Protein tag, tagged protein, and protein purification method

The present invention relates to a protein tag, tagged protein, protein purification method, and the like. More particularly, it relates to a protein tag which insolubilizes a recombinant protein thereby enabling a convenient recovery and the like.

Conventionally, a recombinant protein is purified by allowing a target protein to be expressed as a fusion protein with an affinity tag and utilizing a specific affinity between the affinity tag and other molecule to thereby isolating the fusion protein from a soluble fraction. The affinity tag serves also to increase the solubility of the target protein to allow the fusion protein to be expressed as solubilized.

A commonly employed affinity tag may for example be His tag (histidine tag), GST tag (glutathione-S-transferase tag), MBP tag (maltose-binding protein tag), and FLAG tag. The His tag is a peptide containing about 6 to 10 histidine residues and specifically binds to a metal ion such as nickel ion. The GST tag and the MBP tag specifically bind to low molecular weight compounds such as glutathione and maltose, respectively.

For example, a solution of a protein tagged with the His tag is allowed to pass through a chelate resin having nickel ions immobilized thereon, onto which it is thus adsorbed. Subsequently, the nickel ion or a low molecular weight compound capable of binding to the nickel ion such as imidazole is allowed to pass through the resin, thereby allowing the fusion protein once adsorbed to be eluted and recovered.

The FLAG tag is a tag (epitope tag) utilizing an antigen-antibody reaction, and a protein tagged with the FLAG tag can be isolated from the soluble fraction by allowing it to bind to an anti-FLAG antibody. The epitope tag may otherwise be myc tag and HA tag, and the aforementioned His tag and GST tag can also be employed as epitope tags.

Such an affinity tag is designed also for allowing the fusion protein to be cleaved into a target protein and the tag upon a protease treatment thereby separating the tag from the target protein. For example, a fusion protein once adsorbed onto a resin via a tag is subjected to a protease treatment to separate the target protein and the tag from each other thereby allowing the target protein to be released and recovered exclusively from the resin.

Patent Document 1 discloses a technology for reacting a protein, which was labeled with a composite tag consisting of a His tag and a FLAG tag, specifically with a nickel-binding carrier and an anti-FLAG antibody-binding carrier thereby accomplishing separation and purification of the protein. Patent Document 2 also describes a method for recovering a target protein by using a peptide chain, which contains a cellulose-binding region of a cellulase, as an affinity tag and conduct a protease treatment to separate the tag from the fusion protein.

Patent Literature

[Patent Document 1] JP-A No. 2003-292500

[Patent Document 2] JP-A No. 1106-277088

Non-Patent Literature

[Non-Patent Document 1] “Human protein factory for converting the transcriptome into an in vitro-expressed proteome.” Goshima, N., et al., Nature Methods. 2008, Vol. 5, No. 12, p. 1011-1017

[Non-Patent Document 2] “Molecular Cloning, SECOND EDITION”, 1989, 17.37-17.41, Cold Spring Harbor Laboratory Press

[Non-Patent Document 3] “CURRENT PROTOCOLS IN MOLECULAR BIOLOGY”, 1990, Vol. 2 UNIT 16.5-16.5.4 CURRENT PROTOCOLS

Technical Problem

Since the protein purification of the prior art which employs an affinity tag isolates a fusion protein from a soluble fraction utilizing the specific affinity of the affinity tag, the fusion protein should be expressed in a soluble state and partitioned into the soluble fraction. It is also required that the affinity tag in the fusion protein retains its specific affinity with other molecules.

Nevertheless, some of target proteins are difficult to be solubilized, and the % solubilizability may vary substantially among proteins. In addition, the affinity tag in a fusion protein may sometimes be unsuccessful in exerting its affinity with other molecules sufficiently under the influence of the effect of the target protein.

Accordingly, in the protein purification of the prior art using the affinity tag, the protein yield depends on the solubility level (% solubilizability) of the fusion protein and the affinity level (% retained affinity) of the affinity tag, and the yield may sometime be extremely low. Also in the purification of the prior art, it is difficult to synthesize and purify numerous proteins globally regardless of their % solubilizabilities and the % retained affinities.

Accordingly, a major object of the present invention is to provide a technology which enables a high yield and convenient recovery of a protein and also enables a global protein purification.

Solution to Problem

When recombinant proteins are expressed, some become soluble and others become insoluble, depending on the type of the proteins to be expressed. Moreover, even a single type of the protein may sometimes be separated into 2 fractions, one being soluble fraction and the other being insoluble fraction.

As a result of our intensive studies, an insolubilizing tag was found, which insolubilizes expressed proteins regardless of the type of the proteins, and which allows the expressed proteins to be purified and recovered uniformly from the insoluble fraction. In addition, it was also found that the insolubilizing tag according to the present invention can be solubilized again by a surfactant, which is contained in a solvent for solubilizing a protein of an inclusion body, at a concentration lower than an ordinary concentration.

To overcome the problems described above, the present invention provides a protein tag comprising an amino acid sequence of the full length or a part of MafG protein or an amino acid sequence in which amino acids serving as protease cleavage sites were inserted into the former amino acid sequence.

According to this protein tag, a high insolubility attributable to MafG protein can be imparted to a protein to be tagged.

In this protein tag, the amino acid serving as a protease cleavage site may be arginine which is a trypsin cleavage site.

This protein tag typically comprises the amino acid sequence represented by SEQ ID NO:1 to 4.

Also the present invention provides a protein tagged with a protein tag comprising an amino acid sequence of the full length or a part of MafG protein or an amino acid sequence in which amino acids serving as protease cleavage sites were inserted into the former amino acid sequence.

This tagged protein can be utilized as a protein array formed by immobilizing a protein on a support.

Furthermore, the present invention provides a peptide obtained by a protease treatment of a protein tagged with a protein tag comprising an amino acid sequence in which an amino acid serving as protease cleavage sites were inserted into an amino acid sequence of the full length or a part of MafG protein.

Since the peptide obtained by a protease treatment of a protein tagged with a protein tag comprising an amino acid sequence in which an amino acid serving as protease cleavage sites were inserted can be designed in such a manner that the peptide derived from the tag can extremely be short, it is used preferably as standard peptides for mass spectrometry with regard to the protein subjected to the mass spectrometry.

Furthermore, the present invention provides a vector which expresses the aforementioned protein tag. This vector expresses a fusion protein between a target protein to be tagged and the protein tag.

Furthermore, the present invention provides a protein purification method comprising: a fusion protein preparation procedure for tagging a target protein with a protein tag comprising an amino acid sequence of the full length or a part of MafG protein or amino acids sequences in which amino acids serving as protease cleavage sites were inserted into the former amino acid sequence; and, a purification procedure for recovering the aforementioned target protein which has been tagged into an insoluble fraction.

Since the aforementioned protein tag allows a high insolubility attributable to MafG protein to be imparted to a target protein and allows the fusion protein to be insolubilized, the fusion protein can be recovered into the insoluble fraction at a high yield.

In this protein purification method, the aforementioned fusion protein preparation procedure is a procedure in which a vector expressing a fusion protein between the aforementioned target protein and the aforementioned protein tag is employed in a cell-free protein synthesis system (for example, wheat cell-free protein synthesis system) or a cell-associated protein synthesis system to synthesis the fusion protein. In addition, the aforementioned purification procedure is a procedure for centrifuging the aforementioned target protein which has been tagged.

Moreover, the present invention provides a method for purifying an antibody to a protein, the antibody purification method comprising using a fusion protein between the protein and a protein tag comprising an amino acid sequence of the full length or a part of MafG protein or an amino acid sequence in which amino acids serving as protease cleavage sites were inserted into the former amino acid sequence.

Advantageous Effects of Invention

According to the present invention, a technology which enables a high yield and convenient recovery of a protein and also enables a global protein purification is provided.

FIG. 1 is a view illustrating a protein tag (insolubilizing tag) and a tagged protein according to the invention.

FIG. 2 is a view illustrating an insolubilizing tag and the protease cleavage site of a tagged protein.

FIG. 3 is a view illustrating the design of the protease cleavage sites in an insolubilizing tag.

FIG. 4 is a view illustrating a fundamental sequence of a vector according to the invention.

FIG. 5 is a view illustrating an example of the procedure of the protein purification method according to the invention.

FIG. 6 is a view illustrating the principle of the measurement of a multiple reaction monitoring (MRM).

FIG. 7 is a view illustrating an example of the procedure of the antibody purification method according to the invention.

FIG. 8 is a drawing-substituting photograph showing the results of the purification of a signal transmission protein using an insolubilizing tag (Example 1).

FIG. 9 is a drawing-substituting graph showing the results of the evaluation of the fluorescence of the fusion protein between the insolubilizing tag and a fluorescent protein (Example 2).

FIG. 10 is a drawing-substituting graph showing the results of the evaluation of the enzymatic activity of the fusion protein between the insolubilizing tag and a dephosphorylase (Example 2).

FIG. 11 is a drawing-substituting graph showing the results of the evaluation of the enzymatic activity of the fusion protein between the insolubilizing tag and a phosphorylase (Example 2).

FIG. 12 is a drawing-substituting graph showing the results of the global protein purification using the insolubilizing tag (Example 3).

FIG. 13 is a drawing-substituting photograph showing the results of the detection of the autoantibody using the protein array made in Example 4 (Example 5).

FIG. 14 is a drawing-substituting photograph showing the results of the detection of the autoantibody using the protein array made in Example 4 (Example 5).

FIG. 15 is a drawing-substituting graph showing the results of the detection of the autoantibody using the protein array made in Example 4 (Example 5).

FIG. 16 is a drawing-substituting graph showing the results of the detection of the autoantibody using the protein array made in Example 4 (Example 5).

FIG. 17 is a view illustrating a vector employed in an E. coli cell expression system (Example 6).

FIG. 18 is a drawing-substituting photograph showing the results of the purification of a protein using the insolubilizing tag (Example 6).

FIG. 19 is a view illustrating the domain of the insolubilizing tag (Test Example 1).

FIG. 20 is a drawing-substituting photograph showing the results of domainization of the insolubilizing tag (Test Example 1).

FIG. 21 is a drawing-substituting photograph showing the results of the purification of a protein using an insolubilizing tag into which a protease cleavage site was inserted (Test Example 2).

Preferred embodiments of the present invention are described below with referring to the drawings. These embodiments are merely examples representing the present invention, and are not intended to allow the scope of the present invention to be interpreted narrowly. The description is made in the following order.

1. Protein tag (1-1) Insolubilizing tag (1-2) Designing of protease cleavage site 2. Protein purification method (2-1) Fusion protein preparation procedure (2-1-1) Vector (2-1-2) Expression system (2-2) Purification procedure 3. Tagged protein and its utilization (3-1) Tagged protein (3-2) Standard peptide for mass spectrometry (3-3) Antibody purification

1. Protein Tag

(1-1) Insolubilizing Tag

We employed a cell-free expression system (wheat germ cell-free system) to search for a highly insoluble protein globally. As a result, MafG protein was identified. MafG protein was found to have completely no solubility even when being expressed as a fusion protein with a solubilizing tag employed conventionally such as His tag, GST tag, and the like. MafG protein is one of transcription factors and consists of an amino acid sequence having 162 residues (SEQ ID NO:1). The base sequence of the coding region of MafG gene is represented by SEQ ID NO:5.

The protein tag according to the present invention comprises the full length or a part of the amino acid sequence of the aforementioned MafG protein. This protein tag constituted by comprising the full length or a part of the amino acid sequence of MafG protein imparts a high insolubility attributable to MafG protein to a protein to be tagged. Thus, the protein tag according to the present invention serves as “an insolubilizing tag” which is tagged to a soluble protein, thereby insolubilizing the whole tagged protein integrally (see FIG. 1 ). We also observed that even when a protein such as various enzymes such as kinase or phosphatase or fluorescent protein is expressed as a fusion protein with this insolubilizing tag the fusion protein retains the enzymatic activity or the fluorescence (see Example 2).

The amino acid sequence of MafG protein contained in the insolubilizing tag may be the entire sequence or a partial sequence of the amino acid sequence of MafG protein as long as MafG protein can retain a high insolubility. When a partial sequence is employed, then the partial sequence may be any portion of the full length of the amino acid sequence of MafG protein, and the number of the amino acid residues of the partial sequence is not limited particularly. In addition, the insolubilizing tag may have an amino acid sequence of any number of the residues in addition to the full length or a part of the amino acid sequence of MafG protein at its N terminal or C terminal, as long as it can retain the high insolubility exhibited by MafG protein.

In addition, the amino acid sequence of MafG protein contained in the insolubilizing tag is not limited to the amino acid sequence of human MafG protein (SEQ ID NO:1), and may be an amino acid sequence of the homolog of other species such as mouse or rat.

A typical amino acid sequence of the insolubilizing tag may for example be the amino acid sequence represented by SEQ ID NO:1. This amino acid sequence is identical to the full length of the amino acid sequence of human MafG protein. In addition, the amino acid sequence of the insolubilizing tag may be an amino acid sequence formed by adding 1 or 2 or more amino acids to the N terminal or C terminal of the amino acid sequence represented by SEQ ID NO:1.

In addition, the typical amino acid sequence of the insolubilizing tag may for example be the amino acid sequence represented by SEQ ID NO:2. This amino acid sequence is a partial sequence of human MafG protein, and corresponds to the 56th to 162nd residues from the N terminal of the full length. We observed that the portion of the 56th to 162nd residues from the N terminal in human MafG protein is attributable to the insolubility (see Test Example 1). In addition, the amino acid sequence of the insolubilizing tag may be an amino acid sequence formed by adding 1 or 2 or more amino acids to the N terminal or C terminal of the amino acid sequence represented by SEQ ID NO:2.

(1-2) Designing of Protease Cleavage Site

The insolubilizing tag according to the present invention, for the purpose of utilizing the tagged protein in the preparation of the standard peptide for mass spectrometry described below, preferably comprises an amino acid sequence in which amino acids serving as protease cleavage sites were inserted into the full length or a part of the amino acid sequence of MafG protein. FIG. 2 is a schematic view illustrating the protease cleavage sites of the insolubilizing tag and the tagged protein. The cleavage sites designed in the amino acid sequence of the insolubilizing tag are indicated with the dotted line, while the cleavage sites within the amino acid sequence of the target protein are indicated with the one-dot line. The cleavage sites in the insolubilizing tag indicated with the dotted line include both of the cleavage sites within MafG protein and newly provided cleavage sites.

The amino acid to be inserted can appropriately be selected depending on the type of the protease. For example, when using trypsin which has a cleavage specificity on the side of the carboxyl group of lysine and arginine, lysine or arginine is inserted. The proteases which may be used in addition to trypsin are Lys-C, Glu-C, Asp-N, chymotrypsin, V8, and the like.

While the position at which the amino acids serving as protease cleavage sites to be inserted into the full length or a part of the amino acid sequence of MafG protein is inserted is not limited particularly, it is preferable that the insertion is at a position allowing the length of the tag-derived peptide obtained after protease treatment to be 6 residues or less. Similarly, the number of the insertions of the amino acids serving as cleavage sites is not limited.

When the protease is trypsin, a preferable amino acid sequence of the insolubilizing tag may for example be the amino acid sequence represented by SEQ ID NO:3 (see FIG. 3 ). The amino acid sequence represented by SEQ ID NO:3 is a sequence in which 10 residues of arginine were inserted into the amino acid sequence represented by SEQ ID NO:2, and is a sequence obtained by altering the amino acid sequence represented by SEQ ID NO:2 so that the number of the amino acids between arginine and arginine becomes 6 residues or less. In the amino acid sequence shown in FIG. 3 , an underlined “R” represents arginine within wild-type MafG protein, while an arrowed “R” represents arginine which was inserted additionally (10 in total).

Also, the amino acid sequence represented by SEQ ID NO:4 is the amino acid sequence in which 17 residues of arginine were inserted into the amino acid sequence represented by SEQ ID NO:2, and is a sequence obtained by alteration for further reducing the number of the amino acids between arginine and arginine when compared with the amino acid sequence represented by SEQ ID NO:3. In the amino acid sequence shown in FIG. 3 , an arrowed “R” represents arginine which was further inserted additionally into the amino acid sequence represented by SEQ ID NO:3 (7 in total).

2. Protein Purification Method

(2-1) Fusion Protein Preparation Procedure

The protein purification method using the aforementioned insolubilizing tag is described. The protein purification method includes a fusion protein preparation procedure and a purification procedure.

In the fusion protein preparation procedure, a target protein to be purified is prepared as a fusion protein with an insolubilizing tag. The fusion protein is obtained by ligating a target protein-encoding DNA sequence to an insolubilizing tag-encoding DNA sequence, and introducing a vector constituted so that the target protein can be expressed as a fusion protein with the insolubilizing tag into expression system.

(2-1-1) Vector

FIG. 4 shows an example of the fundamental sequence of the vector. The vector shown in the figure has a constitution in which the amino acid sequence of the insolubilizing tag-encoding DNA sequence (tag sequence) is located upstream (5′ side) of the position at which the amino acid sequence-encoding cDNA of the target protein is inserted (open reading frames (ORFs)). Further upstream of the tag sequence, a transcription promoter sequence and a translation enhancer sequence are positioned. The transcription promoter sequence and the translation enhancer sequence which are exemplified here are SP6 promoter sequence and omega sequence employed frequently in a wheat cell-free protein synthesis system, respectively, to which they are not limited. It is also possible to ligate the tag sequence downstream (3′ side) of the ORFs. Thus, the insolubilizing tag can be used as an N-terminal or a C-terminal tag.

Around the tag sequence and the ORFs, a sequence for gene recombination may be provided. An example illustrated here has a restriction enzyme XhoI site upstream of the tag sequence and a KpnI site positioned downstream, as well as an attB1 site upstream of ORFs and attB2 positioned downstream.

(2-1-2) Expression System

As an expression system, a cell-free system or a cell-associated system can be employed, and a wheat cell-free expression system can especially be employed. Otherwise, the protein synthesis system of the cell-free system may for example be those of E. coli , insects, rabbit reticulocyte, and the like. The protein synthesis system of the cell-associated system may for example be those of E. coli , mammalian cells, insect cells, yeast, and the like.

When expressing a fusion protein in a cell-free system, first the aforementioned vector is subjected to a nucleic acid amplification such as PCR to amplify the target protein and protein tag-encoding DNA sequence (tagged cDNA). Then, from the tagged cDNA, an RNA (tagged RNA) is transcribed by in vitro transcription. Thereafter, the tagged RNA, in the form of a mixture fluid with the wheat cell extract, is subjected to an in vitro translation (see FIG. 5 ).

It is also possible to use, as a protein synthesis system of the cell-associated system, a known system employing E. coli , insect cells, and mammalian cell. Introduction of a gene into a cell can be conducted according to a known transfection method such as calcium phosphate method, electroporation, lipofection, microinjection, and the like.

(2-2) Purification Procedure

Subsequently, in a purification procedure, the target protein expressed as a fusion protein with the insolubilizing tag is recovered into an insoluble fraction. The target protein tagged with the insolubilizing tag is insolubilized integrally as a whole tagged protein. Accordingly, by centrifuging the mixture fluid after the in vitro translation in the cell-free expression system or the cell lysis fluid in the cell-associated expression system, the target protein can be pelleted down into an insoluble fraction, thereby accomplishing the recovery easily (see FIG. 5 ).

While the centrifugation can be conducted according to an ordinary method, a condition can be exemplified which employs 15,000×g, 20 minutes, and 4° C. In order to prevent migration of a soluble protein as a contaminant protein into the insoluble fraction, it is preferable that a surfactant such as Tween 20 is added to the solution subjected to the centrifugation.

It is also possible to use a filter to recover the target protein tagged with the insolubilizing tag into a filter cake fraction. The mixture fluid after the in vitro translation in the cell-free expression system or the cell lysis fluid in the cell-associated expression system are allowed to pass through a filter having a pore size of about 0.22 μm. As a result, the soluble protein which passes through the filter and the target protein which is trapped on the filter can be separated.

The insolubilizing tag according to the present invention can impart a high insolubility attributable to MafG protein to the target protein to be tagged, and, even a highly soluble target protein can be insolubilized integrally as a whole tagged protein. Accordingly, the protein purification method employing this insolubilizing tag enables, unlike to a method of the prior art employing an affinity tag, purification of the target protein at a high yield independently of the solubility level (% solubilizability) thereof.

Also in the protein purification method according to the present invention, the tagged target protein can be recovered by a convenient means such as centrifugation. Accordingly, this method enables, unlike to a method of the prior art employing an affinity tag, purification of the target protein at a high yield anytime regardless of the affinity level (% retained affinity) of the tag in the fusion protein.

In addition, the protein purification method according to the present invention enables purification even of a protein which was difficult to be purified by a method of the prior art due to low % solubilizability or % retained affinity, resulting in a possibility of global synthesis and purification of numerous proteins regardless of the % solubilizability or % retained affinity thereof.

The insolubilizing tag protein thus recovered can be solubilized by treatment with a surfactant, treatment with a protein denaturing agent 7M guanidine hydrochloride or 7M urea, or using acid or alkaline solution. Also, the tagged protein according to the present invention can be solubilized in the presence of sodium dodecyl sulfate (SDS) at a concentration of 0.04 to 1% (w/v) (see Test Examples 3 and 4).

Generally, the protein once insolubilized is solubilized again frequently by using a chaotropic salt having a high denaturing activity such as guanidine hydrochloride (Non-patent Document 2, Non-patent Document 3), or by using an SDS buffer containing 2% SDS or reducing agent for SDS-PAGE. Nevertheless, in a solvent containing the chaotropic salt or surfactant at a high concentration, the target protein may be denatured, and, if the target protein is an enzyme, the enzymatic activity may not be retained. Also in order to use a protein, which was solubilized by a solvent containing the chaotropic salt or surfactant at a high concentration, in a biochemical assay and the like while adding an enzyme such as a protease, it is required to reduce the concentration of the chaotropic salt or surfactant in the solvent by means of dilution, dialysis, ultrafiltration, and the like.

On the contrary, the tagged protein according to the present invention can be solubilized by SDS at a concentration for example of 0.04 to 1% (w/v). As a result, the tagged protein recovered into the insolubilized fraction can be used in the subsequent procedure without conducting complicated operations such as dilution, dialysis, ultrafiltration, and the like. The subsequent procedure means, for example, a protease treatment procedure in utilizing the tagged protein as a standard peptide for mass spectrometry or a procedure for binding the tagged protein to a protein array substrate, as described below.

3. Tagged Protein and its Utilization

(3-1) Tagged Protein

The tagged protein according to the present invention is obtained by the aforementioned protein purification method, and characterized by being tagged with an insolubilizing tag. The tagged protein has an insolubilizing tag ligated at least one of the N-terminal and the C-terminal of the target protein, depending on the structure of the vector employed.

Also the tagged protein according to the present invention can be solubilized from the state exhibiting the insolubility as described above. Accordingly, the present invention may also be a protein production method comprising: a fusion protein preparation procedure for tagging a target protein with a protein tag comprising an amino acid sequence of the full length or a part of MafG protein or an amino acid sequence in which amino acids serving as protease cleavage sites were inserted into the former amino acid sequence; a purification procedure for recovering the aforementioned target protein which has been tagged into an insoluble fraction; and a solubilization procedure for allowing the tagged target protein once recovered in the insoluble fraction to be solubilized again in a solvent. The solvent preferably contains SDS at a concentration of 0.04 to 1% (w/v).

According to the insolubilizing tag according to the present invention, numerous proteins can be synthesized and purified regardless of its % solubilizability and % retained affinity. Accordingly, by inserting a commercially available cDNA library into ORFs of a vector to make an expression clone library and conducting the protein expression and purification, a global protein library (in vitro proteome) can be prepared.

The protein library prepared as an in vitro proteome can be utilized for example in a protein array. A protein is solubilized using a surfactant such as sodium dodecyl sulfate (SDS), a denaturing agent such as guanidine hydrochloride or urea, or an acid or alkaline solution and then immobilized on a support such as a nitrocellulose membrane, and various array substrates to produce a protein array. The protein array can be utilized for example in screening autoantibodies in a serum.

(3-2) Standard Peptide for Mass Spectrometry

The tagged protein according to the present invention can be utilized in producing a standard peptide for mass spectrometry (internal standard peptide). In LC/MS/MS (Liquid chromatography/mass spectrometry/mass spectrometry), CE/MS/MS (capillary electrophoresis/mass spectrometry/mass spectrometry), and GC/MS/MS (gas chromatography/mass spectrometry/mass spectrometry), an analysis referred to as a selected reaction monitoring (SRM) and a multiple reaction monitoring (MRM) is conducted. Since SRM and MRM have extremely high specificities, they are excellent in quantifying ultratrace ingredients.

FIG. 6 shows the principle of measurement of ordinary MRM. In MRM, first, various ions ionized with ionization probes are subjected to the selection of a certain ion (precursor ion) in Q1 (first mass spectrometer). Then, the precursor ion is broken (collision-induced dissociation) in a collision cell (Q2) and a certain ion is detected from the broken ions (product ions) in Q3 (second mass spectrometer).

In MRM, multiple channels can be set in a single measurement, and certain types of proteins can exclusively detected from crude proteins (a crudely purified product) and quantified. The quantification of proteins by MRM can be accomplished by determining the absolute amount of a quantification target peptide based on the peak area ratio between the quantification target peptide and the internal standard stable isotope-labeled peptide together with the calibration curve.

For the purpose of a highly accurate quantification, a peptide having a high ionization efficiency should preliminarily be selected as an internal standard peptide from peptides produced from the protein which is the quantification target. Since the selection of the internal standard peptide is conducted by an actual measurement of the protein which is the quantification target while observing the MS/MS spectrum, a quantification target protein which has been purified for the actual measurement should be ready to use.

Also, the internal standard peptide is subjected to the measurement as being labeled with a stable isotope which enables differentiation from peptides derived from the quantification target protein in a crudely purified product (sample) of the proteins and also as being mixed with the sample. Therefore, it is required for MRM to make the internal standard peptide ready to use upon each measurement.

Since the tagged protein according to the present invention enables synthesis and purification of any protein regardless of the % solubilizability of the protein or the % retained affinity of the fusion protein, it can be utilized preferably as a protein for selecting the aforementioned internal standard peptide and as a protein for preparing the internal standard peptide. Thus, by subjecting a purified tagged protein to protease treatment and then to analysis by LC/MS/MS, an optimal internal standard peptide for MRM can be selected. Moreover, the protease treatment can be conducted while the tag is still being attached without removing the tag preliminarily from the tagged protein. Therefore, there is no need of operation for removing the tag once attached for purification from the purified tagged protein. In addition, the peptide mixture obtained by the protease treatment of the tagged protein labeled with the stable isotope can itself be utilized as an internal standard peptide for MRM.

When using the tagged protein according to the present invention as an internal standard peptide for MRM and the like, a less number of the amino acid residues constituting the protein tag is preferred. A larger number of the amino acid residues constituting the protein tag tends to give longer peptides contained in the protease digestion product, which allows the peptides detected in the mass spectrometry to interfere with the measurement of the target protein more easily. Therefore, it is preferable to use, as a protein tag, a portion containing SEQ ID NO:2 which is involved in the insolubility in the amino acid sequence of MafG protein. By using the amino acid sequence represented by SEQ ID NO:2 as a tag, the number of the amino acid residues of the protein tag can be reduced while retaining the insolubility of the protein tag.

Moreover, a tagged protein synthesized and purified using, as an insolubilizing tag, one which contains an amino acid sequence in which amino acids serving as protease cleavage sites were inserted into the full length or a part of the amino acid sequence of MafG protein (see SEQ ID NO:3, 4) can preferably be utilized.

The amino acids serving as protease cleavage sites have been inserted at the position allowing the length of the peptide obtained after the protease treatment as being derived from the insolubilizing tag (tag-derived peptide) to be 6 residues or less (see FIG. 2 ). By ensuring that the length of the insolubilizing tag-derived peptide obtained after the protease treatment is 6 residues or less, it is possible, in the internal standard peptide obtained by the protease treatment of the tagged protein, to distinguish the tag-derived peptide from the protein-derived peptide clearly. As a result, the risk that the tag-derived peptide is selected mistakenly as an internal standard peptide or that the tag-derived peptide serves as a noise during MRM measurement can be prevented.

For example, when using trypsin as a protease, the amino acid to be inserted into the amino acid sequence of the protein tag according to the present invention is preferably arginine or lysine. A larger amount of arginine or lysine serving as trypsin cleavage sites in the amino acid sequence of the protein tag results in a shorter length of the peptide derived from the protein tag. On the other hand, insertion of arginine or lysine which is a hydrophilic amino acid into an insoluble protein tag may results in a change in the characteristics of the protein tag.

Especially when the number of the arginine residues to be inserted into a tag protein consisting of 107 amino acid residues represented by SEQ ID NO:2 is less than 17, the length of the tag protein-derived peptide can be shorter while retaining the insolubility of the tag protein. Accordingly, the protein tag whose number of the arginine residues inserted into the amino acid sequence represented by SEQ ID NO:2 is less than 17 is employed preferably as a tag for use in purification of an internal standard peptide in mass spectrometry. Specifically, the number of the arginine residues inserted into the amino acid sequence represented by SEQ ID NO:2 is preferably 1 to 16, and especially 6 to 10.

(3-3) Antibody Purification

Also, the tagged protein according to the present invention can be utilized in antibody purification.

Referring to FIG. 7 , the procedure of the antibody purification is described. First, an antibody fluid containing an antibody to be purified (anti-protein T antibody) and a tagged protein T are mixed and centrifuged (for example, 15,000×g, 20 minutes, 4° C.). After centrifugation, the anti-protein T antibody is separated as a complex with the tagged protein T binding it into the sediment. Subsequently, an antibody dissociation buffer is employed to dissociate the anti-protein T antibody from the complex, and centrifugation is conducted again. As a result of this centrifugation, the tagged protein T turns into the sediment and migrates into the insoluble fraction, thereby enabling the recovery of the anti-protein T antibody in the supernatant.

The tagged protein according to the present invention allows all proteins to be synthesized and purified regardless of the % solubilizability of the protein and the % retained affinity of the fusion protein, thus allowing the purification of all anti-protein antibodies to be accomplished. While the separation of the protein-antibody complex by centrifugation was exemplified here, the complex can be separated also by a trap using a filter and the like. By treating the complex trapped on the filter with the antibody dissociation buffer, the anti-protein antibody dissociated from the complex can be solubilized and recovered.

A protein tag of the present invention allows a recombinant protein to be recovered at a high yield in a convenient manner, thereby enabling global protein purification. Accordingly, the protein tag according to the present invention can be utilized in basic and clinical researches using proteins, peptides, anti-protein antibodies, and the like in the medical, pharmaceutical, and biological fields.