JP5645155B2 - Molecular weight marker and method for producing molecular weight marker - Google Patents

Description

  The present invention relates to a molecular weight marker containing a protein having an immunoglobulin binding domain and a method for producing the same.

Conventional protein electrophoresis includes SDS-PAGE and native-PAGE. SDS-PAGE is a method of performing separation depending on molecular weight in a non-natural state by treating a protein with a denaturing agent. According to this method, the molecular weight marker can exhibit accurate molecular mobility. On the other hand, native-PAGE is a method in which a protein is separated in a natural state without being treated with a denaturing agent. According to this method, proteins can be separated in a natural state, but since the mobility varies depending on the isoelectric point, there is a drawback that the mobility depending on the exact molecular weight cannot be shown.
The main method for protein functional analysis is Western blotting. This is a technique using an antibody specific to the target protein (primary antibody) and an antibody (secondary antibody) labeled with the primary antibody. However, Western blotting requires various antibodies for detection of molecular weight markers, and currently there are few useful molecular weight markers.

  In Patent Document 1 below, plasmids in which 1 to 6 genes of the AB domain of protein A are inserted are prepared, and the same amount of protein obtained from E. coli JM109 transformed with these plasmids is mixed and Western blotted. Producing a molecular weight marker for use.

In Patent Document 2 below, formic acid is added to and dissolved in Salmonella flagellar fiber protein, a formic acid solution of BrCN (bromocyan) is added to perform a fragmentation reaction, and a reaction mixture obtained by this fragmentation reaction is used. A method for preparing a molecular weight marker for obtaining proteins having various molecular weights is disclosed. In this method, fragments obtained by chemically or enzymatically fragmenting a protein having one molecular weight are used as marker proteins having different molecular weights. According to this, the trouble of separately generating and mixing a plurality of marker proteins can be omitted, which is advantageous in terms of cost.

Further, Patent Document 3 listed below discloses that purified Type I CAT is allowed to stand for about 30 days in an oxidized state in which a compound having a sulfohydryl group does not exist to produce a molecular weight marker composed of an association of Type I CAT. Yes. By using multimeric proteins composed of the same subunits, the isoelectric points between the subunits can be made the same, and the mobility depending on the molecular weight can be exhibited even in the natural state.
JP-A-5-32699 JP-A-2-25745 JP-A-7-53589

  However, in the molecular weight marker preparation methods disclosed in Patent Documents 1 to 3, the protein must be purified and subjected to various treatments after the purification, and equipment for preparation and a lot of time and cost are required. There is a problem of requiring. That is, in the molecular weight marker preparation method disclosed in Patent Document 1, it is necessary to purify proteins having six kinds of molecular weights, and it is also necessary to mix these proteins. Moreover, in the molecular weight marker preparation method disclosed in Patent Document 2, it is necessary to chemically or enzymatically cleave the protein after purification of the protein. Furthermore, in the molecular weight marker preparation method disclosed in Patent Document 3, it is necessary to associate subunits after protein purification, which takes about 30 days.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a molecular weight marker that can be prepared easily in a short time and at low cost, and a method for producing the same.

  The molecular weight marker and the production method thereof according to the present invention have taken the following means in order to achieve at least a part of the above object.

The molecular weight marker of the present invention includes a polyprotein in which a plurality of immunoglobulin binding domain molecules having an immunoglobulin binding domain are linked via a linker, and a degradation product obtained by cleaving the linker by the expression host of the polyprotein And containing.
Thereby, polyprotein and its degradation product can be obtained simultaneously. As a result, molecular weight markers composed of marker proteins with different molecular weights can be prepared by performing a single chromatographic purification without going through a special purification method. Markers can be made. In addition, since the molecular weight marker is composed of one type of immunoglobulin binding domain molecule, not only when a denaturing agent is used as in SDS-PAGE, but also when no denaturing agent is used as in native-PAGE. Degree can be shown. Furthermore, since the marker protein has an immunoglobulin binding domain, it can be detected by Western blotting. In addition, since the concentration of undegraded polyprotein is large, it appears as a thick band, and since the concentration of degradation products is small, it appears as a thin band. Can have sex.

In addition, the molecular weight marker of the present invention may be characterized in that it contains a multimer in which a Cys residue is introduced into the C-terminus of the polyprotein and the Cys residues are bonded to each other via a disulfide bond.
As a result, polyproteins can be bound to each other via disulfide bonds to produce multimers having multiple immunoglobulin binding domains. As a result, it is possible to eliminate the need to directly produce a large molecular weight marker protein. Here, by introducing a Cys residue into the C-terminus of the polyprotein, the polyproteins can be easily bonded to each other via a disulfide bond.

In the molecular weight marker of the present invention, the polyprotein may be characterized in that 2 to 7 immunoglobulin-binding domain molecules are linked.
Thereby, it can utilize as a molecular weight marker with respect to many target proteins.

  In the molecular weight marker of the present invention, the immunoglobulin binding domain molecule is an antibody binding domain derived from Staphylococcus protein A, a B1 domain derived from Group G Streptococcus G148 G protein (SpG), Fine Gordia -It can also be chosen from the group which consists of L protein derived from Magna (Finegoldia magna).

In the molecular weight marker of the present invention, the antibody binding domain derived from Staphylococcus protein A may be a polypeptide comprising the following amino acid sequence (a) or (b).
(A) the amino acid sequence represented by SEQ ID NO: 1 (b) the amino acid sequence represented by SEQ ID NO: 1, wherein one or several amino acids are deleted, substituted or added

In the molecular weight marker of the present invention, the B1 domain derived from G protein (SpG) of Group G Streptococcus G148 is a polypeptide comprising the following amino acid sequence (a) or (b): You can also.
(A) the amino acid sequence represented by SEQ ID NO: 2 (b) the amino acid sequence represented by SEQ ID NO: 2, wherein one or several amino acids are deleted, substituted or added

Further, in the molecular weight marker of the present invention, the L protein derived from Finegoldia magna may be a polypeptide comprising the following amino acid sequence (a) or (b).
(A) the amino acid sequence represented by SEQ ID NO: 3 (b) the amino acid sequence represented by SEQ ID NO: 3, wherein one or several amino acids have been deleted, substituted or added

In the molecular weight marker production method of the present invention, a polyprotein in which a plurality of immunoglobulin binding domain molecules having an immunoglobulin binding domain are linked via a linker is prepared, and then the expression host of the polyprotein is the linker. The gist is that a molecular weight marker having a plurality of molecular weights is produced by cleaving.
Thereby, polyprotein and its degradation product can be obtained simultaneously. As a result, molecular weight markers composed of marker proteins with different molecular weights can be prepared by performing a single chromatographic purification without going through a special purification method. Markers can be made. In addition, since the molecular weight marker is composed of one type of immunoglobulin binding domain molecule, not only when a denaturing agent is used as in SDS-PAGE, but also when no denaturing agent is used as in native-PAGE. Degree can be shown. Furthermore, since the marker protein has an immunoglobulin binding domain, it can be detected by Western blotting. In addition, since the concentration of undegraded polyprotein is large, it appears as a thick band, and since the concentration of degradation products is small, it appears as a thin band. Can have sex.

As described above, according to the molecular weight marker of the present invention and the production method thereof, polyprotein and its degradation product can be obtained at the same time. Therefore, the molecular weight can be easily and at low cost in a short time without using complicated facilities and processes. Markers can be made. In addition, since the molecular weight marker is composed of one type of immunoglobulin binding domain molecule, not only when a denaturing agent is used as in SDS-PAGE, but also when no denaturing agent is used as in native-PAGE. Degree can be shown. Furthermore, since the marker protein has an immunoglobulin binding domain, it can be detected by Western blotting. In addition, since the concentration of undegraded polyprotein is large, it appears as a thick band, and since the concentration of degradation products is small, it appears as a thin band, so that it can be seen at a glance as a molecular weight marker that shows the molecular weight of the target protein. Can have sex.

It is the figure which showed the marker protein of the monomer of the molecular weight marker of this invention. It is the figure which showed the marker protein of the dimer of the molecular weight marker of this invention. It is the figure which applied the molecular weight marker of this invention to SDS-PAGE, and stained the gel with CBB. It is the figure which performed Western blotting using Zh (5) Cys, Zh (6) Cys, and Zh (7) Cys considered to be especially versatile among the molecular weight markers of the present invention.

  The molecular weight marker of the present invention is composed of marker proteins having different molecular weights. Each marker protein is a polyprotein in which an immunoglobulin binding domain molecule having an immunoglobulin binding domain is linked in multiple tandems, a degradation product of the polyprotein, or a multimer in which they are bound via a disulfide bond. is there. Furthermore, immunoglobulin binding domain molecules are linked via a linker. In addition, Cys for forming a polyploid can be introduce | transduced into a poloprotein.

An immunoglobulin binding domain molecule refers to a molecule comprising a region that can bind an immunoglobulin. For example, an antibody binding domain derived from Staphylococcus protein A, a B1 domain (SpG-B) of Group G Streptococcus G148-derived G protein (SpG), Fine Gordia magna (Peptostreptococcus magnus) Examples include L protein derived from (Finegoldia magna (Peptostreptococcus magnus)), but are not limited thereto.
SEQ ID NO: 1 is an antibody binding domain (Z domain) derived from protein A, SEQ ID NO: 2 is a B1 domain of G protein derived from Streptococcus G148, SEQ ID NO: 3 is an L protein derived from Fine Gordia magna (Peptostreptococcus magnus) Show. In the present specification, the immunoglobulin binding domain molecule includes not only the protein represented by the specific amino acid sequence but also an amino acid sequence in which one or several amino acids are deleted, substituted or added in these amino acid sequences. Polypeptides are included. That is, a polypeptide comprising an amino acid sequence having a homology with the amino acid sequence of 80% or more, preferably 90% or more, more preferably 95% or more is also included.
Figure 1 shows a marker protein (Zh (1) Cys) that has one Z domain and a Cys residue introduced at its C-terminus, and a Cys residue that has two Z domains and its C-terminus. Marker protein (Zh (2) Cys), a marker protein (Zh (3) Cys) that has three Z domains and a Cys residue introduced at its C-terminus, has four Z domains And a marker protein having a Cys residue introduced at its C-terminus (Zh (4) Cys), a marker protein having five Z domains and having a Cys residue introduced at its C-terminus (Zh (5) Cys), a marker protein having six Z domains and having a Cys residue introduced at its C-terminus (Zh (6) Cys), a polyprotein, that is, having seven Z domains and having C at its C-terminus Marker proteins s residues are introduced showing the (Zh (7Cys)).
FIG. 2 shows a marker protein of Zh (1) Cys dimer, marker protein of Zh (2) Cys dimer, marker protein of Zh (3) Cys dimer, marker of Zh (4) Cys dimer Protein, Zh (5) Cys dimer marker protein, Zh (6) Cys dimer marker protein, Zh (7) Cys dimer marker protein. In addition, although a dimer is shown here, it is not limited to a dimer, A multimer more than a trimer may be sufficient.

Here, the linker is a peptide that links immunoglobulin-binding domain molecules, is a peptide that can be cleaved by a protease present in the expression host, and has 5 to 30 amino acid residues, preferably 5 to 10 amino acids. It is a peptide having a residue. For example, a sequence in which GGGGS (G ₄ S) is combined with a sequence containing GPGP, a sequence in which (G ₄ S) is repeated 1 to 4 times ((G ₄ S) ₁ to (G ₄ S) ₃ ), , GPGP is repeated, but is not limited thereto. The linker is not particularly limited except for the cleavage recognition site. The cleavage site is usually considered GPGP, but a small amount of other sites may be mixed. In addition, the number of immunoglobulin binding domain molecules to be linked is preferably 7, which can cover the molecular weight range from 7.2 kDa to 102.0 kDa. Furthermore, a wider range of molecular weights can be covered by increasing the number of immunoglobulin binding domain molecules linked.

Here, the expression host refers to one that expresses a protein based on the introduced gene. For example, prokaryotic organisms (such as Bacillus subtilis and lactic acid bacteria) capable of expressing proteins such as Escherichia coli BL21 (DE3) strain, DH5 alpha strain, JM109 strain, and XL-1 Blue strain, and protein expression systems such as yeast have been established. Examples include, but are not limited to, nuclear organisms (yeast, cultured cells). In addition, it is possible to reduce the molecular weight of a polyprotein chemically or enzymatically, but if an expression host is used, the expression host can cleave the polyprotein with various linkers simply by introducing the gene into the expression host. can do

EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to a following example.

Example 1:
Construction of gene in which immunoglobulin-binding domain molecule is linked in tandem According to the following method, a Z-domain (Zh) which is an antibody-binding domain derived from Staphylococcus protein A is adopted as an immunoglobulin-binding domain molecule. Cloned. Specifically, in order to introduce Cys into the C-terminus of the protein, Cys was introduced into the Z domain using an oligonucleotide primer containing a TGC base sequence so as to optimize the Cys codon of E. coli. The primers used were the sense primer of SEQ ID NO: 4 and the antisense primer of SEQ ID NO: 5. These synthetic oligos were prepared, annealed, introduced into the pCR2.1 vector, and the calcium phosphate method commonly used for E. coli DH5alpha Was transformed by. Furthermore, screening was carried out with ampicillin, which is a drug resistance marker present in the vector, and the target gene-introduced clone was selected to complete the cloning of the Z domain. After confirming the base sequence, a synthetic oligo of the sense primer of SEQ ID NO: 6 and the antisense primer of SEQ ID NO: 7 was prepared, amplified by PCR, treated with restriction enzymes NcoI and XhoI, and then the NcoI and XhoI sites of the pET-21d vector Cloning (subcloning) was completed, and the construction of a gene (pET-21d-Zh (1) Cys) encoding a marker protein having one Z domain and having a Cys residue introduced at the C-terminus thereof was completed. .
Next, a synthetic oligo of the antisense primer of SEQ ID NO: 8 is prepared, and a Z domain gene to be inserted into a plurality of genetically linked forms (tandem) in combination with the previously synthesized sense primer of SEQ ID NO: 6 After amplification by PCR and treatment with NcoI and PciI, cloning into the NcoI site of pET-21d-Zh (1) Cys, confirming the nucleotide sequence, it has two Z domains and the Cys residue at the C-terminus Construction of the gene (pET-21d-Zh (2) Cys) encoding the marker protein into which the group was introduced was completed. By repeating the insertion of the Z domain in this way, a gene (pET-21d-Zh (3) Cys) encoding a marker protein having three Z domains and having a Cys residue introduced at the C-terminus thereof, A gene (pET-21d-Zh (4) Cys) encoding a marker protein having four Z domains and having a Cys residue introduced at the C-terminus, and having five Z domains at the C-terminus Gene encoding a marker protein into which a Cys residue has been introduced (pET-21d-Zh (5) Cys), which encodes a marker protein having six Z domains and having a Cys residue introduced at the C-terminus Gene (pET-21d-Zh (6) Cys), which encodes a marker protein having seven Z domains and having a Cys residue introduced at its C-terminus That was constructed gene (pET-21d-Zh (7) Cys).
The linker responsible for linking these immunoglobulin binding domain molecules is an amino acid sequence consisting of GPPGPH, and this sequence can be specifically recognized by proteases present in E. coli and can cause partial degradation.

Example 2:
Production of Zh (7) Cys After completion of construction of pET-21d-Zh (7) Cys, BL21 (DE3), a protein-expressing Escherichia coli strain, was transformed again by the calcium phosphate method, and after screening with ampicillin, it was a polyprotein. An expression construct for Zh (7) Cys was constructed. For expression of Zh (7) Cys, 10 μl of BL21 (DE3) strain freeze stock containing pET-21d-Zh (7) Cys was inoculated into 100 ml of a general LB-ampicillin-containing medium, and kept at 37 ° C. overnight. Pre-cultured. Subsequently, the cells were planted in 4 250 ml LB-ampicillin media (Total 1 L) and cultured at 37 ° C. for 1.5 hours. At this point, the turbidity OD 600 nm is measured using an absorbance measurement apparatus, and it is confirmed that OD = 0.4 to 0.6, and isopropyl-β-thiogalactopyrano is obtained so that the final concentration is 1 mM. Sid (IPTG) was added and cultured overnight at 37 ° C. to induce protein expression. Thereby, it was possible to obtain a multimeric marker protein in which Cys residues introduced into the polyprotein were bonded via a disulfide bond. Here, in order to induce protein expression in the BL21 (DE3) strain and to bind Cys residues introduced into the polyprotein via disulfide bonds, the experimental temperature is preferably 30 ° C. to 37 ° C. It is necessary to bring the temperature to 37 ° C. After the expression induction, the cells and the culture supernatant were centrifuged at 8000 rpm for 20 minutes, and the culture supernatant was filtered through a 0.45 μm filter to obtain a crudely purified sample.
As a purification method, 10 ml of IgG sepharose resin (GE Healthcare) was packed in an open column to prepare a purification column. Equilibrate until the pH reaches 8.0 with 2-3 times the column volume of TST buffer (pH 8.0) as the column equilibration solution, and then 0.5 to 3 times the column volume to 0.5 M Flow acetic acid solution (pH 3.5), equilibrate until pH is 3.5, repeat this step twice and finally equilibrate with TST buffer (pH 8.0) until pH is 8.0 (TST → acetic acid → TST → acetic acid → TST). The crudely purified sample was naturally dropped or passed through a column with a peristaltic pump or the like, and affinity chromatography was performed.
After chromatographic development of the crudely purified sample, it was washed with 10 volumes of TST buffer (pH 8.0), and then washed with 2 volumes of 0.5 M ammonium acetate solution (pH 5.5). After washing, elution was carried out with 50 ml using an elution buffer (0.5 M acetic acid solution (pH 3.5). Culture supernatant, flow-through fraction, washing fraction, elution fraction each collected during the purification process. The protein was analyzed by 15% acrylamide concentration SDS-PAGE, and the protein expression was confirmed by CBB staining after electrophoresis, and the expression of Zh (7) Cys was confirmed in the eluted fraction. It was concentrated with (Amicon Ultra-4, 30K) and replaced with PBS, and the concentration of Zh (7) Cys fusion protein was determined by BCA method using BCA Protein Assay Reagent (bicinchonic acid) reagent of PIERCE. The total amount of protein recovered from 1 L of culture was 8.18 mg.

Example 3: Confirmation of molecular weight of Zh (7) Cys As a result of SDS-PAGE analysis of Zh (7) Cys, which is an expression-purified polyprotein, Zh (1) Cys to Zh (7) Cys and their It was confirmed that each dimer in which a Cys residue was bonded by a disulfide bond was present. Specifically, as a result of applying Zh (7) Cys at a concentration of 2 μg / lane to 15% acrylamide concentration SDS-PAGE, and performing CBB staining after electrophoresis, the presence of proteins having the molecular weights shown in Table 1 below Was confirmed.

As can be seen from Table 1 above, the monomers are 7.2 kDa which is the molecular weight of Zh (1) Cys, 14.5 kDa which is the molecular weight of Zh (2) Cys, and the molecular weight of Zh (3) Cys. 21.7 kDa, the molecular weight of Zh (4) Cys 29.0 kDa, the molecular weight of Zh (5) Cys 36.3 kDa, the molecular weight of Zh (6) Cys, 43.5 kDa, the molecular weight of Zh (7) Cys The presence of 50.8 kDa protein was confirmed. Furthermore, for each dimer in which the Cys residues of Zh (1) Cys to Zh (7) Cys are linked by a disulfide bond, the molecular weight of Zh (1) Cys dimer is 14.5 kDa, Zh (2) The molecular weight of the Cys dimer is 29.0 kDa, the molecular weight of the Zh (3) Cys dimer is 43.5 kDa, the molecular weight of the Zh (4) Cys dimer is 58.0 kDa, the Zh (5) Cys dimer The presence of a protein of 72.6 kDa which is the molecular weight of the monomer, 87.0 kDa which is the molecular weight of the Zh (6) Cys dimer, and 102.0 kDa which is the molecular weight of the Zh (7) Cys dimer was confirmed.

FIG. 3 is a drawing in which molecular weight markers from Zh (1) Cys to Zh (7) Cys prepared in the same manner were subjected to SDS-PAGE and the gel was stained with CBB. Here, 2 μg of molecular weight markers from Zh (1) Cys to Zh (7) Cys are donated to a 12.5% acrylamide gel, and then stained. Each lane shows the following: That is, Lane M is a molecular weight marker of Bio-Rad, Lane 1 is Zh (1) Cys, Lane 2 is Zh (2) Cys, Lane 3 is Zh (3) Cys, Lane 4 is Zh (4 ) Cys, Lane 5 represents Zh (5) Cys, Lane 6 represents Zh (6) Cys, and Lane 7 represents Zh (7) Cys. A band surrounded by a solid line in each lane indicates a monomeric marker protein. In addition, a band surrounded by a dotted line in each lane indicates a dimeric marker protein. Bands other than those indicating the monomeric and dimeric marker proteins indicate degradation products generated by partial cleavage of the linker present in the monomeric or dimeric marker protein. Moreover, the molecular weight used as a parameter | index is shown on the left side of drawing.

Example 4:
Confirmation of function of immunoglobulin binding domain of Zh (7) Cys Zh (7) Cys, which is a purified polyprotein, is subjected to SDS-PAGE, and the acrylamide gel is transferred to a polyvinylidene difluoride (PVDF) membrane, followed by Western blotting. ) The function of the Cys immunoglobulin binding domain was confirmed. That is, Zh (7) Cys was applied to 15% acrylamide concentration SDS-PAGE at a concentration of 2 μg / lane, and after electrophoresis, the electrophoresis gel was electrically transferred to a PVDF membrane. As conditions for transcription, reaction was carried out at constant 100 mA for 1 hour using EzBlot reagent manufactured by ATTO. The PVDF membrane after transfer is washed with PBST (0.05% Tween-20-containing PBS) for 10 minutes three times, and further washed with PBS for 10 minutes three times. Subsequently, a blocking reaction is performed by reacting 10% skin milk for 1 hour at room temperature. This operation suppresses nonspecific antibody binding. After the blocking reaction, the PVDF membrane is washed with PBST for 10 minutes three times, and further washed with PBS for 10 minutes three times. Subsequently, HRP-labeled immunoglobulin is prepared using a 3% skin milk solution at an appropriate dilution ratio (eg, 4000 times), and allowed to permeate for 30 minutes at room temperature for reaction. Thereafter, washing with PBST for 10 minutes is repeated three times, and further washing with PBS for 10 minutes is performed three times. Expose the x-ray film for an appropriate time using a chemiluminescent HRP substrate. If there is an immunoglobulin binding ability, it can be seen that bands of various molecular weights are detected as signals and function as markers in Western blotting.

FIG. 4 is a drawing of Western blotting using Zh (5) Cys, Zh (6) Cys, and Zh (7) Cys, which are considered to be particularly versatile among the molecular weight markers of the present invention. Here, 2 μg of protein from Zh (5) Cys to Zh (7) Cys was donated to a 12.5% acrylamide gel, subjected to electrophoresis, transferred to a PVDF membrane, 10% skin milk After performing a blocking reaction to suppress non-specific detection with HRP labeled GoatIgG as a detection antibody
Alternatively, the reaction was performed at room temperature for about 30 minutes using HRP-labeled mouse IgG. Thereafter, excess antibody is washed, reacted with a chemical fluorescent substrate, and exposed to an X-ray film for an appropriate time. Each lane shows the following: That is, Lane M is a molecular weight marker of Bio-Rad, Lane 1 is Zh (5) Cys, Lane 2 is Zh (6) Cys, Lane 3 is Zh (7) Cys, Lane D is Oriental Yeast. Each molecular weight marker is indicated. Further, molecular weights serving as indices are shown on the left and right sides of the drawing.

  In the above examples, one kind of gene (pET-21d-Zh (7) Cys) is introduced into the expression host, but a plurality of kinds of genes (for example, pET-21d-Zh (3) Cys and pET-) are introduced. 21d-Zh (7) Cys) can also be introduced into the expression host.

As can be seen from the above results, according to the molecular weight marker of the present invention and the method for producing the same, polyprotein and its degradation product can be obtained at the same time. A molecular weight marker can be produced at a low cost. In addition, since the molecular weight marker is composed of one type of immunoglobulin binding domain molecule, not only when a denaturing agent is used as in SDS-PAGE, but also when no denaturing agent is used as in native-PAGE. Degree can be shown. Furthermore, since the marker protein has an immunoglobulin binding domain, it can be detected by Western blotting. In addition, since the concentration of undegraded polyprotein is large, it appears as a thick band, and since the concentration of degradation products is small, it appears as a thin band, so that it can be seen at a glance as a molecular weight marker that shows the molecular weight of the target protein. Can have sex.
In addition, if a Cys residue is introduced into a polyprotein in the molecular weight marker of the present invention, a polymer can be produced by linking polyproteins via a disulfide bond and retaining an immunoglobulin binding domain in multiples, It is also possible to eliminate the need to directly produce large molecular weight proteins. Here, if a Cys residue is introduced into the C-terminus of the polyprotein, the polyproteins can be easily bonded to each other via a disulfide bond.
Furthermore, in the molecular weight marker of the present invention, if 2 to 7 kinds of immunoglobulin binding domain molecules are linked to a polyprotein, it can be used as a molecular weight marker for many target proteins.
In addition, if multiple types of genes (for example, pET-21d-Zh (6) Cys and pET-21d-Zh (7) Cys) are introduced into the expression host, the concentration of the marker protein, that is, the thickness of the band is adjusted. You can also.

Examples of the use of the molecular weight marker of the present invention include use for protein electrophoresis (SDS-PAGE, native-PAGE) and Western blot analysis. Since the mobility depending on the molecular weight can be realized without being influenced by the isoelectric point, it can be applied to an accurate molecular weight analysis of proteins and multimer-forming proteins that are disintegrated in the presence of denaturation such as SDS.

Claims (8)

A molecular weight marker, a polyprotein that is multiple linked one type of immunoglobulin-binding domain molecules with immunoglobulin-binding domain via a linker, and decomposition products expression hosts said polyprotein has cut the linker A molecular weight marker characterized in that a Cys residue is introduced into the C-terminus of the polyprotein, and the Cys residues are linked via a disulfide bond . 2. The molecular weight marker according to claim 1 , wherein 2 to 7 of the one kind of immunoglobulin binding domain molecules are linked to the polyprotein. 3. The molecular weight marker according to claim 1, wherein the immunoglobulin binding domain molecule is an antibody binding domain derived from Staphylococcus protein A, G protein of Group G Streptococcus G148 (SpG ) -Derived B1 domain, a molecular weight marker selected from the group consisting of L proteins derived from Finegoldia magna. 4. The molecular weight marker according to claim 3 , wherein the antibody binding domain derived from Staphylococcus protein A is a polypeptide having the following amino acid sequence (a) or (b): Molecular weight marker.
(A) amino acid sequence represented by SEQ ID NO: 1 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 The molecular weight marker according to claim 3 , wherein the B1 domain derived from the G protein (SpG) of the Group G Streptococcus G148 is a polypeptide comprising the following amino acid sequence (a) or (b): Characteristic molecular weight marker.
(A) amino acid sequence represented by SEQ ID NO: 2 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 4. The molecular weight marker according to claim 3 , wherein the L protein derived from Finegoldia magna is a polypeptide comprising the following amino acid sequence (a) or (b): Molecular weight marker.
(A) Amino acid sequence represented by SEQ ID NO: 3 (b) Amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 3 The molecular weight marker according to claim 1, which has a monomer and a dimer having the molecular weight described in the following table.
A method for producing a molecular weight marker comprising:
A step of one type of immunoglobulin-binding domain molecules with immunoglobulin-binding domain to create a plurality linked polyprotein via a linker,
Producing a molecular weight marker having a plurality of molecular weights by cleaving the linker from the polyprotein expression host ;
A method for preparing a molecular weight marker , wherein a Cys residue is introduced into the C-terminus of the polyprotein, and the Cys residues are bonded to each other via a disulfide bond .