “UQ-body (registered trademark, the same applies hereinafter)”.

WO2011 / 061944 WO2013 / 065314

Quenching when antigens and ligands are not bound, and releasing the quenching when bound is popular not only in the medical field but also in various fields such as environmental pesticide detection and toxin detection It is expected that. There is a need for a technique for easily producing a fluorescently labeled protein such as Q-body or UQ-body using a protein containing an existing antibody or receptor.

Therefore, an object of the present invention is to provide a fluorescently labeled protein such as Q-body or UQ-body in a short time and in a short time using a protein containing an existing antibody or receptor.

As a result of intensive studies to solve the above problems, the present inventors have made it easy to use fluorescent dyes for proteins such as existing antibodies by binding a fluorescent group to an aldehyde group via a linker having a certain length. It was found that this can be used for fluorescent immunoassay and the like. The present invention has been completed by further studies based on such knowledge.

That is, the present invention
[1] The following general formula (1):

(In the formula, F is a fluorescent group, and R is a linear soft segment composed of a linear hydrocarbon having 5 or more carbon atoms, which may have a substituent, or a part of the main chain carbon. A linker group having a linear soft segment substituted with -O-, -N-, -S-, -CO-, -CONH-, -COO- or -POO-). N-terminal labeling agent for proteins;
[2] The fluorescent group is a rhodamine type, coumarin type, oxazine type, carbopyronine type, cyanine type, pyromesene type, naphthalene type, biphenyl type, anthracene type, phenanthrene type, pyrene type, carbazole type, Cy type, EvoBlue type, The N-terminal labeling agent according to the above [1], which is at least one selected from the group consisting of fluorescein and derivatives thereof;
[3] The N-terminal labeling agent according to [2], wherein the fluorescent group includes at least one selected from the group consisting of carboxytetramethylrhodamine, carboxyfluorescein, ATTO655 (registered trademark) and rhodamine green;
[4] The N-terminal labeling agent according to any one of [1] to [3], wherein the protein is a complete antibody;
[5] The N-terminal labeling agent according to any one of [1] to [3], wherein the protein is an antibody fragment;
[6] A fluorescently labeled protein into which the fluorescent group is introduced using the N-terminal labeling agent according to any one of [1] to [5] at the N-terminus of the protein;
[7] A method for producing a fluorescently labeled protein, the step (a) of reacting the N-terminal labeling agent according to any one of the above [1] to [5] with the N-terminal of the protein,
A method for producing a fluorescently labeled protein, comprising:
[8] The method according to [7], wherein the step (a) is a step of bringing the N-terminal labeling agent into contact with the protein in a solution, and the pH of the solution is pH 3 to 7;
[9] The method according to any of [7] or [8] above, wherein the protein in the step (a) is a complete antibody or a fragment thereof;
[10] A kit for preparing a fluorescently labeled protein,
A kit having the N-terminal labeling agent according to any one of [1] to [5].

According to the present invention, a fluorescent group can be bound to the N-terminus in a short time using a protein such as an existing antibody in a short time. Since the existing protein can be used, it is not necessary to prepare a hybridoma or analyze a gene sequence, and a protein that can be used for a homogeneous fluorescent immunoassay can be easily and efficiently produced.

It is the figure which showed typically one Embodiment of the protein labeled using the N terminal labeling agent of this invention. In this embodiment, fluorescence is quenched (middle) in the absence of antigen (middle). In the presence of antigen, the quench is eliminated and the fluorescent group is exposed from the protein (right side). In this case, the generated fluorescence intensity can be positively correlated with the antigen concentration. It is a figure which shows an example of the manufacturing method of N terminal labeling agent. It is a figure which shows the result of having measured the change of the fluorescence intensity in the fluorescence labeled protein labeled using the N terminal labeling agent. It is a figure which shows the result of having measured the change of the fluorescence intensity in the fluorescence labeled protein which has a linker group of various length. It is a figure which shows the result of having measured the change of the fluorescence intensity in the fluorescence labeled protein which has a linker group of various length. It is a figure which shows the result of having measured the change of the fluorescence intensity in the fluorescence labeled protein which has a linker group of various structures. It is a figure which shows the result of having measured the change of the fluorescence intensity in the fluorescence labeled protein which has various kinds of fluorescent groups. It is a figure which shows the N-terminal labeling agent which has various kinds of fluorescent groups.

Hereinafter, the present invention will be described in detail.
[N-terminal labeling agent]
The present invention provides the following general formula (1):

(In the formula, F is a fluorescent group, and R is a linear soft segment composed of a linear hydrocarbon having 5 or more carbon atoms, which may have a substituent, or a part of the main chain carbon. A linker group having a linear soft segment substituted with -O-, -N-, -S-, -CO-, -CONH-, -COO- or -POO-). It is a protein N-terminal labeling agent. That is, the N-terminal labeling agent in the present invention has an aldehyde group at the terminal, and is bonded to a fluorescent group represented by F via a linker group represented by R. The linker group may have a linear soft segment and other linking groups.

In this specification, the linear soft segment refers to a segment having a linear group in the main chain and having flexibility. Such a linear group is preferably a group in which the main chain rotation and flexibility are not easily inhibited. Examples of the straight chain hydrocarbon include an alkylene group, an alkenylene group, and an alkynylene group.

From the viewpoint of main chain rotation and flexibility, the straight chain hydrocarbon has 5 or more carbon atoms, preferably 6 or more, more preferably 7 or more, still more preferably 8 or more, and even more preferably 9 or more. The above is more preferable, 11 or more is more preferable, 12 or more is particularly preferable, and 13 or more is most preferable. Further, from the viewpoint of the main chain rotation and flexibility, the carbon number in the straight chain hydrocarbon can be 100 or less, preferably 90 or less, more preferably 80 or less, and 70 or less. More preferably. The straight chain soft segment may have a part of the main chain carbon substituted with —O—, —N—, —S—, —CO—, —CONH—, —COO— or —POO—. This includes those having a repeating unit such as an alkylene group.

In the protein labeled with the N-terminal labeling agent, when the fluorescent group is located in the vicinity of the tryptophan residue, the tryptophan residue and the fluorescent group interact to quench the fluorescent group. If the protein is an antibody, fluorescent groups can interact with tryptophan residues in the antigen binding pocket. The principle of quenching is not limited to the interaction between tryptophan residues and fluorescent groups, but the structure and length of linker groups can affect the interaction between tryptophan residues and fluorescent groups. is assumed. For example, if the linker group is too short or too long, the tryptophan residue and the fluorescent group cannot sufficiently interact. In addition, for example, when the linker group includes a plurality of cyclic structures, it is assumed that the rotation and mobility of the linker group are inhibited and the tryptophan residue and the fluorescent group cannot sufficiently interact. The

In this specification, the linear soft segment made of a linear hydrocarbon having 5 or more carbon atoms may have a substituent. In the present specification, the substituent is not limited as long as the rotation and flexibility of the main chain are not hindered. For example, a methyl group, an ethyl group, an n-propyl group, an i-propyl group, a butyl group are not limited. Group, pentyl group, hexyl group, cyclohexylene group, ethenyl group, propenyl group, butenyl group, phenyl group, toluyl group, naphthyl group, pyridyl group, furanyl group, methoxy group, ethoxy group, propoxy group, acetyl group, propanoyl group , Cyclohexanecarbonyl group, benzoyl group, methoxycarbonyl group, ethoxycarbonyl group, hydroxyl group, methylamino group, ethylamino group, dimethylamino group, methylsulfonyl group, ethylsulfonyl group, methylsilyl group, dimethylsilyl group, fluoro group, chloro Group, trifluoromethyl group, amine group, acetal , Ketal group, aldehyde group, ketone group, an imine group, a nitrile group, a carboxyl group, an ether group, an ester group, a nitro group, etc. sulfo group. From the viewpoint of ease of synthesis, when it has a substituent, a methyl group, an ethyl group or the like is preferable.

In the present specification, the linker group may contain another linking group as long as the function of the linear soft segment is not easily inhibited. For example, an arylene group, a heteroarylene group, C ₄ -C ₁₀ Selected from the group consisting of cycloalkyl groups and combinations thereof.

Although not limited, the linker group may have a substituent, imino group, succinylamino group, acetamide group, 2-aminopentanamide, 2 (2 ′)-alkyl-aminoacetyl group, carbamoyl group, Aminoalkyl group, aminoalkylol group, glutaramide group, ornitino group, theopropanoyl group, N- (mercaptomethyl) propionamide group, 2- (1,2-dihydroxy-3-mercaptopropylthio) propanoyl group, It may contain a linking group such as aminoethanethiol group, mercaptopropanol group, (hydroxypropylthio) propanenoyl group, oxy group, succinyl group, acetyl group or oxopentanoyl group.

In addition, although not limited, the linker group may have a substituent, and may include a binding group such as an amino acid linker, a disulfide linker, a phosphate ester linker, or the like containing one or more amino acid repeating units. Good.

When the linker group includes an alkylene group, it is not limited. However, from the viewpoint of remarkably exhibiting the quenching effect of the fluorescent group, a C ₂ to C ₂₀ alkylene group is preferable, and a C ₃ to C ₁₈ alkylene group is preferable. Is more preferable, and a C ₄ to C ₁₆ alkylene group is more preferable.

When the linker group includes a PEG group, the number of repeating units of the PEG group is preferably 3 to 20, more preferably 4 to 18, more preferably 5 to 15 is more preferable, 6 to 12 is particularly preferable, and 2 to 10 is most preferable.

The protein labeled with the N-terminal labeling agent is not particularly limited, but is preferably a protein that can bind to a target substance, and is not limited, but examples thereof include antibodies, protein tags, receptors, and cell adhesion molecules. It is done.

In the present specification, an antibody is a glycoprotein produced by B cells and has a function of recognizing and binding to an antigen. An antibody, also called an immunoglobulin, has two long polypeptide chains (also referred to herein as antibody heavy chains) and two short polypeptide chains (also referred to herein as antibody light chains). Are connected by a disulfide bond. Such an antibody having two antibody heavy chains and two antibody light chains is referred to as a complete antibody in the present specification.

In this specification, when an antibody is used as a protein labeled with an N-terminal labeling agent, either a polyclonal antibody or a monoclonal antibody can be used. Also good. As an antibody isotype, any of IgG, IgA, IgM, IgE, and IgD can be used, and IgG is preferable from the viewpoint of availability. The origin of the antibody is not particularly limited, but is preferably derived from a mammal, more preferably from a human, mouse, rat, dog, cat, monkey, pig, cow, sheep, goat, horse, or dolphin. More preferably derived from human, mouse, rat, sheep or goat, particularly preferably derived from human or mouse, and most preferably derived from human.

The antibody used may be an antibody against various antigens, and examples of the target antigen include proteins, peptides, carbohydrates, lipids, glycolipids, low molecular compounds, and the like. Protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, viruses, animal tissues and the like containing these substances can also be targeted. In addition, chemical substances including low-molecular compounds such as narcotics, explosives, agricultural chemicals, fragrances, and pollutants can be targeted. Examples of such substances include cannabinoids called cannabinoids such as tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A), cannabinol (CBN), cannabidiol (CBD); amphetamine, methamphetamine, morphine , Heroin, codeine and other stimulants and narcotics; fungal toxins such as aflatoxin, sterigmatocystin, neosolanil, nivalenol, fumonisin, ochratoxin, and endophyte-producing toxins; sex hormones such as testosterone and estradiol; feeds such as clenbuterol and ractopamine Additives illegally used in foods; PCBs, gossypol, histamine, benzpyrene, melamine, acrylamide, dioxins and other harmful substances; acetamiprid, imidaclopri Chlorfenapyr, malathion, carbaryl, clothianidin, triflumizole, chlorothalonil, spinosad, Ran'neto, methamidophos, pesticide residues, such as chlorpyrifos; can be mentioned environmental hormones such as bisphenol A and the like. The above substances also include derivatives of each substance.

A complete antibody can be used by cleaving a part thereof by known means. A complete antibody can be separated into two Fab portions and one Fc portion by cleaving with, for example, papain. In addition, a complete antibody can be separated into one F (ab ′) ₂ portion and one Fc ′ portion by cleaving with, for example, pepsin. In the present specification, a part of a complete antibody is referred to as an antibody fragment, and a single chain antibody [single-chain Fv (scFv)], Fab part, F (ab ′) ₂ part, variable region (Fv) part, Fc part , Fc ′ portion and the like. The fragment of the antibody is preferably scFv, Fab portion, F (ab ′) ₂ portion or Fv portion from the viewpoint that it can bind to the antigen. Further, from the viewpoint of being able to bind to an Fc receptor, the antibody fragment is preferably an Fc portion or an Fc ′ portion. Antibody fragments can also be synthesized and used by known means.

The scFv part and the Fab part consist of one polypeptide containing the antibody light chain variable region and one polypeptide containing the antibody heavy chain variable region, and the F (ab ′) ₂ part and the complete antibody contain the antibody light chain variable region. It consists of two polypeptides containing and two polypeptides containing antibody heavy chain variable regions.

Antibody light chain variable region polypeptides, antibody light chain variable region polypeptides, and single chain antibody polypeptides comprising both antibody light chain variable regions and antibody light chain variable regions are known chemical synthesis methods, gene sets It can be prepared using a replacement technique, a method for degrading antibody molecules with proteolytic enzymes, etc., but among them, it is preferable to prepare by a gene recombination technique that can be prepared in large quantities by a relatively easy operation. . When preparing the above-described polypeptide by gene recombination technology, an expression vector obtained by introducing an antibody light chain variable region or a DNA containing a base sequence encoding an amino acid sequence specific for the antibody light chain variable region into a suitable vector And the desired polypeptide can be expressed by an expression system using bacteria, yeast, insects, animal or plant cells as a host, or a cell-free translation system. When expressing a polypeptide in a cell-free translation system, for example, in a reaction solution in which nucleotide triphosphate and various amino acids are added to a cell-free extract such as Escherichia coli, wheat germ, rabbit reticulocyte, etc., the polypeptide is used. Can be expressed.

When an antibody is used as the protein labeled with the N-terminal labeling agent, from the viewpoint of the interaction between the fluorescent group and tryptophan in the variable region of the antibody, the antibody light chain variable region is the number 35 in the Kabat numbering system. It is preferred to have an amino acid sequence in which the second amino acid is tryptophan. The antibody heavy chain variable region preferably has an amino acid sequence in which the 36th, 47th, or 103rd amino acid is tryptophan in the Kabat numbering system.

In this specification, a protein tag refers to a peptide or protein that utilizes affinity with other molecules. The protein tag is not particularly limited as long as it is a protein tag having affinity with other molecules. For example, histidine tag (His tag), glutathione-S-transferase (GST), maltose binding protein (MBP), influenza virus Examples include hemagglutinin peptide sequence tags (HA tags), myc tags, FLAG tags, Biotin Carboxyl Carrier Protein (BCCP) tags (biotinylated peptides), and the like. From the viewpoint of remarkably exhibiting the quenching effect of the fluorescent group, His tag, HA tag, and FLAG tag are preferable. The protein tag may be commercially available or may be obtained by synthesis.

In this specification, a receptor refers to a protein having a binding property to a ligand. The receptor is not particularly limited as long as it is a receptor capable of binding to a ligand, and any of a secretory receptor, a membrane-bound receptor, and an intracellular receptor can be used. Such receptors may be naturally occurring or modified. The receptor may be a commercially available product or a synthesized product. The origin of the receptor is not particularly limited, but is preferably derived from mammals, and more preferably derived from humans, mice, rats, dogs, cats, monkeys, pigs, cows, sheep, goats, horses, or dolphins. Preferably, it is derived from human, mouse, rat, sheep or goat, more preferably derived from human or mouse, and most preferably derived from human. As the receptor, a fragment of the receptor can be used as long as it has binding ability to the ligand.

In the present specification, the secretory receptor refers to a receptor having a structure obtained by removing a transmembrane region and an intracellular region from a transmembrane receptor, and is not particularly limited as long as it has a binding property to a ligand.

The transmembrane receptor is not particularly limited as long as it has a binding property to a ligand. For example, an ion channel-linked receptor, a receptor not linked to an ion channel, a kinase type receptor, Examples include kinase type receptors.

The nuclear receptor is not particularly limited as long as it has a binding property with a ligand, and examples thereof include a thyroid hormone receptor type, a retinoid X receptor type, an estrogen receptor type, and a steroid hormone receptor type.

In the present specification, the cell adhesion molecule refers to a molecule involved in adhesion between cells or between a cell and an extracellular matrix. The cell adhesion molecule is not particularly limited as long as it is a receptor having a binding property with a ligand, and examples thereof include an immunoglobulin superfamily, a cadherin superfamily, an integrin superfamily, a selectin family, and a MAM superfamily. Such cell adhesion molecules may be naturally occurring or modified. Cell adhesion molecules may be commercially available or synthesized. The origin of the cell adhesion molecule is not particularly limited, but is preferably derived from a mammal, and preferably derived from a human, mouse, rat, dog, cat, monkey, pig, cow, sheep, goat, horse or dolphin. More preferably, it is derived from human, mouse, rat, sheep or goat, particularly preferably derived from human or mouse, and most preferably derived from human. As a cell adhesion molecule, a fragment of a cell adhesion molecule can be used as long as it has binding ability to a ligand.

In the present specification, the fluorescent group is not limited as long as the effects of the present invention are exhibited, but the rhodamine system, coumarin system, oxazine system, carbopyronine system, cyanine system, pyromesene system, naphthalene system, biphenyl system, anthracene system, phenanthrene system , Pyrene series, carbazole series, Cy series, EvoBlue series, fluorescein series or derivatives thereof. Among these, from the viewpoint of remarkably exhibiting the quenching effect of the fluorescent group, the fluorescent group is preferably a rhodamine fluorescent group, an oxazine fluorescent group, a Cy system, or a fluorescein system.

Specifically, the fluorescent group is a fluorescent group having, for example, rhodamine, coumarin, oxazine, carbopyronine, cyanine, pyromesene, naphthalene, biphenyl, anthracene, phenanthrene, pyrene, carbazole, Cy, EvoBlue, fluorescein or the like as a basic skeleton, or Derivatives of these fluorescent groups can be mentioned.

More specifically, the fluorescent group is, for example, CR110: carboxyrhodamine 110: Rhodamine Green (trade name), TAMRA: carbocytetremethlrhodamine: TMR, Carboxyrhodamine 6G: CR6G, ATTO655 (trade name), BODIPY FL (trade name): 4, 4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 493/503 (trade name): 4,4-difluoro-1,3,5, 7-tetramethyl-4-bora-3a, 4a-diaza-s-indancene-8-propionicacid, BODIPY R6G (trade name): 4,4-difluoro-5- (4-phenyl-1,3-butadienyl) -4 -bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 558/568 (trade name): 4,4-difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza -s-indancene-3-propionic acid, BODIPY 564/570 (trade name): 4,4-difluoro-5-styryl-4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 576 / 589 (trade name): 4,4-d ifluoro-5- (2-pyrrolyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 581/591 (trade name): 4,4-difluoro-5- (4-phenyl) -1, 3-butadienyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, Cy3 (trade name), Cy3B (trade name), Cy3.5 (trade name), Cy5 (trade mark) Name), Cy5.5 (trade name), EvoBlue 10 (trade name), EvoBlue 30 (trade name), MR121, ATTO 390 (trade name), ATTO 425 (trade name), ATTO 465 (trade name), ATTO 488 (trademark) Name), ATTO 495 (trade name), ATTO 520 (trade name), ATTO 532 (trade name), ATTO Rho 6G (trade name), ATTO 550 (trade name), ATTO 565 (trade name), ATTO Rho 3B (trade name) ), ATTO Rho11 (trade name), A TO Rho12 (trade name), ATTO Thio12 (trade name), ATTO 610 (trade name), ATTO 611X (trade name), ATTO 620 (trade name), ATTO Rho14 (trade name), ATTO 633 (trade name), ATTO 647 (trade name), ATTO 647N (trade name), ATTO 655 (trade name), ATTO Oxa12 (trade name), ATTO 700 (trade name), ATTO 725 (trade name), ATTO 740 (trade name), Alexa Fluor 350 (trade name), Alexa Fluor 405 (trade name), Alexa Fluor 430 (trade name), Alexa Fluor 488 (trade name), Alexa Fluor 532 (trade name), Alexa Fluor ４６ 546 (trade name), Alexa Fluor 555 (trade name), Alexa Fluor 568 (trade name), Alexa 594 (trade name), Alexa Fluor 633 (trade name), Alexa Fluor 647 (trade name), Alexa Fluor 680 (trade name), Alexa Fluor 700 ( Trade name), Alexa Fluor 750 (trade name), Alexa Fluor 790 (trade name), Rhodamine Red-X (trade name), Texas Red-X (trade name), 5 (6) -TAMRA-X (trade name) 5TAMRA (trade name), SFX (trade name), 5-carboxyfluorescein (FAM), 6-carboxyfluorescein, 5,6-dicarboxyfluorescein, 6-carboxy-2 ′, 4,4 ′, 5 ′, 7 , 7'-Hexachlorofluore Cein, 6-carboxy-2 ′, 4,7,7′-tetrachlorofluorescein, 6-carboxy-4 ′, 5′-dichloro-2 ′, 7′-dimethoxyfluorescein, fluorescein-5-isocyanate (FITC), Examples thereof include naphthofluorescein. Among them, the fluorescent group has a rhodamine-based fluorescent group TAMRA, CR110, Rhodamine 商標 Green (trade name), an oxazine-based fluorescent group, ATTO, and a Cy-based fluorescent group, Cy3, from the viewpoint of remarkably exhibiting the quenching effect of the fluorescent group. (Trade name), Cy5 (trade name), and carboxyfluorescein, which is a fluorescein series, are preferable. 5 (6) -TAMRA-X (trade name), 5TAMRA (trade name), ATTO 655 (trade name), Cy3 (trade name) ), Cy5 (trade name), 5-carboxyfluorescein, 6-carboxyfluorescein, and 5,6-dicarboxyfluorescein are more preferable.

When binding the fluorescent group and the linker group, it is possible to carry out a reaction between the reactive end of the compound having the fluorescent group and the end of the compound having the linker group. Examples of such a reaction include amidation by a condensation reaction. In such a reaction, a compound having a linker group may have an aldehyde group at the other end, but after reacting in the form of a hydroxyl group or the like, it may be aldehyded by a reaction such as desmartin oxidation. Is preferred.

[Fluorescent labeled protein]
The present invention relates to a fluorescently labeled protein in which the fluorescent group is introduced at the N-terminus of the protein using the N-terminal labeling agent. Since the N-terminal labeling agent of the present invention has an aldehyde group at one end, it can cause a reductive amination reaction with the N-terminus of the protein in the presence of a reducing agent.

A quencher capable of quenching the fluorescence of the labeled fluorescent group may be further added to the fluorescently labeled protein. As such a quencher, a combination in which the quencher effectively quenches the fluorescent group in the absence of a target substance such as an antigen and does not inhibit the emission of the fluorescent group in the presence of the target substance is appropriately selected. be able to. Examples of such quenchers include NBD: 7-nitrobenzofurazane, DABCYL, BHQ, ATTO, QXL, QSY, Cy, Lowa 'Black, IRDYE, etc., quenching dyes, derivatives of these quenching dyes, and the like. It is done. More specifically, NBD, DABCYL, BHQ-1 (trade name), BHQ-2 (trade name), BHQ-3 (trade name), ATTO 540Q (trade name), ATTO 580Q (trade name), ATTO 612Q (trade name) ), QXL490 (trade name), QXL520 (trade name), QXL 570 (trade name), QXL 610 (trade name), QXL 670 (trade name), QXL 680 (trade name), QSY-35 (trade name), QSY-7 ( Trade name), QSY-9 (trade name), QSY-21 (trade name), Cy5Q (trade name), Cy7Q (trade name), Lowa Black Black FQ (trade name), Lowa Black Black RQ (trade name), IRDYE QC -1 (trade name) and the like, and NBD is preferable from the viewpoint of remarkably exhibiting the effect as a quencher. For example, when a rhodamine fluorescent group is used as the fluorescent group, a combination with NBD can be exemplified.

In this specification, when a fluorescent group is introduced into an antibody using an N-terminal labeling agent, in the absence of an antigen, the fluorescent group is caused by the interaction between the fluorescent group and a tryptophan residue conserved in the antibody variable region. Quenching occurs. When multiple fluorescent groups of the same color are introduced into the antibody, a quenching effect between the fluorescent groups can be obtained. In addition, when a plurality of differently colored fluorescent groups are introduced into an antibody, in addition to quenching by the above tryptophan residues and quenching between fluorescent groups, there is a quenching effect due to the fluorescence resonance energy transfer (FRET) effect. can get. Furthermore, when a fluorescent group and a quencher that quenches the fluorescent group coexist, the dynamic range can be increased by the quenching effect between the fluorescent group and the quencher. FIG. 1 shows an example in which a fluorescent group is introduced into a complete antibody using an N-terminal labeling agent. In this embodiment, fluorescence is quenched (middle) in the absence of antigen (middle). In the presence of antigen, the quench is eliminated and the fluorescent group is exposed from the protein (right side). In such a case, the generated fluorescence intensity can be positively correlated with the antigen concentration.

When a fluorescent group is introduced into a protein tag, receptor or cell adhesion molecule using an N-terminal labeling agent, the hydrophobic group in the protein tag, receptor or cell adhesion molecule is hydrophobically or electrically fluorescent in the absence of the target substance. The part capable of interacting with the fluorescent substance acts as a quencher, and quenching of the fluorescent group can occur. When a plurality of fluorescent groups of the same color are introduced into the protein tag, receptor or cell adhesion molecule, a quenching effect between the fluorescent groups can be obtained. In addition, when a plurality of different-color fluorescent groups are introduced into a protein tag, receptor or cell adhesion molecule, in addition to quenching by the above tryptophan residue and quenching between fluorescent groups, fluorescence resonance energy transfer (FRET) ) Quenching effect can be obtained. Furthermore, when a fluorescent group and a quencher that quenches the fluorescent group coexist, the dynamic range can be increased by the quenching effect between the fluorescent group and the quencher.

As another embodiment, in the fluorescence-labeled protein, quenching of the fluorescent group may occur in the presence of the target substance. When the target substance can interact with the fluorescent group mainly hydrophobicly and electrically, it can be caused by more quenching in the presence of the target substance. In such a case, the generated fluorescence intensity can be negatively correlated with the antigen concentration.

In this specification, the production method of the N-terminal labeling agent can be a known method. For example, when a commercially available fluorescent group is used, the protective group for the fluorescent group is a compound containing the above linker group. Replace. When the substituted reactant has a hydroxy group at the terminal, the terminal can be substituted with an aldehyde group by causing desmartin oxidation using a desmartin reagent, for example. Specifically, it will be described in detail in Examples.

In this specification, a light source and a measuring device used for fluorescence detection can be usually used for measurement of fluorescence. As the light source, any light source capable of irradiating the excitation light wavelength may be used. Specific examples include a mercury lamp, a xenon lamp, an LED (light emitting diode), and laser light. At this time, excitation light having a specific wavelength can be obtained using an appropriate fluorescent filter. As the fluorescence measuring apparatus, for example, a fluorescence microscope equipped with a light source of excitation light and its irradiation system, a fluorescence image acquisition system, and the like can be used. For example, MF20 / FluoroPoint-Light (manufactured by Olympus) or FMBIO- III (manufactured by Hitachi Software Engineering Co., Ltd.). Moreover, you may use the small and portable fluorescence detection apparatus provided with the light source, the irradiation system, and the measurement system. By using such a small device, measurement can be performed at the collection site of the test sample. The fluorescence detection may be detection of a fluorescence spectrum or detection of fluorescence intensity at a specific wavelength.

In addition, when a fluorescently labeled protein is administered to a non-human animal, in addition to collecting the body fluid, tissue, etc., irradiate the detection target region of the non-human animal with BR> Ano excitation light, and the fluorescence of the fluorescent dye 2 Measurement and / or detection can be performed three-dimensionally or three-dimensionally. In this case, examples using a fluorescence microscope, a fluorescence image analyzer, an endoscope equipped with a light source, and the like can be given. In addition, at the time of detection, an image showing the structure of an individual, tissue, or cell of a non-human animal can also be acquired using an endoscope, X-ray, CT, MRI, ultrasound, a microscope, or the like. preferable. When the measured and / or detected fluorescence intensity and the amount of the target substance are positively correlated, the localization (position) of the target substance and / or based on the two-dimensional or three-dimensional image of the detected fluorescence The amount can be known and compared with an image showing the structure. When there is a positive correlation between the fluorescence intensity and the amount of the target substance, a sample for measurement that does not contain a fluorescently labeled protein or does not contain a sample is prepared as a negative control and measured together. And / or can be detected. Further, the amount of target substance can be measured using the fluorescence intensity ratio obtained by dividing the fluorescence measurement value in the negative control by the fluorescence measurement value in the measurement sample. Alternatively, when there is a positive correlation between the fluorescence intensity and the target substance amount, it can also be determined that the target substance is present in the measurement sample when a fluorescence intensity exceeding an appropriately set threshold is obtained.

In this specification, the excitation light to be irradiated and the wavelength of fluorescence to be measured and / or detected may be appropriately selected according to the type of fluorescent group to be used. For example, when TAMRA is used for the fluorescent group, an excitation light wavelength of 530 nm and a fluorescence wavelength of 580 nm can be used, and when ATTO655 is used for the fluorescent group, an excitation light wavelength of 630 nm and a fluorescence wavelength of 680 nm can be used. Moreover, also when using two types of different fluorescent groups, the combination of an excitation light wavelength and a fluorescence wavelength can be selected suitably, and can be used.

[Method for producing fluorescently labeled protein]
The method for producing a fluorescently labeled protein of the present invention includes a step (a) of reacting the N-terminal labeling agent with the N-terminus of the protein. In the step (a), the N-terminal labeling agent can bond the terminal aldehyde group to the N-terminal of the protein by causing a reductive amination reaction. In the step (a), when the N-terminal labeling agent and the protein are brought into contact with each other in the solution, the pH of the solution is preferably pH 3-7. From the viewpoint of efficiently reacting the α-amino group of the protein, the pH of the above solution is more preferably pH 3.5 to 6, further preferably pH 4 to pH 5.5, and pH 4.5 to pH 5. It is particularly preferable to do this. The temperature condition can be, for example, 1 to 30 ° C., preferably 2 to 10 ° C., more preferably 3 to 5 ° C. The reaction time can be, for example, 1 minute to 3 days, preferably 30 minutes to 2 days, and more preferably 1 hour to 1 day.

In the present specification, the reducing agent used in step (a) is not limited as long as it can cause a reductive amination reaction. For example, sodium cyanoborohydride, sodium borohydride, sodium triacetoxyborohydride , 2-picoline borane and the like. From the viewpoint of efficient reaction, sodium cyanoborohydride and 2-picoline borane are preferred.

The method for producing a fluorescently labeled protein can further include a purification step (b). A known method can be used as a purification method of the fluorescently labeled protein, and is not limited. For example, gel filtration chromatography, ultrafiltration, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, adsorption Chromatography, reverse phase chromatography and the like can be mentioned. From the viewpoint of efficient reaction, it is preferable to use gel filtration chromatography and ultrafiltration.

[Kit for creating fluorescently labeled protein]
The present invention also relates to a kit for producing a fluorescently labeled protein, the kit having the above N-terminal labeling agent. Such a kit can further contain usual reagents (reducing agent, pH adjuster, etc.) used for the reductive amination reaction, instruments (equipment relating to purification, etc.), instruction manuals, and the like.

The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.

[Example 1] Synthesis of N-terminal labeling agent using fluorescent group TAMRA-X 1.5 Synthesis of 5 (6) -TAMRA-X-C8-OH 5 (6) -TAMRA- Using X-SE, 5 (6) -TAMRA-X-C8-OH was synthesized. Specifically, it is as follows.
10 μL of 100 mM 5 (6) -TAMRA-X-SE (6- (Tetramethylrhodamine-5- (and-6) -carboxamido) hexanoic acid, succinimidyl ester; manufactured by Molecular Probes), 10 μL of DMSO (Dimethyl sulfoxide), 100 μL Of 100 mM octanolamine and 20 μL of 100 mM NaHCO ₃ hydrate were added to a 1.5 mL low adsorption tube and allowed to react by mixing on ice. The reaction mixture was neutralized by adding 2 μL of 1M acetic acid, and then 140 μL of 0.1% trifluoroacetic acid (TFA) was added. The synthesized product was separated from this solution by high performance liquid chromatography (HPLC) under the following preparative conditions and dried. This dried product was dissolved in DMSO to a predetermined concentration. 671.38 (MH ⁺ , monoisotopic) 5 (6) -TAMRA-X-C8-OH was obtained. Mass spectrometry conditions are as follows.

(HPLC preparative conditions)
Detector: SPD-20A (manufactured by Shimadzu Corporation), 260 nm absorbance detection Column: Xbridge C18 5μm 10x50mm column (manufactured by Waters)
Mobile phase A: 0.1% TFA
Mobile phase B: Acetonitrile (MeCN)
Column temperature: 30 ° C
Gradient condition: 0-100% B in A for 15min
Flow rate: 3.0mL / min

(Mass spectrometric conditions)
Analyzer: Voyager DE Pro (Applied Biosystems)
Matrix: α-CHCA in 0.1% TFA / MeCN (1: 1) solution (10 mg / ml)
Measurement mode: positive External reference material: angiotensin II (1046.5)

2.5 Synthesis of 5 (6) -TAMRA-X-C7-Aldehyde As shown in FIG. 2, using the obtained 5 (6) -TAMRA-X-C8-OH, 5 (6) -TAMRA- X-C7-Aldehyde was synthesized. Specifically, it is as follows.
Add 175 μL of 2 mM 5 (6) -TAMRA-X-C8-OH, 175 μL of DMSO and 350 μL of 500 mM Dess-Martin reagent (manufactured by Tokyo Chemical Industry Co., Ltd.) to a 1.5 mL low adsorption tube and shake at 37 ° C. for 1 hour. Incubated while allowing. 750 μL of 0.1% TFA solution was added, and the synthesized product was separated by HPLC under the above-described preparative conditions and dried. This dried product was dissolved in 100 μL of trifluoroacetic acid / acetonitrile (1: 1), and quantitative analysis was performed using 1 μL thereof. The quantitative analysis was performed by HPLC under the following quantitative / analytical conditions. It was dried again, and the dried product was dissolved in DMSO to a predetermined concentration. As the N-terminal labeling agent, 669.36 (MH ⁺ , monoisotopic) 5 (6) -TAMRA-X-C7-Aldehyde was obtained. Mass spectrometry conditions are as described above.

(HPLC quantification / analysis conditions)
Detector: SPD-20A (manufactured by Shimadzu Corporation), 260 nm absorbance detection column: Xbridge C18 2.5μm 4.6x20mm IS column (manufactured by Waters)
Mobile phase A: 0.1% TFA
Mobile phase B: Acetonitrile (MeCN)
Column temperature: 30 ° C
Gradient condition: 0-100% D in C for 10min
Flow rate: 1.5mL / min

[Example 2] Fluorescent labeling of protein Fluorescence labeling reaction (reductive amination reaction)
Using the N-terminal labeling agent synthesized in Example 1, fluorescent labeling of the N-terminal of the antibody was performed.
9 μL of 1.0 mg / mL antibody solution, 4.5 μL of 0.96 mM 5 (6) -TAMRA-X-C7-aldehyde (50% DMSO solution), 4.5 μL of 192 mM picoline borane (18% DMSO solution), 9 μL of 50 mM quencher Sodium acid buffer (pH 4.8) was added to a 1.5 mL low adsorption tube. This solution was incubated at 4 ° C. for 24 hours to perform a reductive alkylation reaction.

The following antibody solutions were used.
1) Anti-thyroxine antibody (Anti-Thyroxine: HyTest, clone # 1H1)
2) Anti-influenza hemagglutinin antibody (Anti-Influenza Hemagglutinin: MBL, clone # 5D8)
3) Anti-influenza hemagglutinin antibody (Anti-Influenza Hemagglutinin: MBL, clone # TANA2)
4) Anti-FLAG antibody (Sigma, clone # M2)
5) Anti-His Tag antibody (Novagen)
6) scFv derived from anti-BGP antibody (expressed in E. coli with MetAla (methionine and alanine) added to the N-terminus)

2. Gel filtration of fluorescently labeled protein The fluorescently labeled protein was eluted from the resulting reaction solution using a Microspin G25 column (manufactured by GE Healthcare Japan).
Specifically, at first, Microspin G25 column, HKM equilibration buffer (HKM buffer (25 mM HEPES-KOH (pH 7.4), 5 mM MgCl ₂ , 100 mM KCl), 0.1% PEG8000 (manufactured by Sigma), 0.05% Brij (nonionic surfactant, manufactured by Wako Pure Chemical Industries, Ltd.)). For column equilibration, 300 μL of HKM equilibration buffer was added to the column and centrifuged at 3000 rpm for 1 minute at room temperature. This operation was performed 4 times in total. Thereafter, the entire amount of the reaction solution obtained by the above fluorescence labeling reaction was added to the column, and the fluorescence labeled protein was eluted by centrifuging at room temperature and 3000 rpm for 1 minute.

3. Fluorescence measurement of fluorescently labeled protein (antigen titration)
(Preparation of antigen solution)
The antigen was dissolved in pure water or DMSO to prepare a 10 ⁻¹ to 10 ⁻² solution. Then, it diluted in steps to ^10-9 solution using HKM buffer or DMSO. The following antigens were used for various antibodies.
1) Thyroxine antigen (Sigma)
2) Influenza hemagglutinin tag peptide (Wako Pure Chemical Industries, Ltd.)
(The same antigen was used for the fluorescence-labeled proteins using anti-influenza hemagglutinin antibodies clone # 5D8 and TANA2)
3) FLAG peptide antigen (Wako Pure Chemical Industries, Ltd.)
4) His peptide antigen (Wako Pure Chemical Industries, Ltd.)
5) BGP peptide antigen (Genscript)

(Preparation of fluorescence measurement solution)
The sample after gel filtration of 18.9 μL and 926.1 μL of HKM fluorescent buffer (HKM buffer, 0.1% PEG8000, 0.005% Brij) were mixed, and 100 μL each was dispensed into 9 tubes in a 1.5 mL low adsorption tube. Then, add 1.01 μL of HKM buffer or DMSO only to the sample to which no antigen solution is added, add 1.01 μL of serially diluted antigen solution to the remaining sample, mix gently by vortexing, spin down, and spin at 25 ° C. Incubated for 1 hour. Thereafter, 95 μL of each fluorescently labeled protein sample was used for fluorescence measurement.

(Fluorescence measurement)
These fluorescently labeled protein samples were subjected to fluorescence intensity measurement using a fluorescence spectrophotometer (Fluorolog-3; manufactured by Horiba, Ltd.). When TAMRA was used as the fluorescent group, the excitation wavelength (Ex) was set to 550 nm, and the fluorescence intensity at the fluorescence wavelength (Em) of 565 to 700 nm was measured. When comparing the fluorescence intensity, the fluorescence intensity at 580 nm, which is the maximum wavelength, was used.

The measurement results of the fluorescence intensity of fluorescently labeled protein samples using various antibodies are shown in FIG. In each graph, the horizontal axis represents the antigen concentration, and the vertical axis represents the ratio of the fluorescence intensity at each antigen concentration to the fluorescence intensity without the antigen as the fluorescence intensity ratio.

As shown in FIG. 3, the fluorescent group was quenched in the absence of the antigen, but it was confirmed that the quenching of the fluorescent group was canceled and the fluorescence was emitted as the antigen concentration increased. The amplification rate of the fluorescence intensity after addition of the antigen is 1.75 times for the fluorescence-labeled protein sample using the anti-thyroxine antibody, and the anti-influenza hemagglutinin antibody (clone # 5D8). 1.4 times for fluorescent-labeled protein samples using PEG, 1.2 times for fluorescent-labeled protein samples using anti-influenza hemagglutinin antibody (clone # TANA2), 1.6 times for fluorescent-labeled protein samples using anti-FLAG antibody The fluorescence-labeled protein sample using the anti-His tag antibody was 1.2 times, and the fluorescence-labeled protein sample using the anti-BGP antibody-derived scFv was 2.7 times.

[Example 3] Measurement using N-terminal labeling agents having various linker lengths In the following formula (2), n in the linker group was changed to 2, 5, 7, 9 by the same method as in Example 1. Various N-terminal labeling agents were prepared. The N-terminal labeling agent when n = 2 is 5 (6) -TAMRA-X-C2-CHO, and the N-terminal labeling agent when n = 5 is 5 (6) -TAMRA-X-C5-CHO, n = The N-terminal labeling agent for 7 is described as 5 (6) -TAMRA-X-C7-CHO, and the N-terminal labeling agent for n = 9 is described as 5 (6) -TAMRA-X-C9-CHO.

In the same manner as in Example 2, fluorescent labeling of proteins was performed using N-terminal labeling agents having various linker lengths.

Fluorescent labeled protein samples of each linker length were reacted with an antigen, irradiated with excitation light having a wavelength of 550 nm, and the fluorescence spectrum was measured using a fluorescence spectrophotometer (Fluorolog-3).

FIG. 4 or FIG. 5 shows the measurement results of the fluorescence intensity of the fluorescently labeled protein samples of each linker length. FIG. 4 shows the results of using anti-FLAG antibody as an antibody solution and FLAG peptide antigen as an antigen, and FIG. 5 shows the results of using anti-His tag antibody as an antibody solution and His peptide antigen as an antigen. .

As shown in FIG. 4, when an anti-FLAG antibody is used as the antibody solution, when n = 2, 5, 7, or 9 in the above chemical formula (2), the fluorescence intensity of the fluorescence-labeled protein sample in the presence of the antigen It was confirmed that the change could be detected, and a large fluorescence intensity change was detected particularly when n = 5 and 7.

As shown in FIG. 5, when an anti-His tag antibody is used as the antibody solution, when n = 5, 7 in the above chemical formula (2), a high fluorescence intensity change of the fluorescence-labeled protein sample is present in the presence of the antigen. was detected.

[Example 4] Measurement using N-terminal labeling agent having various linker groups N-terminal labeling agents having a PEG group as a linker group were synthesized. This synthesis was the same as the synthesis of TAMRA-X-C2-CHO in Example 3 except that TAMRA-PEO8-SE (manufactured by Biotium) was used instead of 5 (6) -TAMRA-X-SE. I went there.

In the same manner as in Example 2, the protein was fluorescently labeled using an N-terminal labeling agent having a PEG group as a linker group. The antibody solution used was an anti-thyroxine antibody, and the antigen used was a thyroxine antigen.

A fluorescently labeled protein sample having a PEG group as a linker group was reacted with an antigen, irradiated with excitation light having a wavelength of 550 nm, and a fluorescence spectrum was measured using a fluorescence spectrophotometer (Fluorolog-3).

As shown in FIG. 6, it was confirmed that even in a fluorescent labeled protein sample having a PEG group as a linker group, a high fluorescence intensity change of the fluorescent labeled protein sample was detected in the presence of the antigen.

[Example 5] Measurement using N-terminal labeling agent having various fluorescent groups FAM (Molecular Probes), RhodamineRed (Molecular Probes), RhodamineGreen (Molecular Probes), BODIPY FL (Molecular) N-terminal labeling agents were synthesized using Probes), IC3 (Dojin Chemical or Biosearch Technologies), ATTO655 (ATTO Tech) or Cy3 (GE Lifescience). Anti-FLAG antibody was used as the antibody solution, and FLAG peptide antigen was used as the antigen. In the case of FAM, during the oxidation reaction to aldehyde, the concentration of the Dess-Martin reagent was reduced from 500 mM to 15 mM, and the reaction time was extended to 4 hours. Other conditions are the same as in Example 1 above.

Microspin® G25 column was equilibrated with a citric acid equilibration buffer (50 mM sodium citrate buffer (pH 4.8), 100 mM KCl, 0.1% PEG8000, 0.1% Brij35). 300 μL of HKM equilibration buffer was added to the column and centrifuged at 3000 rpm for 1 minute at room temperature. This operation was performed 4 times in total. After that, 1 mg / mL anti-FLAG antibody / 9FLAGμL and 50 mM sodium citrate buffer (pH 4.8) 9 μL mixed solution was added to the column and centrifuged at 3000rpm for 1 minute to exchange the buffer. . To the eluate, add 4.5 μL of N-terminal labeling agent (50% DMSO solution) with 0.96 mM fluorescent group and 4.5 μL of 192 m コリン picoline borane (18% DMSO solution) and incubate at 4 ℃ for 24 hours. A reductive alkylation reaction was carried out. Subsequently, the fluorescently labeled protein was eluted by gel filtration in the same manner as in Example 2.

Fluorescently labeled protein samples with various fluorescent groups are reacted with antigens, and the maximum absorption wavelength of each fluorescent group (TAMRA 550nm, FAM 495nm, RhodamineRed 570nm, RhodamineGreen 495nm, BODIPY FL 495nm, IC3 540nm, ATTO655 650nm, Cy3 540nm ) And the fluorescence spectrum was measured using a fluorescence spectrophotometer (Fluorolog-3). In each graph, the horizontal axis represents the antigen concentration, and the vertical axis represents the ratio of the fluorescence intensity at each antigen concentration to the fluorescence intensity without the antigen as the fluorescence intensity ratio.

As shown in FIG. 7, in fluorescently labeled protein samples having various fluorescent groups, the fluorescent group was quenched in the absence of antigen, but as the antigen concentration increased, the quenching of the fluorescent group was released, It was confirmed to emit fluorescence.

Also when N-terminal labeling agents having various fluorescent groups shown in FIG. 8 were used, fluorescently labeled protein samples were obtained.

Claims

The following general formula (1):

(In the formula, F is a fluorescent group, and R is a linear soft segment composed of a linear hydrocarbon having 5 or more carbon atoms, which may have a substituent, or a part of the main chain carbon. A linker group having a linear soft segment substituted with -O-, -N-, -S-, -CO-, -CONH-, -COO- or -POO-). Protein N-terminal labeling agent.
The fluorescent group is rhodamine, coumarin, oxazine, carbopyronine, cyanine, pyromesene, naphthalene, biphenyl, anthracene, phenanthrene, pyrene, carbazole, Cy, EvoBlue, fluorescein and The N-terminal labeling agent according to claim 1, which is at least one selected from the group consisting of these derivatives.
The N-terminal labeling agent according to claim 2, wherein the fluorescent group contains at least one selected from the group consisting of carboxytetramethylrhodamine, carboxyfluorescein, ATTO655 (registered trademark) and rhodamine green.
The N-terminal labeling agent according to any one of claims 1 to 3, wherein the protein is a complete antibody.
The N-terminal labeling agent according to any one of claims 1 to 3, wherein the protein is an antibody fragment.
A fluorescently labeled protein in which the fluorescent group is introduced at the N-terminus of the protein using the N-terminal labeling agent according to any one of claims 1 to 5.
A method for producing a fluorescently labeled protein, the step of reacting the N-terminal labeling agent according to any one of claims 1 to 5 with the N-terminus of a protein (a),
A method for producing a fluorescently labeled protein, comprising:
The method according to claim 7, wherein in the step (a), the N-terminal labeling agent and the protein are contacted in a solution, and the pH of the solution is from 3 to 7.
The method according to claim 7 or 8, wherein the protein in the step (a) is a complete antibody or a fragment thereof.
A kit for producing a fluorescently labeled protein,
A kit comprising the N-terminal labeling agent according to any one of claims 1 to 5.