WO2011071021A1

WO2011071021A1 - Tag peptide

Info

Publication number: WO2011071021A1
Application number: PCT/JP2010/071846
Authority: WO
Inventors: 隆雄松葉
Original assignee: 東ソー株式会社
Priority date: 2009-12-07
Filing date: 2010-12-06
Publication date: 2011-06-16
Also published as: JP2011140483A; JP5810514B2

Abstract

Provided is a tag peptide which has improved specificity and reactivity and which can be used as an immunoassay reagent, and also provided is a method for purifying and detecting proteins using said peptide. Using an oligopeptide formed from 7 amino acids on the C-terminal side of a B-type natriuretic peptide (BNP) as the tag peptide, proteins are purified and detected by means of a substance that recognizes the tag peptide and the proteins to which the tag peptide has been added. It should be noted that the oligopeptide can be a peptide wherein one or more from among the 1^st through 3^rd amino acids on the N-terminal side are substituted by acidic amino acids, or a peptide wherein one or more from among the 1^st through 3^rd amino acids on the N-terminal side are deleted.

Description

Tag peptide

The present invention relates to a tag peptide for detecting a protein with high sensitivity, and a protein purification / detection method using the tag peptide.

As a technique for easily purifying and detecting genetically expressed protein and immobilizing it on a carrier, a substance that recognizes the peptide, called a tag peptide (for example, when the protein is expressed) There is known a method in which a peptide capable of specifically binding to an antibody or the like is expressed in a state of being added to the protein and then purified, detected, and immobilized using a substance that recognizes the peptide.

Examples of conventionally known tag peptides include a FLAG tag consisting of the amino acid sequence set forth in SEQ ID NO: 1 (see Patent Documents 1 and 2), and a Myc tag consisting of the amino acid sequence set forth in SEQ ID NO: 2 (Non-Patent Document 1). A His tag consisting of the amino acid sequence set forth in SEQ ID NO: 3 (see Patent Documents 3 and 4), an E tag consisting of the amino acid sequence set forth in SEQ ID NO: 4, an HA tag consisting of the amino acid sequence set forth in SEQ ID NO: 5, A T7 tag comprising the amino acid sequence set forth in SEQ ID NO: 6, a Pk tag comprising the amino acid sequence set forth in SEQ ID NO: 7, an HSV tag comprising the amino acid sequence set forth in SEQ ID NO: 8, and a VSV comprising the amino acid sequence set forth in SEQ ID NO: 9. -Partial regions of proteins derived from viruses, phages, and oncogenes that are not originally present in normal cells, such as G tags, and the same amino acids Composed of repeating acid sequences include about 10 amino acid peptide. Of the nine types of tag peptides exemplified above, the FLAG tag (SEQ ID NO: 1) is known as a high-performance tag. However, with the recent increase in sensitivity of experiments, tag peptides with further improved specificity and reactivity are required.

US Pat. No. 4,703,004 Japanese Patent No. 2665359 Japanese Unexamined Patent Publication No. 63-251095 US Pat. No. 5,310,663 JP 2009-024030 A

Considering the principle of tag peptides when investigating new tag peptides, it seems that any peptide consisting of any amino acid sequence can be used as a tag peptide if there is a substance (such as an antibody) that specifically recognizes the sequence. It is. However, it is not easy to determine the sequence of the tag peptide when considering practical use as a tag peptide in the industry. The reason is that, in order to specifically purify and detect the protein with the tag peptide added, the amino acid sequence of the tag peptide is not included in the amino acid sequence of the protein used in a normal experimental system. This is because it is necessary that no similar amino acid sequence exists. In order to confirm the presence / absence of a homologous sequence / similar sequence, homology search using a database such as BLAST (Basic / Local / Alignment / Search / Tool) is possible. However, it is difficult to estimate how much a substance that recognizes the tag peptide (such as an antibody) has cross-reactivity with a peptide having a sequence similar to the amino acid sequence of the tag peptide, Further, it is more difficult to predict the cross-reactivity with proteins not registered in the database.

In that case, amino acids and low molecular weight organic compounds that are not originally incorporated into proteins may be used as tags, but in reality, the cause of cross-reactivity is not only the primary sequence of amino acids but also sequence-specificity. It may be caused by influences such as electrostatic interaction and hydrophobic interaction. In other words, using a non-naturally occurring substance as a tag may make it difficult to predict cross-reactivity. Further, when the amino acid or organic compound is used as a tag, a new problem arises that it is difficult to add the tag to a protein by genetic engineering.

Furthermore, when the tag peptide is used for immunoassay reagents, various components contained in the sample are different between samples, and therefore, there is a possibility of different influences on the tag peptide for each sample. That is, even if an industrially effective tag peptide can be designed, the tag peptide cannot always be used for an immunoassay reagent. The only way to determine whether a designed tag peptide can be used in immunoassay reagent applications is to evaluate a very large number of samples, but to evaluate the usefulness of tag peptides, evaluate a very large number of samples. This is extremely difficult in practice.

Accordingly, an object of the present invention is to provide a tag peptide that has improved specificity and reactivity as compared with the prior art and can be used for immunoassay reagents, and a protein purification / detection method using the tag peptide. is there.

As a result of intensive studies by the inventors in view of the above problems, a tag peptide that has not been reported so far and can be used in immunoassay reagent applications has been found, and the present invention has been completed.

That is, the present invention has the following gist.
(1) An oligopeptide comprising the amino acid sequence set forth in SEQ ID NO: 10 and useful for purifying and detecting a protein.
(2) In the oligopeptide consisting of the amino acid sequence set forth in SEQ ID NO: 10, one or more amino acids from the first cysteine to the third valine are substituted with acidic amino acids to purify and detect the protein It is a useful oligopeptide.
(3) To purify and detect a protein in which one or more amino acids from the first cysteine to the third valine are deleted from the oligopeptide consisting of the amino acid sequence set forth in SEQ ID NO: 10. It is a useful oligopeptide.
(4) A protein purification / detection method using a protein to which the oligopeptide according to any one of (1) to (3) above is added and a substance that recognizes the peptide.
(5) The protein is a protein obtained by adding the oligopeptide according to any one of (1) to (3) on the C-terminal side, and the substance that recognizes the peptide is an antibody that recognizes the peptide, The purification / detection method according to (4).
(6) The protein according to (4) or (5), wherein the protein is a protein obtained by genetically adding the oligopeptide according to any one of (1) to (3). This is a purification / detection method.
(7) The purification according to (4) or (5), wherein the protein is a protein obtained by chemically adding the oligopeptide according to any one of (1) to (3). -It is a detection method.

In the present invention, the length is as short as 7 amino acids, the existence of a substance having cross-reactivity has been denied from the past results, and a substance (antibody) having high affinity and specificity is present in SEQ ID NO: 10. This is a C-terminal 7 amino acid (BNC) of B-type natriuretic peptide (BNP) comprising the described amino acid sequence, and a protein purification / detection method using the same. Conventionally known for purification and detection by adding BNC to the N-terminal side or C-terminal side of the protein to be purified / detected genetically or chemically and binding it to a substance that recognizes BNC. Compared with the purification / detection method using the FLAG tag, specificity and sensitivity can be improved.

The BNC may be a modified BNC obtained by substituting one or more amino acids from the first cysteine to the third valine among amino acids constituting the BNC with an acidic amino acid. It may be a deleted BNC in which one or more of the amino acids up to the valine are deleted. In particular, the modified BNC in which one or more amino acids from the first cysteine to the third valine are substituted with acidic amino acids has improved binding performance (affinity) and is more sensitive / sensitive than BNC. High specific detection and more efficient affinity purification are possible.

FIG. 5 is an SDS-PAGE (SodiumSoDodecyl Sulfate Poly-Acrylamide Gel Electrophoresis) result of maltose binding protein (MBP) to which each tag peptide is added, obtained in Example 2. FIG. In the figure, MBP-BNC indicates MBP in which the C-terminal peptide (BNC) of BNP is added to the C-terminus of MBP. Similarly, MBP-FLAG indicates a FLAG tag, MBP-Myc indicates a Myc tag, MBP-His indicates a MBP with a His tag added to the C-terminal of MBP, respectively. The result of Example 4 is shown. The numerical value in the upper part of the figure shows the number of moles of MBP to which each tag peptide electrophoresed in each well (Well) is added, and the right side shows the assay format of this western blotting. The result of Example 5 is shown. The vertical axis represents the signal obtained by ELISA (Enzyme-Linked ImmunoSorbent Assay), and the horizontal axis represents the added amount (μg / mL) of MBP added with each tag peptide. The numerical values at the top of the figure indicate the number of moles of MBP to which each tag peptide reacted in each well was added, and the right side shows the assay format of this ELISA. The result of Example 6 is shown. The right side of the figure shows the assay format of this ELISA. The result of Example 7 is shown. The right side of the figure shows the assay format of this ELISA. The result of Example 8 is shown. 9 shows a scheme for introducing BNC into an anti-E2 antibody carried out in Example 9. The result of Example 9 is shown. The assay format of this measurement system is shown on the right side of the figure. 2 shows a calibration curve of a recombinant anti-estradiol antibody (RaMoAb-BNC) in which BNC is directly bound to the C-terminal side of the H chain. The left side of the figure shows the fluorescence intensity (FI) measurement ELISA assay format, and the right side of the figure shows the antibody concentration measurement ELISA assay format. 10 shows an outline of the binding performance evaluation using the calibration curve of FIG. Of the results of Example 13, BNC recognition antibody BC2-7 evaluated the oligopeptide in which the first cysteine of BNC was substituted with another amino acid. Of the results of Example 13, oligopeptides obtained by substituting the second lysine of BNC with other amino acids were evaluated with the BNC recognition antibody BC2-7. Of the results of Example 13, oligopeptides obtained by substituting the third valine of BNC with other amino acids were evaluated with the BNC recognition antibody BC2-7. Of the results of Example 13, oligopeptides obtained by substituting the 4th leucine of BNC with other amino acids were evaluated with the BNC recognition antibody BC2-7. Of the results of Example 13, BNC recognition antibody BC2-7 evaluated the oligopeptide in which the 5th arginine of BNC was substituted with another amino acid. Of the results of Example 13, BPC recognition antibody BC2-7 was used to evaluate an oligopeptide obtained by substituting the 6th arginine of BNC with another amino acid. Of the results of Example 13, BPC recognition antibody BC2-7 evaluated the oligopeptide obtained by substituting the 7th histidine of BNC with another amino acid. Of the results of Example 13, BNC recognition antibody BC23-11 evaluated the oligopeptide in which the first cysteine of BNC was substituted with another amino acid. Of the results of Example 13, oligopeptides obtained by substituting the second lysine of BNC with other amino acids were evaluated with the BNC recognition antibody BC23-11. Of the results of Example 13, oligopeptides obtained by substituting the 3rd valine of BNC with other amino acids were evaluated with the BNC recognition antibody BC23-11. Of the results of Example 13, the results were obtained by evaluating the oligopeptide obtained by substituting the 4th leucine of BNC with another amino acid using the BNC recognition antibody BC23-11. Of the results of Example 13, the results are obtained by evaluating the oligopeptide obtained by substituting the 5th arginine of BNC with another amino acid using the BNC recognition antibody BC23-11. Of the results of Example 13, the results were obtained by evaluating the oligopeptide obtained by substituting the 6th arginine of BNC with another amino acid using the BNC recognition antibody BC23-11. Of the results of Example 13, BNC recognition antibody BC23-11 evaluated the oligopeptide obtained by substituting the 7th histidine of BNC with another amino acid. Of the results of Example 13, BNC recognition antibody BC30-73 was used to evaluate an oligopeptide in which the first cysteine of BNC was substituted with another amino acid. Among the results of Example 13, the results are obtained by evaluating an oligopeptide obtained by substituting the second lysine of BNC with another amino acid using the BNC recognition antibody BC30-73. Of the results of Example 13, oligopeptides obtained by substituting the third valine of BNC with other amino acids were evaluated with the BNC recognition antibody BC30-73. Of the results of Example 13, the results were obtained by evaluating the oligopeptide obtained by substituting the 4th leucine of BNC with another amino acid using the BNC recognition antibody BC30-73. Of the results in Example 13, BNC recognition antibody BC30-73 was used to evaluate an oligopeptide obtained by substituting the 5th arginine of BNC with another amino acid. Of the results of Example 13, BNC recognition antibody BC30-73 was used to evaluate an oligopeptide obtained by substituting the 6th arginine of BNC with another amino acid. Among the results of Example 13, the results are obtained by evaluating the oligopeptide obtained by substituting the 7th histidine of BNC with another amino acid using the BNC recognition antibody BC30-73. The result of Example 14 is shown. BC30-73 is used as a BNC recognition antibody. Of the results of Example 13, BNC recognition antibody BC30-73 was used to evaluate oligopeptides in which the first cysteine, the second lysine, or the third valine of BNC were substituted with aspartic acid, respectively. The result of Example 15 is shown. Black circle is BNC (SEQ ID NO: 10), black triangle is N-terminal side 1 amino acid deletion (SEQ ID NO: 25), white circle is N-terminal side 2 amino acid deletion (SEQ ID NO: 26), white triangle is N-terminal side 3 amino acids The result of the deletion product (SEQ ID NO: 27) is shown respectively.

Hereinafter, the present invention will be described in detail.

Tag peptides useful for purifying and detecting proteins include
(1) The length of the amino acid is short in order to reduce the influence on the protein to be fused,
(2) No similar sequence exists and high specificity,
(3) the presence of a substance with high affinity for the peptide,
Is required. Thus, as a result of intensive studies on peptides that satisfy the above requirements, it was found that 7 amino acids on the C-terminal side of B-type natriuretic peptide (BNP) (SEQ ID NO: 10, hereinafter abbreviated as BNC) are useful as tag peptides.

BNP is a cyclic peptide consisting of 32 amino acids. The plasma BNP concentration in healthy individuals is extremely low at 20 pg / mL or less, but it increases markedly in patients with chronic and acute heart failure depending on their severity and is important for understanding the pathophysiology of heart failure by measuring BNP For this reason, an extremely large number of samples have been measured in recent years. Usually, BNP is measured by a sandwich method in which BNP is sandwiched between an antibody that recognizes the C-terminus of BNP and an antibody that recognizes a cyclic portion. BNC consisting of the amino acid sequence set forth in SEQ ID NO: 10 is
(1) The amino acid is short,
(2) The existence of a substance having cross-reactivity with BNC has been denied,
(3) A substance with high affinity and specificity exists.
Therefore, it is useful as a tag peptide.

Many tag peptides reported so far have a length of about 10 amino acids, and the FLAG tag (SEQ ID NO: 1) known as a high-performance tag peptide has 8 amino acids. On the other hand, since BNC is 7 amino acids, it is expected that the effect on the protein to be added is further reduced.

In addition, a BNP measurement system using an antibody that binds to BNC has already been marketed as an immunoassay reagent (for example, E test “TOSOH” II (BNP) manufactured by Tosoh Corporation). In other words, a very large number of specimens have been measured so far, but problems such as an unknown cross-reaction have not occurred. This means that proteins and peptides having BNC or similar sequences are not present in the sample, or the presence does not affect the measurement. The above suggests that BNC is useful as a highly specific tag peptide, and can be used effectively as an immunoassay reagent, as well as a highly specific and reactive tag peptide in normal experiments. Is available.

Furthermore, antibodies that recognize BNC exist as substances having high affinity and specificity for BNC. The antibody does not simply recognize the amino acid sequence of BNC but also recognizes that BNC is present on the C-terminal side of the protein. The existence probability of a protein having the amino acid sequence of BNC on the C-terminal side is extremely low. Therefore, BNC is useful as a tag peptide. By using a protein having BNC added to the C-terminal side and an antibody that recognizes BNC, detection with high specificity and high sensitivity is possible.

When using BNC as a tag peptide, BNP contained in the specimen (blood) may affect the binding between the protein added with BNC on the C-terminal side and the substance that recognizes BNC. However, when the effect of BNP contamination was confirmed in an actual measurement system, BNC was added to the C-terminal side even when 100 ng / mL of BNP much higher than the concentration found in clinical specimens was contained in the specimen. It has been confirmed that it does not affect the binding between the protein and a substance that recognizes BNC (see Example 9). However, antibody and antigen concentrations, blocking agents, buffer conditions, etc. differ depending on the measurement system. Therefore, when a new measurement system is constructed, a large excess of BNP is added to the measurement system, and the effect at what concentration level. It ’s better to check if is starting to appear.

By using BNC as a tag peptide, the protein to which BNC is added can be affinity purified. Affinity purification may be carried out using a substance obtained by binding a substance that recognizes BNC to an appropriate carrier while maintaining the binding property to the protein to which BNC is added. Examples of substances that recognize BNC include monoclonal antibodies that recognize BNC, polyclonal antibodies, and antisera. Monoclonal antibodies that do not require consideration of differences between lots are preferable in terms of stable purification. Elution of proteins with added BNC bound to a substance that recognizes BNC is usually performed by changing the pH, but when purifying proteins that are unstable to pH changes, a large excess of BNC is added. And may be eluted.

As a method of adding BNC to a protein to be expressed, a method of adding it by a genetic engineering method can be mentioned. Specifically, a polynucleotide encoding BNC may be added to the N-terminal side or C-terminal side of the protein to be expressed. In addition, when adding BNC to the protein to be expressed, BNC may be added immediately after the N-terminal side or C-terminal side of the protein to be expressed, or an arbitrary linker for avoiding interaction with the protein to be expressed ( Linker) BNC may be added via a peptide. An example of the linker peptide is a flexible linker peptide consisting of the amino acid sequence shown in SEQ ID NO: 11, but is not limited to the sequence. As another method of adding BNC to the protein to be expressed, a method of chemically adding can be mentioned. As one aspect of the method, a maleimide group is introduced into the protein by reacting the protein to be expressed with a reagent such as SMCC (succinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate), and then reacted with BNC. And a method of chemically introducing BNC. There is no particular limitation on the method for binding the protein to be expressed and BNC. For example, it may be added via a polymer such as polyethylene glycol or polyethyleneimine, or chemically added via a functional group other than a maleimide group. May be.

Examples of functional groups other than the maleimide group include carbodiimide, hydrazide, hydroxymethylphosphine, imide ester, isocyanate, bromoacetate, NHS ester (N- and hydroxysuccinimide ester, pyridyl disulfide, psoralen, and the like.

BNC can be used for the purpose of immobilizing a protein on a carrier in addition to the purpose of purifying and detecting the protein as described above. When a protein is directly immobilized on a water-insoluble carrier, the three-dimensional structure is likely to change and the activity is often lost. Therefore, by immobilizing a substance that recognizes BNC on a water-insoluble carrier, and immobilizing the protein with BNC added to the immobilized carrier, it is immobilized on the carrier in a state in which the three-dimensional structure hardly changes. Can be

Often a problem with immunoassay reagents is heterophilic anti-animal antibodies. For example, when a rabbit-derived polyclonal antibody (antigen recognition) and an anti-rabbit antibody (immobilized on a water-insoluble carrier) are included in the reaction system, if an anti-rabbit antibody is present in the measurement sample, the anti-rabbit in the sample The antibody does not capture the originally expected amount of the rabbit-derived polyclonal antibody on the water-insoluble carrier, and an accurate measurement value cannot be obtained. Therefore, by adding BNC to a rabbit polyclonal antibody and immobilizing the anti-BNC antibody on a water-insoluble carrier, a measurement system that eliminates the influence of heterophilic anti-animal antibodies can be constructed.

BNC is an oligopeptide consisting of 7 amino acids described in SEQ ID NO: 10, but an oligopeptide in which one or more of the amino acids constituting BNC are substituted with other amino acids (hereinafter referred to as modified BNC) is also of the present invention. Included in oligopeptides. Since the modified BNC has changed affinity (binding ability) and / or specificity compared to BNC, an appropriate modified BNC may be selected according to the purpose of use and used as a tag peptide. As a preferred embodiment of the modified BNC, an oligopeptide in which one or more amino acids from the first cysteine to the third valine among the amino acids constituting the BNC are substituted with aspartic acid or glutamic acid (that is, acidic amino acid) can be mentioned. It is done. Furthermore, a more preferable modified BNC can be obtained by combining substitutions with altered affinity and / or specificity among the amino acids constituting BNC. An example of the more preferable modified BNC is an oligopeptide (DDDLRRH, SEQ ID NO: 21) in which all amino acids from the first cysteine to the third valine are substituted with aspartic acid. As described above, affinity purification of BNC-added protein is possible by using BNC as a tag peptide. Therefore, affinity purification of a protein added with BNC can be performed more efficiently by using a modified BNC with improved affinity (binding performance) and / or specificity as a tag peptide.

On the other hand, oligopeptides in which some of the amino acids constituting BNC are deleted (hereinafter referred to as deleted BNC) are also included in the oligopeptide of the present invention. Specifically, in the case of a deleted BNC in which one or more of the amino acids from the first cysteine to the third valine among the amino acids constituting the BNC are deleted, the affinity ( Although the binding performance is reduced, depending on the purpose of use, it has sufficient performance as a tag peptide.

Hereinafter, examples will be shown to describe the present invention in more detail. However, these examples show examples of the present invention, and the present invention is not limited to the examples.

Example 1 Preparation of transformant capable of expressing protein added with tag peptide A transformant capable of expressing a protein added with a tag peptide on the C-terminal side of maltose binding protein (MBP) was prepared by the following method.
(1) The oligonucleotides (homologous strand and complementary strand) encoding each tag peptide are diluted with TE (10 mM Tris-HCl + 1 mM EDTA, pH 8.0) so as to be 10 μM, mixed, and then mixed at 95 ° C. For 5 minutes, and then gradually cooled to 25 ° C. to perform annealing. In the case of the FLAG tag (SEQ ID NO: 1), the oligonucleotides used were SEQ ID NO: 12 (homologous strand) and 13 (complementary strand), and in the case of the Myc tag (SEQ ID NO: 2), SEQ ID NO: 14 (homologous strand). And 15 (complementary chain), and in the case of the His tag (SEQ ID NO: 3), SEQ ID NO: 16 (homologous chain) and 17 (complementary chain) are the C-terminal peptides of BNP (SEQ ID NO: 10, hereinafter abbreviated as BNC). In this case, SEQ ID NOs: 18 (homologous strand) and 19 (complementary strand) were used, respectively.
(2) The oligonucleotide (double-stranded DNA) encoding each tag peptide obtained in (1) was inserted into a pMAL-c4X vector (manufactured by New England Raves) previously treated with BamHI and HindIII. The insertion method is to add 4 μL of DNA ligation kit (manufactured by Takara Bio Inc.) to 67 ng of pMAL-c4X vector treated with restriction enzyme and 100 ng of oligonucleotide (1) and perform a ligation reaction at 16 ° C. for 30 minutes. Inserted with.
(3) Escherichia coli (JM109 strain) was transformed with the pMAL-c4X vector obtained by inserting the oligonucleotide encoding each tag peptide obtained in (2) according to a conventional method.

Example 2 Preparation of protein with tag peptide added The transformant obtained in Example 1 was cultured in LB medium containing 50 μg / mL ampicillin, and when the turbidity at 600 nm reached 0.5, the transformant was terminated. After adding IPTG (isopropyl-β-thiogalactopyranoside) to a concentration of 1 mM, the cells were further cultured at 37 ° C. for 4 hours. Thereafter, Escherichia coli was collected, and maltose binding protein (MBP) to which each expressed tag peptide was added was purified by affinity chromatography using an amylose column. FIG. 1 shows the SDS-PAGE result of the purified protein.

Example 3 Isolation of BNP C-Terminal Peptide (BNC) Recognizing Antibody An antibody recognizing the BNP C-terminal peptide (BNC) was isolated by the following method.
(1) 20 mg of BNC consisting of the amino acid sequence of SEQ ID NO: 10 was bound to 10 mg of ovalbumin (manufactured by PIERCE) activated with 10 mg of maleimide according to the attached instructions. Then, it dialyzed with PBS (Phosphate Buffered Saline), and the density | concentration of the ovalbumin which BNC couple | bonded was adjusted to 1 mg / mL.
(2) The emulsion prepared by mixing equal amounts of the protein prepared in (1) and FCA (Freund's complete adjuvant) was injected intraperitoneally at 100 μL / mouse (first immunization).
(3) From the second time, except that FCA was changed to FICA (Freund's incomplete adjuvant), immunization was performed every week in the same manner as (2), and this was repeated three times.
(4) Splenic B cells were removed from the immunized mice and cell fusion was performed with the myeloma cell line by the PEG method.

By the above operation, six hybridomas (BC2-7, BC23-11, BC25-2, BC25-32, BC30-62 and BC30-73) producing antibodies that specifically react with BNC were obtained.

Example 4 Comparison of detection sensitivity using each tag peptide (part 1)
Detection sensitivity comparison using each tag peptide was performed by Western blotting.
(1) MBP (concentration: 2400 fmol, 800 fmol, 240 fmol, 80 fmol, 24 fmol, 8 fmol, 2.4 fmol, 0.8 fmol) to which each tag peptide purified in Example 2 was added was subjected to SDS-PAGE. In order to prevent loss due to nonspecific adsorption when the amount of sample is small, MBP to which each purified tag peptide is added is obtained by diluting myeloma cells with a supernatant crushed with PBS. It was. Specifically, 4 × 10 ⁷ mouse myeloma cells (SP2 / 0) were suspended in 1 mL of PBS, disrupted with ultrasound, and then centrifuged at 15000 rpm for 10 minutes.
(2) MBP to which each tag peptide was added was transferred to a PVDF (polyvinylidene difluoride) membrane according to a conventional method, and then Western blotting as shown below was performed.
(2-1) The PVDF membrane after transfer was blocked by standing overnight at 4 ° C. in TBS (Tris-buffered saline) containing 5 wt% skim milk.
(2-2) Primary antibody (anti-FLAG antibody: manufactured by SIGMA (M2), anti-Myc antibody: manufactured by Wako Pure Chemical Industries, Ltd. (9E10), anti-His antibody: manufactured by Roche (19224216), BNC recognition antibody: BC2-7 / BC23-11 / BC25-2 / BC25-32 / BC30-62 / BC30-73 (antibodies isolated in Example 3)) at 0.5 μg / mL with TBS containing 5 wt% skim milk. Diluted.
(2-3) HRP (horseradish peroxidase) -labeled anti-mouse antibody that was reacted at room temperature for 2 hours, washed the PVDF membrane 4 times with TBS-T (TBS-Tween 20), and diluted 50000 times with TBS-T (GE Healthcare, NA931) was added and reacted at room temperature for 2 hours.
(2-4) The PVDF membrane was washed 4 times with TBS-T and developed with ECL Plus (GE Healthcare, RPN2132).

The results are shown in FIG. Among the tag peptides generally used, the FLAG tag has the highest performance (detection sensitivity: 24 fmol), and the order of the Myc tag and the His tag follows. On the other hand, when an antibody that recognizes BNC consisting of the amino acid sequence set forth in SEQ ID NO: 10 is used, detection is equivalent to about 10 times more sensitive than detection using a FLAG tag (detection sensitivity: 2.4 to 24 fmol). Turned out to be.

Example 5 Comparison of detection sensitivity using each tag peptide (part 2)
Comparison of detection sensitivity using each tag peptide was performed by ELISA.
(1) The antibody to be evaluated (tag peptide recognition antibody) was immobilized on an ELISA plate at 0.1 μg / well and blocked with 1 wt% skim milk.
(2) MBP to which each tag peptide purified in Example 2 was added and reacted (concentration: 24 pmol / well, 2.4 pmol / well, 240 fmol / well, 24 fmol / well, 2.4 fmol / well, 0 .24 fmol / well).
(3) The amount of MBP added with each tag peptide captured on the solid phase was measured with an HRP-labeled anti-MBP antibody (manufactured by New England Raves, E8038S).

The results are shown in FIG. An antibody (BC2-7 / BC23-11 / BC25-2 / BC25-32 / BC30-62 / BC30-73) that recognizes BNC having the amino acid sequence set forth in SEQ ID NO: 10 is immobilized on a solid phase. Compared to antibodies that recognize FLAG tag (Anti-FLAG) or His tag (Anti-His) immobilized on a solid phase, tag peptide was added especially in the low concentration region (240 fmol / well or less) It was found that the amount of MBP supplemented was increasing. Of the BNC-recognizing antibodies examined this time, those in which BC30-73 was immobilized on the solid phase were 100 times more than those in which the antibody that recognizes the FLAG tag (Anti-FLAG) was immobilized on the solid phase. It has the sensitivity of.

Example 6 Performance as tag for affinity purification (elution by pH change)
The elution of the peptide added with BNC bound to the C-terminal peptide (BNC) recognition antibody of BNP with pH change was confirmed.
(1) Five types (BC2-7 / BC23-11 / BC25-2 / BC30-62 / BC30-73) of the BNC-recognizing antibodies obtained in Example 3 were each fixed to 0.1 μg in a 96-well plate. Turned into.
(2) The amino acid described in SEQ ID NO: 20 (the 9th to 15th amino acids correspond to the amino acid described in SEQ ID NO: 10) after sufficiently blocking with PBS containing 1% by weight skimmed milk and introducing biotin into the N-terminus 1 μg of oligopeptide consisting of the sequence was added and reacted.
(3) After reaction for 1 hour, buffers having different pH (PBS, 100 mM glycine-hydrochloric acid buffer (pH 1.5), 100 mM glycine-hydrochloric acid buffer (pH 2.5), 100 mM glycine-hydrochloric acid buffer (pH 3.0) ) And 100 mM glycine-hydrochloric acid buffer (pH 3.5)).
(4) Streptavidin labeled with alkaline phosphatase was reacted. After washing, p-nitrophenyl phosphate (PNPP) was reacted to measure enzyme activity.

The results are shown in FIG. It can be seen that any of the BNC-recognizing antibodies can completely dissociate the oligopeptide consisting of the amino acid sequence shown in SEQ ID NO: 20 by changing the pH, and the oligopeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 (BNC ) Was found to have sufficient performance as a tag peptide for affinity purification. Among the BNC-recognizing antibodies examined this time, BC2-7 is excellent as an antibody for affinity purification because it can completely dissociate the bound peptide under relatively mild conditions (pH 3.5).

Example 7 Performance as tag for affinity purification (elution by adding peptide)
The elution of BNP-added peptide bound to the BNP C-terminal peptide (BNC) recognition antibody was confirmed by the addition of BNC.
(1) Five types (BC2-7 / BC23-11 / BC25-2 / BC30-62 / BC30-73) of the BNC-recognizing antibodies obtained in Example 3 were each fixed to 0.1 μg in a 96-well plate. Turned into.
(2) After sufficiently blocking with PBS containing 1% by weight skim milk, 1 μg of an oligopeptide consisting of the amino acid sequence shown in SEQ ID NO: 20 having biotin introduced into the N-terminus was added and reacted.
(3) After reaction for 1 hour, 100 μL each of a solution containing BNP C-terminal peptide (BNC) consisting of the amino acid sequence shown in SEQ ID NO: 10 was added (respective peptide concentrations: 1 μg / mL, 10 μg / mL and (100 μg / mL).
(4) After the reaction for 1 hour, it was washed thoroughly and reacted with streptavidin labeled with alkaline phosphatase.
(5) After washing, PNPP was reacted to measure enzyme activity.

The results are shown in FIG. In any case of using any BNC-recognizing antibody, it was found that the addition of BNC can completely dissociate from the bound oligopeptide having the amino acid sequence shown in SEQ ID NO: 20. Since elution of a protein (peptide) to which a tag peptide is added without a change in pH means that it can be applied to a protein that is vulnerable to a change in pH, it is described in SEQ ID NO: 10. It can be seen that an oligopeptide (BNC) comprising an amino acid sequence has a preferable performance as a tag peptide for affinity purification.

Example 8 Purification of Protein Added with BNP C-Terminal Peptide (BNC) The protein was purified using the BNP C-terminal peptide (BNC) consisting of the amino acid sequence shown in SEQ ID NO: 10 as a tag peptide.
(1) The transformant (E. coli) expressing MBP added with BNC obtained in Example 1 was cultured by the method described in Example 2 and then disrupted by ultrasonication to obtain a soluble fraction (lysate )).
(2) Activation of 5 types of BNC recognition antibodies (BC2-7 / BC23-11 / BC25-2 / BC30-62 / BC30-73) obtained in Example 3 with NHS (N-hydroxysuccinimide) The antibody-immobilized column was prepared by immobilizing on the prepared column (GE Healthcare, NHS HP SpinTrap) according to the instruction attached to the product.
(3) By loading the soluble fraction of (1) onto the antibody-immobilized column of (2), MBP added with BNC was adsorbed to the antibody-immobilized column.
(4) The adsorbed MBP was eluted with 100 mM glycine-hydrochloric acid buffer (pH 2.5). In addition, purification of MBP to which BNC was added by affinity chromatography using an amylose column was performed as a comparative example.

The purity test result by SDS-PAGE is shown in FIG. The second lane from the left is the soluble fraction (lysate), and the band at the black triangle is a band corresponding to MBP with BNC added (the other bands are bands corresponding to transformant-derived proteins). . From FIG. 6, it can be seen that the degree of protein purification when MBP added with BNC is purified using a BNC-recognizing antibody-binding column is equivalent to that when an amylose column is used. Therefore, it was found that by using an oligopeptide (BNC) having the amino acid sequence shown in SEQ ID NO: 10 as a tag peptide, the protein can be purified with high purity without contamination.

Example 9 Effect of B-type natriuretic peptide (BNP) concentration When the C-terminal peptide (BNC) of BNP consisting of the amino acid sequence of SEQ ID NO: 10 was used for immunoassay, the concentration of BNP present in the sample In some cases, the binding of BNC and antibody may be competitively inhibited by the BNP. Therefore, the influence of the binding between BNC and BNC-recognizing antibody due to the BNP concentration was examined.
(1) An anti-estradiol antibody (hereinafter abbreviated as anti-E2 antibody) was prepared by the method described in Patent Document 5.
(2) After reacting the anti-E2 antibody prepared in (1) with Sulfo-SMCC (PIERCE, 22322) according to the instructions, the anti-E2 antibody covalently bound to BNC is reacted with a large excess of BNC. It produced (FIG. 7).
(3) A certain amount of the anti-E2 antibody prepared in (1) was reacted with magnetic beads on which 3 μg of BNC-recognizing antibody (BC23-11) was immobilized.
(4) Among the following three components, 50 μL of (b) and (c) was added, and the reaction was started by adding 50 μL of (a). The three ingredients are
(A) BNC-labeled antibody (5 ng / 50 μL),
(B) Estradiol labeled alkaline phosphatase (0.05 mA / 50 μL),
And (c) various concentrations of BNP.
(5) After reacting at 37 ° C. for 10 minutes, the plate was thoroughly washed, and the enzyme activity of alkaline phosphatase (ALP) remaining on the solid phase was measured by a conventional method.

FIG. 8 shows a plot of the signal obtained after the reaction on the vertical axis and the concentration of BNP on the horizontal axis. In FIG. 8, Cal2 to Cal6 are standard products used in a calibration curve of a commercially available immunoassay reagent (manufactured by Tosoh Corporation) (E test “TOSOH” II (BNP)), and each has a concentration of BNP. Are 15.2 pg / mL (Cal2), 42.5 pg / mL (Cal3), 153 pg / mL (Cal4), 643 pg / mL (Cal5), 2450 pg / mL (Cal6). Under the present measurement conditions, even if 100 ng / mL of BNP, which is a higher concentration than the high-concentration standard product in the commercially available reagent, is present in the sample, it affects the binding between the BNC-added protein and the BNC-recognizing antibody. It turns out not to give.

It should be noted that even in a specimen that is determined to have a very high BNP concentration clinically, the BNP concentration rarely exceeds 1000 pg / mL (healthy individuals have a few pg / mL). Since the molecular weight of BNP is 3464, when 1000 pg / mL is converted into a molar concentration, it becomes 0.29 pmol / mL. Assuming that the sample (serum) brought into the reaction is diluted 2-fold with a buffer and 150 μL is used, 0.02175 pmol of BNP will be brought into each well. On the other hand, when estimating the amount of BNC-recognizing antibody that can be immobilized on an ELISA plate (manufactured by NUNC, Maxisorp), the adsorption capacity of the ELISA plate is 650 ng / cm ² and the area per well is 2.7 cm ² (value shown in the catalog) Therefore, the amount of antibody immobilized is 1775 ng / well. Assuming the molecular weight of the antibody is 1.5 × 10 ⁵ , the number of moles of immobilized antibody is calculated to be 11.7 pmol. Since the antibody has two antigen binding sites, the binding site that can participate in the reaction is 23.4 pmol. That is, while there are 23.4 pmol of antigen binding sites, the BNP concentration is 0.02175 pmol (about 1000 times in molar ratio) as described above. Therefore, even if a high concentration of BNP is present in clinical specimens, most of the binding sites remain, and it is considered that there is no influence on the measurement system.

Example 10 Preparation of antibody bound with BNP C-terminal peptide (BNC) monosubstitution One of the amino acids constituting the BNP C-terminal peptide (BNC) consisting of the amino acid sequence set forth in SEQ ID NO: 10 In order to evaluate the binding performance of oligopeptides substituted with other amino acids (hereinafter referred to as monosubstituted BNC), an antibody to which monosubstituted BNC was bound was prepared.
(1) A vector capable of expressing a recombinant anti-estradiol antibody (hereinafter, RaMoAb) in which BNC was directly bound to the heavy chain (H chain) C-terminal side was prepared by genetic engineering techniques.
(2) Among the vectors prepared in (1), mutations were introduced into the oligonucleotide encoding BNC bound to the H-chain C-terminal side of RaMoAb using KOD-PLUS-Mutageness Kit (Toyobo Co., Ltd.). Thus, a vector capable of expressing RaMoAb in which mono-substituted BNC was directly bound to the H chain C-terminal side was prepared (a list of substitution positions and substitution amino acids is shown in Table 1).

(3) Based on the method described in Patent Document 5, using the vector prepared in (1), RaMoAb (hereinafter referred to as RaMoAb-BNC) in which BNC was directly bonded to the C-terminal side of the H chain was prepared in (2). RaMoAb (hereinafter referred to as RaMoAb-mBNC) in which mono-substituted BNC was directly bound to the H-chain C-terminal side was expressed in the culture supernatant using a vector.

Example 11 Quantification of RaMoAb-mBNC The concentration of RaMoAb-mBNC expressed in the culture supernatant in Example 10 is not constant. is required. Therefore, the concentration of RaMoAb-mBNC in the culture supernatant was quantified using an ELISA method.
(1) An anti-rabbit antibody (manufactured by Millipore, AP132) was immobilized on an ELISA plate at 0.1 μg / well and blocked with 1 wt% skim milk.
(2) The culture supernatant containing RaMoAb-mBNC obtained in Example 10 was added to the ELISA plate obtained in (1) and allowed to react.
(3) The amount of RaMoAb-mBNC captured on the solid phase was measured with an alkaline rabbit phosphatase (ALP) labeled anti-rabbit antibody (Millipore, AP132A).
(4) RaMoAb-mBNC concentration was calculated by using the calibration method to measure various concentrations of RaMoAb-BNC in advance by the ELISA methods described in (1) to (3) above.

Example 12 Labeling of BNP C-terminal peptide (BNC) -recognizing antibody Four types (BC2-7 / BC23-11 / BC30-62 / BC30-73) of the BNC-recognizing antibodies obtained in Example 3 were alkaline. ALP was labeled using a phosphatase kit (LK12, manufactured by Dojindo) according to the protocol attached to the kit.

Example 13 Evaluation of binding performance of C-terminal peptide (BNC) monosubstituted product of BNP First, a calibration curve of RaMoAb-BNC was prepared using the ELISA method shown below.
(1) Estradiol-labeled BSA (manufactured by Sigma: E5630) was immobilized on an ELISA plate at 0.1 μg / well and blocked with 1 wt% skim milk.
(2) Various concentrations of RaMoAb-BNC were added to the ELISA plate of (1) and allowed to react.
(3) Among the ALP-labeled BNC-recognizing antibodies prepared in Example 12, those obtained by labeling BC23-11 with ALP 1000-fold with PBS containing 0.1 wt% skim milk and captured by the RaMoAb captured on the solid phase -Reacted with BNC.

Fig. 9 shows the calibration curve of the obtained RaMoAb-BNC. FIG. 9 shows that as the RaMoAb-BNC concentration increases, the amount of RaMoAb-BNC bound to the solid phase increases, so that the fluorescence intensity (FI) derived from ALP-labeled BC23-11 increases.

From this, first of all
(A) The fluorescence intensity derived from the ALP-labeled BNC-recognizing antibody when various concentrations of RaMoAb-BNC were added by the method of (1) to (3) was measured,
(B) A RaMoAb-BNC calibration curve is created by plotting the RaMoAb-BNC concentration on the horizontal axis and the fluorescence intensity measured in (A) on the vertical axis.
next,
(C) Quantifying the concentration of RaMoAb-mBNC in the culture supernatant by the method described in Example 11,
(D) Measure the fluorescence intensity derived from the ALP-labeled BNC-recognizing antibody (the same antibody as (A)) bound to RaMoAb-mBNC by the method of (1) to (3),
(E) The concentration determined in (C) is plotted on the horizontal axis, and the fluorescence intensity measured in (D) is plotted on the vertical axis.
As a result, it is understood that the amino acid substitution improves the binding performance if the plot position of (E) is above the calibration curve created in (B), and the amino acid substitution reduces the binding performance if it is lower ( FIG. 10). The methods (A) to (E) are useful in that the binding performance can be evaluated even when the concentration of RaMoAb-mBNC in the culture supernatant is different.

Therefore, the binding performance of RaMoAb-mBNC was evaluated using the methods shown in (A) to (E) above. The results when BC2-7 is used as a BNC recognition antibody are shown in FIGS. 11 to 17, the results when BC23-11 is used are shown in FIGS. 18 to 24, and the results when BC30-73 is used are shown in FIG. To 31 respectively.

Among the results shown in FIG. 11 to FIG. 31, the results will be described in detail using FIG. 18 as an example. In FIG. 18, the plots indicated by white circles are the results obtained using a dilution series of RaMoAb-BNC purified product, and the calibration curve created using the plots is shown in FIG. The plots indicated by black circles are the results for RaMoAb-mBNC expressed in the culture supernatant by the method of Example 10. The plots indicated by asterisks are the results for RaMoAb-BNC expressed in the culture supernatant by the method of Example 10 performed as a control experiment. Since the plot indicated by the asterisk overlaps with the calibration curve in the error range, it can be seen that this experiment is valid. From the results shown in FIG. 18, when the first cysteine (C) in the BNC consisting of the amino acid sequence shown in SEQ ID NO: 10 is substituted with glutamic acid (E), the binding performance does not change, but aspartic acid (D) It can be seen that the binding performance is slightly reduced when the substitution is made, and the binding performance is almost lost when substitution is made with other amino acids.

The results shown in FIGS. 11 to 31 are summarized below.

(A) When the first cysteine (C) is substituted When BC2-7 is used as a BNC-recognizing antibody (FIG. 11), the binding performance is improved by substitution with glutamic acid (E) or aspartic acid (D). Substitution with other amino acids reduced the binding performance. When BC23-11 was used as a BNC-recognizing antibody (FIG. 18), there was no change in binding performance when substituted with glutamic acid (E), but binding performance decreased slightly when substituted with aspartic acid (D). When the amino acid was substituted, the binding performance was almost lost. When BC30-73 was used as the BNC-recognizing antibody (FIG. 25), the binding performance was slightly improved when substituted with glutamic acid (E) or aspartic acid (D), but the binding performance decreased when substituted with other amino acids.

(B) When the second lysine (K) is substituted When BC2-7 is used as a BNC-recognizing antibody (FIG. 12), the substitution performance is unchanged when arginine (R) is substituted. By substituting with an amino acid, the binding performance was improved, and it was particularly effective when substituting with aspartic acid (D). When BC23-11 was used as a BNC-recognizing antibody (FIG. 19), the substitution performance was slightly lowered when it was substituted with aspartic acid (D), but the substitution performance of other amino acids was not affected. When BC30-73 was used as a BNC-recognizing antibody (FIG. 26), the binding performance was lowered when substituted with arginine (R), but the binding performance was improved by substitution with other amino acids, especially asparagine. Substitution with acid (D) was effective.

(C) When the third valine (V) is substituted When BC2-7 is used as the BNC-recognizing antibody (FIG. 13), aspartic acid (D), alanine (A), glutamic acid (E) or proline (P) The bonding performance was improved by substituting for. When BC23-11 is used as a BNC-recognizing antibody (FIG. 20), there are uniformly amino acid substitutions that improve binding performance, amino acid substitutions that do not change, and amino acid substitutions that decrease, but aspartic acid (D), glutamic acid ( Substituting with E), proline (P) or threonine (T) particularly improved the binding performance. Even when BC30-73 is used as a BNC-recognizing antibody (FIG. 27), there are uniformly amino acid substitutions that improve binding performance, amino acid substitutions that do not change, and amino acid substitutions that decrease, but alanine (A), aspartic acid Substitution with (D) or glutamic acid (E) particularly improved the binding performance.

(D) When the fourth leucine (L) is substituted When BC2-7 is used as a BNC recognition antibody (FIG. 14), phenylalanine (F), proline (P), aspartic acid (D), glutamic acid (E) Alternatively, when the substitution was made with isoleucine (I), the binding performance was not changed, but when the substitution was made with other amino acids, the binding performance was lowered. When BC23-11 is used as a BNC-recognizing antibody (FIG. 21), there are uniformly amino acid substitutions that improve binding performance, amino acid substitutions that do not change, and amino acid substitutions that decrease, but proline (P), aspartic acid ( Substitution with D) or glutamic acid (E) particularly improved the binding performance. When BC30-73 was used as a BNC-recognizing antibody (FIG. 28), the binding performance was almost lost by substitution with other amino acids.

(E) When the 5th arginine (R) is substituted When BC2-7 is used as a BNC-recognizing antibody (FIG. 15), the binding performance does not change when it is substituted with proline (P). When substituted with an amino acid, the binding performance decreased. When BC23-11 was used as the BNC-recognizing antibody (FIG. 22), the binding performance decreased or almost disappeared due to substitution with other amino acids. On the other hand, when BC30-73 was used as a BNC-recognizing antibody (FIG. 29), there was no change in binding performance when substituted with proline (P), but binding performance was improved when substituted with other amino acids.

(F) When the 6th Arginine (R) is Replaced When BC2-7 is used as the BNC recognition antibody (FIG. 16), when BC23-11 is used (FIG. 23), when BC30-73 is used ( 30), in any case, the binding performance was reduced or almost disappeared by substitution with another amino acid.

(G) When the 7th histidine (H) is substituted When BC2-7 is used as a BNC-recognizing antibody (FIG. 17), when BC23-11 is used (FIG. 24), when BC30-73 is used ( In any of the cases shown in FIG. 31), binding performance disappeared due to substitution with other amino acids.

When the results of (a) to (g) are summarized, the first cysteine (C), the second of the BNC consisting of the amino acid sequence shown in SEQ ID NO: 10, although the tendency is partially different depending on the BNC recognition antibody used. By substituting the lysine (K) or the third valine (V) of the amino acid with aspartic acid (D) or glutamic acid (E) (that is, acidic amino acid), the binding performance is improved as compared with BNC. Recognize. On the other hand, when the 6th arginine (R) or the 7th histidine (H) in the BNC consisting of the amino acid sequence set forth in SEQ ID NO: 10 is substituted with another amino acid, the binding performance decreases or disappears. It turns out that amino acid substitution of is not preferable.

Example 14 Evaluation of Binding Performance of BNP C-Terminal Peptide (BNC) Multiple Substituent According to the examination in Example 13, the first cysteine (C) and second of BNC comprising the amino acid sequence set forth in SEQ ID NO: 10 It has been found that the binding performance (affinity) is improved by substituting lysine (K) or the third valine (V) with aspartic acid (D) or glutamic acid (E) (that is, acidic amino acid). Therefore, an oligopeptide (DDDLRRH, SEQ ID NO: 21) in which the first cysteine (C) to the third valine (V) in BNC consisting of the amino acid sequence shown in SEQ ID NO: 10 are all replaced with aspartic acid (D) is obtained. An antibody bound to the C-terminus of the RaMoAb H chain was prepared by mutagenesis using KOD-Plus-Mutageness Kit (manufactured by Toyobo Co., Ltd.). The same as in Example 10, Example 11 and Example 13 The binding performance was evaluated by the method. BC30-73 is used as a BNC recognition antibody.

Results are shown in FIG. In addition, as a control, evaluation results with an oligopeptide (DKVLRH, SEQ ID NO: 22) in which only the first cysteine (C) is replaced with aspartic acid (D) (D plot in FIG. 25), second lysine (K ) Only in the oligopeptide (CDVLRRH, SEQ ID NO: 23) substituted with aspartic acid (D) (plot of D in FIG. 26), only the third valine (V) was substituted with aspartic acid (D). FIG. 33 shows a summary of the evaluation results (Plot of D in FIG. 27) of the oligopeptide (CKDLRRH, SEQ ID NO: 24). Compared with the calibration curve (results with various concentrations of BNC), the binding performance was improved up to about 3 times with mono-substituted BNC (when SEQ ID NO: 23), but the tri-substituted BNC shown in SEQ ID NO: 21 Then, it turned out that it improves to about 4 times. Therefore, it can be seen that by introducing a plurality of specific preferred amino acid substitutions into BNC, the binding performance is further improved.

Example 15 Evaluation of Binding Performance of BNP C-Terminal Peptide (BNC) Deletion Form Among BNCs consisting of the amino acid sequence set forth in SEQ ID NO: 10, one having each amino acid deleted from the N-terminal side was prepared, The binding performance of the oligopeptide was evaluated.
(1) According to the method of Example 1 and Example 2, a fusion protein was produced in which an oligopeptide in which one amino acid was deleted from the C terminus of BNC was bound to the C terminus of maltose binding protein (MBP). The peptide sequences actually bound to the C-terminus of MBP are KVLRRH (SEQ ID NO: 25), VLRRH (SEQ ID NO: 26) and LRRH (SEQ ID NO: 27).
(2) The reactivity with respect to the said oligopeptide was evaluated by ELISA method shown below.
(2-1) The purified protein was adjusted to a concentration of 1 mg / mL with PBS, and a 2-fold dilution series was prepared with PBS based on this. The resulting solution was immobilized on an ELISA plate at 100 μL / well and blocked with 1 wt% skim milk.
(2-2) ALP-labeled BNC-recognizing antibodies (BC2-7, BC30-73, BC30-62, BC23-11) obtained in Example 12 were added 1000 times with PBS containing 0.1% by weight skim milk. The enzyme activity of ALP remaining on the solid phase was measured by a conventional method after dilution.

Results are shown in FIG. Although the tendency varies depending on the BNC-recognizing antibody used, the binding performance is reduced as a result of amino acid deletion compared to the full length of BNC (7 amino acids, SEQ ID NO: 10). However, it can be seen that certain binding performance remains even if the amino acid at the N-terminal side is deleted. Therefore, depending on the purpose of use as a tag peptide, the BNC consisting of the amino acid sequence set forth in SEQ ID NO: 10 contains at least 4 amino acids on the C-terminal side (SEQ ID NO: 27) (ie, from the first cysteine). Any oligopeptide that lacks one or more of the amino acids up to the third valine) can be used as a tag peptide.

By adding a BNP C-terminal peptide consisting of the amino acid sequence shown in SEQ ID NO: 10 to a protein, it is possible to purify the protein with high purity and detect it with high sensitivity. This is an extremely effective method for immobilizing and detecting proteins for immunoassay reagents. Among the BNP C-terminal peptides of the amino acid sequence shown in SEQ ID NO: 10,
(A) an oligopeptide in which one or more amino acids from the first cysteine to the third valine are substituted with acidic amino acids, or (B) one of the amino acids from the first cysteine to the third valine Oligopeptides with one or more deletions,
Even so, the high-purity purification and high-sensitivity detection are possible.

The specification, claims, drawings and abstract of Japanese Patent Application No. 2009-277382 filed on Dec. 7, 2009 and Japanese Patent Application No. 2010-259424 filed on Nov. 19, 2010. Is hereby incorporated by reference as a disclosure of the specification of the present invention.

Claims

An oligopeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 and useful for purifying and detecting a protein.
Of the oligopeptide consisting of the amino acid sequence shown in SEQ ID NO: 10, one or more amino acids from the first cysteine to the third valine are substituted with acidic amino acids, which is useful for purifying and detecting proteins. Oligopeptide.
Oligopeptide useful for purifying and detecting proteins, wherein one or more amino acids from the first cysteine to the third valine are deleted from the oligopeptide consisting of the amino acid sequence set forth in SEQ ID NO: 10 peptide.
A method for purifying and detecting a protein using the protein to which the oligopeptide according to any one of claims 1 to 3 is added and a substance that recognizes the peptide.
The purification according to claim 4, wherein the protein is a protein obtained by adding the oligopeptide according to any one of claims 1 to 3 to the C-terminal side, and the substance that recognizes the peptide is an antibody that recognizes the peptide. -Detection method.
The purification / detection method according to claim 4 or 5, wherein the protein is a protein obtained by genetically adding the oligopeptide according to any one of claims 1 to 3.
The purification / detection method according to claim 4 or 5, wherein the protein is a protein obtained by chemically adding the oligopeptide according to any one of claims 1 to 3.