JPWO2002026964A1 - Method for constructing mutant DNA library and use thereof - Google Patents

Abstract

According to the present invention, (a) the unit region DNAs are ligated in any combination, and (b) the obtained connected unit region DNAs are combined with at least one other type of terminal sequence of all the connected unit region DNAs used. (C) Polymerase \ Chain \ Reaction is performed using the mixture of the connection unit region DNA as a template to obtain a DNA library containing two or more clones. A method for constructing a DNA library is provided. According to the method of the present invention, there is provided a method for constructing a population of genes in which a plurality of DNA fragments encoding an arbitrary amino acid sequence are linked in various orders and lengths.

Description

（実施例２）
（１）単位領域ＤＮＡの作製
ヒトエストロゲン受容体αのリガンド結合ドメイン（以下、「ｈＥＲαＬＢＤ」と称することがある：Ｇｒｅｅｎｅ　ｅｔ　ａｌ．（１９８６）Ｓｃｉｅｎｃｅ，２３１（４７４２），１１５０−１１５４；Ｂｒｚｏｚｏｗｓｋｉ　ｅｔ　ａｌ．（１９９７）Ｎａｔｕｒｅ，３８９（６６５２），７５３−７５８）は、２５８残基のアミノ酸から構成される。このｈＥＲαＬＢＤの全領域を遺伝子のエクソン（Ｐｏｎｇｌｉｋｉｔｍｏｎｇｋｏｌ　ｅｔ　ａｌ．（１９８８）ＥＭＢＯ　Ｊ．，１１，３３８５−３３８８）とタンパク質の立体構造上の二次構造（Ｔａｎｅｎｂａｕｍ　ｅｔ　ａｌ．（１９９８）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ．，９５（１１），５９９８−６００３）の境界で１０個の単位領域領域とした。各単位領域はＮ末端側からそれぞれ、３４、３５、２９、１７、２６、３７、２４、１７、２０残基のアミノ酸をコードする（図４）ように、ｈＥＲαＬＢＤを含むプラスミドを鋳型として各単位領域ＤＮＡ断片の５’側の塩基配列と相同性を有するフォワードプライマーと３’側の塩基配列と相同性を有するリバースプライマー（ダテコンセプトに依頼して作製した）を用いたＰＣＲにより増幅して作製した（本プラスミドはシカゴ大学のＧ．Ｌ．Ｇｒｅｅｎｅ教授から分与されたものである）。
作製した単位領域はＮ末端側から１、２、３、４、５、６、７、８、９、１０とした。プライマーは単位領域ＤＮＡ１については、配列番号２５及び２６、単位領域ＤＮＡ２については配列番号２７及び２８、単位領域ＤＮＡ３については配列番号２９及び３０、単位領域ＤＮＡ４については、配列番号３１及び３２、単位領域ＤＮＡ５については、配列番号３３及び３４、単位領域ＤＮＡ６については、配列番号３５及び３６、単位領域ＤＮＡ７については、配列番号３７及び３８、単位領域ＤＮＡ８については、配列番号３９及び４０、単位領域ＤＮＡ９については、配列番号４１及び４２、また単位領域ＤＮＡ１０については、配列番号４３及び４４を用いた。またこれらの単位領域ＤＮＡ以外に、５’末端共通配列ＤＮＡ断片としてＴ７プロモーター配列を有するＤＮＡ断片（Ｔ７ＯＭ：配列番号４５）、及び３’末端共通配列ＤＮＡ断片（ＣＢＳＨｉｓ：配列番号４６）を化学合成により作製した（ダテコンセプト社に依頼）。この共通配列合成ＤＮＡ断片を鋳型として、各ＤＮＡ断片の５’側の塩基配列と相同性を有するフォワードプライマー（Ｔ７ＯＭ：配列番号４７；ＣＢＳＨｉｓ：配列番号４９）と３’側の塩基配列と相同性を有するリバースプライマー（ダテコンセプトに依頼して作製した）（Ｔ７ＯＭ：配列番号４８；ＣＢＳＨｉｓ：配列番号５０）を用いたＰＣＲにより増幅して共通配列ＤＮＡ断片を作製した。作製した共通配列を有するＤＮＡ断片は５’側をＴ７ＯＭ、３’側をＣＢＳＨｉｓとした。
単位領域ＤＮＡ、及び共通配列ＤＮＡ断片の作製に用いるプライマーはＰＣＲに先立ちリン酸化した。リン酸化反応液（３０μｌ）は、ＤＮＡ溶液（２０ｐｍｏｌ／μｌ）２０μｌ、１０ｍＭ　ＡＴＰ、ポリヌクレオチドリン酸キナーゼ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂ社製）、及び添付の１０×反応緩衝液３μｌを含む。リン酸化反応は３７℃／１２時間によって行った。
リン酸化したプライマーを用いて行ったＰＣＲは、反応液（５０μｌ）としてＤＮＡ溶液（２０ｎｇ／μｌ）１μｌ、各プライマー２０ｐｍｏｌ、ｄＮＴＰｓ２００μＭ、２．５ユニットＰｆｕＤＮＡポリメラーゼ（ＳＴＲＡＴＡＧＥＮＥ社製）、及び添付の１０×ＰｆｕＤＮＡポリメラーゼ反応緩衝液５μｌを用い、反応条件が、第１段階（１サイクル）９５℃／５分、第２段階（３０サイクル）９５℃／３０秒、５６℃／３０秒、第３段階（１サイクル）４℃／１０分で行った。ＰＣＲで増幅した上記した１０個の単位領域ＤＮＡ断片、及び２個の共通配列ＤＮＡ断片はＷｉｚａｒｄ　ＰＣＲ　Ｐｒｅｐｓ（Ｐｒｏｍｅｇａ社製）を用いて調製し、６０μｌの滅菌水に溶解した。
（２）連結単位領域ＤＮＡの作製
上記（１）で作製した１２種類のＤＮＡ断片は以下の組み合わせでＤＮＡリガーゼにより連結させた後に、この連結単位領域ＤＮＡ断片を鋳型として第１の連結単位領域ＤＮＡの５’末端の配列と相同性を有するフォワードプライマー、及び第２の単位領域ＤＮＡの３’末端の配列と相同性を有するリバースプライマーを用いたＰＣＲにより作製した。
Ｔ７ＯＭ−１、Ｔ７ＯＭ−２、Ｔ７ＯＭ−３、Ｔ７ＯＭ−４、Ｔ７ＯＭ−５、Ｔ７ＯＭ−６、Ｔ７ＯＭ−７、Ｔ７ＯＭ−８、Ｔ７ＯＭ−９、Ｔ７ＯＭ−１０、１−２、１−３、１−４、１−５、１−６、１−７、１−８、１−９、１−１０、１−ＣＢＳＨｉｓ、２−３、２−４、２−５、２−６、２−７、２−８、２−９、２−１０、２−ＣＢＳＨｉｓ、３−４、３−５、３−６、３−７、３−８、３−９、３−１０、３−ＣＢＳＨｉｓ、４−５、４−６、４−７、４−８、４−９、４−１０、４−ＣＢＳＨｉｓ、５−６、５−７、５−８、５−９、５−１０、５−ＣＢＳＨｉｓ、６−７、６−８、６−９、６−１０、６−ＣＢＳＨｉｓ、７−８、７−９、７−１０、７−ＣＢＳＨｉｓ、８−９、８−１０、８−ＣＢＳＨｉｓ、９−１０、９−ＣＢＳＨｉｓ
ＤＮＡライゲースによる単位領域ＤＮＡ断片の連結のための反応液はＤＮＡ溶液（１０ｎｇ／μｌ）１μｌ、Ｔ４ＤＮＡリガーゼ４００ユニット（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂ社製）、添付の１０×Ｔ４ＤＮＡリガーゼ反応緩衝液１μｌ、滅菌水６μｌを含み、反応は１６℃で１時間行った。この連結単位領域ＤＮＡ断片を鋳型としたＰＣＲの反応液（５０μｌ）は、各プライマー２０ｐｍｏｌ、ｄＮＴＰｓ　２００μＭ、連結単位領域ＤＮＡを含む上記ＤＮＡリガーゼによる反応液１μｌ、１．５ｍＭ　ＭｇＣｌ_２、Ｔａｑ　ＤＮＡポリメラーゼ（東洋紡社製）１ユニット、及び添付の１０×Ｔａｑ　ＤＮＡポリメラーゼ反応緩衝液５μｌを含む。反応条件は、第１段階（１サイクル）９５℃／５分、第２段階（３０サイクル）９５℃／３０秒、６０℃／３０秒、第３段階（１サイクル）４℃／１０分により行った。
得られた上記連結単位領域ＤＮＡ断片はＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　ＫＩＴ（ＩｎＶｉｔｒｏｇｅｎ社製）を用いてクローニングした。クローニング手順はＫＩＴに添付されているプロトコール（ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｇ，Ｖｅｒ．Ｋ：Ｆｉｖｅ　ｍｉｎｕｔｅｓ　ｃｌｏｎｉｎｇ　ｏｆ　Ｔａｑ　ｐｏｌｙｍｅｒａｓｅ−ａｍｐｌｉｆｉｅｄ　ＰＣＲ　ｐｒｏｄｕｃｔｓ，Ｉｎｖｉｔｒｏｇｅｎ）に従い、ベクターは添付のものを使用した。具体的には、まず、上記で作製した全連結単位領域ＤＮＡ断片のＰＣＲ反応液４μｌ、添付の塩溶液１μｌ、及びＰＣＲ　ＴＯＰＯ　ｖｅｃｔｏｒ　１μｌを混合し、２５℃で５分間インキュベートした。
次に２μｌの上記反応液と添付のコンピテントセル５０μｌを混合し、氷上で３０分間インキュベートした。その後４２℃で３０秒間インキュベートした後、添付のＳＯＣ培地２５０μｌを加え、３７℃で３０分間インキュベートした。このうちの５０μｌを５０μｌ／ｍｌのアンピシリンが予め添加されているＬＢプレートに４０μｇ／ｍｌのイソプロピル−１−チオ−β−ガラクシドガラクトース（ＩＰＴＧ）を２４μｌと、４０μｇ／ｍｌのＸ−ｇａｌ溶液４０μｌを塗布した後、塗布した。
上記ＬＢプレートを１２時間、３７℃でインキュベートした後、プレート上に２００個程度のコロニーを確認した。青色のコロニーを各連結単位領域ＤＮＡにつき３個ずつ選択し、コロニーＰＣＲにより目的の連結単位領域がプラスミド上に含まれているかを確認した。ここで、青色のコロニーを選択した理由は、全ての単位領域ＤＮＡは３の倍数の塩基配列を有しているためｌａｃＺ遺伝子にフレームシフトを起こさせず活性に影響を及ぼさないと予想したからである。上記コロニーＰＣＲの反応液は、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　ＫＩＴに添付されているＭ１３プライマー、及びＭ１３Ｒｅｖ．プライマー（ＩｎＶｉｔｒｏｇｅｎ社製）社製）各２０ｐｍｏｌ、ｄＮＴＰｓ　２００μＭ、１．５ｍＭ　ＭｇＣｌ_２、ＴａｑＤＮＡポリメラーゼ（東洋紡社製）１ユニット、添付の１０×Ｔａｑ　ＤＮＡポリメラーゼ反応緩衝液５μｌ、及びコロニーを滅菌水に懸濁した溶液を含み、反応条件は第１段階（１サイクル）９５℃／５分、第２段階（３０サイクル）９５℃／３０秒、６０℃／３０秒、７２℃／３０秒、第３段階（１サイクル）４℃／１０分によりＰＣＲを行った。
このコロニーＰＣＲ反応液をアガロースゲル電気泳動により解析し、目的の連結単位領域ＤＮＡ断片が増幅されていることを確認した後、生成物をＷｉｚａｒｄ　ＰＣＲ　Ｐｒｅｐｓ　ＤＮＡ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ社製）を用いて精製し、ＤＮＡシーケンサー（ＣＥＱ２０００　ＤＮＡ　Ａｎａｌｙｓｉｓ　Ｓｙｓｔｅｍ：ベックマンコールター社製）による塩基配列解析を行った。塩基配列を解析後、置換、挿入、欠失などの変異がないことを確認した。
ここで正しい塩基配列を有していることが確認された連結単位領域ＤＮＡを含むプラスミドを鋳型として、（１）に示した各連結単位領域ＤＮＡの第１の単位領域ＤＮＡの５’末端の配列と相同性を有するフォワードプライマー、及び第２の単位領域ＤＮＡの３’末端の配列と相同性を有するリバースプライマーを用いたコロニーＰＣＲにより再度連結単位領域ＤＮＡを作製した。ＰＣＲの反応液（５０μｌ）は、各プライマー２０ｐｍｏｌ、ｄＮＴＰｓ　２００μＭ、３．７５ユニットＰｆｕＤＮＡポリメラーゼ（ＳＴＲＡＴＡＧＥＮＥ社製）、及び添付の１０×ＰｆｕＤＮＡポリメラーゼ反応緩衝液５μｌを含み、反応条件は第１段階（１サイクル）９５℃／５分、第２段階（３０サイクル）９５℃／３０秒、５６℃／３０秒、第３段階（１サイクル）４℃／１０分で行った。
上記連結単位領域ＤＮＡを作製したＰＣＲ反応液から連結単位領域ＤＮＡ断片をエタノール沈殿により濃縮し、２０μｌｍの滅菌水に溶解後、低融点アガロースゲル電気泳動により分離し、目的のＤＮＡのバンドを切り出した。このアガロースゲル断片からフェノール／クロロフォルムを用いてＤＮＡを抽出し、これをエタノール沈殿し、６０μｌの滅菌水に融解した。得られた各連結単位領域ＤＮＡ水溶液の濃度は２６０ｎｍの吸光度を測定することにより求めた。ここで得られた連結単位領域ＤＮＡを続く変異ＤＮＡライブラリーを構築するためのＰＣＲの鋳型ＤＮＡとした。
（３）連結単位領域ＤＮＡの分類
上記（２）で得られた全ての連結単位領域ＤＮＡはそれらがコードしている単位領域ＤＮＡによって分類した。分類は上記で作製した連結単位領域ＤＮＡの第１の単位領域ＤＮＡと第２の単位領域ＤＮＡが、それらが存在する構造遺伝子ＤＮＡにおいて何個の単位領域を隔てたものが連結されているかを指標として行った。ここで第１の単位領域ＤＮＡと第２の単位領域ＤＮＡとがそれらが存在する構造遺伝子ＤＮＡにおいて隔てている単位領域の数をＸとすると、作製した連結単位領域ＤＮＡはＸ＝０からＸ＝８の９種類に分類することができた（図５（ａ））。
（４）８個の単位領域ＤＮＡが連結された変異ＤＮＡライブラリーの構築
上記（３）で分類した連結単位領域ＤＮＡ群を鋳型として、８個の単位領域ＤＮＡが連結された変異ＤＮＡライブラリーを作製した。各連結単位領域ＤＮＡ群について、構造遺伝子を分けた連結単位領域の総数（Ｌ）を１０、単位領域の総数と作製するＤＮＡに含まれる単位領域ＤＮＡ数との差（Ｍ）を２とし、第１の単位領域ＤＮＡと第２の単位領域ＤＮＡとがそれらが存在する構造遺伝子ＤＮＡにおいて隔てている単位領域の数（Ｘ）について、下記式（１）
_{Ｌ−Ｘ−１}Ｃ_Ｍ−Ｘ　　　（１）
にあてはめて各連結単位領域ＤＮＡ群のＰＣＲに供するＤＮＡの総量に占める割合を決めた。
上記（３）の分類でＸ＝０の分類に入った連結単位領域ＤＮＡ群は３６、Ｔ７ＯＭ−１は３６、１０−ＣＢＳＨｉｓＸは３６、Ｘ＝１の連結単位領域ＤＮＡ群は８、Ｔ７ＯＭ−２は８、９−ＣＢＳＨｉｓＸは８、Ｘ＝２の連結単位領域ＤＮＡ群は１、Ｔ７ＯＭ−２は１、８−ＣＢＳＨｉｓＸは１となった。
具体的なＤＮＡ量としては、変異ＤＮＡライブラリーを作製するためのＰＣＲにはＤＮＡの総量として６７５ｆｍｏｌを用いたので、Ｘ＝０の連結単位領域ＤＮＡ群のＤＮＡの総量は１８０ｆｍｏｌとなり、該分類に入る連結単位領域ＤＮＡ（１−２、２−３、３−４、５−６、７−８、８−９、９−１０）を２６ｆｍｏｌずつ、Ｔ７ＯＭ−１を１８０ｆｍｏｌ、１０−ＣＢＳＨｉｓＸを１８０ｆｍｏｌ、Ｘ＝１の連結単位領域ＤＮＡ群のＤＮＡの総量は４０ｆｍｏｌとなるので該分類に入る連結単位領域ＤＮＡ（１−３、２−４、３−５、４−６、５−７、６−８、７−９、８−１０）を５ｆｍｏｌずつ、Ｔ７ＯＭ−２を４０ｆｍｏｌ、９−ＣＢＳＨｉｓＸを４０ｆｍｏｌ、Ｘ＝２の連結単位領域ＤＮＡ群のＤＮＡの総量は５ｆｍｏｌとなるので該分類に入る連結単位領域ＤＮＡ（１−４、２−５、３−６、４−７、５−８、６−９、７−１０）を０．７ｆｍｏｌずつ、Ｔ７ＯＭ−３を５ｆｍｏｌ、８−ＣＢＳＨｉｓＸを５ｆｍｏｌずつ混合してＰＣＲの鋳型ＤＮＡとした。
ＰＣＲ反応液（５０μｌ）は、プライマーとしてＴ７ＯＭのフォワードプライマーとＣＢＰＨｉｓのリバースプライマーを各２０ｐｍｏｌ、上記混合鋳型ＤＮＡを６７５ｆｍｏｌ、ｄＮＴＰｓを２００μｌ、ＫＯＤ　Ｄａｓｈ　ＤＮＡポリメラーゼ（東洋紡社製）を１．２５ユニット、添付の１０×ＫＯＤ　Ｄａｓｈ　ＤＮＡポリメラーゼ反応緩衝液５μｌを含み、反応条件が第１段階（１サイクル）９５℃／５分、第２段階（２０サイクル）９５℃／３０秒、６０℃／３０秒、７２℃／３分、第３段階（１サイクル）４℃／１０分によりＰＣＲを行った。
上記ＰＣＲ産物はエタノール沈殿により濃縮後、低融点アガロースゲル電気泳動により解析した結果、意図したとおりの分子量に相当する領域に強いＤＮＡバンドを確認することができた（図６（ａ））。
（５）５個の単位領域ＤＮＡが連結された変異ＤＮＡライブラリーの構築
上記（３）で分類した連結単位領域ＤＮＡ群を鋳型として、５個の単位領域ＤＮＡが連結された変異ＤＮＡライブラリーを作製した。各連結単位領域ＤＮＡ群について、構造遺伝子を分けた連結単位領域の総数（Ｌ）を１０、単位領域の総数と作製するＤＮＡに含まれる単位領域ＤＮＡ数との差（Ｍ）を５とし、第１の単位領域ＤＮＡと第２の単位領域ＤＮＡとがそれらが存在する構造遺伝子ＤＮＡにおいて隔てている単位領域の数（Ｘ）について、上記式（１）にあてはめて各連結単位領域ＤＮＡ群のＰＣＲに供するＤＮＡの総量に占める割合を決めた。
上記（３）の分類でＸ＝０の分類に入った連結単位領域ＤＮＡ群は１２６、Ｔ７ＯＭ−１は１２６、１０−ＣＢＳＨｉｓＸは１２６、Ｘ＝１の連結単位領域ＤＮＡ群は７０、Ｔ７ＯＭ−２は７０、９−ＣＢＳＨｉｓＸは７０、Ｘ＝２の連結単位領域ＤＮＡ群は３５、Ｔ７ＯＭ−３は３５、８−ＣＢＳＨｉｓＸは３５、Ｘ＝３の連結単位領域ＤＮＡ群は１５、Ｔ７ＯＭ−４は１５、７−ＣＢＳＨｉｓＸは１５、Ｘ＝４の連結単位領域ＤＮＡ群は５、Ｔ７ＯＭ−５は５、６−ＣＢＳＨｉｓＸは５、Ｘ＝５の連結単位領域ＤＮＡ群は１、Ｔ７ＯＭ−６は１、５−ＣＢＳＨｉｓＸは１となった。
具体的なＤＮＡ量としては、変異ＤＮＡライブラリーを作製するためのＰＣＲにはＤＮＡの総量として１２６０ｆｍｏｌを用いたので、Ｘ＝０の連結単位領域ＤＮＡ群のＤＮＡの総量は２１０ｆｍｏｌとなり、該分類に入る連結単位領域ＤＮＡ（１−２、２−３、３−４、５−６、７−８、８−９、９−１０）を３０ｆｍｏｌずつ、Ｔ７ＯＭ−１を２１０ｆｍｏｌ、１０−ＣＢＳＨｉｓＸを２１０ｆｍｏｌ、Ｘ＝１の連結単位領域ＤＮＡ群のＤＮＡの総量は１１７ｆｍｏｌとなるので該分類に入る連結単位領域ＤＮＡ（１−３、２−４、３−５、４−６、５−７、６−８、７−９、８−１０）を１４．６ｆｍｏｌずつ、Ｔ７ＯＭ−２を１１７ｆｍｏｌ、９−ＣＢＳＨｉｓＸを１１７ｆｍｏｌ、Ｘ＝２の連結単位領域ＤＮＡ群のＤＮＡの総量は５８ｆｍｏｌとなるので該分類に入る連結単位領域ＤＮＡ（１−４、２−５、３−６、４−７、５−８、６−９、７−１０）を８ｆｍｏｌずつ、Ｔ７ＯＭ−３を５８ｆｍｏｌ、８−ＣＢＳＨｉｓＸを５８ｆｍｏｌ、Ｘ＝３の連結単位領域ＤＮＡ群のＤＮＡの総量は２５ｆｍｏｌとなるので該分類に入る連結単位領域ＤＮＡ（１−５、２−６、３−７、４−８、５−９、６−１０）を４ｆｍｏｌずつ、Ｔ７ＯＭ−４を２５ｆｍｏｌ、７−ＣＢＳＨｉｓＸを２５ｆｍｏｌ、Ｘ＝４の連結単位領域ＤＮＡ群のＤＮＡの総量は８．３ｆｍｏｌとなるので該分類に入る連結単位領域ＤＮＡ（１−６、２−７、３−８、４−９、５−１０）を１．６ｆｍｏｌずつ、Ｔ７ＯＭ−５を８．３ｆｍｏｌ、６−ＣＢＳＨｉｓＸを８．３ｆｍｏｌ、Ｘ＝５の連結単位領域ＤＮＡ群のＤＮＡの総量は１．７ｆｍｏｌとなるので該分類に入る連結単位領域ＤＮＡ（１−７、２−８、３−９、４−１０）を０．４ｆｍｏｌずつ、Ｔ７ＯＭ−６を１．７ｆｍｏｌ、５−ＣＢＳＨｉｓＸを１．７ｆｍｏｌずつ混合してＰＣＲの鋳型ＤＮＡとした。
ＰＣＲ反応液（５０μｌ）は、プライマーとしてＴ７ＯＭのフォワードプライマーとＣＢＰＨｉｓのリバースプライマーを各２０ｐｍｏｌ、上記混合鋳型ＤＮＡを１２６０ｆｍｏｌ、ｄＮＴＰｓを２００μｌ、ＫＯＤ　Ｄａｓｈ　ＤＮＡポリメラーゼ（東洋紡社製）を１．２５ユニット、添付の１０×ＫＯＤ　Ｄａｓｈ　ＤＮＡポリメラーゼ反応緩衝液５μｌを含み、反応条件が第１段階（１サイクル）９５℃／５分、第２段階（２０サイクル）９５℃／３０秒、６０℃／３０秒、７２℃／３分、第３段階（１サイクル）４℃／１０分によりＰＣＲを行った。　上記ＰＣＲ産物はエタノール沈殿により濃縮後、低融点アガロースゲル電気泳動により解析した結果、意図したとおりの分子量に相当する領域に強いＤＮＡバンドを確認することができた。（図６（ｂ））
（６）変異ＤＮＡライブラリーの確認
上記（４）及び（５）で得られたＰＣＲ産物が意図したとおりの配列を有するか否かを調べる目的で上記低融点アガロースゲル電気泳動により得られた強いＤＮＡバンドを示す領域を切り出し、Ｗｉｚａｒｄ　ＰＣＲ　Ｐｒｅｐｓ　ＤＮＡ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｓｙｓｔｅｍ（Ｐｒｏｍｅｇａ社製）を用いてＤＮＡを抽出精製した。続いてＴａｑ　ＤＮＡポリメラーゼを用いてｄＡを３’末端に付加させた後、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　ＫＩＴ（ＩｎＶｉｔｒｏｇｅｎ社製）を用いて上記（２）と同様にクローニングした。この際、ＬＢプレートはＩＰＴＧとＸ−Ｇａｌを塗布していないものを用いた。
このＬＢプレートを１２時間、３７℃でインキュベートした後、プレート上に１００個以上のコロニーを確認できた。４０個以上のコロニーについて、上記（２）と同様の方法で、プラスミド内に含まれる（４）及び（５）で作製したＰＣＲ産物ＤＮＡの塩基配列を解析した。
上記（４）のＰＣＲ産物（８個の単位領域ＤＮＡが結合したもの）については、３８クローンについて塩基配列を解析した。この結果を図７に示した。また（５）のＰＣＲ産物（５個の単位領域が結合したもの）については、３３クローンについて塩基配列を解析した。この結果は図８に示した。いずれのＰＣＲでも得られたＤＮＡ断片は全て５’末端にＴ７ＯＭ配列を３’末端にはＣＢＳＨｉｓ配列を有しており、内部の単位領域ＤＮＡが構造遺伝子において上流から下流にむけての順序を保ったまま様々に連結された構造を有していた。このことは、解析した７１個のＤＮＡ断片の塩基配列中に出現した各単位領域の数が１／３９個、２／３０個、３／５２個、４／３４個、５／４０個、６／４３個、７／５６個、８／３２個、９／４３個、１０／４０個であり、用いた単位領域の出現頻度に著しい偏りがみられなかたことにより確認できた。
また、上記で解析したＤＮＡ断片のうち同じ配列を有するものは図７で示した配列中１組あるのみであった。置換については、７１クローン、４７５３３塩基のうち、置換された塩基は６０個であった。この変異率（１．３×１０^−３）は充分低く、ライブラリー構築の際の障害にはならないと判断した。実際に５０箇所の置換のうち、ストップコドンがあったものは２箇所のみであった。さらに、意図せぬ領域で連結された配列が２種類存在した。１つは上記単位領域ＤＮＡ１と単位領域ＤＮＡ２の両方に共通に存在する「ａｔｇａｔｃ」での相同組換えが誘導されたことにより生じたものであった。もう１つは上記単位領域ＤＮＡ５と単位領域ＤＮＡ４両方に共通に存在する「ｃｃａｇｇｇａ」と「ｃｃａｇｔｇａ」間の相同組換えが誘導されたことにより生じたものであった。その他の配列にはフレームシフトを起こすような変異、つまり欠失や挿入は見られなかった。つまり本発明のＰＣＲによる変異ＤＮＡライブラリー中に含まれるＤＮＡの大部分は正しい読み取り枠を保っていることが示された。
図７に示した配列中には単位領域が５個連結したＤＮＡ断片が３クローン、６個連結したＤＮＡ断片が１０クローン、７個連結したＤＮＡ断片が９クローン、８個連結したＤＮＡ断片が１０クローン、９個連結したＤＮＡ断片が２クローン、１０個連結したＤＮＡ断片が１クローンであった。また、図８に示した配列中には単位領域が２個連結したＤＮＡ断片が１クローン、３個連結したＤＮＡ断片が２クローン、４個連結したＤＮＡ断片が９クローン、５個連結したＤＮＡ断片が１７クローン、６個連結したＤＮＡ断片が４クローンであった。
以上の結果は、意図した単位領域の数より多いまたは少ない数の単位領域ＤＮＡからなるＤＮＡ断片も含まれているが、目的とする数の単位領域ＤＮＡからなるＤＮＡ断片が最も多いことから、該ライブラリーを進化分子工学に適用することにより、有用なタンパク質を創出するという本発明の目的を達するに充分な変異ＤＮＡライブラリーが構築できたことを示している。
（実施例３）
Ｅｒｒｏｒ−ｐｒｏｎｅＰＣＲによって変異ＤＮＡライブラリーを作製した。
実施例２の（１）〜（３）及び（５）の操作と同様にして連結単位領域を混合してＰＣＲを行う際に、該ＰＣＲ反応液に塩化マンガンを０〜１ｍＭの濃度で添加した。増幅されたＤＮＡが有する塩基配列を実施例２の（６）と同様にして解析した。この結果を表２に示す。マンガンイオン濃度の増加に対して、ほぼ直線的に塩基置換の頻度が増加することが分かった。従って、任意の置換率でライブラリーを構築したい場合には、表２のデータに従ってマンガンイオンを添加すればよいことが判明した。

産業上の利用の可能性
本発明の方法によれば、任意のアミノ酸配列をコードする複数のＤＮＡ断片を様々な順序と長さで連結した遺伝子の集団を構築する方法が提供され、該変異ＤＮＡライブラリーから作製される変異プロテインライブラリーにより所望のタンパク質を選択し、さらに進化分子工学を用いることにより、天然タンパク質の部品（単位領域）である二次構造、モジュール、モチーフなどを組み合わせた新しい機能をもつタンパク質を創出するのに極めて有用である。
また、本発明の選択的スプライシングライブラリーから作製される変異プロテインライブラリーからは、分子量が小型化したタンパク質を取得することができる。分子量が２万以下の比較的小さな球状タンパク質の変性は可逆的であることが知られており、一度変性しても適切な条件に戻すことによりその機能を回復することができる。また、このような性質を持つタンパク質は、リボヌクレアーゼに代表されるように、熱や有機溶媒に対する耐性が強い。またこのような酵素を用いたバイオプロセスにおける精製物の回収率も高い。本発明の選択的スプライシングライブラリーから得られる変異タンパク質は、任意のタンパク質をその機能を保持したまま小型化できるため、タンパク質としての応用性を広げるものである。
【配列表】

【図面の簡単な説明】
図１は、本発明の概念を示す図である。３種類の単位領域ＤＮＡをシャッフルする場合を例として示した。２つの単位領域ＤＮＡが連結した連結単位領域ＤＮＡを混合し、Ｔ７のフォワードプライマーとＥｘのリバースプライマーを用いてＰＣＲを行うことにより、３種の単位領域ＤＮＡが、種々の順序で連結された多数のＤＮＡ断片が一度のＰＣＲで合成される。
図２は、ＰＣＲ産物をアガロースゲル（２％）を用いて分離した電気泳動写真である。各レーンは５０μｌのＰＣＲ産物を含む。ラダー状のＤＮＡバンドに対応する配列を右に示した。ＭはＤＮＡマーカーである。
図３は、本発明の変異ＤＮＡライブラリーの構築方法の原理を示す図である。
図４は、ヒューマンエストロゲンレセプターαのリガンド結合ドメインをエクソンと二次構造境界で分断し、１０個の単位領域に分割したことを示す構成図である。
図５は、１０個の単位領域からなる構造遺伝子から本発明の変異遺伝子ライブラリーを構築する際に用いるすべての連結単位領域ＤＮＡを示す図である。各連結単位領域ＤＮＡは、対角線に示されたように、Ｘ＝０からＸ＝８の９通りの連結単位領域ＤＮＡ群に分類できる（ａ）。ある特定の数の単位領域から構成される構造遺伝子の集団中に現れる各連結単位領域ＤＮＡ群ごとの出現頻度を示した（ｂ）。
図６は、本発明の変異ＤＮＡライブラリーを構築した際のＰＣＲ産物を分離したアガロースゲル電気泳動写真である。（ａ）、（ｂ）はそれぞれ８個、及び５個の単位領域ＤＮＡから構成される構造遺伝子の集団を構築した場合のＰＣＲ産物である。図中、Ｍはマーカーを示す。
図７は、８個の単位領域ＤＮＡから構造遺伝子を得るような条件下でＰＣＲを行い得られた本発明の変異遺伝子ライブラリーが有するＤＮＡの構造を示した図である。
図８は、５個の単位領域ＤＮＡから構造遺伝子を得るような条件下でＰＣＲを行い得られた本発明の変異遺伝子ライブラリーが有するＤＮＡの構造を示した図である。Technical field
The present invention relates to a method for constructing a DNA library, which comprises performing Polymerase Chain Reaction (hereinafter, sometimes referred to as “PCR”) using a mixture of DNAs of a linking unit region as a template. More specifically, the present invention relates to a DNA sequence encoding a specific amino acid sequence such as a protein module, a secondary structure, a super secondary structure, a motif, a primitive sequence, an exon, a DNA constituting an expression control region, or an artificial amino acid. A method for constructing a mutant DNA library, wherein PCR is performed using a mixture of linked unit region DNAs to which DNAs encoding the sequence are linked as a template, and obtained by transcribing and translating the DNA library. Regarding protein library. In addition, the present invention provides a method for obtaining a mutant protein having a desired property, which comprises selecting a protein having a desired property using the protein library, and applying a technique of evolutionary molecular engineering. It also relates to the resulting protein with further enhanced desired properties.
Background art
Currently, many enzymes are used in industrial bioprocesses. In relation to environmental issues, energy-efficient bioprocesses are expected to become more widespread in the future. In addition, with the development of genetic engineering, the demand for proteins as pharmaceuticals and biosensors is expected to increase. However, many proteins have low structural stability and are easily denatured by heat or organic solvents. Agglomeration in the manufacturing process causes a decrease in the recovery rate of the synthesized product. Such denaturation and aggregation are often irreversible, and it is difficult to regenerate a once denatured protein to a natural state having a function.
Therefore, attempts have been made to improve heat resistance, organic solvent resistance and the like by protein engineering or evolutionary molecular engineering techniques. For example, in protein engineering, a protein having a known structure is subjected to amino acid substitution by estimating a site likely to contribute to the stabilization of the structure (Applied Chemistry, Protein Engineering, Asakura Shoten, Katsuhide Ikeya, Haruki Nakamura, 1991) ) Method is used. In addition, if the technique of evolutionary molecular engineering is used, a protein with improved function can be obtained by performing site-specific or random amino acid substitution and selecting as an index that the target property has been increased. .
Evolutionary molecular engineering is an excellent technique for creating proteins having specific functions. In the evolutionary molecular engineering of proteins, first, a mutant population containing many proteins (hereinafter, sometimes referred to as “mutant protein library”) is constructed. Next, a mutant protein having a desired function is selected from the library. Furthermore, a mutation is introduced into the DNA encoding the selected mutant protein and expressed, and a mutant protein library is constructed again to select a mutant protein having a further increased desired function. By repeating this operation several times, a protein having excellent functions can be obtained.
In order to obtain a mutant protein by such evolutionary molecular engineering, a library capable of searching a larger sequence space among mutant protein libraries and mutant DNA libraries encoding them is essential. Therefore, the construction method is a very important elemental technology for evolutionary molecular engineering.
In the early stage of constructing a mutant DNA library, a mutagenizing agent for DNA (Myers, RM et al. (1985) Science 229, 242-247; Chu, C.-T. et al. (1979)) ) Virology 98, 168-181) or radiation (Billi, D. (2000) Appl. Environ. Microbiol. 66, 1489-1492), but a method for chemically synthesizing DNA (Sinha, N. D. et al. (1984) Nucleic Acids Res. 12, 4539-4557) and Polymerase Chain Reaction (Saiki, RK et al. (1985) Science 230, 1350-1354). Mutation introduction methods are also methods using recombination technology (Botstein, D. & Shortle, D. (1985) Science 229, 1193-1201), Error-prone PCR (Leung, DW et al. (1989) ) Technique 1,11-15), DNA shuffling (Stemmer, WPC (1994) Proc. Natl. Acad. Sci. USA. 91, 10747-10751), etc. It has become.
In the error-prone PCR, an excess amount of manganese ions and four types of deoxyribonucleotide triphosphates are added at various mixing ratios, and PCR is performed using Taq DNA polymerase. be introduced.
In DNA shuffling, a DNA encoding a protein to be evolved is introduced with a point mutation by error-prone PCR, and then digested with DNase I. Thereafter, by performing PCR in the absence of primers, recombination via homologous sequences occurs, and various point mutations already introduced are combined to construct a DNA library into which various mutations have been introduced. Is done. That is, point mutation can be introduced more efficiently than Error-prone PCR. In DNA shuffling, a sequence in a family protein having relatively high homology in sequence can also be used as a parent sequence.
According to the method described above, it is possible to prepare a mutant DNA library in which a point mutation has been introduced into DNA. However, the amino acid sequence space of a protein is very large. For example, the sequence of a protein consisting of 100 amino acids is 20 ¹⁰⁰ $ 10 ¹³⁰ It is possible. This is the number of atoms in the universe (10 ⁷⁵ More than). It is unlikely that such an enormous sequence has been exhaustively tested in the course of less than 4 billion years of biological evolution. That is, it is highly possible that organisms existing on the earth have sequences with useful functions that have not yet been tried in the vast amino acid sequence space of proteins.
In this regard, Error-prone PCR can only introduce very few point mutations. In addition, since DNA shuffling can only introduce point mutations between sequences having high sequence homology, it is impossible to search a vast sequence space as a mutant with a library constructed by such a method. It is.
In addition, a technique for efficiently constructing a mutant DNA library by randomly combining base sequences encoding any amino acid sequence having no homologous sequence, instead of introducing a point mutation into DNA, has been known. Absent.
As a method of recombining a certain region of such DNA, for example, a method of simply ligating an arbitrary DNA fragment using DNA ligase can be considered. Appears with a half of the probability, the amino acid sequence encoded by the original DNA fragment cannot be obtained, and the termination codon frequently appears, so that the smallest globular protein (from about 100 amino acids It is not suitable as a method for constructing a library, for example, because it is difficult to construct a structural gene corresponding to
As a method of ligating while maintaining the orientation of the DNA, a method of digesting the 5 'end and 3' end of each unit region DNA to be ligated with different restriction enzymes and ligating the fragment using DNA ligase is used. Can be considered. However, in this case, the orientation of each unit region is maintained, but a recognition site (linker) for a restriction enzyme must be artificially added to each unit region. As a result, the specific amino acid sequence can be identified. The problem of insertion of an amino acid occurs.
On the other hand, the present inventors, as one of the methods for introducing site-specific mutations at the unit region level, isolate and isolate a gene DNA encoding a protein for each unit region, and isolate the isolated unit region DNA. Ligated in any combination, the connected unit region DNAs are selected and mixed so that the ends of each unit region DNA overlap in the order in which they are to be replaced, and the mixture of the connected unit region DNAs is used as a template, It discloses a method for constructing a replacement mutant DNA comprising amplifying the DNA using a primer of a terminal unit region DNA (Japanese Patent Application Laid-Open No. 10-014578). However, this method aims at obtaining a replacement mutant DNA obtained by rearranging a predetermined number of unit region DNAs, and further, since only one type of mutant DNA can be obtained per PCR, various proteins can be obtained. A library having a sequence space is not constructed by one PCR.
Disclosure of the invention
An object of the present invention is to provide a method for constructing a library for exploring a vast sequence space of a protein, which cannot be performed by a conventional mutant population construction method, such as Error-prone PCR or DNA shuffling. A region that constitutes a part of a natural protein such as a module, a secondary structure, a super-secondary structure, a motif, a primitive sequence, an exon of DNA, or an expression control region or a region composed of an artificial amino acid sequence is referred to as a unit region (hereinafter referred to as “ This is sometimes referred to as a “unit region”), and is intended to efficiently construct a mutant DNA library in which the DNAs constituting them are linked in an appropriate combination.
Still another object of the present invention is to prepare a protein library by transcribing and translating the above-mentioned DNA library as a method for obtaining a protein having desired properties using the technology of evolutionary molecular engineering. Is to select and obtain a protein having desired properties.
In addition, the present invention provides a mutant protein having a desired property by selecting a protein having a desired property using the protein library, and further applying an evolutionary molecular engineering technique to further improve the desired property. The purpose is to obtain a purified protein.
Further, the present invention provides a method for obtaining a DNA encoding a protein having a desired property by analyzing a DNA encoding a selected protein, and producing a recombinant of the protein having a desired property using the DNA. And so on.
The present inventors have conducted intensive studies to achieve the above object, and as a result, prepared and mixed a plurality of linked unit region DNAs in which DNAs encoding any two types of amino acid sequences were linked, and used this as a template to perform PCR. If performed, DNAs having various sequences in which DNAs encoding arbitrary amino acid sequences are randomly combined are synthesized by a single PCR (hereinafter, this may be referred to as a “random library”). I found that. Furthermore, the present inventors have focused on the fact that, in a eukaryotic protein synthesis system, a plurality of proteins are synthesized by combining limited exons by alternative (alternative) splicing. For the unit region DNA obtained by dividing the structural gene DNA encoding into an arbitrary fragment, a linked unit region DNA is prepared by connecting the structural gene while maintaining the order from upstream to downstream in the structural gene, and these are specified. It has been found that, by mixing the DNA at a ratio to form a template DNA and performing PCR, a DNA sequence group generated by alternative splicing (hereinafter, this may be referred to as “alternative splicing library”) can be constructed. The present invention has been completed based on these findings.
That is, according to the present invention, the following inventions are provided.
(1) (a) ligating the unit region DNAs in an arbitrary combination, and (b) connecting the obtained ligated unit region DNAs to at least one other type of ligated unit in which the terminal sequences of all the ligated unit region DNAs to be used are different. (C) Performing a Polymerase Chain Reaction using the mixture of the linked unit region DNAs as a template to obtain a DNA library containing two or more clones. And a method for constructing a DNA library.
(2) In the step (b), the connecting unit region DNA is used in such a manner that the terminal sequences of all the connecting unit regions used overlap at least the terminal sequences of at least one other type of connecting unit region DNA, and (1) The method according to (1), wherein the sequences of either the 5 ′ end or the 3 ′ end of the connecting unit region DNA are selected and mixed so as to overlap at least the other two types of connecting unit region DNA ends.
(3) The method according to (1) or (2), wherein the mixture of connected unit region DNAs is a mixture of connected unit regions in which two unit region DNAs are connected in all combinations of unit region DNAs used.
(4) The unit region DNA is obtained by dividing a structural gene DNA encoding a protein into L pieces (here, L is an integer of 3 or more), and the ligation method is 3 ′ of an arbitrary unit region DNA. A mixture of the terminal unit and the 5 'end of the unit region DNA present on the C-terminal side of the protein from the unit region so that the amino acid sequence of each unit region is maintained, and a mixture of the linked unit region DNAs Is the number of unit regions separated from each other in the original structural gene by X (where X is an integer from 0 to L-2) as an index. Are mixed using the ratio satisfying the following formula (1) as an index.
_L-X-1 C _MX (1)
(In the formula, L and X have the same meanings as described above, and M represents the difference between the total number of unit regions and the number of unit regions of the mutant DNA to be prepared.)
(5) A unit region DNA in which DNAs encoding a group of proteins having the same tertiary structure but different amino acid sequences are each divided into L unit regions (where L is an integer of 3 or more). The ligation method is such that the 3 ′ end of the arbitrary unit region DNA and the 5 ′ end of the unit region DNA present on the C-terminal side of the above protein group from the unit region have an amino acid sequence of each unit region. And a mixture of linked unit region DNAs is the number of unit regions separated from each other in the original protein group by X (where X is 0 or more and L (2). The method according to (1), wherein the respective linking unit region DNAs classified using the index as an index are mixed using the ratio satisfying the following formula (1) as an index.
_L-X-1 C _MX (1)
(In the formula, L and X have the same meanings as described above, and M represents the difference between the total number of unit regions and the number of unit regions of the mutant DNA to be prepared.)
(6) The method according to any one of (1) to (5), wherein the Polymerase Chain Reaction is performed in the presence of a primer having a sequence complementary to a unit region DNA to be both ends of the DNA to be constructed. .
(7) The method according to any one of (1) to (6), wherein the Polymerase Chain Reaction is performed in the presence of a connecting unit region DNA containing a common DNA sequence and a primer having a sequence complementary to the common DNA sequence. .
(8) A mutant DNA library obtained by any one of the methods (1) to (7).
(9) A protein library obtained by incorporating the DNA library according to (8) into an expression vector, transforming a host cell with the expression vector, culturing the transformant, and collecting protein from the culture. Rally.
(10) An RNA library obtained by transcribing the DNA library according to (8).
(11) A method for producing a protein library, comprising expressing the DNA library according to (8) or the RNA library according to (10) in a cell-free transcription and / or translation system.
(12) A protein library obtained by the method of (11).
(13) A nucleic acid-protein linked molecule library in which a DNA contained in the DNA library according to (7) or an RNA contained in the RNA library according to (9) is bound to a protein encoded by the DNA or RNA. Rally.
(14) A method for obtaining a protein, comprising selecting a protein having desired properties using the library according to (9), (12) or (13).
(15) A protein obtained by the method according to (14).
(16) (a) DNA encoding the protein described in (15) was prepared, (b) a mutation was introduced into the DNA, (c) the DNA having the mutation introduced was amplified, and (d) the DNA was amplified. A method for obtaining a protein, comprising: preparing a protein library by transcribing / translating DNA; and (e) obtaining a protein having desired properties using the library.
(17) A protein obtained by the method according to (16).
(18) A method for obtaining a DNA encoding the protein according to (15) or (17).
(19) DNA obtained by the method according to (18).
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in more detail. In addition, unless otherwise specified in the present specification, the description is common to the random library and the alternative splicing library.
(1) Unit region DNA
The "unit region" used in the present invention refers to a specific region unit having any structural, functional, or evolutionary meaning in a protein, gene, or genomic DNA, or any region not showing such a meaning, or Refers to an expression control region and the like. Examples of the region having a specific meaning in a protein include, for example, protein module, secondary structure, supersecondary structure, structural motif, motif, primitive sequence, artificial amino acid sequence, and the like. And so on. The expression control region specifically refers to a core promoter element, an upstream control element, an enhancer promoter, an enhancer, and the like.
When a random library is prepared, any of the above unit regions can be used. In the case of an alternative splicing library, a fragment obtained by dividing a structural gene encoding a protein is used.
A protein module is a region consisting of 10 to 40 residues of amino acids that is continuous and sterically compact in primary structure in a globular protein or domain. The polypeptides encoded by the exon parts of eukaryotes correspond well to the unit region showing the modular structure.Modules are the basic polypeptides that make up proteins, and are important materials in the early stages of biological evolution. (Go, M. et al. (1987) Cold Spring Harb. Symp. Quant. Biol., 52, 915-924). Specifically, for example, modules constituting lysozyme, hemoglobin, triosephosphate isomerase, and barnase can be mentioned.
The secondary structure of a protein refers to a relatively narrow range (up to about 10 residues) formed by a hydrogen bond between a C 間 の O group and an NH group in the peptide main chain in the three-dimensional structure of the protein. A special three-dimensional structure that is observed and has a regular structure having repetitions such as α-helix, β-sheet, β-turn, or loop structure (Salemme, FR et al. (1983) In biochemistry, ed. Zubay, G., 69-129, Addison-Wesley Publishing Co., Massachusetts), a chameleon sequence (Minor, DL et al., Nature, 380 (6576), 730-734 (1996)), and G-peptide. (Honda, S. et al., J. Mol. Biol., 95 (2), refers to a 269-278 (2000)).
The super-secondary structure of a protein refers to a unit in which a number of secondary structural units such as α-helix and β-sheet structure gather to locally form a compact three-dimensional structure (Salemme, FR. (1983) In biochemistry, ed. Zubay, G., 69-129, Addison-Wesley Publishing Co., Massachusetts). Specific examples include a helix-loop-helix structure.
A motif of a protein is an operation (sequence alignment) in which amino acid sequences of proteins with similar or similar functions are arranged so that amino acid sequences with the same or similar properties are located at the same position. A short but significantly conserved amino acid sequence when compared in sequence. In other words, a motif is a specific site in a protein, which refers to an amino acid sequence or base sequence that has been conserved during evolution. Specific examples include Greek key motifs, and motifs registered in the protein motif database PROSITE (http://expasy.hcuge.ch/sprot/prosite.html).
Primitive sequence refers to the specificity of the transfer RNA as well as the corresponding amino acid sequence. It has been speculated from the comparison of sequence homology of existing proteins that primitive sequences played an important role in diversification of protein functions in the early stages of evolution (Ohnishi, K. (1993) In Endocytobiology V, eds. S. Sato et al., Tubingen University Press, Tubingen, 407-414).
An exon refers to a base sequence translated into a protein in eukaryotic gene DNA. Exons are interrupted by introns whose base sequence is not translated into protein. After the genomic DNA is transcribed into RNA, introns are cut off by splicing, and only exons are joined to form a mature mRNA that is used as information for protein synthesis (Gilbert, W. (1978) Nature, 271, 501).
The term “unit region DNA” used in the present invention means DNA constituting a unit region. When the unit region is a fragment of a structural gene, the base sequence of the original structural gene has a single base sequence. The unit region DNA obtained by dividing the DNA has a single base sequence. When the unit region is not a fragment of the structural gene, the DNA contained in each unit region DNA means a DNA group containing a plurality of homologous base sequences. Here, the term "base sequence having a plurality of homologies" refers to a base sequence having a homology of a certain degree at which PCR described below can be performed and the reading frame of the DNA is retained.
Such a unit region DNA is composed of two complementary strands, and may be DNA that can be separated and isolated from an arbitrary DNA strand or chemically synthesized DNA. The length of the DNA chain is not particularly limited as long as it is a unit having the above structure, but a fragment of 30 to 120 bp encoding 10 to 40 amino acids, or a 5 ′ or 3 ′ terminal A mixture of DNA fragments differing by ± 3 to 15 bp may be used. As an example of such a method for preparing a DNA fragment having a 5 ′ or 3 ′ end differing by ± 3 to 15 bp, a DNA fragment designed to include a sequence shifted by ± 3 to 15 bp adjacent to the base sequence when producing by PCR is used. Using a plurality of primers.
In the method for constructing a mutant DNA library of the present invention, first, this unit region DNA is prepared. The method of preparation may be any method as long as the above-mentioned unit region DNA can be obtained, but for example, a method in which a DNA containing a target DNA sequence is used as a template and obtained by PCR is preferable.
Alternatively, it can be obtained by annealing a complementary strand of DNA produced by chemical synthesis.
(2) Linking unit region DNA
The obtained unit region DNA is ligated with a DNA ligase or the like to prepare a unit region DNA ligated product (hereinafter, this may be referred to as “ligated unit region DNA”). The DNA of the connected unit region can also be amplified by performing PCR using the DNA as a template. At this time, it is necessary to amplify a linked unit region DNA in which each unit region DNA is linked in a direction maintaining the reading frame of amino acids. For this purpose, for example, when amplifying a linked unit region DNA “1-2” obtained by linking “unit region 1” and “unit region 2”, it is complementary to the base sequence at the 5 ′ end of “unit region 1”. PCR is carried out using a forward primer having a property and a reverse primer having complementarity with the negative base sequence at the 3 ′ end of “unit region 2”. By selecting the primers in this manner, the linked unit region DNA that has been inverted and ligated by ligation with DNA ligase or the like can be automatically deleted.
The DNA of the linking unit region is subjected to a reaction with DNA ligase or the like or the above-mentioned PCR, and then the reaction solution is separated by agarose gel electrophoresis or the like, and a gel containing a target DNA is cut out, and the gel is cut out. Can be obtained by recovery and purification using a method known per se. Further, it is also possible to prepare and obtain a full-length DNA of the above-mentioned connecting unit region DNA by chemical synthesis.
Regarding the method of selecting unit region DNA and the order of ligation for preparing the linked unit region DNA, when a random library is prepared, any unit region DNA can be used as the unit region DNA to be linked. There is no particular limitation on the number of unit areas. However, it is preferable to prepare a linked unit region DNA in which two unit region DNAs are linked from the viewpoint of the efficiency of diversification of the components of the library to be constructed. The unit region DNAs to be connected and the order of the connection are not particularly limited. However, when the connected unit region DNAs of all combinations of the unit region DNAs to be used are prepared, a DNA library prepared by a method described below using the same is used. Thus, it is possible to obtain a DNA in which all the unit regions used are combined. Specifically, in the case of producing a connected unit region DNA in which two unit regions are connected using n types of unit region DNA, the maximum is _n C ₂ Ligation reaction can be performed.
In addition, in the case of ligation, ligation may be carried out such that DNAs encoding about 1 to 10, preferably about 1 to 5 random amino acid residues are interposed between unit region DNAs. Such a linking unit region DNA can be prepared by PCR using appropriate primers.
When preparing an alternative splicing library, the connected unit region DNA is selected from two unit region DNAs obtained by dividing a single structural gene DNA in the above (1) and connected in a specific order. Can be obtained. The first unit region DNA to be selected can be selected from the unit region DNA obtained by dividing the single structural gene DNA in the above (1), except for the one located at the most C-terminal side of the encoded protein. The second unit region DNA to be bound to the linking unit region may be any unit region DNA that encodes a unit region located on the C-terminal side of the first unit region in the protein encoded by these units. Good. The order of the ligation is as follows: the 3 ′ end of the first unit region DNA and the 5 ′ end of the second unit region DNA encoding the unit region present on the C-terminal side in the protein encoded by these units. The procedure is performed so that the amino acid sequence in the sample is maintained.
As described above, the linking unit region, which is linked so as to maintain the order from upstream to downstream in the encoded protein, is prepared by dividing the structural gene into L unit region DNAs. In this case, a combination of all L-1 units selected as the first unit region DNA and all the unit regions selectable as the second connected unit region DNA is prepared. That is, when the structural gene DNA is divided into L unit region DNAs, the connected unit region DNAs _L C ₂ Make a kind.
(3) Classification of linking unit region DNA
The resulting linked unit region can be used as a template to perform a PCR to produce the mutant DNA library of the present invention. However, when an alternative splicing library is to be prepared, The connected unit region DNA obtained as described above is used as an index to indicate how many unit regions in the structural gene DNA where the first unit region DNA and the second unit region DNA exist are connected. Need to be classified as Here, X, which is the number of unit regions separating the first unit region DNA and the second unit region DNA in the structural gene DNA in which they exist, is 0 or more when L divided unit regions are L. -2 or less.
Specifically, when a DNA encoding a certain protein is divided into five unit region DNAs, and then a linked unit region DNA is prepared, the unit region DNAs are divided into 1, 2, 3 in order from the N-terminal side of the structural gene. , 4, and 5, the connection unit region DNAs are 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, 4 Ten types of -5 are produced. Among these, those in which the first unit region DNA and the second unit region DNA are separated by 0 unit regions in the structural gene DNA where they exist (X = 0) are 1-2, 2-3, 3-4 and 4-5. Similarly, one unit area (X = 1) is 1-3, 2-4, 3-5, and two unit areas (X = 2) are 1-4, 2-5, 3 Those separated from each other (X = 3) can be classified as 1-5. The unit region DNAs belonging to each group of the connected unit region DNAs classified in this way may be collectively referred to as a “connected unit region DNA group”.
(4) PCR using ligation unit region DNA as template
According to the method of the present invention, the linked unit region DNAs thus obtained are mixed, and a PCR reaction is carried out using the mixed unit region DNAs as a template. A mutant DNA library consisting of genes can be constructed by a single PCR.
The ligation unit region DNAs used as templates in this PCR are selected and mixed so that the terminal sequences of all the ligation unit region DNAs used overlap at least the end sequences of at least one other type of ligation unit region DNA. Here, when a random library is prepared, the connecting unit region DNA used as a template in this PCR is such that the terminal sequence of all the connecting unit region DNAs used is at least the terminal sequence of at least one other type of connecting unit region DNA. And at least one of the 5′-terminal and 3′-terminal sequences of at least one type of the linking unit region DNA is selected so as to overlap with at least the terminal sequence of the other two types of the linking unit region DNA. I do. The type and number of the connecting unit region DNA used for PCR in the present method are not particularly limited as long as the above conditions are satisfied.
For the purpose of obtaining a DNA in which all the unit regions used in the random library are combined, the connected unit region DNAs connected by all the combinations of the unit region DNAs to be used are selected and mixed. PCR may be performed using as a template. In this case, it is preferable to use a connected unit region DNA in which two unit region DNAs are connected. Specifically, when a linked body of n unit region DNAs is produced by using a linked unit region DNA obtained by connecting two unit regions using n types of unit region DNAs, n ² Since there are different types of linked unit region DNAs in different combinations, if PCR is performed using all these linked unit region DNAs as templates, it is possible to obtain a mutant DNA library linked by all combinations of unit region DNAs used. it can.
In the random library, the appearance frequency of a specific unit region DNA can be adjusted by the concentration of the linked unit region DNA containing the unit region DNA. Specifically, when it is expected that a specific unit region DNA appears twice more than that of the specific unit region DNA, double the amount of the unit unit DNA which does not include the linked unit region containing the unit region DNA may be added.
In the case of preparing an alternative splicing library, the DNA of the linking unit region classified in the above (3) can be used as a template for PCR. The amount of DNA of the DNA of the linking unit region to be subjected to the PCR is appropriately 300 to 1500 fmol, preferably 500 to 1300 fmol, as a total of each DNA of the linking unit region. The amount of DNA occupied by each connected unit region DNA group in the template DNA is the number of unit regions in which the first unit region DNA and the second unit region DNA are separated in the structural gene DNA in which they are present. Regarding a certain X, L which is the total number of divided unit regions, and M which is the difference between the total number of unit regions and the number of unit region DNAs of the mutant DNA to be prepared, the following formula (1)
_L-X-1 C _MX (1)
(In the formula, L and X have the same meaning as described above, and M indicates the difference between the total number of unit regions and the number of unit region DNAs of the mutant DNA to be prepared.)
Can be calculated by The value calculated in this manner indicates the ratio of the total amount of DNA in each of the connected unit region DNA groups to the total amount of the template DNA. Further, it is preferable that the calculated amount of DNA of each connected unit region DNA in the total amount of DNA of each connected unit region DNA group is equal to the number of molecules.
Specifically, when the total number of the divided unit regions is 5 (L = 5) and the mutant DNA to be prepared has 3 (M = 3), the above-described classification is performed. In the DNA group of the connection unit region where X = 0, ₄ C ₂ = 6, X = 1 ₃ C ₁ = 3, X = 2 in the connected unit region DNA group ₂ C ₀ = 1, X = 4 ₀ C _-1 = 0, that is, the sum of the amounts of DNA obtained by mixing equal amounts of 1-2, 2-3, 3-4, and 4-5 classified as X = 0, and 1-3 as the DNA classified as X = 1. The total amount of DNA obtained by mixing equal amounts of 2-4 and 3-5 and the total amount of DNA obtained by mixing equal amounts of 1-4 and 2-5 classified as X = 2 are 6: 3: 1. And PCR is performed using this as a template.
More specifically, for example, when the total amount of DNA as a template is 500 fmol, the total amount of DNA obtained by mixing equal amounts of 1-2, 2-3, 3-4, and 4-5 classified as X = 0 Is 300 fmol, and each of the above-mentioned ligation unit region DNAs is subjected to PCR as a template DNA by 75 fmol. Similarly, the total amount of DNA obtained by mixing equal amounts of 1-3, 2-4, and 3-5 classified as X = 1 is 150 fmol, and each ligation unit region DNA is subjected to PCR as a template DNA by 50 fmol. The total amount of DNA obtained by mixing equal amounts of 1-4 and 2-5 classified as X = 2 is 50 fmol, and 25 fmol of each connected unit region DN is used as a template DNA for PCR.
Thus, by performing PCR using the connected unit region DNA mixed in an appropriate amount as a template based on the value satisfying the above formula (1), the order of these unit region DNAs from upstream to downstream in the structural gene was maintained. As it is, a mutant DNA library in which specific unit region DNA is selectively deleted and ligated can be constructed by a single PCR.
Appropriate known PCR reaction buffers and enzymes can be used. Preferably, when a random library is prepared, Vent DNA Polymerase (manufactured by NEB) or an alternative splicing livestock is used. When preparing a rally, KOD Dash Polymerase (manufactured by Toyobo Co., Ltd.) is used.
There is no particular limitation on the amount of DNA in the linking unit region to be subjected to PCR, as long as the DNA amplification reaction occurs. Specifically, when PCR is performed on a reaction scale of 50 μl, the total amount is 300 to 1500 fmol, preferably 500 to 1300 fmol.
The PCR may be performed under any conditions as long as a DNA amplification reaction occurs using the DNA of the connected unit region as a template. Depending on the length of the overlapping sequence of each linking unit region DNA, the length of the unit region DNA, or the length of the DNA to which the connecting unit region to be finally constructed is linked, Bio Experiment Illustrated 3, Cell Engineering Separate Volume, written by Hiroki Nakayama, Shujunsha (1993) Chapter 1, p. It can be appropriately selected and adjusted as described in 13-43 and used. Specifically, a DNA denaturation reaction is performed at 94 to 96 ° C, preferably 95 ° C for 15 to 60 seconds, preferably 20 to 40 seconds, and primer binding (annealing) is performed at 50 to 70 ° C, preferably 55 to 70 ° C. Performing at 65 ° C. for 15 to 60 seconds, preferably 20 to 40 seconds, followed by a polymerase elongation reaction at 70 to 74 ° C., preferably 72 to 73 ° C. for 1 to 5 minutes, preferably 2 to 3 minutes. Is a cycle, and it is appropriate to carry out 15 to 40 cycles, preferably 20 to 30 cycles.
Primers are not particularly required in the above-mentioned PCR, but can be carried out using appropriate primers. The advantage of performing PCR using primers is that DNA having a sequence complementary to the primers at both ends is selectively amplified, so that any unit region DNA or common DNA can be added to both ends of the constructed DNA. That is, it is possible to selectively obtain a substance having a unit region containing the DNA, and to increase the amount of the constructed DNA so that it is easy to see when separating a PCR reaction product. The amount of the primer is appropriately 10 to 60 pmol, preferably 20 to 40 pmol for the forward and reverse primers, respectively.
When constructing a mutant DNA library in which unit region DNA to be both ends of DNA to be constructed is fixed, a primer having a sequence complementary to unit region DNA to be both ends of DNA to be constructed Is preferably performed in the presence of
By performing PCR as described above, a DNA library containing two or more clones can be obtained. The “DNA library containing two or more clones” means a library consisting of two or more clones in which the DNA contained in the obtained library has a different sequence.
The presence of an appropriate manganese ion in the PCR reaction buffer can simultaneously induce base substitution at a certain frequency in each DNA sequence when preparing the DNA library of the present invention. Specific examples of the manganese ion source include manganese chloride. The manganese ion concentration in the PCR buffer is selected according to the frequency of base substitution to be induced. The relationship between the frequency of base substitution and the manganese ion concentration was determined by performing PCR with the manganese ion concentration varied, analyzing the base sequence of the DNA fragment contained in the obtained DNA library, and detecting the base substitution induced in the DNA. It can be specified by examining the rate. As this function, for example, the values described in Table 2 of the third embodiment can be used. As another method, a base substitution can be induced in each DNA sequence contained in the mutant DNA library to be prepared by changing the composition of dNTP.
(5) PCR using as a template a DNA of a connecting unit region having a common DNA sequence
As a preferred embodiment of the PCR using the above-mentioned ligation unit region DNA as a template, a ligation unit region DNA in which different common sequences are added to both ends of the DNA to be constructed is selected and used as a template, and A method of performing PCR using a primer having a sequence complementary to a DNA sequence is exemplified. By this method, a mutant DNA library having a common DNA sequence at both ends can be selectively constructed and obtained.
The common DNA sequence may be an appropriate sequence having a length of about 15 to 100 base pairs. However, when the DNA to be constructed is used for preparing a mutant protein library to be described later, the 5′-terminal common sequence is appropriately used. It is preferable to use a simple promoter sequence.
The promoter sequence used as the common DNA sequence is not particularly limited as long as the protein encoded by the DNA is expressed in a host that expresses the DNA. Specifically, when the host microorganism is Escherichia coli, T3, T7, SP6, tac, or lac promoter can be used, and when yeast is used, the nmt1 promoter, Gal1 promoter, and the like can be used. In the case of cultured animal cells, SV40 promoter, CMV promoter and the like can be mentioned.
As a method for adding the common DNA sequence to the linked unit region DNA, a double-stranded DNA having a common sequence as one of the unit region DNAs is prepared in the same manner as the method for preparing the unit region DNAs described in (1) above. This can be ligated in the same manner as in the method for preparing a ligation unit region DNA described in (2) above. Specifically, the desired 5 'terminal common DNA sequence and the unit region DNA, and the 3' terminal common DNA sequence and the unit region DNA are ligated by the same operation as when the above linked unit region DNA was prepared. Next, with respect to the obtained ligated product of the 5 ′ terminal common DNA sequence and the unit region DNA, and the ligated product of the 3 ′ terminal common DNA sequence and the unit region DNA, the 5 ′ common DNA sequence was forwarded with a forward primer, The gene sequence at the 3 'end of the unit region DNA linked thereto is used as a reverse primer, and the 3' common DNA sequence is used as the reverse primer at the 3 'end, and the 5' end DNA sequence of the unit region DNA linked thereto is used as the reverse primer. It is prepared by performing PCR as a word primer.
In addition, the primer may be added by using a primer having complementarity with the terminal sequence of the unit region DNA to which a common sequence is added at the 5 ′ or 3 ′ end as a primer in the PCR for preparing the above-described linked unit region DNA. Can be.
The ligation unit region DNA containing the common DNA sequence thus prepared is mixed with other ligation unit region DNAs and subjected to PCR. The method of selecting other linking unit region DNAs, the mixing ratio, and the like can be performed in the same manner as described in (4) above, and are described in detail below.
Mutant DNA which has a common sequence at the 5 'end or 3' end in a random library among mutant DNA libraries prepared by the method of the present invention, and in which all combinations of unit region DNAs used for recombination have been performed For the purpose of obtaining the above, a connecting unit region DNA connected by all combinations of unit region DNAs to be used and a connecting unit obtained by adding a common sequence to the 5 ′ end or 3 ′ end of all the unit region DNAs to be used Regions may be selected and mixed, and PCR may be performed using this as a template. In this case, it is preferable to use a connected unit region DNA in which two unit region DNAs are connected. Specifically, a linked DNA of n unit region DNAs having a common sequence at the 5 ′ end or 3 ′ end is formed by connecting unit region DNAs connecting two unit regions using n types of unit region DNAs. Is prepared by PCR, n ² Is mixed with 2n linked unit region DNAs having a common sequence at the 5 'end or 3' end, and PCR is performed using the mixed unit region DNA as a template, all combinations of unit region DNAs used are obtained. And a random DNA library having a common sequence at the 5 ′ end or 3 ′ end can be obtained.
In the alternative splicing library, the DNA of the linked unit region containing the common DNA sequence can be constructed in a single PCR by mixing the DNA at a specific ratio and performing PCR using the mixed DNA as a template.
The unit DNA obtained by connecting the obtained common DNA sequence and each unit region DNA is separated by the number of unit regions (X: where X is a fragment) which is separated in the structural gene DNA between the unit regions to be connected to the common DNA sequence. (Where L is an integer of 0 or more and L-1 or less when the number of unit regions obtained is L), the mixing ratio is calculated as described in (3) above, and mixed with the other connected unit region DNA groups. To perform PCR.
Specifically, when a DNA encoding a certain protein is divided into five unit region DNAs and then a linked unit region DNA is prepared, the unit region DNAs are sequentially arranged from the N-terminal side of the structural gene to 1, 2, 3, 4,. Assuming that the 5 ′ common DNA sequence is T and the 3 ′ common DNA sequence is C, the linked unit region DNA in which the common DNA sequence T or C is linked to the unit region DNA is T-1, T-2, T -3, T-4, T-5, 1-C, 2-C, 3-C, 4-C, and 5-C are produced. Among these, those in which the common DNA sequence and the unit region DNA are separated by 0 unit regions in the structural gene DNA where they exist (X = 0) are T-1, 5-C. Similarly, one unit region (X = 1) is T-2, C-4, two units (X = 2) are T-3, 3-C, three units (X = 3) is T-4, 2-C, and those separated by four (X = 4) are T-5, 1-C. Therefore, mixing of the linked unit region DNA to be subjected to PCR from each value of X The ratio can be calculated.
In the PCR using the unit region DNA group containing the unit region DNA having the common sequence selected and mixed as described above as a template, a primer having a sequence complementary to the common DNA sequence added to the 5 ′ end or 3 ′ end It is necessary to use
The PCR reaction buffer, enzymes, reaction conditions and the like can be determined according to the method described in the above (4). However, as for the DNA of the linking unit region containing the common sequence, the total amount is preferably 5 to 30 fmol, and more preferably 10 to 20 fmol when the PCR is performed on a 50 μl reaction scale.
(6) Principle of alternative splicing library construction method
Among the mutant DNA libraries of the present invention, the principle of the method for constructing an alternative splicing library will be described using a protein composed of five unit regions in FIG. 3 as an example. In this case, the linking unit region DNA obtained by linking these two by two so as to maintain the order from upstream to downstream in the structural gene is ₅ C ₂ = 10 possible. These linked unit region DNAs are classified into four types according to the unit region that they encode (FIG. 3b). If the selectively mutated protein to be constructed by deleting a suitable unit region is a protein containing three unit regions selected from five unit regions, the structure of the mutated protein is ₅ C ₃ = 10 cases (FIG. 3c).
Next, the connection unit regions found in the ten types of sequences shown above are counted, and they are classified based on FIG. 3b. As a result, when N, N + 1, N + 2, N + 3, and N + 4 are sequentially set from the N-terminal side of the protein encoding the unit region, N-N + 1 (the number of separated unit regions (X) = 0) is 12, and N There are six −N + 2, two N−N + 3, and zero N−N + 4.
By referring to this value, a DNA library encoding a group of proteins (in this case, 10 types) linked by deleting appropriate two unit regions from a protein composed of five unit regions can be efficiently prepared. In order to obtain it, the connection unit region DNA, N-N + 1 (the number of separated unit regions (X) = 0): ratio of N-N + 2: N-N + 3: N-N + 4 = 6: 3: 1: 0 It can be seen that mixing may be performed and PCR may be performed using these as templates.
Applying the above to the general formula, the structural gene DNA encoding the protein was divided into L unit region DNAs (where L is an arbitrary integer), and M were selectively deleted and linked. When a mutant DNA library is prepared by PCR, a connected unit region DNA in which two unit region DNAs serving as templates are connected so as to maintain an order from upstream to downstream in a structural gene is separated from each other on the structural gene. When the number of unit regions (X) is present, the ratio of the DNA can be calculated by the following equation (1).
_L-X-1 C _MX (1)
(7) Purification of mutant DNA library
The DNA fragment contained in the DNA library amplified and constructed by the PCR described in the above (4) or (5) can be confirmed by electrophoresis using an agarose gel or the like.
Further, by electrophoresis using the above agarose gel or the like, a mutant DNA library consisting only of DNA of an arbitrary length is obtained from a mixture of DNAs in which the unit region is linked in various numbers, Can obtain a mutant DNA library containing the largest number of target ligated DNAs. In this case, it is preferable to use a low melting point agarose gel, the DNA is electrophoresed in the gel, a portion containing DNA of an arbitrary length is cut out, and the DNA is cut out from the cut out gel by a method known per se. Can be extracted and purified.
The mutant DNA library thus obtained can be used as a DNA library for analyzing protein-nucleic acid interaction or an RNA library. The DNA contained in the DNA library may be coded by a method such as introducing this into an appropriate vector and transforming the recombinant vector into an appropriate host, or expressing it in a cell-free translation system after transcription. (Hereinafter, this may be referred to as a “mutated protein library”).
(8) Introduction of a mutant DNA library into a vector and preparation of a mutant protein library
The vector used for the production of the recombinant vector is not particularly limited as long as it can express the DNA contained in the mutant DNA library of the present invention in the host microorganism or cultured cells. A commercially available protein expression vector into which a promoter suitable for a cell is inserted can be used. On the other hand, as described above, when a 5′-end of a linking unit region DNA used as a template for PCR when constructing a mutant DNA library is added with an appropriate promoter sequence as a common DNA sequence, Normal cloning vectors can be used. Even in the case of a mutant DNA library in which the promoter sequence DNA is not added to the 5 ′ end as described above, if a suitable promoter sequence DNA is inserted so as to be linked to the 5 ′ end, ordinary cloning can be performed. Vectors can be used.
When the host microorganism is Escherichia coli, examples of the protein expression vector include pET3, pET11 (Staratagene), pGEX (Amersham Pharmacia Biotech) and the like, and when the host is yeast, pESP-I expression vector. (Manufactured by Stratagene) and the like. When animal cultured cells are used as a host, ZAP Express (manufactured by Stratagene), pSVK3 (manufactured by Amersham Pharmacia Biotech) and the like, and when insect cells are used, BacPAK6 (manufactured by Clontech) can be used. .
As a promoter for expressing DNA contained in the mutant DNA library of the present invention, a promoter possessed by a host microorganism or a cultured cell can be generally used, but is not limited thereto. Specifically, when the host microorganism is Escherichia coli, T3, T7, SP6, tac, and lac promoters can be used. When yeast is used as a host, examples include the nmt1 promoter and Gal1 promoter. In the case of animal cultured cells, SV40, CMV promoter and the like can be mentioned.
Insertion of the DNA contained in the mutant DNA library of the present invention into these vectors can be performed by ligation using DNA ligase or the like such that the DNA fragment is present downstream of the above promoter in the vector. .
A transformant can be produced by introducing the thus obtained recombinant vector into an appropriate host by an appropriate method known per se. Specific methods for introduction into a host include the heat shock method (J. Mol. Biol., 53, 154- (1970)), the calcium phosphate method (Science, 221, 551- (1983)), and the DEAE dextran method (Science). 215, 166- (1982)), electric pulse method (Proc. Natl. Acad. Sci. USA, 81, 7161 (1984)), in vitro packaging method (Proc. Natl. Acad. Sci. USA, 72, 581). (1975)) or the virus vector method (Cell, 37, 1053 (1984)).
At this time, the host into which the DNA is introduced may be any host as long as the protein encoded by the DNA can be expressed in the host by a protein expression recombinant vector containing the DNA. , Yeast, baculovirus (arthropod polyhedrosis virus)-insect cells, animal cells, animal cultured cells, and the like.
Specifically, BL21 and XL-1-Blue (manufactured by Stratagene) are used for Escherichia coli, SP-Q01 (manufactured by Stratagene) is used for yeast, and AcNPV is used for baculovirus (J. Biol. Chem., 263, 7406). 1988)) and its host Sf-9 (J. Biol. Chem., 263, 7406 (1988)). Examples of animal cultured cells include mouse fibroblast C127 (J. Viol., 26, 291 (1978)), Chinese hamster ovary cell CHO (Proc. Natl. Acad. Sci. USA., 77, 4216 (1980)) and the like. African green monkey kidney cells COS (ATCC: CRL1651) and the like.
By culturing the thus obtained transformant in a suitable medium by a method suitable for each, the expression of the protein encoded by the DNA of the mutant DNA library can be induced. The medium used for the culture contains a carbon source, a nitrogen source, an inorganic substance, a vitamin, serum, a drug used for resistance screening, and the like necessary for the growth of the transformant. Specifically, when the host of the transformant is Escherichia coli, for example, LB medium (manufactured by Nacalai Tesque) or the like, for yeast, YPD medium (Genetic Engineering, 1, 117, Plenum Press (1979)). In the case of insect cells, animal cells, and animal cultured cells, MEM medium, DMEM medium, RPMI1640 medium (manufactured by Sigma) containing 20% or less of fetal bovine serum can be used.
The cultivation of the transformant is usually performed at a temperature of 20 to 45 ° C. and a pH of 5 to 8, and aeration and stirring are performed as necessary. There is no particular limitation as long as the transformant grows under a medium composition or culture conditions other than these, and a protein encoded by the introduced DNA is produced.
Cells or cells are collected from the resulting culture by a method such as centrifugation, suspended in a suitable buffer, and disrupted by a suitable method known per se such as ultrasonication, lysozyme and / or freeze-thawing. After that, a crudely purified protein solution is obtained by centrifugation, filtration, or the like, and a purified protein can be obtained by further combining an appropriate purification method. Thus, a mutant protein library is created.
(9) Expression of mutant DNA library by cell-free translation system and preparation of mutant protein library
In addition to the above-described expression method using a protein expression recombinant vector, protein expression is induced by subjecting an RNA library obtained by transcribing a mutant DNA library to a cell-free translation system to prepare a mutant protein library. can do. The transcription of the DNA library can be carried out by a commonly used method known per se, but can be carried out simultaneously with the translation using a cell-free transcription / translation system. The cell-free transcription / translation system used in the present invention is a system containing all elements necessary for transcription from DNA to mRNA and translation from mRNA to protein, and the DNA is coded by adding DNA thereto. Refers to any system by which the protein in question is synthesized.
Specific examples of the cell-free transcription / translation system include a transcription / translation system prepared based on extracts from eukaryotic cells and bacterial cells or a part thereof, and particularly preferred specific examples include rabbit reticulocytes, A transcription translation system prepared based on an extract from wheat germ and Escherichia coli (Escherichia coli S30 extract) may be mentioned.
Separation and purification of the protein encoded by the mutant DNA library from the obtained transcription-translation product of the cell-free transcription / translation system can be carried out by a method known per se and generally used. Specifically, for example, a DNA region encoding an epitope peptide, a polyhistidine peptide, glutathione-S-transferase (GST), a maltose binding protein, or the like is introduced into the DNA to be transcribed and translated, and expressed as described above. The protein can be purified by utilizing the affinity of the protein with a substance having affinity.
(10) Screening of mutant protein library
The mutant protein library obtained in the above (8) or (9) can be subjected to screening using desired properties as an index. A protein having desired properties is a protein that binds to a desired target molecule, a protein that catalyzes a desired chemical reaction, or a protein whose binding ability, catalytic ability, substrate specificity, thermal stability, and environmental conditions are altered as described above. Protein, etc.
Methods for selecting a protein having desired properties depending on the desired biological activity include a method for selecting using a binding ability to a target molecule as an index and a method for selecting using a catalytic activity as an index. Using these binding capacities or catalytic activities as indices, for example, it is possible to select a protein having desired properties using an affinity resin adsorption method, an immunoprecipitation method, an electrophoresis method, a chromatography method, a flow cytometry method, or the like. it can. More specifically, target molecules such as proteins, nucleic acids (DNA or RNA), carbohydrates, and lipids are previously bound to a solid phase such as a microplate or a bead, and the mutant protein library constructed above is added thereto. After adding rally and reacting under a suitable temperature condition for a certain period of time, washing is performed, and a mutant protein bound to the target molecule is selected. This method can be carried out in accordance with the already established Enzyme Linked Immunosorbent Assay (ELISA) (Crowther, JR, 1995, Method in Molecular Biology, Vol. 42, Humana Press Inc.).
When selecting a protein having desired properties, for example, an enzyme protein, using the catalytic activity as an index, for example, the following two methods can be used. One is a method of selecting a protein that binds to a resin on which a transition state analog of a desired catalytic reaction is immobilized as in the case of a catalytic antibody (Tramontano, A. et al. (1986) Science, 234, 1566-1570). The other is a method in which proteins contained in a mutant protein library are bound to and dissociated from a resin by binding and cleavage of molecules by catalytic activity, as in the case of an RNA enzyme (ribozyme) (Gold). , L. et al. (1995) Annu. Rev. Biochem., 64, 763-797). The former selection method is difficult to increase the enzymatic activity turnover, while the latter selection method is based on catalytic activity rather than binding, as shown in many successful examples of ribozymes. The latter method is effective.
In addition, the fact that the catalytic antibody is restricted by the antibody structure is also one of the reasons why one having the activity as high as that of the natural enzyme has not been obtained. In the present invention, a ribozyme type selection method can be used, and the selection range of the protein structure is not limited to an antibody, and therefore, it can be expected to greatly contribute to the creation of a novel catalytic protein that exceeds conventional catalytic antibodies.
(11) Obtaining a protein having desired properties improved
The protein selected by the screening method described in (10) above is prepared by preparing DNA encoding the same, introducing a mutation into the DNA, amplifying the DNA having the mutation introduced therein, expressing the amplified DNA, By preparing a mutant protein library and using the library to select a protein using the desired property as an index, a protein having the desired property improved can be obtained. In addition, by repeating the above operation, a protein having desired properties can be obtained from the thus obtained protein.
As a method for preparing a DNA encoded by a protein selected as an indicator having desired properties, the DNA is recovered from a transformant producing the protein by a method known per se and amplified using PCR or the like. A method can be used. For amplification of the DNA encoding the desired protein by PCR, a primer having a sequence complementary to the sequence of the vector into which the mutant DNA has been inserted, or a common DNA sequence when a linking unit region having a common DNA sequence is used. A primer having a sequence complementary to the above may be used.
In addition, as a method for inducing a mutation in the amplified DNA, an established error-prone PCR (Leung, DW et al. (1989) J. Methods Cell. Mol. Biol., 1, 11). -15) and Sexual PCR (Stemmer, WPC (1994) Proc. Natl. Acad. Sci. USA, 91, 10747-10751) can be used.
The thus-obtained protein having further improved desired properties can be separated and purified by a method known per se and obtained.
(12) Obtaining a Protein with Improved Desired Properties Using a Nucleic Acid-Protein Linking Molecule
The method for obtaining a protein having an improved desired property described in the above (11) is more efficiently performed by using a nucleic acid-protein linking molecule in which a protein and a nucleic acid encoding the protein are linked and linked. Can be. Nucleic acid-protein linking molecules can be obtained by the phage display method (Smith, GP (1985) Science 228, 1315-1317), the ribosome display method (Hanes, J. and Pluckthun, A. (1997) Proc. Natl. Acad.Sci.USA 94, 4937-4942), in vitro virus method (Nemoto, N. et al. (1997) FEBS Lett. 414, 405-408; Roberts, RW and Szostak, JW. Natl. Acad. Sci. USA 94, 12297-12302; WO 98/16636), STABLE method (Doi, N. et al. (1999) FEBS Lett. 457, 227-230). It can be prepared by methods.
The phage display method is a method that utilizes phages that infect Escherichia coli and proliferate. A phage particle is in a form in which a coat protein constituting the phage wraps a DNA encoding itself. By inserting DNA into the phage coat protein gene and expressing it, a phage library in which the peptide encoded by the DNA is displayed on the surface of phage particles can be prepared. In other words, although indirectly, the protein displayed on the surface of the phage particle is associated with the DNA present in the phage. The DNA encoding the displayed peptide can be obtained by recovering the DNA from the phage selected from the displayed peptides having the desired property as an index.
In the ribosome display method, when the mRNA excluding the stop codon is translated using a cell-free translation system or the like, the synthesized protein is trapped on the ribosome. As a result, a protein-ribosome-mRNA complex is produced and can be used as a protein-nucleic acid linking molecule. DNA encoding the protein can be obtained from the ribosome to which a protein selected from the proteins trapped on the ribosome and having the desired property as an index is bound.
In the in vitro virus method, when expressing mRNA in a cell-free protein synthesis system or the like, it is possible to construct an mRNA-protein linked molecule in which mRNA and protein are chemically bonded on ribosomes. Further, the mRNA-protein linked molecule (in vitro virus) can be selected by an in vitro selection method, and the mRNA portion of the selected in vitro virus can be amplified and obtained by reverse transcription PCR.
In the STABLE method, a protein specifically binding to a ligand substance such as biotin previously bound to DNA, for example, an adapter protein such as streptavidin is expressed as a fusion protein with a protein encoded by the DNA, and the ligand and the adapter protein are expressed. This is a method for binding a protein and a nucleic acid via the binding of Specifically, for example, biotin is chemically bound to the end of DNA in advance, and the DNA is expressed in a cell-free transcription / translation system or the like. At this time, one molecule of DNA is expressed in a state isolated in a microcapsule or the like. The produced protein binds to the DNA present in the microcapsule via a ligand, for example, biotin, and an adapter protein, for example, expressed as a fusion protein, for example, streptavidin. Is produced. The DNA encoding the selected protein can be obtained by amplifying, by PCR or the like, the DNA portion of the connected molecule selected by using the protein portion of the connected molecule having desired properties as an index.
A DNA encoding a protein having desired properties obtained by using such a nucleic acid-protein linking molecule was also introduced with a mutation in the same manner as in (11) above, and the amplified DNA was amplified and amplified. By expressing a DNA to prepare a new mutant protein library and selecting a protein using the library based on the fact that the library has desired properties, a protein having desired properties improved can be obtained. In addition, by repeating the above operation, a protein having desired properties can be obtained from the thus obtained protein. The thus-obtained protein having further improved desired properties can be separated and purified by the above-mentioned method known per se and obtained.
Screening for a protein having desired properties may be performed in the same manner as in the method described in (10) above.
(13) Obtaining DNA encoding a protein having desired properties
By analyzing a protein having the desired properties described in the above (10), (11) or (12) and obtaining a DNA encoding the same, a recombinant of the protein can be produced.
In the case of a protein obtained by the screening method described in the above (10) or (11), the DNA encoding the protein can be analyzed and obtained by appropriately combining commonly used methods known per se. Specifically, in the case of a protein obtained from an alternative splicing library, for example, (1) the molecular weight of the protein is measured using a mass spectrometer or the like, and (2) the unit region contained in the protein is determined from the molecular weight. Can be identified by calculating the type and number of The DNA fragment having the identified DNA sequence can be prepared in the same manner as in the method for preparing the DNA of the linking unit region described in (2) above.
In the case of a protein obtained from a random library, for example, it is possible to analyze the amino acid sequence of the protein by a known method, and to identify the type and the binding order of the unit region contained from the obtained amino acid sequence. it can. The DNA fragment having the identified DNA sequence can be prepared in the same manner as in the method for preparing the DNA of the linking unit region described in (2) above.
In the case of the protein obtained by the screening method described in (12) above, the protein can be obtained by amplifying the nucleic acid portion of the obtained nucleic acid-protein linked molecule by reverse transcription PCR or PCR.
In this specification, unless otherwise specified, procedures for isolating, preparing, ligating, and synthesizing DNA, or for transforming, constructing a plasmid, and culturing a transformant are described in Sonnbrook, J. Am. et al. (1989) Molecular Cloning, 2 ^nd The method can be carried out by the method described in Edition, Cold Spring Harbor Laboratory Press or a method analogous thereto.
The contents described in the specifications of Japanese Patent Application Nos. 2000-293692 and 2001-29138, which are the basis of the priority claimed by the present application, are all incorporated herein as a part of the disclosure of the present specification. Shall be quoted inside.
Hereinafter, the present invention will be described in more detail with reference to examples.However, the following examples should be regarded as helping to obtain specific recognition of the present invention, and the scope of the present invention is not limited by the following examples. Not something.
Example
(Example 1)
(1) Preparation of unit region DNA
The entire region of the dinucleotide binding site of glutaminyl-tRNA synthetase and part of the acceptor domain (Rould, MA et al. (1989) Science 246, 1135-1142) are composed of 150 amino acids. (Hoben, P. et al. (1982) J. Biol. Chem., 257, 11644-11650). A DNA encoding 25 amino acid residues obtained by dividing the entire region of the dinucleotide binding site and a region corresponding to a part of the acceptor domain into six, and 75 base pairs as a unit region DNA (1 to 6; SEQ ID NO: 1) ~ 6). The unit region DNA fragment was prepared by chemical synthesis (Espec Oligo Service). In addition, a DNA fragment (T7; SEQ ID NO: 19) containing a T7 promoter sequence as a 5 ′ consensus sequence was prepared by chemical synthesis (Espec Oligo Service). Similarly, the 3 'consensus sequence (Ex; SEQ ID NO: 20) was also prepared by chemical synthesis (Espec Oligo Service).
Each unit region synthetic DNA fragment and common sequence synthetic DNA fragment are annealed to form a double strand, and have a forward primer (F1 to F6; SEQ ID NO: 7 to SEQ ID NO: 7) having homology to the 5 ′ base sequence of each unit region DNA. 12) and a reverse primer (R1 to R6; SEQ ID NOS: 8 to 18) having homology with the 3 ′ nucleotide sequence, and a forward primer (FT7; sequence) having homology with the 5 ′ nucleotide sequence of each common sequence No. 21, FEx; SEQ ID NO: 23) and a reverse primer (RT7; SEQ ID NO: 22, REx; SEQ ID NO: 24) having homology to the 3′-side nucleotide sequence (date concept).
Each primer was phosphorylated prior to PCR. The phosphorylation reaction solution (30 μl) contains 20 μl of the DNA solution, 10 mM ATP, 1 unit of polynucleotide phosphate kinase (New England Biolab), and 3 μl of the attached 10 × reaction buffer. The PCR reaction solution (50 μl) contains 20 pmol of each primer, 200 μM of dNTPs, 1 unit vent DNA polymerase (manufactured by New England Biolab), and 5 μl of the attached 10 × vent DNA polymerase buffer. The reaction conditions are as follows: the first stage (1 cycle) at 95 ° C for 5 minutes; the second stage (30 cycles) at 95 ° C for 30 seconds, 60 ° C for 30 seconds; and the third stage (1 cycle) at 4 ° C for 10 minutes. Was. A total of eight types of DNA fragments, that is, 6 unit region DNA fragments amplified by PCR, 5 ′ common sequence DNA fragment, and 3 ′ common sequence DNA fragment, were extracted using phenol and chloroform, and then ethanol-precipitated. Dissolved in TE buffer.
(2) Preparation of DNA for linked unit region
The eight types of DNA fragments obtained in the above (1) were ligated in the following combinations.
T7-1, T7-2, T7-3, T7-4, T7-5, T7-6, 1-2, 1-3, 1-4, 1-5, 1-6, 1-Ex, 2- 3, 2-4, 2-5, 2-6, 2-Ex, 3-4, 3-5, 3-6, 3-Ex, 4-5, 4-6, 4-Ex, 5-6, 5-Ex, 6-Ex.
The reaction solution contains 10 μl of the DNA solution and 10 μl of ligation high (manufactured by Toyobo). The reaction was performed at 16 ° C. for 1 hour. The solution containing the ligation unit region DNA that has undergone this reaction is referred to as “ligation reaction solution”.
Using the above ligation reaction product as a template DNA, the ligation unit region DNA in which the two unit region DNAs were linked was amplified using the appropriate primers used in (1) above. The amplified ligated single-region DNAs were T7-1, T7-2, T7-3, T7-4, T7-5, T7-6, 1-1, 1-2, 1-3, 1-4, 1- 5, 1-6, 1-Ex, 2-1 2-2, 2-3, 2-4, 2-5, 2-6, 2-Ex, 3-1 -3-2, 3-3, 3-4, 3-5, 3-6, 3-Ex, 4-1 4-2, 4-3, 4-4, 4-5, 4-6, 4-Ex, 5-1 and 5- 2,5-3,5-4,5-5,5-6,5-Ex, 6-1,6-2,6-3,6-4,6-5,6-6,6-Ex There are 48 types.
The PCR reaction solution (50 μl) was composed of 20 pmol of each primer, 200 μM of dNTPs, 1 μl of a ligation reaction solution, 1.5 mM MgCl 2 ₂ 1 unit of Taq DNA polymerase (manufactured by Toyobo Co., Ltd.) and an attached 10 × Taq DNA polymerase buffer. The reaction conditions are as follows: the first stage (1 cycle) at 95 ° C for 5 minutes; the second stage (30 cycles) at 95 ° C for 30 seconds, 60 ° C for 30 seconds; and the third stage (1 cycle) at 4 ° C for 10 minutes. Was. These PCR reaction solutions were precipitated with ethanol, dissolved in 20 μl of sterilized water, and electrophoresed using a low-melting point agarose gel. After electrophoresis, Objective The DNA band was cut out, extracted with phenol and chloroform, precipitated with ethanol, and dissolved in 60 μl of sterilized water. The concentration of the obtained linked unit region DNA was determined by measuring the absorbance at 260 nm. The concentration of all the linking unit region DNAs was about 60 to 100 ng / μl.
The obtained 48 kinds of DNAs of the connection unit region were cloned using TOPO TA Cloning KIT (manufactured by Invitrogen). The cloning procedure followed the protocol attached to KIT (TOPO TA Cloning Ver. K: Five Minutes Cloning of Taq Polymerase-Amplified PCR Products, Invitrogen). First, 4 μl of the PCR product of the ligation unit region DNA, 1 μl of the salt solution attached to KIT, and 1 μl of PCR-TOPO vector were mixed and incubated at 25 ° C. for 5 minutes. Next, 2 μl of the reaction solution and 50 μl of the competent cells attached to KIT were mixed and incubated on ice for 30 minutes. Then, after incubating at 42 ° C. for 30 seconds, all the competent cells were added to 250 μl of SOC medium attached to KIT, and cultured at 37 ° C. for 30 minutes. Thereafter, 50 μl of SOC was applied to an LB plate to which 50 μg / ml of ampicillin was added. The LB plate used was coated with 24 μl of 40 μg / ml isopropyl-1-thio-β-D-galactoside galactose (IPTG) and 40 μl of a 40 μg / ml X-gal solution in advance.
After incubating the above LB plate for 12 hours at 37 ° C., about 200 colonies were confirmed on the plate. Three blue colonies were selected for each ligation unit region DNA, and it was examined by colony PCR whether or not the desired ligation unit region DNA was contained on the plasmid. Here, the reason why blue colonies were selected instead of white was that all unit region DNAs and common sequence DNAs consisted of multiples of 3 base pairs, did not cause a frame shift in the lacZ gene, and affected the activity. I expected that there would be none. The reaction solution (50 μl) for colony PCR was composed of 20 pmol of each primer, 200 μM of dNTP, 1.5 mM MgCl ₂ 1 unit of Taq DNA polymerase (manufactured by Toyobo Co., Ltd.), and 5 μl of the attached 10 × Taq DNA polymerase buffer. As a template DNA, a colony suspended in sterile water was used. The reaction conditions are as follows: 1st stage (1 cycle) 95 ° C for 5 minutes; 2nd stage (30 cycles) 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds; 3rd stage (1 cycle) 4 ° C for 10 minutes. The colony PCR product was analyzed by agarose electrophoresis, and after confirming that the target ligation unit region DNA was amplified, the product was purified using Wizard PCR Preps DNA Purification System (Promega), and the DNA sequencer was used. (CEQ2000 DNA Analysis System; Beckman Coulter, Inc.) was used to analyze the nucleotide sequence. By analyzing the base sequence, it was confirmed that there was no mutation such as substitution, insertion, or deletion in the prepared DNA of the connection unit region, and the DNA was used as a template DNA for the subsequent PCR.
(3) Preparation of mutant DNA library
PCR was performed by mixing all of the DNAs of the connection unit region prepared in the above (2). A PCR reaction solution (50 μl) was prepared by combining a forward primer of T7 (SEQ ID NO: 21) and a reverse primer of Ex (SEQ ID NO: 24) as primers in 20 pmol each, and a total of 36 kinds of linked unit region DNAs including unit regions 1 to 6 in total. 750 fmol, 15 fmol in total of 12 kinds of DNAs of the connecting unit region containing T7 and Ex, 200 μM of dNTPs, 1 unit of Vent DNA polymerase (manufactured by New England Biolab), and 5 μl of the attached 10 × vent DNA polymerase buffer. The reaction conditions are as follows: 1st stage (1 cycle) 95 ° C for 5 minutes; 2nd stage (20 cycles) 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 3 minutes; 3rd stage (1 cycle) 4 ° C for 10 minutes. The resulting PCR product was concentrated by ethanol precipitation, and then electrophoresed on a low-melting point agarose gel, whereby 15 or more ladder-like bands could be confirmed (FIG. 2). Since the interval between the ladders is equivalent to 75 base pairs, these ladders are T7- (X) -Ex and T7- (X) in order of decreasing molecular weight. ² -Ex, T7- (X) ³ -Ex, ..., T7- (X) ^Fifteen -Ex, ... ("X" is any of the unit areas 1 to 6). That is, the possibility that six unit regions are connected in various orders and numbers inside T7 which is a 5 ′ common sequence and Ex which is a 3 ′ common sequence was shown.
(4) Confirmation of mutant DNA library
In order to check whether the PCR product obtained in the above (2) has the sequence as predicted above, T7- (X) ⁶ The DNA band corresponding to -Ex was cut out, and after cloning, the nucleotide sequence was analyzed. First, T7- (X) was prepared using Wizard PCR Preps DNA Purification System (manufactured by Promega). ⁶ The DNA band corresponding to -Ex was purified. Subsequently, dA was added to the 3 ′ end using Taq DNA polymerase, followed by cloning using TOPO TA Cloning KIT (Invitrogen). The cloning procedure is the same as that described in the above (2) except that an LB plate to which IPTG and X-gal were not applied was used.
After incubating the LB plate for 12 hours at 37 ° C., about 100 colonies were confirmed on the plate. Colony PCR was performed on all colonies to check whether insert DNA was contained. The reaction solution (50 μl) of the colony PCR was prepared by adding 20 pmol of each of the M13 forward and reverse primers attached to Kit, 200 μM of dNTP, 1.5 mM MgCl 2. ₂ 1 unit of Taq DNA polymerase (manufactured by Toyobo Co., Ltd.), and 5 μl of the attached 10 × Taq DNA polymerase buffer. As a template DNA, a colony suspended in sterile water was used. The reaction conditions are as follows: first stage (1 cycle) 95 ° C. for 5 minutes; second stage (30 cycles) 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 30 seconds; third stage (1 cycle) 4 ° C for 10 minutes. As a result of analyzing the colony PCR product by agarose electrophoresis, it was confirmed that 66 colonies contained the insert DNA. These colony PCR products were purified using Wizard PCR Preps DNA Purification System (Promega), and the nucleotide sequence was analyzed by a DNA sequencer (CEQ2000 DNA Analysis System, Beckman Coulter).
Table 1 shows the results of analyzing 66 sequences in which the insert DNA was confirmed. The numbers in the table indicate the order of ligation of the DNA fragments 1 to 6. Of the 66 sequences, 64 sequences had T7 at the 5 'end and Ex at the 3' end, and had a structure in which the internal unit regions were linked in various orders. That is, it was confirmed that the reaction as estimated above occurred. The breakdown of the 66 sequences is T7- (X) ⁹ -One Ex, T7- (X) ⁷ -One Ex, T7- (X) ⁶ -Ex is 50 pieces, T7- (X) ⁵ -Ex is 5 pieces, T7- (X) ⁴ -Ex 4 pieces, T7- (X) ³ T7- (X) having three Ex and no Ex on the 3 ′ side ⁷ 2 had the above sequence. Of these, only two sets had the same sequence, and 64 different sequences were confirmed. That is, it was confirmed that in one PCR, many structural genes in which six kinds of unit region DNAs were linked in various different orders were synthesized at the same time. Since the sequence analyzed after cloning using Escherichia coli is only a small part of the DNA produced by the PCR, more types of structural genes are constructed in the mutant DNA library produced by the PCR. It is assumed that there is.
The analyzed base sequence contains T7- (X) ⁶ The reason that longer DNA or shorter DNA other than -Ex was contained was that T7- (X) cut out by low melting point agarose electrophoresis. ⁶ This is probably because a small number of DNAs having different lengths were present in the DNA band corresponding to -Ex. These DNAs also had T7 on the 5 ′ side and Ex on the 3 ′ side, and had sequences in which unit region DNAs were linked in various orders.
In addition, in the case of two types of sequences having no Ex at the 3 'end, it is presumed that the sequence is generated due to the connection unit region DNA itself to be internally connected serving as a primer. In the sequence of T7-3-4-6-5-1-2-5-Ex, a large deletion was observed between 65. This is because the sequence “gTACgACT” from the 15th position on the 5 ′ side of the unit region DNA 6 is identical to the sequence “gTACgACT” from the 3rd position on the 5 ′ side of the unit region DNA5. It is considered that the replacement occurred.
Substitutions were found at nine of the 40299 base pairs analyzed. That is, the frequency of substitution is about 1/5000, and the frequency of substitution in normal PCR using Vent DNA polymerase (Mattila et al. (1991) Nucl. Acids. Res., 19, 4967-4973). In comparison, the frequency was 2.5 to 5 times higher. However, this value is sufficiently acceptable for the purpose of constructing a library for evolutionary molecular engineering.
In the agarose electrophoresis of the PCR product in this example, 15 or more ladder-like DNA bands were confirmed, and most of the analyzed DNA sequences had different sequences, and frame shifts such as deletions were observed. When six types of unit regions were used, a single PCR resulted in ^Fifteen ≒ 5 × 10 ¹¹ It is possible to construct a structural gene that does not contain more than one type of stop codon.

(Example 2)
(1) Preparation of unit region DNA
The ligand binding domain of the human estrogen receptor α (hereinafter, may be referred to as “hERαLBD”: Greene et al. (1986) Science, 231 (4742), 1150-1154; Brzozooski et al. (1997) Nature, 389 ( 6652), 753-758) are composed of 258 amino acids. The entire region of this hERαLBD is expressed by the exon of the gene (Ponglikitmongol et al. (1988) EMBO J., 11, 3385-3388) and the secondary structure in the three-dimensional structure of the protein (Tanenbaum et al. (1998) Proc. Natl. Acad. Sci.USA., 95 (11), 5998-6003), and 10 unit area regions were defined. Each unit region encodes 34, 35, 29, 17, 26, 37, 24, 17, and 20 amino acids from the N-terminal side (FIG. 4), and the plasmid containing hERαLBD is used as a template. Amplified and prepared by PCR using a forward primer having homology to the base sequence on the 5 'side and a reverse primer having homology to the base sequence on the 3' side of the region DNA fragment (prepared by the date concept) (This plasmid was kindly provided by Professor GL Greene of the University of Chicago).
The prepared unit regions were 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 from the N-terminal side. Primers are SEQ ID NOS: 25 and 26 for unit region DNA1, SEQ ID NOs: 27 and 28 for unit region DNA2, SEQ ID NOs: 29 and 30 for unit region DNA3, SEQ ID NOs: 31 and 32 for unit region DNA4, unit region For DNA5, SEQ ID NOS: 33 and 34, for unit region DNA6, SEQ ID NOS: 35 and 36, for unit region DNA7, SEQ ID NOs: 37 and 38, for unit region DNA8, SEQ ID NOs: 39 and 40, and for unit region DNA9 Used SEQ ID NOS: 41 and 42, and for the unit region DNA 10, SEQ ID NOs: 43 and 44. In addition to these unit region DNAs, a DNA fragment having a T7 promoter sequence (T7OM: SEQ ID NO: 45) and a 3 ′ terminal common sequence DNA fragment (CBSHis: SEQ ID NO: 46) are chemically synthesized as 5 ′ terminal common sequence DNA fragments. (Requested to Date Concept) Using this common sequence synthesized DNA fragment as a template, homology to the 3 ′ nucleotide sequence with a forward primer (T7OM: SEQ ID NO: 47; CBSHis: SEQ ID NO: 49) having homology to the 5 ′ nucleotide sequence of each DNA fragment (T7OM: SEQ ID NO: 48; CBSHis: SEQ ID NO: 50) was amplified by PCR using a reverse primer having the following sequence: The prepared DNA fragment having the common sequence was T7OM on the 5 ′ side and CBSHis on the 3 ′ side.
The primers used for preparing the unit region DNA and the common sequence DNA fragment were phosphorylated prior to PCR. The phosphorylation reaction solution (30 μl) contains 20 μl of a DNA solution (20 pmol / μl), 10 mM ATP, polynucleotide phosphate kinase (manufactured by New England Biolab), and 3 μl of an attached 10 × reaction buffer. The phosphorylation reaction was performed at 37 ° C. for 12 hours.
The PCR performed using the phosphorylated primers was performed as follows: 1 μl of a DNA solution (20 ng / μl) as a reaction solution (50 μl), 20 pmol of each primer, 200 μM of dNTPs, 2.5 units of Pfu DNA polymerase (manufactured by STRATAGENE), and the attached 10 × Using 5 μl of Pfu DNA polymerase reaction buffer, the reaction conditions were as follows: the first step (1 cycle): 95 ° C./5 minutes, the second step (30 cycles): 95 ° C./30 seconds, 56 ° C./30 seconds, the third step (1 cycle) (Cycle) 4 ° C./10 minutes. The above-mentioned ten unit region DNA fragments and two common sequence DNA fragments amplified by PCR were prepared using Wizard PCR Preps (manufactured by Promega) and dissolved in 60 μl of sterilized water.
(2) Preparation of DNA for linked unit region
The twelve kinds of DNA fragments prepared in the above (1) were ligated by DNA ligase in the following combinations, and then, using this ligation unit region DNA fragment as a template, the sequence was homologous to the sequence at the 5 ′ end of the first ligation unit region DNA. And a reverse primer having homology to the sequence at the 3 'end of the second unit region DNA.
T7OM-1, T7OM-2, T7OM-3, T7OM-4, T7OM-5, T7OM-6, T7OM-7, T7OM-8, T7OM-9, T7OM-10, 1-2, 1-3, 1- 4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-CBSHis, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-CBSHis, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-CBSHis, 4- 5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-CBSHis, 5-6, 5-7, 5-8, 5-9, 5-10, 5-CBSHis, 6-7, 6-8, 6-9, 6-10, 6-CBSHis, 7-8, 7-9, 7-10, 7-CBSHis, 8-9, 8-10, 8-CBSHis, 9- 10, 9 CBSHis
A reaction solution for ligation of unit region DNA fragments by DNA ligase was 1 μl of a DNA solution (10 ng / μl), 400 units of T4 DNA ligase (manufactured by New England Biolab), 1 μl of an attached 10 × T4 DNA ligase reaction buffer, and 6 μl of sterilized water. And the reaction was carried out at 16 ° C. for 1 hour. A PCR reaction solution (50 μl) using the DNA fragment of the ligation unit region as a template was 1 μl of a reaction solution of the above DNA ligase containing 20 pmol of each primer, 200 μM of dNTPs, and ligation unit region DNA, and 1.5 mM MgCl 2. ₂ , 1 unit of Taq DNA polymerase (manufactured by Toyobo Co., Ltd.), and 5 μl of the attached 10 × Taq DNA polymerase reaction buffer. The reaction conditions are as follows: the first stage (one cycle) at 95 ° C./5 minutes, the second stage (30 cycles) at 95 ° C./30 seconds, 60 ° C./30 seconds, and the third stage (one cycle) at 4 ° C./10 minutes. Was.
The obtained DNA fragment of the linking unit region was cloned using a TOPO TA Cloning KIT (manufactured by InVitrogen). The cloning procedure was in accordance with the protocol attached to the KIT (TOPO TA Cloning, Ver. K: Five minutes cloning of Taq polymerase-amplified PCR products, Invitrogen), and the vector used was the one attached thereto. Specifically, 4 μl of the PCR reaction solution, 1 μl of the attached salt solution, and 1 μl of the PCR TOPO vector of the DNA fragment of the entire ligation unit region prepared above were mixed and incubated at 25 ° C. for 5 minutes.
Next, 2 μl of the above reaction solution and 50 μl of the attached competent cells were mixed and incubated on ice for 30 minutes. After incubating at 42 ° C. for 30 seconds, the attached SOC medium (250 μl) was added and incubated at 37 ° C. for 30 minutes. 50 μl of this was added to an LB plate to which 50 μl / ml ampicillin had been added in advance, 24 μl of 40 μg / ml isopropyl-1-thio-β-galactoside galactose (IPTG) and 40 μl of a 40 μg / ml X-gal solution. Was applied and then applied.
After incubating the LB plate for 12 hours at 37 ° C., about 200 colonies were confirmed on the plate. Three blue colonies were selected for each ligation unit region DNA, and it was confirmed by colony PCR whether the desired ligation unit region was contained on the plasmid. Here, the reason why the blue colonies were selected was that all the unit region DNAs had a base sequence that was a multiple of 3 and were expected not to cause a frame shift in the lacZ gene and not to affect the activity. is there. The reaction solution of the colony PCR was prepared using the M13 primer attached to the TOPO TA Cloning KIT and the M13 Rev. Primers (manufactured by InVitrogen) 20 pmol each, dNTPs 200 μM, 1.5 mM MgCl ₂ , 1 unit of Taq DNA polymerase (manufactured by Toyobo Co., Ltd.), 5 μl of an attached 10 × Taq DNA polymerase reaction buffer, and a solution in which colonies were suspended in sterile water. The reaction conditions were the first stage (one cycle) at 95 ° C./5 PCR was carried out in the second step (30 cycles) at 95 ° C./30 seconds, 60 ° C./30 seconds, 72 ° C./30 seconds, and the third step (1 cycle) at 4 ° C./10 minutes.
The colony PCR reaction solution was analyzed by agarose gel electrophoresis, and after confirming that the target ligation unit region DNA fragment was amplified, the product was purified using Wizard PCR Preps DNA Purification System (Promega). Then, the nucleotide sequence was analyzed using a DNA sequencer (CEQ2000 DNA Analysis System: manufactured by Beckman Coulter, Inc.). After analyzing the nucleotide sequence, it was confirmed that there was no mutation such as substitution, insertion, or deletion.
Using the plasmid containing the linking unit region DNA confirmed to have the correct nucleotide sequence as a template, the sequence at the 5 'end of the first unit region DNA of each linking unit region DNA shown in (1) Ligation unit region DNA was prepared again by colony PCR using a forward primer having homology with the reverse primer and a reverse primer having homology with the sequence at the 3 ′ end of the second unit region DNA. The PCR reaction solution (50 μl) contained 20 pmol of each primer, 200 μM of dNTPs, 3.75 units of Pfu DNA polymerase (manufactured by STRATAGENE), and 5 μl of the attached 10 × Pfu DNA polymerase reaction buffer. The cycle was performed at 95 ° C./5 minutes, the second stage (30 cycles) at 95 ° C./30 seconds, 56 ° C./30 seconds, and the third stage (one cycle) at 4 ° C./10 minutes.
From the PCR reaction solution in which the above-mentioned ligation unit region DNA was prepared, the ligation unit region DNA fragment was concentrated by ethanol precipitation, dissolved in 20 μl of sterilized water, separated by low melting point agarose gel electrophoresis, and the band of the target DNA was cut out. . DNA was extracted from the agarose gel fragment using phenol / chloroform, ethanol-precipitated, and dissolved in 60 μl of sterile water. The concentration of the obtained aqueous DNA solution of each linking unit region was determined by measuring the absorbance at 260 nm. The DNA of the connected unit region obtained here was used as a template DNA for PCR for constructing a subsequent mutant DNA library.
(3) Classification of linking unit region DNA
All the linked unit region DNAs obtained in the above (2) were classified according to the unit region DNAs they encoded. The classification is based on how many unit regions in the structural gene DNA in which the first unit region DNA and the second unit region DNA of the above-mentioned connected unit region DNA exist are connected. Went as. Here, assuming that the number of unit regions separating the first unit region DNA and the second unit region DNA in the structural gene DNA in which they exist is X, the created connected unit region DNA is from X = 0 to X = 8 (9) (FIG. 5 (a)).
(4) Construction of a mutant DNA library in which eight unit region DNAs are linked
A mutant DNA library in which eight unit region DNAs were linked was prepared using the linked unit region DNA group classified in the above (3) as a template. For each of the connected unit region DNA groups, the total number (L) of connected unit regions obtained by dividing the structural gene is set to 10, and the difference (M) between the total number of unit regions and the number of unit region DNAs contained in the DNA to be prepared is set to 2, Regarding the number (X) of unit regions in which the first unit region DNA and the second unit region DNA are separated in the structural gene DNA in which they exist, the following formula (1)
_L-X-1 C _MX (1)
And the proportion of each ligation unit region DNA group to the total amount of DNA to be subjected to PCR was determined.
In the classification of the above (3), 36 are the connecting unit region DNA groups that fall into the classification of X = 0, T7OM-1 is 36, 10-CBSHisX is 36, X is 1 the connecting unit region DNA group is 8, and T7OM-2 Was 8, 9-CBSHisX was 8, the DNA group of the linked unit region where X = 2 was 1, T7OM-2 was 1, and 8-CBSHisX was 1.
As the specific amount of DNA, 675 fmol was used as the total amount of DNA in PCR for preparing a mutant DNA library, so that the total amount of DNA in the linked unit region DNA group of X = 0 was 180 fmol, 26 fmol each of the connecting unit region DNAs (1-2, 2-3, 3-4, 5-6, 7-8, 8-9, 9-10), 180 fmol of T7OM-1 and 180 fmol of 10-CBSHisX, Since the total amount of the DNAs in the DNA group of the connecting unit region where X = 1 is 40 fmol, the connecting unit region DNAs (1-3, 2-4, 3-5, 4-6, 5-7, 6-8) which fall into this category are included. , 7-9, 8-10) in 5 fmol increments, T7OM-2 in 40 fmol, 9-CBSHisX in 40 fmol, and the total amount of DNA in the linked unit region DNA group of X = 2 was 5 fmol. Therefore, the linking unit region DNAs (1-4, 2-5, 3-6, 4-7, 5-8, 6-9, 7-10) which fall into the classification are each 0.7 fmol, and T7OM-3 is 5 fmol, 8-CBSHisX was mixed at 5 fmol each to obtain a template DNA for PCR.
The PCR reaction solution (50 μl) includes T7OM forward primer and CBPHis reverse primer, each 20 pmol, 675 fmol of the above-mentioned mixed template DNA, 200 μl of dNTPs, and 1.25 units of KOD Dash DNA polymerase (manufactured by Toyobo Co., Ltd.). 5 × 1 × 10 × KOD Dash DNA polymerase reaction buffer, the reaction conditions were the first stage (1 cycle) 95 ° C./5 min, the second stage (20 cycles) 95 ° C./30 sec, 60 ° C./30 sec, 72 PCR was carried out at a third stage (1 cycle) of 4 ° C./10 minutes at 3 ° C./3 minutes.
The PCR product was concentrated by ethanol precipitation and analyzed by low-melting point agarose gel electrophoresis. As a result, a strong DNA band was confirmed in a region corresponding to the intended molecular weight (FIG. 6 (a)).
(5) Construction of a mutant DNA library in which five unit region DNAs are linked
A mutant DNA library in which five unit region DNAs were linked was prepared using the linked unit region DNA group classified in the above (3) as a template. For each connected unit region DNA group, the total number (L) of connected unit regions obtained by dividing the structural gene is set to 10, and the difference (M) between the total number of unit regions and the number of unit region DNAs contained in the DNA to be prepared is set to 5, Regarding the number (X) of unit regions in which the first unit region DNA and the second unit region DNA are separated in the structural gene DNA in which they exist, the above expression (1) was applied to the PCR of each connected unit region DNA group. The ratio of the DNA to the total amount of DNA to be subjected to was determined.
In the classification of (3) above, 126 were the connecting unit region DNA groups that entered the classification of X = 0, 126 were T7OM-1 and 126 were 10-CBSHisX, 70 were the connecting unit region DNA group of X = 1, and T7OM-2. Is 70, 9-CBSHisX is 70, the linking unit region DNA group of X = 2 is 35, T7OM-3 is 35, 8-CBSHisX is 35, the connecting unit region DNA group of X = 3 is 15, and T7OM-4 is 15 , 7-CBSHisX is 15, the linking unit region DNA group of X = 4 is 5, T7OM-5 is 5, the 6-CBSHisX is 5, the connecting unit region DNA group of X = 5 is 1, and the T7OM-6 is 1,5. -CBSHisX was 1.
As the specific amount of DNA, since 1260 fmol was used as the total amount of DNA in PCR for preparing a mutant DNA library, the total amount of DNA in the DNA unit of the linked unit region region where X = 0 was 210 fmol, and 30 fmol of the connecting unit region DNA (1-2, 2-3, 3-4, 5-6, 7-8, 8-9, 9-10), 210 fmol of T7OM-1 and 210 fmol of 10-CBSHisX, Since the total amount of DNA in the DNA group of the connection unit region where X = 1 is 117 fmol, the DNA of the connection unit region (1-3, 2-4, 3-5, 4-6, 5-7, 6-8) included in the classification is included. , 7-9, 8-10) in 14.6 fmol increments, T7OM-2 in 117 fmol, 9-CBSHisX in 117 fmol, and the total amount of DNA in the linked unit region DNA group of X = 2 is: Since 8 fmol is obtained, 8 fmol each of the connected unit region DNAs (1-4, 2-5, 3-6, 4-7, 5-8, 6-9, 7-10) and 58 fmol of T7OM-3 are included. , 8-CBSHisX is 58 fmol, and the total amount of DNA of the connected unit region DNA group of X = 3 is 25 fmol, so that the connected unit region DNAs (1-5, 2-6, 3-7, 4-8, 5-9, 6-10) in 4 fmol increments, T7OM-4 in 25 fmol, 7-CBSHisX in 25 fmol, and the total amount of DNA in the DNA group of the connection unit region where X = 4 is 8.3 fmol. Linkage of 1.6 fmol of the region DNA (1-6, 2-7, 3-8, 4-9, 5-10), 8.3 fmol of T7OM-5, 8.3 fmol of 6-CBSHisX, and X = 5 unit Since the total amount of the DNAs in the range DNA group is 1.7 fmol, the connecting unit region DNAs (1-7, 2-8, 3-9, 4-10) included in the classification are 0.4 fmol each, and T7OM-6 is 1 fmol. 1.7 fmol and 1.7 fmol of 5-CBSHisX were mixed at a time to obtain a template DNA for PCR.
The PCR reaction solution (50 μl) includes T7OM forward primer and CBPHis reverse primer of 20 pmol each, 1260 fmol of the above-mentioned mixed template DNA, 200 μl of dNTPs, 1.25 units of KOD Dash DNA polymerase (Toyobo), attached 5 × 1 × 10 × KOD Dash DNA polymerase reaction buffer, the reaction conditions were the first stage (1 cycle) 95 ° C./5 min, the second stage (20 cycles) 95 ° C./30 sec, 60 ° C./30 sec, 72 PCR was carried out at a third stage (1 cycle) of 4 ° C./10 minutes at 3 ° C./3 minutes. The PCR product was concentrated by ethanol precipitation and analyzed by low-melting point agarose gel electrophoresis. As a result, a strong DNA band was confirmed in a region corresponding to the intended molecular weight. (FIG. 6 (b))
(6) Confirmation of mutant DNA library
In order to check whether or not the PCR products obtained in the above (4) and (5) have the intended sequence, a region showing a strong DNA band obtained by the low melting point agarose gel electrophoresis was cut out, and Wizard was used. The DNA was extracted and purified using PCR Preps DNA Purification System (Promega). Subsequently, dA was added to the 3 'end using Taq DNA polymerase, and then cloned using the TOPO TA Cloning KIT (manufactured by InVitrogen) in the same manner as in (2) above. At this time, the LB plate used was not coated with IPTG and X-Gal.
After incubating the LB plate for 12 hours at 37 ° C., 100 or more colonies were confirmed on the plate. For 40 or more colonies, the nucleotide sequence of the PCR product DNAs prepared in (4) and (5) contained in the plasmid was analyzed in the same manner as in (2) above.
With regard to the PCR product of (4) above (to which eight unit region DNAs were bound), the nucleotide sequence of 38 clones was analyzed. The result is shown in FIG. Regarding the PCR product of (5) (in which five unit regions were bound), the nucleotide sequence of 33 clones was analyzed. The result is shown in FIG. All of the DNA fragments obtained by any of the PCRs have a T7OM sequence at the 5 'end and a CBSHis sequence at the 3' end, and the internal unit region DNA maintains the order from upstream to downstream in the structural gene. It had a structure that was variously connected as it was. This means that the number of each unit region that appeared in the base sequence of the analyzed 71 DNA fragments was 1/39, 2/30, 3/52, 4/34, 5/40, 6 / 43, 7/56, 8/32, 9/43, and 10/40, which were confirmed by the fact that there was no significant bias in the frequency of appearance of the unit regions used.
Further, among the DNA fragments analyzed above, there was only one set having the same sequence in the sequences shown in FIG. Regarding the substitution, out of 71 clones and 47533 bases, 60 bases were replaced. This mutation rate (1.3 × 10 ^-3 ) Was determined to be sufficiently low that it would not hinder the construction of the library. Of the 50 substitutions, only two had a stop codon. Furthermore, there were two types of sequences linked in unintended regions. One was caused by induction of homologous recombination at "atgatc" which is present in both the unit region DNA1 and the unit region DNA2. The other was caused by the induction of homologous recombination between “ccagggga” and “ccagtga” which are commonly present in both the unit region DNA5 and the unit region DNA4. The other sequences did not show any frameshift mutations, ie, no deletions or insertions. That is, it was shown that most of the DNA contained in the mutant DNA library obtained by PCR of the present invention maintained the correct reading frame.
In the sequence shown in FIG. 7, 3 clones of DNA fragments each having 5 linked unit regions, 10 clones of 6 linked DNA fragments, 9 clones of 7 linked DNA fragments, and 10 clones of 8 linked DNA fragments Two clones and nine cloned DNA fragments were one clone, and ten cloned DNA fragments were one clone. Further, in the sequence shown in FIG. 8, one DNA fragment in which two unit regions are linked, two clones in which three DNA fragments are linked, nine clones in which four DNA fragments are linked, and five DNA fragments in which five linked. Were 17 clones, and 4 cloned DNA fragments were 4 clones.
The above results include DNA fragments composed of a larger or smaller number of unit region DNAs than the intended number of unit regions. This shows that by applying the library to evolutionary molecular engineering, a mutant DNA library sufficient to achieve the object of the present invention to create a useful protein could be constructed.
(Example 3)
A mutant DNA library was prepared by Error-prone PCR.
When performing PCR by mixing the connection unit regions in the same manner as in the operations (1) to (3) and (5) of Example 2, manganese chloride was added to the PCR reaction solution at a concentration of 0 to 1 mM. . The nucleotide sequence of the amplified DNA was analyzed in the same manner as in Example 2, (6). Table 2 shows the results. It was found that the frequency of base substitution increased almost linearly with an increase in manganese ion concentration. Therefore, when it was desired to construct a library at an arbitrary substitution rate, it was found that manganese ions should be added according to the data in Table 2.

Industrial potential
According to the method of the present invention, there is provided a method for constructing a population of genes in which a plurality of DNA fragments encoding an arbitrary amino acid sequence are linked in various orders and lengths, and a mutant prepared from the mutant DNA library is provided. By selecting a desired protein using a protein library and using evolutionary molecular engineering, we can create proteins with new functions that combine secondary structures, modules, motifs, etc., which are parts (unit regions) of natural proteins. Is extremely useful.
Further, a protein having a reduced molecular weight can be obtained from a mutant protein library prepared from the alternative splicing library of the present invention. Denaturation of relatively small globular proteins having a molecular weight of 20,000 or less is known to be reversible, and once denatured, its function can be restored by returning to appropriate conditions. In addition, proteins having such properties have strong resistance to heat and organic solvents, as represented by ribonuclease. In addition, the recovery rate of a purified product in a bioprocess using such an enzyme is high. The mutant protein obtained from the alternative splicing library of the present invention can reduce the size of any protein while maintaining its function, and thus expands its application as a protein.
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing the concept of the present invention. The case where three types of unit region DNAs are shuffled is shown as an example. By mixing the linked unit region DNAs in which the two unit region DNAs are linked and performing PCR using the forward primer of T7 and the reverse primer of Ex, a large number of the three unit region DNAs linked in various orders Is synthesized by one PCR.
FIG. 2 is an electrophoretic photograph obtained by separating PCR products using an agarose gel (2%). Each lane contains 50 μl of PCR product. The sequence corresponding to the ladder-like DNA band is shown on the right. M is a DNA marker.
FIG. 3 is a diagram showing the principle of the method for constructing a mutant DNA library of the present invention.
FIG. 4 is a block diagram showing that the ligand binding domain of human estrogen receptor α is divided at exon and secondary structure boundaries and divided into ten unit regions.
FIG. 5 is a diagram showing all connected unit region DNAs used when constructing the mutant gene library of the present invention from a structural gene consisting of 10 unit regions. Each connecting unit region DNA can be classified into nine types of connecting unit region DNA groups from X = 0 to X = 8 as indicated by diagonal lines (a). The appearance frequency of each connected unit region DNA group appearing in a population of structural genes composed of a specific number of unit regions is shown (b).
FIG. 6 is an agarose gel electrophoresis photograph obtained by separating a PCR product when the mutant DNA library of the present invention was constructed. (A) and (b) are PCR products when constructing a group of structural genes composed of 8 and 5 unit region DNAs, respectively. In the figure, M indicates a marker.
FIG. 7 is a diagram showing the structure of the DNA of the mutant gene library of the present invention obtained by performing PCR under such conditions that a structural gene is obtained from eight unit region DNAs.
FIG. 8 is a diagram showing the structure of the DNA of the mutant gene library of the present invention, which was obtained by performing PCR under conditions that obtain a structural gene from five unit region DNAs.

Claims (19)

(A) the unit region DNAs are ligated in any combination, and (b) the obtained connected unit region DNAs are combined with at least one other type of the connected unit region DNAs at all the terminal sequences of all the connected unit region DNAs used. (C) performing a Polymerase \ Chain \ Reaction using the mixture of ligation unit region DNAs as a template to obtain a DNA library containing two or more clones. How to build a library. In the step (b), the connecting unit region DNA is prepared by combining at least one type of connecting unit region with the terminal sequence of at least one other type of connecting unit region DNA being used. The method according to claim 1, wherein the DNA is selected and mixed so that the sequence at either the 5 'end or the 3' end of the DNA overlaps at least the ends of the other two types of connecting unit region DNA. The method according to claim 1 or 2, wherein the mixture of linked unit region DNAs is a mixture of linked unit regions in which two unit region DNAs are linked in all combinations of unit region DNAs used. The unit region DNA is obtained by dividing a structural gene DNA encoding a protein into L pieces (here, L is an integer of 3 or more). The 5 ′ end of the unit region DNA present on the C-terminal side of the protein from the unit region is linked so that the amino acid sequence of each unit region is maintained, and the mixture of linked unit region DNAs is linked. Each of the linked unit region DNAs classified using X as an index (where X is an integer of 0 or more and L-2 or less), which is the number of unit regions separated from each other in the original structural gene, is as follows: The method according to claim 1, wherein the mixture is performed using a ratio satisfying the expression (1) as an index.
_L-X-1 C _MX (1)
(In the formula, L and X have the same meanings as described above, and M represents the difference between the total number of unit regions and the number of unit regions of the mutant DNA to be prepared.) Unit region DNA is a DNA obtained by dividing DNAs encoding proteins having similar tertiary structures but different amino acid sequences into L unit regions (where L is an integer of 3 or more). The ligation method is such that the amino acid sequence of each unit region is maintained at the 3 'end of the arbitrary unit region DNA and the 5' end of the unit region DNA located on the C-terminal side in the protein group from the unit region. X (where X is 0 or more and L-2 or less) is a mixture of linked unit region DNAs, and the mixture of the linked unit region DNAs is the number of unit regions separated in the original protein group. The method according to claim 1, wherein each of the connected unit region DNAs classified using an index as an index is a mixture obtained by using an index that satisfies the following formula (1) as an index.
_L-X-1 C _MX (1)
(In the formula, L and X have the same meanings as described above, and M represents the difference between the total number of unit regions and the number of unit regions of the mutant DNA to be prepared.) The method according to any one of claims 1 to 5, wherein the Polymerase Chain Reaction is performed in the presence of a primer having a sequence complementary to a unit region DNA to be both ends of the DNA to be constructed. The method according to any one of claims 1 to 6, wherein the Polymerase Chain Reaction is performed in the presence of a connecting unit region DNA containing a common DNA sequence and a primer having a sequence complementary to the common DNA sequence. A mutant DNA library obtained by the method according to claim 1. A protein library obtained by incorporating the DNA library according to claim 8 into an expression vector, transforming a host cell with the expression vector, culturing the transformant, and collecting a protein from the culture. An RNA library obtained by transcribing the DNA library according to claim 8. A method for producing a protein library, comprising expressing the DNA library according to claim 8 or the RNA library according to claim 10 in a cell-free transcription and / or translation system. A protein library obtained by the method of claim 11. A nucleic acid-protein linked molecule library in which a DNA contained in the DNA library according to claim 7 or an RNA contained in the RNA library according to claim 9 is bound to a protein encoded by the DNA or RNA. A method for obtaining a protein, comprising selecting a protein having desired properties using the library according to claim 9, 12 or 13. A protein obtained by the method according to claim 14. (A) preparing a DNA encoding the protein of claim 15, (b) introducing a mutation into the DNA, (c) amplifying the DNA having the mutation introduced therein, and (d) transcribing the amplified DNA. / A method of preparing a protein library by performing translation, and (e) obtaining a protein having desired properties using the library. A protein obtained by the method according to claim 16. A method for obtaining a DNA encoding the protein according to claim 15. A DNA obtained by the method according to claim 18.

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