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WO2005033307A1 - Nouvelle methode de repli et proteine ainsi obtenue - Google Patents

Nouvelle methode de repli et proteine ainsi obtenue Download PDF

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Publication number
WO2005033307A1
WO2005033307A1 PCT/JP2004/014801 JP2004014801W WO2005033307A1 WO 2005033307 A1 WO2005033307 A1 WO 2005033307A1 JP 2004014801 W JP2004014801 W JP 2004014801W WO 2005033307 A1 WO2005033307 A1 WO 2005033307A1
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WIPO (PCT)
Prior art keywords
protein
receptor
refolding
ligand
refolded
Prior art date
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PCT/JP2004/014801
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English (en)
Japanese (ja)
Inventor
Izuru Ohki
Shin-Ichi Tate
Original Assignee
Kyowa Hakko Kogyo Co., Ltd.
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Publication date
Application filed by Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Priority to JP2005514502A priority Critical patent/JPWO2005033307A1/ja
Publication of WO2005033307A1 publication Critical patent/WO2005033307A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1136—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel refolding method. Further, the present invention relates to a protein obtained by the refolding method of the present invention.
  • the protein obtained by the refolding method of the present invention has a high purity and a high homogeneity.
  • the present invention also provides a protein crystal obtained by the refolding method of the present invention.
  • the present invention provides a receptor chip, a receptor force ram, and a ligand prepared by immobilizing a ligand binding fragment of a receptor protein obtained by applying the refolding method of the present invention on a solid phase.
  • the present invention relates to a removal material and a therapeutic material containing such a ligand removal material.
  • the refolding method of the present invention after expressing a region related to the ligand recognition site of a swivel receptor used as a receptor fragment in a cell as an inclusion body, the refolding method of the present invention is used. Then, a receptor chip prepared by immobilizing the refolded protein on a solid phase is provided. For example, after the extracellular region of a scavenger receptor or a C-type lectin-like region (abbreviated as CTLD) is expressed in a cell as an inclusion body, the refolding method of the present invention is applied, and the refolded protein is applied.
  • CTLD C-type lectin-like region
  • LDL Low Density Lipoprotein
  • a highly sensitive receptor chip can be provided.
  • the extracellular region or CTLD of the scavenger-I receptor prepared using the refolding method of the present invention has high purity and homogeneity, so that non-specific binding to substances other than the target ligand is small.
  • CTLD sclerobens monoreceptor
  • a crystal using the protein prepared by the refolding method of the present invention is provided.
  • BACKGROUND ART As a method for preparing a desired protein in a large amount, a protein recombinant expression method is well known. Recombinant expression has made it possible to prepare large quantities of proteins that are difficult to prepare from natural sources.
  • a receptor present on the cell surface is a protein that specifically binds to a ligand corresponding to the receptor and transmits various signals into cells.
  • the receptors present on the cell surface are diverse and their corresponding ligands are different, a large number of receptors that specifically bind to the ligand are prepared in order to detect and / or quantify the specific ligand. It is useful to use By immobilizing the receptor or receptor fragment prepared in a large amount on a solid phase and preparing a receptor chip using a diagnostic marker for abnormal cells or disease as a ligand, the presence of abnormal cells in a cell population can be detected, or However, it is expected that useful tools for diagnosing diseases can be provided. For example, abnormal cells such as denatured LDL and apoptotic cells and senescent erythrocytes accumulated in the living body, and bacteria that have entered the living body are recognized and formed.
  • abnormal cells such as denatured LDL and apoptotic cells and senescent erythrocytes accumulated in the living body, and bacteria that have entered the living body are recognized and formed.
  • the binding between the receptor and its corresponding ligand is specific, it is necessary to provide a material that removes ligands by immobilizing a large amount of expressed receptor or its ligand-binding fragment on a solid phase. Is possible. For example, by using a receptor for denatured LDL to remove denatured LDL in the blood, it is possible to treat diseases such as arteriosclerosis and hyperlipidemia caused by abnormal LDL dynamics. Accordingly, the present invention also provides a therapeutic material containing the modified LDL ligand removal material.
  • Various host cells and vectors used to recombinantly express a protein are well known. Host cells for recombinant expression include bacterial cells, animal cells, plant cells, fungal cells, and the like.
  • bacterial cells such as Escherichia coli are widely used as host cells because of their rapid growth rate, relatively simple operation, and low preparation cost, and especially on an industrial scale. Suitable for production.
  • the product protein is often accumulated as an insoluble substance in the cell as an inclusion body. This inclusion body is insoluble and requires solubilization and refolding because the three-dimensional structure of the inclusion body protein differs from that of the native protein.
  • the conventional refolding method involves, for example, adsorbing a receptor to a resin, contacting the adsorbed resin with a buffer solution containing a denaturant, and then gradually reducing the concentration of the denaturant. All of these methods were complicated, such as contacting with a solution (JP-A-2003-1696993). Furthermore, when refolding is performed while immobilized on a solid phase, it is necessary to go through steps such as elution and extraction from the solid phase after refolding, which increases the complexity and reduces the yield. There was a problem. Furthermore, in the conventional refolding method using a surfactant (Japanese Patent Application Laid-Open No.
  • the conventional method has problems in that the substances used in the solubilization and refolding steps are mixed in the refolded protein, resulting in reduced purity and inhomogeneity due to the presence of incompletely refolded protein. It is.
  • a ligand-binding fragment of a receptor when refolded and used as a receptor chip for ligand detection, conventional refolding methods cannot be used to prepare highly pure and homogeneous refolded proteins. , Resulting in reduced sensitivity in qualitative and quantitative detection. It can also be used to refold ligand binding fragments of the receptor, e.g., when used as a material for removing ligands present in blood, when the purity and homogeneity of the refolded protein is low. Will remove substances other than the target ligand by interacting with other factors in the blood.
  • An object of the present invention is to provide a refolding method for preparing a highly pure, homogeneous and properly folded protein from a denatured protein.
  • a further object of the present invention is to provide a highly pure, homogeneous and properly folded protein by using the refolding method of the present invention.
  • Another object of the present invention is to prepare a receptor chip by preparing a highly pure, homogeneous and properly folded protein from the inclusion body protein and immobilizing the protein on a solid phase.
  • a further object of the present invention is to provide a detection kit and a detection method using such a chip.
  • a further object of the present invention is to provide a method for detecting a denatured LDL, an abnormal cell, and a bacterium by preparing a receptor chip using a squirrel receptor, A large amount of soluble ligand recognition region that can be immobilized on a solid phase (so that the ligand binding site is located outside) is prepared in large quantities, and a part of the sensor that utilizes the ligand recognition characteristics is constructed.
  • An object of the present invention is to provide a method and a detection kit for detecting abnormal cells such as denatured LDL and apoptotic cells and senescent erythrocytes, and bacteria invading the living body.
  • a further object of the present invention is to provide a ligand removing material using a scavenger receptor for removing denatured LDL, abnormal cells and bacteria in blood, and blood using such a material.
  • An object of the present invention is to provide a method and a material for removing denatured LDL, abnormal cells and bacteria therein.
  • the present invention has solved the above-mentioned problems by using a step of dropping a denatured protein into a refolding buffer containing arginine, reduced daltuthione, and oxidized daltathione.
  • the present invention also provides methods and materials for removing denatured LDL, abnormal cells and bacteria in blood by using LOX-1 ligand binding fragments as refolded proteins.
  • the above-mentioned problem of providing is solved.
  • the present invention provides the following.
  • a method of refolding a denatured protein comprising: a) dropping the denatured protein into a refolding buffer containing arginine, reduced daltathione, and oxidized daltathione;
  • a method comprising:
  • the denatured protein is a denatured protein obtained by solubilizing an inclusion body protein expressed in bacteria using a soluble Ech buffer containing an SH protection reagent and a denaturant.
  • Item 16 The method described in.
  • Scavenger receptor receptor belonging to the insulin receptor family, receptor belonging to the EGF receptor family, receptor belonging to the PDGF receptor family, receptor belonging to the VEGF receptor family, receptor belonging to the FGF receptor family, Growth factor receptor of the NGF receptor family, receptor belonging to the TGF-; 8 super family, receptor belonging to the To 11-1 ike receptor family, receptor belonging to the LDL receptor-related protein family, and G Receptors belonging to the protein-coupled receptor family 31.
  • the refolded evening protein of item 31 having a purity of 98% or more.
  • the refolded protein of item 31 having a purity that provides a single crystal that provides an X-ray diffraction image that provides a resolution of 2.5 angstroms.
  • 34. The refolded protein according to item 31, wherein a solid phase immobilization site is added.
  • tag sequence is a tag sequence selected from the group consisting of:
  • His tag His tag, GST tag, myc tag, cellulose binding domain tag, cell source binding peptide tag, calmodulin binding peptide tag, S protein binding peptide tag, T7 tag or a combination thereof.
  • the receptor chip according to item 37 which is suitable for detection by surface plasmon resonance, quartz crystal microbalance, or mass spectrometer.
  • a detection kit comprising the receptor chip according to item 38.
  • a ligand-removing material comprising a refolding protein immobilized on a solid phase via the solid phase immobilization site according to item 34.
  • a ligand-removing material for removing ligand in blood comprising the ligand-removing material according to item 41.
  • 4 3 A therapeutic material for treating arteriosclerosis caused by degenerative LDL dyskinetics, comprising the material for removing a ligand according to item 41. 4.
  • a therapeutic material for treating hyperlipidemia caused by denatured LDL dyskinetics comprising the ligand-removing material according to item 41.
  • a pharmaceutical composition for preventing or treating arteriosclerosis comprising the refolded protein according to item 31.
  • the present invention provides a ligand-binding fragment of a receptor protein which specifically binds to a desired ligand in a highly pure and homogeneous state, and binds the fragment to a solid phase to thereby form the desired ligand.
  • the present invention also provides a method for detecting the desired ligand using such a receptor chip.
  • the present invention provides a method for preparing a ligand-binding fragment of a receptor protein that specifically binds to a desired ligand in a highly pure and homogeneous state, and binding the fragment to a solid phase to obtain the desired ligand.
  • a ligand removing material for specifically removing a ligand.
  • the present invention also provides a method of removing the desired ligand from the blood, comprising the steps of:
  • the blood treated by the above method is returned to the subject.
  • the present invention provides a method for removing pathogens such as bacteria and / or viruses in a sample, the method comprising the following steps: (1) A method for removing pathogens is provided. Obtaining a sample; and (2) a step of bringing the sample into contact with a ligand-removing material under conditions where the pathogen in the sample specifically binds to the ligand-removing material.
  • the sample treated by the above method is injected into a subject.
  • the present invention provides a method for detecting a modified LDL, an abnormal cell or a bacterium by an intermolecular interaction analysis method, which comprises using a recombinant protein obtained by expressing a region related to ligand recognition of a receptor in a cell. Provide a way.
  • the present invention also provides a method for expressing a region involved in ligand recognition of a receptor as a biotinylated protein in a cell, and then expressing the expressed biotinylated protein on an immobilized solid phase via avidin or streptavidin.
  • the present invention provides a method for detecting a ligand by an intermolecular interaction analysis method, wherein the method is characterized by using the immobilized protein.
  • the present invention provides a method for expressing a region related to ligand recognition of a receptor as a tagged protein in a cell, and then directing the expressed tagged protein via a factor that specifically binds to the tag.
  • the present invention provides a method for detecting a ligand by a molecular interaction analysis method, wherein the method is characterized by using a protein immobilized on a solid phase while maintaining the same.
  • the present invention further provides a reconstituted protein obtained by reconstituting an extracellular region or a ligand recognition region of a receptor accumulated in Escherichia coli by refolding to a correct three-dimensional structure and using the reconstituted protein. It provides a method for detecting denatured LDL, abnormal cells or bacteria by an action analysis method.
  • the present invention relates to a method for remodeling a recombined biotinylated extracellular region or a biotinylated ligand recognition region of a receptor accumulated in Escherichia coli by refolding into a correct three-dimensional structure, and then reconstituting the reconstituted biotinylated protein with avidin or streptococcus.
  • a method for detecting denatured LDL, abnormal cells or bacteria by intermolecular interaction analysis which is characterized in that it is immobilized on a solid phase while maintaining its orientation via avidin and the immobilized protein is used. .
  • the present invention provides a method for tagging the extracellular region of the receptor or the ligand-recognizing region of the receptor accumulated in Escherichia coli by refolding and reconstituting the correct three-dimensional structure.
  • a method for detecting denatured LDL, abnormal cells or bacteria by an intermolecular interaction analysis method comprising immobilizing the protein on a solid phase while maintaining its orientation via a factor that specifically binds to the protein, and using the immobilized protein. I will provide a.
  • the present invention also provides a modified LDL containing a protein in which the extracellular region or ligand recognition region of a receptor accumulated as an aggregate in Escherichia coli is unraveled with a denaturing agent and then refolded into a correct three-dimensional structure, Or provide a kit for detecting bacteria.
  • the present invention also provides proteins for producing a vaccine composition for treating various diseases (eg, atherosclerosis).
  • vaccine compositions for treating atherosclerosis may include, for example, lipoproteins such as denatured LDL, receptors belonging to the Tolike receptor family, and scavenger receptors such as LOX-1. May contain protein.
  • a novel refolding method and a protein obtained by the method are provided.
  • the refolding method of the present invention makes it possible to obtain a highly pure and homogeneous refolded protein.
  • a highly sensitive receptor chip sensor for a desired ligand is also provided.
  • FIG. 1 shows that the protein obtained by the refolding method of the present invention has a narrower molecular weight than the conventional refolding method using cyclic carbohydrate cycloamylose, and thus has a higher purity. Indicates that it has been purified.
  • FIG. 1 shows that the protein obtained by the refolding method of the present invention has a narrower molecular weight than the conventional refolding method using cyclic carbohydrate cycloamylose, and thus has a higher purity. Indicates that it has been purified.
  • FIG. 2 shows that the protein obtained by the refolding method of the present invention has been purified to a higher degree by using a conventional cyclic carbohydrate cycloamylose with fewer contaminants than the refolding method.
  • Fig. 3 shows that the protein obtained by the refolding method of the present invention is refolding as a single molecular species, whereas the protein obtained by the conventional method is a mixture of incomplete refolding proteins. Is shown.
  • FIG. 4 shows that the proteins obtained by the refolding method of the present invention do not contain incomplete refolded proteins.
  • FIG. 5 shows that LOX-1 CTLD refolded using the refolding method of the present invention has a similar affinity for oxidized LDL and acetylated LDL as native LOX-1.
  • Figure 6 shows that the protein obtained as a result of refolding the full-length extracellular domain of LOX-1 by the refolding method of the present invention is a dimer, similar to the mode of LOX-1 present on the cell surface. Is shown. In the figure, i8Me (+) indicates that ⁇ -mercaptoethanol was added. / 3Me (—) is / 3 Mercapto Yuno Indicates that no tools were added.
  • Figure 7 shows that the acetylated LDL immobilized on the sensor chip via the apoBlOO antibody interacts with the full-length L0X-1 extracellular domain forming a dimeric structure and the ligand-binding domain CTLD present as a monomer.
  • the result of the experiment which was made to act is shown. It has been shown that recognition of acetylated LDL requires that the ligand binding domain adopt a dimeric structure.
  • receptor is a biological structure comprising one or more binding domains that reversibly and specifically complex with one or more ligands, wherein This complex has a biological structure.
  • Receptors can be completely extracellular (extracellular receptors), in the cell membrane (but directing parts of the receptor to the extracellular environment and cytosol), or completely intracellular (intracellular) Receptor). They can also function independently of cells. Reception in the cell membrane The body allows cells to communicate with space outside their boundaries (eg, signal transduction) and to function in the transport of molecules and ions into and out of cells.
  • a receptor can be a full length receptor or a fragment of a receptor.
  • a site related to ligand recognition of the receptor protein is used.
  • the site involved in ligand recognition of the receptor protein can be identified as follows.
  • homology search or domain search the ligand recognition region can be estimated from the structure of proteins having high homology and similarity in function. For example, when amino acid sequences of different receptor molecules that specifically bind to the same ligand are calculated using BLAST default parameters, 50% or more, preferably 55% or more, more preferably 60% or more More preferably, a region showing homology of 65% or more is estimated as a ligand recognition region.
  • those skilled in the art can easily transiently express, in animal cells and the like, a gene encoding a mutant receptor into which a deletion mutation, amino acid substitution, or the like has been introduced, and determine a region essential for its function.
  • ligand is a binding partner for a specific receptor or family of receptors.
  • the ligand may be an endogenous ligand for the receptor, or alternatively, a synthetic ligand for the receptor, such as a drug, drug candidate, or pharmacological tool.
  • protein protein
  • polypeptide oligopeptide
  • peptide refers to a polymer of amino acids of any length. This polymer may be linear, branched or cyclic. Amino acids may be natural or non-natural, and may be modified amino acids. The term can also be assembled into a complex of multiple polypeptide chains. The term also includes naturally or artificially modified amino acid polymers. Such modifications include: For example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component).
  • This definition also includes, for example, polypeptides containing one or more analogs of an amino acid (eg, including unnatural amino acids, etc.), peptidomimetic compounds (eg, peptides), and those known in the art. Other modifications are included.
  • polynucleotide As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “derivative oligonucleotide” or “derivative polynucleotide”. “Derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has unusual linkages between nucleotides, and is used interchangeably.
  • oligonucleotides include 2′-0-methyl monoliponucleotides, derivative oligonucleotides in which a phosphoric diester bond in an oligonucleotide is converted to a phosphorothioate bond, and oligonucleotides in an oligonucleotide.
  • nucleic acid sequence also includes conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Is contemplated. Specifically, degenerate codon substitutions create a sequence in which the third position of one or more selected (or all) codons has been replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucl Acids Res. 19: 5081 (1991); Oh tsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al. Mo 1. Cell. 8: 91-98 (1994)).
  • degenerate codon substitutions create a sequence in which the third position of one or more selected (or all) codons has been replaced with a mixed base and / or deoxyinosine residue.
  • nucleic acid is also used interchangeably herein with genes, cDNAs, mRNAs, oligonucleotides, and polynucleotides. Particular nucleic acid sequences also include “splice variants.” Similarly, a particular protein encoded by a nucleic acid implicitly includes any protein encoded by a splice variant of the nucleic acid. As the name suggests, a “splice variant” is a gene The After transcription, an initial nucleic acid transcript can be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. The mechanism of production of splice variants varies, but involves alternative splicing of exons. Another polypeptide derived from the same nucleic acid by read-through transcription is also included in this definition. Any product of a splicing reaction, including recombinant forms of the splice product, is included in this definition.
  • gene refers to a factor that defines a genetic trait. Usually they are arranged in a certain order on the chromosome. Genes that define the primary structure of proteins are called structural genes, and regulatory genes that control their expression. As used herein, “gene” may refer to "polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein””polypeptide””oligopeptide” and “peptide” . As used herein, “homology” of a gene refers to two or more Means the degree of identity of the gene sequences to each other. Thus, the higher the homology between two genes, the higher the identity or similarity between their sequences.
  • Whether the two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, the hybridization method under stringent conditions.
  • the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably Genes are homologous if they are at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical.
  • the comparison of the identity of the nucleotide sequences and the calculation of the homology are calculated by using BLAST, which is a sequence analysis tool, using default parameters.
  • expression of a gene, polynucleotide, polypeptide, or the like means that the gene or the like undergoes a certain action in vivo and takes on another form.
  • it means that a gene, a polynucleotide, or the like is transcribed and translated to form a polypeptide.
  • transcription and production of mRNA may also be an aspect of expression. More preferably, such polypeptide forms may be post-translationally processed.
  • amino acid may be natural or non-natural.
  • “Derivative amino acids” or “amino acid analogs” refer to amino acids that differ from naturally occurring amino acids but have a similar function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art.
  • the term "natural amino acid” refers to the L-isomer of a naturally occurring amino acid. Natural amino acids include glycine, alanine, palin, leucine, isoleucine, serine, methionine, threonine, fenylalanine, tyrosine, tributofan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, dalsamine.
  • Carboxyglutamic acid, arginine, orditin, and lysine are in the L-form.
  • Unnatural amino acid refers to an amino acid not normally found in nature in proteins. Examples of unnatural amino acids include norleucine, paranitrophenylalanine, homophenylalanine, parafluorophenylalanine, 3-amino-3-benzylpropionic acid, D- or L-form of homoarginine, and D-phenylalanine.
  • No. “Amino acid analog” refers to a molecule that is not an amino acid, but that is similar in physical properties and / or function to the amino acid.
  • Amino acid analogs include, for example, etyonin, kynapananin, 2-methylglutamine and the like.
  • Amino acid mimetics are compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC—IUB Biochemica l Nomencl lateur Comm i s s sion. Nucleotides may also be referred to by the generally accepted one-letter code.
  • corresponding amino acid refers to a protein or polypeptide molecule that has, or is expected to have, the same action as a predetermined amino acid in a protein or polypeptide that is a reference for comparison.
  • an enzyme molecule it refers to an amino acid that is present at a similar position in the active site and has a similar contribution to catalytic activity.
  • nucleotide may be natural or non-natural.
  • “Derivative nucleotide” or “nucleotide analog” refers to one that is different from a naturally occurring nucleotide but has the same function as the original nucleotide.
  • Such derivative nucleotides and nucleotide analogs are well-known in the art. Examples of such derivative nucleotides and nucleotide analogs include phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2-dimethyl ribonucleotide, peptide mononucleic acid (PNA), It is not limited to these.
  • fragment refers to a polypeptide or polynucleotide having a sequence length of 1 to n ⁇ 1 with respect to a full-length polypeptide or polynucleotide (length is n).
  • the length of the fragment can be appropriately changed depending on the purpose.
  • the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10 for a polypeptide.
  • 15, 20, 25, 30, 40, 50 and more amino acids, and lengths represented by integers not specifically listed herein (eg, 11) are also suitable as lower limits. possible.
  • polynucleotides 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides can be mentioned.
  • the receptor “fragment” specifically binds to a ligand to which the full-length receptor can specifically bind.
  • a preferred fragment of the lectin-like oxidized LDL receptor is a fragment comprising the C-type lectin-like domain (CTLD).
  • a method for producing the polypeptide of the present invention for example, a bacterium that is a prokaryote that produces the polypeptide is cultured, the recombinant receptor protein is accumulated in the bacterium as an inclusion body, and the host bacterium is destroyed.
  • a method for obtaining the polypeptide can be mentioned.
  • the amino acid sequence of the biotinylation motif for biotinylation of protein in E. coli is as follows:
  • the amino acid sequence “GLND I FEAQK I EWHEJ (SEQ ID NO: 6) is also available as a biotinylation motif. Even if a mutation is introduced into a region other than a K (lysine) residue, the sequence does not greatly affect the activity of the biotinylation. Therefore, a sequence in which a region other than the lysine residue is substituted can also be used as a biotinylation motif.
  • KI G, KI, KIA, KIE, KI GDP (SEQ ID NO: 7), KLWS I (SEQ ID NO: 8), KL G, KVG” containing ⁇ ⁇ ⁇ ⁇ that actually undergoes biotinylation are added to the C terminal. By doing so, it is also possible to carry out biotinylation.
  • Transformant refers to all or part of a living organism such as a cell produced by transforming a host cell. Prokaryotic cells are exemplified as transformants. A transformant is also referred to as a transformed cell, a transformed tissue, a transformed host, and the like depending on the subject, and includes all of these forms in the present specification. May refer to a form.
  • the host bacterial cell for obtaining the transformant is not particularly limited as long as it expresses a polypeptide that retains biological activity, and various types of host bacterial cells conventionally used in genetic engineering can be used.
  • Prokaryotic cells include prokaryotic cells belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynepacterium, Microbacterium, Pseudomonas, etc., for example, Eschelium richiacoli XL 1—Blue, E sc he richiacoli XL2-B lue, Es che richiacoli DHl, E schericiacoli MC 1000, E sc he richiaco
  • Examples include TCC 15354, Pseudomonassp.D—0110.
  • one or more amino acids in the amino acid sequence may be replaced, added and / or deleted, as long as they have substantially the same action as the naturally occurring polypeptide.
  • the sugar chain may be substituted, added and / or deleted.
  • Certain amino acids are substituted for other amino acids in a protein structure, such as the binding site of a ligand molecule, without appreciable loss or loss of interaction binding capacity. Can be It is the interaction capacity and properties of a protein that define the biological function of a protein. Thus, certain amino acid substitutions can be made in the amino acid sequence, or at the level of its DNA coding sequence, resulting in a protein that retains its original properties after the substitution. Thus, various modifications may be made in the peptide disclosed herein or the corresponding DNA encoding this peptide without any apparent loss of biological utility.
  • the hydropathic index of amino acids can be considered.
  • the importance of the hydrophobic amino acid index in conferring interactive biological functions on proteins is generally recognized in the art (Kyte. J and Doo 1 ittie, RFJ Mo 1. Biol). 157 (1): 105—132, 1 982).
  • the hydrophobic nature of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).
  • Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); fenilalanine (+2.5.
  • Tributofan (-0.9); Tyrosine (1.3); Proline (-1.6); Histidine (1.3.2); Glutamic acid (-3.5); Glutamine (-3.5) Aspartic acid (-3.5); Asparagine (1.3.5); Lysine (1.
  • one amino acid can be replaced by another amino acid having a similar hydrophobicity index, and still yield a protein having a similar biological function (eg, a protein equivalent in ligand binding ability).
  • the hydrophobicity index is preferably within ⁇ 2, more preferably within soil 1, and even more preferably within ⁇ 0.5. Is more preferable. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.
  • an amino acid can be substituted for another that has a similar hydrophilicity index and still provide a bioisostere.
  • the hydrophilicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • conservative substitution refers to a substitution in an amino acid substitution in which the original amino acid and the amino acid to be substituted have a similar hydrophilicity index or hydrophobicity index as described above.
  • conservative substitutions are well known to those skilled in the art and include, for example, substitutions within each of the following groups: arginine and lysine; glutamic and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. , Etc., but are not limited to these.
  • variant refers to a substance in which a substance such as an original polypeptide or a polynucleotide is partially modified. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. Alleles are genetic variants that belong to the same locus and are distinct from each other. Therefore, “Allelic variant” refers to a variant that has an allelic relationship to a gene. “Species homologue or homolog (homo 1 og)” refers to homology (preferably 60% or more homology, more preferably 80% 85% or more, 90% or more, 95% or more homology). Methods for obtaining such species homologs will be apparent from the description herein.
  • ortholog ort ho 1 og
  • orthologous gene ort holo ogou s gene
  • orthologous gene ort holo ogou s gene
  • “Conservatively modified” applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, or essentially the same if the nucleic acid does not encode the amino acid sequence. Sequence. Because of the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at every position where an alanine is specified by a codon, that codon can be changed to any of the corresponding codons described without changing the encoded polypeptide.
  • nucleic acid variation is a "silent modification (mutation)," a species of conservatively modified mutation.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid (AUG, which is usually the only codon for methionine, and only one codon, usually for tryptophan) It is understood that the codons (excluding TGG) can be modified to produce functionally identical molecules.
  • each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • such modifications can be made to avoid substitution of the amino acid cystine, which greatly affects the conformation of the polypeptide.
  • amino acid additions, deletions, or modifications can be made in addition to amino acid substitutions to produce functionally equivalent polypeptides.
  • substitution of an amino acid refers to substitution of the original peptide with one or more, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3 amino acids.
  • the addition of an amino acid refers to adding one or more, for example, 1 to 10, preferably 1 to 5, and more preferably 1 to 3, amino acids to the original peptide chain.
  • Deletion of an amino acid refers to deletion of one or more, for example, 1 to 10, preferably;! To 5, more preferably 1 to 3 amino acids from the original peptide.
  • Amino acid modifications include, but are not limited to, amidation, hepoxylation, sulphation, halogenation, alkylation, dalicosylation, phosphorylation, hydroxylation, acylyl (eg, acetylation), and the like.
  • the amino acid to be substituted or added may be a natural amino acid, an unnatural amino acid, or an amino acid analog. Natural amino acids are preferred.
  • Such a nucleic acid can be obtained by the well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method, a hybridization method, or the like.
  • substitution, addition, or deletion of a polypeptide or polynucleotide means that an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, corresponds to an original polypeptide or a polynucleotide.
  • Replacement, addition or removal Techniques for such substitution, addition or deletion are well known in the art, and examples of such techniques are provided. Examples include site-directed mutagenesis techniques. Substitutions, additions or deletions
  • the number may be any number as long as it is one or more, and such a number retains the desired function (for example, one cancer marker, one neurological marker, etc.) in a variant having the substitution, addition or deletion. As much as you can.
  • a number may be one or several, and preferably may be within 20%, within 10%, or under 100, 50, 25, etc. of the total length .
  • Macromolecular structures can be described for various levels of organization. For a general discussion of this construction, see, for example, A 1 berts et al., Mo 1 ecu 1 ar Biol ogy oft he Cell (3rd ed., 1994), and Cantor and Schimme 1, B i ophy sica 1 Chemistry Part I: T e Confo rma ti on of B ioloical Ma cromo lecules (1980).
  • Primary structure refers to the amino acid sequence of a particular peptide.
  • “Secondary structure” refers to a locally arranged three-dimensional structure within a polypeptide. These structures are commonly known as domains.
  • a domain forms a compact unit of a polypeptide, and is that portion of the polypeptide that is typically 50-350 amino acids in length.
  • a typical domain is made up of parts such as ⁇ -sheets (such as / 3 strands) and the strain of a single helix (streetch).
  • Tetiary structure refers to the complete three-dimensional structure of a polypeptide monomer.
  • Quaternary structure refers to a three-dimensional structure formed by the noncovalent association of independent tertiary units. Terms related to anisotropy are used in the same way as those known in the energy field.
  • a “vector” refers to a vector that can transfer a target polynucleotide sequence into a target cell.
  • examples of such a vector include a vector capable of autonomous replication in a bacterial host cell and containing a promoter at a position suitable for transcription of the polynucleotide of the present invention.
  • “Expression vector” refers to a nucleic acid sequence in which, in addition to a structural gene and a promoter that regulates its expression, various regulatory elements are operably linked in a host cell.
  • the regulatory element may preferably include a terminator and a selection mechanism. It is well known to those skilled in the art that the type of expression vector and the type of regulatory element used may vary depending on the host bacterial cell.
  • Recombinant vector refers to a vector capable of transferring a target polynucleotide sequence into a target cell.
  • examples of such a vector include those capable of autonomous replication in a prokaryotic host cell and containing a promoter at a position suitable for transcription of the polynucleotide of the present invention.
  • Examples of the “recombinant vector” for prokaryotic cells include pBTr p2, pBTacl, and pBTac2 (all sold by TfJ from Roc Mole Cu lar Bi ochemi ca 1 s), pKK233—2 (Pharma cia), p SE 280 (Invitrogen), p GEMEX-1 (Promomega), pQE-8 (QI AG EN), pKYP 10 (JP-A-58-110600), pKYP 200 (Ag ri c. Biol. Chem., 48, 669 (1984)), pLS Al (Agric. Biol. Chem., 53, 277 (1989)), pGEL1 (Proc.
  • promoter refers to a region on DNA that determines the transcription initiation site of a gene and that directly regulates the frequency of transcription, and a nucleotide sequence to which RNA polymerase starts transcription by binding. It is.
  • the putative promoter region varies for each structural gene, but is usually located upstream of the structural gene, but is not limited thereto, and may be located downstream of the structural gene.
  • solid phase refers to a support to which a molecule such as an antibody can be immobilized.
  • the shape of the solid phase may be planar, spherical, or any other shape. Further, the solid phase of the present invention may be in a gel state.
  • the solid phase when detection is performed using the surface plasmon resonance principle, the solid phase is preferably a glass substrate base material having a metal thin film containing gold, silver or aluminum on one surface.
  • a frequency conversion element for example, a crystal oscillator or a surface acoustic wave element
  • a receptor is directly bound.
  • substrate is a planar solid phase, which refers to a material (preferably a solid) from which a chip or array of the invention is constructed. Therefore, the substrate is included in the concept of the solid phase.
  • the material of the substrate may be any covalent or non-covalent bond that has the property of binding to the biomolecule used in the present invention or that may be derivatized to have such property. Solid materials are mentioned.
  • Such materials for use as the solid phase and substrate can be any material that can form a solid surface, including, for example, glass, silica, silicone, ceramic, silicon dioxide, plastic, metal (alloys). ), Natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon), including but not limited to:
  • the substrate may be formed from a plurality of different material layers.
  • inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used.
  • a membrane used for blotting such as a nylon membrane, a nitrocellulose membrane, or a PVDF membrane
  • a material having hardness such as glass
  • a preferable material for the substrate varies depending on various parameters such as a measuring instrument, and a person skilled in the art can appropriately select an appropriate material from the various materials described above.
  • chip or “microchip” is used interchangeably, refers to a microminiature integrated circuit that has various functions and becomes a part of a system.
  • a solid phase on which a biotinylated receptor is immobilized is referred to as a receptor chip and / or a receptor microchip.
  • array refers to a pattern or a substrate (for example, a chip) having a pattern in which one or more (for example, 100 or more) receptors are arranged and arranged.
  • Arrays that are patterned on a small substrate eg, 10 ⁇ 10 mm
  • microarrays and arrays are used interchangeably herein. Therefore, even a pattern that is larger than the above-mentioned substrate is sometimes called a microarray.
  • an array consists of a set of desired receptors that are themselves immobilized on a solid surface or membrane.
  • Array preferably comprises at least 0 two identical or different receptors, more preferably at least 1 0 3, and more preferably at least 1 0 4, even more preferably at least 1 0 5 a. These receptors are preferably arranged with a surface of 125 x 80 mm, more preferably 10 x 10 mm.
  • microplates such as a 96-well microphone plate and a 384-well microtiter plate can be used.
  • the receptor to be immobilized may be one kind or plural kinds. The number of such types can be any number from one to the number of spots. For example, about 100, about 100, about 500, and about 1000 receptors can be immobilized.
  • any number of biomolecules eg, receptor
  • any number of biomolecules may be arranged, usually per one substrate 1, 1 0 8 biological molecules or in, until 1 0 7 biological molecules in another embodiment, 1 0 6 biological molecules, 1 0 up to five biomolecules, up to 1 0 4 biomolecules, 1 0 3 biomolecules up, or it may be arranged pieces of biomolecules to 1 0 2 biological molecules, but 1 0 8 biological More biomolecules than molecules may be located.
  • the size of the substrate is preferably smaller.
  • the size of a biomolecule receptor spot can be as small as the size of a single biomolecule (which can be on the order of 1-2 nm).
  • the minimum substrate area is in some cases determined by the number of biomolecules on the substrate.
  • the factor that specifically binds to cells is usually immobilized by covalent bond or physical interaction in the form of a spot of 0.01 to 10 mm.
  • spots of biomolecules may be arranged.
  • “Spot” refers to a certain set of biomolecules. As used herein, “spotting” refers to producing a spot of a certain biomolecule on a certain substrate or solid phase. Spotting can be performed in any manner, for example, by pitting or the like, or can be performed by an automated device, and such methods are well known in the art. As used herein, a biomolecule is a receptor, a fragment of a receptor, or a variant of a receptor.
  • the term "address” refers to a unique location on a substrate, which may be distinguishable from other unique locations.
  • the address is appropriate for associating with the spot with that address, and the entity at every address can be distinguished from the entity at another address (eg, optically) by any shape. Can be taken.
  • the shape defining the address can be, for example, circular, oval, square, rectangular, or irregular. Therefore, “address” indicates an abstract concept, and “spot” may be used to indicate a specific concept. However, when there is no need to distinguish between the two, in this specification,
  • the size defining each address is, among other things, the size of the substrate, the number of addresses on a particular substrate, the amount of analyte and / or reagents available, the size of the microparticles, and any Depends on the degree of resolution required for the method
  • the size can be, for example, in the range of 1-2 nm to several cm, but can be any size consistent with the application of the array.
  • the spatial arrangement and shape defining the address are designed to suit the particular application in which the microarray is used.
  • the addresses can be closely spaced, widely distributed, or sub-grouped into a desired pattern appropriate for a particular type of analyte.
  • GAT C Generic anisy s ys e s t c cn o ol ogy c on s or ti um
  • a method using a microphone-opening patterned surface of an aluminum thiol monomolecular film. is there.
  • a monomolecular film of alkanethiol having a hydrophobic functional group such as a methyl group or a fluoromethyl group is formed on a glass substrate having a gold thin film deposited on one surface.
  • the monomolecular film is overlaid with a photomask in which a large number of light-transmitting spots having a diameter of several m to about 1 mm are arranged, and irradiated with ultraviolet rays.
  • the alkanethiol in the irradiated portion can be decomposed and removed in the form of spots.
  • the reactive functional group introduced into the spot immobilize streptavidin, avidin and other proteins that specifically bind to biotin, or immobilize factors that specifically bind to tags.
  • the receptor protein can be maintained in a directed state under mild conditions without chemical treatment. Immobilization is completed.
  • a carboxyl group is converted to an active ester using N-hydroxysuccinimide, avidin or streptavidin is immobilized, and a small amount of a biomolecule-containing solution is applied to each spot. Immobilization can be performed by dropping.
  • the hydrophobic monolayer formed around the spot is effective for suppressing the diffusion of the solution. Blocking with an inactive protein such as serum albumin or a hydrophilic macromolecule such as Polyethylene Render Recall to reduce non-specific interaction of the analyte with the background area around the spot.
  • microarrays can perform high-throughput analysis of many samples on a single substrate, making them extremely effective Means.
  • the present invention applies the concept of such a microarray to quickly measure the interaction between various types of biomolecules and cells.
  • microarray integration is important in order to analyze a very large number of specimens simultaneously and to minimize the amount of biomolecules and cells required for analysis.
  • data with large errors can be obtained unless measurement is performed on a population consisting of a certain number of cells.
  • the size of each spot constituting the microarray is desirably at least tens to thousands of cells capable of interacting with each other.
  • the diameter of the microarray is about several m to lmm
  • various methods such as a microcontact printing method and an optical lithography method can be used for the production of a microarray. This is a method utilizing a micropatterned surface of a molecular film.
  • biomolecule refers to a molecule associated with a living organism.
  • organ refers to a biological organism, including, but not limited to, animals, plants, fungi, viruses, and the like.
  • Biomolecules include, but are not limited to, molecules extracted from living organisms, and are included in the definition of biomolecules as long as they can affect living organisms.
  • Such biomolecules include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA, genomic DNA, and R such as mRNA).
  • NA polysaccharides
  • oligosaccharides lipids
  • small molecules for example, hormones, ligands, signal transducers, organic small molecules, combinatorial library compounds, etc.
  • composite molecules of these but are not limited thereto.
  • Book Preferred biomolecules herein are receptors and receptor fragments and their ligands.
  • an agent that specifically binds to biotin refers to any agent that can specifically bind to biotin.
  • the binding of biotin and a factor that specifically binds to biotin may be reversible or irreversible.
  • Factors that specifically bind to biotin include, but are not limited to, avidin and streptavidin, and variants thereof.
  • an agent that specifically binds to a tag refers to any factor that can specifically bind to a tag.
  • the binding of the tag to an agent that specifically binds to the tag may be reversible or irreversible.
  • Factors that specifically bind to tags vary depending on the type of tag and are well known in the art.
  • a factor that specifically binds to the tag is, for example, daltathione; when tagging my c protein, a factor that specifically binds to the tag is: For example, an anti-myc antibody; when using 6 consecutive histidine residues as a tag, a factor that specifically binds to the tag is, for example, a nickel chelate column; When a cellulose-binding domain tag is used, a factor that specifically binds to the tag is, for example, cellulose; when a calmodulin-binding peptide tag such as SEQ ID NO: 12 is used, it specifically binds to evening.
  • the factor is, for example, calmodulin; when an S protein-binding peptide tag is used, such as SEQ ID NO: 13, the factor specifically binds to the tag. Is, for example, an S protein; when a T7 tag such as SEQ ID NO: 14 is used, a factor that specifically binds to the tag is, for example, an anti-T7 antibody.
  • SPR Surface plasmon resonance
  • a method of arranging a prism composed of a medium with a high refractive index K retschmann arrangement
  • injecting laser light and LED light is used.
  • the wave number of the plasmon wave changes due to a change in the dielectric constant of the medium in contact with the metal surface opposite to the prism. That is, when a substance approaches a metal surface, the incident angle of light that gives surface plasmon resonance shifts. By utilizing this fact, it becomes possible to sense the coating of the metal surface with the substance.
  • This measurement method has excellent resolution in the vertical direction of the surface (on the order of 0.1 nm), and is capable of observing the amount of substances existing on the surface in the order of ng to pcm 2 in real time.
  • the ability to measure in aqueous media is also a great advantage in investigating the behavior of biomolecules such as proteins.
  • a measuring device utilizing this has been developed as a biomolecular interaction measuring device, and has been applied to the analysis of interactions between proteins and DNA.
  • the crystal resonator microbalance chemically couples and fixes one of the bonding pairs on the electrode of the frequency conversion element, immerses the frequency conversion element in water, and specifically binds the bonding pair to the corresponding bonding pair.
  • the frequency change of the frequency conversion element caused by the mass change caused by the coupling is measured to detect the presence or absence of the coupling (for example, JP-A-6-94591).
  • Examples of the frequency conversion element include a crystal oscillator and a surface acoustic wave element (SAW).
  • the receptor chip of the present invention can also be used as a mass spectrometer chip for a mass spectrometer.
  • mass spectrometry analysis involves the vaporization and ionization of a small sample using a high energy source, such as a laser, including a laser beam.
  • a high energy source such as a laser, including a laser beam.
  • the material is vaporized by the laser beam from the surface of the tip of the mass spectrometry chip into a gas or gas phase, and during this process, some of the individual molecules take in protons and are ionized.
  • the molecules ionized to these positive charges are then accelerated by a short high-voltage electric field, guided to a high-vacuum chamber (drift), and then subjected to sensitivity. Collides with the surface of a high detection device.
  • drift high-vacuum chamber
  • time of flight is a function of the mass of the ionized molecule
  • time that elapses between ionization and collision can be used to determine the mass of that molecule, and that molecular mass is then It can be used to determine whether known molecules are present (time-of-flight mass spectrometry (TOF)).
  • TOF time-of-flight mass spectrometry
  • Methods for producing gas phase ions include a desorption / ionization method obtained from the impact of particles on a sample. This method includes fast atom bombardment (FAB-bombarding neutral particles (neutrals) on a sample suspended in a volatile matrix), secondary ion mass spectrometry (S IMS-keV-secondary ion Impacts the surface to generate secondary ions), liquid S IMS (similar to F AB except that the primary species of LS IMS are ions), plasma desorption mass spectrometry (Me V— Except for using it, the same as S IMS), mass cluster impact method (MC I—similar to S IMS using large class primary ions), laser desorption Z ionization method (LDI—surface using laser light Matrix-assisted laser desorption Z-ionization method (MALD I—Similar to LDI except for desorption and ionization of species from a matrix that can assist in desorption and ionization events) and so on.
  • Typical mass spectrometry methods
  • a measurement method using a mass spectrometry chip to which a molecule such as a receptor that performs affinity binding is bound in a mass spectrometer is disclosed in, for example, JP-A-9-1501489 as follows: Exposing the surface of the mass spectrometric chip on which the receptor is immobilized to a source of the analyte molecules (for example, a mixture containing ligands) so that the analyte molecules are bound; and Placing the tip of the mass spectrometry chip to which the analyte molecules are attached at one end of a time-of-flight mass spectrometer, and applying a vacuum and an electric field to create an accelerating potential in the spectrometer; One or more laser pulses are applied to at least a portion of the analyte bound to the induced mass spectrometer tip in the spectrometer to desorb the analyte molecule ions.
  • a source of the analyte molecules for example, a mixture containing
  • the mass spectrometer uses the mass spectrometer to detect the mass of the ion by time of flight; and displaying the mass thus detected.
  • the mass of ions of a molecule for example, a ligand that specifically binds to a receptor bound to the mass spectrometry chip can be detected.
  • the mass of the analyte molecule can be measured by laser desorption ionization and time-of-flight mass spectrometry, and this method facilitates the desorption and ionization of the analyte.
  • an energy absorbing substance eg, sinapinic acid, cinnamamide, cinnamyl bromide, 2,5-dihydroxybenzoic acid, and ⁇ -cyano 4-hydroxycaic acid
  • sinapinic acid, cinnamamide, cinnamyl bromide, 2,5-dihydroxybenzoic acid, and ⁇ -cyano 4-hydroxycaic acid can be used together with the analyte.
  • a further measurement method using a mass spectrometry chip in which a molecule that performs affinity binding such as a receptor is immobilized in a mass spectrometer is disclosed in JP-T-Hei-11-512125.
  • an affinity binding molecule such as a receptor
  • a chip typically on a support having a hydrogel, and more particularly, a polysaccharide hydrogel, such as carboxymethylated dextran, and analyzed.
  • a substance eg, a ligand
  • a preferred receptor is L ⁇ X-1, which belongs to the receptor of the LDL receptor-related protein family 1.
  • LOX-1 is an abbreviation for lectin-like oxidized LDL receptor-1, and refers to the receptor for the family of LDL receptor-related proteins, which are a type of skeletal venja receptor. Point. LOX-1 is a very inducible gene, and its expression is induced under conditions such as hypertension, hyperlipidemia, and diabetes that promote arteriosclerosis. In addition, when oxidized LDL acts on vascular endothelial cells via LOX-1, it causes the production of reactive oxygen and consequent reduction of NO release. It is also said that the expression of the cell adhesion molecule ⁇ chemokine is induced, which causes a state of endothelial dysfunction.
  • LDL low-density lipoprotein, which is a type of serum protein that is a promoting factor of arteriosclerosis. Serum contains about 300 mg / dl, contains about 50% cholesterol, and contains 20% of a protein called apo B.
  • refolding of a receptor protein expressed as an inclusion body is performed by dropping a denatured protein into a refolding buffer containing arginine, reduced dal thione, and oxidized dal thione.
  • a denatured protein is dropped into a refolding buffer containing arginine, reduced dal thione, and oxidized dal thione.
  • neither the denatured protein nor the refolding buffer contains a detergent.
  • the term "denatured” It means that the primary structure of the protein remains unchanged, only the higher-order structure is ruptured, and its natural state and physical properties change.
  • the term "denaturing agent” refers to an agent that causes protein denaturation.
  • Modifiers include, but are not limited to, guanidine hydrochloride, urea, dioxane, alcohol, ethylene glycol, and the like.
  • concentration using guanidine hydrochloride is preferably 4-8 M, more preferably 6 M.
  • the term "refolding” refers to obtaining a protein that has recovered its original tertiary structure, biological activity, etc., from a denatured protein.
  • the biological activity is, for example, the ligand binding activity.
  • ⁇ inclusion body '' refers to a protein that is produced when large amounts of a foreign gene are recombinantly expressed in a bacterium such as Escherichia coli. Refers to a structure.
  • SH protection reagent refers to a reagent that prevents a thiol group (SH group) in a protein from reacting with a substance that is reactive with the SH group.
  • SH protecting reagents include, but are not limited to, dithiothreyl], 3-mercaptoethanol, and daltathione.
  • the refolding of the receptor protein expressed as an inclusion body is performed by dropping the denatured protein into a refolding buffer containing arginine, reduced daluzion, and oxidized daluzion.
  • the dropping rate is a rate of 10 to 301 minutes, and more preferably a rate of 20 to 301 / minute.
  • the ⁇ ⁇ of the refolding buffer preferably ranges from 7.5 to 9.5, more preferably 8. 0-8. It is well known in the art to change the appropriate pH, depending on the nature of the protein to be refolded, which is in the range of 5.
  • the refolding buffer contains Tris-HCl, preferably the concentration of Tris-HCl is 5-10 OmM.
  • the concentration of arginine in the refolding buffer is preferably 10
  • the ratio of reduced daltathione to oxidized glutathione is preferably 1: 5 to 1:12, more preferably 1: 5 to 1:10, and most preferably 1: 5. 5 More preferably, the concentration of reduced daltathione is 1-2 OmM and the concentration of oxidized daltathione is 0.2-2 mM.
  • the ratio of the volume of the denatured protein solution to be dropped and the volume of the refolding buffer is preferably 1:50 to 1: 100.
  • the protein concentration in the refolding buffer after the dropwise addition of the denatured protein solution to be dropped is 5 to 500 g / mL, more preferably 5 to 250 gZmL, and still more preferably, 20-250 gZmL.
  • the denatured protein used in the refolding method of the present invention is a protein denatured by a denaturing agent, heat, ultraviolet irradiation, or irradiation, but is preferably a protein denatured by a denaturing agent. It is.
  • the denatured protein used in the refolding method of the present invention is preferably a denatured protein obtained by solubilizing an inclusion body protein expressed in bacteria using a solubilization buffer containing an SH protection reagent and a denaturant. It is.
  • the solubilization to obtain the denatured protein is performed with protein at a concentration of 1-5 mg ZmL. More preferably, the solubilization to obtain the denatured protein is performed using a protein at a concentration of 2.0 to 3.0 Omg / mL.
  • the pH of the solubilization buffer is between 7.0 and 9.0, and in one aspect, the solubilization buffer is 5-10 OmM Tris-HCl buffer.
  • the SH protecting reagent is dithiothreitol, more preferably, the concentration of dithiothreitol is 5-20 OmM, and even more preferably, the concentration of dithiothreitol is 50-10 OmM.
  • a step of stirring the solution is performed after the denatured protein solution is dropped into the refolding buffer. More preferably, this stirring step is performed for 10 to 48 hours, and even more preferably, this stirring step is performed for 10 to 14 hours. This stirring can be performed at 4 ° C to room temperature, but is preferably performed at 4 ° C.
  • the refolding method of the present invention it is possible to obtain a more pure and more homogeneous protein than the protein prepared using the conventional refolding method. For example, by using the refolding method of the present invention, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% Thus, a protein having a purity of 99% or more can be obtained. Further, when the protein obtained by using the refolding method of the present invention is analyzed by mass spectrometry, the width of mZz by MS spectrum is 70 or less, 60 or less, 50 or less, 40 or less, 30 or less. A protein having a purity of 20 or less can be obtained.
  • “prophylaxis or prevention” refers to treating a disease or disorder before such condition is caused so that the condition does not occur.
  • treatment refers to preventing the deterioration of a disease or disorder when such a condition occurs, preferably maintaining the status quo, more preferably reducing the disease, More preferably, it refers to fluctuating.
  • vaccine refers to an agent that provides immunological protection against a disease. Such immunological prophylaxis may be humoral or cellular immunity.
  • a vaccine composition can include, for example, an antigen that elicits an immune response associated with a disease.
  • Antigens included in the vaccine composition include, but are not limited to, proteins, peptides, nucleic acids, nucleotides, nucleosides, amino acids, saccharides, and the like.
  • a preferred antigen contained in the vaccine is a protein antigen, more preferably a protein refolded by the method of the present invention.
  • a "kit” includes a plurality of containers and manufacturer's instructions, and each container contains a pharmaceutical composition of the present invention, other agents, and a carrier. Refers to products containing.
  • a "subject” is a subject to which the pharmaceutical composition of the present invention is administered, and includes, but is not limited to, animals such as humans, mice, mice, and chickens Not done.
  • pharmaceutically acceptable carrier refers to a substance used when producing agricultural chemicals such as pharmaceuticals or veterinary drugs, which does not adversely affect the active ingredient.
  • pharmaceutically acceptable carriers include, for example, These include, but are not limited to: antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, excipients. Form and / or pharmaceutical adjuvant.
  • the type and amount of the drug used in the treatment method of the present invention are determined based on the information obtained by the method of the present invention (eg, information on a disease), the purpose of use, the target disease (type, severity, etc.) It can be easily determined by those skilled in the art in consideration of the patient's age, weight, sex, medical history, form or type of the site of the subject to be administered, and the like.
  • the frequency of applying the monitoring method of the present invention to the subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, and course of treatment. A person skilled in the art can easily determine this in consideration of such factors.
  • the frequency of monitoring disease status may include, for example, daily—once every few months (eg, once a week—once a month). It is preferable to monitor once a week—monthly while monitoring the progress.
  • more than one drug may be used in the treatment of the present invention.
  • substances having similar properties or origins may be used, or drugs having different properties or origins may be used.
  • Information regarding disease levels for methods of administering more than one such drug can also be obtained by the methods of the present invention.
  • candidate compound refers to a candidate compound that can be used for treating a disease or disorder of interest.
  • a compound may be called a candidate compound if it is expected to be effective for the disease or disorder of interest.
  • compound species refers to one compound having desired properties, such as having a specific target activity, in a certain set of compounds. For example, in a set of compounds that modulate LOX-1 activity, modulate LOX-1 activity. Where a nodal compound is specified, such compound may be referred to as a compound species. In this specification, it is simply referred to as a compound.
  • library refers to a certain set of compounds and the like for screening.
  • the library 1 may be a set of compounds having similar properties or a set of random compounds. Preferably, but not limited to, a collection of compounds predicted to have similar properties is used.
  • the refolded protein of the present invention can also be used as a component of a pharmaceutical composition for treating, preventing, diagnosing, or prognosing atherosclerosis.
  • the term “effective amount” of a drug refers to an amount by which the drug can exert a desired drug effect. In the present specification, among such effective amounts, the lowest concentration may be referred to as the minimum effective amount. Such minimum effective amounts are well known in the art, and usually the minimum effective amount of a drug is determined by those skilled in the art, or can be determined by the skilled artisan as appropriate. In determining such an effective amount, an animal model can be used in addition to the actual administration. The present invention is also useful in determining such an effective amount.
  • pharmaceutically acceptable carrier refers to a substance used when producing agricultural chemicals such as pharmaceuticals or veterinary drugs, which does not adversely affect the active ingredient.
  • pharmaceutically acceptable carriers include, but are not limited to, for example, antioxidants, preservatives, coloring agents, flavorings, and diluents, emulsifiers, suspending agents, Solvents, fillers, bulking agents, buffers, delivery vehicles, excipients and / or agricultural or pharmaceutical adjuvants.
  • the type and amount of the drug used in the treatment method of the present invention are determined based on the information obtained by the method of the present invention (for example, information on a disease), the purpose of use, and the target disease (type, severity, etc.).
  • the patient's age, weight, gender, medical history Those skilled in the art can easily determine the form in consideration of the form or type of the site.
  • the frequency of applying the monitoring method of the present invention to the subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, and course of treatment. A person skilled in the art can easily determine this in consideration of such factors.
  • the frequency of monitoring the disease state may be, for example, once every few months (eg, once a week, once a month). It is preferable to conduct monitoring once a week-once a month while monitoring the progress.
  • the “instruction” describes the treatment method and the like of the present invention for a person who administers such as a doctor or a patient.
  • This instruction sheet describes, for example, a word indicating that the medicament or the like of the present invention is to be administered immediately after or immediately before radiation treatment (for example, within 24 hours).
  • These instructions are prepared and prepared according to the format prescribed by the competent authority of the country in which the invention is implemented (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States). It will be specified that it has been approved by the government. Instructions are so-called package inserts, which are usually provided on paper, but are not limited to them, such as electronic media (eg, homepages provided on the Internet, e-mail), etc. It can also be provided in form.
  • more than one drug may be used in the treatment of the present invention.
  • substances having similar properties or origins may be used, or drugs having different properties or origins may be used.
  • Information regarding disease levels for methods of administering more than one such drug can also be obtained by the methods of the present invention.
  • the present invention will be described in detail with reference to Examples and the like, but the present invention is not limited thereto.
  • a human LOX-1 CTLD (143-273) having the following sequence was incorporated into the multi-cloning site (Ndel-Xhol) of pET28a, a histidine-tag fusion protein expression vector manufactured by Novagen, and E. coli BL2 1 (DE3) and expressed in large quantities:
  • Escherichia coli transformed with the LOX-1 expression vector was cultured at 37 ° C in 8 L of M9 minimal medium containing 50 ⁇ gZm1 of kanamycin, and the OD value at 660 nm reached 0.5.
  • IPTG was added to a final concentration of ImM, culture was further continued at 37 ° C for 4 hours, and the cells were recovered by centrifugation at 3,50 OXg for 30 minutes.
  • the collected cells were washed with 5 ml of lysis buffer (50 mM Tris / HC1 (pH 8.0), 400 mM KC1, 0.1% TritonX-1000) per 1 g of cells. After suspending, add Comp1 et eMini protease inhibitor (Roche, using 0.5 ml of bullet per gram of cells), and lyse the cells at 4 ° C using an Astrason ultrasonic crusher. The pellet was crushed, and the pellet was collected by centrifugation (10,000 X g, 30 minutes, ° C). Resuspend pellet in lysis buffer, supersonic Wave treatment and centrifugation were repeated twice to wash well, recovered as an insoluble inclusion body, and stored at 180 ° C until the next solubilization operation was performed.
  • lysis buffer 50 mM Tris / HC1 (pH 8.0), 400 mM KC1, 0.1% TritonX-1000
  • the column was washed with a column equilibration buffer (50 mM Tris / HC 1 (pH 7.5), 10 OmM NaC 1, 10 mM imidazole), and the imidazole concentration was increased with a linear gradient to 50 OmM.
  • a column equilibration buffer 50 mM Tris / HC 1 (pH 7.5), 10 OmM NaC 1, 10 mM imidazole
  • the imidazole concentration was increased with a linear gradient to 50 OmM.
  • the eluted protein was concentrated to 2 ml with Centricon 10 (Millipore), and 10 cleavage units of bovine thrombin protease were added per 1 mg of protein, followed by reaction at 4 ° C for 5 hours to cleave the histidine tag.
  • benzamiji Thrombin was removed by passing through a Sepharose column (manufactured by Pharmacia), and PMSF was added to a final concentration of 0.1 ImM to stop the protease reaction.
  • the protein solution was then applied to a Superdex 75 gel filtration column (Pharmacia) equilibrated with a gel filtration column equilibration buffer (1 OmM Tris / HC1 (pH 7.5), NaC 150 mM). The fraction with the correct molecular weight was collected.
  • Solubilization and refolding procedures were performed in the same manner as LOX-1 CTLD.
  • the composition of the dialysis buffer was adjusted to 25 mM Tris / HC 1 (pH 7.5), The gel filtration power was adjusted to 10 OmM NaC1, the composition of the ram equilibration buffer was eluted to 10 mM Tris / HC1 (pH 7.5), NaC140 OmM, and further from the Ni-chelating Sepharose column.
  • the procedure was the same as for LOX-1 CTLD, except that the concentration of the protein solution was added after NaC1 was added to a final concentration of 40 OmM.
  • the extracellular region of hLOX-1 or the DNA fragment encoding CTLD was prepared by a conventional PCR method. Nr'ul on the 5 'side and EcoRV restriction enzyme sites on the 3' side were added to both ends. After extracting the PCR product and treating it with both restriction enzymes, it was inserted into pBS, a cloning vector, and the DN.A sequencer was used to confirm that there was no error in the gene sequence.
  • the nucleotide sequence and amino acid sequence of the extracellular region of hLOX-1 are shown in SEQ ID NOS: 1 and 2, respectively, and the nucleotide sequence and amino acid sequence of hLOX-1 CTLD are shown in SEQ ID NOs: 3 and 4, respectively.
  • a gene encoding the protein whose sequence has been confirmed is excised with a restriction enzyme, and a plasmid vector encoding a polypeptide known to undergo biotinylation in Escherichia coli PinPinint Xa (Promega Manufactured by the company).
  • a transformant that correctly incorporated the target gene was selected.
  • the cells were centrifuged at 000 rpm for several minutes to collect the cells. After disrupting the collected cells by sonication, centrifuged at 20,000 g for 30 minutes. The supernatant (soluble surface) and the precipitate (insoluble surface) obtained are suspended in SDS sample buffer, and the suspension is recovered at 95 ° C. Treated with C for 4 minutes. Next, the proteins were separated by 12% SDS-PAGE and then electrically transferred to a nitrocellulose membrane.
  • the nitrocellulose membrane was stained with Ponce S-S to confirm the protein band position, and then TBS-Tween (20 mM Tris, 15 OmM NaCl, pH 7.6, 0.1% Tween 20) The mixture was gently stirred at room temperature for 60 minutes. Next, the reaction was carried out in streptavidin-labeled alkaline phosphatase at room temperature for 30 minutes. Next, the nitrocellulose membrane after the reaction is washed with TBS-Tweeen, and then an NBTZBC IP solution, which is a substrate for alkaline phosphatase, is added. The reaction is performed at room temperature until the band of the biotinylated protein is detected. I let you. As a result, in the insoluble fraction, a remarkable band of the biotinylated protein was detected at the position corresponding to the molecular weight of the biotinylated extracellular region and the biotinylated CTLD.
  • TBS-Tween 20 mM Tris, 15 OmM NaCl,
  • Om 1 solubilization buffer (10 OmM Tris / HC 1 (pH 8.0), 6M The solution was suspended in guanidine hydrochloride (5 mM DTT) (final protein concentration: 3 mg Zm1), centrifuged to remove the precipitate, and allowed to stand at room temperature for 4 hours.
  • the protein solution was then applied to a Superdex 75 gel filtration column (Pharmacia) equilibrated with a gel filtration column equilibration buffer (10 mM Tris / HC 1 (pH 7.5), NaC 15 OmM). The fraction with the correct molecular weight was collected.
  • a protein refolded by a conventional refolding method using cyclic carbohydrate cycloamylose and a surfactant was prepared as follows.
  • Inclusion bodies are treated with 6 M guanidine hydrochloride solution containing 40 mM DTT at room temperature Processing for 1 hour at, completely dislodged the wrong structure. Subsequently, a 70-fold volume of a surfactant solution (0.1% CTAB or SB3-14, PBS (-) solution containing 2 mM DL-cysteine at a final concentration) was added, and reacted at room temperature for 1 hour. After the reaction, 24 ml of the reaction solution was taken out, 6 ml of a 3% CA solution was added, and the mixture was further reacted at room temperature for 1 hour.
  • a surfactant solution 0.1% CTAB or SB3-14, PBS (-) solution containing 2 mM DL-cysteine at a final concentration
  • biotinylated CTLD When the refolded biotinylated extracellular region, biotinylated CTLD, was immobilized on streptavidin beads and the binding of Di I AcLDL, which fluorescently labeled acetylated LDL, one of the ligands, was confirmed. Fluorescence was observed on the immobilized beads on which the biotinylated extracellular region or the biotinylated CTLD region was immobilized, indicating that both recovered the ligand binding ability.
  • the refolding method of the present invention is a method for preparing a protein with higher purity than the conventional method (FIG. 1).
  • the purity of the protein prepared by the refolding method of the present invention is about 99% or more. Is shown.
  • the protein from which the tag has been removed from the histidine-tagged LOX-1 CTLD protein is dissolved in a 0.1% TFA (trifluoroacetic acid) solution, and the solution is added to a 30% acetonitrile solution containing 0.1 TFA.
  • TFA trifluoroacetic acid
  • the dissolved sinapinic acid solution was added and mixed.
  • the dissolved sample was analyzed using a MALD I-T ⁇ F mass spectrometer (Voyager Elite, Perspective Biostems).
  • Protein purity assay results of refolded LOX-1 CTLD by mass spectrometry The untagged histidine-tagged LOX-1 CTLD protein obtained by the refolding method of the present invention gave a narrow width mass spectrometric spectrum with an m / z ratio (mass / charge) of about 50. The result of this narrow width indicates that the product is purified to a higher degree of purity with less contaminants than the conventional refolding method using cyclic carbohydrate cycloamylose (FIG. 2).
  • the tag was removed from the protein obtained in Example 1, and the protein was solubilized in 20 mM Tris HC1 buffer (pH 7.0) and 5 OmM NaCl to a concentration of ImM. 1 H— 15 N HSQC (Heteronuc lear Single le Quantum um Coher enc ec orrelati on spectroscopy) The root ij of the two-dimensional correlation spectrum was determined at 25 ° C.
  • CTLD was used as a sample.
  • the crystallization of CTLD was performed using an 8 mg / mL IL IL CTLD solution (1 OmM Tris-HC1, pH 7.5, 5 OmM NaCl, 1 OmM zinc acetate was used as a buffer to dissolve the protein). Then, 1 L of 0.1 M citrate buffer (pH 3.0-4.0) was added, and this solution was added to 0.1 M citrate buffer (pH '3.0-4.0). Obtained by vapor diffusion for 2-4 days.
  • LOX-11CTLD obtained by the refolding method of the present invention was of such high purity that crystallization was possible.
  • the histidine-tagged extracellular region or the histidine-tagged CTLD that has been successfully refolded or the histidine-tagged CTLD is used as a sensor to detect denatured LDL, etc.
  • the immobilized protein was immobilized, and the actual binding of the ligand was examined.
  • BI Acore manufactured by BI Acore was used as the surface plasmon resonance device.
  • Example 1 The refolded histidine-modified extracellular region prepared in Example 1 was immobilized on a BI ACORE sensor chip such that the portion involved in ligand recognition faces outward. Specifically, the NTA sensor chip (B IAc ore Inc.) surface was washed with PBS (phosphate buffered saline) containing 0. 35M EDTA, a 500 MN i C 1 2 to inject, The protein was immobilized on the sensor chip. The histidine-tagged CTLD prepared in Example 1 was immobilized on this chip. The amount of protein to be injected was adjusted so that the immobilization amount was 1000 RU or less.
  • PBS phosphate buffered saline
  • oxidized LDL concentration of purified LDL and copper sulfate solution was adjusted so as to be 3 mg / m L and 75 M, respectively and incubated for 20 hours in C0 2 incubator within one. Then, it was dialyzed against a 0.15 M sodium chloride solution containing EDTA to obtain oxidized LDL.
  • acetylated LDL A sodium acetate solution was added to a final concentration of 50% with respect to the purified LDL, and the mixture was cooled to 0 ° C. At this time, the whole volume was adjusted to 1 mL. Acetic anhydride 1 was added 5 times at 10 minute intervals while stirring on ice, and the mixture was further cooled and stirred for 30 minutes to complete the reaction.
  • Binding of various LDLs to the receptor chip was detected as follows. At a flow rate of 2 minutes, LDL and denatured LDL (acetylated LDL and oxidized LDL) were injected for 2 minutes and changes in the sensorgram were recorded. In the case of a device that uses the surface plasmon resonance principle, the binding of the ligand is detected as a change in the sensorgram indicating an increase in resonance unit: RU. Thereafter, the sensor chip was regenerated by injecting 1M NaCl 1-83 buffer (117.4) for 30 seconds and detaching the bound LDL. This cycle is repeated five times, and after confirming that the baseline of the sensor chip and the response level of the sensorgram are stable and reproducible, the binding constant and the amount of binding are calculated from changes in the sensorgram. did.
  • histidine-tagged LOX-1 CTLD refolded using the refolding method of the present invention has a similar affinity to oxidized LDL and acetylated LDL as to natural LOX-1 This was confirmed ( Figure 5).
  • the refolded LOX-1 CTLD showed binding ability to LDL which was two orders of magnitude lower than that of the modified LDL (data not shown).
  • the immobilization buffer was allowed to flow for 20 minutes, and proteins not bound to the chip were washed away. Even after washing for 600 minutes, the decrease in RU value was 5% or less.
  • the binding of LDL and denatured LDL was tested in the same manner as in (1.2) and (1.3) above.
  • biotinylated CTLD had the same affinity for oxidized LDL and acetylated LDL as natural LOX-1.
  • LOX-1 protein forms dimers. Therefore, it was confirmed using gel filtration, PAGE, and mass spectrometry whether or not LOX-1 refolded by the refolding method of the present invention forms a dimer.
  • the full-length extracellular domain (61-27) obtained according to the method of Example 2 was used. After expression with histidine tag in 3), refolding was performed, and then the protein without histidine tag was used.
  • Sinapic acid solution dissolved in a 30% acetate nitrile solution containing 0.1 TFA in the histidine-tagged LOX-1 extracellular domain prepared in Example 1 dissolved in 0.1% TFA (trifluoroacetic acid) solution was mixed.
  • the dissolved sample was analyzed by a MALD I-TOF mass spectrometer (VoyagerElite, Perpstechtiv eBiostems).
  • a sample buffer containing 20% / 3_mercaptoethanol in L ⁇ X_1 protein obtained by the refolding method of the present invention (25 OmM Tris HCl, pH 6.8, 8% SDS, 20% sucrose, 0% 02% bromophenol blue (BPB)) was added, and the sample was boiled at 100 for 5 minutes to obtain a sample in a reduced state.
  • a sample obtained by adding a sample buffer obtained by removing j3-mercaptoethanol from the above sample buffer and reacting at room temperature for 30 minutes was used as a non-reduced sample.
  • Each sample was stained with Coomassie brilliant blue (CBB) after 15% polyacrylamide gel electrophoresis.
  • CBB Coomassie brilliant blue
  • sample buffer for SDS-PAGE 25 OmM Tris HC1, pH 6.8, 8% SDS, 20% sucrose, 0.02% bromophenol blue (BPB), 20%
  • BPB bromophenol blue
  • the protein obtained as a result of refolding the full-length extracellular domain of L—X-1 by the refolding method of the present invention was found to be similar to the mode of LOX-1 existing on the cell surface. It was shown to be a dimer ( Figure 6).
  • LOX-1 with the N-terminus tagged with a biotinylated tag prepared in Example 3 was immobilized on a microplate (Biocoat (registered trademark), BD Biosciece, Inc.) coated with streptavidin via a biotin. Become Blood is collected in a test tube containing heparin to avoid unnecessary blood coagulation (heparin blood collection) . The LDL fraction obtained by centrifugation from the obtained human plasma is dispensed, and at 4 ° C Leave for 1 hour. Thereafter, the cells were washed three times with 0.05% Tween 20-TBS (NaCl 1 140 mM, KC 12.
  • LOX_1 having the N-terminal with a biotinylated tag prepared in Example 3 is bound to porcelain beads (Dynabeads M-280 Streptavidin, Veritas Co., Ltd.) coated with streptavidin.
  • porcelain beads Dynabeads M-280 Streptavidin, Veritas Co., Ltd.
  • streptavidin a biotinylated tag prepared in Example 3
  • LOX-1 ligand, acetylated LDL is immobilized on an SPR chip with an LDL antibody, and LOX-1 protein is flown there.
  • the binding activity of LOX-1 to acetylated LDL was examined by observing nulls.
  • the Biacore 2000 system manufactured by Biacore was used for the SPR measurement device.
  • a goat anti-ApoB antibody (Rock 1 and Co.) was immobilized on a Biacore CM3 chip using the amine coupling method, and acetylated LDL (Molecular probes) was added thereto. To bind to the antibody. The addition amount was adjusted so that the amount of acetylated LDL bound to the CM3 chip was about 3,000 ResponseUnit. After that, the chip was equilibrated with a measurement buffer (1 OmM Hepes, pH 7.5, 15 OmM NaCl), and LOX-1 extracellular domain and LOX-1 CTLD (protein concentration: 10 mM) suspended in the same buffer. nM-2020) was added at a flow rate of 20 1 / min, and the SPR signal was observed. Based on the obtained data, the reaction rate constant was calculated using BIAeVa1uation software (Biacore).
  • Figure 7 shows the results of one gram of the sensor and the calculated values of Ka and Kd.
  • the protein refolded by the refolding method of the present invention is a natural protein. It is considered to have substantially the same structure.
  • the full length of the extracellular region forms a stable homodimer by SS binding and exhibits binding activity to acetylated LDL, but the binding activity is lower than that of LOX-11 present on cells.
  • dimerization of CTLD is essential for binding of CTLD to acetylated LDL, but its binding activity is not sufficient if merely dimerization of CTLD is sufficient.
  • the binding capacity of simply dimerized CTLD is about four orders of magnitude lower than that of LOX-1 present on cells. From these facts, it is considered that LOX-1 extracellular domains are further associated with each other and cooperatively bind to acetylated LDL on cells.
  • L ⁇ X-1 forms an aggregate of at least four molecules (dimer X2) on cells. Therefore, based on these experimental results, CTLD as a monomer has a binding ability to acetylated LDL, assuming responses such as measurement of oxidized LDL content in blood, removal of oxidized LDL from blood, and use of vaccines. This indicates that it cannot be used because it does not have it, and that the dimer structure obtained by this refolding is required as the minimum unit.
  • the refolding method of the present invention has made it possible to obtain a highly pure and homogeneous refolded protein.
  • a highly sensitive receptor chip can be produced.
  • the protein obtained by the refolding method of the present invention it is possible to provide a ligand-removing material in which non-specific binding is reduced as compared with the conventional method.

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Abstract

L'invention concerne une méthode de repli permettant d'élaborer une protéine extrêmement pure et homogène en comparaison à celles obtenues à l'aide des méthodes existantes, ainsi qu'une protéine élaborée au moyen de cette méthode de repli. Notamment, une méthode de repli d'une protéine dénaturée, telle qu'un corps d'inclusion solubilisé, consiste à a) introduire la protéine dénaturée dans une solution tampon de repli contenant de l'arginine, du glutathione réduit, et du glutathione oxydé. Cette invention a aussi trait à une protéine préparée au moyen de cette méthode et à une composition pharmaceutique la contenant.
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EP1630173A3 (fr) * 2004-08-27 2006-03-08 Bioceuticals Arzneimittel AG Procédé de récupération du G-CSF humain sous une forme biologiquement active à partir de corps d'inclusion
JP2008546670A (ja) * 2005-06-17 2008-12-25 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト 少なくとも１つの非天然のシステインを含んでいる操作されたタンパク質の選択的な還元および誘導体化
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