JP2002014102A - Method of detecting protein, and method of detecting probe peptide and insulin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To readily detect an object protein in a solution without using chromatography, electrophoresis, immunoreaction and the like. SOLUTION: In this method, an object protein is detected using a probe peptide bonded with at least two molecules taking action relations of inclusion on and an included compound and immobilized on a solid phase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a technique for detecting a protein.

[0002]

2. Description of the Related Art Proteins are the main substances forming the basic structure of living organisms, and many proteins are contained in living organisms and substances derived from living organisms. The amino acid sequence of a protein in a living body is determined from the nucleotide sequence of a gene, and is extremely diverse. About 20 proteins
Although it is a polymer in which species of amino acids are linked linearly, its functions are various, such as structural proteins that form the skeleton of living organisms, and physiologically active substances that act as enzymes and hormones, and play an essential and important role in living organisms. I am carrying it.

[0003] Since proteins play an extremely important role in living organisms, confirmation of existence and function of proteins in living organisms is an important task. Among them, in recent years, techniques for treating proteins as disease marker substances have attracted attention. According to this technology, in the body of a patient affected by a certain disease, a protein specific to the disease is produced in the affected part, and the disease is diagnosed by examining the presence or amount of the protein by a blood test or the like. Technology. For example, typical tumor markers are also referred to as cancer markers and are proteins abnormally produced by in vivo tissue formation of cancer, such as AFP (α-fetoprotein), CEA (carcinoembryonic antigen), and BFP (base Fetal antigens), PIVKA-II (abnormal prothrombin) and the like are well known. Measuring the amounts of these proteins has made it possible to diagnose cancer. Specific proteins produced for various diseases other than tumor markers have been elucidated, and many of them have been applied to diagnosis and put to practical use.

[0004] The importance of specific protein detection technology has long been recognized and studied. However, the proteins that exist in the living body have an amino acid sequence determined from the base sequence of the gene, and are extremely diverse. They have a large molecular weight and are often structurally or chemically unstable. In many cases, only a very small amount is present, and the work of distinguishing and detecting them is often difficult. This is because proteins are usually composed of about 20 types of L-amino acids, and different types of proteins only differ in amino acid sequence. Therefore, when viewed macroscopically, the physical properties are extremely similar to each other, and the similarity brings difficulties in detecting and identifying a specific protein by a physicochemical method such as column chromatography.

[0005] On the other hand, methods for detecting a specific protein by a biological technique have been developed and put to practical use. As a typical technique, there is a technique that utilizes an antigen-antibody reaction in a living body. This technique uses an antibody that specifically binds to a target protein (antigen) to be detected, separates the obtained antigen-antibody complex, and labels previously bound to the antibody, for example, a fluorescent dye or iodine. This is a method for detecting a target protein by measuring a radioisotope such as 125. In particular, a method of labeling an antibody with an enzyme and causing color development is called an enzyme immunoassay, which is widely and generally used from a research level to a practical level.

[0006] However, although detection using antibodies has advantages such as specificity of detection and high sensitivity, the production of antibodies requires an immune response of rabbits, mice, goats, etc., and directly treats organisms. It has the disadvantage of requiring a lot of time and effort, such as being necessary. Therefore, there are many obstacles in terms of operation complexity and cost in performing industrial applications.

As a detection method by a biological technique without using an antibody, there is a method using a short-chain peptide having an affinity binding to a target protein. In this method, the detection of a target protein by an antibody is performed in the same manner by substituting a short-chain peptide having the same level of binding strength as that of the antibody, and the peptide is obtained by chemical synthesis without using an animal immune reaction. It has the advantage of being The detection of a protein with a short-chain peptide is often performed specifically as follows.

[0008] First, the amino acid sequence of a peptide that has an affinity for the target protein is determined. There are several methods for determining the amino acid sequence, such as a peptide library method in which a peptide library synthesized on the surface of a bead is used to determine an affinity-binding peptide by amino acid analysis, and a phage that infects Escherichia coli. There is a phage display library method in which an amino acid sequence is determined by genetic analysis using a peptide on the surface, and a peptide is synthesized based on the amino acid sequence obtained therefrom. The synthesized peptide is bound to the surface of a latex or polystyrene bead using a terminal functional group or the like, or to a solid phase such as a glass or plastic plate. If a peptide can be directly synthesized on a solid phase, it is used. The protein serving as the specimen is labeled in advance with a fluorescent dye such as fluorescein or rhodamine or a radioactive isotope such as iodine, and allowed to act on the peptide on the solid phase.
Peptides on the solid phase that bind affinity to the target protein include:
The target protein is affinity-bound, and a labeling agent such as a fluorescent dye bound to the protein is simultaneously immobilized on the solid phase.
Subsequently, the target protein can be detected by measuring the information signal obtained from the labeling agent on the solid phase, that is, the fluorescence intensity or radiation.

In the detection of a protein using a short-chain peptide, a peptide serving as a detection probe can be easily obtained as compared with an antibody or the like, and the physical and chemical stability is higher than that of an antibody. It has the advantage that it can be used in a form. Peptides also have the advantage of excellent productivity such as being able to be synthesized by an automatic synthesizer and mass production on an industrial scale, and their application as a sensor as a device has attracted attention in recent years. For example, JP-A-5-322901 describes detection of endothelin-1 using a 6-mer peptide. In the present invention, a 6-mer peptide that binds to a target protein with affinity is determined based on the amino acid sequence of a receptor that binds to the target protein with affinity and is used as a detection probe. Without using a specific protein.

[0010]

Here, when a target protein is detected using a peptide having an affinity binding to the target protein, a method for confirming whether or not the target protein has an affinity binding to the peptide is used. In the related art, a target protein is labeled with a fluorescent dye, a radioisotope, or the like, and information generated from the label is measured. However, labeling a specimen not only causes an increase in cost due to the complicated process itself, but also causes various obstacles as described below in terms of measurement accuracy and reliability.

[0011] The mechanism of affinity binding of a peptide as a probe to a target protein is complicated, but in general, based on properties such as the three-dimensional structure of the protein surface, charge, hydrophobicity or hydrophilicity, other than covalent bonding. It binds by intermolecular interaction. Labeling with fluorescent dyes, radioisotopes, etc.
Often performed for functional groups such as carboxyl groups and thiol groups, which block the affinity binding site of the peptide as a probe in the target protein or change the three-dimensional structure of the entire target protein molecule due to the binding of a label There is a risk of letting them do that. In the case of a protein having a plurality of sites that can be labeled, a plurality of labels may be formed on one molecule of the protein, which also affects the measurement accuracy. On the other hand, there may be a problem that the target protein is not labeled due to a lack of a labeling site or an obstacle such as inability to label due to steric structural restrictions. Conversely, protein molecules other than the target protein contained in the sample may be labeled, and may be non-specifically adsorbed to the peptide as a probe. Thus, molecules other than the target protein may be detected.

An object of the present invention is to solve these problems and to provide a simple protein detection method capable of accurately detecting a specific protein.

[0013]

Means for Solving the Problems As a result of repeated studies to solve the above-mentioned problems, the present inventors have found that a peptide which has an affinity binding to a target protein to be detected is located at an appropriate position. By using at least two or more molecules capable of taking an action relationship between inclusion and inclusion, and using a probe peptide immobilized on a solid phase such as a glass plate or a resin plate or a bead surface, We found that we could solve the problem.

That is, the present invention provides a method for detecting a target protein using a probe peptide immobilized on a solid phase, in which at least two or more molecules capable of taking an action relationship of inclusion and inclusion are bound. Detecting a change in the spectroscopic characteristics of the probe peptide caused by the probe peptide having an affinity binding to the target protein, or detecting the target protein by measuring an amount of the change. This is a method for detecting a target protein.

More specifically, this method comprises synthesizing a peptide that binds to a target protein with affinity, and forming at least two types of molecules capable of forming an inclusion complex at appropriate positions in the peptide molecule, At least two or more molecules having an action and molecules having an inclusion function are bound to each other, and a probe peptide immobilized on a solid phase is produced. An inclusion complex of a molecule having an inclusion function and a molecule having an inclusion function is formed in the molecule of the probe peptide. It is characterized in that the target protein is detected using the probe peptide forming the inclusion complex. In an environment where the target protein does not exist, the molecule having an inclusion effect and the molecule having an inclusion effect bound to the probe peptide still form an inclusion complex in the molecule. On the other hand, in the presence of the target protein, the probe peptide changes the three-dimensional structure so as to bind to the target protein, and the dissociation of the inclusion complex occurs accordingly. For example, when a fluorescent dye is used as a molecule having an inclusion function and cyclodextrin is bonded as a molecule having an inclusion function, in a state where the fluorescent dye is included in the cyclodextrin, the fluorescent molecule is located inside the cyclodextrin. It is placed in a hydrophobic environment, and its molecular movement is three-dimensionally restricted, and exhibits spectral characteristics according to the environment. Generally, a fluorescent substance has a property that the fluorescence intensity is increased when the surrounding environment is hydrophobic, and the fluorescence intensity is increased even if the molecular motion is restricted. On the other hand, when the inclusion is released due to a change in the three-dimensional structure of the probe peptide, the fluorescent dye is exposed to the external hydrophilic environment, and at the same time, the degree of freedom of molecular movement is increased, and the spectral characteristics are correspondingly increased. Also change.

Therefore, in the present invention, a sample solution containing the target protein is added to the probe peptide of the present invention immobilized on a solid phase such as a glass plate or a resin bead surface by utilizing the above-mentioned properties. The target protein in the sample is detected by detecting a change in spectral characteristics such as fluorescence intensity on a solid phase such as a glass plate or the like, or measuring the amount of the change. FIG. 1 shows a conceptual diagram of detection of a target protein using the molecule having an inclusion effect according to the present invention.

[0017]

DETAILED DESCRIPTION OF THE INVENTION Terms used in the present invention are defined as follows. The term “detection” shall mean not only the detection of the target protein in its original meaning, but also the measurement of the abundance, concentration, etc. of the target protein. The “probe peptide” refers to a peptide that has an affinity binding with a target protein to be detected and that has been chemically modified and used to detect a target protein. “Affinity binding of a probe peptide to a target protein” means that the probe peptide binds to the target protein by intermolecular interactions other than covalent bonds based on the surface structure, charge, hydrophobicity, or hydrophilicity of the target protein. Specifically binds, and has the same meaning as a term generally used in the life science field. When two molecules that can have an inclusion-inclusion action relationship, that is, a molecule having an inclusion action and a molecule having an inclusion action, bind to a peptide, those molecules are no longer originally called molecules but what are they? Although they are referred to as residues (for example, amino acids constituting a peptide are referred to as amino acid residues), in the present invention, they will be referred to as molecules for convenience. In the inclusion / inclusion action relationship, a molecule having an inclusion action is sometimes referred to as a “host”, and a molecule having an inclusion action is referred to as a “guest”. Molecule with inclusion function of “host” and “guest”
Is unified as a molecule having an inclusion function. The term “inclusion complex” refers to a state in which a molecule having an inclusion function and a molecule having an inclusion function are in an inclusion / inclusion action relationship.

The length of the peptide constituting the probe peptide of the present invention is 4 to 40 mer, preferably 5 to 20 m.
er, more preferably 7 to 18 mer. 25me
If it exceeds r, it takes a long time to synthesize the peptide, which adds complexity. It also adds complexity to the modification of peptides with molecules that have inclusion and inclusion functions.

The present invention is accomplished by the following steps. (1) A step of searching for a peptide having a strong affinity for the protein to be detected. (2) A step of synthesizing a probe peptide in which a molecule having an inclusion function and a molecule having an inclusion function are bound at appropriate positions. (3) A step of immobilizing the probe peptide on a solid phase. (4) A step of allowing the probe peptide immobilized on the solid phase to act on the target protein, and detecting the target protein from the change or the amount of change in the spectroscopic properties of the molecule having an inclusion function bound to the probe peptide . Further, if necessary, a step may be added between the above (3) and (4) in which a molecule having an inclusion function and a molecule having an inclusion function form an intramolecular inclusion complex. Although the probe peptide of the present invention originally forms an intramolecular inclusion complex when synthesized, it may not.

In the first step of the present invention, the search for a peptide that has an affinity for a target protein can be easily performed by, for example, a phage display library method. Phage display library method is B
A kit commercially available from ioLab, for example, Ph.D-1
2 By using a Pharge Display Peptide Library kit or the like and following a predetermined operation method, the amino acid sequence that binds to a specific protein can be known. In addition to the phage display library method, it is also possible to select a peptide having an affinity binding to a target protein by using a peptide library. The peptide library is obtained by artificially synthesizing a peptide of about 10 mer on a bead-shaped resin, and contains a peptide having any sequence of about 20 amino acids. 1
Since one kind of peptide exists in one bead, it is made to act on the target protein, select a bead resin with strong affinity, and analyze the amino acid sequence of the peptide on the beads. You can choose. The phage display library method requires the target protein to be adsorbed on a microtiter plate or the like, whereas the latter method using a peptide library allows manipulation with a free protein. In either method, there is a possibility that a plurality of peptides having affinity binding may be obtained. However, if necessary, one or a plurality of peptides may be selected.

Even if the above method is not carried out, if the peptide which binds to the target protein with affinity is known in the literature, the amino acid sequence may be determined based on the information. For example, if the recognition site, three-dimensional structure, recognition mechanism, etc. of the antibody or enzyme acting on the target protein are known, the amino acid sequence of the peptide that binds to the protein based on that information is determined. Is also good.

Next, a peptide is synthesized based on the amino acid sequence obtained as described above. It can be done in a conventional manner, which is commonly done. For example, F on resin
Examples of the method include a moc solid-phase synthesis method and a Boc solid-phase synthesis method. In the synthesis process, it is sufficient that a molecule having an inclusion function or a molecule having an inclusion function can be introduced into the peptide on the solid phase.
Peptide synthesis is performed with a sequence in which an amino acid having a preferred functional group is inserted as necessary depending on the bonding form, and then a molecule having an inclusion function and a molecule having an inclusion function may be bound to each functional group. . For example, when an amino group is to be introduced, lysine may be introduced, and when a thiol group is to be introduced, cysteine may be introduced. If synthesis is possible, a molecule other than an amino acid may be bound. The site of introduction of the molecule having an inclusion function or the molecule having an inclusion function is preferably at the end of the peptide in order to maintain the affinity binding site of the probe peptide to the target protein. Is not necessarily limited to the terminal, and may be introduced into the middle of the sequence of the peptide. In addition, if a linker having an appropriate molecular length or an appropriate length is required to facilitate inclusion, a necessary amino acid or the like may be separately introduced.

The molecules having an inclusion action include α, β,
Gamma cyclodextrin, crown ether, porphyrin and the like are generally known and can be used in the present invention together with derivatives of these compounds, but are not necessarily limited to these compounds. Of these, preferred examples include α, β, and γ cyclodextrins and crown ethers. In addition, as the molecule having an inclusion function, a compound which can take an inclusion function by interacting with a molecule having an inclusion function bound to a peptide, such as various dyes, dyes, and aromatic compounds, can be used. However, a compound whose spectroscopic properties change greatly depending on the form of interaction with a molecule having an inclusion function is desirable. Among these, azo dyes, anthraquinone dyes, fluorescent dyes and the like are preferable. Specific examples thereof include methyl red, fluorescein, rhodamine, acridine orange, and pyrene.

The step of immobilizing the probe peptide prepared as described above on a solid phase is as follows.
Known solid phases used in the prior art can be used as the solid phase. For example, a glass plate, a metal plate, a plastic plate made of a synthetic resin, or beads can be used. Among these, a particularly preferred one is a glass plate. Immobilization may utilize bonding such as adsorption to a solid phase, or may utilize covalent bonding to a solid phase using a functional group. When a covalent bond is used, for example, a maleimide group, a succinimide group, or the like is previously introduced into a solid phase such as a glass plate using a silane coupling agent, a cross-linking agent, and the like, and a probe is provided for these functional groups. A method in which amino groups and thiol groups of the peptide are reacted and covalently bonded may be employed. In this case, an appropriate linker may be interposed between the solid phase and the probe peptide. Examples of the linker include a polymer of ethylene glycol, a peptide chain, and a methylene chain. Among them, preferred is an ethylene glycol polymer. This addition of a linker is particularly suitable when the three-dimensional structure of the target protein changes from the structure of the solid phase when the probe peptide of the present invention and the target protein are affinity-bound.

The method of the present invention can be applied to any kind of target protein.
When affinity binding with the immobilized probe peptide, the three-dimensional structure of the target protein is hardly changed from the structure of the solid phase. For example, those having a tight three-dimensional structure, small molecules, linear peptides and the like can be mentioned. More specifically, low molecular proteins such as insulin, hormones and the like can be mentioned. However, it is not necessarily limited to this.

The step of forming an intramolecular inclusion complex between the molecule having the inclusion function and the molecule having the inclusion function is to place the probe peptide in a hydrophilic environment or to change the solvent composition. In general, since both the inclusion part of the molecule having the inclusion function and the molecule having the inclusion function often have hydrophobic properties, the probe peptide is placed in a hydrophilic environment, so Form a molecular inclusion complex.
However, otherwise, by changing the solvent composition, an intramolecular inclusion complex is formed. To confirm that the obtained probe peptide has an inclusion complex and a molecule having an inclusion complex with each other, an inclusion complex is formed in the molecule. Can be confirmed by measuring through a glass plate or the like which is a solid phase. Alternatively, the reflection spectrum of a solution containing the immobilized probe peptide may be measured. For example, the interior of clathrates such as cyclodextrins are in a hydrophobic environment, and the inclusions limit the movement of molecules sterically, resulting in different characteristics from those in a hydrophilic environment. A spectrum is obtained. You.

In order to detect a target protein using the immobilized probe peptide of the present invention, a sample solution containing the target protein may directly act on the probe peptide. When the sample solution is allowed to act on the probe peptide, it is desirable to perform the reaction under the same conditions as when the molecule having the inclusion function bound to the probe peptide and the molecule having the inclusion function form an intramolecular inclusion complex. But that is not the case.

As an example of the conditions, first, a solution such as a buffer solution is added to the immobilized probe peptide. By this operation, the molecule having the inclusion function and the molecule having the inclusion function bound to the probe peptide form an intramolecular inclusion complex. As the buffer, those generally used in the field of biochemical research, such as a phosphate buffer, a Tris-HCl buffer, and a Good's buffer, may be used. A preferred example is a phosphate buffer. The concentration of the buffer is 1-100 mM, preferably 10-100 mM. Its pH value is between 3 and 9, preferably between 6 and 8. Of course, the present invention can be achieved even in simple water such as distilled water.

In order to promote the affinity binding between the probe peptide and the target protein, it is generally preferable that the buffer or water does not contain a metal ion.
If the binding is not specifically inhibited, it will not be removed. Next, a sample solution containing the target protein is added.

When the target protein-containing specimen contains a protein that binds to the probe peptide with affinity,
The probe peptide changes the molecular structure so as to bind to the target protein with affinity. At the same time, the inclusion state (inclusion complex) of the molecule having the inclusion function and the molecule having the inclusion function that are bound to the probe peptide changes (dissociates),
The spectroscopic properties of the molecular compound having the inclusion function, for example, the light absorption and the fluorescence spectrum or the intensity thereof change. The measurement is performed before and after the sample is added, and the target protein contained in the sample is detected from the change or the amount of change. It is also effective to adjust the pH of the reaction system so that the light absorption intensity and the fluorescence intensity can be measured more strongly.

[0031]

EXAMPLES The present invention will be specifically described below with reference to examples. Example 1 Synthesis Example of Probe Peptide to which Pyrene and Cyclodextrin are Bound [1] Search for Peptide Affinity-Binding to Insulin
Phase Display Peptide commercially available from oLab
Performed with Library Kit. The kit used was 12mer
Is a library in which 2
× 10 ^{9 A} library containing ⁹ types of peptides having different sequences. As the insulin, a product commercially available from Sigma (Catalog No. I-0259) was used. Using this kit, a peptide having an affinity for insulin was searched according to the protocol attached to the kit. As a result, one 12-mer peptide was obtained. The amino acid sequence of the obtained one kind of peptide is shown in the following [SEQ ID NO: 1]. [SEQ ID NO: 1] (N-terminal) Trp-His-Trp-Pro-Trp-Tyr-Gln-Gly-Gln-Leu-
Trp-Pro (C-terminal)

[2] Synthesis of Peptide Having a Binding Site for Pyrene as a Molecule Having an Inclusion Function and Cyclodextrin as a Molecule Having an Inclusion Function The peptide of [SEQ ID NO: 1] includes pyrene and cyclodextrin. It must be bound and further bound to a solid phase. Therefore, to provide each binding site in the peptide of [SEQ ID NO: 1], an amino acid sequence (N-terminal) Gly-Gly-Gly-Glu-Glu-Ser-Cys (C-terminal) was added to the C-terminal of [SEQ ID NO: 1]. 18me having the amino acid sequence of [SEQ ID NO: 2]
The r peptide was synthesized. [SEQ ID NO: 2] (N-terminal) Trp-His-Trp-Pro-Trp-Tyr-Gln-Gly-Gln-Leu-
Trp-Pro-Gly-Gly-Gly-Glu-Ser-Cys (C-terminal)

The peptide was synthesized as follows. Fmoc-NH-SAL-Resin2 manufactured by Watanabe Chemical Industry
00 mg was swollen with NMP (N-methylpyrrolidone), and Fm was added with a 20% NMP solution of PPD (piperidine).
The oc group (fluorenylmethoxycarbonyl group) was removed. After washing the resin with NMP to completely remove PPD,
Fmoc amino acid, condensing agent PyBOP {benzotriazole 1-yloxy (trispirolidi) phosphonium hexafluorophosphate}, catalyst HoBt (N-hydroxybenzotriazole) 3 equivalents each, and diisopropylethylamine 6 equivalents were added, and the coupling reaction of amino acids was performed. went. Kaiser test (E.kaiser, et al., Anal. Biochem., 34, 595)
P. 1970) to confirm the introduction of amino acids.
When the residual amino group was confirmed by the Kaiser test, the reaction was carried out again and repeated until the amino group completely reacted. As the amino acid requiring side chain functional group protection, an amino acid protected with a protecting group that can be deprotected with trifluoroacetic acid was used as necessary. The procedure from deprotection of the Fmoc group to amino acid binding was repeated for each amino acid to synthesize the target peptide. Cleavage of the peptide from the resin is performed using m-cresol, thioanisole,
Performed using trifluoroacetic acid in the presence of ethanediol. Since each protecting group of the amino acid was deprotected by trifluoroacetic acid, all the protecting groups of each amino acid were removed at the same time as the cleavage from the resin. The excised peptide was precipitated in diethyl ether and washed repeatedly by decantation.

The obtained crude peptide was used for HPLC column W
The column was purified by an HPLC apparatus (Waters HPLC system) equipped with akosil DNA (Wako Pure Chemical Industries, Ltd.) so as not to exceed the retention amount of the column. As a developing solvent, a mixed solvent of water and acetonitrile was used in the presence of 0.1% TFA. The peptide was detected by measuring the absorbance at 220 nm and 280 nm to obtain 40 mg of the target purified peptide.

[3] Binding reaction of pyrene to the peptide of [SEQ ID NO: 2] (synthesis of probe peptide) Pyrene was bound to the N-terminal of the 17-mer peptide of [SEQ ID NO: 2]. 20 mg of the peptide was added to DMF (N,
N'-dimethylformamide) and dissolved sufficiently in 100 µl, and then 1-Pyrenebutanoic Acid Succinimidyl E
15 mg of ster was added. The reaction was carried out at room temperature for 6 hours with stirring. The reaction was followed by HPLC in the same manner as described above, and it was confirmed that a target compound having both pyrene group absorption (wavelength 340 nm) and peptide absorption (wavelength 215 nm) was formed. After confirming that the reaction had progressed for the most part, the column was purified in the same manner as described above by dividing the column so as not to exceed the holding capacity of the column.
15 mg of a purified peptide having pyrene bound to the end was obtained. The structure of the obtained compound was as shown in the following (a). It consists of elemental analysis and UV, IR, NM
R, CMR, and MS were measured, and their spectra were analyzed.

[0036]

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[4] Mono-6-O-ethylenediamine-
Synthesis of β-cyclodextrin (ethylenediamine cyclodextrin) The synthesis of β-cyclodextrin (hereinafter referred to as CD) derivatives was carried out according to the method of Ueno et al. (Akihiko Ueno et al., Cyclodextrin Basics and Applications, Sangyo Tosho, 1995). Β-CD was reacted with p-tosyl chloride in pyridine to synthesize monotosylated β-CD in which the hydroxyl group at the 6-position was tosylated at only one position. Subsequently, a DMF solution containing a large excess of ethylenediamine was placed in an oil bath at 60 ° C.
Reflux the DM of monotosylated β-CD while stirring.
The F solution (100 mg / ml) was slowly added dropwise. The reaction was monitored by TLC by coloring with concentrated sulfuric acid ("Guidance for Instrumental Analysis", Kagaku Dojin, 1986), and it was confirmed that the reaction had progressed almost completely in about 3 hours. After concentrating the reaction solution with an evaporator and distilling out most of the ethylenediamine, the remaining solution was slowly dropped into a large amount of stirred acetone to precipitate the desired product. Was obtained. Purification of ethylenediamine CD was performed using a desalted cation exchange gel (CM-25) column manufactured by Pharmacia. The structure of ethylenediamine CD is shown in (b) below. Its structure is based on elemental analysis and UV, IR, NM
R, CMR, and MS were measured, and their spectra were analyzed.

[0038]

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[5] Synthesis of Probe Peptide of the Present Invention 10 mg of the compound obtained in [3] and 5 mg of the compound obtained in [4] were dissolved in 500 μl of DMF, and it was confirmed that the compound was completely dissolved. And cooled in an ice bath. 5 mg of dicyclohexylcarbodiimide and 3 mg of HoBt were added, and the mixture was stirred in an ice bath for 2 hours, and then reacted at room temperature overnight.
After confirming the progress of the reaction by TLC, the reaction solution was poured into acetone to precipitate the desired product. The obtained crude product was dissolved in a mixed solvent of water and acetonitrile (2: 1 v / v), purified by HPLC in the same manner as in [2], and [Sequence 2]
7 mg of the probe peptide of the present invention in which pyrene and β-cyclodextrin were bonded to the peptide of No. 1 was obtained. This probe peptide is a compound shown in the following (c) in which the carboxyl group at the C-terminus of the synthesized product shown in the above (a) and the amino group of the ethylenediamine CD in the above (b) are acid-amide bonded. . It consists of elemental analysis and UV, IR,
NMR, CMR, and MS were measured, and their spectra were analyzed.

[0040]

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Example 2 Immobilization of Probe Peptide on Glass Plate [1] Surface Treatment of Glass Plate The treatment of a glass plate to be a solid phase is described in JP-A-11-18790.
It carried out according to the method of the Example described in No. 0 publication. That is, a 1 inch × 1 inch quartz glass plate was ultrasonically cleaned using a cleaning detergent and an aqueous alkaline solution. Subsequently, a silane coupling agent aqueous solution (K from Shin-Etsu Chemical Co., Ltd.)
An amino group was introduced into the glass surface by treating the surface of the glass with BM603). The glass plate treated with the silane coupling agent was fixed by baking sufficiently at 120 ° C. Subsequently, in a mixed solvent of DMSO (dimethyl sulfoxide) and ethanol (DMSO:
Ethanol = 1: 1 v / v), EMCS on glass plate
(N-succiimidyl 6-maleimidohexanoate)
(Dojin Co., divalent cross-linking agent) was reacted to obtain a glass plate having a maleimide bonded to the surface via a silane coupling agent and EMCS.

[2] Immobilization of Probe Peptide on Glass Plate Immobilization of the peptide on the glass plate was performed by bonding a thiol group, which is a side chain of the C-terminal cysteine of the peptide, to a maleimide group on the glass plate. . The peptide obtained in Example 1 was dissolved at a concentration of 3 mg / ml in a 1: 1 mixed solvent of water and acetonitrile, and a glass plate treated with [1] was immersed in 1 ml of the solution, and the solution was allowed to stand at room temperature. The reaction was performed for 2 hours. After the reaction, the glass plate was washed twice using a mixed solvent of DMSO and ethanol (DMSO: ethanol = 1: 1 v / v), finally washed once with ethanol, and air-dried.

Example 3 Measurement of Fluorescence Intensity of Glass Plate [1] Measurement of Fluorescence Intensity of Glass Plate The surface of the glass plate on which the probe peptide was immobilized was covered with a cover glass, and p
H7.4 10 mM phosphate buffer (PBS) was infiltrated. The fluorescence intensity of the probe peptide-immobilized surface of the glass plate was measured using an upright fluorescence microscope (manufactured by Nikon Corporation) connected to an image analysis device (trade name: ARGUS 50 manufactured by Hamamatsu Photonics KK). During the measurement,
A filter set suitable for pyrene fluorescence measurement was attached to a slot in the microscope, and the fluorescence intensity was measured according to a known method.

[2] Results As a result of the measurement using ARGUS 50, a constant fluorescence intensity could be measured. Since the target protein does not exist in the measurement system, this measurement is in a state in which pyrene is included in the cyclodextrin and is in a hydrophobic environment.

Example 4 Detection of Insulin (Target Protein) The insulin used in Example 1 was dissolved in the same PBS as described above to a concentration of 100 μg / ml. The immobilized surface of the probe peptide on the glass plate prepared in Example 2 was immersed in 1 ml of the insulin solution,
Left for hours. Thereafter, the immobilized surface was washed with PBS. Further, in the same manner as in Example 3, the fixed surface was covered with a cover glass, and the space between the fixed surface and the cover glass was infiltrated with the same PBS as described above. The fluorescence intensity of the probe peptide-immobilized surface was measured by a fluorescence microscope in the same manner as described above. From these results, it was found that insulin, which is the target protein, had affinity-bonded to the probe peptide immobilized on the glass plate. As a result, the inclusion complex of pyrene and cyclodextrin was dissociated, and the surrounding environment of pyrene became hydrophobic. It was found that the environment became hydrophilic.

Comparative Example 1 α-Chymotrypsin (Type II) manufactured by Sigma
(Catalog No. C-4129) was dissolved in PBS as described above to a concentration of 100 μg / ml. The fluorescence intensity was measured in the same manner as in Example 4 except that the prepared chymotrypsin solution was used instead of the insulin solution of Example 4. As a result, the same phenomenon as in Example 3 was observed. This result indicates that pyrene and cyclodextrin bound to the probe peptide still form an inclusion complex. Since the fluorescence intensity changes specifically for insulin, the protein detection method according to the present invention can be said to be a very effective detection method as a specific protein sensor.

[0047]

EFFECT OF THE INVENTION In the detection method for detecting a target protein by immobilizing a short-chain peptide on a solid phase, the method of the present invention makes it possible to easily detect the target protein without labeling it with a fluorescent dye or the like. became. In addition, since the detection method according to the present invention performs detection in a solid phase system,
In addition to easy B / F separation, the target protein contained in the sample can be concentrated on the solid phase, and the target protein contained in the sample can be diluted without labeling the target protein. It can be detected with high sensitivity.

[Brief description of the drawings]

FIG. 1 shows that a probe peptide of the present invention in which a molecule having an inclusion effect and a molecule having an inclusion effect are bound to a peptide having an affinity binding to a target protein and immobilized on a solid phase has an affinity for the target protein. It is a conceptual diagram showing that a three-dimensional structure is changed with sexual coupling.

[Explanation of symbols]

 101: Peptide 102: Solid phase 103: Molecule having inclusion activity 104: Molecule having inclusion activity (fluorescent dye) 105: Target protein

Claims (19)

[Claims] 1. A method for detecting a target protein by using a probe peptide to which at least two or more molecules capable of taking an action relationship of inclusion and inclusion are bound and immobilized on a solid phase. Detecting a change in the spectroscopic characteristics of the probe peptide caused by affinity binding of the probe peptide to the target protein, or detecting the target protein by measuring the amount of the change. A method for detecting a target protein. 2. The method according to claim 1, wherein the change in the spectroscopic properties of the probe peptide or the amount of the change is caused by a change in the molecular structure of the probe peptide caused by the affinity binding of the probe peptide to a target protein. 2. The method for detecting a target protein according to item 1. 3. The target protein according to claim 2, wherein the change in the molecular structure is a change in the inclusion state of at least two or more molecules in an operative relationship of inclusion and inclusion bound to the probe peptide. Detection method of detection. 4. The method for detecting a target protein according to any one of claims 1 to 3, wherein the molecule having an inclusion effect bound to the probe peptide is cyclodextrin. 5. The method for detecting a target protein according to claim 4, wherein the cyclodextrin is any one of α, β, and γ. 6. The method for detecting a target protein according to claim 1, wherein the molecule having an inclusion action bound to the probe peptide is a crown ether. 7. The method for detecting a target protein according to any one of claims 1 to 6, wherein the molecule having an inclusion function bound to the probe peptide is an aromatic compound. 8. The method according to claim 1, wherein the molecule having an inclusion function bound to the probe peptide is a dye.
2. The method for detecting a target protein according to item 1. 9. The method for detecting a target protein according to claim 8, wherein the dye is a fluorescent dye. 10. The method for detecting a target protein according to claim 7, wherein the aromatic compound is pyrene. 11. The protein detection method according to claim 1, wherein the solid phase on which the probe peptide is immobilized is a glass plate. 12. The method according to claim 1, wherein the solid phase to which the probe peptide is immobilized is a plate made of a synthetic resin. 13. The protein detection method according to claim 1, wherein the solid phase on which the probe peptide is immobilized is a synthetic resin bead. 14. The method for detecting a target protein according to claim 1, wherein the spectroscopic property is fluorescence or light absorption. 15. The probe peptide according to any one of claims 1 to 13. 16. The method for detecting a target protein according to claim 1, wherein the target protein to be detected is insulin. 17. A method for detecting insulin by using a probe peptide to which at least two or more molecules capable of taking an action relationship of inclusion and inclusion are bound and immobilized on a solid phase. 17. The method for detecting insulin according to claim 16, wherein the probe peptide chain has a peptide having the amino acid sequence of [SEQ ID NO: 1]. [SEQ ID NO: 1] (N-terminal) Trp-His-Trp-Pro-Trp-Tyr-Gln-Gly-Gln-Leu-
Trp-Pro (C-terminal) 18. The method for detecting insulin according to claim 17, wherein the two molecules capable of taking the relation of inclusion and inclusion are cyclodextrin and pyrene, respectively. 19. The probe peptide according to claim 17, which has the amino acid sequence of [SEQ ID NO: 2]. [SEQ ID NO: 2] (N-terminal) Trp-His-Trp-Pro-Trp-Tyr-Gln-Gly-Gln-Leu-
Trp-Pro-Gly-Gly-Gly-Glu-Ser-Cys (C-terminal)