JP2003024060A - Method for obtaining useful protein from fusion protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for selective cutting in order to obtain a protein kinase from a fusion protein, and a method for obtaining a protein kinase from the fusion protein by enzymatically cutting an amino-acid sequence at its linkage part or the periphery of the linkage part. SOLUTION: This method for obtaining a desired protein kinase is carried out by cutting the linkage part or its periphery without cutting the sequence in the protein part of a fusion protein prepared by linking a tag and a protein kinase. In the process, a nonspecific endoprotease is used.

Description

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【発明の属する技術分野】本発明は、融合蛋白質よりプ
ロテインキナーゼを取得するための選択的な切断方法に
関する。また、本発明は連結部分やその周辺部分のアミ
ノ酸配列を酵素的に切断することを特徴とする融合蛋白
質よりプロテインキナーゼを取得する方法に関する。本
発明は、通常の方法では選択的に切断し難い融合蛋白質
などに有用である。また、本発明によって、非常に安価
に克つ簡便に融合蛋白質よりプロテインキナーゼを調製
することが可能となる。TECHNICAL FIELD The present invention relates to a selective cleavage method for obtaining a protein kinase from a fusion protein. The present invention also relates to a method for obtaining a protein kinase from a fusion protein, which is characterized by enzymatically cleaving the amino acid sequence of the connecting portion and its peripheral portion. INDUSTRIAL APPLICABILITY The present invention is useful for fusion proteins and the like that are difficult to selectively cleave by ordinary methods. Further, according to the present invention, it becomes possible to prepare a protein kinase from a fusion protein easily at a very low cost.

【０００２】[0002]

【従来の技術】遺伝子工学の進歩は、これまで天然物か
ら精製していた蛋白質を遺伝子レベルで解析し、目的蛋
白質を人工的に増幅させることを可能にした。その後発
明されたチオレドキシン（特表平５−５０７２０９）
や、グルタチオン−Ｓ−トランスフェラーゼ（ＧＳＴ）
（特表平１−５０３４４１）等を融合させたＤＮＡ配列
の応用によって、本来発現しにくいような蛋白質をも発
現させることができるようになり、融合蛋白質を発現さ
せる技術は広く用いられるようになった。更に、これら
の融合蛋白質は融合させたペプチドに結合する化合物を
担体として利用することにより簡便に精製することが可
能であり、精製のステップを大幅に簡略化できる。この
ような目的から、融合蛋白質調製に用いることを目的と
した特定のアミノ酸配列のペプチド（タグ）が種々開発
されてきた。結果として、隣接するヒスチジン残基を含
む親和性ペプチド（Ｈｉｓ−ｔａｇ）（特開昭６３−２
５１０９５）やマルトース結合蛋白質（特開平１−２０
０９４）などと所望の蛋白質を連結部分を介して融合さ
せ、作成した融合蛋白質をアフィニティークロマトグラ
フィー技術等を用いて簡便に精製する技術が確立してい
る。しかしながら、融合蛋白質より所望の蛋白質を取得
するためには融合させたペプチドを切断、除去する必要
がある。融合蛋白質が所望の蛋白質と実質的に同等の特
性、生理活性を有する場合は、融合蛋白質をそのまま応
用に用いることが可能であるが、このようなことは稀で
ある。多くの融合蛋白質は融合させたペプチドの影響を
受け、その特性や生理活性が少なからず変化する。融合
させたペプチドを除去する方法としては酵素的に切断す
る方法と化学反応による方法が考えられるが、所望の蛋
白質を安定な状態で取得するため一般的には酵素的に切
断する方法が採用される。酵素的切断方法とは、所望の
蛋白質と融合させたペプチドとの間の連結部分に配列特
異的プロテアーゼの認識アミノ酸配列をあらかじめ入れ
ておき、対応する配列特異的プロテアーゼを作用させて
融合させたペプチドを除去する方法である。配列特異的
プロテアーゼは特定のアミノ酸配列のみを認識し切断す
ることができる酵素で、所望の蛋白質を切断してしまう
心配が無い。しかしながら、配列特異的プロテアーゼは
非常に高価であり実用的酵素の創製に本方法を用いるこ
とは現実的ではなく、安価でしかも配列特異的プロテア
ーゼと同等の効果と簡便性が得られる新しい方法が望ま
れていた。2. Description of the Related Art Advances in genetic engineering have made it possible to artificially amplify a target protein by analyzing, at the gene level, a protein that has been purified from a natural product. Thioredoxin invented after that (Tokuhyo 5-507209)
And glutathione-S-transferase (GST)
By applying a DNA sequence in which (Table 1-503441) and the like are fused, it becomes possible to express even a protein that is difficult to express originally, and the technique for expressing a fusion protein has become widely used. It was Furthermore, these fusion proteins can be easily purified by using a compound that binds to the fused peptide as a carrier, and the purification step can be greatly simplified. For this purpose, various peptides (tags) having a specific amino acid sequence have been developed for use in the preparation of fusion proteins. As a result, an affinity peptide (His-tag) containing adjacent histidine residues (JP-A-63-2).
51095) and maltose binding protein (JP-A 1-20
094) and the like and a desired protein are fused via a connecting portion, and a technique for easily purifying the produced fusion protein using an affinity chromatography technique or the like has been established. However, in order to obtain the desired protein from the fusion protein, it is necessary to cleave and remove the fused peptide. When the fusion protein has substantially the same properties and physiological activities as the desired protein, the fusion protein can be directly used for the application, but this is rare. Many fusion proteins are affected by the fused peptides, and their properties and physiological activities are considerably changed. As a method for removing the fused peptide, an enzymatic cleavage method or a chemical reaction method can be considered.However, in order to obtain the desired protein in a stable state, an enzymatic cleavage method is generally adopted. It Enzymatic cleavage method is a peptide fused with a sequence-specific protease recognition amino acid sequence that has been inserted in advance at the junction between the desired protein and the fused peptide, and the corresponding sequence-specific protease is allowed to act. Is a method of removing. A sequence-specific protease is an enzyme capable of recognizing and cleaving only a specific amino acid sequence, and there is no fear of cleaving a desired protein. However, sequence-specific proteases are extremely expensive, and it is not practical to use this method to create a practical enzyme, and a new method that is inexpensive and has the same effect and convenience as sequence-specific proteases is desired. It was rare.

【０００３】[0003]

【発明が解決しようとする課題】このような理由から、
安価で簡便に融合蛋白質の選択的な切断をおこなう方法
が求められていた。すなわち本発明の目的は、安価に克
つ簡便な方法で融合蛋白質を選択的に切断する方法を提
供することである。For these reasons,
There has been a need for an inexpensive and convenient method for selectively cleaving the fusion protein. That is, an object of the present invention is to provide a method for selectively cleaving a fusion protein by a simple method that is inexpensive and convenient.

【０００４】[0004]

【課題を解決するための手段】我々は、非特異的エンド
プロテアーゼを用いることにより、タグとプロテインキ
ナーゼとを連結した融合蛋白質を切断することが可能と
なることを見出し、本発明に至った。さらに詳しくは、
非特異的エンドプロテアーゼを用いることにより、連結
部分のアミノ酸配列が特定のアミノ酸配列を有する融合
蛋白質よりプロテインキナーゼを取得することが可能と
なることを見出し、本発明に至った。これまで、非特異
的エンドプロテアーゼは特定の配列を認識せず蛋白質を
複数の箇所で切断すると考えられてきたため、融合蛋白
質を選択的に切断する方法に用いることは不可能と思わ
れていた。しかしながら、我々は種々の検討をおこな
い、非特異的エンドプロテアーゼを用いて連結部分のア
ミノ酸配列が特定のアミノ酸配列を有する融合蛋白質よ
りプロテインキナーゼを取得することを可能とした。We have found that it is possible to cleave a fusion protein in which a tag and a protein kinase are linked by using a non-specific endoprotease, and arrived at the present invention. For more details,
The present inventors have found that the use of a non-specific endoprotease makes it possible to obtain a protein kinase from a fusion protein having a specific amino acid sequence in the linking portion, and completed the present invention. Until now, it has been considered that non-specific endoprotease does not recognize a specific sequence and cleaves the protein at a plurality of sites, and thus it has been considered impossible to use it for a method of selectively cleaving a fusion protein. However, we conducted various studies and made it possible to obtain a protein kinase from a fusion protein having a specific amino acid sequence in the connecting portion using a non-specific endoprotease.

【０００５】すなわち本発明は、以下の構成からなる。 ［１］タグとプロテインキナーゼとを連結した融合蛋白
質を、非特異的エンドプロテアーゼで切断する方法。 ［２］タグとプロテインキナーゼとを連結した融合蛋白
質に対し、蛋白質部分の配列を切断することなく、連結
部分またはその周辺部分の配列を切断し所望のプロテイ
ンキナーゼを取得する工程において、非特異的エンドプ
ロテアーゼを用いることを特徴とする融合蛋白質よりプ
ロテインキナーゼを取得するための方法。 ［３］非特異的エンドプロテアーゼにより認識されやす
い配列が式（１）で表されることを特徴とする［２］に
記載の方法。 X-Met-Phe-Ser-(Xaa)n-Y（１） [式中、Xはプロテインキナーゼを意味しYはタグを意味
する。Xaaは任意のアミノ酸残基を意味する。nは０〜５
０の整数である。] ［４］非特異的エンドプロテアーゼにより認識されやす
い配列が式（２）で表されることを特徴とする［２］に
記載の方法。 X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys-(Xaa)n-Y（２） [式中、Xはプロテインキナーゼを意味しYはタグを意味
する。Xaaは任意のアミノ酸残基を意味する。nは０〜４
０の整数である。] ［５］非特異的エンドプロテアーゼにより認識されやす
い配列が式（３）で表されることを特徴とする［２］に
記載の方法。 X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys-Y（３） [式中、Xはプロテインキナーゼを意味しYはタグを意味
する。] ［６］タグがグルタチオン−Ｓ−トランスフェラーゼで
あることを特徴とする［１］〜［５］に記載の方法。 ［７］非特異的エンドプロテアーゼがプロティナーゼ
Ｋ、トリプシン、キモトリプシン、Ｖ８プロテアーゼよ
り選ばれることを特徴とする［１］〜［６］に記載の方
法。 ［８］非特異的エンドプロテアーゼがプロティナーゼＫ
であることを特徴とする［７］に記載の方法。That is, the present invention has the following configuration. [1] A method of cleaving a fusion protein in which a tag and a protein kinase are linked with a non-specific endoprotease. [2] Non-specific for a fusion protein in which a tag and a protein kinase are ligated, in a step of obtaining a desired protein kinase by cleaving the sequence of the ligation part or its peripheral part without cleaving the sequence of the protein part A method for obtaining a protein kinase from a fusion protein characterized by using an endoprotease. [3] The method according to [2], wherein the sequence easily recognized by the non-specific endoprotease is represented by the formula (1). X-Met-Phe-Ser- (Xaa) nY (1) [In the formula, X means a protein kinase and Y means a tag. Xaa means any amino acid residue. n is 0-5
It is an integer of 0. [4] The method according to [2], wherein the sequence that is easily recognized by the non-specific endoprotease is represented by the formula (2). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys- (Xaa) nY (2) [where X is a protein kinase and Y is a tag means. Xaa means any amino acid residue. n is 0-4
It is an integer of 0. [5] The method according to [2], wherein the sequence easily recognized by the non-specific endoprotease is represented by the formula (3). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys-Y (3) [wherein X means a protein kinase and Y means a tag. [6] The method according to [1] to [5], wherein the tag is glutathione-S-transferase. [7] The method according to [1] to [6], wherein the non-specific endoprotease is selected from proteinase K, trypsin, chymotrypsin, and V8 protease. [8] Proteinase K is a non-specific endoprotease
The method according to [7], wherein

【０００６】本発明において、非特異的エンドプロテア
ーゼとは、特定の配列を認識せず蛋白質を複数の箇所で
切断できるプロテアーゼをいう。詳しくは、蛋白質と反
応させたときには特定の配列を認識せず蛋白質を複数の
箇所で切断するが、本発明による該蛋白質の融合蛋白質
と反応させたときには、蛋白質部分の配列を切断するこ
となく、連結部分またはその周辺部分の配列を切断する
プロテアーゼをいう。In the present invention, the non-specific endoprotease refers to a protease which does not recognize a specific sequence and can cleave a protein at a plurality of sites. Specifically, when it reacts with a protein, it does not recognize a specific sequence and cleaves the protein at a plurality of locations, but when it reacts with the fusion protein of the protein according to the present invention, it does not cleave the sequence of the protein part, It refers to a protease that cleaves the sequence of the connecting portion or its peripheral portion.

【０００７】さらに詳しくは、下記のいずれかの性質を
有するプロテアーゼである。例えば、（ａ）１ｇ／Ｌの
プロテアーゼを２０ｍＭ、ｐＨ７．５のTris−HCｌ緩衝
液中で３ｇ／Ｌの蛋白質と３７℃で３０分反応させたと
きの収率が６０％以上であるが、（ｂ）同条件で本発明
による該蛋白質の融合蛋白質と反応させたときの所望の
蛋白質の収率が４０％を超える様な性質をもつプロテア
ーゼである。好ましくは、（ａ）において、蛋白質の収
率が８０％以上であるが、（ｂ）において本発明による
所望の蛋白質の収率が７０％を超える様な性質をもつプ
ロテアーゼであり、さらに好ましくは、（ａ）におい
て、蛋白質の収率が９５％以上であるが、（ｂ）におい
て本発明による所望の蛋白質の収率が９０％を超える様
な性質をもつプロテアーゼである。収率は、酵素などの
場合は活性を測定すること等により、あるいはイムノア
ッセイなどにより求めることが出来る。すなわち、プロ
テアーゼで処理する前後の活性あるいはイムノアッセイ
値の比率を求めることにより、収率を求めることができ
る。More specifically, it is a protease having any of the following properties. For example, (a) yield of 60% or more when 1 g / L protease is reacted with 3 g / L protein in 20 mM Tris-HCl buffer at pH 7.5 at 37 ° C. for 30 minutes, (B) A protease having the property that the yield of the desired protein exceeds 40% when it is reacted with the fusion protein of the protein according to the present invention under the same conditions. Preferably, the protease has a property such that the yield of the protein in (a) is 80% or more, but the yield of the desired protein according to the present invention in (b) exceeds 70%, more preferably , (A), the yield of the protein is 95% or more, while in (b), the yield of the desired protein of the present invention is 90% or more. The yield can be determined by measuring the activity in the case of an enzyme or the like, or by an immunoassay or the like. That is, the yield can be determined by determining the activity or the ratio of immunoassay values before and after the treatment with protease.

【０００８】あるいは、例えば、１ｇ／Ｌのプロテアー
ゼを２０ｍＭ、ｐＨ７．５のTris-HCl緩衝液中で３ｇ／
Ｌの蛋白質と３７℃で３０分反応させたとき、生成する
分解産物のうち最も量の多い分解産物の全体に対する比
率が４０％を超えない様な性質をもつプロテアーゼであ
る。好ましくは、最も量の多い分解産物の全体に対する
比率が２０％を超えない様な性質をもつプロテアーゼで
ある。さらに好ましくは、最も量の多い分解産物の全体
に対する比率が１０％を超えない様な性質をもつプロテ
アーゼである。分解産物の比率は例えばＳＤＳ−ＰＡＧ
Ｅなどのデータから計算により求めることが出来る。す
なわち、反応液のＳＤＳ−ＰＡＧＥの結果を一次元画像
解析装置、デンシトメーターにかけ、各分解産物のバン
ドの濃度を数値として算出することにより比率を求める
ことができる。Alternatively, for example, 1 g / L of protease is added to 3 g / L in 20 mM Tris-HCl buffer of pH 7.5.
It is a protease having such a property that, when it is reacted with L protein at 37 ° C. for 30 minutes, the ratio of the largest amount of degradation products to the total degradation products does not exceed 40%. Preferred is a protease having properties such that the ratio of the highest amount of degradation products to the whole does not exceed 20%. More preferably, it is a protease having properties such that the ratio of the highest amount of degradation products to the whole does not exceed 10%. The ratio of degradation products is, for example, SDS-PAG.
It can be calculated from data such as E. That is, the ratio can be determined by applying the SDS-PAGE result of the reaction solution to a one-dimensional image analyzer and a densitometer and calculating the band concentration of each degradation product as a numerical value.

【０００９】本発明の方法において対象となるプロテイ
ンキナーゼの性質については特に問わないが、特定の分
子量を有する単一の蛋白質であることが好ましい。The property of the protein kinase to be used in the method of the present invention is not particularly limited, but a single protein having a specific molecular weight is preferable.

【００１０】本発明における融合蛋白質の選択的な切断
方法は、以下の工程によって行われるのが好ましい。即
ち、（１）タグと対象となる蛋白質とを連結した融合蛋
白質をコードする遺伝子の作成。（２）融合蛋白質の合
成。（３）融合蛋白質の調製。（４）融合蛋白質の連結
部分またはその周辺部分の配列の酵素分解。（５）所望
の蛋白質の調製。ただ、（４）〜（５）の工程はまとめ
ても実質的に同様の結果が得られることになると思われ
る。The method for selectively cleaving the fusion protein in the present invention is preferably carried out by the following steps. That is, (1) preparation of a gene encoding a fusion protein in which a tag and a protein of interest are linked. (2) Synthesis of fusion protein. (3) Preparation of fusion protein. (4) Enzymatic degradation of the sequence of the fusion protein connecting region or its peripheral region. (5) Preparation of desired protein. However, even if the steps (4) to (5) are put together, substantially the same result will be obtained.

【００１１】本発明において、用いられる融合蛋白質の
合成手段は特に問わない。方法としては、従来からおこ
なわれてきた遺伝子工学的手法やその変法、および蛋白
質工学的手法などを用いることができる。具体的には、
タグと蛋白質とを遺伝子レベルで連結し、作成した融合
蛋白質遺伝子を遺伝子組換え技術を用いて組換え菌で発
現させて合成することができる。更に、培養・精製技術
を用いることにより融合蛋白質の純度を高め、実質的に
単一の蛋白質標品を得ることも可能である。微生物の遺
伝子操作系を用いて目的の蛋白質を得る一般的な方法を
以下に記す。In the present invention, the means for synthesizing the fusion protein used is not particularly limited. As the method, conventionally used genetic engineering methods, modified methods thereof, and protein engineering methods can be used. In particular,
The tag and the protein can be linked at the gene level, and the produced fusion protein gene can be expressed in a recombinant bacterium using a gene recombination technique and synthesized. Furthermore, it is also possible to increase the purity of the fusion protein and obtain a substantially single protein preparation by using a culture / purification technique. A general method for obtaining a target protein using a microbial gene manipulation system is described below.

【００１２】本発明の蛋白質の遺伝子を得る方法として
は、例えば染色体ＤＮＡを分離、精製した後、超音波破
砕、制限酵素処理等を用いてＤＮＡを切断したものと、
リニヤーな発現ベクターとを両ＤＮＡの平滑末端または
接着末端においてＤＮＡリガーゼなどにより結合閉鎖さ
せて組換えベクターを構築する。こうして得られた組換
えベクターは複製可能な宿主微生物に移入した後、ベク
ターのマーカーと酵素活性の発現を指標としてスクリー
ニングして、所望の蛋白質をコードする遺伝子を含有す
る組換えベクターを保持する微生物を得る。次いで該微
生物を培養し、該培養菌体から該組換えベクターを分離
・精製し、該組換えベクターから所望の蛋白質の遺伝子
を採取すれば良い。The method for obtaining the gene of the protein of the present invention includes, for example, a method in which chromosomal DNA is separated and purified, and then the DNA is cleaved using ultrasonic disruption, restriction enzyme treatment, or the like.
A recombinant expression vector is constructed by ligating and closing a linear expression vector at the blunt ends or cohesive ends of both DNAs with DNA ligase or the like. The recombinant vector thus obtained is transferred into a replicable host microorganism and then screened using the expression of the vector marker and the enzyme activity as an index, and a microorganism carrying the recombinant vector containing the gene encoding the desired protein. To get Then, the microorganism may be cultured, the recombinant vector may be separated and purified from the cultured cells, and the gene of the desired protein may be collected from the recombinant vector.

【００１３】染色体ＤＮＡは、具体的には、以下のよう
に採取される。すなわち、供与生物を例えば１〜３日間
撹拌培養して得られた培養物を遠心分離にて集菌し、次
いでこれを溶菌させることにより遺伝子の含有溶菌物を
調製することができる。溶菌の方法としては、例えばリ
ゾチームやβ−グルカナーゼ等の溶菌酵素により処理が
施され、必要に応じてプロテアーゼや他の酵素やラウリ
ル硫酸ナトリウム（SDS)等の界面活性剤が併用され、さ
らに凍結融解やフレンチプレス処理のような物理的破砕
方法と組み合わせても良い。このようにして得られた溶
菌物からＤＮＡを分離・精製するには常法に従って、例
えばフェノール処理やプロテアーゼ処理による除蛋白処
理や、リボヌクレアーゼ処理、アルコール沈澱処理など
の方法を適宜組み合わせることによりおこなうことがで
きる。分離・精製されたＤＮＡを切断する方法は、例え
ば超音波処理、制限酵素処理などにより行うことができ
る。好ましくは特定のヌクレオチド配列に作用するII型
制限酵素が適している。The chromosomal DNA is specifically collected as follows. That is, the gene-containing lysate can be prepared by collecting the culture obtained by stirring and culturing the donor organism for 1 to 3 days by centrifugation, and then lysing the culture. As a lysing method, for example, treatment is performed with a lysing enzyme such as lysozyme or β-glucanase, and if necessary, a protease or other enzyme and a surfactant such as sodium lauryl sulfate (SDS) are used together, and further freeze-thawing It may be combined with a physical crushing method such as or French press treatment. In order to separate and purify DNA from the lysate thus obtained, a conventional method may be used, for example, deproteinization treatment by phenol treatment or protease treatment, ribonuclease treatment, alcohol precipitation treatment and the like are appropriately combined. You can The method of cleaving the separated / purified DNA can be carried out, for example, by ultrasonic treatment or restriction enzyme treatment. Type II restriction enzymes that act on specific nucleotide sequences are preferred.

【００１４】ベクターとしては、宿主微生物内で自律的
に増殖し得るファージまたはプラスミドから遺伝子組換
え用として構築されたものが適している。ファージとし
ては、例えばエシェリヒア・コリー（Escherichia col
i）を宿主微生物とする場合には、Lambda-gt10、Lambda
-gt11などが使用できる。またプラスミドとしては、例
えばエシェリヒア・コリーを宿主微生物とする場合に
は、ｐＢＲ３２２，ｐＵＣ１９、pBluescript 、ｐＬＥ
Ｄ−Ｍ１(Journal of Fermentation and Bioenzineerin
g, Vol.76, 265-269 (1993))などが使用できる。このよ
うなベクターを、上述した染色ＤＮＡの切断に使用した
制限酵素で切断してベクター断片を得ることができる
が、必ずしも染色体ＤＮＡの切断に使用した制限酵素と
同一の制限酵素を用いる必要はない。染色体ＤＮＡ断片
とベクターＤＮＡ断片とを結合させる方法は、公知のＤ
ＮＡリガーゼを用いる方法であれば良く、例えば染色体
ＤＮＡ断片の接着末端とベクター断片の接着末端とのア
ニーリングの後、適当なＤＮＡリガーゼの使用により染
色体ＤＮＡ断片とベクターＤＮＡ断片との組換えベクタ
ーを作成する。必要なら、アニーリングの後、宿主微生
物に移入して生体内のＤＮＡリガーゼを利用し組換えベ
クターを作成することもできる。Suitable vectors are those constructed for gene recombination from phages or plasmids capable of autonomous growth in host microorganisms. Examples of phages include Escherichia col
When i) is used as a host microorganism, Lambda-gt10, Lambda
-gt11 etc. can be used. As a plasmid, for example, when Escherichia coli is used as a host microorganism, pBR322, pUC19, pBluescript, pLE
D-M1 (Journal of Fermentation and Bioenzineerin
g, Vol.76, 265-269 (1993)) can be used. Such a vector can be cleaved with the above-mentioned restriction enzyme used for the cleavage of the stained DNA to obtain a vector fragment, but it is not always necessary to use the same restriction enzyme as that used for the cleavage of the chromosomal DNA. . The method for ligating the chromosomal DNA fragment and the vector DNA fragment is known in the art.
A method using NA ligase may be used, for example, after the cohesive ends of the chromosomal DNA fragment and the cohesive ends of the vector fragment are annealed, a recombinant vector of the chromosomal DNA fragment and the vector DNA fragment is prepared by using an appropriate DNA ligase. To do. If necessary, after annealing, it can be transferred into a host microorganism and utilized in vivo DNA ligase to prepare a recombinant vector.

【００１５】宿主微生物としては、組換えベクターが安
定、かつ自律増殖可能で外来性遺伝子の形質発現できる
ものであれば良く、一般的にはエシェリヒア・コリーW3
110、エシェリヒア・コリーC600、エシェリヒア・コリ
ーHB101、エシェリヒア・コリーJM109などを用いること
ができる。宿主微生物に組換えベクターを移入する方法
としては、例えば宿主微生物がエシェリヒア・コリーの
場合には、カルシウム処理によるコンピテントセル法や
エレクトロポレーション法などが用いることができる。
このようにして得られた形質転換体である微生物は、栄
養培地で培養されることにより、多量の蛋白質を安定に
生産し得る。宿主微生物への目的組換えベクターの移入
の有無についての選択は、目的とするＤＮＡを保持する
ベクターの薬剤耐性マーカーを発現する微生物を検索す
れば良い。上記の方法により得られた組換えベクター
は、形質転換微生物から取り出され、他の微生物に移入
することも容易に実施することができる。また、制限酵
素やＰＣＲにより蛋白質をコードする遺伝子であるＤＮ
Ａを回収し、他のベクター断片と結合させ、宿主微生物
に移入することも容易に実施できる。The host microorganism may be any as long as the recombinant vector is stable, capable of autonomous growth, and capable of expressing a foreign gene, and generally Escherichia coli W3.
110, Escherichia coli C600, Escherichia coli HB101, Escherichia coli JM109 and the like can be used. As a method of transferring the recombinant vector into the host microorganism, for example, when the host microorganism is Escherichia coli, the competent cell method by calcium treatment or the electroporation method can be used.
The thus obtained transformant microorganism can stably produce a large amount of protein by culturing in a nutrient medium. The selection as to whether or not the target recombinant vector has been transferred into the host microorganism can be carried out by searching for a microorganism that expresses the drug resistance marker of the vector carrying the target DNA. The recombinant vector obtained by the above method can be easily taken out from a transformed microorganism and transferred to another microorganism. In addition, DN, which is a gene encoding a protein by restriction enzyme or PCR
It is also easy to collect A, combine it with another vector fragment, and transfer it to a host microorganism.

【００１６】形質転換体である宿主微生物の培養形態
は、宿主の栄養生理的性質を考慮して培養条件を選択す
ればよく、通常多くの場合は液体培養で行うが、工業的
には通気撹拌培養を行うのが有利である。培地の栄養源
としては、微生物の培養に通常用いられるものが広く使
用され得る。炭素源としては資化可能な炭素化合物であ
ればよく、例えばグルコ−ス、シュークロース、ラクト
ース、マルトース、フラクトース、糖蜜、ピルビン酸な
どが使用される。窒素源としては利用可能な窒素化合物
であればよく、例えばペプトン、肉エキス、酵母エキ
ス、カゼイン加水分解物、大豆粕アルカリ抽出物などが
使用される。その他、リン酸塩、炭酸塩、硫酸塩、マグ
ネシウム、カルシウム、カリウム、鉄、マンガン、亜鉛
などの塩類、特定のアミノ酸、特定のビタミンなどが必
要に応じて使用される。培養温度は菌が発育し、目的の
蛋白質を生産する範囲で適宜変更し得るが、エシェリヒ
ア コリーの場合、好ましくは20〜42℃程度である。培
養時間は条件によって多少異なるが、目的の蛋白質が最
高収量に達する時期を見計らって適当時期に培養を終了
すればよく、通常は６〜４８時間程度である。培地ｐＨ
は菌が発育し目的の蛋白質を生産する範囲で適宜変更し
得るが、特に好ましくはｐＨ６．０〜９．０程度であ
る。Regarding the culture form of the host microorganism which is a transformant, the culture conditions may be selected in consideration of the nutritional physiological properties of the host. Usually, in most cases, liquid culture is carried out, but industrially aeration stirring is carried out. It is advantageous to carry out culturing. As the nutrient source of the medium, those usually used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, and for example, glucose, sucrose, lactose, maltose, fructose, molasses, pyruvic acid, etc. are used. Any available nitrogen compound may be used as the nitrogen source, and for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkali extract, etc. are used. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids, specific vitamins and the like are used as necessary. The culturing temperature may be appropriately changed within the range in which the bacterium grows and produces the desired protein, but in the case of Escherichia coli, it is preferably about 20 to 42 ° C. Although the culturing time varies slightly depending on the conditions, it may be completed at a suitable time in consideration of the time when the desired protein reaches the maximum yield, and it is usually about 6 to 48 hours. Medium pH
Can be appropriately changed within the range in which the bacterium grows and produces the desired protein, but the pH is preferably about 6.0 to 9.0.

【００１７】培養物中の目的の蛋白質を生産する菌体を
含む培養液をそのまま採取し利用することもできるが、
一般には、常法に従って目的の蛋白質が培養液中に存在
する場合は濾過、遠心分離などにより、目的の蛋白質を
含有する溶液と微生物菌体とを分離した後に利用され
る。目的の蛋白質が菌体内に存在する場合には、得られ
た培養物から濾過または遠心分離などの手段により菌体
を採取し、次いでこの菌体を機械的方法またはリゾチー
ムなどの酵素的方法で破壊し、また必要に応じてＥＤＴ
Ａ等のキレート剤及びまたは界面活性剤を添加して目的
の蛋白質を可溶化し、水溶液として分離採取する。The culture solution containing the cells producing the desired protein in the culture can be directly collected and used.
Generally, when the target protein is present in the culture solution according to a conventional method, it is used after separating the solution containing the target protein from the microbial cells by filtration, centrifugation, or the like. When the target protein is present in the microbial cells, the microbial cells are collected from the obtained culture by means such as filtration or centrifugation, and then the microbial cells are destroyed by a mechanical method or an enzymatic method such as lysozyme. And EDT if necessary
A target protein is solubilized by adding a chelating agent such as A and / or a surfactant, and separated and collected as an aqueous solution.

【００１８】このようにして得られた目的の蛋白質を含
有する溶液を、例えば減圧濃縮、膜濃縮、更に硫酸アン
モニウム、硫酸ナトリウムなどの塩析処理、或いは親水
性有機溶媒、例えばメタノール、エタノール、アセトン
などによる分別沈澱法により沈澱せしめればよい。ま
た、加熱処理や等電点処理も有効な精製手段である。そ
の後、吸着剤或いはゲル濾過剤などによるゲル濾過、吸
着クロマトグラフィー、イオン交換クロマトグラフィ
ー、アフィニティークロマトグラフィーを行うことによ
り、精製された目的の蛋白質を得ることができる。例え
ば、セファデックス(Sephadex) G-25 （ファルマシア
バイオテク）などによるゲルろ過、DEAEセファロースCL
-6B(ファルマシア バイオテク）、オクチルセファロー
スCL6-B(ファルマシア バイオテク）カラムクロマトグ
ラフィーにより分離・精製し、精製酵素標品を得ること
ができる。この精製酵素標品は、電気泳動(SDS-PAGE)的
に、ほぼ単一のバンドを示す程度に純化されている。The solution containing the desired protein thus obtained is concentrated under reduced pressure, membrane concentration, salting out with ammonium sulfate, sodium sulfate or the like, or a hydrophilic organic solvent such as methanol, ethanol or acetone. It may be precipitated by the fractional precipitation method according to. Further, heat treatment and isoelectric point treatment are also effective refining means. Then, gel filtration using an adsorbent or a gel filter, adsorption chromatography, ion exchange chromatography, or affinity chromatography can be performed to obtain the purified target protein. For example, Sephadex G-25 (Pharmacia
Biotech) gel filtration, DEAE Sepharose CL
-6B (Pharmacia Biotech), octyl sepharose CL6-B (Pharmacia Biotech) column chromatography can be separated and purified to obtain a purified enzyme preparation. This purified enzyme preparation has been purified by electrophoresis (SDS-PAGE) so as to show an almost single band.

【００１９】また、無細胞蛋白質合成法を用いて融合蛋
白質を合成しても良い。無細胞蛋白質合成法を用いた蛋
白質の合成は、近年のバイオテクノロジーの進展にとも
ないその利用価値が急速に注目されるようになりつつあ
る。無細胞蛋白質合成は生体の蛋白質合成に関係する成
分を生体外へ取り出し、目的蛋白質をコードするmRNAと
ATP、GTPなどのエネルギー源を添加することにより、半
人工的に蛋白質を合成する手段である。その生体材料と
しては、大腸菌、網状赤血球や、コムギ胚芽が用いられ
ることが多く、現在までに様々な調製方法や利用方法が
検討されている。組換え微生物や培養細胞などを用いる
蛋白質の合成方法に対し無細胞蛋白質合成法の利点は、
１）生体に負に作用するような蛋白質（翻訳系に影響を
及ぼすものは除く）の合成が比較的容易である、２）簡
便に条件を決定することができる（微生物などを用いる
場合、最低一ヶ月程度を要するのが普通である）、３）
非天然型アミノ酸を用いることができる、ことなどを挙
げることができる。Alternatively, the fusion protein may be synthesized using a cell-free protein synthesis method. With the progress of biotechnology in recent years, the utility value of protein synthesis using the cell-free protein synthesis method is rapidly attracting attention. Cell-free protein synthesis takes out components related to protein synthesis in the living body to the outside of the body, and extracts mRNA that encodes the target protein.
It is a means to synthesize proteins artificially by adding energy sources such as ATP and GTP. Escherichia coli, reticulocytes, and wheat germ are often used as the biomaterials, and various preparation methods and utilization methods have been studied so far. The advantages of cell-free protein synthesis method over protein synthesis methods using recombinant microorganisms and cultured cells are:
1) Proteins that negatively act on the body (excluding those that affect the translation system) are relatively easy to synthesize. 2) Conditions can be easily determined (minimum when using microorganisms). It usually takes about a month) 3)
It can be mentioned that non-natural amino acids can be used.

【００２０】本発明において、融合蛋白質の連結部分ま
たはその周辺部分の配列を酵素的に分解するために使用
される配列非特異的プロテアーゼの由来は特に問わない
が、セリンプロテアーゼ、チオールプロテアーゼなどに
属するプロテアーゼ、具体的にはプロティナーゼＫ、ト
リプシン、キモトリプシン、サチライシン、Ｖ８プロテ
アーゼなどを用いることが好ましい。プロテアーゼによ
る分解反応の条件は各プロテアーゼの酵素的性質に依存
するが、一般的にはＧＯＯＤバッファーやリン酸バッフ
ァーなどの緩衝液で反応液のｐＨを固定し、１０〜９０
℃で５分間〜２４時間反応させる。詳しくは、例えば、
中性プロテアーゼを弱酸性〜弱アルカリ性のTris−HCｌ
緩衝液中で２０〜８０℃で１０分〜１６時間反応させ
る。 あるいは、アルカリプロテアーゼをｐＨ８〜１１
のＧＯＯＤバッファー中で１０〜６０℃で３０分〜１６
時間反応させる。In the present invention, the origin of the sequence-nonspecific protease used for enzymatically degrading the sequence of the connecting portion of the fusion protein or its peripheral portion is not particularly limited, but belongs to serine protease, thiol protease and the like. It is preferable to use protease, specifically proteinase K, trypsin, chymotrypsin, subtilisin, V8 protease and the like. The condition of the decomposition reaction by protease depends on the enzymatic properties of each protease, but generally, the pH of the reaction solution is fixed at 10 to 90 by fixing the pH of the reaction solution with a buffer solution such as GOOD buffer or phosphate buffer.
The reaction is performed at 5 ° C. for 5 minutes to 24 hours. For details, for example,
Neutral protease is weakly acidic to weakly alkaline Tris-HCl
The reaction is carried out in the buffer at 20 to 80 ° C. for 10 minutes to 16 hours. Alternatively, use alkaline protease at pH 8-11.
30 to 16 minutes at 10 to 60 ° C. in GOOD buffer of
React for hours.

【００２１】本発明で使用される融合蛋白質は合成した
後その溶液をそのまま使用しても良いが、精製したもの
を用いても良い。好ましくは、イオン交換クロマトグラ
フィー、疎水クロマトグラフィー、アフィニティークロ
マトグラフィー、ゲル濾過、ヒドロキシアパトイト、ま
たは等電点クロマトグラフィーを用いて高純度に精製す
る。さらに好ましくは、チオレドキシンやＧＳＴ、Ｈｉ
ｓ−ｔａｇ、マルトース結合蛋白質など融合させる特定
のアミノ酸配列のペプチドを特異的に認識し吸着するこ
とを利用した精製方法によりアフィニティー精製するこ
ともできる。The fusion protein used in the present invention may be used as it is after its synthesis after synthesis, or may be purified. Preferably, it is purified to high purity using ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, hydroxyapatite, or isoelectric focusing chromatography. More preferably, thioredoxin, GST, Hi
It is also possible to carry out affinity purification by a purification method utilizing specific recognition and adsorption of a peptide having a specific amino acid sequence to be fused such as s-tag and maltose binding protein.

【００２２】本発明の一態様は、融合蛋白質より所望の
蛋白質を簡便かつ安価に取得する方法である。また、本
発明の別な態様は、蛋白質精製・構造解析の研究に関す
る試薬系である。本発明を使用することにより、今まで
の技術では安価に精製することが困難であった蛋白質の
簡便な調製が可能となりうる。また、構造解析の材料供
給において効率よく目的の蛋白質を調製することが可能
となる。One aspect of the present invention is a method for easily and inexpensively obtaining a desired protein from a fusion protein. Another aspect of the present invention is a reagent system for research on protein purification / structural analysis. By using the present invention, it may be possible to easily prepare a protein, which has been difficult to purify at low cost by conventional techniques. In addition, it becomes possible to efficiently prepare a target protein in supplying materials for structural analysis.

【００２３】[0023]

【実施例】以下に、本発明の実施例を例示することによ
って、本発明の効果をより一層明確なものとするが、こ
れに限定されるものではない。 実施例 非特異的プロテアーゼによる処理とプロテイン
キナーゼの調製 新規な方法の実施例として、非特異的プロテアーゼによ
る処理とプロテインキナーゼの調製をあげることができ
る。ＧＳＴ−プロテインキナーゼ融合蛋白質をコードす
る遺伝子の配列を配列表の配列番号１に、ＧＳＴ−プロ
テインキナーゼ融合蛋白質のアミノ酸配列を配列表の配
列番号２に、それぞれ記載した。組換えプラスミドでエ
シェリヒア・コリーJM109のコンピテントセル（Compete
nt High（東洋紡製））を形質転換し、ＧＳＴ−プロテ
インキナーゼ融合蛋白質生産組換え菌を調製した。これ
を５０ｍｌ・ＴＢ−ｂｒｏｔｈを含む５００ｍｌ容量フ
ラスコに植菌し、３７℃で１７時間培養した。培養液よ
り菌体を遠心分離にて集菌し結合バッファー（１０ｍＭ
Ｎａ２ＨＰＯ４，１．８ｍＭ ＫＨ２ＰＯ４,１４０
ｍＭ ＮａＣｌ，２．７ｍＭ ＫＣｌ（ｐＨ７．３））
にて懸濁後、超音波破砕にて蛋白質を抽出した。破砕上
清をプレパック・カラムクロマトグラフィー（GSTrap
FF（アマーシャム・ファルマシア社製））に通し、製造
者のプロトコールに従い溶出バッファー（１０ｍＭ 還
元型グルタチオン，５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ
８．０））にて溶出した。更に、溶出液をＳｅｐｈａｄ
ｅｘＧ−２５脱塩・カラムクロマトグラフィー（Hitrap
-Desalting-（アマーシャム・ファルマシア社製））に
通し、製造者のプロトコールに従いバッファーＣ（２０
ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５））にて溶出、バ
ッファー置換し、融合蛋白質の精製標品を調製した。融
合蛋白質の精製標品を、プロティナーゼＫ（ナカライテ
スク社製）の２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．
５）溶液にて処理した。５０μｌ融合蛋白質：約２ｍｇ
／μｌに対しプロティナーゼＫ：約０．１〜１μｇ／ｍ
ｌの終濃度で添加し、３７℃で３０分間処理した。反応
は、プロティナーゼＫの阻害剤であるＰＭＳＦ（フッ化
フェニルメチルスルホニル（ナカライテスク社製））を
終濃度１ｍＭ分添加して終結させた。結果として、プロ
ティナーゼＫの処理濃度に伴い融合蛋白質の連結部位が
選択的に切断され、所望の蛋白質であるプロテインキナ
ーゼを得ることができた。EXAMPLES The effects of the present invention will be further clarified by exemplifying the examples of the present invention, but the present invention is not limited thereto. Examples Treatment with non-specific protease and preparation of protein kinase As an example of the novel method, treatment with non-specific protease and preparation of protein kinase can be mentioned. The sequence of the gene encoding the GST-protein kinase fusion protein is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the GST-protein kinase fusion protein is shown in SEQ ID NO: 2 in the sequence listing. Competitive cells of Escherichia coli JM109 (Compete
nt High (manufactured by Toyobo) was transformed to prepare a GST-protein kinase fusion protein-producing recombinant bacterium. This was inoculated into a 500 ml volumetric flask containing 50 ml TB-broth and cultured at 37 ° C. for 17 hours. The bacterial cells were collected from the culture solution by centrifugation and combined with a binding buffer (10 mM
Na2HPO4, 1.8 mM KH2PO4, 140
mM NaCl, 2.7 mM KCl (pH 7.3))
After suspending in, the protein was extracted by ultrasonication. The crushed supernatant is subjected to prepack column chromatography (GSTrap
Elution buffer (10 mM reduced glutathione, 50 mM Tris-HCl (pH) through FF (Amersham Pharmacia) according to the manufacturer's protocol.
It was eluted at 8.0)). In addition, the eluate is Sephad
exG-25 desalting / column chromatography (Hitrap
-Desalting- (manufactured by Amersham Pharmacia) according to the manufacturer's protocol.
Elution with mM Tris-HCl (pH 7.5)) and buffer replacement were performed to prepare a purified preparation of the fusion protein. A purified preparation of the fusion protein was treated with Proteinase K (Nacalai Tesque) 20 mM Tris-HCl (pH 7.
5) Treated with solution. 50 μl fusion protein: about 2 mg
/ Μl proteinase K: about 0.1 to 1 μg / m
It was added at a final concentration of 1 and treated at 37 ° C. for 30 minutes. The reaction was terminated by adding PMSF (phenylmethylsulfonyl fluoride (manufactured by Nacalai Tesque)), which is an inhibitor of proteinase K, at a final concentration of 1 mM. As a result, the ligation site of the fusion protein was selectively cleaved with the treatment concentration of proteinase K, and the desired protein kinase could be obtained.

【００２４】参考例１ 融合蛋白質をコードする遺伝子
の作成 所望の蛋白質として、尿酸測定臨床検査薬に使用する実
用的酵素であるバチルス・エスピー（Bacillus sp.）TB
90株由来ウリカーゼ（尿酸オキシダーゼ,EC1.7.3.3）を
取り上げた。特定のアミノ酸配列を有するペプチドとし
て、隣接するヒスチジン残基を含む親和性ペプチド、具
体的には６つのヒスチジン残基が並んだ６ｘＨｉｓ−ｔ
ａｇを取り上げ、これがウリカーゼのカルボキシル末端
に連結部分を介して結合した融合蛋白質をコードする遺
伝子の作成を下記の手順にておこなった。作成の流れ
は、図1においても示した。ウリカーゼ遺伝子を含むプ
ラスミドｐＵＯＤ（特開平２−５３４８８広報）(Journ
al of Biochemistry, Vol.119, 80-84 （1996）)（バ
イオインダストリー,Vol.13, 20-28（1996））を鋳型
として、以下の組成および条件にてポリメラーゼ・チェ
イン・リアクション（ＰＣＲ）をおこない融合蛋白質遺
伝子を作成するためのウリカーゼ遺伝子を調製した。 ＤＮＡポリメラーゼ（ＫＯＤ−Ｐｌｕｓ−（東洋紡製）） １μｌ 鋳型（ｐＵＯＤ） ０．０５ｎｇ プライマー（１）（配列表の配列番号３記載） ２５ｐｍｏｌｅ プライマー（２）（配列表の配列番号４記載） ２５ｐｍｏｌｅ ｘ１０ バッファー（東洋紡製） ５μｌ ２ｍＭ ｄＮＴＰ（東洋紡製） ５μｌ ２５ｍＭ ＭｇＳＯ４（東洋紡製） ４μｌ 全液量：５０μｌ ＰＣＲ条件： ９４℃,２分間 ↓ ９４℃,１５秒間→５０℃,３０秒間→６８℃,１分間 （３０サイクル） ↓ ６８℃,２分間 ＰＣＲ産物として取得したウリカーゼ遺伝子の配列を配
列表の配列番号５に、ウリカーゼのアミノ酸配列を配列
表の配列番号６に、それぞれ記載した。取得したウリカ
ーゼ遺伝子はＰＣＲ産物精製キット（MagExtractor-PCR
&Gel Clean Up-（東洋紡製））にてプロトコール通りに
精製し、制限酵素EcoRIとPstIにて切断し、同様にEcoRI
とPstIにて切断した発現ベクターｐＫＫ２２３−３（ア
マーシャム・ファルマシア社製）とＴ４ＤＮＡリガーゼ
を用いて連結した。得られたウリカーゼ遺伝子の発現プ
ラスミドをｐＵＯＤＳＰ１と命名した（図１）。ｐＵＯ
ＤＳＰ１を制限酵素SmaIとPstIで切断し、ウリカーゼ遺
伝子のカルボキシル末端側を開裂した。これに配列表の
配列番号７および８記載の配列を有する合成オリゴヌク
レオチドをＴ４ＤＮＡリガーゼを用いて連結し、最終的
に６つのヒスチジン残基が並んだ６ｘＨｉｓ−ｔａｇが
ウリカーゼのカルボキシル末端に連結部分を介して結合
した融合蛋白質をコードする遺伝子を含む発現プラスミ
ド、ｐＵＯＤＳＰ１−ＨＴを作成することができた（図
１）。作成した融合蛋白質をコードする遺伝子の配列を
配列表の配列番号９に、融合蛋白質のアミノ酸配列を配
列表の配列番号１０に、それぞれ記載した。Reference Example 1 Preparation of Gene Encoding Fusion Protein As a desired protein, Bacillus sp. TB which is a practical enzyme used in a clinical test drug for measuring uric acid.
Uricase from 90 strains (urate oxidase, EC 1.7.3.3) was taken up. As a peptide having a specific amino acid sequence, an affinity peptide containing adjacent histidine residues, specifically 6xHis-t in which 6 histidine residues are arranged.
Taking ag, a gene encoding a fusion protein in which this was bound to the carboxyl terminus of uricase via a linking portion was prepared by the following procedure. The flow of creation is also shown in FIG. Plasmid pUOD containing uricase gene (publication of JP-A-2-53488) (Journ
al of Biochemistry, Vol.119, 80-84 (1996)) (Bioindustry, Vol.13, 20-28 (1996)) as a template, and polymerase chain reaction (PCR) under the following composition and conditions. The uricase gene for preparing the fusion protein gene was prepared. DNA polymerase (KOD-Plus- (manufactured by Toyobo)) 1 μl Template (pUOD) 0.05 ng Primer (1) (described in SEQ ID NO: 3 in the sequence listing) 25 pmole primer (2) (described in SEQ ID NO: 4 in the sequence listing) 25 pmole x10 buffer (Toyobo) 5 μl 2 mM dNTP (Toyobo) 5 μl 25 mM MgSO4 (Toyobo) 4 μl Total solution volume: 50 μl PCR conditions: 94 ° C., 2 minutes ↓ 94 ° C., 15 seconds → 50 ° C., 30 seconds → 68 ° C., 1 minute (30 cycles) ↓ 68 ° C., 2 minutes The sequence of the uricase gene obtained as a PCR product is shown in SEQ ID NO: 5 in the sequence listing, and the amino acid sequence of uricase is shown in SEQ ID NO: 6 in the sequence listing. The obtained uricase gene is a PCR product purification kit (MagExtractor-PCR
& Gel Clean Up- (manufactured by Toyobo) according to the protocol, and cut with restriction enzymes EcoRI and PstI.
And the expression vector pKK223-3 (manufactured by Amersham Pharmacia) cleaved with PstI and T4 DNA ligase were used for ligation. The resulting uricase gene expression plasmid was named pUODSP1 (FIG. 1). pUO
DSP1 was cleaved with restriction enzymes SmaI and PstI to cleave the carboxyl terminal side of the uricase gene. Synthetic oligonucleotides having the sequences shown in SEQ ID NOs: 7 and 8 in the sequence listing were ligated to this using T4 DNA ligase, and finally 6xHis-tag in which 6 histidine residues were aligned was linked to the carboxy terminal of uricase. An expression plasmid, pUODSP1-HT, containing a gene encoding the fusion protein bound via the plasmid could be constructed (FIG. 1). The sequence of the gene encoding the prepared fusion protein is shown in SEQ ID NO: 9 in the sequence listing, and the amino acid sequence of the fusion protein is shown in SEQ ID NO: 10 in the sequence listing.

【００２５】参考例２ 融合蛋白質の調製 ｐＵＯＤＳＰ１−ＨＴでエシェリヒア・コリーJM109の
コンピテントセル（Competent High（東洋紡製））を形
質転換し、融合蛋白質生産組換え菌JM109/ｐＵＯＤＳＰ
１−ＨＴを調製した。これを５０ｍｌ・ＴＢ−ｂｒｏｔ
ｈを含む５００ｍｌ容量フラスコに植菌し、３７℃で１
７時間培養した。培養液より菌体を遠心分離にて集菌し
バッファーＡ（２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．
５）,３５０ｍＭ ＮａＣｌ）にて懸濁後、超音波破砕
にて蛋白質を抽出した。破砕上清をニッケルキレート・
カラムクロマトグラフィー（HiTrap-Chelating-（アマ
ーシャム・ファルマシア社製））に通し、製造者のプロ
トコールに従いバッファーＢ（２０ｍＭ Ｔｒｉｓ−Ｈ
Ｃｌ（ｐＨ７．５）, ３５０ｍＭ ＮａＣｌ,５００ｍ
Ｍ イミダゾール）にて溶出した。更に、溶出液をＳｅ
ｐｈａｄｅｘＧ−２５脱塩・カラムクロマトグラフィー
（Hitrap-Desalting-（アマーシャム・ファルマシア社
製））に通し、製造者のプロトコールに従いバッファー
Ｃ（２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５））にて
溶出、バッファー置換し、融合蛋白質の精製標品を調製
した。破砕上清および精製蛋白質のＳＤＳ−ポリアクリ
ルアミドゲルによる分析結果を、図２に示した。Reference Example 2 Preparation of Fusion Protein A competent cell of Escherichia coli JM109 (Competent High (manufactured by Toyobo)) was transformed with pUODSP1-HT to prepare a fusion protein producing recombinant bacterium JM109 / pUODSP.
1-HT was prepared. 50 ml of this, TB-brot
Inoculate into a 500 ml volumetric flask containing h and incubate at 37 ° C for 1
Cultured for 7 hours. The bacterial cells were collected from the culture solution by centrifugation, and buffer A (20 mM Tris-HCl (pH 7.
5), after suspending with 350 mM NaCl), the protein was extracted by ultrasonication. Crush the supernatant with nickel chelate
Pass through column chromatography (HiTrap-Chelating- (manufactured by Amersham Pharmacia)) and follow buffer B (20 mM Tris-H) according to the manufacturer's protocol.
Cl (pH 7.5), 350 mM NaCl, 500 m
(M imidazole). Furthermore, the eluate is Se
It is passed through a phasex G-25 desalting / column chromatography (Hitrap-Desalting- (manufactured by Amersham Pharmacia)), eluted with buffer C (20 mM Tris-HCl (pH 7.5)) according to the manufacturer's protocol, and the buffer is replaced. , A purified preparation of the fusion protein was prepared. The analysis results of the disrupted supernatant and the purified protein by SDS-polyacrylamide gel are shown in FIG.

【００２６】参考例３ 非特異的プロテアーゼによる処
理と所望の蛋白質の調製 融合蛋白質の精製標品を、プロティナーゼＫ（ナカライ
テスク社製）の２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．
５）溶液にて処理した。５０μｌ融合蛋白質：約２ｍｇ
／μｌに対しプロティナーゼＫ：約０．１〜１μｇ／ｍ
ｌの終濃度で添加し、３７℃で３０分間処理した。反応
は、プロティナーゼＫの阻害剤であるＰＭＳＦ（フッ化
フェニルメチルスルホニル（ナカライテスク社製））を
終濃度１ｍＭ分添加して終結させた。結果として、プロ
ティナーゼＫの処理濃度に伴い融合蛋白質のカルボキシ
ル末端が選択的に切断され、所望の蛋白質であるウリカ
ーゼを得ることができた。融合蛋白質のプロティナーゼ
Ｋ処理産物のＳＤＳ−ポリアクリルアミドゲルによる分
析結果を、図２に示した。融合蛋白質の連結部分がプロ
ティナーゼＫの酵素分解作用により切断を受け、６ｘＨ
ｉｓ−ｔａｇペプチドが除かれ、ウリカーゼが調製され
ていく様子が分析結果より明らかとなった。非特異的プ
ロテアーゼによる融合蛋白質の連結部分の切断は、ＭＡ
ＬＤＩ−ＴＯＦＦ−ＭＡＳＳによる質量分析によっても
確認された。非特異的プロテアーゼによる融合蛋白質の
選択的切断は連結部分または連結部分とその周辺部分の
構造的な特徴にあると推察されるが、現時点では詳細な
基礎的メカニズムは明らかとなっておらず、応用面での
有用性のみが判明している。従って、連結部分のアミノ
酸配列が式（１）で表されることを特徴とする、 X-Met-Phe-Ser-(Xaa)n-Y（１） [式中、Xは特定分子量の蛋白質を意味しYは特定アミノ
酸配列のペプチドを意味する。Xaaは任意のアミノ酸残
基を意味する。nは０〜５０の整数である。]或いは連結
部分のアミノ酸配列が式（２）で表されることを特徴と
する、 X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys-(Xaa)n-Y（２） [式中、Xは特定分子量の蛋白質を意味しYは特定アミノ
酸配列のペプチドを意味する。Xaaは任意のアミノ酸残
基を意味する。nは０〜４０の整数である。]或いは連結
部分のアミノ酸配列が式（３）で表されることを特徴と
する、 X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys-Y（３） [式中、Xは特定分子量の蛋白質を意味しYは特定アミノ
酸配列のペプチドを意味する。] 新規な方法としての情報が参考例として開示された。Reference Example 3 Treatment with Non-Specific Protease and Preparation of Desired Protein A purified preparation of the fusion protein was treated with proteinase K (Nacalai Tesque) 20 mM Tris-HCl (pH 7.
5) Treated with solution. 50 μl fusion protein: about 2 mg
/ Μl proteinase K: about 0.1 to 1 μg / m
It was added at a final concentration of 1 and treated at 37 ° C. for 30 minutes. The reaction was terminated by adding PMSF (phenylmethylsulfonyl fluoride (manufactured by Nacalai Tesque)), which is an inhibitor of proteinase K, at a final concentration of 1 mM. As a result, the carboxyl terminus of the fusion protein was selectively cleaved with the treatment concentration of proteinase K, and uricase, which was the desired protein, could be obtained. The analysis results of the proteinase K-treated product of the fusion protein by SDS-polyacrylamide gel are shown in FIG. The connecting portion of the fusion protein is cleaved by the enzymatic degradation of proteinase K to give 6xH.
The analysis results revealed that the uricase was being prepared by removing the is-tag peptide. Cleavage of the junction of the fusion protein by a non-specific protease
It was also confirmed by mass spectrometry by LDI-TOFF-MASS. It is speculated that the selective cleavage of the fusion protein by a non-specific protease is due to the structural characteristics of the ligation part or the ligation part and its peripheral part, but at this time, the detailed basic mechanism has not been clarified, and its application. Only its utility in terms of aspects is known. Therefore, X-Met-Phe-Ser- (Xaa) nY (1) is characterized in that the amino acid sequence of the linking portion is represented by formula (1) [wherein X represents a protein of a specific molecular weight] Y means a peptide having a specific amino acid sequence. Xaa means any amino acid residue. n is an integer of 0 to 50. ] Alternatively, the amino acid sequence of the connecting portion is represented by the formula (2), X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys- (Xaa) nY (2) [In the formula, X means a protein having a specific molecular weight, and Y means a peptide having a specific amino acid sequence. Xaa means any amino acid residue. n is an integer of 0-40. Alternatively, the amino acid sequence of the connecting portion is represented by the formula (3), X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys- Y (3) [In the formula, X means a protein having a specific molecular weight, and Y means a peptide having a specific amino acid sequence. ] Information as a novel method was disclosed as a reference example.

【００２７】参考例４ 融合蛋白質のアフィニティー・
ビーズへの結合と非特異的プロテアーゼ処理による所望
の蛋白質の調製 本特許の方法を利用すれば、従来は費用面から実質的に
困難であった融合蛋白質をアフィニティー・ビーズへ結
合させ配列選択的切断により所望の蛋白質を取得すると
いう手法が可能になる。この手法が可能となることによ
り、蛋白質のハイスループット精製が簡便に実施可能と
なる。本特許の方法による蛋白質のハイスループット精
製のフローの一例を図３に示した。参考例１と同様に、
融合蛋白質生産組換え菌JM109/ｐＵＯＤＳＰ１−ＨＴを
ＴＢ−ｂｒｏｔｈに植菌し、３７℃で１７時間培養し
た。培養液より菌体を遠心分離にて集菌しバッファーＡ
にて懸濁後、超音波破砕にて蛋白質を抽出した。破砕液
をニッケルキレート・磁性体ビーズ（MagExtractor-His
-tag-（東洋紡製））に吸着させ、製造者のプロトコー
ルに従い添付バッファーにてビーズを洗浄した。この操
作で、融合蛋白質が吸着した磁性体ビーズが調製され
る。この磁性体ビーズをプロティナーゼＫ（ナカライテ
スク社製）の２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．
５）溶液にて処理し、所望の蛋白質を溶出させた。融合
蛋白質吸着磁性ビーズ２０μｌ容量に対しプロティナー
ゼＫ溶液を約１〜４μｇ／ｍｌの濃度で１００μｌ添加
し、３７℃で６０分間処理した。結果として、プロティ
ナーゼＫの処理濃度に伴い融合蛋白質のカルボキシル末
端が選択的に切断され、所望の蛋白質であるウリカーゼ
を得ることができた。磁性体ビーズのプロティナーゼＫ
処理産物のＳＤＳ−ポリアクリルアミドゲルによる分析
結果を、図３に示した。融合蛋白質の連結部分がプロテ
ィナーゼＫの酵素分解作用により切断を受け、６ｘＨｉ
ｓ−ｔａｇペプチドが磁性体ビーズに吸着した状態で所
望の蛋白質が溶出され、固液分離により所望の蛋白質を
含む溶液が調製されていく様子が分析結果より明らかと
なった。非特異的プロテアーゼによる融合蛋白質の連結
部分の切断は、ＭＡＬＤＩ−ＴＯＦＦ−ＭＡＳＳによる
質量分析によっても確認された。なお、本参考例では磁
性体ビーズを用いているため、固液分離は遠心分離をお
こなわずとも磁石を用いて非常に簡便に実施することが
できる。磁石を用いた磁性体ビーズの固液分離システム
は自動化も容易であり、実際、自動化装置が既に開発、
販売されている（MFX-2000, MFX-6000, MFX-9600（東
洋紡製））。従って、これらの装置と本特許の方法を組
合せることにより、蛋白質のハイスループット精製は容
易に実現できる。Reference Example 4 Affinity of Fusion Protein
Preparation of desired protein by binding to beads and treatment with non-specific protease By using the method of this patent, it is possible to bind a fusion protein to affinity beads, which has been substantially difficult in terms of cost, by sequence selective cleavage. This makes it possible to obtain a desired protein. By enabling this technique, high-throughput purification of proteins can be easily carried out. An example of the flow of high throughput protein purification by the method of this patent is shown in FIG. Similar to Reference Example 1,
The fusion protein producing recombinant bacterium JM109 / pUODSP1-HT was inoculated into TB-broth and cultured at 37 ° C. for 17 hours. The bacterial cells are collected from the culture solution by centrifugation to collect the buffer A.
After suspending in, the protein was extracted by ultrasonication. Disintegrate liquid with nickel chelate / magnetic beads (MagExtractor-His
-tag- (manufactured by Toyobo)), and the beads were washed with the attached buffer according to the manufacturer's protocol. By this operation, magnetic beads to which the fusion protein is adsorbed are prepared. The magnetic beads were used as proteinase K (manufactured by Nacalai Tesque, Inc.) 20 mM Tris-HCl (pH 7.
5) Treatment with the solution to elute the desired protein. 100 μl of proteinase K solution was added to a volume of 20 μl of the fusion protein-adsorbed magnetic beads at a concentration of about 1 to 4 μg / ml, and the mixture was treated at 37 ° C. for 60 minutes. As a result, the carboxyl terminus of the fusion protein was selectively cleaved with the treatment concentration of proteinase K, and uricase, which was the desired protein, could be obtained. Proteinase K for magnetic beads
The analysis result of the treated product by SDS-polyacrylamide gel is shown in FIG. The connecting portion of the fusion protein is cleaved by the enzymatic decomposition action of proteinase K to give 6xHi.
The analysis results revealed that the desired protein was eluted with the s-tag peptide adsorbed on the magnetic beads, and a solution containing the desired protein was prepared by solid-liquid separation. Cleavage of the fusion protein junction by a non-specific protease was also confirmed by mass spectrometry by MALDI-TOFF-MASS. Since magnetic beads are used in this reference example, solid-liquid separation can be performed very easily using a magnet without centrifugation. The solid-liquid separation system for magnetic beads using a magnet is easy to automate, and in fact, an automation device has already been developed,
Sold (MFX-2000, MFX-6000, MFX-9600 (manufactured by Toyobo)). Therefore, by combining these apparatuses with the method of the present patent, high-throughput purification of proteins can be easily realized.

【００２８】[0028]

【発明の効果】本発明により、今までの技術では困難で
あった非特異的プロテアーゼによる融合蛋白質の選択的
な切断が可能となる。これにより、融合蛋白質より所望
の蛋白質を簡便克つ安価に調製することが可能となる。
また、本方法のコスト面での優位性を活かして、従来は
コスト的に難しかった蛋白質のハイスループット・スク
リーニングへの応用が期待できる。INDUSTRIAL APPLICABILITY According to the present invention, it is possible to selectively cleave a fusion protein with a non-specific protease, which has been difficult with conventional techniques. This makes it possible to easily and inexpensively prepare a desired protein from the fusion protein.
Further, by taking advantage of the cost advantage of this method, it can be expected to be applied to high-throughput screening of proteins, which was conventionally difficult in terms of cost.

【００２９】[0029]

【配列表】 <110> Toyo Boseki Kabushiki Kaisya <120> Specific cleavage method of fusion protein <130> 01-0506 <141> 2001-07-10 <160> 8 <170> PatentIn version 2.0 <210> 1 <211> 1899 <212> DNA <213> Homo sapiens <400> 1 atg tcc cct ata cta ggt tat tgg aaa att aag ggc ctt gtg caa ccc 48 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 act cga ctt ctt ttg gaa tat ctt gaa gaa aaa tat gaa gag cat ttg 96 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 tat gag cgc gat gaa ggt gat aaa tgg cga aac aaa aag ttt gaa ttg 144 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 ggt ttg gag ttt ccc aat ctt cct tat tat att gat ggt gat gtt aaa 192 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 tta aca cag tct atg gcc atc ata cgt tat ata gct gac aag cac aac 240 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 atg ttg ggt ggt tgt cca aaa gag cgt gca gag att tca atg ctt gaa 288 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 gga gcg gtt ttg gat att aga tac ggt gtt tcg aga att gca tat agt 336 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 aaa gac ttt gaa act ctc aaa gtt gat ttt ctt agc aag cta cct gaa 384 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 atg ctg aaa atg ttc gaa gat cgt tta tgt cat aaa aca tat tta aat 432 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 ggt gat cat gta acc cat cct gac ttc atg ttg tat gac gct ctt gat 480 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 gtt gtt tta tac atg gac cca atg tgc ctg gat gcg ttc cca aaa tta 528 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 gtt tgt ttt aaa aaa cgt att gaa gct atc cca caa att gat aag tac 576 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 ttg aaa tcc agc aag tat ata gca tgg cct ttg cag ggc tgg caa gcc 624 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 acg ttt ggt ggt ggc gac cat cct cca aaa tcg gat ctg atc gaa ggt 672 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Ile Glu Gly 210 215 220 cgt ggg atc ccc gaa ttc cca acc ctt atg ttc tct gat gaa ccc gat 720 Arg Gly Ile Pro Glu Phe Pro Thr Leu Met Phe Ser Asp Glu Pro Asp 225 230 235 240 cat aaa gga gca ctt aaa ccc ggt atg agc aga agc aag cgt gac aac 768 His Lys Gly Ala Leu Lys Pro Gly Met Ser Arg Ser Lys Arg Asp Asn 245 250 255 aat ttt tat agt gta gag att gga gat tct aca ttc aca gtc ctg aaa 816 Asn Phe Tyr Ser Val Glu Ile Gly Asp Ser Thr Phe Thr Val Leu Lys 260 265 270 cga tat cag aat tta aaa cct ata ggc tca gga gct caa gga ata gta 864 Arg Tyr Gln Asn Leu Lys Pro Ile Gly Ser Gly Ala Gln Gly Ile Val 275 280 285 tgc gca gct tat gat gcc att ctt gaa aga aat gtt gca atc aag aag 912 Cys Ala Ala Tyr Asp Ala Ile Leu Glu Arg Asn Val Ala Ile Lys Lys 290 295 300 cta agc cga cca ttt cag aat cag act cat gcc aag cgg gcc tac aga 960 Leu Ser Arg Pro Phe Gln Asn Gln Thr His Ala Lys Arg Ala Tyr Arg 305 310 315 320 gag cta gtt ctt atg aaa tgt gtt aat cac aaa aat ata att ggc ctt 1008 Glu Leu Val Leu Met Lys Cys Val Asn His Lys Asn Ile Ile Gly Leu 325 330 335 ttg aat gtt ttc aca cca cag aaa tcc cta gaa gaa ttt caa gat gtt 1056 Leu Asn Val Phe Thr Pro Gln Lys Ser Leu Glu Glu Phe Gln Asp Val 340 345 350 tac ata gtc atg gag ctc atg gat gca aat ctt tgc caa gtg att cag 1104 Tyr Ile Val Met Glu Leu Met Asp Ala Asn Leu Cys Gln Val Ile Gln 355 360 365 atg gag cta gat cat gaa aga atg tcc tac ctt ctc tat cag atg ctg 1152 Met Glu Leu Asp His Glu Arg Met Ser Tyr Leu Leu Tyr Gln Met Leu 370 375 380 tgt gga atc aag cac ctt cat tct gct gga att att cat cgg gac tta 1200 Cys Gly Ile Lys His Leu His Ser Ala Gly Ile Ile His Arg Asp Leu 385 390 395 400 aag ccc agt aat ata gta gta aaa tct gat tgc act ttg aag att ctt 1248 Lys Pro Ser Asn Ile Val Val Lys Ser Asp Cys Thr Leu Lys Ile Leu 405 410 415 gac ttc ggt ctg gcc agg act gca gga acg agt ttt atg atg acg cct 1296 Asp Phe Gly Leu Ala Arg Thr Ala Gly Thr Ser Phe Met Met Thr Pro 420 425 430 tat gta gtg act cgc tac tac aga gca ccc gag gtc atc ctt ggc atg 1344 Tyr Val Val Thr Arg Tyr Tyr Arg Ala Pro Glu Val Ile Leu Gly Met 435 440 445 ggc tac aag gaa aac gtt gac att tgg tca gtt ggg tgc atc atg gga 1392 Gly Tyr Lys Glu Asn Val Asp Ile Trp Ser Val Gly Cys Ile Met Gly 450 455 460 gaa atg atc aaa ggt ggt gtt ttg ttc cca ggt aca gat cat att gat 1440 Glu Met Ile Lys Gly Gly Val Leu Phe Pro Gly Thr Asp His Ile Asp 465 470 475 480 cag tgg aat aaa gtt att gaa cag ctt gga aca cca tgt cct gaa ttc 1488 Gln Trp Asn Lys Val Ile Glu Gln Leu Gly Thr Pro Cys Pro Glu Phe 485 490 495 atg aag aaa ctg caa cca aca gta agg act tac gtt gaa aac aga cct 1536 Met Lys Lys Leu Gln Pro Thr Val Arg Thr Tyr Val Glu Asn Arg Pro 500 505 510 aaa tat gct gga tat agc ttt gag aaa ctc ttc cct gat gtc ctt ttc 1584 Lys Tyr Ala Gly Tyr Ser Phe Glu Lys Leu Phe Pro Asp Val Leu Phe 515 520 525 cca gct gac tca gaa cac aac aaa ctt aaa gcc agt cag gca agg gat 1632 Pro Ala Asp Ser Glu His Asn Lys Leu Lys Ala Ser Gln Ala Arg Asp 530 535 540 ttg tta tcc aaa atg ctg gta ata gat gca tct aaa agg atc tct gta 1680 Leu Leu Ser Lys Met Leu Val Ile Asp Ala Ser Lys Arg Ile Ser Val 545 550 555 560 gat gaa gct ctc caa cac ccg tac atc aat gtc tgg tat gat cct tct 1728 Asp Glu Ala Leu Gln His Pro Tyr Ile Asn Val Trp Tyr Asp Pro Ser 565 570 575 gaa gca gaa gct cca cca cca aag atc cct gac aag cag tta gat gaa 1776 Glu Ala Glu Ala Pro Pro Pro Lys Ile Pro Asp Lys Gln Leu Asp Glu 580 585 590 agg gaa cac aca ata gaa gag tgg aaa gaa ttg ata tat aag gaa gtt 1824 Arg Glu His Thr Ile Glu Glu Trp Lys Glu Leu Ile Tyr Lys Glu Val 595 600 605 atg gac ttg gag gag aga acc aag aat gga gtt ata cgg ggg cag ccc 1872 Met Asp Leu Glu Glu Arg Thr Lys Asn Gly Val Ile Arg Gly Gln Pro 610 615 620 tct cct tta gca cag gtg cag cag tga 1899 Ser Pro Leu Ala Gln Val Gln Gln 625 630 [Sequence list] <110> Toyo Boseki Kabushiki Kaisya <120> Specific cleavage method of fusion protein <130> 01-0506 <141> 2001-07-10 <160> 8 <170> PatentIn version 2.0 <210> 1 <211> 1899 <212> DNA <213> Homo sapiens <400> 1 atg tcc cct ata cta ggt tat tgg aaa att aag ggc ctt gtg caa ccc 48 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro   1 5 10 15 act cga ctt ctt ttg gaa tat ctt gaa gaa aaa tat gaa gag cat ttg 96 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu              20 25 30 tat gag cgc gat gaa ggt gat aaa tgg cga aac aaa aag ttt gaa ttg 144 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu          35 40 45 ggt ttg gag ttt ccc aat ctt cct tat tat att gat ggt gat gtt aaa 192 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys      50 55 60 tta aca cag tct atg gcc atc ata cgt tat ata gct gac aag cac aac 240 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn  65 70 75 80 atg ttg ggt ggt tgt cca aaa gag cgt gca gag att tca atg ctt gaa 288 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu                  85 90 95 gga gcg gtt ttg gat att aga tac ggt gtt tcg aga att gca tat agt 336 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser             100 105 110 aaa gac ttt gaa act ctc aaa gtt gat ttt ctt agc aag cta cct gaa 384 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu         115 120 125 atg ctg aaa atg ttc gaa gat cgt tta tgt cat aaa aca tat tta aat 432 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn     130 135 140 ggt gat cat gta acc cat cct gac ttc atg ttg tat gac gct ctt gat 480 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 gtt gtt tta tac atg gac cca atg tgc ctg gat gcg ttc cca aaa tta 528 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu                 165 170 175 gtt tgt ttt aaa aaa cgt att gaa gct atc cca caa att gat aag tac 576 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr             180 185 190 ttg aaa tcc agc aag tat ata gca tgg cct ttg cag ggc tgg caa gcc 624 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala         195 200 205 acg ttt ggt ggt ggc gac cat cct cca aaa tcg gat ctg atc gaa ggt 672 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Ile Glu Gly     210 215 220 cgt ggg atc ccc gaa ttc cca acc ctt atg ttc tct gat gaa ccc gat 720 Arg Gly Ile Pro Glu Phe Pro Thr Leu Met Phe Ser Asp Glu Pro Asp 225 230 235 240 cat aaa gga gca ctt aaa ccc ggt atg agc aga agc aag cgt gac aac 768 His Lys Gly Ala Leu Lys Pro Gly Met Ser Arg Ser Lys Arg Asp Asn                 245 250 255 aat ttt tat agt gta gag att gga gat tct aca ttc aca gtc ctg aaa 816 Asn Phe Tyr Ser Val Glu Ile Gly Asp Ser Thr Phe Thr Val Leu Lys             260 265 270 cga tat cag aat tta aaa cct ata ggc tca gga gct caa gga ata gta 864 Arg Tyr Gln Asn Leu Lys Pro Ile Gly Ser Gly Ala Gln Gly Ile Val         275 280 285 tgc gca gct tat gat gcc att ctt gaa aga aat gtt gca atc aag aag 912 Cys Ala Ala Tyr Asp Ala Ile Leu Glu Arg Asn Val Ala Ile Lys Lys     290 295 300 cta agc cga cca ttt cag aat cag act cat gcc aag cgg gcc tac aga 960 Leu Ser Arg Pro Phe Gln Asn Gln Thr His Ala Lys Arg Ala Tyr Arg 305 310 315 320 gag cta gtt ctt atg aaa tgt gtt aat cac aaa aat ata att ggc ctt 1008 Glu Leu Val Leu Met Lys Cys Val Asn His Lys Asn Ile Ile Gly Leu                 325 330 335 ttg aat gtt ttc aca cca cag aaa tcc cta gaa gaa ttt caa gat gtt 1056 Leu Asn Val Phe Thr Pro Gln Lys Ser Leu Glu Glu Phe Gln Asp Val             340 345 350 tac ata gtc atg gag ctc atg gat gca aat ctt tgc caa gtg att cag 1104 Tyr Ile Val Met Glu Leu Met Asp Ala Asn Leu Cys Gln Val Ile Gln         355 360 365 atg gag cta gat cat gaa aga atg tcc tac ctt ctc tat cag atg ctg 1152 Met Glu Leu Asp His Glu Arg Met Ser Tyr Leu Leu Tyr Gln Met Leu     370 375 380 tgt gga atc aag cac ctt cat tct gct gga att att cat cgg gac tta 1200 Cys Gly Ile Lys His Leu His Ser Ala Gly Ile Ile His Arg Asp Leu 385 390 395 400 aag ccc agt aat ata gta gta aaa tct gat tgc act ttg aag att ctt 1248 Lys Pro Ser Asn Ile Val Val Lys Ser Asp Cys Thr Leu Lys Ile Leu                 405 410 415 gac ttc ggt ctg gcc agg act gca gga acg agt ttt atg atg acg cct 1296 Asp Phe Gly Leu Ala Arg Thr Ala Gly Thr Ser Phe Met Met Thr Pro             420 425 430 tat gta gtg act cgc tac tac aga gca ccc gag gtc atc ctt ggc atg 1344 Tyr Val Val Thr Arg Tyr Tyr Arg Ala Pro Glu Val Ile Leu Gly Met         435 440 445 ggc tac aag gaa aac gtt gac att tgg tca gtt ggg tgc atc atg gga 1392 Gly Tyr Lys Glu Asn Val Asp Ile Trp Ser Val Gly Cys Ile Met Gly     450 455 460 gaa atg atc aaa ggt ggt gtt ttg ttc cca ggt aca gat cat att gat 1440 Glu Met Ile Lys Gly Gly Val Leu Phe Pro Gly Thr Asp His Ile Asp 465 470 475 480 cag tgg aat aaa gtt att gaa cag ctt gga aca cca tgt cct gaa ttc 1488 Gln Trp Asn Lys Val Ile Glu Gln Leu Gly Thr Pro Cys Pro Glu Phe                 485 490 495 atg aag aaa ctg caa cca aca gta agg act tac gtt gaa aac aga cct 1536 Met Lys Lys Leu Gln Pro Thr Val Arg Thr Tyr Val Glu Asn Arg Pro             500 505 510 aaa tat gct gga tat agc ttt gag aaa ctc ttc cct gat gtc ctt ttc 1584 Lys Tyr Ala Gly Tyr Ser Phe Glu Lys Leu Phe Pro Asp Val Leu Phe         515 520 525 cca gct gac tca gaa cac aac aaa ctt aaa gcc agt cag gca agg gat 1632 Pro Ala Asp Ser Glu His Asn Lys Leu Lys Ala Ser Gln Ala Arg Asp     530 535 540 ttg tta tcc aaa atg ctg gta ata gat gca tct aaa agg atc tct gta 1680 Leu Leu Ser Lys Met Leu Val Ile Asp Ala Ser Lys Arg Ile Ser Val 545 550 555 560 gat gaa gct ctc caa cac ccg tac atc aat gtc tgg tat gat cct tct 1728 Asp Glu Ala Leu Gln His Pro Tyr Ile Asn Val Trp Tyr Asp Pro Ser                 565 570 575 gaa gca gaa gct cca cca cca aag atc cct gac aag cag tta gat gaa 1776 Glu Ala Glu Ala Pro Pro Pro Lys Ile Pro Asp Lys Gln Leu Asp Glu             580 585 590 agg gaa cac aca ata gaa gag tgg aaa gaa ttg ata tat aag gaa gtt 1824 Arg Glu His Thr Ile Glu Glu Trp Lys Glu Leu Ile Tyr Lys Glu Val         595 600 605 atg gac ttg gag gag aga acc aag aat gga gtt ata cgg ggg cag ccc 1872 Met Asp Leu Glu Glu Arg Thr Lys Asn Gly Val Ile Arg Gly Gln Pro     610 615 620 tct cct tta gca cag gtg cag cag tga 1899 Ser Pro Leu Ala Gln Val Gln Gln 625 630

【００３０】 <210> 2 <211> 632 <212> PRT <213> Homo sapiens <400> 2 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Ile Glu Gly 210 215 220 Arg Gly Ile Pro Glu Phe Pro Thr Leu Met Phe Ser Asp Glu Pro Asp 225 230 235 240 His Lys Gly Ala Leu Lys Pro Gly Met Ser Arg Ser Lys Arg Asp Asn 245 250 255 Asn Phe Tyr Ser Val Glu Ile Gly Asp Ser Thr Phe Thr Val Leu Lys 260 265 270 Arg Tyr Gln Asn Leu Lys Pro Ile Gly Ser Gly Ala Gln Gly Ile Val 275 280 285 Cys Ala Ala Tyr Asp Ala Ile Leu Glu Arg Asn Val Ala Ile Lys Lys 290 295 300 Leu Ser Arg Pro Phe Gln Asn Gln Thr His Ala Lys Arg Ala Tyr Arg 305 310 315 320 Glu Leu Val Leu Met Lys Cys Val Asn His Lys Asn Ile Ile Gly Leu 325 330 335 Leu Asn Val Phe Thr Pro Gln Lys Ser Leu Glu Glu Phe Gln Asp Val 340 345 350 Tyr Ile Val Met Glu Leu Met Asp Ala Asn Leu Cys Gln Val Ile Gln 355 360 365 Met Glu Leu Asp His Glu Arg Met Ser Tyr Leu Leu Tyr Gln Met Leu 370 375 380 Cys Gly Ile Lys His Leu His Ser Ala Gly Ile Ile His Arg Asp Leu 385 390 395 400 Lys Pro Ser Asn Ile Val Val Lys Ser Asp Cys Thr Leu Lys Ile Leu 405 410 415 Asp Phe Gly Leu Ala Arg Thr Ala Gly Thr Ser Phe Met Met Thr Pro 420 425 430 Tyr Val Val Thr Arg Tyr Tyr Arg Ala Pro Glu Val Ile Leu Gly Met 435 440 445 Gly Tyr Lys Glu Asn Val Asp Ile Trp Ser Val Gly Cys Ile Met Gly 450 455 460 Glu Met Ile Lys Gly Gly Val Leu Phe Pro Gly Thr Asp His Ile Asp 465 470 475 480 Gln Trp Asn Lys Val Ile Glu Gln Leu Gly Thr Pro Cys Pro Glu Phe 485 490 495 Met Lys Lys Leu Gln Pro Thr Val Arg Thr Tyr Val Glu Asn Arg Pro 500 505 510 Lys Tyr Ala Gly Tyr Ser Phe Glu Lys Leu Phe Pro Asp Val Leu Phe 515 520 525 Pro Ala Asp Ser Glu His Asn Lys Leu Lys Ala Ser Gln Ala Arg Asp 530 535 540 Leu Leu Ser Lys Met Leu Val Ile Asp Ala Ser Lys Arg Ile Ser Val 545 550 555 560 Asp Glu Ala Leu Gln His Pro Tyr Ile Asn Val Trp Tyr Asp Pro Ser 565 570 575 Glu Ala Glu Ala Pro Pro Pro Lys Ile Pro Asp Lys Gln Leu Asp Glu 580 585 590 Arg Glu His Thr Ile Glu Glu Trp Lys Glu Leu Ile Tyr Lys Glu Val 595 600 605 Met Asp Leu Glu Glu Arg Thr Lys Asn Gly Val Ile Arg Gly Gln Pro 610 615 620 Ser Pro Leu Ala Gln Val Gln Gln 625630[0030] <210> 2 <211> 632 <212> PRT <213> Homo sapiens <400> 2 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro   1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu              20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu          35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys      50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn  65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu                  85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser             100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu         115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn     130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu                 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr             180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala         195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Ile Glu Gly     210 215 220 Arg Gly Ile Pro Glu Phe Pro Thr Leu Met Phe Ser Asp Glu Pro Asp 225 230 235 240 His Lys Gly Ala Leu Lys Pro Gly Met Ser Arg Ser Lys Arg Asp Asn                 245 250 255 Asn Phe Tyr Ser Val Glu Ile Gly Asp Ser Thr Phe Thr Val Leu Lys             260 265 270 Arg Tyr Gln Asn Leu Lys Pro Ile Gly Ser Gly Ala Gln Gly Ile Val         275 280 285 Cys Ala Ala Tyr Asp Ala Ile Leu Glu Arg Asn Val Ala Ile Lys Lys     290 295 300 Leu Ser Arg Pro Phe Gln Asn Gln Thr His Ala Lys Arg Ala Tyr Arg 305 310 315 320 Glu Leu Val Leu Met Lys Cys Val Asn His Lys Asn Ile Ile Gly Leu                 325 330 335 Leu Asn Val Phe Thr Pro Gln Lys Ser Leu Glu Glu Phe Gln Asp Val             340 345 350 Tyr Ile Val Met Glu Leu Met Asp Ala Asn Leu Cys Gln Val Ile Gln         355 360 365 Met Glu Leu Asp His Glu Arg Met Ser Tyr Leu Leu Tyr Gln Met Leu     370 375 380 Cys Gly Ile Lys His Leu His Ser Ala Gly Ile Ile His Arg Asp Leu 385 390 395 400 Lys Pro Ser Asn Ile Val Val Lys Ser Asp Cys Thr Leu Lys Ile Leu                 405 410 415 Asp Phe Gly Leu Ala Arg Thr Ala Gly Thr Ser Phe Met Met Thr Pro             420 425 430 Tyr Val Val Thr Arg Tyr Tyr Arg Ala Pro Glu Val Ile Leu Gly Met         435 440 445 Gly Tyr Lys Glu Asn Val Asp Ile Trp Ser Val Gly Cys Ile Met Gly     450 455 460 Glu Met Ile Lys Gly Gly Val Leu Phe Pro Gly Thr Asp His Ile Asp 465 470 475 480 Gln Trp Asn Lys Val Ile Glu Gln Leu Gly Thr Pro Cys Pro Glu Phe                 485 490 495 Met Lys Lys Leu Gln Pro Thr Val Arg Thr Tyr Val Glu Asn Arg Pro             500 505 510 Lys Tyr Ala Gly Tyr Ser Phe Glu Lys Leu Phe Pro Asp Val Leu Phe         515 520 525 Pro Ala Asp Ser Glu His Asn Lys Leu Lys Ala Ser Gln Ala Arg Asp     530 535 540 Leu Leu Ser Lys Met Leu Val Ile Asp Ala Ser Lys Arg Ile Ser Val 545 550 555 560 Asp Glu Ala Leu Gln His Pro Tyr Ile Asn Val Trp Tyr Asp Pro Ser                 565 570 575 Glu Ala Glu Ala Pro Pro Pro Lys Ile Pro Asp Lys Gln Leu Asp Glu             580 585 590 Arg Glu His Thr Ile Glu Glu Trp Lys Glu Leu Ile Tyr Lys Glu Val         595 600 605 Met Asp Leu Glu Glu Arg Thr Lys Asn Gly Val Ile Arg Gly Gln Pro     610 615 620 Ser Pro Leu Ala Gln Val Gln Gln 625630

【００３１】 <210> 3 <211> 21 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide for PCR primer, described in example No.1 <400> 3 aaacagaatt cgagctcgcg c 21[0031] <210> 3 <211> 21 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide for PCR primer, described in example No.1 <400> 3   aaacagaatt cgagctcgcg c 21

【００３２】 <210> 4 <211> 41 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide for PCR primer, described in example No.1 <400> 4 gatatctgca gtcacccggg tttaagtgct cctttatgat c 41[0032] <210> 4 <211> 41 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide for PCR primer, described in example No.1 <400> 4   gatatctgca gtcacccggg tttaagtgct cctttatgat c 41

【００３３】 <210> 5 <211> 1083 <212> DNA <213> Bacillus sp.TB-90 <400> 5 aaacagaatt cgagctcgcg caatgtttcg tttgcaagaa atatttcgtg aaggagagaa 60 taattcg atg acc aaa cac aaa gaa aga gtg atg tat tat gga aaa ggt 109 Met Thr Lys His Lys Glu Arg Val Met Tyr Tyr Gly Lys Gly 1 5 10 gac gta ttt gct tat cgc acc tat tta aaa cca ctt act gga gtt aga 157 Asp Val Phe Ala Tyr Arg Thr Tyr Leu Lys Pro Leu Thr Gly Val Arg 15 20 25 30 acg att cct gaa tct cca ttt tcc ggt cga gat cat att ctt ttt gga 205 Thr Ile Pro Glu Ser Pro Phe Ser Gly Arg Asp His Ile Leu Phe Gly 35 40 45 gta aat gta aaa atc tca gta gga gga aca aaa ttg ctg acc tcc ttt 253 Val Asn Val Lys Ile Ser Val Gly Gly Thr Lys Leu Leu Thr Ser Phe 50 55 60 acg aaa ggg gat aac agc tta gtc gtt gca aca gac tcg atg aaa aac 301 Thr Lys Gly Asp Asn Ser Leu Val Val Ala Thr Asp Ser Met Lys Asn 65 70 75 ttt ata caa aaa cat tta gct agt tat aca gga aca acg ata gaa ggt 349 Phe Ile Gln Lys His Leu Ala Ser Tyr Thr Gly Thr Thr Ile Glu Gly 80 85 90 ttt tta gaa tat gta gct act tct ttt ttg aag aaa tat tct cat att 397 Phe Leu Glu Tyr Val Ala Thr Ser Phe Leu Lys Lys Tyr Ser His Ile 95 100 105 110 gaa aag att tcg ttg ata gga gag gaa att ccc ttt gaa aca act ttt 445 Glu Lys Ile Ser Leu Ile Gly Glu Glu Ile Pro Phe Glu Thr Thr Phe 115 120 125 gca gta aag aat gga aat aga gca gct agt gag cta gta ttt aaa aaa 493 Ala Val Lys Asn Gly Asn Arg Ala Ala Ser Glu Leu Val Phe Lys Lys 130 135 140 tca cga aat gaa tat gcc acc gct tat ttg aat atg gtt cgt aat gaa 541 Ser Arg Asn Glu Tyr Ala Thr Ala Tyr Leu Asn Met Val Arg Asn Glu 145 150 155 gat aac acc cta aac att act gaa caa caa agc gga ctt gct ggt ctt 589 Asp Asn Thr Leu Asn Ile Thr Glu Gln Gln Ser Gly Leu Ala Gly Leu 160 165 170 caa tta ata aaa gtc agc gga aat tcc ttt gtc ggt ttt att cgt gac 637 Gln Leu Ile Lys Val Ser Gly Asn Ser Phe Val Gly Phe Ile Arg Asp 175 180 185 190 gaa tac aca act ctt cca gag gat tca aac cgc cct cta ttt gtt tac 685 Glu Tyr Thr Thr Leu Pro Glu Asp Ser Asn Arg Pro Leu Phe Val Tyr 195 200 205 tta aac atc aaa tgg aag tac aaa aac acg gaa gac tca ttt gga acg 733 Leu Asn Ile Lys Trp Lys Tyr Lys Asn Thr Glu Asp Ser Phe Gly Thr 210 215 220 aat cca gaa aat tat gtt gca gct gaa caa att cgc gac atc gcc act 781 Asn Pro Glu Asn Tyr Val Ala Ala Glu Gln Ile Arg Asp Ile Ala Thr 225 230 235 tcc gta ttt cat gaa acc gag acg ctt tcc atc caa cat tta att tat 829 Ser Val Phe His Glu Thr Glu Thr Leu Ser Ile Gln His Leu Ile Tyr 240 245 250 tta atc ggc cgc aga ata tta gaa aga ttc cct caa ctt caa gaa gtt 877 Leu Ile Gly Arg Arg Ile Leu Glu Arg Phe Pro Gln Leu Gln Glu Val 255 260 265 270 tac ttc gaa tct caa aat cat aca tgg gat aaa ata gtg gag gaa att 925 Tyr Phe Glu Ser Gln Asn His Thr Trp Asp Lys Ile Val Glu Glu Ile 275 280 285 cct gaa tca gaa ggg aaa gta tat aca gaa ccg cga ccg cca tat gga 973 Pro Glu Ser Glu Gly Lys Val Tyr Thr Glu Pro Arg Pro Pro Tyr Gly 290 295 300 ttt caa tgc ttt act gtc acc caa gaa gac ttg cca cac gaa aac att 1021 Phe Gln Cys Phe Thr Val Thr Gln Glu Asp Leu Pro His Glu Asn Ile 305 310 315 ctt atg ttc tct gat gaa ccc gat cat aaa gga gca ctt aaa ccc ggg 1069 Leu Met Phe Ser Asp Glu Pro Asp His Lys Gly Ala Leu Lys Pro Gly 320 325 330 tgactgcaga tatc 1083[0033] <210> 5 <211> 1083 <212> DNA <213> Bacillus sp.TB-90 <400> 5 aaacagaatt cgagctcgcg caatgtttcg tttgcaagaa atatttcgtg aaggagagaa 60 taattcg atg acc aaa cac aaa gaa aga gtg atg tat tat gga aaa ggt 109         Met Thr Lys His Lys Glu Arg Val Met Tyr Tyr Gly Lys Gly          1 5 10 gac gta ttt gct tat cgc acc tat tta aaa cca ctt act gga gtt aga 157 Asp Val Phe Ala Tyr Arg Thr Tyr Leu Lys Pro Leu Thr Gly Val Arg  15 20 25 30 acg att cct gaa tct cca ttt tcc ggt cga gat cat att ctt ttt gga 205 Thr Ile Pro Glu Ser Pro Phe Ser Gly Arg Asp His Ile Leu Phe Gly                  35 40 45 gta aat gta aaa atc tca gta gga gga aca aaa ttg ctg acc tcc ttt 253 Val Asn Val Lys Ile Ser Val Gly Gly Thr Lys Leu Leu Thr Ser Phe              50 55 60 acg aaa ggg gat aac agc tta gtc gtt gca aca gac tcg atg aaa aac 301 Thr Lys Gly Asp Asn Ser Leu Val Val Ala Thr Asp Ser Met Lys Asn          65 70 75 ttt ata caa aaa cat tta gct agt tat aca gga aca acg ata gaa ggt 349 Phe Ile Gln Lys His Leu Ala Ser Tyr Thr Gly Thr Thr Ile Glu Gly      80 85 90 ttt tta gaa tat gta gct act tct ttt ttg aag aaa tat tct cat att 397 Phe Leu Glu Tyr Val Ala Thr Ser Phe Leu Lys Lys Tyr Ser His Ile  95 100 105 110 gaa aag att tcg ttg ata gga gag gaa att ccc ttt gaa aca act ttt 445 Glu Lys Ile Ser Leu Ile Gly Glu Glu Ile Pro Phe Glu Thr Thr Phe                 115 120 125 gca gta aag aat gga aat aga gca gct agt gag cta gta ttt aaa aaa 493 Ala Val Lys Asn Gly Asn Arg Ala Ala Ser Glu Leu Val Phe Lys Lys             130 135 140 tca cga aat gaa tat gcc acc gct tat ttg aat atg gtt cgt aat gaa 541 Ser Arg Asn Glu Tyr Ala Thr Ala Tyr Leu Asn Met Val Arg Asn Glu         145 150 155 gat aac acc cta aac att act gaa caa caa agc gga ctt gct ggt ctt 589 Asp Asn Thr Leu Asn Ile Thr Glu Gln Gln Ser Gly Leu Ala Gly Leu     160 165 170 caa tta ata aaa gtc agc gga aat tcc ttt gtc ggt ttt att cgt gac 637 Gln Leu Ile Lys Val Ser Gly Asn Ser Phe Val Gly Phe Ile Arg Asp 175 180 185 190 gaa tac aca act ctt cca gag gat tca aac cgc cct cta ttt gtt tac 685 Glu Tyr Thr Thr Leu Pro Glu Asp Ser Asn Arg Pro Leu Phe Val Tyr                 195 200 205 tta aac atc aaa tgg aag tac aaa aac acg gaa gac tca ttt gga acg 733 Leu Asn Ile Lys Trp Lys Tyr Lys Asn Thr Glu Asp Ser Phe Gly Thr             210 215 220 aat cca gaa aat tat gtt gca gct gaa caa att cgc gac atc gcc act 781 Asn Pro Glu Asn Tyr Val Ala Ala Glu Gln Ile Arg Asp Ile Ala Thr         225 230 235 tcc gta ttt cat gaa acc gag acg ctt tcc atc caa cat tta att tat 829 Ser Val Phe His Glu Thr Glu Thr Leu Ser Ile Gln His Leu Ile Tyr     240 245 250 tta atc ggc cgc aga ata tta gaa aga ttc cct caa ctt caa gaa gtt 877 Leu Ile Gly Arg Arg Ile Leu Glu Arg Phe Pro Gln Leu Gln Glu Val 255 260 265 270 tac ttc gaa tct caa aat cat aca tgg gat aaa ata gtg gag gaa att 925 Tyr Phe Glu Ser Gln Asn His Thr Trp Asp Lys Ile Val Glu Glu Ile                 275 280 285 cct gaa tca gaa ggg aaa gta tat aca gaa ccg cga ccg cca tat gga 973 Pro Glu Ser Glu Gly Lys Val Tyr Thr Glu Pro Arg Pro Pro Tyr Gly             290 295 300 ttt caa tgc ttt act gtc acc caa gaa gac ttg cca cac gaa aac att 1021 Phe Gln Cys Phe Thr Val Thr Gln Glu Asp Leu Pro His Glu Asn Ile         305 310 315 ctt atg ttc tct gat gaa ccc gat cat aaa gga gca ctt aaa ccc ggg 1069 Leu Met Phe Ser Asp Glu Pro Asp His Lys Gly Ala Leu Lys Pro Gly     320 325 330 tgactgcaga tatc 1083

【００３４】 <210> 6 <211> 334 <212> PRT <213> Bacillus sp.TB-90 <400> 6 Met Thr Lys His Lys Glu Arg Val Met Tyr Tyr Gly Lys Gly Asp Val 1 5 10 15 Phe Ala Tyr Arg Thr Tyr Leu Lys Pro Leu Thr Gly Val Arg Thr Ile 20 25 30 Pro Glu Ser Pro Phe Ser Gly Arg Asp His Ile Leu Phe Gly Val Asn 35 40 45 Val Lys Ile Ser Val Gly Gly Thr Lys Leu Leu Thr Ser Phe Thr Lys 50 55 60 Gly Asp Asn Ser Leu Val Val Ala Thr Asp Ser Met Lys Asn Phe Ile 65 70 75 80 Gln Lys His Leu Ala Ser Tyr Thr Gly Thr Thr Ile Glu Gly Phe Leu 85 90 95 Glu Tyr Val Ala Thr Ser Phe Leu Lys Lys Tyr Ser His Ile Glu Lys 100 105 110 Ile Ser Leu Ile Gly Glu Glu Ile Pro Phe Glu Thr Thr Phe Ala Val 115 120 125 Lys Asn Gly Asn Arg Ala Ala Ser Glu Leu Val Phe Lys Lys Ser Arg 130 135 140 Asn Glu Tyr Ala Thr Ala Tyr Leu Asn Met Val Arg Asn Glu Asp Asn 145 150 155 160 Thr Leu Asn Ile Thr Glu Gln Gln Ser Gly Leu Ala Gly Leu Gln Leu 165 170 175 Ile Lys Val Ser Gly Asn Ser Phe Val Gly Phe Ile Arg Asp Glu Tyr 180 185 190 Thr Thr Leu Pro Glu Asp Ser Asn Arg Pro Leu Phe Val Tyr Leu Asn 195 200 205 Ile Lys Trp Lys Tyr Lys Asn Thr Glu Asp Ser Phe Gly Thr Asn Pro 210 215 220 Glu Asn Tyr Val Ala Ala Glu Gln Ile Arg Asp Ile Ala Thr Ser Val 225 230 235 240 Phe His Glu Thr Glu Thr Leu Ser Ile Gln His Leu Ile Tyr Leu Ile 245 250 255 Gly Arg Arg Ile Leu Glu Arg Phe Pro Gln Leu Gln Glu Val Tyr Phe 260 265 270 Glu Ser Gln Asn His Thr Trp Asp Lys Ile Val Glu Glu Ile Pro Glu 275 280 285 Ser Glu Gly Lys Val Tyr Thr Glu Pro Arg Pro Pro Tyr Gly Phe Gln 290 295 300 Cys Phe Thr Val Thr Gln Glu Asp Leu Pro His Glu Asn Ile Leu Met 305 310 315 320 Phe Ser Asp Glu Pro Asp His Lys Gly Ala Leu Lys Pro Gly 325 330[0034] <210> 6 <211> 334 <212> PRT <213> Bacillus sp.TB-90 <400> 6  Met Thr Lys His Lys Glu Arg Val Met Tyr Tyr Gly Lys Gly Asp Val  1 5 10 15  Phe Ala Tyr Arg Thr Tyr Leu Lys Pro Leu Thr Gly Val Arg Thr Ile               20 25 30  Pro Glu Ser Pro Phe Ser Gly Arg Asp His Ile Leu Phe Gly Val Asn           35 40 45  Val Lys Ile Ser Val Gly Gly Thr Lys Leu Leu Thr Ser Phe Thr Lys       50 55 60  Gly Asp Asn Ser Leu Val Val Ala Thr Asp Ser Met Lys Asn Phe Ile   65 70 75 80  Gln Lys His Leu Ala Ser Tyr Thr Gly Thr Thr Ile Glu Gly Phe Leu                   85 90 95  Glu Tyr Val Ala Thr Ser Phe Leu Lys Lys Tyr Ser His Ile Glu Lys              100 105 110  Ile Ser Leu Ile Gly Glu Glu Ile Pro Phe Glu Thr Thr Phe Ala Val          115 120 125  Lys Asn Gly Asn Arg Ala Ala Ser Glu Leu Val Phe Lys Lys Ser Arg      130 135 140  Asn Glu Tyr Ala Thr Ala Tyr Leu Asn Met Val Arg Asn Glu Asp Asn  145 150 155 160  Thr Leu Asn Ile Thr Glu Gln Gln Ser Gly Leu Ala Gly Leu Gln Leu                  165 170 175  Ile Lys Val Ser Gly Asn Ser Phe Val Gly Phe Ile Arg Asp Glu Tyr              180 185 190  Thr Thr Leu Pro Glu Asp Ser Asn Arg Pro Leu Phe Val Tyr Leu Asn          195 200 205  Ile Lys Trp Lys Tyr Lys Asn Thr Glu Asp Ser Phe Gly Thr Asn Pro      210 215 220  Glu Asn Tyr Val Ala Ala Glu Gln Ile Arg Asp Ile Ala Thr Ser Val  225 230 235 240  Phe His Glu Thr Glu Thr Leu Ser Ile Gln His Leu Ile Tyr Leu Ile                  245 250 255  Gly Arg Arg Ile Leu Glu Arg Phe Pro Gln Leu Gln Glu Val Tyr Phe              260 265 270  Glu Ser Gln Asn His Thr Trp Asp Lys Ile Val Glu Glu Ile Pro Glu          275 280 285  Ser Glu Gly Lys Val Tyr Thr Glu Pro Arg Pro Pro Tyr Gly Phe Gln      290 295 300  Cys Phe Thr Val Thr Gln Glu Asp Leu Pro His Glu Asn Ile Leu Met  305 310 315 320  Phe Ser Asp Glu Pro Asp His Lys Gly Ala Leu Lys Pro Gly                  325 330

【００３５】 <210> 7 <211> 29 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide, described in example No.1 <400> 7 ggtcatcacc atcaccatca ctgactgca 21[0035] <210> 7 <211> 29 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide, described in example No.1 <400> 7   ggtcatcacc atcaccatca ctgactgca 21

【００３６】 <210> 8 <211> 25 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide, described in example No.1 <400> 8 gtcagtgatg gtgatggtga tgacc 25[0036] <210> 8 <211> 25 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide, described in example No.1 <400> 8   gtcagtgatg gtgatggtga tgacc 25

【００３７】 <210> 9 <211> 1101 <212> DNA <213> Bacillus sp.TB-90 <400> 9 aaacagaatt cgagctcgcg caatgtttcg tttgcaagaa atatttcgtg aaggagagaa 60 taattcg atg acc aaa cac aaa gaa aga gtg atg tat tat gga aaa ggt 109 Met Thr Lys His Lys Glu Arg Val Met Tyr Tyr Gly Lys Gly 1 5 10 gac gta ttt gct tat cgc acc tat tta aaa cca ctt act gga gtt aga 157 Asp Val Phe Ala Tyr Arg Thr Tyr Leu Lys Pro Leu Thr Gly Val Arg 15 20 25 30 acg att cct gaa tct cca ttt tcc ggt cga gat cat att ctt ttt gga 205 Thr Ile Pro Glu Ser Pro Phe Ser Gly Arg Asp His Ile Leu Phe Gly 35 40 45 gta aat gta aaa atc tca gta gga gga aca aaa ttg ctg acc tcc ttt 253 Val Asn Val Lys Ile Ser Val Gly Gly Thr Lys Leu Leu Thr Ser Phe 50 55 60 acg aaa ggg gat aac agc tta gtc gtt gca aca gac tcg atg aaa aac 301 Thr Lys Gly Asp Asn Ser Leu Val Val Ala Thr Asp Ser Met Lys Asn 65 70 75 ttt ata caa aaa cat tta gct agt tat aca gga aca acg ata gaa ggt 349 Phe Ile Gln Lys His Leu Ala Ser Tyr Thr Gly Thr Thr Ile Glu Gly 80 85 90 ttt tta gaa tat gta gct act tct ttt ttg aag aaa tat tct cat att 397 Phe Leu Glu Tyr Val Ala Thr Ser Phe Leu Lys Lys Tyr Ser His Ile 95 100 105 110 gaa aag att tcg ttg ata gga gag gaa att ccc ttt gaa aca act ttt 445 Glu Lys Ile Ser Leu Ile Gly Glu Glu Ile Pro Phe Glu Thr Thr Phe 115 120 125 gca gta aag aat gga aat aga gca gct agt gag cta gta ttt aaa aaa 49 3 Ala Val Lys Asn Gly Asn Arg Ala Ala Ser Glu Leu Val Phe Lys Lys 130 135 140 tca cga aat gaa tat gcc acc gct tat ttg aat atg gtt cgt aat gaa 541 Ser Arg Asn Glu Tyr Ala Thr Ala Tyr Leu Asn Met Val Arg Asn Glu 145 150 155 gat aac acc cta aac att act gaa caa caa agc gga ctt gct ggt ctt 589 Asp Asn Thr Leu Asn Ile Thr Glu Gln Gln Ser Gly Leu Ala Gly Leu 160 165 170 caa tta ata aaa gtc agc gga aat tcc ttt gtc ggt ttt att cgt gac 637 Gln Leu Ile Lys Val Ser Gly Asn Ser Phe Val Gly Phe Ile Arg Asp 175 180 185 190 gaa tac aca act ctt cca gag gat tca aac cgc cct cta ttt gtt tac 685 Glu Tyr Thr Thr Leu Pro Glu Asp Ser Asn Arg Pro Leu Phe Val Tyr 195 200 205 tta aac atc aaa tgg aag tac aaa aac acg gaa gac tca ttt gga acg 733 Leu Asn Ile Lys Trp Lys Tyr Lys Asn Thr Glu Asp Ser Phe Gly Thr 210 215 220 aat cca gaa aat tat gtt gca gct gaa caa att cgc gac atc gcc act 781 Asn Pro Glu Asn Tyr Val Ala Ala Glu Gln Ile Arg Asp Ile Ala Thr 225 230 235 tcc gta ttt cat gaa acc gag acg ctt tcc atc caa cat tta att tat 829 Ser Val Phe His Glu Thr Glu Thr Leu Ser Ile Gln His Leu Ile Tyr 240 245 250 tta atc ggc cgc aga ata tta gaa aga ttc cct caa ctt caa gaa gtt 877 Leu Ile Gly Arg Arg Ile Leu Glu Arg Phe Pro Gln Leu Gln Glu Val 255 260 265 270 tac ttc gaa tct caa aat cat aca tgg gat aaa ata gtg gag gaa att 925 Tyr Phe Glu Ser Gln Asn His Thr Trp Asp Lys Ile Val Glu Glu Ile 275 280 285 cct gaa tca gaa ggg aaa gta tat aca gaa ccg cga ccg cca tat gga 973 Pro Glu Ser Glu Gly Lys Val Tyr Thr Glu Pro Arg Pro Pro Tyr Gly 290 295 300 ttt caa tgc ttt act gtc acc caa gaa gac ttg cca cac gaa aac att 1021 Phe Gln Cys Phe Thr Val Thr Gln Glu Asp Leu Pro His Glu Asn Ile 305 310 315 ctt atg ttc tct gat gaa ccc gat cat aaa gga gca ctt aaa ccc ggt 1069 Leu Met Phe Ser Asp Glu Pro Asp His Lys Gly Ala Leu Lys Pro Gly 320 325 330 cat cac cat cac cat cac tgactgcaga tatc 1101 His His His His His His 335 340 [0037] <210> 9 <211> 1101 <212> DNA <213> Bacillus sp.TB-90 <400> 9 aaacagaatt cgagctcgcg caatgtttcg tttgcaagaa atatttcgtg aaggagagaa 60 taattcg atg acc aaa cac aaa gaa aga gtg atg tat tat gga aaa ggt 109         Met Thr Lys His Lys Glu Arg Val Met Tyr Tyr Gly Lys Gly          1 5 10 gac gta ttt gct tat cgc acc tat tta aaa cca ctt act gga gtt aga 157 Asp Val Phe Ala Tyr Arg Thr Tyr Leu Lys Pro Leu Thr Gly Val Arg 15 20 25 30 acg att cct gaa tct cca ttt tcc ggt cga gat cat att ctt ttt gga 205 Thr Ile Pro Glu Ser Pro Phe Ser Gly Arg Asp His Ile Leu Phe Gly                 35 40 45 gta aat gta aaa atc tca gta gga gga aca aaa ttg ctg acc tcc ttt 253 Val Asn Val Lys Ile Ser Val Gly Gly Thr Lys Leu Leu Thr Ser Phe             50 55 60 acg aaa ggg gat aac agc tta gtc gtt gca aca gac tcg atg aaa aac 301 Thr Lys Gly Asp Asn Ser Leu Val Val Ala Thr Asp Ser Met Lys Asn         65 70 75 ttt ata caa aaa cat tta gct agt tat aca gga aca acg ata gaa ggt 349 Phe Ile Gln Lys His Leu Ala Ser Tyr Thr Gly Thr Thr Ile Glu Gly      80 85 90 ttt tta gaa tat gta gct act tct ttt ttg aag aaa tat tct cat att 397 Phe Leu Glu Tyr Val Ala Thr Ser Phe Leu Lys Lys Tyr Ser His Ile 95 100 105 110 gaa aag att tcg ttg ata gga gag gaa att ccc ttt gaa aca act ttt 445 Glu Lys Ile Ser Leu Ile Gly Glu Glu Ile Pro Phe Glu Thr Thr Phe                 115 120 125 gca gta aag aat gga aat aga gca gct agt gag cta gta ttt aaa aaa 49 3 Ala Val Lys Asn Gly Asn Arg Ala Ala Ser Glu Leu Val Phe Lys Lys             130 135 140 tca cga aat gaa tat gcc acc gct tat ttg aat atg gtt cgt aat gaa 541 Ser Arg Asn Glu Tyr Ala Thr Ala Tyr Leu Asn Met Val Arg Asn Glu         145 150 155 gat aac acc cta aac att act gaa caa caa agc gga ctt gct ggt ctt 589 Asp Asn Thr Leu Asn Ile Thr Glu Gln Gln Ser Gly Leu Ala Gly Leu     160 165 170 caa tta ata aaa gtc agc gga aat tcc ttt gtc ggt ttt att cgt gac 637 Gln Leu Ile Lys Val Ser Gly Asn Ser Phe Val Gly Phe Ile Arg Asp 175 180 185 190 gaa tac aca act ctt cca gag gat tca aac cgc cct cta ttt gtt tac 685 Glu Tyr Thr Thr Leu Pro Glu Asp Ser Asn Arg Pro Leu Phe Val Tyr                 195 200 205 tta aac atc aaa tgg aag tac aaa aac acg gaa gac tca ttt gga acg 733 Leu Asn Ile Lys Trp Lys Tyr Lys Asn Thr Glu Asp Ser Phe Gly Thr             210 215 220 aat cca gaa aat tat gtt gca gct gaa caa att cgc gac atc gcc act 781 Asn Pro Glu Asn Tyr Val Ala Ala Glu Gln Ile Arg Asp Ile Ala Thr         225 230 235 tcc gta ttt cat gaa acc gag acg ctt tcc atc caa cat tta att tat 829 Ser Val Phe His Glu Thr Glu Thr Leu Ser Ile Gln His Leu Ile Tyr     240 245 250 tta atc ggc cgc aga ata tta gaa aga ttc cct caa ctt caa gaa gtt 877 Leu Ile Gly Arg Arg Ile Leu Glu Arg Phe Pro Gln Leu Gln Glu Val 255 260 265 270 tac ttc gaa tct caa aat cat aca tgg gat aaa ata gtg gag gaa att 925 Tyr Phe Glu Ser Gln Asn His Thr Trp Asp Lys Ile Val Glu Glu Ile                 275 280 285 cct gaa tca gaa ggg aaa gta tat aca gaa ccg cga ccg cca tat gga 973 Pro Glu Ser Glu Gly Lys Val Tyr Thr Glu Pro Arg Pro Pro Tyr Gly             290 295 300 ttt caa tgc ttt act gtc acc caa gaa gac ttg cca cac gaa aac att 1021 Phe Gln Cys Phe Thr Val Thr Gln Glu Asp Leu Pro His Glu Asn Ile         305 310 315 ctt atg ttc tct gat gaa ccc gat cat aaa gga gca ctt aaa ccc ggt 1069 Leu Met Phe Ser Asp Glu Pro Asp His Lys Gly Ala Leu Lys Pro Gly     320 325 330 cat cac cat cac cat cac tgactgcaga tatc 1101 His His His His His His 335 340

【００３８】 <210> 10 <211> 340 <212> PRT <213> Bacillus sp.TB-90 <400> 10 Met Thr Lys His Lys Glu Arg Val Met Tyr Tyr Gly Lys Gly Asp Val 1 5 10 15 Phe Ala Tyr Arg Thr Tyr Leu Lys Pro Leu Thr Gly Val Arg Thr Ile 20 25 30 Pro Glu Ser Pro Phe Ser Gly Arg Asp His Ile Leu Phe Gly Val Asn 35 40 45 Val Lys Ile Ser Val Gly Gly Thr Lys Leu Leu Thr Ser Phe Thr Lys 50 55 60 Gly Asp Asn Ser Leu Val Val Ala Thr Asp Ser Met Lys Asn Phe Ile 65 70 75 80 Gln Lys His Leu Ala Ser Tyr Thr Gly Thr Thr Ile Glu Gly Phe Leu 85 90 95 Glu Tyr Val Ala Thr Ser Phe Leu Lys Lys Tyr Ser His Ile Glu Lys 100 105 110 Ile Ser Leu Ile Gly Glu Glu Ile Pro Phe Glu Thr Thr Phe Ala Val 115 120 125 Lys Asn Gly Asn Arg Ala Ala Ser Glu Leu Val Phe Lys Lys Ser Arg 130 135 140 Asn Glu Tyr Ala Thr Ala Tyr Leu Asn Met Val Arg Asn Glu Asp Asn 145 150 155 160 Thr Leu Asn Ile Thr Glu Gln Gln Ser Gly Leu Ala Gly Leu Gln Leu 165 170 175 Ile Lys Val Ser Gly Asn Ser Phe Val Gly Phe Ile Arg Asp Glu Tyr 180 185 190 Thr Thr Leu Pro Glu Asp Ser Asn Arg Pro Leu Phe Val Tyr Leu Asn 195 200 205 Ile Lys Trp Lys Tyr Lys Asn Thr Glu Asp Ser Phe Gly Thr Asn Pro 210 215 220 Glu Asn Tyr Val Ala Ala Glu Gln Ile Arg Asp Ile Ala Thr Ser Val 225 230 235 240 Phe His Glu Thr Glu Thr Leu Ser Ile Gln His Leu Ile Tyr Leu Ile 245 250 255 Gly Arg Arg Ile Leu Glu Arg Phe Pro Gln Leu Gln Glu Val Tyr Phe 260 265 270 Glu Ser Gln Asn His Thr Trp Asp Lys Ile Val Glu Glu Ile Pro Glu 275 280 285 Ser Glu Gly Lys Val Tyr Thr Glu Pro Arg Pro Pro Tyr Gly Phe Gln 290 295 300 Cys Phe Thr Val Thr Gln Glu Asp Leu Pro His Glu Asn Ile Leu Met 305 310 315 320 Phe Ser Asp Glu Pro Asp His Lys Gly Ala Leu Lys Pro Gly His His 325 330 335 His His His His 340[0038] <210> 10 <211> 340 <212> PRT <213> Bacillus sp.TB-90 <400> 10 Met Thr Lys His Lys Glu Arg Val Met Tyr Tyr Gly Lys Gly Asp Val  1 5 10 15  Phe Ala Tyr Arg Thr Tyr Leu Lys Pro Leu Thr Gly Val Arg Thr Ile               20 25 30  Pro Glu Ser Pro Phe Ser Gly Arg Asp His Ile Leu Phe Gly Val Asn           35 40 45  Val Lys Ile Ser Val Gly Gly Thr Lys Leu Leu Thr Ser Phe Thr Lys       50 55 60  Gly Asp Asn Ser Leu Val Val Ala Thr Asp Ser Met Lys Asn Phe Ile   65 70 75 80  Gln Lys His Leu Ala Ser Tyr Thr Gly Thr Thr Ile Glu Gly Phe Leu                   85 90 95  Glu Tyr Val Ala Thr Ser Phe Leu Lys Lys Tyr Ser His Ile Glu Lys              100 105 110  Ile Ser Leu Ile Gly Glu Glu Ile Pro Phe Glu Thr Thr Phe Ala Val          115 120 125  Lys Asn Gly Asn Arg Ala Ala Ser Glu Leu Val Phe Lys Lys Ser Arg      130 135 140  Asn Glu Tyr Ala Thr Ala Tyr Leu Asn Met Val Arg Asn Glu Asp Asn  145 150 155 160  Thr Leu Asn Ile Thr Glu Gln Gln Ser Gly Leu Ala Gly Leu Gln Leu                  165 170 175  Ile Lys Val Ser Gly Asn Ser Phe Val Gly Phe Ile Arg Asp Glu Tyr              180 185 190  Thr Thr Leu Pro Glu Asp Ser Asn Arg Pro Leu Phe Val Tyr Leu Asn          195 200 205  Ile Lys Trp Lys Tyr Lys Asn Thr Glu Asp Ser Phe Gly Thr Asn Pro      210 215 220  Glu Asn Tyr Val Ala Ala Glu Gln Ile Arg Asp Ile Ala Thr Ser Val  225 230 235 240  Phe His Glu Thr Glu Thr Leu Ser Ile Gln His Leu Ile Tyr Leu Ile                  245 250 255  Gly Arg Arg Ile Leu Glu Arg Phe Pro Gln Leu Gln Glu Val Tyr Phe              260 265 270  Glu Ser Gln Asn His Thr Trp Asp Lys Ile Val Glu Glu Ile Pro Glu          275 280 285  Ser Glu Gly Lys Val Tyr Thr Glu Pro Arg Pro Pro Tyr Gly Phe Gln      290 295 300  Cys Phe Thr Val Thr Gln Glu Asp Leu Pro His Glu Asn Ile Leu Met  305 310 315 320  Phe Ser Asp Glu Pro Asp His Lys Gly Ala Leu Lys Pro Gly His His                  325 330 335  His His His His              340

【図面の簡単な説明】[Brief description of drawings]

【図１】融合蛋白質をコードする遺伝子の作成を示す
図。FIG. 1 is a diagram showing the construction of a gene encoding a fusion protein.

【図２】融合蛋白質とその選択的な切断の様子を分析し
たＳＤＳ−ポリアクリルアミドゲルの結果を示す図。FIG. 2 is a view showing the results of SDS-polyacrylamide gel in which a fusion protein and its selective cleavage state were analyzed.

【図３】アフィニティー・ビーズを用いた蛋白質のハイ
スループット精製のフローを示す図。FIG. 3 is a diagram showing a flow of high-throughput purification of a protein using affinity beads.

【図４】融合蛋白質とその選択的な切断の様子を分析し
たＳＤＳ−ポリアクリルアミドゲルの結果を示す図。FIG. 4 is a view showing the results of SDS-polyacrylamide gel in which the fusion protein and its selective cleavage state were analyzed.

【手続補正書】[Procedure amendment]

【提出日】平成１３年８月８日（２００１．８．８）[Submission date] August 8, 2001 (2001.8.8)

【手続補正１】[Procedure Amendment 1]

【補正対象書類名】明細書[Document name to be amended] Statement

【補正対象項目名】特許請求の範囲[Name of item to be amended] Claims

【補正方法】変更[Correction method] Change

【補正内容】[Correction content]

【特許請求の範囲】[Claims]

Claims (8)

【特許請求の範囲】[Claims] 【請求項１】タグとプロテインキナーゼとを連結した融
合蛋白質を、非特異的エンドプロテアーゼで切断する方
法。1. A method for cleaving a fusion protein in which a tag and a protein kinase are linked with a non-specific endoprotease. 【請求項２】タグとプロテインキナーゼとを連結した融
合蛋白質に対し、蛋白質部分の配列を切断することな
く、連結部分またはその周辺部分の配列を切断し所望の
プロテインキナーゼを取得する工程において、非特異的
エンドプロテアーゼを用いることを特徴とする融合蛋白
質よりプロテインキナーゼを取得するための方法。2. A fusion protein in which a tag and a protein kinase are ligated to each other in the step of obtaining a desired protein kinase by cleaving the sequence of the ligation part or its peripheral part without cleaving the sequence of the protein part. A method for obtaining a protein kinase from a fusion protein, which comprises using a specific endoprotease. 【請求項３】非特異的エンドプロテアーゼにより認識さ
れやすい配列が式（１）で表されることを特徴とする請
求項２に記載の方法。 X-Met-Phe-Ser-(Xaa)n-Y（１） [式中、Xはプロテインキナーゼを意味しYはタグを意味
する。Xaaは任意のアミノ酸残基を意味する。nは０〜５
０の整数である。]3. The method according to claim 2, wherein the sequence easily recognized by the non-specific endoprotease is represented by the formula (1). X-Met-Phe-Ser- (Xaa) nY (1) [In the formula, X means a protein kinase and Y means a tag. Xaa means any amino acid residue. n is 0-5
It is an integer of 0. ] 【請求項４】非特異的エンドプロテアーゼにより認識さ
れやすい配列が式（２）で表されることを特徴とする請
求項２に記載の方法。 X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys-(Xaa)n-Y（２） [式中、Xはプロテインキナーゼを意味しYはタグを意味
する。Xaaは任意のアミノ酸残基を意味する。nは０〜４
０の整数である。]4. The method according to claim 2, wherein the sequence easily recognized by the non-specific endoprotease is represented by the formula (2). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys- (Xaa) nY (2) [where X is a protein kinase and Y is a tag means. Xaa means any amino acid residue. n is 0-4
It is an integer of 0. ] 【請求項５】非特異的エンドプロテアーゼにより認識さ
れやすい配列が式（３）で表されることを特徴とする請
求項２に記載の方法。 X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys-Y（３） [式中、Xはプロテインキナーゼを意味しYはタグを意味
する。]5. The method according to claim 2, wherein the sequence that is easily recognized by the non-specific endoprotease is represented by the formula (3). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys-Y (3) [wherein X means a protein kinase and Y means a tag. ] 【請求項６】タグがグルタチオン−Ｓ−トランスフェラ
ーゼであることを特徴とする請求項１〜５に記載の方
法。6. The method according to claim 1, wherein the tag is glutathione-S-transferase. 【請求項７】非特異的エンドプロテアーゼがプロティナ
ーゼＫ、トリプシン、キモトリプシン、Ｖ８プロテアー
ゼより選ばれることを特徴とする請求項１〜６に記載の
方法。7. The method according to claim 1, wherein the non-specific endoprotease is selected from proteinase K, trypsin, chymotrypsin and V8 protease. 【請求項８】非特異的エンドプロテアーゼがプロティナ
ーゼＫであることを特徴とする請求項７に記載の方法。8. The method according to claim 7, wherein the non-specific endoprotease is proteinase K.