JP2004170194A - Screening method of glycosaminoglycan bonding material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new means for screening a glycosaminoglycan bonding material. <P>SOLUTION: A screening method of the glycosaminoglycan bonding material uses a fat bonding glycosaminoglycan having fat bonded to the glycosaminoglycan for a glycosaminoglycan bonding material acquisition material, and has processes (1), (2). In the process (1), the glycosaminoglycan bonding material in a specimen and a fat bonding glycosaminoglycan complex is brought into contacts. In the process (2), a complex of the glycosaminoglycan bonding material and the fat bonding glycosaminoglycan generated by the contact is detected. <P>COPYRIGHT: (C)2004,JPO

Description

【００８９】
【発明の効果】
本発明により、グリコサミノグリカンに脂質が結合してなる「脂質結合グリコサミノグリカン」をグリコサミノグリカン結合性物質捕捉材として使用した「グリコサミノグリカン結合性物質のスクリーニング方法」が提供され、グリコサミノグリカン結合性を有する物質のスクリーニングを容易に行うことが可能となる。
【図面の簡単な説明】
【図１】クローン１の発現産物のグリコサミノグリカンへの結合性を示す図である。図中、Ｈｅｐ−ＰＥ、ＨＳ−ＰＥ、ＨＡ−ＰＥ、ＣＳＡ−ＰＥ、ＤＳ−ＰＥ、ＣＳＣ−ＰＥ、ＣＳＤ−ＰＥ、ＣＳＥ−ＰＥ、及びＢＳＡは実施例中に記載した略号と同義である。
【図２】クローン２の発現産物のグリコサミノグリカンへの結合性を示す図である。図中、Ｈｅｐ−ＰＥ、ＨＳ−ＰＥ、ＨＡ−ＰＥ、ＣＳＡ−ＰＥ、ＤＳ−ＰＥ、ＣＳＣ−ＰＥ、ＣＳＤ−ＰＥ、ＣＳＥ−ＰＥ、及びＢＳＡは実施例中に記載した略号と同義である。[0001]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to a “screening method for a substance having glycosaminoglycan binding property” using a glycosaminoglycan to which a lipid is bound as a glycosaminoglycan binding substance capturing material.
[0002]
[Prior art]
Lipid-bound glycosaminoglycans in which a lipid is bound to glycosaminoglycans are known, and are disclosed, for example, in Patent Document 1, Patent Document 2, Patent Document 3, Non-Patent Documents, and the like.
[0003]
A method has been developed in which a phospholipid, phosphatidylethanolamine, is covalently bound to the reducing end of glycosaminoglycan. Further, the phospholipid-bound glycosaminoglycan derivative obtained by such a method is used in a plastic culture dish. Hepatocytes are cultured in solid phase as a culture substrate, and cultured for spheroidization to form an artificial liver or the like (Patent Document 4), or to produce an antibody against hyaluronic acid. In order to immunize a mouse with HA-phosphatidylethanolamine in which HA and phosphatidylethanolamine are covalently bound as an antigen or to measure the titer of an antibody present in the serum of the immunized mouse, HA-phosphatidylethanolamine was distilled water. There is an example in which a substance dissolved in a solution is used as an antigen solution (Patent Document 5).
[0004]
Patent Document 6 discloses a method in which a lipid-bound glycosaminoglycan is immobilized on a solid phase, and a glycosaminoglycan-binding substance is measured using the lipid-bound glycosaminoglycan and an antibody. However, a method of screening for a glycosaminoglycan binding substance from an unknown sample, or expressing the protein by genetic engineering using a nucleic acid encoding a protein whose binding to glycosaminoglycan is unknown. No method for screening a nucleic acid encoding a glycosaminoglycan binding protein is disclosed or suggested.
[0005]
[Patent Document 1] JP-A-4-80201
[Patent Document 2] JP-A-4-80202
[Patent Document 3] JP-A-9-30979
[Patent Document 4] JP-A-5-236951
[Patent Document 5] JP-A-9-126000
[Patent Document 6] JP-A-2000-65837
[Non-Patent Document] Biol. Chem. , (1993) 268, 15779-15787.
[0006]
[Problems to be solved by the invention]
In recent years, a large number of nucleic acids encoding unknown proteins have been discovered. On the other hand, finding a glycosaminoglycan affinity (binding) substance that plays a very important role in the living body is important in elucidating the function of glycosaminoglycan in the living body, and is also unknown. It is extremely important in elucidating the functions of proteins and nucleic acids.
[0007]
Therefore, development of a method for efficiently screening a substance having glycosaminoglycan binding properties from a specimen has been strongly desired.
[0008]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in order to solve the above-described problems, and as a result, by using the lipid-bound glycosaminoglycan as a glycosaminoglycan-binding substance-trapping material, it is possible to use a glycosaminoglycan with an extremely simple operation. The present inventors have found that "screening of a binding substance" can be achieved, and by using this method, a nucleic acid encoding an unknown protein has been screened and found to be useful for elucidating its function, thereby completing the present invention.
[0009]
That is, the present invention is as follows.
(1) Screening for "glycosaminoglycan-binding substance" characterized by using "lipid-bound glycosaminoglycan" in which lipid is bound to glycosaminoglycan as a glycosaminoglycan-binding substance-trapping material Method".
(2) The “screening method for glycosaminoglycan-binding substances” according to (1), comprising at least the following steps (a) and (b):
(A) contacting a “glycosaminoglycan-binding substance” in a sample with “lipid-bound glycosaminoglycan” as a glycosaminoglycan-binding substance capturing material;
(B) a step of detecting a “complex of a glycosaminoglycan-binding substance and a lipid-bound glycosaminoglycan” generated by the contact;
(3) The “screening method for a glycosaminoglycan-binding substance” according to (1) or (2), wherein the “glycosaminoglycan-binding substance” is a protein.
(4) The “screening method for a glycosaminoglycan binding substance” according to any one of (1) to (3), wherein the “sample” is a “biologically-derived sample”.
(5) The "glycosami" described in any one of (1) to (4), wherein the "lipid-bound glycosaminoglycan" is a solid-phased lipid-bound glycosaminoglycan bound to a solid phase. Method for Screening Noglycan-binding Substance ".
(6) A “screening method for a nucleic acid encoding a glycosaminoglycan-binding protein”, comprising at least all of the following steps (a) to (d).
(A) a step of expressing the protein in a cell using a nucleic acid encoding a “protein whose binding property to glycosaminoglycan is unknown” to obtain a specimen containing the protein;
(B) contacting the protein in a sample with “lipid-bound glycosaminoglycan” as a glycosaminoglycan-binding protein-capture material;
(C) detecting a “complex of the protein and lipid-bound glycosaminoglycan” generated by the contact;
(D) converting the “protein having unknown binding ability to glycosaminoglycan” encoded by the nucleic acid, which is determined to have binding property to glycosaminoglycan by the detection step, into “glycosaminoglycan binding property”; Step of selecting as "protein".
(7) The “glycosaminoglycan-binding property” according to (6), wherein the “lipid-bound glycosaminoglycan” is a “solid-phased lipid-bound glycosaminoglycan” bound to a solid phase. Method for screening nucleic acid encoding protein ".
(8) A method for producing a glycosaminoglycan-binding substance, comprising the following steps (a) to (c).
(A) A “sample containing a glycosaminoglycan-binding substance” and a “sample comprising a lipid-bound glycosaminoglycan bound to a solid phase as a glycosaminoglycan-binding substance capturing material” Contacting the “glycosaminoglycan-binding substance” in the sample with the “immobilized lipid-bound glycosaminoglycan” in the sample;
(B) a step of separating “immobilized lipid-bound glycosaminoglycan bound to a glycosaminoglycan-binding substance” by a solid-liquid separation means;
(C) a step of isolating the “glycosaminoglycan-binding substance” from the “immobilized lipid-bound glycosaminoglycan bound to the glycosaminoglycan-binding substance”.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail by embodiments of the present invention.
1. Screening method 1 of the present invention
The screening method 1 of the present invention is a method for screening a glycosaminoglycan-binding substance, which comprises using a lipid-linked glycosaminoglycan as a glycosaminoglycan-binding substance capturing material.
[0011]
Here, the lipid-bound glycosaminoglycan is a substance in which lipid is bound to glycosaminoglycan, preferably a substance in which lipid is chemically bound to glycosaminoglycan. 4-80202 and JP-A-9-30979.
[0012]
Glycosaminoglycans contained in the “lipid-bound glycosaminoglycan” used as a glycosaminoglycan-binding substance-capturing material in the screening method 1 of the present invention include D-glucosamine or D-galactosamine, D-glucuronic acid, It is a polysaccharide composed of a repeating unit of disaccharide of L-iduronic acid and / or D-galactose as a basic skeleton, extracted from natural products such as animals, obtained by culturing microorganisms, and chemically. Alternatively, any of those enzymatically synthesized can be used. Specifically, for example, hyaluronic acid, chondroitin, chondroitin sulfate (so-called chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate E, chondroitin sulfate K, etc.), chondroitin polysulfate, and dermatan sulfate (also commonly referred to as chondroitin sulfate B) ), Heparin, keratan sulfate, keratan polysulfate, etc., but are not limited thereto. These glycosaminoglycans may be commonly used salts.
[0013]
Further, as the lipid to be bound to the glycosaminoglycan, a complex lipid or a simple lipid derived from a natural product such as an animal, a plant, or a microorganism, or chemically or enzymatically synthesized or partially decomposed may be used. And glycerolipids such as phospholipids, long-chain fatty acids, long-chain fatty acid amines, cholesterols, sphingolipids, ceramides, and the like. In particular, glycerolipids such as phosphatidylethanolamine, phosphatidylserine, phosphatidyltreonine, ethanolamine plasmalogen, serine plasmalogen, lysophosphatidylcholine, phospholipids such as lysophosphatidylinositol, monoacylglycerol, and neutral lipids such as diacylglycerol are preferable. . Among these, a phospholipid having a primary amino group is particularly preferred. The chain length and the degree of unsaturation of the acyl group in the lipid are not particularly limited, but those having 6 or more carbon atoms are preferred. Examples of the acyl group include palmitoyl (hexadecanoyl) and stearoyl (octadecanoyl). In addition, these lipids may be commonly used salts.
[0014]
The binding position of the glycosaminoglycan to the lipid is not particularly limited, but in light of the purpose of the present invention of screening for a glycosaminoglycan binding substance, the possibility of preventing the binding of glycosaminoglycan is low. The terminal part of noglycan is preferable, and in particular, the bond to the "reducing terminal of glycosaminoglycan" is preferable from the viewpoint of ease of reaction. The form of the bond is particularly preferably a chemical bond, and among them, a bond by a covalent bond is most preferable.
[0015]
In the case of a lipid-bound glycosaminoglycan in which a glycosaminoglycan and a lipid are covalently bonded, a carboxyl group (including a lactone), a formyl group (including a hemiacetal group), a hydroxyl group or a primary amino group of the glycosaminoglycan, Alternatively, an acid amide bond formed between the group separately introduced into glycosaminoglycan and the carboxyl group, formyl group or primary amino group of lipid, or the group separately introduced into lipid (-CO- NH-), an ester bond or an aminoalkyl bond (-CH₂Those covalently bonded by -NH-) are preferable.
[0016]
In particular, the pyranose ring at the reducing end of glycosaminoglycan is opened, and the carboxyl group (including lactone) of glycosaminoglycan formed by chemical treatment is reacted with the primary amino group of lipid to form. Acid amide bond (—CO—NH—), an acid amide bond (—CO—NH—) formed by the reaction between the carboxyl group of the uronic acid portion of glycosaminoglycan and the primary amino group of the lipid, or The pyranose ring at the reducing end of glycosaminoglycan is opened, and the Schiff base formed by the reaction between the formyl group of glycosaminoglycan formed by chemical treatment and the primary amino group of lipid is reduced. The aminoalkyl bond formed (-CH₂-NH-) is preferred.
[0017]
The amino group, carboxyl group, formyl group (including hemiacetal group), and hydroxyl group involved in the covalent bond are those originally present in glycosaminoglycans or lipids, and are formed by subjecting them to chemical treatment. Or a spacer compound having the above-mentioned functional group at the terminal, which has been separately introduced in advance by reacting with a glycosaminoglycan or a lipid in advance.
[0018]
Particularly preferred methods for producing lipid-bound glycosaminoglycans, which are lipid-bound glycosaminoglycans, in which a lipid is covalently bonded to the reducing end of glycosaminoglycan, include, for example, JP-A-4-80201 and JP-A-4-80202. And a method disclosed in JP-A-9-30979, and more specifically, for example, the following method can be used.
[0019]
Reducing terminal limited oxidation method
This method reduces the pyranose ring at the reducing end by reducing the galactose residue, uronic acid residue, or hexosamine residue, which is the sugar residue at the reducing end of glycosaminoglycan, and subjecting it to limited oxidation (partial oxidation). In addition to ring opening (cleavage), a formyl group is formed at the reducing end of the glycosaminoglycan to form an aldehyde compound, and the formyl group of the aldehyde compound is reacted with a primary amino group of lipid to form a Schiff base. And then reduce the Schiff base to form an aminoalkyl linkage (-CH₂-NH-) to form a covalent bond between glycosaminoglycan and lipid.
[0020]
The reduction of the sugar residue at the reducing end of glycosaminoglycan may be carried out by reducing the amount of the reducing agent (such as sodium borohydride, sodium cyanoborohydride, etc.) to about 5 to 50 equivalents, preferably 25 to 30 equivalents, relative to glycosaminoglycan. The reaction can be carried out in an appropriate aqueous solvent (eg, water, borate buffer, etc.) at 10 to 30 ° C., preferably 15 to 25 ° C., using an aqueous borohydride salt.
[0021]
After the above reduction, limited oxidation is performed to produce an aldehyde compound having a formyl group specifically at the reducing end of glycosaminoglycan. In the limited oxidation, 1 to 10 equivalents, preferably 3 to 6 equivalents, of an oxidizing agent (eg, alkali periodate such as sodium periodate and potassium periodate) is used based on the reduced glycosaminoglycan. , Usually at 0 to 10 ° C, preferably at 0 to 4 ° C.
[0022]
The reaction of reacting the obtained aldehyde compound with a lipid having a primary amino group (such as a phospholipid such as phosphatidylethanolamine) to form a Schiff base can be carried out by using an aqueous solvent (water, phosphate buffer, etc.) or an appropriate solvent. A solution in which the above aldehyde compound is dissolved in a suitable organic solvent (dimethylformamide, dimethylsulfoxide, etc.) and a solution in which the lipid is dissolved in a suitable organic solvent (chloroform, methanol, etc.) are mixed, usually at a temperature of 15 to 60 ° C. Can be reacted. At the time of this reaction or after completion of the reaction, a suitable reducing agent (eg, an alkali salt of borohydride such as sodium borohydride, sodium cyanoborohydride, etc.) is allowed to act to reduce the Schiff base.
[0023]
When producing lipid-bound glycosaminoglycan, instead of a lipid having a primary amino group, a bifunctional spacer compound having a primary amino group (for example, an alkylenediamine such as ethylenediamine, or lysine or the like) ) And the above aldehyde compound to form an aminoalkyl bond (-CH₂-NH-), and then a lipid (e.g., a monoacyl such as a monoacylglycerol succinate) having a functional group (e.g., a carboxyl group) capable of reacting with the other functional group (e.g., an amino group) of the spacer compound. (Glycerol dicarboxylate).
[0024]
Reduction terminal lactonization method
In this method, a pyranose ring at a reducing end is specifically opened (cleaved) by oxidizing a galactose residue, a uronic acid residue, or a hexosamine residue, which is a sugar residue at the reducing end of glycosaminoglycan. To form a carboxyl group at the reducing end of the glycosaminoglycan, followed by a lactone formation reaction to make the reducing end of the glycosaminoglycan into a lactone structure, thereby reacting the lactone with a primary amino group of lipid. In this method, a glycosaminoglycan and a lipid are covalently bonded by forming an acid amide bond (—CO—NH—).
[0025]
Oxidation of the sugar residue at the reducing end of glycosaminoglycan is carried out by using an oxidizing agent (iodine, bromine, etc.) of about 2 to 20 equivalents, preferably about 5 to 15 equivalents to glycosaminoglycan. The reaction can be carried out in an aqueous solvent (eg, water, phosphate buffer, etc.) at a temperature of usually 0 to 40 ° C, preferably 15 to 30 ° C.
[0026]
After the above oxidation reaction, a strongly acidic cation exchange resin, for example, Dowex 50 (trade name, manufactured by Dow Chemical Co., Ltd.), Amberlite IR-120 (trade name, manufactured by Organo Co., Ltd.) and / or acid (inorganic such as hydrochloric acid and sulfuric acid) By treatment with an acid or an acid anhydride of an organic acid such as acetic acid, citric acid, and succinic acid), a lactone compound in which the reducing end of glycosaminoglycan is specifically lactonized can be produced.
[0027]
The reaction between the obtained lactone compound and a lipid having a primary amino group (phospholipid such as phosphatidylethanolamine) is carried out by a suitable aqueous solvent (water, phosphate buffer, etc.) or a suitable organic solvent (dimethylformamide, A solution in which a lactone compound is dissolved in dimethyl sulfoxide) and a solution in which a lipid is dissolved in an appropriate organic solvent (chloroform, methanol, etc.) are mixed and reacted at a temperature of 5 to 80 ° C, preferably 30 to 60 ° C. Just do it.
[0028]
As in the case of the reduction terminal-limited oxidation method, instead of a lipid having a primary amino group, a difunctional spacer compound having a primary amino group is reacted with the lactone compound to form an acid amide bond ( —CO—NH—) to react the other functional group of the spacer compound with a functional group of the lipid (for example, a carboxyl group).
[0029]
Hemiacetal method
The reducing terminal sugar of the sugar chain is in an equilibrium state between a cyclic structure and a ring-opened structure. In the ring-opened structure, an aldehyde group is formed, and when a lipid having an amino group is added thereto, a Schiff base is formed. This reaction is also an equilibrium reaction, and its ratio varies greatly depending on the reaction conditions. When a selective reducing agent is added here, the Schiff base is reduced (hydrogenated) to form an aminoalkyl-type conjugate.
[0030]
Specifically, when glycosaminoglycan is an alkali metal salt such as a sodium salt, the organic solvent-soluble salt can be obtained by the following method. That is, Dowex 50W-X8, Dowex 50W-X12, Dowex 50W-X4 (all manufactured by Muromachi Chemical Co., Ltd.), Amberlite IR-120, Amberlite IRC-50 (all manufactured by Organo), Duolite 225, Duolite C26C (all) H such as Bio-Rex 70, Chelex 100, AG 50W-X8 (all manufactured by Bio-Rad), SE Cellulose (manufactured by Whatman) and the like.⁺The solution is decationized by passing through a cation exchange resin of the type to form a free form. Immediately, an organic amine such as tetrabutylammonium or an aqueous solution thereof is added to adjust the pH from weakly acidic to neutral. This solution is concentrated under reduced pressure, and freeze-dried at room temperature for about 3 days to obtain a glycosaminoglycan organic amine salt dry powder that is soluble in an organic solvent.
[0031]
Then, an organic solvent-soluble salt of glycosaminoglycan and a lipid having a primary amino group are combined with a polar organic solvent capable of dissolving both the organic solvent-soluble salt of glycosaminoglycan and the lipid under anhydrous conditions. And reacting in the presence of a reducing agent to form an aminoalkyl-type bond between the reducing terminal sugar aldehyde group of the glycosaminoglycan and the primary amino group of the lipid, thereby synthesizing a lipid-bound glycosaminoglycan. Specifically, for example, an anhydrous polar organic solvent is dissolved in the above-mentioned organic amine salt dry powder, and the resulting solution is added to a solution of a lipid such as phosphatidylethanolamine in an anhydrous polar organic solvent. After stirring the obtained mixture at 20 to 120 ° C for 1 to 24 hours under a nitrogen atmosphere, a reducing agent is added, and stirring is further continued at 20 to 120 ° C for 1 to 24 hours. The above reducing agent is further added twice every about 24 hours, and reacted at 20 to 120 ° C. for 1 to 72 hours to complete the Schiff base formation and reductive amination reaction.
[0032]
However, a method for producing a lipid-bound glycosaminoglycan in which a lipid is covalently bonded to the reducing end of glycosaminoglycan is not limited to these methods, and a method capable of binding a lipid to the reducing end of glycosaminoglycan. Then, it can be manufactured by any other method.
[0033]
As a method for introducing a lipid to a position other than the terminal of glycosaminoglycan, for example, a carboxyl group of a uronic acid portion of glycosaminoglycan is reacted with a primary amino group of lipid to form an acid amide bond (—CO—NH— ) Is formed.
[0034]
In the above reaction, an acid amide bond (—CO—NH—) is formed using a condensing agent (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, dicyclohexylcarbodiimide, or the like), or a uronic acid moiety. Is reacted with an activator (for example, N-hydroxysuccinimide, p-nitrophenol, N-hydroxybenzotriazole, etc.) in the presence of the condensing agent to form an active ester, and then reacted with the lipid. An acid amide bond (—CO—NH—) can be formed.
[0035]
In the above reaction, the uronic acid portion of the glycosaminoglycan is converted to a salt that can be dissolved in an organic solvent (e.g., an amine salt such as triethylamine and tributylamine), and the reaction is performed in an organic solvent (dimethylformamide, dimethylsulfoxide, pyridine, etc.). It is preferably carried out in
[0036]
Examples of lipid-bound glycosaminoglycan salts include alkali metal salts such as sodium and potassium; alkaline earth metal salts such as calcium and magnesium; amine salts such as trialkylamines; and salts with organic bases such as pyridine. Although not particularly limited, an alkali metal salt is particularly preferred, and a sodium salt is most preferred.
[0037]
Specific examples of the lipid-linked glycosaminoglycans as described above include dipalmitoyl-L- (α-phosphatidyl) ethanolamine-bonded hyaluronic acid, dipalmitoyl-L- (α-phosphatidyl) ethanolamine-bonded chondroitin sulfate, and stearoyl palmitoyl Phosphatidylserine-bound chondroitin sulfate, monostearoylglycerol / succinate-bound chondroitin sulfate, dipalmitoyl-L- (α-phosphatidyl) ethanolamine-bound hyaluronic acid, and the like, and more specific examples of these lipid-bound glycosaminoglycans Details of the structure, the manufacturing method, and the like are described in JP-A-4-80201, JP-A-4-80202, and JP-A-9-30979.
[0038]
More specifically, the screening method 1 of the present invention has at least the following steps (1) and (2).
(1) a step of bringing a “glycosaminoglycan-binding substance” in a specimen into contact with “lipid-bound glycosaminoglycan” as a glycosaminoglycan-binding substance capturing material;
(2) Means for detecting “complex of glycosaminoglycan-binding substance and lipid-bound glycosaminoglycan” generated by the contact.
[0039]
That is, the substance in the sample having affinity with the `` glycosaminoglycan part '' of the `` lipid-bound glycosaminoglycan '' is bound to form a complex, and the presence or absence of the complex is confirmed. It is a feature of the screening method 1 of the present invention that it is concluded that the sample in which the complex was formed and the complex was detected contained a glycosaminoglycan-binding substance.
[0040]
The "glycosaminoglycan-binding substance" screened by the screening method 1 of the present invention is not particularly limited, but is preferably a protein.
[0041]
The “sample” in the above step (1) is preferably a liquid sample, and more preferably a sample containing water. Glycosaminoglycan is a biological substance, and it is considered that a substance having a binding property to the substance often exhibits a binding property in the presence of water. Specific examples include a specimen such as a culture solution of cells and tissues, and a specimen extracted from a living body such as blood, urine, and tears, and are not particularly limited, but a culture solution of cells and tissues is preferable.
[0042]
In the screening method 1 of the present invention, “contacting” in the above step (1) may include adding a lipid-bound glycosaminoglycan to a sample, but preferably, a lipid-bound glycosaminoglycan fixed to a solid phase and a sample. For example, the lipid-bound glycosaminoglycan bound to a carrier such as a gel is mixed with the carrier together with the specimen, the specimen is passed through a column filled with the carrier, or a microplate or the like. A lipid-bound glycosaminoglycan is immobilized on the sample, and the sample is dispensed there.
[0043]
The “detection of a complex of a glycosaminoglycan-binding substance and a lipid-bound glycosaminoglycan” in the above step (2) in the screening method 1 of the present invention includes, for example, the step of binding to the lipid portion of the lipid-bound glycosaminoglycan. A method of visualization using an antibody or the like, or a method of detection by absorption of ultraviolet light or the like, and the like. When using a method in which lipid-bound glycosaminoglycan is fixed to a solid phase, it is possible to detect the labeled substance fixed to the solid phase and perform the above detection by labeling the substance in the sample in advance. It becomes possible. Such labeling can be performed by a known method using a known substance.
[0044]
2. The screening method 2 of the present invention
The screening method 2 of the present invention is a method for screening a nucleic acid encoding a glycosaminoglycan-binding protein, which comprises at least all of the following steps (1) to (4).
(1) a step of expressing the protein in a cell using a nucleic acid encoding “a protein whose binding property to glycosaminoglycan is unknown” to obtain a specimen containing the protein;
(2) a step of bringing the protein in a sample into contact with “lipid-bound glycosaminoglycan” as a glycosaminoglycan-binding protein capturing material;
(3) detecting a “complex of the protein and lipid-bound glycosaminoglycan” generated by the contact;
(4) The “glycosaminoglycan binding property” of the “protein having unknown binding property to glycosaminoglycan” encoded by the nucleic acid, which is determined to have binding property to glycosaminoglycan by the detection step. Step of selecting as "protein".
[0045]
In the screening method 2 of the present invention, “glycosaminoglycan”, “lipid-bound glycosaminoglycan”, and “lipid” in the “lipid-bound glycosaminoglycan” are all the same as the words in the screening method 1 of the present invention. .
[0046]
In the screening method 2 of the present invention, the “nucleic acid” in the above step (1) may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), but is DNA in terms of easy handling. Is preferred. Such a nucleic acid can be prepared according to a conventional method. Although cDNA is prepared from mRNA generated upon expression of DNA, it has a structure that does not contain introns and contains the genetic code of the encoded protein in a form that can be easily expressed. Preferably, cDNA is used.
[0047]
Expression of the “protein having unknown binding property to glycosaminoglycan” encoded by the nucleic acid can be easily performed by those skilled in the art according to a conventional method. For example, only the coding region of such a nucleic acid is amplified, the amplified product is inserted into a vector suitable for the host cell used for expression to prepare an expression vector, and the expression vector is introduced into the host cell to obtain a recombinant. The expression can be carried out as follows. Further, when constructing a vector, by using a vector constructed so as to express the above-mentioned "protein having unknown binding ability to glycosaminoglycan" as a fusion protein with a labeled peptide in advance, the fusion protein and lipid It is more preferable because the complex with the bound glycosaminoglycan can be easily detected.
[0048]
Examples of such "identification peptides" include, for example, a signal peptide (15 to 30 amino acid residues present at the N-terminus of many proteins and functioning in the cell for the purpose of protein selection in the intracellular membrane permeation mechanism) A peptide consisting of, for example, OmpA, OmpT, Dsb, etc.), protein kinase A, protein A (a component of the cell wall of S. aureus having a molecular weight of about 42,000), glutathione S-transferase, His tag (histidine residue of 6 To 10), myc tag (13 amino acid sequence derived from cMyc protein), FLAG peptide (analytical marker consisting of 8 amino acid residues), T7 tag (comprising the first 11 amino acid residues of gene10 protein) , S-tag (consisting of 15 amino acid residues from pancreatic RNaseA) ), HSV tag, pelB (22 amino acid sequence of E. coli outer membrane protein pelB), HA tag (consisting of 10 amino acid residues derived from hemagglutinin), Trx tag (thioredoxin sequence), CBP tag (calmodulin binding peptide), CBD tag ( Cellulose-binding domain), CBR tag (collagen-binding domain), β-lac / blu (β-lactamase), β-gal (β-galactosidase), luc (luciferase), HP-Thio (His-patch thioredoxin), HSP (heat shock) Peptide), Lnγ (laminin γ peptide), Fn (fibronectin partial peptide), GFP (green fluorescent peptide), YFP (yellow fluorescent peptide), CFP (cyan fluorescent peptide), BFP (blue fluorescent peptide), DsRed, DsRe 2 (red fluorescent peptide), MBP (maltose binding peptide), LacZ (lactose operator), IgG (immunoglobulin G), avidin, protein G and the like, and any discriminating peptide can be used. It is possible. Among them, especially signal peptide, protein kinase A, protein A, glutathione S transferase, His tag, myc tag, FLAG peptide, T7 tag, S tag, HSV tag, GFP, YFP, CFP, BFP, DsRed, DsRed3, pelB or The HA tag is preferable because the detection of the complex of the protein and the lipid-bound glycosaminoglycan in the screening method 2 of the present invention by the genetic engineering technique becomes easier. Particularly, GFP, YFP, CFP, BFP, DsRed, and DsRed3 are preferable. It is preferable to express "a protein whose binding property to glycosaminoglycan is unknown" as a fusion protein with a fluorescent peptide such as described above because of its excellent ease of detection.
[0049]
The binding position of the “labeled peptide” to the “protein having unknown binding property to glycosaminoglycan” does not prevent the “labeled peptide” from binding to the protein and the “lipid-bound glycosaminoglycan”. As far as it is, it may be C-terminal or N-terminal.
[0050]
The host cell used in such a step may be a prokaryotic cell (eg, Bacillus subtilis, Escherichia coli, etc.) or a eukaryotic cell (eg, yeast, a cultured cell derived from a mammal, a cultured cell derived from an insect, etc.). There may be. Those skilled in the art can easily select these host cells according to the purpose, and can easily select a vector suitable for the host cells.
[0051]
Further, the insertion of a nucleic acid encoding "a protein whose binding property to glycosaminoglycan is unknown" into the above-mentioned vector can also be carried out by forming an appropriate restriction enzyme end or the like at the end of the nucleic acid according to the structure of the vector. It is possible to perform according to.
[0052]
The “contact” in the above step (2) in the screening method 2 of the present invention refers to, for example, a culture solution containing “a protein whose binding property to glycosaminoglycan is unknown” expressed in a cell in the step (1). The test can be performed by adding lipid-bound glycosaminoglycan as a glycosaminoglycan-binding protein-capturing material as a sample, or by adding the above culture solution or the like to a solution of lipid-bound glycosaminoglycan or the like. You can also. However, it is preferably carried out by contacting the above-mentioned culture solution or the like with the lipid-bound glycosaminoglycan fixed to the solid phase.For example, the lipid-bound glycosaminoglycan bound to a carrier such as a gel is added to the culture solution together with the carrier. It can be performed by mixing and suspending, passing the culture solution through a column filled with the above carrier, or fixing lipid-linked glycosaminoglycan on a microplate or the like, and dispensing the culture solution there. it can.
[0053]
The “detection of a complex of a protein and a lipid-bound glycosaminoglycan” in the step (3) in the screening method 2 of the present invention is performed when the fusion protein with the labeled peptide is expressed in the step (1). The detection can be easily performed by detecting the labeled peptide. For example, a set in which "a nucleic acid encoding a protein whose binding to glycosaminoglycan is unknown" is introduced into a vector expressing the inserted gene as a fusion protein with "GFP", and the gene is introduced by the vector. When the fusion protein is expressed in step (1) using the recombinant, "detection" in step (3) can be performed by detecting fluorescence.
[0054]
The step (4) in the screening method 2 of the present invention is performed when the “complex of a protein having unknown binding property to glycosaminoglycan and a lipid-bound glycosaminoglycan” is detected in the step (3). Means that the “protein whose binding property to glycosaminoglycan is unknown” has binding property to glycosaminoglycan, and the nucleic acid encoding the protein has “binding property to glycosaminoglycan. It may be a nucleic acid encoding a "protein having".
[0055]
3. Production method of the present invention
The production method of the present invention is a “method of producing a glycosaminoglycan-binding substance”, which includes all of the following steps (1) to (3).
(1) The “material containing a glycosaminoglycan-binding substance” is brought into contact with “a solid-phased lipid-bound glycosaminoglycan as a glycosaminoglycan-binding substance-capturing material”, and the A step of binding the “glycosaminoglycan-binding substance” to the “immobilized lipid-bound glycosaminoglycan”;
(2) a step of separating “immobilized lipid-bound glycosaminoglycan bound to a glycosaminoglycan-binding substance” by a solid-liquid separation means;
(3) A step of isolating the “glycosaminoglycan-binding substance” from the “immobilized lipid-bound glycosaminoglycan bound to the glycosaminoglycan-binding substance”.
[0056]
In the production method of the present invention, “glycosaminoglycan”, “lipid-bound glycosaminoglycan”, and “lipid” in the above “lipid-bound glycosaminoglycan” are all synonymous with the terms in the screening method 1 of the present invention.
[0057]
The solid phase in step (1) in the method of the present invention is exemplified by a carrier such as a gel (eg, agarose) or a microplate. In particular, a gel is preferable in terms of facilitating the operation of the method of the present invention, but is not limited thereto.
[0058]
The “sample” in the step (1) of the production method of the present invention is not particularly limited as long as it contains a glycosaminoglycan-binding substance. For example, it is known that the binding property to glycosaminoglycan is known. Culture supernatants of cultured cells that secrete proteins (fibroblast growth factor (FGF), hepatocyte growth factor (HGF), hyaluronic acid binding protein (HABP), midkine (MK), etc.) You. In addition, “a nucleic acid encoding a protein having a binding property to glycosaminoglycan” obtained by the above-described screening method 2 of the present invention is introduced, and a “protein having a binding property to a glycosaminoglycan” encoded by the nucleic acid is introduced. It is needless to say that even a recombinant supernatant prepared so as to express large amounts of "" can be used.
[0059]
In addition, it can be easily understood by those skilled in the art that the "glycosaminoglycan-binding substance" obtained by the production method of the present invention differs depending on the type of glycosaminoglycan used for preparing the lipid-bound glycosaminoglycan. By the way, those skilled in the art can appropriately select a lipid-bound glycosaminoglycan to be used as a glycosaminoglycan-binding substance capturing material according to the glycosaminoglycan-binding property of a substance to be obtained.
[0060]
The “contact” in the step (1) in the production method of the present invention refers to, for example, a column in which a lipid-bound glycosaminoglycan bound to a gel carrier is mixed and suspended in a culture solution together with the carrier, or a column filled with the gel carrier. The culture can be performed by passing the culture solution through a tube, or fixing the lipid-bound glycosaminoglycan on a microplate or the like, and dispensing the culture solution there.
[0061]
The solid-liquid separation means in step (2) in the production method of the present invention can use a conventional method. For example, filtration, centrifugation and the like may be performed according to the solid phase on which the lipid-bound glycosaminoglycan is immobilized. Can be appropriately selected. When the lipid-bound glycosaminoglycan is fixed to the microplate, the solid phase is extremely recovered by separating the microplate and the solution, preferably by subjecting the solution to solid-liquid separation means such as washing. It can be easily performed and is preferable, but not limited thereto.
[0062]
The “elution of a glycosaminoglycan-binding substance from the solid phase” in the step (3) in the production method of the present invention can be performed using, for example, a change in pH, a change in salt concentration, or the like. Elution can be easily carried out by using a change in salt concentration, which is a technique preferably used for ordinary affinity columns.
[0063]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
Preparation Example 1: Preparation of lipid-bound glycosaminoglycan
Heparin (derived from pig small intestine: Scientific Protein Laboratories), heparan sulfate (derived from cow kidney: manufactured by Seikagaku Corporation), hyaluronic acid (derived from cockscomb: manufactured by Seikagaku Corporation), chondroitin sulfate A (whale cartilage) Origin: Seikagaku Corporation; Dermatan Sulfuricide (from chicken crown: Seikagaku Corporation); Chondroitin Sulfate C (Shark Cartilage: Seikagaku Corporation); Chondroitin Sulfate D (Shark Cartilage: Biochemistry) Lipid-bound glycosaminoglycans were prepared using Chondroitin Sulfate E (from Squid Cartilage; manufactured by Seikagaku Corporation). Phosphatidylethanolamine was used as a lipid. Specifically, synthesis was performed by the following method.
[0064]
According to the method described in Example 1 of JP-A-4-80201, a lipid-bound glycosaminoglycan was prepared, for example, as follows.
[0065]
500 mg of hyaluronic acid (sodium salt: derived from cockscomb, molecular weight: about 23,000, HA) was dissolved in 10 ml of water, 5 ml of a 0.1 mol / l iodine methanol solution was added, and the mixture was reacted at room temperature for 6 hours. Thereafter, about 5 ml of 0.1 mol / l potassium hydroxide was added to the reaction solution to eliminate the color of free iodine. To this solution was added potassium acetate-saturated ethanol, and the resulting precipitate was collected by filtration, sufficiently washed with ethanol, and dried under reduced pressure to obtain 423 mg of reduced terminal oxidized hyaluronic acid (sodium salt) having a weight average molecular weight of about 23,000. .
[0066]
400 mg of the above-mentioned reduced terminal oxidized hyaluronic acid is dissolved in 10 ml of water, and 50 ml of a strongly acidic ion exchange resin (Dowex 50 (H⁺)) Over 1 hour to obtain an aqueous solution containing 390 mg of reduced terminal lactone hyaluronic acid.
[0067]
The above aqueous solution was neutralized in tri-n-butylamine and freeze-dried to obtain 400 mg of a tri-n-butylamine salt of reduced terminal lactone hyaluronic acid.
[0068]
400 mg of the above-prepared reduced-terminal lactone hyaluronic acid tri-n-butylamine salt was dissolved in 200 ml of dimethylformamide, and 27.6 mg of L- (α-phosphatidyl) ethanolamine / dipalmitoyl solution in chloroform was added thereto. The mixture was reacted at 2 ° C. for 2 hours, chloroform was distilled off, and an excess sodium acetate aqueous solution was added to make a sodium salt, and then sodium acetate-saturated ethanol was added. The resulting precipitate was collected by filtration, dissolved in a 0.3 mol / l ammonium acetate solution, adsorbed on a hydrophobic chromatography column (TSKgel Phenyl Toyopearl 650M, 400 ml), and thoroughly washed with a 0.3 mol / l aqueous ammonium acetate solution. The desired substance was eluted with a 30% aqueous methanol solution. The fraction eluted with a 30% aqueous methanol solution was concentrated under reduced pressure, freeze-dried and purified to obtain 36 mg of L- (α-phosphatidyl) ethanolamine / dipalmitoyl-bound hyaluronic acid (hereinafter abbreviated as “HA-PE”). Was.
[0069]
Similarly, according to the method described in Example 1 of JP-A-4-80201, L- (α-phosphatidyl) ethanolamine / dipalmitoyl-bonded chondroitin sulfate A (hereinafter abbreviated as “CSA-PE”), L- (α -Phosphatidyl) ethanolamine / dipalmitoyl-bonded chondroitin sulfate C (hereinafter abbreviated as “CSC-PE”), L- (α-phosphatidyl) ethanolamine / dipalmitoyl-bonded dermatan sulfate (hereinafter abbreviated as “DS-PE”) , L- (α-phosphatidyl) ethanolamine / dipalmitoyl-bonded chondroitin sulfate D (hereinafter abbreviated as “CSD-PE”), L- (α-phosphatidyl) ethanolamine / dipalmitoyl-bonded chondroitin sulfate E (hereinafter “CHE- PE "), L- (α-phosphatidyl) Ethanolamine, dipalmitoyl binding heparin (hereinafter abbreviated as "Hep-PE"), (hereinafter abbreviated as "HS-PE") L-(alpha-phosphatidyl) ethanolamine, dipalmitoyl bound heparan sulfate were synthesized.
[0070]
Preparation Example 2: Preparation of plate bound with lipid-bound glycosaminoglycan
A plate to which each lipid-bound glycosaminoglycan prepared in Preparation Example 1 was bound was prepared. That is, a solution prepared by dissolving the lipid-bound glycosaminoglycan in PBS at a concentration of 20 μg / ml was added to each well of an EasyView glass-bottom 96-well plate (manufactured by Iwaki Glass Co., Ltd.) in an amount of 50 μl, and this was added at 4 ° C. Incubated for 10 hours. Thereafter, the plate was washed twice with PBS (-), and the plate was blocked with PBS (-) containing bovine serum albumin (hereinafter also referred to as "BSA") at 1%, and each lipid-bound glycosamino. Glycan fixed plates were prepared.
[0071]
Preparation Example 3: Construction of Expression Vector
A plasmid for expressing the protein was created.
(1) Preparation of pCIkana
pEGFP-N1 (manufactured by Clontech) was cut at the NdeI site and NotI site, and a fragment containing the neomycin / kanamycin resistance gene was purified. In addition, pCIneo (Promega) was cut at the NdeI site and NotI site, and the fragment containing the intron was purified. The fragment containing this intron was inserted between the NdeI site and the NotI site of the fragment containing the neomycin / kanamycin resistance gene of pEGFP-N1, to obtain a plasmid pCIkana.
[0072]
(2) Preparation of pCIkana-hrGFP2xMyc
In order to amplify hrGFP cDNA, pIRES-hrGFP-2a (manufactured by Stratagene) was used as a template, and a 5 ′ primer consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a 3 ′ primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 were used. To perform polymerase chain reaction (hereinafter also referred to as “PCR”).
[0073]
The 5 'primer contains a GTG sequence one codon 3' from the translation initiation codon of hrGFP cDNA, and further has a multiple cloning site on the 5 'side. In addition, the 3 ′ primer is a sequence encoding two myc tags consisting of 19 bases and 10 amino acids including a GTG sequence located one codon upstream from the stop codon of hrGFP cDNA in tandem, and A PspOMI site for incorporating a gene fragment into plasmid pCIkana is arranged in tandem.
[0074]
PCR using the above primers was carried out using an Expand high-fidelity PCR system (manufactured by Roche) according to the protocol. The amplified fragment obtained by this PCR was inserted into pCR 2.1-TOPO (manufactured by Invitrogen), and Escherichia coli (One ShotTOP10 Chemically competent E. coli, manufactured by Invitrogen) was transformed. From the transformed Escherichia coli, pCR2.1-TOPO into which the amplified fragment was inserted was extracted, cut with restriction enzymes EcoRI and PspOMI, and a fragment containing cDNA in which hrGFP and two Myc tags were linked was purified. This DNA fragment was inserted between the EcoRI site and the NotI site of the plasmid pCIkana obtained in the above (1) according to a conventional method to obtain a plasmid pCIkana-hrGFP2xMyc.
[0075]
(3) Preparation of pCIkanaEGFP3xMyc
In order to amplify the EGFP cDNA, PCR was performed using pEGFP-N1 as a template and the 5 'primer shown in SEQ ID NO: 3 and the 3' primer shown in SEQ ID NO: 4.
[0076]
The 5 ′ primer contains a GTG sequence which is one codon 3 ′ from the translation initiation codon of the EGFP cDNA, and a BstBI site for integration into the plasmid pCIkana-hrGFP2xMyc obtained in (2) above is located on the 5 ′ side. It is arranged. The 3 'primer contains a CTG sequence one codon upstream of the stop codon of the EGFP cDNA, and further encodes a myc tag consisting of 10 amino acids, and the 3' primer obtained in the above (2). The BstBI site for incorporation into pCIkana-hrGFP2xMyc was arranged in tandem.
[0077]
PCR using the above primers was performed according to the protocol using Expand high-fidelity PCR system (manufactured by Roche). The amplified fragment obtained by the PCR was inserted into pCR 2.1-TOPO (manufactured by Invitrogen), and Escherichia coli (One Shot TOP10 Chemically competent E. coli, manufactured by Invitrogen) was transformed. A DNA fragment containing EGFP cDNA was purified from the transformed E. coli. This fragment was inserted between the two BstBI sites of the above-mentioned pCIkana-hrGFP2xMyc to prepare a plasmid pCIkanaEGFP3xMyc.
[0078]
The above plasmid pCIkanaEGFP3xMyc is a plasmid consisting of 5122 base pairs, pUCori, SV40 promoter, and Kan.^r/ Neo^r, HSTK polyA, CMV promoter, intron, multiple cloning site, EGFP, 3xMyc tag, SV40 polyA. The pUCori is a sequence necessary for replicating the plasmid. Kan above^r/ Neo^rIs the kanamycin / neomycin resistance gene. The SV40 promoter is a promoter sequence for expressing a kanamycin / neomycin resistance gene.
[0079]
The multi-cloning site is a sequence having a plurality of restriction enzyme regions, and is used for inserting a gene or cDNA to be expressed. The restriction enzyme regions arranged at the multiple cloning site are NheI, XhoI, EcoRI, SalI, BglII, NotI, and BstBI in order from the 5 'side.
[0080]
The CMV promoter is a promoter sequence for expressing a gene or cDNA inserted into a multicloning site. The above-mentioned EGFP is a gene encoding the fluorescently labeled protein GFP. The 3xMyc tag has three consecutive base sequences encoding the Myc tag as a labeling protein, and a stop codon is provided in a downstream region of the 3xMyc tag. Therefore, when the gene inserted into the multi-cloning site is expressed, the protein is produced in a form in which EGFP and the 3xMyc tag are fused in the downstream region of the C-terminal of the protein.
[0081]
Example
Screening of DNA encoding glycosaminoglycan binding protein
(1) Construction of vector and preparation of recombinant
Using a C57BL / 6J mouse (10-day-old fetus), a cDNA library was prepared according to a conventional method. From this cDNA library, GenBank accession numbers AK01323 (hereinafter referred to as “clone 1”) and AK012169 (hereinafter referred to as “clone 2”) were amplified by polymerase chain reaction (PCR). SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: 7 and SEQ ID NO: 8 were used as combinations of 5 'and 3' primers for amplifying each. The reaction conditions of the PCR method for amplifying these were as follows: a cycle of 94 ° C. for 3 minutes, a cycle of 94 ° C. for 30 seconds, 60 ° C. for 60 seconds and 72 ° C. for 4 minutes was repeated 30 times. The obtained PCR product was collected according to a conventional method, and digested with restriction enzymes BglII and NotI, respectively.
[0082]
The PCR product thus treated was purified using a Multiscreen-PCR 96-well plate (manufactured by Millipore) and digested with the restriction enzymes BglII and NotI to obtain a fragment between the BglII and NotI regions of the plasmid pCIkanaEGFP3xMyc (see Preparation Example 3). To prepare recombinant vectors expressing each cDNA.
[0083]
Using this recombinant vector, One Shot TOP10 Chemically competent E.C. coli. (Invitrogen) was transformed according to the instructions. The transformed Escherichia coli was cultured and propagated, the introduced expression vector was amplified, extracted, purified, and introduced into human 239T cells using FuGene6 (Roche) to obtain a recombinant.
[0084]
0.3% (CO 2₂/ Air) at 37 ° C. in a DMEM medium (containing 10% FCS, 5 μg / ml penicillin, and 5 μg / ml streptomycin), and collected the culture supernatant. After centrifugation at 3,000 × g, only the supernatant was collected and used as a sample for screening.
[0085]
(2) Screening of samples using plates
After the plate prepared in Preparation Example 2 was washed twice with PBS (-), the sample prepared in (1) was added in an amount of 50 µl each, and incubated at 4 ° C for 10 hours. After the incubation, the plate was washed three times with PBS (-), and the "complex of each clone and GFP fluorescent protein" fixed on the plate was measured using Fluoroscent (Dainippon Pharmaceutical Co., Ltd.) according to the instruction manual. . As a negative control, the extracellular secretory GFP protein gene was expressed, and the measured value obtained by adding the expression product to the plate was used, and the measured value of the negative control was compared with the measured value of each sample (clone 1). : FIG. 1, clone 2: FIG. 2). As a control, an experimental group to which BSA used for blocking was fixed without using various lipid-bound glycosaminoglycans was used.
[0086]
As a result, it was found that clone 1 exhibited strong binding to hyaluronic acid. Clone 2 was found to exhibit binding properties to chondroitin sulfate C (chondroitin sulfate derived from shark cartilage), and was also found to have some binding property to hyaluronic acid.
[0087]
From these results, it was clarified that the use of the screening method of the present invention can efficiently detect a DNA encoding a protein having a binding property to glycosaminoglycan.
[0088]
[Sequence list]

[0089]
【The invention's effect】
According to the present invention, there is provided a "screening method for a glycosaminoglycan-binding substance" using a "lipid-bound glycosaminoglycan" in which a lipid is bound to glycosaminoglycan as a glycosaminoglycan-binding substance capturing material. This makes it possible to easily screen for a substance having glycosaminoglycan binding properties.
[Brief description of the drawings]
FIG. 1 shows the binding of the expression product of clone 1 to glycosaminoglycans. In the figure, Hep-PE, HS-PE, HA-PE, CSA-PE, DS-PE, CSC-PE, CSD-PE, CSE-PE, and BSA are the same as the abbreviations described in the examples.
FIG. 2 is a graph showing the binding of the expression product of clone 2 to glycosaminoglycan. In the figure, Hep-PE, HS-PE, HA-PE, CSA-PE, DS-PE, CSC-PE, CSD-PE, CSE-PE, and BSA are the same as the abbreviations described in the examples.

Claims (8)

A "screening method for a glycosaminoglycan-binding substance", comprising using a "lipid-bound glycosaminoglycan" in which a lipid is bound to glycosaminoglycan as a glycosaminoglycan-binding substance-trapping material. 2. The "screening method for glycosaminoglycan-binding substances" according to claim 1, comprising at least the following steps (1) and (2).
(1) a step of bringing a “glycosaminoglycan-binding substance” in a specimen into contact with “lipid-bound glycosaminoglycan” as a glycosaminoglycan-binding substance capturing material;
(2) a step of detecting a “complex of a glycosaminoglycan-binding substance and a lipid-bound glycosaminoglycan” generated by the contact; The "screening method for a glycosaminoglycan-binding substance" according to claim 1 or 2, wherein the "glycosaminoglycan-binding substance" is a protein. The "screening method for glycosaminoglycan-binding substances" according to any one of claims 1 to 3, wherein the "sample" is a "sample derived from a living body". The "glycosaminoglycan" according to any one of claims 1 to 4, wherein the "lipid-bound glycosaminoglycan" is "a solid-phased lipid-bound glycosaminoglycan" in a state of being bound to a solid phase. Screening method for glycan binding substance ". A "screening method for a nucleic acid encoding a glycosaminoglycan-binding protein", comprising at least all of the following steps (1) to (4):
(1) a step of expressing the protein in a cell using a nucleic acid encoding “a protein whose binding property to glycosaminoglycan is unknown” to obtain a specimen containing the protein;
(2) a step of bringing the protein in a sample into contact with “lipid-bound glycosaminoglycan” as a glycosaminoglycan-binding protein capturing material;
(3) detecting a “complex of the protein and lipid-bound glycosaminoglycan” generated by the contact;
(4) The “protein having unknown binding property to glycosaminoglycan” encoded by the nucleic acid, which is determined to have binding property to glycosaminoglycan by the detection step, is referred to as “glycosaminoglycan binding property”. Step of selecting as "protein". The "lipid-bound glycosaminoglycan" is a "solid-phased lipid-bound glycosaminoglycan" in a state of being bound to a solid phase, wherein the "glycosaminoglycan-binding protein" is coded. Screening method for nucleic acid to be used ". "A method for producing a glycosaminoglycan-binding substance", comprising the following steps (1) to (3).
(1) A “sample containing a glycosaminoglycan-binding substance” and a “sample containing a lipid-bound glycosaminoglycan bound to a solid phase as a glycosaminoglycan-binding substance capturing material” Contacting the “glycosaminoglycan-binding substance” in the sample with the “immobilized lipid-bound glycosaminoglycan” in the sample;
(2) a step of separating “immobilized lipid-bound glycosaminoglycan bound to a glycosaminoglycan-binding substance” by a solid-liquid separation means;
(3) A step of isolating the “glycosaminoglycan-binding substance” from the “immobilized lipid-bound glycosaminoglycan bound to the glycosaminoglycan-binding substance”.

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