JPH1080281A - New protein and its production - Google Patents
New protein and its productionInfo
- Publication number
- JPH1080281A JPH1080281A JP8257644A JP25764496A JPH1080281A JP H1080281 A JPH1080281 A JP H1080281A JP 8257644 A JP8257644 A JP 8257644A JP 25764496 A JP25764496 A JP 25764496A JP H1080281 A JPH1080281 A JP H1080281A
- Authority
- JP
- Japan
- Prior art keywords
- protein
- kda
- seq
- amino acid
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、人乳に由来するシ
ステインプロテアーゼ阻害活性を有する新規蛋白質及び
その製造方法に関する。本発明の蛋白質は、その活性よ
り、抗体産生促進効果及び破骨細胞の骨吸収作用に対す
る阻害効果を有し、抗ウイルス剤、抗アレルギー剤、あ
るいは骨粗鬆症などの骨疾患の予防及び／又は治療剤と
して有用である。TECHNICAL FIELD The present invention relates to a novel protein derived from human milk and having a cysteine protease inhibitory activity, and a method for producing the same. Due to its activity, the protein of the present invention has an antibody production promoting effect and an inhibitory effect on bone resorption of osteoclasts, and is an antiviral agent, an antiallergic agent, or an agent for preventing and / or treating bone diseases such as osteoporosis. Useful as
【０００２】[0002]
【従来の技術】システインプロテアーゼ阻害剤は、活性
中心にＳＨ基を持ったシステインプロテアーゼの蛋白質
分解活性を阻害する物質であり、動物組織、細胞、血液
中や尿中に見出されている。このようなシステインプロ
テアーゼ阻害剤であって蛋白性の物質はシスタチンと総
称されている。これまでに見出されたシスタチンは、そ
の分子構造から３つのファミリーに分類されている（Bi
ochem.J., 236, 312 (1986))。この文献によれば、ファ
ミリー１（ステフィンファミリー）にはラット肝臓由来
のシスタチンβ（Biochem. Biophys. Res. Commun., 11
5, 902 (1983))、ラット表皮由来のシスタチンα（Bioc
hem. Biophys. Res. Commun., 121, 149 (1984))やヒト
の白血球に見出されたステフィンＡ(Hoppe-Seyler's Z.
Physiol.Chem.,364,1487 (1983))が含まれている。こ
れらのシスタチンはいずれも分子量約12kDa を示し、糖
を持たない。又、ファミリー２にはヒト尿由来のシスタ
チンＣ(Proc. Natl. Acad. Sci. U.S.A.79, 3024 (198
2))、卵白シスタチン(Hoppe-Seyler's Z. Physiol. Che
m.,364, 1487 (1983)) が分類されており、またウシ初
乳由来のシスタチン(FEBS Lett., 186, 41 (1985))もこ
のファミリーに属する。このファミリーに属するものは
分子量約13〜15KDa を示し、ファミリー１と同様に糖を
有していない。さらに、血漿蛋白質であるヒトキニノー
ゲン (Biochemistry, 23, 5691 (1984))、ラットのＴキ
ニノーゲン（Biochem. Biophys. Res. Commun., 129, 2
80 (1985))など、キニノーゲン類がファミリー３に属し
ている。キニノーゲン類は分子量 78KDaの高分子キニノ
ーゲンと分子量 45kDaの低分子キニノーゲンが存在する
ことが知られている。本発明者らは既に、牛初乳由来の
糖鎖を有する分子量約57kDa 及び16±2 kDa又は13±2 k
Da の新規な２種のシスティンプロテアーゼ阻害物質を
単離した。そして、牛初乳由来の糖鎖を有する分子量約
57kDa の新規なシステインプロテアーゼ阻害剤（特開平
7-2896号公報）、及び、牛初乳由来の分子量16±2kDa又
は13±2kDaの新規なシステインプロテアーゼ阻害剤（特
開平7-126294号公報）を見出している。2. Description of the Related Art Cysteine protease inhibitors are substances that inhibit the proteolytic activity of cysteine proteases having an SH group at the active center, and have been found in animal tissues, cells, blood and urine. Such cysteine protease inhibitors and proteinaceous substances are collectively referred to as cystatins. Cystatins found so far have been classified into three families based on their molecular structures (Bi
ochem. J., 236, 312 (1986)). According to this document, family 1 (the stefin family) includes cystatin β derived from rat liver (Biochem. Biophys. Res. Commun., 11) .
5, 902 (1983)), cystatin α derived from rat epidermis (Bioc
Chem. Biophys. Res. Commun., 121, 149 (1984)) and Stefin A (Hoppe-Seyler's Z. et al.) found in human leukocytes.
Physiol. Chem., 364, 1487 (1983)). Each of these cystatins has a molecular weight of about 12 kDa and has no sugar. Family 2 includes cystatin C derived from human urine (Proc. Natl. Acad. Sci. USA 79 , 3024 (198
2)), egg white cystatin (Hoppe-Seyler's Z. Physiol.
m., 364, 1487 (1983)), and cystatin from bovine colostrum (FEBS Lett., 186, 41 (1985)) also belongs to this family. Members belonging to this family have a molecular weight of about 13 to 15 KDa and do not have sugars as in Family 1. Furthermore, human kininogen which is a plasma protein (Biochemistry, 23 , 5691 (1984)) and rat T kininogen (Biochem. Biophys. Res. Commun., 129, 2)
80 (1985)), and kininogens belong to Family 3. It is known that kininogens include a high molecular weight kininogen having a molecular weight of 78 kDa and a low molecular weight kininogen having a molecular weight of 45 kDa. The present inventors have already found that the sugar chain derived from bovine colostrum has a molecular weight of about 57 kDa and 16 ± 2 kDa or 13 ± 2 kDa.
Two new cysteine protease inhibitors of Da were isolated. And, the molecular weight of sugar chain derived from bovine colostrum is about
A novel 57kDa cysteine protease inhibitor (Japanese
No. 7-2896) and a novel cysteine protease inhibitor derived from bovine colostrum and having a molecular weight of 16 ± 2 kDa or 13 ± 2 kDa (JP-A-7-126294).
【０００３】システインプロテアーゼ阻害剤の有用な作
用の１つとして、ウィルスの増殖阻害作用が確認されて
いる (Biochem. Biophys. Res. Commun., 127, 1072
(1985))。又、骨疾患モデルの動物の骨からのカルシウ
ムの遊離を抑制する作用を示すことから、特開平2-2235
29号公報には抗アレルギー剤や骨疾患治療剤としての用
途が開示されている。又、特開昭61-225130 号公報には
卵白由来のシスタチンを精製し、これを抗ウイルス剤と
して使用することが開示されている。又、特開昭64-258
2 号公報にはシスタチンαの合成遺伝子、特開平1-7149
2 号公報にはシスタチンＢの合成遺伝子、特開平1-1573
90号公報にはシスタチンＡの合成遺伝子がそれぞれ開示
されており、遺伝子操作によりシスタチンを生産するこ
とも可能となってきている。As one of the useful effects of cysteine protease inhibitors, a virus growth inhibitory effect has been confirmed (Biochem. Biophys. Res. Commun., 127, 1072).
(1985)). Further, since it exhibits an action of suppressing the release of calcium from the bone of an animal of a bone disease model, Japanese Patent Application Laid-Open No. 2-2235
No. 29 discloses use as an antiallergic agent or a therapeutic agent for bone disease. JP-A-61-225130 discloses that cystatin derived from egg white is purified and used as an antiviral agent. Also, JP-A 64-258
No. 2 discloses a cystatin α synthetic gene,
No. 2 discloses a cystatin B synthetic gene,
No. 90 discloses cystatin A synthetic genes, respectively, and it has become possible to produce cystatin by genetic manipulation.
【０００４】近年、高齢化の進展にともない、破骨細胞
の骨吸収に起因する骨粗鬆症が急増している。現在、破
骨細胞の吸収活性を抑える医薬として、カルシトニン製
剤がある。しかしこの製剤は、医薬品として、サケやウ
ナギ由来のホルモンを利用したホルモン製剤であり、食
品素材から得られ、食品素材として使えるような安全な
物質についてはあまり検討されていないのが現状であ
る。特開平7-126294号公報では、牛由来の新規シスタチ
ンが骨吸収阻害因子であることが示されているが、医薬
品として用いる場合、ヒト型のシスタチンとの構造の違
いからアレルゲンとなる可能性もある。[0004] In recent years, osteoporosis caused by bone resorption of osteoclasts has been rapidly increasing with aging. At present, there is a calcitonin preparation as a medicine for suppressing the resorption activity of osteoclasts. However, this preparation is a hormone preparation using a hormone derived from salmon or eel as a drug, and at present, safe substances obtained from food materials and usable as food materials have not been studied much. Japanese Patent Application Laid-Open No. 7-126294 discloses that a novel bovine cystatin is a bone resorption inhibitor, but when used as a pharmaceutical, it may be an allergen due to a difference in structure from human cystatin. is there.
【０００５】[0005]
【発明が解決しようとする課題】本発明者らは、抗原性
のない、安全な素材として使える新規なシステインプロ
テアーゼ阻害物質を求め鋭意探索した結果、人乳中に従
来のものと明らかに異なる、システインプロテアーゼ阻
害活性を有する新規な蛋白質を見出した。従って本発明
は、人乳由来のシステインプロテアーゼ阻害活性を有す
る新規蛋白質及びその製造方法を提供することを課題と
する。DISCLOSURE OF THE INVENTION The present inventors have intensively searched for a novel cysteine protease inhibitor which can be used as a safe material without antigenicity. A novel protein having cysteine protease inhibitory activity was found. Accordingly, an object of the present invention is to provide a novel protein having a cysteine protease inhibitory activity derived from human milk and a method for producing the same.
【０００６】[0006]
【課題を解決するための手段】本発明は、システインプ
ロテアーゼ阻害活性を有する新規蛋白質及びその製造方
法に関する。本発明の蛋白質は、その活性より、抗体産
生促進効果及び破骨細胞に対する骨吸収作用に対する阻
害効果を示し、抗ウイルス剤、抗アレルギー剤、あるい
は骨粗鬆症などの骨疾患の予防及び／又は治療剤として
有用である。本発明により提供される人乳由来のシステ
インプロテアーゼ阻害活性を有する新規蛋白質は、下記
の特性により特定される。 (1) 還元及び非還元条件下のSDS-PAGEによる分子量測
定で、13±2 kDa 又は16±2 kDa を示す。 (2) 13±2 kDa の蛋白質は PAS染色陰性で等電点 4.8
〜5.0 を示す。16±2 kDa の蛋白質は PAS染色陽性で等
電点 4.4〜4.6 を示す。 (3) 配列表配列番号１に示されるＮ末端アミノ酸配列
を有する。 (4) 固定化したカルボキシメチル化パパインに結合性
を有する。 (5) カテプシンＬに特異的な阻害活性を有する。 また、本発明は、人乳をカルボキシメチルパパイン固定
化担体を用いてアフィニティクロマトグラフィーにかけ
てシスタチン画分を吸着させ、この吸着画分をアルカリ
条件下で溶出させ、ゲルろ過及び逆相クロマトグラフィ
ーにより精製することを特徴とするシステインプロテア
ーゼ阻害活性を有する蛋白質の製造法に関する。SUMMARY OF THE INVENTION The present invention relates to a novel protein having cysteine protease inhibitory activity and a method for producing the same. Due to its activity, the protein of the present invention exhibits an antibody production promoting effect and an inhibitory effect on bone resorption to osteoclasts, and is used as an antiviral agent, an antiallergic agent, or a preventive and / or therapeutic agent for bone diseases such as osteoporosis. Useful. The novel protein having a cysteine protease inhibitory activity derived from human milk provided by the present invention is specified by the following properties. (1) Measurement of molecular weight by SDS-PAGE under reducing and non-reducing conditions shows 13 ± 2 kDa or 16 ± 2 kDa. (2) 13 ± 2 kDa protein was negative for PAS staining and had an isoelectric point of 4.8.
~ 5.0. The protein of 16 ± 2 kDa is positive for PAS staining and has an isoelectric point of 4.4 to 4.6. (3) It has the N-terminal amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing. (4) It has a binding property to immobilized carboxymethylated papain. (5) It has a cathepsin L-specific inhibitory activity. In addition, the present invention, human milk is subjected to affinity chromatography using carboxymethylpapain-immobilized carrier to adsorb the cystatin fraction, the adsorbed fraction is eluted under alkaline conditions, purified by gel filtration and reverse phase chromatography And a method for producing a protein having cysteine protease inhibitory activity.
【０００７】[0007]
【発明の実施の形態】本発明の蛋白質は人乳から容易に
回収することができる。本発明の蛋白質は、泌乳期間の
うち特に初乳中に大量に含有されるが、通常の乳中にも
含有される。これらの人乳を原料として、上述の文献に
記載されているように、代表的なシステインプロテアー
ゼであるパパインなどに対する親和性を利用してアフィ
ニティークロマトグラフィー等のクロマトグラフィー操
作により分離することができる。即ち、ヒト脱脂初乳に
終濃度0.5MとなるようにNaClを添加し、これをカルボキ
シメチル化パパイン固定化担体を用いたアフィニティー
クロマトグラフィーに供し、ヒト初乳シスタチン類の濃
縮画分を調製する。この画分は既知のシスタチンと本発
明の物質を含有している。特に、NaClの添加により非特
異的吸着を抑制し、比活性の高い濃縮画分を調製でき
る。あるいは、超遠心分離によりホエー画分を調製し、
このホエー画分をエタノールにより分別沈澱し、さらに
凍結乾燥によりエタノールを除去したものを出発材料と
して、カルボキシメチル化パパイン固定化担体を用いた
アフィニティークロマトグラフィーに供し、ヒト初乳シ
スタチン類の濃縮画分を調製する。さらに、この画分を
イオン交換クロマトグラフィーやゲル濾過クロマトグラ
フィー等のクロマトグラフィーによって、本発明の蛋白
質と既知のシスタチンとを分離することができる。さら
に、蛋白質の精製方法として知られている逆相クロマト
グラフィーやＨＰＬＣなどの方法を組み合わせても良
い。BEST MODE FOR CARRYING OUT THE INVENTION The protein of the present invention can be easily recovered from human milk. The protein of the present invention is contained in a large amount especially in colostrum during the lactation period, but is also contained in ordinary milk. As described in the above-mentioned literature, these human milks can be separated by a chromatographic operation such as affinity chromatography utilizing affinity for papain or the like which is a typical cysteine protease. That is, NaCl was added to human defatted colostrum so as to have a final concentration of 0.5 M, and this was subjected to affinity chromatography using a carboxymethylated papain-immobilized carrier to prepare a concentrated fraction of human colostrum cystatins. . This fraction contains the known cystatin and the substance according to the invention. In particular, by adding NaCl, non-specific adsorption can be suppressed, and a concentrated fraction having high specific activity can be prepared. Alternatively, prepare a whey fraction by ultracentrifugation,
The whey fraction was fractionated and precipitated with ethanol, and then subjected to affinity chromatography using a carboxymethylated papain-immobilized carrier, starting from a material from which ethanol was removed by freeze-drying, to concentrate human colostrum cystatins. Is prepared. Further, the protein of the present invention and known cystatins can be separated from this fraction by chromatography such as ion exchange chromatography and gel filtration chromatography. Further, methods such as reverse phase chromatography and HPLC known as protein purification methods may be combined.
【０００８】本発明の上述のようにして得られた天然型
の蛋白質のアミノ酸配列に基づいてこの蛋白質をコード
するｃＤＮＡをクローニングし、得られるｃＤＮＡを用
いて遺伝子工学的手法により本発明のシステインプロテ
アーゼ阻害活性を有する蛋白質を得ることができる。即
ち、配列表配列番号１と配列表配列番号３に示したアミ
ノ酸配列をもとにＰＣＲ用デジェネレーションプライマ
ーを合成し、ヒト乳腺上皮細胞より調製したＲＮＡから
逆転写反応により合成したｃＤＮＡを鋳型としてＰＣＲ
反応を行い、本発明蛋白質の遺伝子のクローニングを行
う。次いでクローニングされたｃＤＮＡ断片をもとに、
3'ＲＡＣＥ法及び5'ＲＡＣＥ法により、本発明蛋白質の
全長ｃＤＮＡをクローニングする。得られた本発明蛋白
質全長ｃＤＮＡを発現ベクターに挿入して、本発明蛋白
質の発現プラスミドを作製し、これを各種の細胞又は菌
株に導入して形質転換させることにより、組み換え型の
本発明蛋白質を発現させることができる。[0008] Based on the amino acid sequence of the natural protein obtained as described above of the present invention, a cDNA encoding the protein is cloned, and the resulting cysteine protease of the present invention is genetically engineered using the obtained cDNA. A protein having an inhibitory activity can be obtained. That is, degeneration primers for PCR were synthesized based on the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 in the Sequence Listing, and cDNA synthesized by reverse transcription from RNA prepared from human mammary epithelial cells was used as a template. PCR
The reaction is performed to clone the gene of the protein of the present invention. Then, based on the cloned cDNA fragment,
The full-length cDNA of the protein of the present invention is cloned by the 3′RACE method and the 5′RACE method. The obtained full-length cDNA of the protein of the present invention is inserted into an expression vector to prepare an expression plasmid for the protein of the present invention, and the resulting plasmid is introduced into various cells or strains to transform the same. Can be expressed.
【０００９】本発明の蛋白質は、乳由来のシスタチンＣ
として知られている物質やその他のシスタチンファミリ
ーの物質と比較して、そのＮ末端アミノ酸配列や内部の
アミノ酸配列が明らかに異なっており、又、遺伝子配列
のホモロジー検索を行ってもこのような配列を報告した
文献はなく、新規な蛋白質である。本発明の蛋白質の分
子量は、上記したようにSDS-PAGEの還元及び非還元条件
下のいずれにおいても、16±２kDa 又は13±２kDa のい
ずれかである。このうち、16±2kDa (以後、単に16kDa
という）を示す物質はＰＡＳ染色陽性であることから糖
鎖の存在が推定され、一方、13±2kDa (以後、単に13kD
a という) を示す物質はＰＡＳ染色陰性であることか
ら、糖鎖を有しない。この16kDa を示す物質を、Ｎ−グ
リカナーゼを用い常法によって脱グリコシレーション処
理を行い、SDS-PAGEにより分子量の変化を分析したとこ
ろ、処理後の蛋白質の移動度が13kDa の移動度と一致し
たことから、16kDa の物質は、13kDa の物質に糖鎖が付
加したものと考えられる。[0009] The protein of the present invention is derived from cystatin C derived from milk.
The N-terminal amino acid sequence and the internal amino acid sequence are clearly different from those known as cystatin family members and other cystatin family members. There is no literature reporting this, and it is a novel protein. As described above, the molecular weight of the protein of the present invention is either 16 ± 2 kDa or 13 ± 2 kDa under both reducing and non-reducing conditions of SDS-PAGE. Of these, 16 ± 2 kDa (hereinafter simply 16 kDa
Is positive for PAS staining, the presence of sugar chains is presumed. On the other hand, 13 ± 2 kDa (hereinafter simply referred to as 13 kD)
a) does not have a sugar chain since it is negative for PAS staining. The substance exhibiting 16 kDa was subjected to deglycosylation by a conventional method using N-glycanase, and the change in molecular weight was analyzed by SDS-PAGE. As a result, the mobility of the protein after the treatment coincided with the mobility of 13 kDa. Therefore, it is considered that a substance of 16 kDa has a sugar chain added to a substance of 13 kDa.
【００１０】本発明の蛋白質は、固定化したカルボキシ
メチル化パパインによるアフィニティークロマトグラフ
ィーとゲル濾過により、分子量の異なる２つの画分とし
て分離される。この分離した画分をゲルろ過及び逆相ク
ロマトグラーフィーに付すことにより、それぞれ単一の
ピークとして回収することができる。この逆相クロマト
グラフィー単一ピークをSDS-PAGEに付しても、やはり単
一のバンドとして検出される。このバンドの分子量は、
13±２kDa 及び16±２kDa の値を示す。逆相クロマトグ
ラフィーで単離した前記 13kDaと16kDa の各成分を、気
相法によるアミノ酸シーケンサーによりＮ末端アミノ酸
配列を分析すると、両者共に配列表配列番号１に記載し
た配列を示す。又、逆相クロマトグラフィーで単離した
各成分を還元ピリジルエチル化し、アルギニルエンドペ
プチダーゼを用いて常法によって加水分解を行い、再度
逆相クロマトグラフィーで分離し、特定のピークを分取
して内部アミノ酸配列を分析すると、配列表配列番号３
〜６に記載した配列が得られる。さらに後述するよう
に、本発明の蛋白質は乳中から分離されるシスタチンフ
ァミリー２の物質と比較して、同程度のシステインプロ
テアーゼ阻害活性を示す。さらに、本発明の蛋白質はゲ
ル等電点電気泳動法により等電点を測定すると、前記16
kDa の物質は 4.4〜4.6 を示し、13kDa の物質は 4.8〜
5.0 を示す。これらの特性により、本発明の蛋白質は特
定される。[0010] The protein of the present invention is separated into two fractions having different molecular weights by affinity chromatography and gel filtration using immobilized carboxymethylated papain. By subjecting the separated fractions to gel filtration and reversed-phase chromatography, each can be collected as a single peak. When a single peak of this reverse phase chromatography is subjected to SDS-PAGE, it is still detected as a single band. The molecular weight of this band is
The values are 13 ± 2 kDa and 16 ± 2 kDa. When the N-terminal amino acid sequence of each of the 13 kDa and 16 kDa components isolated by reverse phase chromatography is analyzed using an amino acid sequencer by a gas phase method, both of them show the sequences described in SEQ ID NO: 1 in the Sequence Listing. In addition, each component isolated by reverse phase chromatography is reduced to pyridylethyl, hydrolyzed by arginyl endopeptidase by a conventional method, separated again by reverse phase chromatography, and a specific peak is collected. When the internal amino acid sequence is analyzed,
6 are obtained. As described further below, the protein of the present invention exhibits the same degree of cysteine protease inhibitory activity as compared to cystatin family 2 substances isolated from milk. Further, when the protein of the present invention was measured for its isoelectric point by gel isoelectric focusing,
The kDa substance shows 4.4 to 4.6, and the 13 kDa substance shows 4.8 to 4.6.
Indicates 5.0. These properties define the protein of the present invention.
【００１１】また、本発明の蛋白質のパパインに対する
阻害活性は Barrettらの方法（Methods in Enzymology,
Vol.80, pp771(1981)）に準じて測定することができ
る。即ち、0.1Mベンゾイル−Ｄ，Ｌ−アルギニン−４−
ニトロアニリド (Benzoil-D,L-arginine-4-nitroanilid
e)のジメチルスルホキシド溶液を基質とし、測定用の緩
衝液として、２mMのＥＤＴＡを含む20mMリン酸緩衝液
に、使用直前にシステインを終濃度８mMとなるように添
加した溶液を用いて、比色法により酵素の活性を測定す
ることにより酵素の阻害率を求めることができる。The inhibitory activity of the protein of the present invention on papain is determined by the method of Barrett et al. (Methods in Enzymology,
Vol.80, pp771 (1981)). That is, 0.1 M benzoyl-D, L-arginine-4-
Nitroanilide (Benzoil-D, L-arginine-4-nitroanilid
e) Using the dimethyl sulfoxide solution as a substrate, and using a solution in which cysteine was added to a 20 mM phosphate buffer solution containing 2 mM EDTA to a final concentration of 8 mM immediately before use as a buffer for measurement, using a colorimetric method. The enzyme inhibition rate can be determined by measuring the activity of the enzyme by the method.
【００１２】本発明の蛋白質は、微生物あるいはウイル
スなどによる感染症、アレルギー、あるいは骨粗鬆症な
どの骨疾患の治療及び改善を目的とした医薬組成物とし
て、あるいはこのような疾患の免疫学的診断を確立する
ための抗原として有用である。本発明の蛋白質を含む製
剤は、医薬組成物あるいは食品等に配合することによ
り、ヒト及び動物に対して安全に投与されるものであ
る。医薬組成物の形態としては、注射剤、点滴用液剤、
坐剤、経鼻剤、舌下剤、経皮吸収剤などが挙げられる。
これらの製剤は、本発明の蛋白質の薬理学的有効量と製
剤学的に許容されうる担体との混合物であり、担体とし
てアミノ酸、糖類、セルロース誘導体及びその他の有機
又は無機化合物などの、一般的に添加される賦形剤又は
賦活剤を用いることができる。又、必要に応じてｐＨ調
整剤、緩衝剤、安定化剤、可溶化剤などを添加すること
ができる。The protein of the present invention is used as a pharmaceutical composition for the treatment and improvement of bone diseases such as infectious diseases caused by microorganisms or viruses, allergies, or osteoporosis, or has established an immunological diagnosis of such diseases. It is useful as an antigen for The formulation containing the protein of the present invention can be safely administered to humans and animals by blending it into a pharmaceutical composition or food. Examples of the form of the pharmaceutical composition include injections, infusion solutions,
Suppositories, nasal preparations, sublinguals, transdermal absorbents and the like can be mentioned.
These preparations are a mixture of a pharmacologically effective amount of the protein of the present invention and a pharmaceutically acceptable carrier, and include carriers such as amino acids, saccharides, cellulose derivatives and other organic or inorganic compounds, An excipient or an activator added to the mixture can be used. If necessary, a pH adjuster, a buffer, a stabilizer, a solubilizer, and the like can be added.
【００１３】以下の実施例によって本発明をより詳細に
説明するが、これらは単に例示するのみであり、これら
によって本発明が限定されるものではない。The present invention will be described in more detail with reference to the following examples, which are merely illustrative and do not limit the present invention.
【００１４】[0014]
【実施例１】システインプロテアーゼ阻害活性を有する蛋白質の単離 （１）システインプロテアーゼ阻害活性測定法 システインプロテアーゼに対する阻害活性は、パパイン
に対する阻害活性を Barrettらの方法（Methods in Enz
ymology, Vol.80, pp771(1981)）に準じて測定した。即
ち、0.1Mベンゾイル−Ｄ，Ｌ−アルギニン−４−ニトロ
アニリド(Benzoil-D,L-arginine-4-nitroanilide、シグ
マ社) ジメチルスルホキシド溶液を基質とし、測定用の
緩衝液として、２mMのＥＤＴＡを含む20mMリン酸緩衝液
に、使用直前にシステインを終濃度８mMとなるように添
加した。パパイン（シグマ社）を0.5 mg/ml の濃度で、
システインを含まない測定用緩衝液に溶解した。又、反
応停止用溶液として30％酢酸溶液を使用した。測定は、
以下の手順で行った。測定用チューブに１mlのシステイ
ンを含むリン酸緩衝液と0.1 mlのパパイン溶液を入れ、
37℃で５分間インキュベートした。次いで、試料溶液0.
1 ml及び基質溶液30μｌを加え攪拌した。37℃で30分間
反応後、0.2 mlの反応停止液を加え、410nm の吸光度を
測定した。インヒビター活性は次式（Ｉ）より求めた。Example 1 Isolation of Protein Having Cysteine Protease Inhibitory Activity (1) Method of Measuring Cysteine Protease Inhibitory Activity The inhibitory activity on cysteine protease was determined by the method of Barrett et al.
ymology, Vol.80, pp771 (1981)). That is, 0.1 mM benzoyl-D, L-arginine-4-nitroanilide (Benzoil-D, L-arginine-4-nitroanilide, Sigma) dimethyl sulfoxide solution was used as a substrate, and 2 mM EDTA was used as a measurement buffer. Immediately before use, cysteine was added to a final 20 mM phosphate buffer solution to a final concentration of 8 mM. Papain (Sigma) at a concentration of 0.5 mg / ml,
The cells were dissolved in a measurement buffer containing no cysteine. A 30% acetic acid solution was used as a reaction stopping solution. The measurement is
The procedure was as follows. In a measuring tube, put 1 ml of phosphate buffer containing cysteine and 0.1 ml of papain solution.
Incubated at 37 ° C for 5 minutes. Then, the sample solution 0.
1 ml and 30 μl of the substrate solution were added and stirred. After reacting at 37 ° C. for 30 minutes, 0.2 ml of a reaction stop solution was added, and the absorbance at 410 nm was measured. The inhibitor activity was determined by the following formula (I).
【００１５】 比活性[unit/mg] ＝｛ (A0-AI ) ×1.43｝／｛ 8.8×30× WS ｝ （Ｉ） A0 : インヒビターフリーの吸光度 AI : サンプルの吸光度 WS : サンプル溶液中の蛋白質量[mg]Specific activity [unit / mg] = ｛(A 0 -A I ) × 1.43｝ / ｛8.8 × 30 × W S ｝ (I) A 0 : Inhibitor-free absorbance A I : Sample absorbance W S : Protein content in sample solution [mg]
【００１６】(2) システインプロテアーゼ阻害活性を有
する蛋白質の精製 1) エタノール沈澱 分娩後３日以内に搾乳したヒト初乳２ｌから遠心分離に
より脱脂乳を調製し、次いで超遠心分離によりホエーを
調製した。得られたホエーを氷冷しながら、終濃度30％
(v/v) になるようにエタノールを添加し、生じた沈殿を
遠心分離により除去した。さらに終濃度60％(v/v) とな
るようにエタノールを添加し、生じた沈殿を遠心分離に
より回収した。回収した沈殿に含まれるエタノールを除
去するため、沈澱を凍結乾燥した。 (2) It has cysteine protease inhibitory activity
Skim milk was prepared by centrifugation from the protein purification 1) human colostrum 2l which milked within ethanol precipitation postpartum 3 days to then prepare a whey by ultra-centrifugation. While cooling the obtained whey on ice, the final concentration is 30%
(v / v) was added to ethanol, and the resulting precipitate was removed by centrifugation. Further, ethanol was added to a final concentration of 60% (v / v), and the resulting precipitate was collected by centrifugation. The precipitate was freeze-dried to remove ethanol contained in the collected precipitate.
【００１７】2) カルボキシメチル化パパインアフィニ
ティークロマトグラフィー カルボキシメチル化パパインをトレシルトヨパール（Tr
esyl-Toyopearl, 東ソー社) に結合させた担体を26φ×
150mm のカラムへ充填後、50mMリン酸−0.5M NaCl 緩衝
液 (pH7.0)で平衡化した。このカラムに、先の凍結乾燥
物をカラムの平衡化緩衝液に溶解後通液し、システイン
プロテアーゼ阻害活性を有する蛋白質を吸着させた。通
液後、平衡化に使用した緩衝液に 0.1％ Tween-20 を添
加した溶液でカラムを洗浄した。次いで、50mMリン酸ナ
トリウム−0.5M NaCl 緩衝液 (pH11.5) で該蛋白質を溶
出させ、溶出画分を１M グリシン塩酸緩衝液 (pH3.0)で
直ちに中和した。 2) Carboxymethylated papain affinity
Tea chromatography carboxymethylated papain is treated with Tresil Toyopearl (Tr
(esyl-Toyopearl, Tosoh Corporation)
After packing into a 150 mm column, the column was equilibrated with 50 mM phosphate-0.5 M NaCl buffer (pH 7.0). The lyophilized product was dissolved in the equilibration buffer of the column and passed through the column to adsorb a protein having cysteine protease inhibitory activity. After the passage, the column was washed with a solution obtained by adding 0.1% Tween-20 to the buffer used for equilibration. Next, the protein was eluted with 50 mM sodium phosphate-0.5 M NaCl buffer (pH 11.5), and the eluted fraction was immediately neutralized with 1 M glycine hydrochloride buffer (pH 3.0).
【００１８】3) ゲルろ過 アフィニティークロマトグラフィーで調製した画分に対
し、セファクリルS-200 HR（ファルマシア社）を用いた
ゲルフィルトレーションを実施した。各画分の活性は実
施例１(1) に示したように、インヒビターフリーの吸光
度 (Ａ0)及びサンプルの吸光度（Ａ1)を求め、次式(II)
によって相対活性値を求めた。結果を図１に示す。 3) The fraction prepared by gel filtration affinity chromatography was subjected to gel filtration using Sephacryl S-200 HR (Pharmacia). The activity of each fraction was determined by measuring the inhibitor-free absorbance (A 0 ) and the sample absorbance (A 1 ) as shown in Example 1 (1).
The relative activity was determined by The results are shown in FIG.
【００１９】 活性〔％〕＝｛（Ａ0 −Ａ1 ）／Ａ0 ｝×１００ (II) Activity [%] = {(A 0 −A 1 ) / A 0 } × 100 (II)
【００２０】4) 逆相クロマトグラフィー ゲルろ過で調製した画分を分画分子量６kDa のフォロー
ファイバー型ＵＦ膜（旭化成製）により濃縮した。0.15
M の塩化ナトリウムを含む20mMリン酸ナトリウム緩衝液
で平衡化したカラム (26φ×750mm)に通液した。溶出液
を分取し、阻害活性を示す画分のうち、既知のシスタチ
ンと明らかに異なる二つのピークで、図１に矢印で示し
た画分 (本発明の 16kDa及び13kDa の蛋白質) を回収
し、それぞれ、逆相クロマトグラフィーに供した。VYDA
C 214TP54 (Vydac社) カラム(4.6mm×250mm)を用い、逆
相クロマトグラフィーを実施した。カラムの平衡化に
は、0.08％ ＴＦＡ (トリフロロ酢酸）を添加した５％
CH3 CN-95 ％Ｈ2Oを、溶離液として、0.06％のＴＦＡを
添加した80％ CH3 CN-20 ％ Ｈ2Oを使用し、１％／分
の溶出速度で直線グラジエントによって溶出した。その
結果を図２及び図３に示した。図２は、13kDa の蛋白質
の溶出パターンを、図３は、16kDa の蛋白質の溶出パタ
ーンをそれぞれ示す。さらに、各ピークを分取し、阻害
活性を測定した。図中にＰ37又はＰ38で示した各ピーク
に活性が認められた。この活性画分の一部を同一条件で
逆相クロマトグラフィーに供した結果、それぞれ単一の
ピークを示した。２つの活性画分中には、本発明の 16k
Da及び 13kDaの蛋白質が、それぞれ38μg 及び57μg 含
まれていた。 4) The fraction prepared by reverse phase chromatography gel filtration was concentrated by a follow fiber type UF membrane (Asahi Kasei) having a molecular weight cut off of 6 kDa. 0.15
The solution was passed through a column (26φ × 750 mm) equilibrated with a 20 mM sodium phosphate buffer containing M sodium chloride. The eluate was collected, and among the fractions showing the inhibitory activity, the fractions indicated by arrows in FIG. 1 (the 16 kDa and 13 kDa proteins of the present invention) were collected with two peaks clearly different from known cystatins. , Respectively, were subjected to reverse phase chromatography. VYDA
Reversed phase chromatography was performed using a C 214TP54 (Vydac) column (4.6 mm × 250 mm). For column equilibration, 5% with 0.08% TFA (trifluoroacetic acid) was added.
Eluted with a linear gradient at an elution rate of 1% / min using CH 3 CN-95% H 2 O and 80% CH 3 CN-20% H 2 O with 0.06% TFA as eluent. . The results are shown in FIGS. FIG. 2 shows the elution pattern of the 13 kDa protein, and FIG. 3 shows the elution pattern of the 16 kDa protein. Furthermore, each peak was collected and the inhibitory activity was measured. Activity was observed at each peak indicated by P37 or P38 in the figure. A part of this active fraction was subjected to reverse phase chromatography under the same conditions, and as a result, each showed a single peak. In the two active fractions, the 16k
Da and 13 kDa proteins were contained at 38 μg and 57 μg, respectively.
【００２１】[0021]
【実施例２】本発明蛋白質の特性 実施例１で得られた16kDa 及び13kDa の本発明の蛋白質
をそれぞれ 5mgづつ用い、特性値を測定した。（１）SDS-PAGE 実施例１(2)4) で得られた、逆相クロマトグラフィーで
精製した２種類の本発明の蛋白質をSDS-PAGE(Fast Syst
em, ファルマシア社) に供した。結果を図４に示す。16
kDa 及び13kDa の本発明の蛋白質は還元状態及び非還元
状態のいずれにおいても共に単一バンドを示し、不純物
は認められなかった。又、還元及び非還元条件下でも変
化が無いことから、両者共に単一のポリペプチドから成
ることが示された。又、これらの分子量は、それぞれ約
16kDa 及び約13kDa であることが確認された。Example 2 Characteristics of the Protein of the Present Invention Using 5 mg each of the 16 kDa and 13 kDa proteins of the present invention obtained in Example 1, characteristic values were measured. (1) SDS-PAGE Two kinds of the proteins of the present invention, which were obtained in Example 1 (2) 4) and purified by reverse phase chromatography, were subjected to SDS-PAGE (Fast Syst
em, Pharmacia). FIG. 4 shows the results. 16
The kDa and 13 kDa proteins of the present invention showed a single band in both the reduced state and the non-reduced state, and no impurities were observed. Also, there was no change under reducing and non-reducing conditions, indicating that both consist of a single polypeptide. These molecular weights are about
It was confirmed to be 16 kDa and about 13 kDa.
【００２２】（２）パパイン阻害活性 精製した 16kDa及び13kDa の本発明の蛋白質、及び公知
のヒトシスタチンＣのパパインに対する阻害活性を、実
施例１(1) の阻害活性測定法に従って比較した。その結
果を表１に示す。表１に示されるように、本発明の蛋白
質は、公知のヒトシスタチンＣと同等及びそれ以上の比
活性を示した。 (2) Papain Inhibitory Activity The inhibitory activities of the purified 16 kDa and 13 kDa proteins of the present invention and the known human cystatin C on papain were compared according to the inhibitory activity measurement method of Example 1 (1). Table 1 shows the results. As shown in Table 1, the protein of the present invention exhibited specific activity equivalent to or higher than that of known human cystatin C.
【００２３】[0023]
【表１】 ─────────────────────── 比活性(unit/mg) ─────────────────────── 天然型本発明蛋白質(16KDa) 0.294 天然型本発明蛋白質(13KDa) 0.302 ヒトシスタチンＣ 0.283 ───────────────────────[Table 1] ─────────────────────── Specific activity (unit / mg) ──────────────── ─────── Native type protein of the present invention (16KDa) 0.294 Natural type protein of the present invention (13KDa) 0.302 Human cystatin C 0.283 ───────────────────── ──
【００２４】（３）Ｎ末端アミノ酸配列 精製した16kDa 、13kDa の分子量を有する本発明の蛋白
質のＮ末端アミノ酸配列分析を、気相法によるアミノ酸
シーケンサー（120 Ａ型アナライザー、ＡＢＩ社）を用
いて実施した。両者とも１８残基まで同定でき、両者は
完全に一致した。このことから、両画分とも同一のＮ末
端配列を有するペプチド鎖を含んでいることが確認され
た。この配列を配列表配列番号１に示した。又、比較の
ために公知のヒトシスタチンＣの配列を配列表配列番号
２に示した。公知のヒトシスタチンＣと本発明の蛋白質
（16±2kDa及び13±2kDa) は、全く異なる配列であっ
た。 (3) N-Terminal Amino Acid Sequence The N-terminal amino acid sequence of the purified protein of the present invention having a molecular weight of 16 kDa or 13 kDa was analyzed using an amino acid sequencer (120 A type analyzer, ABI) by a gas phase method. did. In both cases, up to 18 residues could be identified, and both were completely identical. From this, it was confirmed that both fractions contained a peptide chain having the same N-terminal sequence. This sequence is shown in SEQ ID NO: 1 in the Sequence Listing. For comparison, the sequence of a known human cystatin C is shown in SEQ ID NO: 2 in the Sequence Listing. Known human cystatin C and the protein of the present invention (16 ± 2 kDa and 13 ± 2 kDa) had completely different sequences.
【００２５】（４）精製した13kDa 及び16kDa の蛋白質
の内部アミノ酸配列分析 精製した本発明の蛋白質（16kDa 及び13kDa)の内部アミ
ノ酸配列の分析を行った。即ち、精製した本発明の蛋白
質をそれぞれ還元ピリジルエチル化し、アルギニルエン
ドペプチダーゼ（宝酒造社）で加水分解し、ついで逆相
クロマトグラフィーでペプチドマッピングを行った。結
果を図５(13kDaの蛋白質) 及び図６(16kDa の蛋白質)
に示す。それぞれの図のAP-3及び AP-3'で示したピーク
の溶出時間 (AP-3: 50.4分, AP-6: 61.3分, AP-3': 46.
4 分，AP-6': 61.4 分) やピークの形状が全く異なって
いたがその他のピークには顕著な差は認められなかっ
た。そこでAP-6及び AP-6'とともに、アミノ酸配列を解
析した。アミノ酸配列の分析は実施例２(3) と同様に行
った。これらの配列を配列表配列番号３から６に示す。
AP-6 (配列番号３) とAP-6'(配列番号４) のペプチドの
配列は完全に一致していた。又、 AP-3(配列番号５) と
AP-3'(配列番号６) ではAP-3' の未同定な１残基を除き
一致していた。AP-3' の未同定アミノ酸残基及びその前
後部分に相当するAP-3の配列は-Asn-Ser-Ser- であり、
この配列はＮ結合型糖鎖が結合するコンセンサス配列、
即ち -Asn-Xaa-Ser/Thr-(Xaaは任意のアミノ酸残基）と
一致していた。又、糖鎖が結合しているAsn 残基は通常
のアミノ酸配列分析では同定できないこと、及び16kDa
の蛋白質が糖蛋白質であることから、AP-3' の未同定の
アミノ酸残基は糖鎖を結合したAsn 残基と推定される。 (4) Purified 13 kDa and 16 kDa proteins
The internal amino acid sequence of the purified protein of the present invention (16 kDa and 13 kDa) was analyzed. That is, each of the purified proteins of the present invention was subjected to reductive pyridylethylation, hydrolyzed with arginyl endopeptidase (Takara Shuzo), and then subjected to peptide mapping by reverse phase chromatography. The results are shown in FIG. 5 (13 kDa protein) and FIG. 6 (16 kDa protein).
Shown in The elution time of the peaks indicated by AP-3 and AP-3 'in each figure (AP-3: 50.4 minutes, AP-6: 61.3 minutes, AP-3': 46.
(4 minutes, AP-6 ': 61.4 minutes) and the shape of the peaks were completely different, but no significant difference was observed in the other peaks. Therefore, the amino acid sequence was analyzed together with AP-6 and AP-6 '. The analysis of the amino acid sequence was performed in the same manner as in Example 2 (3). These sequences are shown in SEQ ID NOs: 3 to 6 in the sequence listing.
The sequences of the peptides AP-6 (SEQ ID NO: 3) and AP-6 '(SEQ ID NO: 4) were completely identical. Also, AP-3 (SEQ ID NO: 5)
AP-3 '(SEQ ID NO: 6) was identical except for one unidentified residue of AP-3'. The sequence of AP-3 corresponding to the unidentified amino acid residue of AP-3 'and the region before and after it is -Asn-Ser-Ser-,
This sequence is a consensus sequence to which N-linked sugar chains bind,
That is, it was consistent with -Asn-Xaa-Ser / Thr- (Xaa is an arbitrary amino acid residue). In addition, the Asn residue to which the sugar chain is bound cannot be identified by ordinary amino acid sequence analysis, and 16 kDa
Since this protein is a glycoprotein, the unidentified amino acid residue of AP-3 ′ is assumed to be a sugar chain-bound Asn residue.
【００２６】（５）糖鎖の有無 精製した16kDa 及び13kDa の本発明の蛋白質の糖鎖の有
無を、ＰＡＳ染色法で確認した。その結果、16kDa の蛋
白質のみが陽性を示し、糖鎖を含有していることが確認
された。 (5) Presence or Absence of Sugar Chain The presence or absence of sugar chains of the purified 16 kDa and 13 kDa proteins of the present invention was confirmed by PAS staining. As a result, it was confirmed that only the 16 kDa protein was positive and contained a sugar chain.
【００２７】（６）等電点 両画分をゲル等電点電気泳動法（Fast system 、ファル
マシア社) に供し、ｐＩ値（等電点）を求めた。その結
果、16kDa の蛋白質のｐＩ値は4.4-4.6 を示し、13kDa
の蛋白質のｐＩ値は、4.8-5.0 を示した。この結果か
ら、本発明蛋白質は、糖鎖を含有する分子量16kDa と糖
鎖を有しない13kDa の２つの分子量を持つ物質であるこ
とが確認された。 (6) Both fractions were subjected to gel isoelectric focusing (Fast system, Pharmacia) to determine the pI value (isoelectric point). As a result, the pI value of the 16 kDa protein was 4.4 to 4.6, and the pI value was 13 kDa.
The pI value of the protein was 4.8-5.0. From these results, it was confirmed that the protein of the present invention was a substance having two molecular weights of a sugar chain-containing 16 kDa and a sugar chain-free 13 kDa.
【００２８】[0028]
【実施例３】本発明蛋白質の遺伝子クローニング （１）本発明蛋白質の遺伝子クローニング アミノ酸配列分析の結果をもとに、ＰＣＲ反応用のプラ
イマーを合成した。即ち、配列表配列番号１に示したＮ
末端アミノ酸配列をコードするＤＮＡ配列、及び配列表
配列番号３から６に示した内部アミノ酸配列をコードす
るＤＮＡ配列を推定して、デジェネレートプライマーを
合成した。デジェネレートプライマーの配列を、表２に
示す。又、鋳型として、正常ヒト乳腺上皮細胞 (Mammar
y Pack、クラボウ社）より調製したｍＲＮＡ由来一本鎖
ｃＤＮＡを用い、ＰＣＲ反応を行った。反応終了後、反
応液を２％アガロースゲル電気泳動に付したところ、約
200bp の顕著なバンドが認められた。このフラグメント
を回収し、 DNA BluntingKit （宝酒造社）によりフラ
グメントの末端を平滑化した後、あらかじめ制限酵素Hi
ncIIで切断処理したプラスミドベクターpUC119（宝酒造
社）とライゲーションさせた。次いで、ライゲーション
混合物により大腸菌ＪＭ109 （宝酒造社）を形質転換し
た。形質転換によりアンピシリン耐性株となった中から
ＰＣＲ法により200bp の挿入フラグメントを有する株を
選定した。選定した株からプラスミドを精製し、挿入フ
ラグメントの塩基配列を解析したところ、実施例２(3)
又は(4) で示した本発明の蛋白質のアミノ酸配列の一部
をコードしていた。Example 3 Gene Cloning of the Protein of the Present Invention (1) Gene Cloning of the Protein of the Present Invention Based on the results of amino acid sequence analysis, primers for a PCR reaction were synthesized. That is, N shown in SEQ ID NO: 1 in the sequence listing
A DNA sequence encoding a terminal amino acid sequence and a DNA sequence encoding an internal amino acid sequence shown in SEQ ID NOs: 3 to 6 in the Sequence Listing were estimated to synthesize degenerate primers. Table 2 shows the sequences of the degenerate primers. As a template, normal human mammary epithelial cells (Mammar
PCR was performed using mRNA-derived single-stranded cDNA prepared from Y Pack (Kurabo Co., Ltd.). After completion of the reaction, the reaction solution was subjected to 2% agarose gel electrophoresis.
A remarkable band of 200 bp was observed. This fragment is recovered, and the ends of the fragment are blunt-ended with DNA Blunting Kit (Takara Shuzo).
It was ligated with plasmid vector pUC119 (Takara Shuzo) digested with ncII. Next, E. coli JM109 (Takara Shuzo) was transformed with the ligation mixture. From the transformed ampicillin-resistant strains, a strain having a 200 bp insert was selected by PCR. Purification of the plasmid from the selected strain and analysis of the nucleotide sequence of the inserted fragment revealed that Example 2 (3)
Or, it encoded a part of the amino acid sequence of the protein of the present invention shown in (4).
【００２９】[0029]
【表２】 [Table 2]
【００３０】（２）ＲＡＣＥ法による3'末端領域の塩基
配列解析 実施例３(1) で得られたｃＤＮＡ配列をもとにプライマ
ーを合成し、 3' ＲＡＣＥ法とネステッドＰＣＲ法を組
み合わせた方法により、本発明蛋白質の3'末端領域の塩
基配列を解析した。一連の操作に用いるプライマーとし
て、本発明の蛋白質の固有なアミノ酸配列をコードする
プライマー＃１（配列表配列番号９）及びプライマー＃
２（配列表配列番号１０）を、逆転写用のプライマーと
してはプライマー＃３（配列表配列番号１１）を、ＲＡ
ＣＥ反応用としてプライマー＃４（配列表配列番号１
２）をそれぞれ合成した。プライマー＃２には制限酵素
EcoRI 及びKpnIの切断サイトを、プライマー＃３及び＃
４には制限酵素SalI, PstI,HindIII の切断サイトをデ
ザインした。実施例３(1) で使用したヒト乳腺上皮細胞
由来のｍＲＮＡから、プライマー＃３を用いて一本鎖ｃ
ＤＮＡを合成し、これを鋳型としてプライマーをセンス
プライマー、プライマー＃４をアンチセンスプライマー
としてＰＣＲ反応を行った。反応後の反応液の一部を取
り1000倍に希釈して次に行うネステッドＰＣＲ反応の鋳
型とし、プライマーには＃２及び＃４を用いた。ネステ
ッドＰＣＲ反応後, 反応液を２％アガロースゲル電気泳
動に付したところ、 300bpの顕著なバンドが認められ
た。このフラグメントを回収し、制限酵素EcoRI 及びHi
ndIII で処理後、あらかじめ制限酵素 EcoRI及びHindII
I で切断しておいたプラスミドベクターpUC18 （宝酒造
社）とライゲーションさせた。以後は実施例３(1) と同
様にして、挿入フラグメントの配列を解析した。得られ
た塩基配列は、実施例２(4) で示した本発明の蛋白質の
内部アミノ酸配列をコードしていた。 (2) Base in 3 ′ terminal region by RACE method
Sequence analysis A primer was synthesized based on the cDNA sequence obtained in Example 3 (1), and the nucleotide sequence of the 3 'end region of the protein of the present invention was analyzed by a method combining the 3' RACE method and the nested PCR method. did. Primers # 1 (SEQ ID NO: 9) encoding a unique amino acid sequence of the protein of the present invention and primer #
2 (SEQ ID NO: 10) as primer for reverse transcription, primer # 3 (SEQ ID NO: 11) as RA
For the CE reaction, primer # 4 (SEQ ID NO: 1 in the Sequence Listing)
2) was synthesized respectively. Restriction enzyme for primer # 2
Insert the EcoRI and KpnI cleavage sites with primers # 3 and #
In No. 4, cleavage sites for restriction enzymes SalI, PstI and HindIII were designed. From the mRNA derived from human mammary epithelial cells used in Example 3 (1), single-stranded c
DNA was synthesized, and a PCR reaction was performed using the DNA as a template and a primer as a sense primer and primer # 4 as an antisense primer. A part of the reaction solution after the reaction was taken and diluted 1000-fold and used as a template for the next nested PCR reaction, and # 2 and # 4 were used as primers. After the nested PCR reaction, the reaction mixture was subjected to 2% agarose gel electrophoresis. As a result, a remarkable 300 bp band was observed. This fragment was recovered and the restriction enzymes EcoRI and Hi were used.
After treatment with ndIII, use the restriction enzymes EcoRI and HindII in advance.
The plasmid was ligated with the plasmid vector pUC18 (Takara Shuzo) cut with I. Thereafter, the sequence of the inserted fragment was analyzed in the same manner as in Example 3 (1). The obtained nucleotide sequence encoded the internal amino acid sequence of the protein of the present invention shown in Example 2 (4).
【００３１】（３）ＲＡＣＥ法による5'末端領域の塩基
配列 ＲＡＣＥ法によって本発明の蛋白質遺伝子の5'末端領域
の塩基配列を決定した。デザインしたプライマーは、プ
ライマー＃５（配列表配列番号１３）及びプライマー＃
６（配列表配列番号１４）であり、これらは本発明蛋白
質に固有なアミノ酸配列をコードする。又、目的の遺伝
子を検索するプローブとして、ＤＩＧ標識したｃＤＮＡ
を調製した。即ち、(1) で得られた配列をもとにして、
プライマー＃７（配列表配列番号１５）及びプライマー
＃８（配列表配列番号１６）を合成し、実施例３(1) で
得られたプラスミドを鋳型としてＰＣＲ反応によってプ
ローブを調製した。ＰＲＣ反応で、 DIG DNA Labeling
Mix （ベーリンガーマンハイム社）を反応液に添加し
て、ＰＣＲ産物をDIG 標識しプローブとした。ＰＲＣ反
応終了後、バイオスピンカラムＰ６（バイオラット社）
を用いて目的のプローブを精製した。 5' ＲＡＣＥは以
下の手順で行った。まずヒト乳腺上皮細胞よりｍＲＮＡ
を精製し、プライマー＃５を用いて一本鎖ｃＤＮＡを合
成し、その5'末端にデオキシリボヌクレオチド末端転位
酵素（ギブコ社）を用いて、ポリＡテイルを付加させ
た。次にポリＡをテイル付加した一本鎖ｃＤＮＡを鋳型
として、プライマー＃６及び＃３をプライマーとしてＰ
ＣＲ反応を行った。反応液の一部を２％アガロースゲル
電気泳動に付した。又、ニトロセルロースメンブランに
転写後、ＤＩＧプローブを用いて目的の遺伝子をサザン
ブロッティングで検索したところ、 250bpのバンドが検
出された。このフラグメントを回収し、制限酵素EcoRI
及びHindIII で末端を処理した後実施例３(2) と同様に
サブクローニングして挿入断片の配列を解析した。得ら
れた塩基配列は、実施例２(4) で示した本発明蛋白質の
アミノ酸配列をコードしていた。 (3) Base at 5 ′ end region by RACE method
The nucleotide sequence of the 5 ′ terminal region of the protein gene of the present invention was determined by the sequence RACE method. The designed primers include primer # 5 (SEQ ID NO: 13 in the Sequence Listing) and primer #
6 (SEQ ID NO: 14), which codes for an amino acid sequence unique to the protein of the present invention. As a probe for searching for a target gene, DIG-labeled cDNA was used.
Was prepared. That is, based on the sequence obtained in (1),
Primer # 7 (SEQ ID NO: 15) and Primer # 8 (SEQ ID NO: 16) were synthesized, and a probe was prepared by PCR using the plasmid obtained in Example 3 (1) as a template. DIG DNA Labeling in PRC reaction
Mix (Boehringer Mannheim) was added to the reaction solution, and the PCR product was DIG-labeled and used as a probe. After completion of the PRC reaction, Biospin Column P6 (Biorat)
Was used to purify the target probe. 5 'RACE was performed according to the following procedure. First, mRNA from human mammary epithelial cells
Was purified, a single-stranded cDNA was synthesized using primer # 5, and a poly-A tail was added to the 5 'end of the cDNA using deoxyribonucleotide terminal transferase (Gibco). Next, the single-stranded cDNA to which poly A was added was used as a template, and primers # 6 and # 3 were used as primers.
A CR reaction was performed. A part of the reaction solution was subjected to 2% agarose gel electrophoresis. After transfer to a nitrocellulose membrane, the target gene was searched by Southern blotting using a DIG probe, and a band of 250 bp was detected. This fragment is recovered, and the restriction enzyme EcoRI is used.
After the ends were treated with HindIII, the clone was subcloned and the sequence of the inserted fragment was analyzed in the same manner as in Example 3 (2). The obtained nucleotide sequence encoded the amino acid sequence of the protein of the present invention shown in Example 2 (4).
【００３２】（４）本発明の蛋白質全長遺伝子のクロー
ニング ＰＣＲ法により、本発明の蛋白質の全長遺伝子をクロー
ニングした。プライマーにプライマー＃９（配列表配列
番号１７）及びプライマー＃１０（配列表配列番号１
８）を用い、制限酵素によって容易に切り出せるように
デザインした。実施例３(1) で用いた一本鎖ｃＤＮＡを
鋳型として、ＰＣＲ法によりクローニングした。ＰＣＲ
法、サザンブロッティング、塩基配列等の解析によっ
て、目的の遺伝子を挿入断片として有するクローンを選
別した。得られたクローンには、Ｎ末端のArg 残基から
121 番目のMet 残基までをコードする遺伝子を有してい
た。このクローンより得られたプラスミドを、pUC-hcm1
と命名した。配列表配列番号７に本発明蛋白質のｃＤＮ
Ａ配列を、配列表配列番号８にそれがコードするアミノ
酸配列を示す。 (4) Clones of the full-length gene of the protein of the present invention
The training PCR and cloned full-length gene of the protein of the present invention. Primers # 9 (SEQ ID NO: 17) and primer # 10 (SEQ ID NO: 1)
8) was designed so that it could be easily cut out with restriction enzymes. Using the single-stranded cDNA used in Example 3 (1) as a template, it was cloned by PCR. PCR
A clone having the target gene as an inserted fragment was selected by analysis of the method, Southern blotting, nucleotide sequence and the like. The resulting clone contains the N-terminal Arg residue
It had a gene encoding up to the 121st Met residue. Plasmid obtained from this clone was called pUC-hcm1
It was named. SEQ ID NO: 7 shows the cDN of the protein of the present invention.
The A sequence shows the amino acid sequence encoded by it in SEQ ID NO: 8 in the sequence listing.
【００３３】[0033]
【発明の効果】本発明によりシステインプロテアーゼ阻
害活性を有する新規蛋白質及びその製造方法が提供され
る。本発明の蛋白質は、その活性より、抗体産生促進効
果及び破骨細胞の骨吸収作用に対する阻害効果を示し、
抗ウイルス剤、抗アレルギー剤、あるいは骨粗鬆症など
の骨疾患の予防及び／又は治療剤として有用である。Industrial Applicability The present invention provides a novel protein having cysteine protease inhibitory activity and a method for producing the same. The protein of the present invention exhibits an antibody production promoting effect and an inhibitory effect on bone resorption of osteoclasts from its activity.
It is useful as an antiviral agent, an antiallergic agent, or an agent for preventing and / or treating bone diseases such as osteoporosis.
【００３４】[0034]
配列番号：１ 配列の長さ：１８ 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 配列 Arg Pro Gln Glu Arg Met Val Gly Glu Leu Arg Asp Leu Ser Pro Asp 1 5 10 15 Asp Pro SEQ ID NO: 1 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Arg Pro Gln Glu Arg Met Val Gly Glu Leu Arg Asp Leu Ser Pro Asp 1 5 10 15 Asp Pro
【００３５】配列番号：２ 配列の長さ：１８ 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 配列 Ser Ser Pro Gly Lys Pro Pro Pro Arg Leu Val Gly Gly Pro Met Asp 1 5 10 15 Ala Ser SEQ ID NO: 2 Sequence length: 18 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Ser Ser Pro Gly Lys Pro Pro Arg Leu Val Gly Gly Pro Met Asp 1 5 10 15 Ala Ser
【００３６】配列番号：３ 配列の長さ：２８ 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 配列 Asp Thr His Ile Ile Lys Ala Gln Ser Gln Leu Val Ala Gly Ile Lys 1 5 10 15 Lys Try Phe Leu Thr Met Glu Met Gly Ser Thr Asp Cys 20 25 SEQ ID NO: 3 Sequence length: 28 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Asp Thr His Ile Ile Lys Ala Gln Ser Gln Leu Val Ala Gly Ile Lys 1 5 10 15 Lys Try Phe Leu Thr Met Glu Met Gly Ser Thr Asp Cys 20 25
【００３７】配列番号：４ 配列の長さ：２８ 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 配列 Asp Thr His Ile Ile Lys Ala Gln Ser Gln Leu Val Ala Gly Ile Lys 1 5 10 15 Tyr Phe Leu Thr Met Glu Met Gly Ser Thr Asp Cys 20 25 SEQ ID NO: 4 Sequence length: 28 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Asp Thr His Ile Ile Lys Ala Gln Ser Gln Leu Val Ala Gly Ile Lys 1 5 10 15 Tyr Phe Leu Thr Met Glu Met Gly Ser Thr Asp Cys 20 25
【００３８】配列番号：５ 配列の長さ：２２ 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 配列 Cys Asp Phe Glu Val Leu Val Val Pro Trp Gln Asn Ser Ser Gln Leu 5 10 15 Leu Lys His Asn Cys Val 20 SEQ ID NO: 5 Sequence length: 22 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Cys Asp Phe Glu Val Leu Val Val Pro Trp Gln Asn Ser Ser Gln Leu 5 10 15 Leu Lys His Asn Cys Val 20
【００３９】配列番号：６ 配列の長さ：２２ 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 配列 Cys Asp Phe Glu Val Leu Val Val Pro Trp Gln Xaa Ser Ser Gln Leu 1 5 10 15 Leu Lys His Asn Cys Val 20 SEQ ID NO: 6 Sequence length: 22 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Cys Asp Phe Glu Val Leu Val Val Pro Trp Gln Xaa Ser Ser Gln Leu 1 5 10 15 Leu Lys His Asn Cys Val 20
【００４０】配列番号：７ 配列の長さ：４５０ 配列の型：核酸 鎖の数：二本鎖 トポロジー：直鎖状 配列の種類：cDNA to mRNA 配列の特徴： 特徴を表す記号： sig peptide 存在位置： 1..84 特徴を決定した方法：Ｅ 特徴を表す記号： CDS 存在位置： 85..447 特徴を決定した方法：Ｅ 特徴を表す記号： terminator 存在位置： 448..450 特徴を決定した方法：Ｅ 配列 atggcgcgtt cgaacctccc gctggcgctg ggcctggccc tggtcgcatt ctgcctcctg 60 gcgctgccac gcgacgcccg ggcccggccg caggagcgca tggtcggaga actccgggac 120 ctgtcgcccg acgacccgca ggtgcagaag gcggcgcagg cggccgtggc cagctacaac 180 atgggcagca acagcatcta ctacttccga gacacgcaca tcatcaaggc gcagagccaa 240 ctggtggccg gcatcaagta cttcctgacg atggagatgg ggagcacaga ctgccgcaag 300 accagggtca ctggagacca cgtcgacctc accacttgcc ccctggcagc aggggcgcag 360 caggagaagc tgcgctgtga ctttgaggtc cttgtcgttc cctggcagaa ctcctctcag 420 ctcctaaagc acaactgtgt gcagatgtga 450SEQ ID NO: 7 Sequence length: 450 Sequence type: number of nucleic acid chains: double-stranded Topology: linear Sequence type: cDNA to mRNA Sequence characteristics: Characteristic symbol: sig peptide 1..84 Method for determining the feature: E Symbol representing the feature: CDS Location: 85..447 Method for determining the feature: E Symbol for representing the feature: terminator Location: 448..450 Method for determining the feature : E SEQ atggcgcgtt cgaacctccc gctggcgctg ggcctggccc tggtcgcatt ctgcctcctg 60 gcgctgccac gcgacgcccg ggcccggccg caggagcgca tggtcggaga actccgggac 120 ctgtcgcccg acgacccgca ggtgcagaag gcggcgcagg cggccgtggc cagctacaac 180 atgggcagca acagcatcta ctacttccga gacacgcaca tcatcaaggc gcagagccaa 240 ctggtggccg gcatcaagta cttcctgacg atggagatgg ggagcacaga ctgccgcaag 300 accagggtca ctggagacca cgtcgacctc accacttgcc ccctggcagc aggggcgcag 360 caggagaagc tgcgctgtga ctttgaggtc cttgtcgttc cctggcagaa ctcctctcag 420 ctcctaaagc acaactgtgt gcagatgtga 450
【００４１】配列番号：８ 配列の長さ：１４９ 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：タンパク質 配列 Met Ala Arg Ser Asn Leu Pro Leu Ala Leu Gly Leu Ala Leu Val Ala -25 -20 -15 Phe Cys Leu Leu Ala Leu Pro Arg Asp Ala Arg Ala Arg Pro Gln Glu -10 -5 -1 1 Arg Met Val Gly Glu Leu Arg Asp Leu Ser Pro Asp Asp Pro Gln Val 5 10 15 20 Gln Lys Ala Ala Gln Ala Ala Val Ala Ser Tyr Asn Met Gly Ser Asn 25 30 35 Ser Ile Tyr Tyr Phe Arg Asp Thr His Ile Ile Lys Ala Gln Ser Gln 40 45 50 Leu Val Ala Gly Ile Lys Tyr Phe Leu Thr Met Glu Met Gly Ser Thr 55 60 65 Asp Cys Arg Lys Thr Arg Val Thr Gly Asp His Val Asp Leu Thr Thr 70 75 80 Cys Pro Leu Ala Ala Gly Ala Gln Gln Glu Lys Leu Arg Cys Asp Phe 85 90 95 100 Glu Val Leu Val Val Pro Trp Gln Asn Ser Ser Gln Leu Leu Lys His 105 110 115 Asn Cys Val Gln Met 120SEQ ID NO: 8 Sequence length: 149 Sequence type: amino acid Topology: linear Sequence type: protein sequence Met Ala Arg Ser Asn Leu Pro Leu Ala Leu Gly Leu Ala Leu Val Ala -25 -20- 15 Phe Cys Leu Leu Ala Leu Pro Arg Asp Ala Arg Ala Arg Pro Gln Glu -10 -5 -1 1 Arg Met Val Gly Glu Leu Arg Asp Leu Ser Pro Asp Asp Pro Gln Val 5 10 15 20 Gln Lys Ala Ala Gln Ala Ala Val Ala Ser Tyr Asn Met Gly Ser Asn 25 30 35 Ser Ile Tyr Tyr Phe Arg Asp Thr His Ile Ile Lys Ala Gln Ser Gln 40 45 50 Leu Val Ala Gly Ile Lys Tyr Phe Leu Thr Met Glu Met Gly Ser Thr 55 60 65 Asp Cys Arg Lys Thr Arg Val Thr Gly Asp His Val Asp Leu Thr Thr 70 75 80 Cys Pro Leu Ala Ala Gly Ala Gln Gln Glu Lys Leu Arg Cys Asp Phe 85 90 95 100 Glu Val Leu Val Val Pro Trp Gln Asn Ser Ser Gln Leu Leu Lys His 105 110 115 Asn Cys Val Gln Met 120
【００４２】配列番号：９ 配列の長さ：２０ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 agcaacagca tctactactt 20SEQ ID NO: 9 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence agcaacagca tctactactt 20
【００４３】配列番号：１０ 配列の長さ：３４ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 gagaattcgg taccatcaag tacttcctga cgat 34SEQ ID NO: 10 Sequence length: 34 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence gagaattcgg taccatcaag tacttcctga cgat 34
【００４４】配列番号：１１ 配列の長さ：３４ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 gagtcgactg cagaagcttt tttttttttt tttt 34SEQ ID NO: 11 Sequence length: 34 Sequence type: number of nucleic acid chains: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence gagtcgactg cagaagcttt tttttttttt tttt 34
【００４５】配列番号：１２ 配列の長さ：１８ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 gagtcgactg cagaagct 17Sequence number: 12 Sequence length: 18 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence gagtcgactg cagaagct 17
【００４６】配列番号：１３ 配列の長さ：２０ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 tcaaagtcac agcgcagctt 20SEQ ID NO: 13 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence tcaaagtcac agcgcagctt 20
【００４７】配列番号：１４ 配列の長さ：３４ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 gagaattcgg taccatcgtc aggaagtact tgat 34SEQ ID NO: 14 Sequence length: 34 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence gagaattcgg taccatcgtc aggaagtact tgat 34
【００４８】配列番号：１５ 配列の長さ：２０ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 aggagcgcat ggtcggagaa 20SEQ ID NO: 15 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence aggagcgcat ggtcggagaa 20
【００４９】配列番号：１６ 配列の長さ：２０ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 ctggtcttgc ggcagtctgt 20SEQ ID NO: 16 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ctggtcttgc ggcagtctgt 20
【００５０】配列番号：１７ 配列の長さ：２９ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 gaattcaggc ctcggccgca ggagcgcat 29SEQ ID NO: 17 Sequence length: 29 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence gaattcaggc ctcggccgca ggagcgcat 29
【００５１】配列番号：１８ 配列の長さ：２４ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成ＤＮＡ 配列 aagcttcatc tgcacacagt tgtg 24SEQ ID NO: 18 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence aagcttcatc tgcacacagt tgtg 24
【図１】本発明蛋白質のゲル濾過による分画プロファイ
ルを示す。FIG. 1 shows the fractionation profile of the protein of the present invention by gel filtration.
─●−：活性 ───：ＯＤ280nm の吸光度 ─ ●-: Activity ───: Absorbance at OD280nm
【図２】ゲル濾過によって得られた分子量13kDa の本発
明蛋白質の、逆相ＨＰＬＣにおける溶出プロファイルを
示す。FIG. 2 shows an elution profile of a protein of the present invention having a molecular weight of 13 kDa obtained by gel filtration, which is measured by reversed-phase HPLC.
【図３】ゲル濾過によって得られた分子量16kDa の本発
明蛋白質の、逆相ＨＰＬＣにおける溶出プロファイルを
示す。FIG. 3 shows an elution profile of a protein of the present invention having a molecular weight of 16 kDa obtained by gel filtration, which is measured by reversed-phase HPLC.
【図４】ＨＰＬＣにより分画した本発明の蛋白質の、SD
S-PAGEの泳動パターンを示す。FIG. 4: SD of the protein of the present invention fractionated by HPLC
1 shows an S-PAGE electrophoresis pattern.
１：分子量16kDa の本発明蛋白質の還元条件下における
パターン ２：分子量13kDa の本発明蛋白質の還元条件下における
パターン ３：分子量16kDa の本発明蛋白質の非還元条件下におけ
るパターン ４：分子量13kDa の本発明蛋白質の非還元条件下におけ
るパターン1: Pattern under the reducing conditions of the protein of the present invention having a molecular weight of 16 kDa 2: Pattern under the reducing conditions of the protein of the present invention having a molecular weight of 13 kDa 3: Pattern under the non-reducing conditions of the protein of the present invention having a molecular weight of 16 kDa 4: The present invention having a molecular weight of 13 kDa Patterns of proteins under non-reducing conditions
【図５】分子量13kDa の本発明の蛋白質加水分解物の、
逆相ＨＰＬＣにおける溶出プロファイルを示す。FIG. 5 shows the protein hydrolyzate of the present invention having a molecular weight of 13 kDa.
Fig. 3 shows an elution profile in reverse phase HPLC.
【図６】分子量16kDa の本発明の蛋白質加水分解物の、
逆相ＨＰＬＣにおける溶出プロファイルを示す。FIG. 6 shows the protein hydrolyzate of the present invention having a molecular weight of 16 kDa.
Fig. 3 shows an elution profile in reverse phase HPLC.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｐ 21/02 Ｃ１２Ｐ 21/02 Ｃ // Ａ６１Ｋ 38/55 ＡＢＦ Ｃ１２Ｎ 9/99 ＡＢＪ Ａ６１Ｋ 37/64 ＡＢＦ ＡＤＹ ＡＢＪ Ｃ１２Ｎ 9/99 ＡＤＹ (Ｃ１２Ｐ 21/02 Ｃ１２Ｒ 1:19） ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 6 Identification code Agency reference number FI Technical display location C12P 21/02 C12P 21/02 C // A61K 38/55 ABF C12N 9/99 ABJ A61K 37/64 ABF ADY ABJ C12N 9/99 ADY (C12P 21/02 C12R 1:19)
Claims (7)
阻害活性を有する蛋白質。 (1) 還元及び非還元条件下のSDS-PAGEによる分子量測
定で、13±2kDa又は16±2kDaを示す。 (2) 13±2kDaの蛋白質はPAS 染色陰性で、等電点 4.8
〜5.0 を示す。16±2 kDa の蛋白質は PAS染色陽性で等
電点 4.4〜4.6 を示す。 (3) 配列表配列番号１に示されるＮ末端アミノ酸配列
を有する。 (4) 固定化したカルボキシメチル化パパインに結合性
を有する。 (5) カテプシンＬに特異的な阻害活性を有する。1. A protein having the following properties and having cysteine protease inhibitory activity. (1) Measurement of molecular weight by SDS-PAGE under reducing and non-reducing conditions shows 13 ± 2 kDa or 16 ± 2 kDa. (2) The 13 ± 2 kDa protein was negative for PAS staining and had an isoelectric point of 4.8.
~ 5.0. The protein of 16 ± 2 kDa is positive for PAS staining and has an isoelectric point of 4.4 to 4.6. (3) It has the N-terminal amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing. (4) It has a binding property to immobilized carboxymethylated papain. (5) It has a cathepsin L-specific inhibitory activity.
アミノ酸配列を有する、請求項１記載の蛋白質。2. The protein according to claim 1, which has an internal amino acid sequence shown in SEQ ID NOs: 3 and 5 in the sequence listing.
アミノ酸配列を有する、請求項１記載の蛋白質。3. The protein according to claim 1, which has an internal amino acid sequence shown in SEQ ID NOs: 4 and 6 in the sequence listing.
列を有する、請求項１〜３のいずれかに記載の蛋白質。4. The protein according to claim 1, which has an amino acid sequence represented by SEQ ID NO: 7 in the sequence listing.
列をコードするｃＤＮＡ。5. A cDNA encoding the amino acid sequence represented by SEQ ID NO: 7 in the sequence listing.
有する、請求項１〜４のいずれかに記載の蛋白質をコー
ドするＤＮＡ。6. A DNA encoding the protein according to any one of claims 1 to 4, which has the nucleotide sequence shown in SEQ ID NO: 8 in the sequence listing.
化担体を用いたアフィニティクロマトグラフィーにかけ
てシスタチン画分を吸着させ、この吸着画分をアルカリ
条件下で溶出させ、ゲルろ過及び逆相クロマトグラフィ
ーにより精製することを特徴とする請求項１記載のシス
テインプロテアーゼ阻害活性を有する蛋白質の製造法。7. A human milk is subjected to affinity chromatography using a carboxymethylated papain-immobilized carrier to adsorb the cystatin fraction, and the adsorbed fraction is eluted under alkaline conditions, and purified by gel filtration and reverse phase chromatography. The method for producing a protein having cysteine protease inhibitory activity according to claim 1, wherein
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8257644A JPH1080281A (en) | 1996-09-06 | 1996-09-06 | New protein and its production |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8257644A JPH1080281A (en) | 1996-09-06 | 1996-09-06 | New protein and its production |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH1080281A true JPH1080281A (en) | 1998-03-31 |
Family
ID=17309114
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8257644A Pending JPH1080281A (en) | 1996-09-06 | 1996-09-06 | New protein and its production |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH1080281A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1040833A1 (en) * | 1999-03-30 | 2000-10-04 | Snow Brand Milk Products, Co., Ltd. | Bone resorption suppressing agent |
| EP1161881A3 (en) * | 2000-06-09 | 2004-05-06 | Snow Brand Milk Products, Co., Ltd. | Method of producing fractions containing a high concentration of milk basic cystatin and decomposition products thereof |
| WO2004050116A1 (en) * | 2002-11-29 | 2004-06-17 | Morinaga Milk Industry Co., Ltd. | Protease inhibitor |
| EP1602284A1 (en) * | 2000-06-09 | 2005-12-07 | Snow Brand Milk Products, Co., Ltd. | Method of producing fractions containing a high concentration of milk basic cystatin and decomposition products thereof |
-
1996
- 1996-09-06 JP JP8257644A patent/JPH1080281A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1040833A1 (en) * | 1999-03-30 | 2000-10-04 | Snow Brand Milk Products, Co., Ltd. | Bone resorption suppressing agent |
| US6607743B1 (en) | 1999-03-30 | 2003-08-19 | Snow Brand Milk Products Co., Ltd. | Bone resorption suppressing agent |
| EP1161881A3 (en) * | 2000-06-09 | 2004-05-06 | Snow Brand Milk Products, Co., Ltd. | Method of producing fractions containing a high concentration of milk basic cystatin and decomposition products thereof |
| EP1602284A1 (en) * | 2000-06-09 | 2005-12-07 | Snow Brand Milk Products, Co., Ltd. | Method of producing fractions containing a high concentration of milk basic cystatin and decomposition products thereof |
| WO2004050116A1 (en) * | 2002-11-29 | 2004-06-17 | Morinaga Milk Industry Co., Ltd. | Protease inhibitor |
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