WO2013150680A1 - プロテインタグ、タグ化タンパク質及びタンパク質精製方法 - Google Patents
プロテインタグ、タグ化タンパク質及びタンパク質精製方法 Download PDFInfo
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- WO2013150680A1 WO2013150680A1 PCT/JP2012/080133 JP2012080133W WO2013150680A1 WO 2013150680 A1 WO2013150680 A1 WO 2013150680A1 JP 2012080133 W JP2012080133 W JP 2012080133W WO 2013150680 A1 WO2013150680 A1 WO 2013150680A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
Definitions
- the present invention relates to a protein tag, a tagged protein, a protein purification method, and the like. More specifically, the present invention relates to a protein tag that enables simple recovery by insolubilizing a recombinant protein.
- the purification of a recombinant protein involves expressing the target protein as a fusion protein with an affinity tag, and using the specific affinity that the affinity tag exhibits with other molecules to remove the fusion protein from the soluble fraction. It is done by isolating.
- the affinity tag also functions to increase the solubility of the target protein and solubilize and express the fusion protein.
- a His tag (histidine tag), a GST tag (glutathione-S-transferase tag), an MBP tag (maltose binding protein tag), a FLAG tag, and the like are widely used.
- the His tag is a peptide containing about 6 to 10 histidine residues and specifically binds to a metal ion such as nickel.
- the GST tag and the MBP tag specifically bind to low molecular weight compounds such as glutathione and maltose, respectively.
- a protein solution tagged with a His tag is passed through a chelate resin immobilizing nickel ions and adsorbed. Subsequently, if a low molecular compound that binds to nickel ions or nickel ions such as imidazole is passed through the resin, the adsorbed fusion protein can be eluted and recovered.
- the FLAG tag is a tag (epitope tag) using an antigen-antibody reaction, and the protein tagged with the FLAG tag can be isolated from the soluble fraction by binding to the anti-FLAG antibody.
- Other epitope tags include myc tag and HA tag, and the above-mentioned His tag and GST tag can also be used as the epitope tag.
- these affinity tags are designed so that the fusion protein can be cleaved into the target protein and the tag by protease treatment so that the tag can be separated from the target protein. For example, if the fusion protein adsorbed to the resin through the tag is treated with protease and the target protein and the tag are cleaved, only the target protein can be released from the resin and recovered.
- Patent Document 1 discloses a technique for separating and purifying a protein by specifically reacting a protein labeled with a composite tag composed of a His tag and a FLAG tag with a nickel binding carrier and an anti-FLAG antibody binding carrier.
- Patent Document 2 describes a method in which a peptide chain containing a cellulose-binding region of a cellulolytic enzyme is used as an affinity tag, and the target protein is recovered by separating the tag from the fusion protein by protease treatment.
- the fusion protein is isolated from the soluble fraction using the specific affinity of the affinity tag. It is necessary to fractionate.
- the affinity tag in the fusion protein needs to maintain specific affinity with other molecules.
- the affinity tag in the fusion protein may not exhibit sufficient affinity with other molecules under the influence of the target protein.
- the protein recovery rate depends on the degree of solubility of the fusion protein (solubilization rate) and the affinity level of the affinity tag (affinity maintenance rate), and the recovery rate is In some cases, it would be very low.
- affinity maintenance rate affinity level of the affinity tag
- the main object of the present invention is to provide a technique that enables easy recovery of a protein in a high yield and enables comprehensive protein purification.
- proteins that are soluble and insoluble depending on the type of protein to be expressed.
- even one type of protein may be separated into two fractions, soluble and insoluble.
- the present inventors have found an insolubilized tag that insolubilizes the expressed protein regardless of the type of protein, and enables the expressed protein to be uniformly purified and recovered from the insoluble fraction. . Further, it has been found that the insolubilizing tag according to the present invention can be resolubilized at a lower concentration than the general concentration of the surfactant contained in the solvent for solubilizing the inclusion body protein. .
- the present invention provides a protein tag comprising a full-length or partial amino acid sequence of the MafG protein, or an amino acid sequence in which an amino acid serving as a cleavage site by a protease is inserted into the amino acid sequence.
- a protein tag comprising a full-length or partial amino acid sequence of the MafG protein, or an amino acid sequence in which an amino acid serving as a cleavage site by a protease is inserted into the amino acid sequence.
- high insolubility derived from MafG protein can be imparted to the protein to be tagged.
- the amino acid that becomes the cleavage site by the protease can be arginine that is the cleavage site of trypsin.
- the protein tag can comprise the amino acid sequences set forth in SEQ ID NOs: 1 to 4.
- the present invention also provides a protein tagged with a protein tag comprising the full-length or partial amino acid sequence of the MafG protein, or an amino acid sequence in which an amino acid serving as a cleavage site by a protease is inserted into the amino acid sequence.
- This tagged protein can be applied, for example, as a protein array in which the protein is immobilized on a support.
- the present invention provides a peptide obtained by subjecting a protein tagged with a protein tag comprising an amino acid sequence in which an amino acid serving as a cleavage site by a protease is inserted into the full length or a part of the amino acid sequence of the MafG protein to a protease. Also provide.
- the peptide obtained by protease treatment of a protein tagged with a protein tag containing an amino acid sequence inserted with an amino acid that becomes a cleavage site by a protease can be designed so that the peptide derived from the tag is very short, It is suitable as a standard peptide for mass spectrometry for proteins to be subjected to mass spectrometry.
- the present invention also provides a vector that expresses the protein tag.
- This vector expresses a fusion protein of a target protein to be tagged and a protein tag.
- the present invention tags the target protein with a protein tag comprising the full-length or partial amino acid sequence of the MafG protein, or an amino acid sequence in which an amino acid serving as a cleavage site by a protease is inserted into the amino acid sequence.
- a protein purification method comprising a fusion protein preparation procedure and a purification procedure for recovering the tagged target protein in an insoluble fraction.
- high insolubility derived from the MafG protein can be imparted to the target protein and the fusion protein can be insolubilized, so that the fusion protein can be recovered in a high yield in the insoluble fraction.
- the fusion protein preparation procedure is performed by using a cell-free protein synthesis system (for example, a wheat cell-free protein synthesis system) or a cell protein synthesis system by using a vector that expresses a fusion protein of the target protein and the protein tag. Used to synthesize the fusion protein.
- the purification procedure is a procedure of centrifuging the tagged target protein.
- the present invention is a method for purifying an antibody against a protein, wherein the protein and the full-length or partial amino acid sequence of the MafG protein, or an amino acid in which an amino acid serving as a cleavage site by a protease is inserted into the amino acid sequence
- a method for purifying an antibody which comprises using a fusion protein of a protein tag comprising a sequence.
- FIG. 10 is a drawing-substituting photograph showing the detection results of autoantibodies by the protein array prepared in Example 4 (Example 5).
- 10 is a drawing-substituting photograph showing the detection results of autoantibodies by the protein array prepared in Example 4 (Example 5).
- FIG. 9 is a drawing-substituting graph showing the detection results of autoantibodies by the protein array prepared in Example 4 (Example 5).
- FIG. 9 is a drawing-substituting graph showing the detection results of autoantibodies by the protein array prepared in Example 4 (Example 5). It is a figure explaining the vector used by E. coli cell expression system (Example 6).
- Protein Tag (1-1) Insolubilized Tag
- the inventors comprehensively searched for proteins showing high insolubility using a cell-free expression system (wheat cell-free system). As a result, MafG protein was found. It has been revealed that the MafG protein does not exhibit any solubility when expressed as a fusion protein with a solubilization tag used conventionally such as a His tag or a GST tag.
- MafG protein is one of transcription factors and consists of an amino acid sequence of 162 residues (SEQ ID NO: 1). The base sequence of the coding region of the MafG gene is shown in SEQ ID NO: 5.
- the protein tag according to the present invention comprises the full-length or partial amino acid sequence of the above-mentioned MafG protein.
- This protein tag including the full length or a part of the amino acid sequence of the MafG protein imparts high insolubility derived from the MafG protein to the tagged protein. That is, the protein tag according to the present invention functions as an “insolubilized tag” that is tagged with a soluble protein and insolubilized as a whole of the tagged protein (see FIG. 1).
- the present inventors have found that various proteins such as kinases and phosphatases, fluorescent proteins, etc., when these proteins are expressed as fusion proteins with this insolubilizing tag, the fusion proteins maintain enzymatic activity or fluorescence. (See Example 2).
- the amino acid sequence of the MafG protein contained in the insolubilization tag may be the entire sequence or a partial sequence of the amino acid sequence of the MafG protein as long as the high insolubility exhibited by the MafG protein can be maintained.
- the said partial sequence may be arbitrary parts of the amino acid sequence full length of MafG protein, and the number of amino acid residues of the said partial sequence is not specifically limited, either.
- the insolubilizing tag has an amino acid sequence of an arbitrary number of residues at the N-terminal or C-terminal in addition to the full-length or partial amino acid sequence of the MafG protein as long as the high insolubility exhibited by the MafG protein can be maintained. You may have.
- amino acid sequence of the 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 the amino acid sequence of other homologues including mouse and rat.
- amino acid sequence of the insolubilizing tag examples include the amino acid sequence shown in SEQ ID NO: 1. This amino acid sequence is identical to the full length amino acid sequence of human MafG protein.
- the amino acid sequence of the insolubilizing tag may be an amino acid sequence in which one or two or more amino acids are added to the N-terminal or C-terminal of the amino acid sequence shown in SEQ ID NO: 1.
- the specific amino acid sequence of the insolubilizing tag may be the amino acid sequence shown in SEQ ID NO: 2, for example.
- This amino acid sequence is a partial sequence of the human MafG protein and corresponds to 56 to 162 residues from the full length N-terminus. The present inventors have found that a portion of residues 56 to 162 from the N-terminal of human MafG protein contributes to insolubility (see Test Example 1).
- the amino acid sequence of the insolubilizing tag may be an amino acid sequence in which one or two or more amino acids are added to the N-terminal or C-terminal of the amino acid sequence shown in SEQ ID NO: 2.
- the tagged protein the entire amino acid sequence of the MafG protein or a part of Preferably, the amino acid sequence comprises an amino acid sequence in which an amino acid serving as a cleavage site by a protease is inserted.
- FIG. 2 is a schematic diagram showing the protease cleavage site of the insolubilized tag and the tagged protein.
- a cleavage site designed in the amino acid sequence of the insolubilizing tag is indicated by a dotted line, and a cleavage site inherent in the amino acid sequence of the target protein is indicated by a one-dot broken line.
- the cleavage site of the insolubilizing tag indicated by the dotted line includes both a cleavage site inherent in the MafG protein and a newly provided cleavage site.
- the amino acid to be inserted can be appropriately selected depending on the type of protease.
- protease having cleavage specificity on the carboxyl group side of lysine and arginine
- lysine or arginine is inserted.
- the protease LysC, GluC, AspN, chymotrypsin, V8 and the like can be used in addition to trypsin.
- the insertion position of the amino acid serving as the cleavage site by the protease inserted into the full length or a part of the amino acid sequence of the MafG protein is not particularly limited, but the length of the tag-derived peptide obtained after protease treatment is 6 residues or less It is preferable to be inserted at such a position. In addition, the number of amino acid insertions serving as cleavage sites is not particularly limited.
- a suitable amino acid sequence of the insolubilizing tag includes the amino acid sequence shown in SEQ ID NO: 3 (see FIG. 3).
- the amino acid sequence shown in SEQ ID NO: 3 is a sequence in which 10 residues of arginine is inserted into the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 2 has 6 amino acid residues between arginine and arginine. It is a sequence modified to be below the group.
- the underlined “R” indicates the arginine inherent in the wild-type MafG protein, and the “R” with an arrow indicates the arginine inserted and added (10). Indicates.
- the amino acid sequence shown in SEQ ID NO: 4 is a sequence obtained by inserting 17 residues of arginine into the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence between arginine and arginine as compared to the amino acid sequence shown in SEQ ID NO: 3. The sequence is modified so that the number is further reduced.
- “R” with an arrow indicates arginine (7) further inserted and added to the amino acid sequence shown in SEQ ID NO: 3.
- the protein purification method includes a fusion protein preparation procedure and a purification procedure.
- the target protein to be purified is prepared as a fusion protein with an insolubilizing tag.
- a fusion protein is obtained by connecting a DNA sequence encoding an insolubilizing tag to a DNA sequence encoding the target protein, and introducing a vector configured to express the target protein as a fusion protein with the insolubilizing tag into an expression system. .
- FIG. 4 shows an example of the basic sequence of the vector.
- the vector shown in the figure is a DNA sequence (tag sequence) encoding the amino acid sequence of the insolubilized tag upstream (5 'side) of the position where the cDNA encoding the amino acid sequence of the target protein is inserted (open reading frames (ORFs)).
- a transcription promoter sequence and a translation enhancer sequence are located further upstream of the tag sequence. Examples of the transcription promoter sequence and the translation enhancer sequence include, but are not limited to, an SP6 promoter sequence and an omega sequence often used in a wheat cell-free protein synthesis system, respectively.
- the tag sequence can be connected to the downstream (3 ′ side) of the ORFs. That is, the insolubilizing tag can be used as both an N-terminal tag and a C-terminal tag.
- a sequence for gene recombination may be provided before and after the tag sequence and ORFs.
- a restriction enzyme XhoI site is provided upstream of the tag sequence
- a KpnI site is provided downstream
- an attB1 site upstream of the ORFs is shown.
- (2-1-2) Expression system As the expression system, a cell-free system or a cell system can be used, and in particular, a wheat cell-free expression system can be used.
- Other cell-free protein synthesis systems include E. coli, insects, rabbit reticulocytes, and the like. Examples of cell-based protein synthesis systems include Escherichia coli, mammalian cells, insect cells, and yeast.
- a DNA sequence (tagged cDNA) encoding a target protein and a protein tag is amplified from the above vector by a nucleic acid amplification method such as PCR.
- RNA (tagged RNA) is transcribed from the tagged cDNA by in vitro transcription.
- in vitro translation is performed using the tagged RNA as a mixed solution with the wheat cell extract (see FIG. 5).
- a conventionally known system using Escherichia coli, insect cells and mammalian cells can be used.
- Introduction of a gene into a cell can be performed according to a known transfection method such as a calcium phosphate method, an electroporation method, a lipofection method, or a microinjection method.
- the target protein expressed as a fusion protein with the insolubilized tag is recovered in an insoluble fraction.
- the target protein tagged with the insolubilizing tag is insolubilized as a whole of the tagged protein. Therefore, if the mixed solution after in vitro translation in the cell-free expression system or the cell lysate in the cell expression system is centrifuged, the target protein can be pelleted down into the insoluble fraction and easily recovered (FIG. 5). reference).
- Centrifugation can be carried out according to a conventional method, and as an example, conditions of 15,000 ⁇ g, 20 minutes and 4 ° C. can be mentioned.
- a surfactant such as Tween 20
- the target protein tagged with the insolubilizing tag can be recovered in a filter-impermeable fraction using a filter.
- the mixed solution after in vitro translation in the cell-free expression system or the cell lysate in the cell expression system is passed through a filter having a pore size of about 0.22 ⁇ m.
- acquired by a filter are separable.
- the insolubilizing tag according to the present invention can impart high insolubility derived from the MafG protein to the target protein to be tagged, and even a highly soluble target protein can be insolubilized as a whole of the tagged protein. . Therefore, according to the protein purification method using the insolubilizing tag, unlike the conventional method using the affinity tag, the target protein can be purified at a high recovery rate without depending on the degree of solubility (solubilization rate).
- the tagged target protein can be recovered by a simple technique called centrifugation. Therefore, in this method, unlike the conventional method using an affinity tag, the target protein can be purified with a high recovery rate without any problem in the degree of affinity (affinity maintenance rate) of the tag in the fusion protein.
- proteins that were difficult to purify due to the low solubilization rate or affinity maintenance rate of the conventional method can be purified.
- Comprehensive synthesis and purification are possible regardless of the solubilization rate and affinity maintenance rate.
- the recovered insolubilized tag protein can be solubilized by treatment with a surfactant, treatment with a protein denaturing agent such as 7M guanidine hydrochloride or 7M urea, or an acid or alkaline solution.
- a protein denaturing agent such as 7M guanidine hydrochloride or 7M urea
- an acid or alkaline solution 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).
- SDS sodium dodecyl sulfate
- resolubilization of insolubilized proteins uses chaotropic salts with high denaturing power such as guanidine hydrochloride (Non-patent Documents 2 and 3), or about 2% SDS for SDS-PAGE.
- SDS buffer to which a reducing agent is added is used.
- the target protein may be denatured.
- the enzyme activity may not be maintained.
- the tagged protein according to the present invention can be solubilized, for example, at a concentration of 0.04 to 1% (w / v) SDS. Therefore, the tagged protein recovered in the insolubilized fraction can be used in the next procedure without performing complicated operations such as dilution, dialysis, and ultrafiltration.
- the next procedure is, for example, a protease treatment procedure in use of a tagged protein described later as a standard peptide for mass spectrometry, or a procedure for binding a tagged protein to a protein array substrate.
- Tagged protein and its application (3-1) Tagged protein
- the tagged protein according to the present invention is obtained by the above-described protein purification method, and is characterized by being tagged with an insolubilizing tag.
- the tagged protein has an insolubilizing tag linked to at least one of the N-terminus and C-terminus of the target protein depending on the structure of the vector used.
- the tagged protein according to the present invention can be solubilized from the insoluble state as described above.
- the present invention tags the target protein with a protein tag comprising the full-length or partial amino acid sequence of the MafG protein, or an amino acid sequence in which an amino acid serving as a cleavage site by a protease is inserted into the amino acid sequence.
- a fusion protein preparation procedure, a purification procedure for recovering the tagged target protein in an insoluble fraction, and a solubilization procedure for re-solubilizing the tagged target protein recovered in the insoluble fraction in a solvent It can also be a protein production method.
- a solvent containing SDS at a concentration of 0.04 to 1% (w / v) is preferable.
- a large number of proteins can be synthesized and purified without depending on their solubilization rate and affinity maintenance rate. Therefore, a comprehensive protein library (in vitro proteome) can be prepared by inserting a generally available cDNA library into an ORF of a vector to prepare an expression clone library, and performing protein expression and purification.
- a protein library prepared as an in vitro proteome can be applied to, for example, a protein array.
- Proteins are 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 immobilized on a support such as a nitrocellulose membrane or various array substrates.
- SDS sodium dodecyl sulfate
- a denaturing agent such as guanidine hydrochloride or urea
- an acid or alkaline solution an acid or alkaline solution
- Standard peptide for mass spectrometry The tagged protein according to the present invention can be applied to the production of a standard peptide for mass spectrometry (internal standard peptide). Selection 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) Analyzes called reaction monitoring (SRM) and multiple reaction monitoring (MRM) have been performed. Since SRM and MRM are extremely specific, they are excellent in quantitative properties of ultra-trace components.
- SRM reaction monitoring
- MRM multiple reaction monitoring
- FIG. 6 shows a general MRM measurement principle.
- MRM MRM
- specific ions precursor ions
- Q1 first mass spectrometer
- Q2 collision cell
- specific ions are detected from the broken ions (product ions) in Q3 (second mass spectrometer).
- MRM a plurality of channels can be set by a single measurement, and it is possible to detect and quantify only a few specific types of proteins from crude proteins (protein crude purified product).
- Protein quantification by MRM is performed by determining the absolute amount of the peptide to be quantified from the peak area ratio and calibration curve of the peptide to be quantified and the stable isotope-labeled peptide that is the internal standard.
- the internal standard peptide is labeled with a stable isotope so as to be distinguishable from the peptide derived from the protein to be quantified in the crude protein (sample), mixed with the sample, and used for measurement. For this reason, in MRM, it is necessary to prepare an internal standard peptide for each measurement.
- the tagged protein according to the present invention can be synthesized and purified for all proteins regardless of the solubilization rate of the protein and the affinity maintenance rate of the fusion protein. It can be suitably used as a protein for preparing a protein and a peptide for internal standard. That is, by purifying the purified tagged protein with protease and analyzing it by LC / MS / MS, it is possible to select an internal standard peptide optimal for MRM. Moreover, the protease treatment can be performed with the tag added without removing the tag from the tagged protein in advance. For this reason, the operation
- the number of amino acid residues constituting the protein tag is preferably small. This is because as the number of amino acid residues constituting the protein tag increases, the peptide contained in the protease digestion product tends to be longer, and the peptide detected in mass spectrometry tends to interfere with the measurement of the target protein. Therefore, in the amino acid sequence of the MafG protein, it is preferable to use a portion containing SEQ ID NO: 2 involved in insolubility as a protein tag. By using the amino acid sequence shown in SEQ ID NO: 2 as a tag, the number of amino acid residues of the protein tag can be reduced and the insolubility of the protein tag can be maintained.
- an insolubilizing tag synthesized using a tag comprising an amino acid sequence (see SEQ ID NOs: 3 and 4) in which an amino acid serving as a cleavage site by a protease is inserted into the full-length or part of the amino acid sequence of the MafG protein.
- the purified tagged protein can be suitably used.
- the amino acid that becomes the cleavage site by the protease is inserted at a position where the length of the peptide (tag-derived peptide) obtained after protease treatment derived from the insolubilized tag is 6 residues or less (see FIG. 2).
- the length of the insolubilized tag-derived peptide obtained after protease treatment is 6 residues or less, the peptide derived from the tag and the protein-derived peptide in the peptide for internal standard in which the tagged protein is treated with protease Clearly distinguishable.
- arginine or lysine is preferable as the amino acid inserted into the amino acid sequence of the protein tag according to the present invention.
- arginine or lysine which is a trypsin cleavage site
- the length of the peptide derived from the protein tag can be shortened.
- arginine or lysine which is a hydrophilic amino acid
- the properties of the protein tag may change.
- the number of arginine residues inserted into the tag protein consisting of 107 amino acid residues shown in SEQ ID NO: 2 is particularly less than 17, the length of the peptide derived from the tag protein is shortened while retaining the insolubility of the tag protein. be able to. Therefore, a protein tag in which less than 17 arginine residues are inserted into the amino acid sequence shown in SEQ ID NO: 2 is suitable as a tag used for purifying an internal standard peptide in mass spectrometry.
- the number of arginine residues inserted in the amino acid sequence shown in SEQ ID NO: 2 is preferably 1 or more and 16 or less, and particularly preferably 6 or more and 10 or less.
- the tagged protein according to the present invention can be applied to antibody purification.
- the antibody solution containing the antibody to be purified (anti-protein T antibody) and the 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 into the sediment as a complex bound to the tagged protein T. Next, the anti-protein T antibody is dissociated from the complex using an antibody dissociation buffer, and centrifuged again. By this centrifugation, the tagged protein T becomes a sediment and moves to the insoluble fraction, so that the anti-protein T antibody can be recovered in the supernatant.
- the tagged protein according to the present invention can be synthesized and purified for all proteins regardless of the solubilization rate of proteins and the affinity maintenance rate of fusion proteins, purification of all anti-protein antibodies can be realized.
- the complex can also be separated by trapping with a filter or the like. By treating the complex trapped by the filter with an antibody dissociation buffer, the anti-protein antibody dissociated from the complex can be eluted and recovered.
- Example 1 Purification of signal transduction proteins using insolubilized tags (wheat cell-free expression system)
- synthesis and purification of a fusion protein of an insolubilizing tag and a signal transduction protein according to the present invention were performed using a wheat cell-free expression system.
- Non-Patent Document 1 The method described in Non-Patent Document 1, using an entry clone in which an open reading frame (ORF) sequence of a signal transduction protein is cloned and a destination vector for insolubilizing tag fusion for a wheat cell-free expression system shown in FIG. Then, protein synthesis using a wheat germ extract (WEPRO7240, Cell Free Science) was performed.
- Table 1 shows the gene symbol of the signal transduction protein (Gene symbol), the accession number of the public database (GenBank: http://www.ncbi.nlm.nih.gov/genbank/) and the entry clone number (ID). ⁇ Shown in Table 3.
- the solution after synthesis was diluted 4 times with PBS in order to reduce contaminating proteins mixed during purification, and centrifuged at 15,000 ⁇ g for 20 minutes at 4 ° C. The supernatant fraction was removed and the resulting precipitate (insoluble fraction) was used as the purified protein fraction.
- protein synthesis was similarly performed using a destination vector for GST tag fusion.
- the synthesized solution was diluted 4-fold with PBS, adsorbed on glutathione resin (GE Pharmacia), and eluted with 0.8 M glutathione to recover the purified protein.
- FIG. 8A shows the results of SDS-PAGE of a protein purified using a GST tag.
- FIG. 8B shows the results of SDS-PAGE of the protein purified using the insolubilizing tag.
- bands can be confirmed for all proteins, and the bands of each protein are observed darker than FIG. 8A. From this result, it was shown that the protein can be purified at a high rate and in a high yield by using the insolubilizing tag as compared with the conventional purification method using the GST tag.
- Example 2 Purification of fluorescent protein or enzyme using insolubilizing tag
- the fusion protein of the insolubilizing tag according to the present invention and the fluorescent protein or enzyme is purified, and the purified fusion protein maintains fluorescence or enzyme activity. I confirmed.
- Example 2 In the same manner as in Example 1, a fusion protein of a fluorescent protein and an insolubilizing tag was synthesized.
- the fluorescent protein mVenus (Venus A206K, GenBank accession No. DQ092360.1) was used.
- FIG. 9 shows the results of measurement of fluorescence (excitation wavelength: 515 nm, fluorescence wavelength: 528 nm) of the crude protein solution.
- Example 2 a phosphatase and a fusion protein of a phosphatase and an insolubilizing tag were synthesized.
- DUSP3, PTPN1, and PTPN6 were used as phosphatases.
- tyrosine kinases WEE1 and Hck1 were used as phosphorylating enzymes.
- Table 4 shows the accession numbers of the public databases for each enzyme.
- the phosphatase activity was measured by the following method.
- the crude protein solution was centrifuged to obtain a purified protein fraction.
- a buffer solution 50 mM Tris-HCl, pH 7.5
- centrifugation were repeated twice to remove contaminating proteins.
- 3.0 ⁇ l of a buffer solution 50 mM Tris-HCl, pH 7.5
- the phosphatase activity was measured by absorbance measurement according to a conventional method using the suspension and pNpp chromogenic substrate.
- the tyrosine kinase activity was measured by the following method.
- the purified protein fraction obtained by centrifuging the crude protein solution in the same manner as in Example 1 was dissolved in LDS sample buffer (Life Technologies), then subjected to electrophoresis and Western blotting using NuPAGE electrophoresis system (Life Technologies), The autophosphorylation ability of tyrosine kinase was evaluated.
- An ECL-PLUS chemiluminescence detection kit using P-Tyr-100 antibody (mouse IgG, Cell Signaling) as the primary antibody and anti-mouse IgG antibody (sheep IgG, HRP label, GE Healthcare) as the secondary antibody Detection was performed using (GE Healthcare).
- Lane 1 is the crude protein fraction of untagged WEE1
- lane 2 is the crude protein solution of insolubilized tag-fused WEE1
- lane 3 is the centrifuged supernatant fraction of insolubilized tag-fused WEE1
- lane 4 is the insolubilized tag-fused WEE1. It is the purified protein fraction of the centrifuged precipitate.
- Lane 5 is a crude protein fraction of Hck1 that is not tagged
- Lane 6 is a crude protein solution of insolubilized tag fusion Hck1
- Lane 7 is a supernatant fraction obtained by centrifugation of insolubilized tag fusion Hck1
- Lane 8 is an insoluble tag fusion.
- Example 3 Exhaustive protein purification using insolubilizing tags
- exhaustive protein purification was performed using the insolubilizing tags according to the present invention.
- FIG. 12 shows the results of electrophoretic quantification of the purified protein by the protein fluorescence prelabeling method.
- the protein fluorescence prelabeling method was performed by introducing the fluorescent dye Cy5 into a lysine residue by amine coupling.
- a protein array was produced by binding a protein purified using an insolubilizing tag according to the present invention to a substrate.
- Example 2 In the same manner as in Example 1, an entry clone in which the ORF sequence of the protein shown in Table 5 was cloned was used to synthesize a fusion protein of the protein (see Table 5) and the insolubilized tag, and the precipitate (insoluble fraction) was centrifuged. Min) as a purified protein fraction.
- the purified protein fraction is suspended in a lysis solution (0.04% SDS (w / v), 0.1 M phosphate buffer (pH 7.8)) and mixed with shaking for about 1 minute.
- sonication high frequency output 160 W, 40 kHz
- Example 5 Detection of autoantibodies by protein array Using the protein array prepared in Example 4, autoantibodies in serum were detected.
- a human serum containing anti-TRIM21 antibody and anti-CT45A5 antibody was prepared as an autoantibody.
- This human serum was diluted 1000 times with TBST containing 3% (w / v) skim milk to obtain a primary antibody solution.
- the protein array produced in Example 4 was previously immersed in TBST containing 3% (w / v) skim milk at room temperature for 1 hour to block the substrate surface. After blocking, the surface of the substrate was immersed in the primary antibody solution and placed at room temperature for 1 hour while shaking the primary antibody solution. Thereafter, the substrate surface was cleaned using TBST.
- an anti-human IgG antibody labeled with the fluorescent dye Alexa647 is prepared as a secondary antibody, and this anti-human IgG antibody is mixed with 3% (w / v) skim milk.
- the secondary antibody solution was diluted 1000-fold with TBST.
- the surface of the substrate was immersed in the secondary antibody solution and placed at room temperature for 1 hour while shaking the secondary antibody solution. Thereafter, the substrate surface was cleaned using TBST.
- the excitation wavelength is set to 635 nm
- the SuperNHS protein array has a PMT of 420 V
- the FAST Slide substrate has a PMT of 200 V. It was measured.
- FIGS. FIG. 13 and FIG. 14 show the measurement results of fluorescence emitted from spots on the substrate where the proteins are attached.
- FIG. 13 shows the result of using SuperNHS as the substrate
- FIG. 14 shows the result of using FAST Slide 1-Pad as the substrate.
- 15 is a graph in which the fluorescence intensity of each spot shown in FIG. 13 is digitized
- FIG. 16 is a graph in which the fluorescence intensity of each spot shown in FIG. 14 is digitized. Note that the spot indicated by the arrowhead in FIG. 14 was excluded from the fluorescence measurement target because the protein was insufficiently bound to the substrate.
- secondary antibody-derived fluorescence was measured at the spots of TRIM21 and CT45A5 in which autoantibodies are contained in the serum.
- the measured fluorescence intensity was proportional to the amount of protein attached to the substrate. This indicates that the primary antibody is specifically bound to the protein bound to the substrate.
- the fluorescence intensity measured for the spot of MGLL which is a protein whose serum does not contain an antibody, was lower than the fluorescence intensity measured for the spot of TRIM21 or CT45A5.
- the spot of purified human IgG as a positive control
- the fluorescence intensity comparable to that of TRIM21 was measured.
- the spot of Venus as a negative control
- the measured fluorescence intensity was comparable to that of the above-mentioned MGLL spot.
- the protein purified using the insolubilizing tag according to the present invention was purified to a level suitable for specific detection by an antibody in the analysis using a protein array. Therefore, the protein purified using the insolubilizing tag according to the present invention can be used in a protein array by binding on a substrate.
- Example 6 Protein purification using insolubilizing tags (E. coli intracellular expression system)
- insolubilizing tags E. coli intracellular expression system
- a fusion protein of the insolubilizing tag and GST according to the present invention was purified using a colon cell expression system.
- FIG. 17A is a vector for synthesizing GST to which an insolubilizing tag is added.
- FIG. 17B shows a vector for synthesizing only GST.
- the vectors shown in FIGS. 17A and 17B were introduced into Escherichia coli to obtain transformed Escherichia coli.
- the transformed E. coli is cultured overnight at 37 ° C., and the resulting E. coli culture solution is diluted so that the OD 600 is 0.5, and then the concentration is 0.1% in this diluted solution.
- L-arabinose was added to initiate expression induction. Dilutions were collected immediately after expression induction (0 hour) and 3 hours after expression induction, and the cells were collected by centrifugation.
- FIG. 17 Three hours after expression, in the suspension obtained from Escherichia coli into which the vector shown in FIG. 17A was introduced, a band was observed at the position of molecular weight determined to be GST with an insolubilizing tag added to the C-terminal (FIG. 17). 18A, T). On the other hand, in the suspension obtained from Escherichia coli into which the vector shown in FIG. 17B was introduced, a band was observed at the molecular weight determined to be GST (see FIG. 18B and T).
- the insolubilizing tag according to the present invention insolubilized the fusion protein to which the insolubilizing tag was added even when the protein was synthesized using an E. coli cell expression system.
- domain A is amino acids 1 to 55 from the N terminus
- domain B is amino acids 56 to 109 from the N terminus
- domain C is 110 to 162 from the N terminus. Amino acids were used (see FIG. 19).
- a vector capable of expressing five types of insolubilizing tags consisting of the full length, only domain A, only domain A and domain B, only domain B, only domain B and domain C, and domain C was constructed. Methionine is added to the N-terminus of each insolubilizing tag.
- ORFs fluorescent protein mVenus or GST cDNA, which is a highly soluble protein, was inserted. Using these vectors, a fusion protein was synthesized in a wheat cell-free system, centrifuged, and then subjected to SDS-PAGE.
- FIG. 20 shows the result of comparison of the recovery amount among the 5 types of insolubilizing tags.
- the insolubilization tag (B + C) consisting of the amino acid sequences of domain B and domain C is recovered in the same amount as the insolubilization tag (MG) consisting of the full-length amino acid sequence. there were.
- the insolubilizing tag consisting of the amino acid sequence of only domain A (A), domain A and domain B (A + B), only domain B (B), and only domain C (C)
- the insolubilizing tag consisting of the full-length amino acid sequence The amount recovered was less than (MG).
- domain B and domain C in particular contribute to insolubility among the full-length amino acids of the MafG protein, and that the insolubilizing tag can function even if it is reduced to 107 residues of domain B and domain C.
- ⁇ Test Example 2> 8 Insertion of protease cleavage site
- an amino acid serving as a cleavage site by a protease was inserted into the amino acid sequences of domain B and domain C obtained in Test Example 1 to modify the amino acid sequence of the insolubilizing tag.
- the insolubilizing tag consisting of the full-length amino acid (SEQ ID NO: 1) of the MafG protein is version 1, version 2 (SEQ ID NO: 3) in which 10 arginines are inserted into the amino acid sequences of domain B and domain C, and 7 more arginines are inserted in version 2
- An insolubilizing tag of Version 3 (SEQ ID NO: 4) was prepared.
- FIG. 3 can be referred to for the specific amino acid sequence of each insolubilizing tag.
- the version 2 insolubilizing tag is obtained by inserting and adding arginine so that the length of the tag-derived peptide obtained after trypsin treatment is 6 residues or less.
- the insolubilizing tag of Version 3 is obtained by inserting and adding more arginine.
- Protein synthesis is performed in a wheat cell-free system using vectors capable of expressing fusion proteins of these insolubilizing tags and 12 types of proteins (see Table 6), and after centrifugation, SDS-PAGE is performed to recover the fusion protein. The amount was compared.
- FIG. 21A shows version 1
- FIG. 21B shows version 2
- FIG. 21C shows the result of SDS-PAGE of the fusion protein with the insolubilization tag of version 3.
- the Version 2 insolubilization tag a protein recovery rate equivalent to that of the Version 1 insolubilization tag was obtained.
- the Version 3 insolubilization tag the protein recovery rate was slightly reduced as compared with the Version 1 insolubilization tag. From this result, when the shortest amino acid sequence (107 residues) domained in Test Example 1 is used as the amino acid sequence of the insolubilizing tag, the preferred number of insertions of amino acids serving as cleavage sites by proteases is less than 17. It was suggested.
- Example 2 a fusion protein of a fluorescent protein mVenus (Venus A206K, GenBank accession No. DQ092360.1) and an insolubilized tag was synthesized using a wheat cell-free expression system.
- an entry clone entity clone ID FLJ40036AAAF
- the ORF sequence of immunoglobulin heavy chain gamma 3 constant region IgHG3, GenBank accession No. AK097355
- the suspension containing the synthesized tagged protein was centrifuged at 19,000 ⁇ g for 20 minutes at 4 ° C., and the tagged protein was recovered as an insolubilized fraction.
- any one of the solvents shown in Table 7 was added, mixed by shaking for 1 minute, and then subjected to ultrasonic treatment for 1 minute (high frequency output 160 W, 40 kHz) three times. It was.
- the suspension containing the tagged protein was centrifuged again at 19,000 ⁇ g for 20 minutes at 4 ° C., and the supernatant was used as a tagged protein lysate.
- tagged protein lysate SDS-PAGE was performed, the gel after electrophoresis was stained with Coomassie Brilliant Blue (CBB), and the color intensity of the band derived from the fusion protein was measured.
- CBB Coomassie Brilliant Blue
- a calibration curve showing the color development intensity of CBB with respect to the amount of protein was obtained using a BSA solution containing bovine serum albumin (BSA) at a predetermined concentration. Based on this calibration curve, the amount of tagged protein contained in the tagged protein lysate was calculated, and the proportion of tagged protein that was recovered in the insolubilized fraction and then solubilized was examined.
- BSA bovine serum albumin
- the solubilization rate in the solvents 16 to 20 when using a solvent having an SDS concentration in the range of 0.04 to 1% (w / v), the Venus recovered in the insolubilized fraction and Almost all fusion proteins with insolubilized tags were resolubilized (Table 8). Furthermore, as shown in the solubilization rate in solvents 21 to 24, the solvent containing SDS at a concentration of 0.04% (w / v) has a solubilization rate of the tagged protein at pH 7.8 to pH 9.2. The fusion protein of IgHG3 and the insolubilized tag recovered in the high insolubilized fraction was almost completely resolubilized (Table 8).
- Example 2 In the same manner as in Example 1, an entry clone in which the ORF sequence of the protein shown in Table 9 was cloned was used to synthesize a fusion protein of the protein (see Table 9) and the insolubilized tag, and the precipitate (insoluble fraction) was centrifuged. Min) as a purified protein fraction. The solvent 23 was added to the purified protein fraction in the same manner as in Test Example 3 to obtain a tagged protein solution. The quantification of the tagged protein contained in the lysate was performed in the same manner as in Test Example 3.
- the 28 types of proteins to which the insolubilizing tag was added (Table 9) were resolubilized with the solvent 23 in almost all amounts relative to the amount contained in the insolubilized fraction. From the results of this test example, the tagged protein to which the protein tag according to the present invention is added is recovered in the insolubilized fraction, and then a solvent containing SDS at a concentration of 0.04% (w / v) is used. It was confirmed that re-solubilization was possible.
- the recombinant protein can be easily recovered in a high yield, and comprehensive protein purification becomes possible. Therefore, the protein tag and the like according to the present invention can be used for basic research and clinical research using proteins, peptides, anti-protein antibodies and the like in the fields of medicine, pharmacy and biology.
Abstract
Description
このプロテインタグによれば、MafGタンパク質に由来する高い不溶性をタグ化されるタンパク質に付与できる。
このプロテインタグにおいて、プロテアーゼによる切断部位となるアミノ酸は、トリプシンの切断部位であるアルギニンとできる。
このプロテインタグは、具体的には配列番号1~4に記載のアミノ酸配列を含んでなるものとできる。
また、本発明は、MafGタンパク質の全長又は一部のアミノ酸配列、又は該アミノ酸配列にプロテアーゼによる切断部位となるアミノ酸が挿入されたアミノ酸配列、を含んでなるプロテインタグによりタグ化されたタンパク質を提供する。
このタグ化タンパク質は、例えばタンパク質を支持体に固定したプロテインアレイとして応用が可能である。
さらに、本発明は、MafGタンパク質の全長又は一部のアミノ酸配列にプロテアーゼによる切断部位となるアミノ酸が挿入されたアミノ酸配列を含んでなるプロテインタグによりタグ化されたタンパク質をプロテアーゼ処理して得られるペプチドをも提供する。
プロテアーゼによる切断部位となるアミノ酸が挿入されたアミノ酸配列を含んでなるプロテインタグによりタグ化されたタンパク質をプロテアーゼ処理して得られるペプチドは、タグ由来のペプチドが非常に短くなるように設計できるため、質量分析の対象とするタンパク質についての質量分析用標準ペプチドとして好適である。
上記プロテインタグによれば、MafGタンパク質に由来する高い不溶性を目的タンパク質に付与し、融合タンパク質を不溶化させることができるため、融合タンパク質を不溶画分に高い収率で回収できる。
このタンパク質精製方法において、前記融合タンパク質調製手順は、無細胞タンパク質合成系(例えばコムギ無細胞タンパク質合成系)又は細胞タンパク質合成系において、前記目的タンパク質と前記プロテインタグとの融合タンパク質を発現するベクターを用いて、該融合タンパク質を合成する手順とされる。また、前記精製手順は、タグ化された前記目的タンパク質を遠心分離する手順とされる。
1.プロテインタグ
(1-1)不溶化タグ
(1-2)プロテアーゼによる切断部位の設計
2.タンパク質精製方法
(2-1)融合タンパク質調製手順
(2-1-1)ベクター
(2-1-2)発現系
(2-2)精製手順
3.タグ化タンパク質とその応用
(3-1)タグ化タンパク質
(3-2)質量分析用標準ペプチド
(3-3)抗体精製
(1-1)不溶化タグ
本発明者らは、無細胞発現系(コムギ無細胞系)を用いて高い不溶性を示すタンパク質を網羅的に検索した。その結果、MafGタンパク質を見出した。MafGタンパク質は、HisタグやGSTタグ等の従来用いられている可溶化タグとの融合タンパク質として発現させた場合にも全く可溶性を示さないことが明らかになった。MafGタンパク質は、転写因子の一つであり、162残基のアミノ酸配列(配列番号1)からなる。MafG遺伝子のコーディング領域の塩基配列を配列番号5に示す。
本発明に係る不溶化タグは、タグ化されたタンパク質を後述する質量分析用標準ペプチドの作成に適用するため、MafGタンパク質のアミノ酸配列の全長又は一部のアミノ酸配列にプロテアーゼによる切断部位となるアミノ酸が挿入されたアミノ酸配列を含んでなることが好ましい。図2は、不溶化タグ及びタグ化タンパク質のプロテアーゼ切断部位を示す模式図である。不溶化タグのアミノ酸配列中に設計された切断部位を点線で、目的タンパク質のアミノ酸配列に内在する切断部位を一点破線でそれぞれ示す。点線で示す不溶化タグの切断部位には、MafGタンパク質に内在する切断部位と、新たに設けられた切断部位との両方が含まれる。
(2-1)融合タンパク質調製手順
上述の不溶化タグを用いたタンパク質精製方法について説明する。タンパク質精製方法は、融合タンパク質調製手順と精製手順とを含む。
図4に、ベクターの基本配列の一例を示す。図に示すベクターは、目的タンパク質のアミノ酸配列をコードするcDNAが挿入される位置(オープンリーディングフレーム(ORFs))の上流(5´側)に、不溶化タグのアミノ酸配列をコードするDNA配列(タグ配列)が配置された構成を有している。タグ配列のさらに上流には、転写プロモーター配列と翻訳エンハンサー配列が位置している。転写プロモーター配列及び翻訳エンハンサー配列は、ここではそれぞれSP6プロモーター配列及びコムギ無細胞タンパク質合成系でよく用いられるオメガ配列を例示するが、これらに限定されない。また、タグ配列は、ORFsの下流(3´側)に繋ぐこともできる。すなわち、不溶化タグは、N末端タグとしてもC末端タグとしても使用できる。
発現系には、無細胞系あるいは細胞系を用いることができ、特にコムギ無細胞発現系を用いることができる。無細胞系のタンパク質合成系としては、他に、大腸菌、昆虫、ウサギ網状赤血球などが挙げられる。細胞系のタンパク質合成系としては、例えば、大腸菌、哺乳類細胞、昆虫細胞、酵母等が挙げられる。
次に、精製手順において、不溶化タグとのフュージョンタンパク質として発現された目的タンパク質を不溶画分に回収する。不溶化タグによりタグ化された目的タンパク質は、タグ化タンパク質全体として不溶化する。従って、無細胞発現系におけるインビトロトランスレーション後の混合液又は細胞発現系における細胞溶解液などを遠心分離すれば、目的タンパク質を不溶画分にペレットダウンして簡単に回収することができる(図5参照)。
(3-1)タグ化タンパク質
本発明に係るタグ化タンパク質は、上述のタンパク質精製方法によって得られ、不溶化タグによりタグ化されていることを特徴とする。タグ化タンパク質は、使用するベクターの構造によって、目的タンパク質のN末端及びC末端の少なくとも一方に不溶化タグが繋がれたものとされる。
本発明に係るタグ化タンパク質は、質量分析用標準ペプチド(内部標準用ペプチド)の作成に応用できる。LC/MS/MS(Liquid chromatography/mass spectrometry/mass spectrometry)、CE/MS/MS(capillary electrophoresis/mass spectrometry/mass spectrometry)及びGC/MS/MS(gas chromatography/mass spectrometry/mass spectrometry)において、選択反応モニタリング(SRM:selected reaction monitoring)及び多重反応モニタリング(MRM:multiple reaction monitoring)と称される分析が行われている。SRM及びMRMは、極めて特異性が高いため、超微量成分の定量性に優れている。
また、本発明に係るタグ化タンパク質は、抗体の精製に応用できる。
1.不溶化タグを用いたシグナル伝達タンパク質の精製(コムギ無細胞発現系)
本実施例では、コムギ無細胞発現系を用いて、本発明に係る不溶化タグとシグナル伝達タンパク質との融合タンパク質の合成及び精製を行った。
合成後の溶液(クルードタンパク質溶液)を、精製時に混入する共雑タンパク質を少なくするためにPBSで4倍に希釈し、15,000xg、20分間、4℃で遠心分離した。上清画分を除き、得られた沈渣(不溶画分)を精製タンパク質画分とした。
比較のため、GSTタグ融合用デスティネーションベクターを用いて同様にタンパク質合成を行った。合成後の溶液をPBSで4倍希釈し、グルタチオンレジン(GEファルマシア)に吸着させ、0.8Mグルタチオンで溶出し、精製タンパク質を回収した。
2.不溶化タグを用いた蛍光タンパク質又は酵素の精製
本実施例では、本発明に係る不溶化タグと蛍光タンパク質又は酵素との融合タンパク質を精製し、精製後の融合タンパク質が蛍光性又は酵素活性を維持していることを確認した。
3.不溶化タグを用いた網羅的なタンパク質精製
本実施例では、本発明に係る不溶化タグを用いて、網羅的なタンパク質精製を行った。
4.プロテインアレイの作製
本実施例では、本発明に係る不溶化タグを用いて精製されたタンパク質を基板に結合して、プロテインアレイを作製した。
5.プロテインアレイによる自己抗体の検出
実施例4において作製されたプロテインアレイを用いて、血清中の自己抗体の検出を行った。
6.不溶化タグを用いたタンパク質の精製(大腸菌細胞内発現系)
本実施例では、大腸細胞内発現系を用いて、本発明に係る不溶化タグとGSTとの融合タンパク質の精製を行った。
7.不溶化タグのドメイン化
本試験例では、MafGタンパク質のアミノ酸全長のうち、特に不溶性に寄与している部分配列(ドメイン)を同定した。
8.プロテアーゼ切断部位の挿入
本試験例では、試験例1で得られたドメインB及びドメインCのアミノ酸配列にプロテアーゼによる切断部位となるアミノ酸を挿入し、不溶化タグのアミノ酸配列の改変を行った。
9.タグ化タンパク質の再可溶化条件の検討
不溶化画分として回収された不溶化タグが付加された融合タンパク質(タグ化タンパク質)の再可溶化について、適切な溶媒を検討した。
10.溶媒による不溶化タグとの融合タンパク質の再可溶化
試験例3において検討された溶媒23(表7参照)について、さらに複数の、不溶化タグとの融合タンパク質(タグ化タンパク質)の再可溶化に適しているか検証した。
Claims (16)
- MafGタンパク質の全長又は一部のアミノ酸配列、又は該アミノ酸配列にプロテアーゼによる切断部位となるアミノ酸が挿入されたアミノ酸配列、を含んでなるプロテインタグ。
- トリプシンの切断部位であるアルギニンが挿入された請求項1記載のプロテインタグ。
- 配列番号1~4に記載のアミノ酸配列を含んでなる請求項1又は2記載のプロテインタグ。
- 配列番号3又は4に記載のアミノ酸配列を含んでなる請求項3記載のプロテインタグ。
- MafGタンパク質の全長又は一部のアミノ酸配列、又は該アミノ酸配列にプロテアーゼによる切断部位となるアミノ酸が挿入されたアミノ酸配列、を含んでなるプロテインタグによりタグ化されたタンパク質。
- 請求項5記載のタンパク質を支持体に固定したプロテインアレイ。
- MafGタンパク質の全長又は一部のアミノ酸配列にプロテアーゼによる切断部位となるアミノ酸が挿入されたアミノ酸配列を含んでなるプロテインタグによりタグ化されたタンパク質をプロテアーゼ処理して得られるペプチド。
- 前記プロテインタグが配列番号3又は4に示すアミノ酸配列を含んでなる請求項7記載のペプチド。
- 質量分析の目的タンパク質と、MafGタンパク質の全長又は一部のアミノ酸配列にプロテアーゼによる切断部位となるアミノ酸が挿入されたアミノ酸配列を含んでなるプロテインタグと、の融合タンパク質をプロテアーゼ処理して得られるペプチドを含む、前記目的タンパク質の質量分析用標準ペプチド。
- 請求項1記載のプロテインタグを発現するベクター。
- タグ化する目的タンパク質と前記プロテインタグとの融合タンパク質を発現する請求項10記載のベクター。
- MafGタンパク質の全長又は一部のアミノ酸配列、又は該アミノ酸配列にプロテアーゼによる切断部位となるアミノ酸が挿入されたアミノ酸配列、を含んでなるプロテインタグにより、目的タンパク質をタグ化する融合タンパク質調製手順と、
タグ化された前記目的タンパク質を不溶画分に回収する精製手順と、を含むタンパク質精製方法。 - 前記融合タンパク質調製手順は、無細胞タンパク質合成系又は細胞タンパク質合成系において、前記目的タンパク質と前記プロテインタグとの融合タンパク質を発現するベクターを用いて、該融合タンパク質を合成する手順である請求項12記載のタンパク質精製方法。
- 前記無細胞タンパク質合成系が、コムギ無細胞タンパク質合成系である請求項13記載のタンパク質精製方法。
- 前記精製手順は、タグ化された前記目的タンパク質を遠心分離する手順である請求項12~14のいずれか一項に記載のタンパク質精製方法。
- タンパク質に対する抗体を精製する方法であって、
前記タンパク質と、MafGタンパク質の全長又は一部のアミノ酸配列、又は該アミノ酸配列にプロテアーゼによる切断部位となるアミノ酸が挿入されたアミノ酸配列、を含んでなるプロテインタグと、の融合タンパク質を用いることを特徴とする抗体精製方法。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000006763A1 (en) * | 1998-07-31 | 2000-02-10 | Pierce Chemical Company | Fusion products containing insoluble proteinaceous tag |
JP2006145470A (ja) * | 2004-11-24 | 2006-06-08 | Toyobo Co Ltd | 相互作用測定方法 |
US20060263855A1 (en) * | 2005-03-14 | 2006-11-23 | Wood David W | Intein-mediated protein purification using in vivo expression of an elastin-like protein |
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CA2446886A1 (en) | 2001-04-06 | 2002-10-17 | Biovision Ag | Method for detecting chronic dementia diseases, and corresponding peptides and detection reagents |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000006763A1 (en) * | 1998-07-31 | 2000-02-10 | Pierce Chemical Company | Fusion products containing insoluble proteinaceous tag |
JP2006145470A (ja) * | 2004-11-24 | 2006-06-08 | Toyobo Co Ltd | 相互作用測定方法 |
US20060263855A1 (en) * | 2005-03-14 | 2006-11-23 | Wood David W | Intein-mediated protein purification using in vivo expression of an elastin-like protein |
Non-Patent Citations (6)
Title |
---|
INOUE A ET AL.: "Role of the nucleoplasmin 2 C-terminal domain in the formation of nucleolus-like bodies in mouse oocytes", FASEB J., vol. 24, no. 2, 2010, pages 485 - 494 * |
LECHERTIER T ET AL.: "A B23-interacting sequence as a tool to visualize protein interactions in a cellular context", J. CELL SCI., vol. 120, 2007, pages 265 - 275 * |
MASATOSHI MORI ET AL.: "Tanpakushitsu no Kan'i Seisei o Kano ni suru Fuyoka Tag no Kaihatsu to Riyo", ANNUAL MEETING OF THE MOLECULAR BIOLOGY SOCIETY OF JAPAN PROGRAM YOSHISHU, 21 November 2011 (2011-11-21), Retrieved from the Internet <URL:http://www.aeplan.co.jp/mbsj2011> [retrieved on 20111121] * |
NAOKI GOSHIMA ET AL.: "Akusei Shuyo ni Tomonau Shinkeikin Shogai Jiko Kotai Profiling", GEKKAN MEDICAL SCIENCE DIGEST, vol. 37, no. 4, 25 April 2011 (2011-04-25), pages 132 - 135 * |
PENG CC ET AL.: "A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides", J. BIOTECHNOL., vol. 111, no. 1, 2004, pages 51 - 57, XP002659232, DOI: doi:10.1016/J.JBIOTEC.2004.03.013 * |
SHIN'ICHI OHASHI ET AL.: "Purification of dihydrofolate reductase-polypeptide fused proteins", JISEDAI SANGYO KIBAN GIJUTSU SYMPOSIUM BIO TECHNOLOGY YOKOSHU, vol. 7, no. 2, 1989, pages 53 - 67 * |
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