JP2011184418A - Affinity variable antibody - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a modified antibody having affinity to antigens variable by a mild condition change such as calcium concentration change. <P>SOLUTION: There is provided a fused protein obtained by preparing a polynucleotide encoding a fused protein composed of an antibody VH region-calmodulin-M13 peptide-antibody VL region and transforming an E. coli with an expression plasmid containing the polynucleotide. The fused protein develops high affinity to an antigen in the absence of calcium, however, the affinity is remarkably lowered by addition of ≥1 μM of calcium ion. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention can be used for applications such as pharmaceuticals, clinical diagnostics, biosensors, affinity ligands (separating agents), etc., and is a modification that changes the affinity with an antigen in response to changes in the external environment, in particular, changes in calcium concentration. Type antibodies.

  Using the property of binding specifically to a specific substance, antibodies can be used for disease diagnostic tests, antibody drugs, environmental monitoring, food quality control, antigen removal from specimens, protein affinity purification, and cell and virus recovery. Application to such as has been attempted. Currently, methods for producing antibodies that bind to various substances including proteins have been established. That is, it can be said that there is no substance that cannot be detected / purified using antibodies. In recent years, with the development of genetic engineering, antibody engineering has rapidly developed since it has become easier to obtain antibody genes. This makes it possible to introduce and express antibody genes not only in animal cells but also in yeast (patent document 1) and E. coli (non-patent document 1), and mass production of antibodies is facilitated.

  An antibody is generally a tetrameric protein consisting of two heavy chains (H chains) and two light chains (L chains). However, when an antibody is produced by gene recombination, the entire protein must be expressed. For example, if only the ability to bind to an antigen is required, only the Fab part or Fv part containing the variable region may be expressed, and if only the cytotoxicity or complement activation ability is required, Fc Only the part needs to be expressed.

  In addition, since Fab and Fv are formed from two polypeptides, there is a problem in expressing and associating both genes equally. However, both polypeptides are artificially composed of several amino acid residues called linkers. A method of functioning as a single polypeptide called scFv by linking with a polypeptide has also been developed (Patent Document 2). Furthermore, an attempt has been made to bind a physiologically active polypeptide or low molecular weight compound to this antibody fragment so that the pharmaceutical agent acts only on a specific tissue or cell (Patent Document 3).

  Fv is composed of two polypeptides, VH and VL fragments, each of which has the ability to bind to an antigen and is the smallest polypeptide having an antibody function. When VH and VL are prepared and mixed, both polypeptides exist as monomers, but it is known to form Three-Hybrid when an antigen is mediated, and an open sandwich immunoassay method using this Has been reported (Non-Patent Document 2). This method has made it possible to measure a low molecular weight compound that could not be measured by the conventional sandwich method with high sensitivity.

  Furthermore, using this property, two fusion proteins in which DNA-binding protein or transcriptional activity regulator is bound to VH and VL, respectively, and when the antigen is present, both fusion proteins are complexed via the VH and VL regions. An application has been devised such as promoting or suppressing gene expression by forming a body and allowing this complex to act on a gene expression vector using a promoter that responds to a DNA binding protein (Patent Document 4).

  One of the uses of antibodies is to specifically recover proteins, viruses or cells from a culture solution or the like. In this application, high selectivity for antigen is utilized, and the target substance such as protein is adsorbed on the carrier carrying the antibody, and impurities that are not adsorbed are washed away from the carrier, and then an acidic buffer or protein denaturant is used as the carrier. It is passed through to dissociate the antibody and the target substance, and elution is recovered. This method is a very effective purification method because a high-purity target product can be recovered by a simple operation in one step. However, since the binding force between the antibody and the target substance is extremely strong, a strongly acidic condition around pH 3 and a very high concentration of a protein denaturant are required for dissociation. For this reason, the target is often deactivated, and there is a drawback that it can be used only for a highly stable target. Further, since the immobilized antibody itself is gradually deactivated, there is a drawback that the number of times it can be used repeatedly is limited. That is, there has been a demand for an antibody that has high selectivity and strong binding force when the target substance is adsorbed and whose binding force decreases due to milder change of conditions during elution.

  Furthermore, for example, if an antibody whose affinity for an antigen is lowered by the action of a compound different from the antigen on the antibody is provided, a new application development as a sensor or a diagnostic reagent becomes possible. For example, by modifying an antibody against an arbitrary substance serving as a reporter, creating an antibody whose affinity for the reporter substance is reduced by the binding of the heavy metal, and detecting the heavy metal using a complex of the modified antibody and the reporter molecule Possible uses. If such an antibody is created, a simple biosensor can be provided by detecting the release of the reporter molecule from the complex by the action of heavy metal.

JP-A-6-030778 Japanese Patent Laid-Open No. 9-220092 Japanese Patent Application Laid-Open No. 6-087898 JP 2009-201504 A

Itoh, et al. (1993) J. Am. Biol. Chem. , 268, 16639-16647 Ueda, et al. (1996) Nature Biotechnol. , 14, 1714-1718 Kaihara et al. , (2008) Anal. Sci. , 24, 1704-1408 Wada, et al. (2003) J. Am. Am. Chem. Soc. , 125, 16228-16234 D. M.M. Hoover et al. (2002) Nucleic Acid Res. 30, e43 Gang Wu et al. (2006) Protein Expr Purif. 47, 441-445 T.A. Kajino et al. , (2000) Appl. Environ. Microbiol. , 66, 638-642

  The present invention can be used for applications such as pharmaceuticals, clinical test drugs, biosensors, affinity ligands (separating agents), and the like, and the affinity with an antigen changes in response to changes in the external environment, particularly calcium concentration. It is to provide a modified antibody.

  As a result of intensive investigations on the above problems, the inventors of the present application fused a protein whose conformation is changed by binding of a low molecular weight substance, for example, calmodulin whose conformation is changed by binding of calcium ions, and an antibody. The inventors have found that the affinity of an antibody can be reduced by changing the conditions such that the protein, cell or virus is not inactivated, and the present invention has been completed.

That is, the present invention includes the following inventions.
(1) A fusion protein represented by the general formula, XYZ, wherein X is a peptide fragment containing a VH region or a VL region of an antibody, and Y is a low molecular weight compound bound by a specific low molecular weight compound. It is a peptide fragment having a three-dimensional structure different from the case where it does not exist, and Z is a peptide fragment containing the antibody VH region or VL region that is different from X, and formed by X and Z by the binding of the low molecular weight compound A fusion protein in which the affinity with a specific antigen for an antibody to be increased or decreased.
(2) The fusion according to (1), wherein Y is a fusion protein represented by the general formula Y1-Y2, Y1 is calmodulin, Y2 is a calmodulin-binding peptide, and the specific low-molecular compound is a calcium ion. protein.
(3) The fusion protein according to (2), wherein Y2 is a partial structure of myosin light chain kinase.
(4) The fusion protein according to (2) or (3), wherein Y2 is peptide M13 derived from the calcium binding domain of myosin light chain kinase.
(5) The fusion protein according to (2) to (4), wherein Y1 and Y2 are linked by a Gly-Gly linker.
(6) The fusion protein according to (1) to (5), wherein X is a peptide fragment containing a VH region of an antibody, and Z is a peptide fragment containing an antibody VL region.
(7) The VH region is a VH region of an anti-lysozyme antibody comprising the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence in which one or several amino acids are substituted, deleted or added in the sequence, and the VL region Is a VL region of an anti-lysozyme antibody consisting of the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence in which one or several amino acids are substituted, deleted or added in the sequence, and calcium ion of 1 μM or more is added Thus, the fusion protein according to any one of (1) to (6), wherein the lysozyme binding ability is reduced in the absence of calcium.

  The fusion protein of the present invention can change the binding property of the fusion protein to a target substance by changing the concentration of a specific low molecular compound such as calcium ion. Changes in the concentration of a specific low molecular weight compound are milder than the denaturation conditions normally required to release the antigen bound to the antibody, and it is possible to avoid deactivation of the target product. .

It is a process figure to intermediate | middle vector pBS-CaM-gly-gly-M13-VL among the processes of expression vector pET-trx-His-VH-CaM-gly-gly-M13-VL preparation of this fusion protein. It is process drawing after intermediate | middle vector pBS-CaM-gly-gly-M13-VL among the processes of expression vector pET-trx-His-VH-CaM-gly-gly-M13-VL preparation of this fusion protein. The result of having tested the influence of the calcium ion with respect to the binding ability to the lysozyme of this fusion protein by ELISA method is shown. In the figure, “Ca ²⁺ (+)” indicates the results of ELISA in the presence of 10 mM calcium ion, and “Ca ²⁺ (−)” indicates the results of ELISA in the absence of calcium. In addition, “without lysozyme” indicates the results of the blank test when lysozyme was not bound to the solid phase, and “without trx” indicates that the fusion protein was not added. It is a figure which shows the calcium concentration dependence of the binding ability to the lysozyme of this fusion protein.

Hereinafter, the present invention will be described in detail.
Examples of the protein that changes the three-dimensional structure by changing the concentration of the low-molecular compound used in the present invention include calmodulin shown in SEQ ID NO: 1 (non-patent document 3), maltose-binding protein whose three-dimensional structure changes by the binding of maltose, and glutamine by binding of glutamine. A binding protein etc. can be used (nonpatent literature 4). Among these, calmodulin has a molecular weight of about 17 kDa and is relatively small, and the factor causing the conformational change is inexpensive and highly stable calcium, so that it can be used more suitably. Calmodulin has four calcium ion binding sites in the molecule, and the three-dimensional structure of calmodulin is greatly changed by the binding of calcium to these sites. (Non Patent Literature 3)

  In the present invention, a gene fragment encoding the entire antibody H chain or a part including the VH region, or a gene fragment encoding the entire L chain or a part including the VL region is added to the 5 'end of the calmodulin gene. On the other hand, at the 3 ′ end, peptide M13 (SEQ ID NO: 13) derived from the calcium binding domain of myosin light chain kinase and the gene fragment encoding the entire antibody L chain or a part containing the VL region, or the entire H chain or Of the gene fragment encoding a part containing the VH region, the one different from the gene fragment added to the 5 ′ end side of the calmodulin gene (for example, encoding the entire antibody H chain or a part containing the VH region on the 5 ′ end side) When a gene fragment is added, a gene to which the entire antibody L chain or a part including the VL region is added) is introduced into an arbitrary cell using an arbitrary expression vector, and the cell is cultured. To produce the desired fusion protein.

  At the time of production, the calmodulin gene can be used by changing to any nucleotide sequence within the sequence of SEQ ID NO: 1 within the range in which the encoded amino acid does not change, and in particular, the codon of the cell expressing the target fusion protein. It can be changed according to the frequency of use. Such a codon change may dramatically increase the expression level when gene expression is performed using prokaryotic cells such as Escherichia coli and Bacillus subtilis. Information on codon usage can be obtained from, for example, a public database (http://www.kazusa.or.jp/codon/).

  The base sequence can be converted by using a known mutagenesis method such as the site-directed mutagenesis method, the DNAWorks method (Non-patent Document 5) in which a synthetic oligonucleotide and PCR are combined, or the Synthetic Gene Designer method (Non-patent document). Document 6) can be used. In the above method, a full-length gene can be prepared by synthesizing an oligonucleotide group consisting of several tens of bases based on the amino acid sequence encoding the polypeptide and assembling the synthetic oligonucleotide by the PCR method.

  Furthermore, as long as the property of changing the three-dimensional structure due to the binding of calcium is not lost, a part or all of it can be replaced with another amino acid, or a part can be deleted and used. Such modification can be expected to have an effect of increasing or decreasing the calcium concentration necessary for the structural change and improving the stability of calmodulin. When the fusion protein is used for protein purification, the molecular weight of the fusion protein is preferably small in order to increase the amount of binding to the carrier, and the effect of reducing the molecular weight of the fusion protein can also be obtained.

  Note that the above-described codon conversion and gene modification may be similarly performed for the M13 gene (SEQ ID NO: 13) added to the C-terminal side of calmodulin.

  The antibody gene used for fusion for the same purpose can also be modified so as to substitute, delete and / or add a part of the amino acid constituting the target compound without losing the binding ability to the target compound. Although the full lengths of the H chain and L chain can be used as the antibody gene, it is preferable that the antibody gene is miniaturized within a range that does not lose its function in the sense of reducing the molecular weight. It is also possible to delete several residues at the N-terminal or C-terminal of VH and VL within a range not losing the function.

  Here, the antibody to be used can be used without limitation as long as the gene has been prepared. As an example, when used for recovering lysozyme, the antibodies shown in SEQ ID NO: 6 and SEQ ID NO: 9 can be used, but are not limited thereto. SEQ ID NO: 6 shows the VL region of the anti-lysozyme antibody, and SEQ ID NO: 9 also shows the VH region. In addition, for example, a portion corresponding to SEQ ID NOs: 6 and 9 of an anti-insulin antibody can be used for purification of insulin, and a portion corresponding to SEQ ID NOs: 6 and 9 of an anti-interferon antibody can be used for purification of interferon.

  In preparing the gene of this fusion protein, the antibody portion and the calmodulin portion or M13 portion can be directly bonded, but can also be bonded via a linker sequence encoding several amino acid residues. Examples of the linker sequence include Gly-Gly, Gly-Ser, Gly-Gly-Ser, or a sequence obtained by repeating these two to three times.

  In order to facilitate the purification of the fusion protein, an oligonucleotide encoding a peptide serving as a tag may be added. Examples of the tag peptide include polyhistidine tag (His-tag), Mick tag (C-myc tag) and the like. These tag peptides can be added to any position on the N-terminal side, C-terminal side, or central part of the fusion protein molecule as long as the biological activity of the above-mentioned polypeptide is not impaired. The addition of the oligonucleotide encoding the tag peptide to the oligonucleotide encoding the protein of the present invention can be carried out by genetic engineering by a method well known to those skilled in the art.

  Furthermore, in order to facilitate the expression of the fusion protein in cells, an arbitrary polypeptide can be added. For example, when expressed in Escherichia coli, it is known that productivity is improved by adding thioredoxin (SEQ ID NO: 12), glutathione-S-transferase, or the like. In addition, in an expression system using a Bacillus genus bacterium known for its high protein secretory expression ability, when Brevibacillus choshinensis is used as a host as an example, fusion with protein disulfide isomerase (PDI) derived from filamentous fungi An example in which the productivity of geranylgeranyl diphosphate synthase derived from a hyperthermophilic bacterium is improved (Non-patent Document 7). These arbitrary polypeptides can be selected and used according to the type of cells to be expressed. These optional polypeptides are usually added to the N-terminal side of the fusion protein.

  It is also possible to add a signal peptide for secretory production outside the cell. A signal peptide is a polypeptide that allows a protein expressed in the cytoplasm to pass through the cell membrane and be secreted outside the cell membrane, and is usually present on the N-terminal side of the protein. Cleaved by enzymes.

  There are no restrictions on the cells used for gene expression, and any cells can be used. Examples of E. coli include K12 strains, JM series strains thereof, and B21 strains such as BL21 strains, Examples of Bacillus subtilis include M168 strain and its derivatives MT-2 strain, DB104 strain, DB117 strain, etc. In addition, Brevibacillus choshinensis closely related to Bacillus subtilis, Saccharomyces cerevisiae, which is a budding yeast, and Schizosaccharomyces, which is a fission yeast. And bacilli such as Pichia pastoris and Aspergillus oryzae of methanol-utilizing yeast, animal cells, insect cells, and the like, and further using a cell-free protein expression system using an extract such as wheat germ You can also

  As a vector used for gene introduction into a cell, a vector suitable for the cell to be used can be arbitrarily selected and introduced into the cell. As such a vector, for example, when Escherichia coli is used for gene expression, vectors such as pBR, pUC and pET can be used. Also, when using Bacillus subtilis, pUB110 or the like, when using Brevibacillus genus bacteria, pNCMO2 or pNY326 (Takara Bio Inc.), etc., when using Saccharomyces genus yeast, pAUT101, pAUR123 (Takara Bio Inc.) or pTEF1 / Bsd (manufactured by Invitrogen) or the like, pPHIL-D2, pAO815 or pPICZ (Invitrogen) or the like can be used when using yeast belonging to Pichia, and pUNA or pUSA can be used when using koji molds.

  The method for culturing the transformant of the present invention is not particularly limited, and a normal liquid medium or a solid medium obtained by solidifying it with agar may be used. The shape of the solid medium is not limited, and may be a slant medium or a flat medium. The composition of the medium is not particularly limited as long as the transformant of the present invention can proliferate and express the fusion protein. As the carbon source, molasses, glucose, fructose, maltose, sucrose, starch, lactose, glycerol, acetic acid and the like are used. As the nitrogen source, ammonium acetate, ammonium chloride, ammonium sulfate, peptone, corn steep liquor, yeast extract and the like are used. As the inorganic salt, phosphates such as sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, and the like are used. Metal ions include magnesium chloride, magnesium sulfate, iron (II) sulfate, iron (III) sulfate, iron (II) chloride, iron (III) chloride, iron citrate, iron iron sulfate, calcium chloride dihydrate, Calcium sulfate, zinc sulfate, zinc chloride, copper sulfate, copper chloride, manganese sulfate, manganese chloride, etc. are used. As vitamins, yeast extract, biotin, nicotinic acid, thiamine, riboflavin, inositol, pyridoxine and the like are used. When a solid medium is used, a solidifying agent such as agar or gellan gum is dissolved in a medium having the above composition by heating, and then dispensed into a test tube or petri dish used for culture, and further cooled to a target temperature and solidified. Can be obtained.

  The culture temperature depends on the cells used for expression, but usually 15 to 40 ° C. is preferable when E. coli is used, and the pH is preferably 6 to 8. Although the culture time can be arbitrarily set, it is preferably a time during which the present fusion protein is sufficiently produced. When E. coli is used, it is usually set between several hours and 200 hours.

  The method for analyzing the fusion protein obtained by the production method of the present invention is not particularly limited as long as it is a method that can be stably and efficiently quantified from the culture solution. The ELISA method (enzyme-linked immunosorbent method) or the Western blot method. Can be given.

  The fusion protein obtained by the production method of the present invention can be used in the state of a culture solution, can be used in a highly purified form, and has various purification degrees with intermediate purity. Can also be used. The fusion protein may be purified by combining various methods such as centrifugation, ultrafiltration, ammonium sulfate precipitation fractionation, and column chromatography until the desired purity is achieved. When the polynucleotide of the present invention contains a polynucleotide encoding a tag sequence such as a His tag sequence or a c-myc antigen sequence, the fusion protein expressed by the transformant of the present invention has its N-terminal side or Since the tag sequence is added to the C-terminal side, the tag sequence can be easily purified using affinity chromatography that specifically recognizes the tag sequence.

  The fusion protein obtained by the production method of the present invention can be used as an affinity ligand (separation agent) for purifying biopharmaceuticals, cells, and viruses, as well as for various uses such as pharmaceuticals, clinical diagnostics, and biosensors. Can be used.

  By fusing calmodulin and the variable region of an antibody, a modified antibody whose affinity with an antigen changes depending on the calcium concentration is provided. This fusion protein can be used in various applications such as pharmaceuticals, clinical diagnostics, biosensors, or affinity ligands (separating agents). In particular, when using ordinary antibodies for affinity ligands, the use of this fusion protein is considered because the use of strongly acidic conditions or protein denaturing agents in the recovery of the target compound causes inactivation of the target compound. In such a case, recovery can be performed under mild conditions such as a change in calcium ion concentration, so that it is possible to avoid deactivation of the target product.

  The present invention will be described below with reference to examples. However, the present invention is not limited to these examples, and it goes without saying that the present invention can be arbitrarily changed without departing from the gist of the invention. Absent.

Example 1 Preparation of Fusion Protein Expression Plasmid (1) A PCR reaction was performed using the polynucleotide of SEQ ID NO: 1 as a template and the oligonucleotides of SEQ ID NO: 2 and SEQ ID NO: 3 as primers to obtain an amplification product. This amplified product was treated with restriction enzymes SpeI and BamHI.
(2) The commercially available vector pBluescript II SK (+) (Stratadine) was cleaved with restriction enzymes SpeI and BamHI, and then dephosphorylated by alkaline phosphatase treatment.
(3) The amplified product of (1) and the vector of (2) were ligated with T4 DNA ligase to obtain plasmid pBS-CaM.
(4) The pBS-CaM was cleaved with the restriction enzyme NcoI, and then the synthetic oligonucleotides of SEQ ID NOs: 4 and 5 were ligated to prepare a plasmid pBS-CaM- (NcoI) -M13.
(5) The pBS-CaM- (NcoI) -M13 was treated with Mung Bean nuclease and then self-ligated to obtain a plasmid pBS-CaM-gly-gly-M13.
(6) The above-mentioned pBS-CaM-gly-gly-M13 was treated with restriction enzymes BamHI and NotI, and further treated with alkaline phosphatase to dephosphorylate.
(7) The anti-lysozyme antibody VL region of SEQ ID NO: 6 is amplified by PCR reaction using the oligonucleotides of SEQ ID NO: 7 and SEQ ID NO: 8 as primers, treated with restriction enzymes BamHI and NotI, and then cleaved with restriction enzymes BamHI and NotI in advance. The plasmid was further ligated to the plasmid pBluescript II SK (+) that had been dephosphorylated to obtain a plasmid pBS-VL.
(8) The plasmid pBS-VL of (7) is treated with restriction enzymes BamHI and NotI to recover the VL region fragment, and ligated to the vector prepared in (6) with T4 DNA ligase to obtain plasmid pBS-CaM-gly- gly-M13-VL was obtained.
(9) The above-mentioned pBS-CaM-gly-gly-M13-VL was cleaved with restriction enzymes KpnI and XbaI and then dephosphorylated.
(10) The anti-lysozyme antibody VH region of SEQ ID NO: 9 is amplified by PCR reaction using the oligonucleotides of SEQ ID NO: 10 and SEQ ID NO: 11 as primers, treated with restriction enzymes KpnI and XbaI, and then cleaved with restriction enzymes KpnI and XbaI in advance The plasmid was further ligated to the plasmid pBluescript II SK (+) that had been dephosphorylated to obtain a plasmid pBS-VH.
(11) The pBS-VH is cleaved with restriction enzymes KpnI and XbaI to recover the VH region, and ligated to the plasmid of (9) with T4 DNA ligase to obtain plasmid pBS-VH-CaM-gly-gly-M13-VL. Obtained.
(12) The above plasmid was treated with restriction enzymes NcoI and NotI to obtain a DNA fragment encoding a fusion protein from the VH region to the VL region.
(13) A commercial vector pET32c (Novagen) was treated with restriction enzymes NcoI and NotI, and then dephosphorylated. Expression vector pET-trx-His-VH-CaM-gly-gly- in which a fusion protein gene in which the DNA fragment obtained in (12) is linked and thioredoxin and His-tag gene are added to the 5 'end is cloned here M13-VL was prepared. The above process is shown in FIGS.

Example 2 Transformation into E. coli and Preparation of Fusion Protein (1) A commercially available competent cell (Takara Bio Inc.) of E. coli BL21 (DE3) strain was transformed with the expression vector prepared in Example 1. This recombinant strain was inoculated into LB medium and cultured at 37 ° C. for 2 hours, and then isopropylthiogalactosylpyranoside (IPTG, final concentration 1 mM) was added and further cultured at 25 ° C. overnight.
(2) After completion of the culture, the cells were collected by centrifugation and suspended in 10 mM MOPS buffer (pH 7.4) containing 100 mM KCl. The suspension was sonicated to disrupt the cells, and the soluble fraction was collected by centrifugation.
(3) The fusion protein is adsorbed by passing the soluble fraction obtained in (2) through a column packed with His-Select Nickel Affinity Gel (SIGMA) equilibrated in advance with 10 mM MOPS buffer containing 100 mM KCl. After washing with the equilibration buffer, the purified product of the fusion protein was obtained by elution with 10 mM MOPS buffer containing 100 mM KCl containing 200 mM imidazole.

Example 3 Affinity of fusion protein in the presence of calcium (1) The fusion protein solution prepared in Example 2 was dialyzed against 10 mM MOPS buffer containing 10 mM EGTA and 100 mM KCl for 1 hour, and further 2.5 mM EGTA. And 1 hour against 10 mM MOPS buffer containing 100 mM KCl, 1 hour against 10 mM MOPS buffer containing 100 mM KCl, and then overnight dialysis against the same buffer to obtain 60.0 μg / mL fusion protein. A solution was prepared.
(2) 200 μL of the solution of (1) was applied to the wells of a 96-well titer plate, and incubated at 37 ° C. for 1.5 hours for immobilization.
(3) The well of (2) was washed 3 times with 10 mM MOPS buffer containing 0.01% Tween 20 and 100 mM KCl, and then 300 μL of Block Ace (DS Pharma Biomedical) was applied and blocked at 37 ° C. for 1 hour. Went.
(4) Lysozyme was dissolved in a 10 mM MOPS buffer containing 10 mM CaCl ₂ and 100 mM KCl to a final concentration of 200 μg / L.
(5) The well of (3) was washed 3 times with 10 mM MOPS buffer containing 0.01% Tween 20 and 100 mM KCl, 200 μL of the lysozyme solution of (4) was added, and the mixture was incubated at 37 ° C. for 1.5 hours. Lysozyme was bound to the solid phase fusion protein.
(6) After washing 3 times with 10 mM MOPS buffer containing 0.01% Tween 20 and 100 mM KCl, commercially available HRP-labeled anti-lysozyme polyantibody (GeneTex) was diluted 10,000 times and added at 37 ° C. It was incubated for 5 hours and bound to solid phase lysozyme.
(7) After washing twice with 10 mM MOPS buffer containing 0.01% Tween 20 and 100 mM KCl and once with 10 mM MOPS buffer containing 100 mM KCl, 100 μL of TMB substrate solution (KPL) was added and reacted for 1 minute. I let you.
(8) 100 μL of 1M sulfuric acid was added to stop the reaction, and the absorbance at 450 nm was measured. The absorbance at this time was 0.25. The results are shown in FIG.

Example 4 Affinity of fusion protein in the absence of calcium Among the steps shown in Example 3, the same procedure was performed except that a buffer containing no CaCl ₂ was used in (4). The absorbance in 8) was 1.25 (FIG. 3). This result shows that the fusion protein of the present invention binds a larger amount of lysozyme in the absence of calcium than in the presence of 10 mM calcium. That is, it was confirmed that the affinity of the fusion protein of the present invention with lysozyme is greatly reduced by adding 10 mM calcium.

Comparative Example 1 Blank Test Of the steps shown in Example 3, the same operation was performed except that, in (5), a 10 mM MOPS buffer containing 100 mM KCl was used instead of the lysozyme solution. Absorbance was 0.1. (Figure 3)

Moreover, when the same operation was performed in (2) except that a 10 mM MOPS buffer containing 100 mM KCl was used instead of the fusion protein solution of (1) in the same step, the absorbance in (8) was 0. .02. (Figure 3)
This result confirmed that the absorbance in Example 3 and Example 4 was due to the binding of the fusion protein and lysozyme, and not due to non-specific reaction.

Example 5 Calcium concentration dependency In the step (4) in Example 3, the same operation was performed except that the calcium concentration of the buffer was changed to 0, 10, 100, 1000 or 10000 μM. As a result, it was confirmed that the absorbance in (8) was 1.15 at a calcium concentration of 0 μM, but decreased to 0.35 at a concentration of 10 μM, and further, the absorbance decreased in a concentration-dependent manner at higher concentrations. . The results are shown in FIG. From this result, it was confirmed that this fusion protein shows a high affinity for lysozyme at a calcium concentration of 0 μM, but is greatly reduced by adding 10 μM or less of calcium.

Claims (8)

  A fusion protein represented by the general formula, XYZ, wherein X and Z are peptide fragments containing a VH region or a VL region of an antibody, provided that Z is a VL region when X is a VH region. A peptide fragment having a steric structure different from that in the absence of the low molecular weight compound by binding to a low molecular weight compound when X is a VL region and Y is a bond with the low molecular weight compound; A fusion protein that enhances or decreases the affinity of a specific antigen for an antibody formed by X and Z by binding to.   The fusion protein according to claim 1, wherein Y is a fusion protein represented by the general formula Y1-Y2, Y1 is calmodulin, Y2 is a calmodulin-binding peptide, and the specific low-molecular compound is a calcium ion.   The fusion protein according to claim 2, wherein Y2 is a partial structure of myosin light chain kinase.   The fusion protein according to claim 2 or 3, wherein Y2 is peptide M13 derived from a calcium binding domain of myosin light chain kinase.   The fusion protein according to any one of claims 2 to 4, wherein Y1 and Y2 are linked by a Gly-Gly linker.   The fusion protein according to any one of claims 1 to 5, wherein X is a peptide fragment containing an antibody VH region, and Z is a peptide fragment containing an antibody VL region.   The VH region comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence, and the VL region is shown in SEQ ID NO: 9 Or a fusion protein according to any one of claims 1 to 6, which comprises an amino acid sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence, and has an ability to bind an anti-lysozyme antibody. .   The fusion protein according to claim 7, wherein the lysozyme binding ability decreases from the absence of calcium by adding 1 μM or more of calcium ions.

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