JP2006266919A - Labeling method for measuring single protein molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new protein labeling method for controlling the number of labeling molecules per labeled protein molecule and an introduction region of the molecule, and labeling the protein molecule. <P>SOLUTION: The method includes a process for creating a fusing protein for containing a target protein and a binding protein for refining an affinity, a process for binding the fusion protein with an affinity binding group of an affinity carrier as the specific binding protein, a process for replacing the affinity binding group, binding an eluted material bound to the fusion protein with a labeling reagent and creating a labeling eluted material, and a process for reacting the labeling eluted material to the fusion protein, dissolving the fusion protein from the affinity carrier and creating the labeled fusion protein as the fusion protein labeled by the labeling reagent by binding the labeling eluted material. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for labeling a protein. More specifically, it is a method in which a labeled molecule can be controlled and introduced into a single protein molecule to be measured, and in particular, to a single molecule measurement technique such as fluorescence correlation spectroscopy (hereinafter abbreviated as FCS). The present invention relates to a labeling method useful for the application of the above.

  Biomolecule labeling methods are implemented in various ways depending on the type of biomolecule. There are various labeling modes, but a non-controllable mode in which an unspecified number of labeling reagents are randomly introduced at unspecified sites of a single target molecule, and a desired number of labeling reagents in a desired part of the target molecule. Can be roughly divided into control styles that can be controlled and introduced. The labeling method is appropriately implemented by selecting an appropriate method according to the purpose of labeling. That is, for the purpose of detecting a labeled molecule, the target molecule may be labeled and detected by a detection method based on the label. However, when considering the quantitative property of the labeled molecule, or when measuring the behavior of a single labeled molecule in a solution, it is preferable to achieve a more homogeneous labeling state using a control mode.

  In the case of labeling a nucleic acid, a primer or the like can be labeled during synthesis. At this time, it is possible to arbitrarily set which base in the primer is labeled and how many bases are labeled. It is possible to grasp how many labeled molecules exist in which region of one molecule of a PCR product to be measured by performing a PCR reaction using the primer thus prepared. As described above, when labeling a nucleic acid, there is a method capable of labeling by controlling the number of labeled molecules per molecule to be labeled, the introduction site, and the like.

  As a conventional technique for labeling proteins, for example, a labeling reagent active group in which an active group capable of forming a bond with a specific functional group and a labeling reagent such as a fluorescent substance are combined with a target protein to be labeled are randomly mixed. There are ways to label. In this case, the active group and the functional group of the protein that can react with the active group bind to each other, and as a result, a labeling reagent is introduced into the protein to achieve labeling. Known examples of the active group include N-hydroxyl succinimide (NHS) that reacts with an amino group and maleimide that reacts with a sulfhydryl group. Also known is a method of labeling by cross-linking a labeling reagent and a target protein via a crosslinker using various crosslinkers having similar active groups at both ends. However, as described above, these methods are all non-controlling methods for labeling proteins at random, and there is currently no method for controlling and labeling proteins.

  When a protein is labeled by an uncontrolled method, the active site of the target protein may be labeled because the labeling site is random. As a result, the activity of the labeled protein is inhibited, and it is extremely suspicious that the measurement result using the labeled protein gives accurate information.

  In addition, since labeling by a non-control method varies in the number of labeled molecules introduced into one protein molecule to be measured, single molecule analysis such as fluorescence correlation spectroscopy (hereinafter abbreviated as FCS). It is not preferable as an object to be analyzed by technology. This is because FCS is a method for measuring physical quantities such as the number of molecules and the size or shape of molecules using the Brownian motion of labeled molecules carried by the reaction behavior of molecules in a reaction vessel. Therefore, when the number of molecules of the labeling reagent to be introduced varies, the analysis result can be obtained only as an average of each molecule. Therefore, the protein labeling method of the prior art cannot fully exhibit the analysis ability inherent in FCS, which allows single molecule measurement.

In the prior art related to protein labeling, as described above, a method using a reagent in which a labeled molecule is bonded to an active group chemically bonded to a functional group, a method using a crosslinker, etc. It is a controlled way.
An object of the present invention is to provide a novel protein labeling means capable of controlling and labeling the number of labeled molecules per molecule of the protein molecule to be labeled and the introduction site in the molecule.
More specifically, it is to provide a protein labeling method particularly useful for application to a single molecule measurement technique such as fluorescence correlation spectroscopy (hereinafter abbreviated as FCS).

The present invention solves the problem by providing the following means. That is,
A method for labeling the protein while controlling the introduction of a labeling reagent in order to enable highly accurate single-molecule measurement of the protein,
Creating a fusion protein comprising a target protein portion to be measured and a binding protein portion used for affinity purification;
Binding the fusion protein to an affinity binding group of an affinity carrier to which the binding protein moiety can specifically bind;
Creating a labeled elution substance by binding a labeling reagent to an elution substance that displaces the affinity binding group of the affinity carrier and binds to the fusion protein;
Causing the labeled elution substance to act on the fusion protein bound to the affinity carrier to dissociate the fusion protein from the affinity carrier, and simultaneously bind the labeled elution substance to the dissociated fusion protein. Producing a labeled fusion protein in which the fusion protein is labeled with the labeling reagent;
A method comprising:

In addition, in order to enable highly accurate single molecule measurement of a protein, the protein is labeled while controlling the introduction of a labeling reagent,
By manipulating the amino acid residue at the desired position of the target protein to be measured by genetic engineering techniques, insertion of cysteine at that position, substitution of other amino acids with cysteine, substitution of cysteine with other amino acids Alternatively, deletion of cysteine results in the creation of a modified target protein portion so that the desired number of cysteines are retained in the desired position in the target protein, and the sulfhydryl group of the cysteine placed in the portion (target molecule) A step of making a disulfide bondable with another molecule via a sulfhydryl group),
Producing a fusion protein comprising the target protein portion and a binding protein portion used for affinity purification;
Producing a tag protein comprising a sulfhydryl group (tag protein sulfhydryl group) and a functional group that can be labeled with a labeling reagent;
Labeling the labelable functional group of the tag protein with a labeling reagent to form a labeled tag protein;
Binding the fusion protein to an affinity binding group of an affinity carrier to which the binding protein moiety can specifically bind;
The labeled tag protein is allowed to act appropriately on the fusion protein bound to the affinity binding group of the affinity carrier, and the target molecule sulfhydryl group and the tag protein sulfhydryl group are disulfide bonded to produce a labeled fusion protein. Process,
Cleaving the labeled fusion protein at an appropriate cleavage site and isolating a labeled fragment comprising at least the target protein portion and the tag protein;
A method comprising:

  Labeling can be performed by controlling the number of labeled molecules per molecule of the protein molecule to be labeled and the introduction site in the molecule. More specifically, a labeled protein that is particularly useful for application to single-molecule measurement techniques such as FCS can be produced.

The present invention controls the introduction of a labeling reagent so as to enable highly accurate single molecule measurement when applied to a protein labeling method that overcomes the above-described problems of the invention, particularly a single molecule measurement method such as FCS. Thus, a method for labeling a protein is provided. According to the present invention, since the number of label molecules for one molecule of the target protein is constant, it is possible to achieve a homogeneous label state. In addition, since the target protein is not directly treated with a chemical used in a labeling process such as a labeling reagent, there is almost no influence of the chemical. Furthermore, since the site to be labeled is determined in advance, introduction of a labeling reagent into an inappropriate site hardly causes changes in protein activity and / or conformation. Therefore, the measurement result using the protein labeled according to the present invention gives more accurate information.
Hereinafter, typical embodiments of the present invention will be exemplified and described in detail.

<Embodiment 1>
[Overview]
The outline of this embodiment is described below.
A fusion protein of a protein to be labeled and a binding protein used during affinity purification is prepared. This is concentrated with an affinity column to remove contaminants that do not bind to the column. Then, by using the labeled elution substance when eluting the fusion protein from the affinity column, the fusion protein bound to the affinity column is labeled by binding the labeled elution substance at the same time as elution from the affinity column. Method (see FIG. 1).
A detailed description of this embodiment is given below.

[Detailed description]
Each process of this aspect is demonstrated with reference to the description demonstrated below.
Step 1: A step of producing a fusion protein comprising a target protein part to be measured and a binding protein part used for affinity purification.
In this embodiment, the “fusion protein” is a fusion protein including at least a “target protein portion” and a “binding protein portion”, and other functional regions such as a region consisting of an amino acid sequence having affinity for a specific antibody. May also be included. The target protein portion is a protein to be labeled and actually measured. This protein may be the entire protein encoded by a specific gene, or a fragment thereof, and an appropriate region is appropriately selected according to the purpose.

  The binding protein portion is a protein that can specifically bind to an affinity binding group immobilized on an affinity carrier. That is, the binding protein portion and the affinity binding group are substances that form a pair that specifically binds. Specifically, both are substances that form a pair that specifically bind to each other, such as a substrate or coenzyme for an enzyme, a ligand for a receptor, and an antibody for an antigen. Furthermore, as the binding protein portion, a protein encoded by a nucleic acid that is any one of the pair and can be manipulated by genetic engineering can be used. The other of the pair corresponding to one of the pair used for the binding protein portion is the affinity binding group. The above definition will be described using an example of an enzyme protein that specifically binds to a non-protein substrate. Since the substrate in this case is not a protein, a fusion protein cannot be produced. Therefore, an enzyme is used as the binding protein. Therefore, the other paired substrate is automatically used as the affinity binding group.

  More specifically, the binding protein portion may be GST (glutathione S transferase), MBP (maltose binding protein), His tag (oligopeptide consisting of histidine) or the like. The affinity carrier will be described in detail in the section “Description of matters common to each aspect” described below.

  A person skilled in the art can produce the fusion protein of this embodiment by appropriately using various genetic engineering techniques. For example, the nucleic acid encoding the target protein portion can be obtained from a cDNA library or the like by PCR amplification using appropriate primers. Next, the nucleic acid is appropriately inserted into a vector for expressing various fusion proteins, and the vector into which the binding protein portion is incorporated. Various expression vectors incorporating GST or the like that can serve as the binding protein part are commercially available (for example, pGEX vector, Amersham Pharmacia), and a fusion protein can be prepared by appropriately incorporating a nucleic acid encoding a protein to be fused thereto. It may be created. The vector can then be introduced into a suitable host to express the fusion protein.

  Step 2: A step of binding the fusion protein to an affinity binding group of an affinity carrier to which the binding protein portion can specifically bind. This step is a step of binding the fusion protein expressed in the above step to an affinity binding group immobilized on an affinity carrier. This step may be performed according to a well-known method for affinity purification of protein. An example of the operation procedure briefly summarized is as follows.

A lysis operation for lysing a host cell expressing the fusion protein with an appropriate lysis buffer;
A clarification operation for removing the insoluble fraction of the cell lysate in which the cells are lysed by centrifugation or the like;
A binding operation in which the soluble fraction of the lysate is bound to a column packed with the affinity carrier, or bound by a batch method using the affinity carrier;
A washing operation of washing with an appropriate washing solution to remove contaminants non-specifically bound to the affinity carrier;

Subsequently, the process may be shifted to the next step, or the following operation may be added as desired when the fusion protein is once stored. That is,
Elution operation for eluting the fusion protein bound to the column with the eluent;
Buffer exchange operations such as gel filtration, dialysis, and ultrafiltration to eliminate the eluate mixed with the eluted fusion protein in the eluate;
Concentration operation for concentrating the fusion protein by ultrafiltration or the like.
Next, in order to store the fusion protein under appropriate conditions and perform the labeling of this embodiment again, this step is repeated from the beginning, that is, it is bound again to the affinity binding group and this step is terminated.

Step 3: A step of producing a labeled elution substance by binding a labeling reagent to an elution substance that substitutes the affinity binding group of the affinity carrier and binds to the fusion protein.
In this embodiment, the “labeled eluting substance” is created by binding the “labeling reagent” to the “eluting substance”.

    The eluting substance is a substance having a specific affinity for the binding protein portion and a substance having the same basic structure as the affinity binding group. Therefore, the elution substance as well as the affinity binding group is determined corresponding to the binding protein portion to be used. Therefore, if the binding protein portion is, for example, a GST, MBP, or His tag, glutathione, maltose, imidazole or a derivative thereof is used as the corresponding elution substance.

    The labeling reagent is a main body that is actually measured by the measurement means of the present invention described below. The “labeling reagent” and “measuring means” of the present invention will be described in the following section “Description of matters common to each embodiment”.

  In this step, a labeled elution substance is produced by binding a labeling reagent to the elution substance. This bonding can be performed using an active group such as succinimide and maleimide described above, or a crosslinker having these active groups at both ends. What is necessary is just to make it react on the conditions which use these reactive groups and a crosslinker on the conditions (for example, PIERCE) which supplies these.

  Moreover, it is preferable that selection of a reactive group and a crosslinker is suitably selected according to the available functional group which exists in the said elution substance. The usable functional group is generally a functional group in the elution substance that does not participate in binding with the binding protein moiety, and as a result, the labeled elution substance to which a labeling reagent is bound becomes the binding protein moiety. It is only necessary to retain specific affinity for. In addition, for example, a labeling reagent into which an active group has been introduced, such as FITC maleimide (PIERCE) in which maleimide has been introduced into FITC, which is a fluorescent reagent, is also commercially available. Good. For example, when glutathione is used as the eluting substance, the labeled eluting substance can be produced by labeling glutathione with FITC using the FITC maleimide (PIERCE).

  Further, at the end of this step, an operation of removing the labeling reagent that has not reacted with the eluting substance may be added.

  Step 4: The labeled elution substance is allowed to act on the fusion protein bound to the affinity carrier to dissociate the fusion protein from the affinity carrier, and at the same time, the labeled elution substance is applied to the dissociated fusion protein. Producing a labeled fusion protein in which the fusion protein is labeled with the labeling reagent by binding.

  In this embodiment, the “labeled fusion protein” is a protein in which the labeled elution substance is bound to the fusion protein, and as a result, the fusion protein is a protein labeled with the labeling reagent. This labeled fusion protein is used for actual measurement, and the labeling state in which the number of labeled molecules per molecule, the introduction site in the molecule, and the like, which is the subject of the present invention, is achieved. It is a protein.

  This step can be performed according to a method for eluting a substance specifically bound in affinity purification. Specifically, the labeled eluent may be allowed to act on the fusion protein bound to the affinity carrier to dissociate the fusion protein from the affinity carrier. This dissociation is a phenomenon that occurs when the labeled eluent substitutes for the solid-phase affinity binding group to which the binding protein portion is bound. Therefore, dissociation from the affinity carrier and elution into the solution means that the labeled elution substance is bound to the binding protein portion in the fusion protein. Therefore, the fusion protein eluted in this step is labeled with the labeling reagent.

  Furthermore, in this step, an operation of removing the unreacted labeled eluent by gel filtration, dialysis, ultrafiltration, or the like may be added.

<Embodiment 2>
[Overview]
The outline of this embodiment is described below.
Cysteine is added to the protein to be labeled by genetic engineering to enable disulfide bonds. In addition, a tag protein having a functional group capable of disulfide bond and labeling is produced by genetic manipulation or chemical synthesis. The tag protein is concentrated and labeled on the labelable functional group of the tag protein. Next, the protein to be labeled and the tag protein are labeled by bonding with a disulfide bond (see FIG. 2).
A detailed description of this embodiment is given below.

[Detailed description]
Each process of this aspect is demonstrated with reference to the description demonstrated below.
Step 1: Manipulating an amino acid residue at a desired position of a target protein to be measured by a genetic engineering technique to insert a cysteine at that position, replace another amino acid with cysteine, and replace another amino acid with cysteine Substitution or deletion of cysteine results in the creation of a modified target protein portion so that the desired number of cysteines are retained in the desired position in the target protein, and the sulfhydryl group of cysteine placed in the portion. A step of making a disulfide bondable with another molecule via (target molecule sulfhydryl group). This step is a step of creating the target protein portion by genetic manipulation so that cysteine is present at the position where the labeling reagent for the target protein is introduced.

    The target protein portion is a protein to be measured that the practitioner wants to label, and has been subjected to the operations described below for the DNA encoding the entire protein or a fragment thereof. That is, by manipulating the amino acid residue at a desired position of the target protein to be measured by a genetic engineering technique, at that position, cysteine insertion, substitution of another amino acid with cysteine, cysteine other amino acid As a result, a target protein portion modified so that a desired number of cysteines are retained at a desired position in the target protein is created by substitution or substitution of cysteine, and a sulfhydryl of cysteine placed in the portion is created. This is an operation to make a disulfide bondable with another molecule through a group (target molecule sulfhydryl group). If cysteine originally present in the target protein to be measured may be used, in this case, the unprocessed target protein that is not subjected to the above operation is used as the target protein portion.

Step 2: creating a fusion protein comprising the target protein portion and a binding protein portion used for affinity purification.
In this embodiment, the “fusion protein” is a fusion protein including at least a “target protein portion” and a “binding protein portion”, and other functional regions such as a region consisting of an amino acid sequence having affinity for a specific antibody. May also be included. As the binding protein portion, the same binding protein portion as defined in Embodiment 1 above can be used. However, the binding protein portion in this embodiment may be genetically manipulated with respect to cysteine in the same manner as the target protein described in Step 1 above. That is, as a result, a modified binding protein moiety is created that retains the desired number of cysteines in the desired position in the binding protein moiety. When the binding protein portion thus modified is used as the binding protein portion, the presence of cysteine in the whole fusion protein is controlled.

  This step creates a fusion protein of this embodiment in which the target protein portion and the binding protein portion are fused using genetic manipulation or the like. A person skilled in the art can produce the fusion protein of this embodiment by appropriately using various genetic engineering techniques. For example, it can be produced by the method described below. The nucleic acid encoding the target protein portion can be obtained from a cDNA library or the like by PCR amplification using appropriate primers. Next, the nucleic acid is appropriately inserted into a vector for expressing various fusion proteins, and the vector into which the binding protein portion is incorporated. Various expression vectors incorporating GST or the like that can serve as the binding protein portion are commercially available, and a fusion protein may be produced by appropriately incorporating a gene encoding a protein to be fused thereto. Next, this vector may be introduced into an appropriate host to express the fusion protein.

Step 3: A step of producing a tag protein having a sulfhydryl group (tag protein sulfhydryl group) and a functional group that can be labeled with a labeling reagent.
This step is a step of producing a tag protein. The tag protein is a protein having a sulfhydryl group (tag protein sulfhydryl group) and a functional group that can be labeled with a labeling reagent, or a fragment thereof, and preferably a chemically synthesized peptide. The labelable functional group is a functional group that can be introduced by covalently bonding a labeling reagent to the functional group, preferably a functional group that can react with the above-mentioned active groups such as succinimide and maleimide. . Accordingly, the labelable functional group is an amino group or a sulfhydryl group, preferably an amino group or a sulfhydryl group possessed by lysine, cysteine or the like.

    The labeling reagent is a main body that is actually measured by the measurement means of the present invention described below. The “labeling reagent” and “measuring means” of the present invention will be described in the following section “Description of matters common to each embodiment”.

Step 4: A step of labeling the labelable functional group of the tag protein with a labeling reagent to form a labeled tag protein.
In this step, a labeling tag protein is produced by binding a labeling reagent to a labelable functional group of the tag protein. This bonding can be performed using an active group such as succinimide and maleimide described above, or a crosslinker having these active groups at both ends. The conditions for using these active groups and the crosslinker may be reacted under the conditions recommended by the manufacturer (for example, PIERCE) supplying them. The selection of the active group and the crosslinker is appropriately selected depending on the labelable functional group of the tag protein.
Furthermore, at the end of this step, operations such as gel filtration, dialysis, and ultrafiltration may be added to remove the labeling reagent that has not reacted with the tag protein.

  Step 5: binding the fusion protein to an affinity binding group of an affinity carrier to which the binding protein moiety can specifically bind. What is necessary is just to implement this process similarly to the process 2 of the above-mentioned Embodiment 1. FIG.

  Step 6: The labeled tag protein is allowed to act appropriately on the fusion protein bound to the affinity binding group of the affinity carrier, and the target molecule sulfhydryl group and the tag protein sulfhydryl group are disulfide-bonded to form a labeled fusion protein. The process of creating.

  In this embodiment, the “labeled fusion protein” is a protein in which the labeled tag protein is disulfide bonded to the fusion protein, and as a result, the fusion protein is a protein labeled with the labeling reagent. The binding between the fusion protein and the labeled tag protein is achieved by disulfide bonding of both sulfhydryl groups. More specifically, this is achieved by disulfide bonding between the target molecule sulfhydryl group in the fusion protein and the tag protein sulfhydryl group in the tag protein. This binding is preferably achieved in a state where the fusion protein is bound to the affinity binding group of the affinity carrier, but may be achieved by reacting after the dissociation. Next, the labeled fusion protein is preferably washed with an appropriate washing solution to remove the labeled tag protein that has not reacted. The labeled fusion protein can then be eluted from the affinity carrier with the eluent and used for measurement. This labeled fusion protein is a protein that has achieved a labeling state in which the number of labeled molecules per molecule, the introduction site in the molecule, and the like, which are the subject of the present invention, are controlled.

  Step 7: cleaving the labeled fusion protein at an appropriate cleavage site, and isolating a labeled fragment containing at least the target protein portion and the tag protein. This step is a step of cleaving and eluting a portion essential for measurement while the labeled fusion protein is bound to the affinity carrier. In this embodiment, the labeled fusion protein produced in the above step 6 may be the subject of measurement, but the labeled fragment produced in this step may be the subject of measurement.

  In this embodiment, the “appropriate cleavage site” may be such that the labeled fragment containing at least the target protein portion and the tag protein is generated when cleavage is performed at that site. As described above, the labeled fragment is also a protein in which a labeling state in which the number of labeled molecules per molecule and the introduction site in the molecule are controlled, which is the subject of the present invention, is achieved. The cleavage site is a site containing a specific amino acid sequence that is recognized and cleaved by a protease, and may be introduced into a portion of the fusion protein to be cleaved by genetic manipulation or the like, preferably in the binding protein portion, more preferably It is introduced into the connecting portion between the binding protein portion and the target protein portion. As the protease, for example, a protease that recognizes a specific amino acid sequence such as Factor X and decomposes the sequence portion can be preferably used.

<Description of matters common to each aspect>
[Affinity carrier]
As the affinity carrier used in the present invention, a carrier having the affinity binding group immobilized thereon can be used. This carrier may be any carrier that is usually used for protein purification. For example, cross-linked products of various polysaccharides such as agarose and cellulose can be suitably used.

[Measuring means]
As the measuring means for measuring the protein labeled by the method of the present invention, a measuring means capable of measuring the “labeling reagent” used in the present invention described below can be used. In addition, one of the problems of the present invention is “to provide a protein labeling method useful for application to a single molecule measurement technique”, and single molecule measurement based on FCS, polarization, flow cytometry, scattered light, and the like. The technique is a suitable means for measuring proteins labeled according to the present invention.

  Next, an FCS suitable as a measuring means of the present invention will be described. The basic feature of FCS is that the concentration and intermolecular action of molecules labeled with a labeling reagent such as a fluorescent reagent contained in a homogeneous solution are averaged in a very small area under a microscope field. It is possible to monitor through the “fluctuation” of the light generated from the labeling reagent derived from the Brownian motion. Furthermore, it is also characterized in that it can be monitored in real time without going through a physical separation process. In this way, FCS is a technology that is expected to be applicable to a wide range of research subjects because it detects free molecular motion in solution.

The features of the FCS measurement unit will be described next. The FCS uses a confocal optical system excited by a laser beam as a sample measuring unit, captures light there by a detector, and then records and analyzes data by a digital correlator. Laser light as excitation light is concentrated at one point in the sample solution, and light from that point is captured by the detection system due to the characteristics of the confocal optical system. The actual measurement region in the solution is not an ideal point but a cylindrical region, and the size of the region is, for example, a diameter of about 400 nm, an axial length of about 2000 nm, and a volume of about femtoliter (10 ⁻¹⁵ L). is there. An object to be measured by the FCS is a solution, and the labeling reagent molecules present in the measurement region in the solution perform Brownian motion. Therefore, the number of molecules in a certain measurement region is not always constant, but fluctuates around a certain value, resulting in “number fluctuation”. Furthermore, due to this number fluctuation, “intensity fluctuation” occurs in the intensity of light emitted from the labeled reagent molecule to be measured. By analyzing the fluctuation of the light intensity, it is possible to obtain information on the diffusion rate and the number of molecules.

[Labeling reagent]
As the labeling reagent used in the present invention, a labeling reagent that can be measured by the measuring means can be used. When the measurement means is FCS, any labeling reagent that can be measured optically may be used, but a fluorescent reagent such as FITC, Cy3, Cy5, rhodamine, etc. can be preferably used. In addition, reagents using chemiluminescence or bioluminescence such as enzymes (for example, POD, ALP, luciferase) as luminescent reagents and substrates (for example, luminol / H ₂ O ₂ , AMPDD, luciferin) corresponding to these enzymes are used. May be. Further, when the measuring means is a measuring means based on polarized light, a reagent such as gold colloid or latex can be used.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1]
A protein labeling method according to Embodiment 1 is shown in this example.
In this example, glutathione S transferase (GST) is used as the binding protein, and c-Jun (1-79) is used as the target protein. That is, the fusion protein of Embodiment 1 is a fusion protein (GST-c-Jun) of glutathione S transferase (GST) and c-Jun (1-79).

<Method>
GST-c-Jun (1-79) fusion protein (UBI) was used in glutathione-conjugated beads and T-TBS [0.1% Triton X-100, TBS pH7.4 (10 mM Tris-HCl, 150 mM NaCl)]. The reaction is performed at 4 ° C. for 1 hour to specifically bind the fusion protein to the beads. The fusion protein that did not bind to the beads was removed by washing with T-TBS.

  On the other hand, reduced glutathione was labeled with FITC maleimide, and unreacted FITC maleimide was removed by column operation. This FITC labeling was performed using fluorescein maleimide (PIERCE) according to the instructions of PIERCE.

  A 10 mM FITC-labeled glutathione solution was added to the beads associated with the fusion protein and reacted at room temperature for 1 hour. As a result of this operation, glutathione immobilized on the beads is replaced with FITC-labeled glutathione, whereby one molecule of GST-c-Jun fusion protein is labeled with one molecule of FITC. At the same time, the GST-c-Jun fusion protein dissociates from glutathione on the beads, so that it is released from the beads and eluted in the solution.

  The solution from which the fusion protein was eluted was collected in another tube. Furthermore, in order to remove unreacted FITC-labeled glutathione, gel filtration was performed using a PD-10 gel filtration column (Amersham), and the FITC-labeled GST-c-Jun fusion protein was recovered.

<Result>
The results of measuring the FITC-labeled GST-c-Jun fusion protein produced in this way by FCS are shown in FIG.

[Example 2]
A protein labeling method based on Embodiment 2 is shown in this example.
In this example, the fusion protein of embodiment 2 is a fusion protein (GST-c-Jun) of glutathione S transferase (GST), which is a binding protein portion, and c-Jun (1-79), which is a target protein portion. -Cys). Similarly, the labeled tag protein of embodiment 2 is a FITC labeled peptide.

<Labeled protein>
First, a nucleic acid encoding c-Jun (1-79) is amplified by PCR. Next, the nucleic acid is inserted into a GST fusion protein expression vector (pGEX vector, Amersham Pharmacia) so that the reading frame at the time of translation is the same, and the GST-c-Jun (1-79) fusion protein is encoded. Create a fusion protein gene. Furthermore, the following modifications were made to produce GST-c-Jun-Cys, which is a target to be labeled in this example. Specifically, GST-c-Jun-Cys was created by substituting all cysteine residues present in the region encoding Jun amino acids 1-79 with methionine residues and further substituting the C-terminal amino acid with cysteine. Therefore, the cysteine residue in GST-c-Jun-Cys has the following (a) and (b).

(A) A cysteine residue present in the GST region. A cysteine residue present in this region is a cysteine residue originally present in the GST protein.
(B) One cysteine residue newly introduced at the C-terminus of the c-Jun region.
As explained below, the GST region in the GST-c-Jun-Cys fusion protein is cleaved by factor X and is not brought into the analysis step by FCS. Therefore, in this example, the target of the analysis is the cysteine labeling state (b). Since only one cysteine is present in (b), only the cysteine can react with the labeled tag protein of embodiment 2.

<Method>
Expression of the above GST-c-Jun-Cys fusion protein was induced using E. coli, which was collected by centrifugation and dissolved. After lysing the lysate, the supernatant was diluted with T-TBS and reacted with glutathione-conjugated beads for 1 hour at 4 ° C. Thereafter, the GST-c-Jun-Cys fusion protein was specifically bound to the beads, and the unreacted fusion protein was washed with T-TBS.

  On the other hand, a peptide consisting of one cysteine at the N-terminus, six histidines at the center, and one lysine at the C-terminus [C (6xH) K, where C is a cysteine, FI represents a ε-amino group of lysine to produce a FITC-labeled peptide. H represents histidine and K represents lysine.

  The FIST labeled peptide was reacted with the GST-c-Jun-Cys fusion protein bound to the beads, and the cysteine of the fusion protein and the cysteine of the FITC labeled peptide were disulfide bonded. Thereafter, the unreacted peptide was washed with T-TBS. Next, factor X is reacted with the factor X cleavage site present in the GST region, and the C-terminal portion of GST of the fusion protein is cut off. As a result of this treatment, a complex of c-Jun and a fluorescently labeled histidine peptide (FITC-labeled c-Jun protein) dissociated from the beads and was recovered.

<Result>
The results of measuring the FITC-labeled c-Jun protein thus prepared by FCS are shown in Table 2.

It is a figure which shows the method of Embodiment 1 of this invention. It is a figure which shows the method of Embodiment 2 of this invention. It is a graph which shows the result of having measured the protein labeled in Example 1 by FCS.

Claims (6)

A method for labeling the protein while controlling the introduction of a labeling reagent in order to enable highly accurate single-molecule measurement of the protein,
Producing a fusion protein comprising a target protein portion to be measured and a binding protein portion used for affinity purification;
Binding the fusion protein to an affinity binding group of an affinity carrier to which the binding protein moiety can specifically bind;
Creating a labeled elution substance by binding a labeling reagent to an elution substance that displaces the affinity binding group of the affinity carrier and binds to the fusion protein;
Causing the labeled elution substance to act on the fusion protein bound to the affinity carrier to dissociate the fusion protein from the affinity carrier, and simultaneously bind the labeled elution substance to the dissociated fusion protein. Producing a labeled fusion protein in which the fusion protein is labeled with the labeling reagent;
A method comprising:   2. The method according to claim 1, wherein the binding protein is GST, MBP, or His tag, and the eluent corresponding to the binding protein is glutathione, maltose, or imidazole, respectively. A method for labeling the protein while controlling the introduction of a labeling reagent in order to enable highly accurate single-molecule measurement of the protein,
By manipulating the amino acid residue at the desired position of the target protein to be measured by genetic engineering techniques, insertion of cysteine at that position, substitution of other amino acids with cysteine, substitution of cysteine with other amino acids Alternatively, deletion of cysteine results in the creation of a modified target protein portion so that the desired number of cysteines are retained in the desired position in the target protein, and the sulfhydryl group of the cysteine placed in the portion (target molecule) A step of making a disulfide bondable with another molecule via a sulfhydryl group),
Producing a fusion protein comprising the target protein portion and a binding protein portion used for affinity purification;
Producing a tag protein comprising a sulfhydryl group (tag protein sulfhydryl group) and a functional group that can be labeled with a labeling reagent;
Labeling the labelable functional group of the tag protein with a labeling reagent to form a labeled tag protein;
Binding the fusion protein to an affinity binding group of an affinity carrier to which the binding protein moiety can specifically bind;
The labeled tag protein is allowed to act appropriately on the fusion protein bound to the affinity binding group of the affinity carrier, and the target molecule sulfhydryl group and the tag protein sulfhydryl group are disulfide bonded to produce a labeled fusion protein. Process,
Cleaving the labeled fusion protein at a suitable cleavage site and isolating a labeled fragment comprising at least the target protein portion and the tag protein;
A method comprising:   The method according to claim 3, wherein the tag protein is a chemically synthesized peptide.   The method according to any one of claims 1 to 4, wherein the labeling reagent is FITC.   A method for measuring a single molecule of protein with high accuracy, wherein the protein labeled by the method according to any one of claims 1 to 5 is compliant with Fluorescence Correlation Spectroscopy (FCS). How to measure.

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