JP2004524811A - Combinatorial screening of mixed populations of organisms - Google Patents

Abstract

A method is provided for screening a gene library from a mixed population of organisms for the biological activity of a given biomolecule. The mixed sample of organisms can be, for example, a cultured or uncultured population from the environment. Further, a method for screening a segregant or enriched population of an organism is provided, wherein the segregate comprises, for example, a spatial, temporal, or hierarchical population of a particular species, genus, family, or class of the organism. including. Identified clones containing a given biomolecule or biological activity can be further altered, or alter the DNA contained in the clone, to form a new biomolecule or biological activity. It is possible.

Description

テストパッケージ及びプレート。ライゲーション反応は、製造業者のプロトコルに従ってパッケージする。パッケージング反応は、５００μｌのＳＭバッファにより停止させ、同じライゲーションに由来するパッケージングによりプールされる。各プール済み反応の１μｌを適切な宿主上で滴定する（ＯＤ_６００＝１．０）（ＸＬ１−Ｂｌｕｅ ＭＲＦ）。２００μｌの宿主（ＭｇＳＯ_４内）を、Ｆａｌｃｏｎ ２０５９チューブに追加し、１μｌのパッケージしたファージを接種し、３７℃で１５分間培養する。約３ｍｌの４８℃トップアガー（１５０μｌ ＩＰＴＧ（０．５Ｍ）及び３００μｌ Ｘ−ＧＡＬ（３５０ｍｇ／ｍｌ）を含む５０ｍｌストック）を追加し、１００ｍｍプレート上で平板培養する。プレートは、３７℃で一晩培養する。
【０３２９】
ライブラリの増幅（各ライブラリから５．０×１０^５の組み換え）。約３．０ｍｌの宿主細胞（ＯＤ_６００＝１．０）を、二本の５０ｍｌ円錐管に追加し、円錐管当たり２．５×１０^５ｐｆｕのファージを接種し、その後、３７℃で２０分間培養する。トップアガーを各管に追加し、最終体積を４５ｍｌにする。各管は、五枚の１５０ｍｍプレートで平板培養する。プレートは、３７℃で６乃至８時間、或いはプラークがピンヘッドのサイズになるまで培養する。プレートにｍ８乃至１０ｍｌのＳＭバッファを被せ、４℃で一晩置く（可能な場合は穏やかに揺らす）。
【０３３０】
ファージの回収。ファージの懸濁は、ＳＭバッファを各プレートから５０ｍｌ円錐管に注ぐことで回収される。約３ｍｌのクロロホルムを追加し、強く振り、室温で１５分間培養する。この管を２０００ｒｐｍで１０分間遠心分離し、細胞残屑を取り除く、上澄みを無菌フラスコに注ぎ、５００μｌクロロホルムを追加し、４℃で保存する。
【０３３１】
滴定量増幅ライブラリ。回収したファージの連続希釈を行い（例えば、１０^−５＝１ｍｌのＳＭバッファ内に１μｌの増幅ファージ、１０^−６＝１ｍｌのＳＭバッファ１μｌの１０^−３稀釈物、及びその他）、２００μｌの宿主（１０ｍＭ ＭｇＳＯ_４内）を二本の管に追加する。一方の管には、１０μｌの１０^−６稀釈物（１０^−５）を接種する。他方の管には、１μｌの１０^−６稀釈物（１０^−６）を接種し、３７℃で１５分間培養する。
【０３３２】
約３ｍｌの４８℃トップアガー（１５０μｌ ＩＰＴＧ（０．５Ｍ）及び３７μｌ Ｘ−ＧＡＬ（３５０ｍｇ／ｍｌ）を含む５０ｍｌストック）を各管に追加し、１００ｍｍプレートで平板培養する。プレートは、３７℃で一晩培養する。
【０３３３】
ＺＡＰ ＩＩライブラリは、製造業者のプロトコル（Ｓｔｒａｔａｇｅｎｅ）に従って、ｐＢＬＵＥＳＣＲＩＰＴライブラリを形成するために切除される。
【０３３４】
ＤＮＡライブラリは、宿主細胞（Ｅ．ｃｏｌｉ等）で形質転換させ、クローンの発現ライブラリを生成することができる。
【０３３５】
実施例２
平均化
ライブラリ生成の前に、精製ＤＮＡは、平均化することができる。ＤＮＡは、以下のプロトコルに従って断片化される。ゲノムＤＮＡを含む試料は、塩化セシウム勾配で精製される。塩化セシウム（Ｒｆ＝１．３９８０）溶液は、０．２μｌフィルタで濾過し、１５ｍｌを３５ｍｌ ＯｐｔｉＳｅａｌ管（Ｂｅｃｋｍａｎ）に入れる。ＤＮＡを追加し、完全に混合する。１０マイクログラムのビスベンズイミド（Ｓｉｇｍａ、Ｈｏｅｃｈｓｔ ３３２５８）を追加し、完全に混合する。この管は、次に、濾過した塩化セシウム溶液で満たし、Ｂｅｃｋｍａｎ Ｌ８−７０ ＵｌｔｒａｃｅｎｔｒｉｆｕｇｅのＢｔｉ５０ロータにおいて、３３０００ｒｐｍで７２時間回転させる。遠心分離に続いて、シリンジポンプ及び精留塔（Ｂｒａｎｄｅｌ Ｍｏｄｅｌ １８６）を使用して、２８０ｎｍに設定したＩＳＣＯ ＵＡ−５ＵＶ吸光度検出器に勾配を通す。環境試料に存在する有機体からのＤＮＡを表すピークが取得される。真正細菌の配列は、増幅のための以下のプライマを使用して、Ｅ．ｃｏｌｉのピークの１０倍稀釈からのｒＲＮＡを符号化するＤＮＡをＰＣＲ増幅することで検出できる。
【０３３６】
フォワードプライマ：５’−ＡＧＡＧＴＴＴＧＡＴＣＣＴＧＧＣＴＣＡＧ−３’（ＳＥＱ ＩＤ ＮＯ：４）
リバースプライマ：５’−ＧＧＴＴＡＣＣＴＴＧＴＴＡＣＧＡＣＴＴ−３’（ＳＥＱ ＩＤ ＮＯ：５）
回収されたＤＮＡは、３乃至６ｋｂの断片に剪断又は酵素的に消化される。ローンリンカプライマが連結され、ＤＮＡは、サイズ選択される。サイズ選択ＤＮＡは、必要な場合は、ＰＣＲによって増幅される。
【０３３７】
平均化は、その後、二本鎖ＤＮＡ試料をハイブリッド形成バッファ（０．１２Ｍ ＮａＨ_２ＰＯ_４、ｐＨ６．８／０．８２Ｍ ＮａＣｌ／１ｍＭ ＥＤＴＡ／０．１％ＳＤＳ）において再懸濁することで達成される。この試料は、鉱油を被せ、１０分間沸騰させることで変性させる。この試料は、６８℃で１２乃至３６時間培養する。二本鎖ＤＮＡは、６０℃のヒドロキシアパタイトにおいて、標準のプロトコル（サンブルック，１９８９）に従って、一本鎖ＤＮＡから分離される。一本鎖ＤＮＡ断片は、脱塩し、ＰＣＲにより増幅する。このプロセスは、更に数ラウンド（五回まで又はそれ以上）繰り返される。
【０３３８】
実施例３
酵素活性検定
以下は、ヒドロラーゼ活性に関して、例１に従って準備された発現ライブラリをスクリーニングする代表的な手順である。
【０３３９】
例１において説明したように準備したライブラリのプレートは、各ウェルに２００μｌのＬＢ Ａｍｐ／Ｍｅｔｈのグリセロールを含む単一プレートを多重接種するのに使用される。このステップは、Ｂｅｃｋｍａｎ ＢＩＯＭＥＫ ＲＴＭのＨｉｇｈ Ｄｅｎｓｉｔｙ Ｒｅｐｌｉｃａｔｉｎｇ Ｔｏｏｌ（ＨＤＲＴ）を使用して、１％漂白剤、水、イソプロパノール、各接種間の空気乾燥殺菌と共に実行される。この単一プレートは、二時間３７℃で成長させ、その後、各ウェルに２５０μｌのＬＢ Ａｍｐ／Ｍｅｔｈのグリセロールを含む二枚の白色９６ウェルＤｙｎａｔｅｃｈマイクロタイタドータープレートを接種するのに使用される。オリジナルの単一プレートは、３７℃で１８時間培養し、その後、−８０℃で保管する。二枚の凝縮ドータープレートは、３７℃で同じく１８時間培養する。凝縮ドータープレートは、その後、７０℃で４５分間加熱して細胞を殺し、宿主Ｅ．ｃｏｌｉ酵素を不活性化する。ＤＭＳＯ内の５ｍｇ／ｍＬモルフォウレアフェニルアラニル−７−アミノ−４−トリフルオロメチルクマリン（ＭｕＰｈｅＡＦＣ、「基質」）のストック溶液は、０．６ｍｇ／ｍＬの界面活性ドデシルマルトシドを含む５０ｍＭ ｐＨ７．５ Ｈｅｐｅｓバッファにより、６００μＭまで稀釈する。６００μＭのＭｕＰｈｅＡＦＣ溶液５０μｌを、白色凝縮プレートのウェルのそれぞれに、ＢＩＯＭＥＫを使用して、１００μｌ混合サイクルで追加し、基質の最終濃度を約１００μＭにする。蛍光値は、基質の追加直後
【０３４０】
【化】

（ｔ＝０）にプレート読み出し蛍光光度計により記録する。このプレートは、７０℃で１００分間培養し、その後、周囲温度で更に１５分間冷却させる。蛍光値を、再度記録する（ｔ＝１００）。ｔ＝０の値をｔ＝１００の値から減算し、活性クローンが存在するかどうかを判断する。
【０３４１】
ＭｕＰｈｅＡＦＣ
このデータは、特定のウェル内のクローンの一つが基質を加水分解したかどうかを示すことになる。活性を備える個別のクローンを判定するために、ソースライブラリを解凍し、個別のクローンを使用して、ＬＢ Ａｍｐ／Ｍｅｔｈのグリセロールを含む新しいプレートに単独で接種する。前記のように、このプレートは、３７℃で培養して細胞を成長させ、７０℃で加熱して宿主酵素を不活性化し、Ｂｉｏｍｅｋを使用して６００μＭのＭｕＰｈｅＡＦＣ５０μｌを追加する。
【０３４２】
基質の追加後、前記のように、ｔ＝０の蛍光値を記録し、プレートを７０℃で培養し、ｔ＝１００の値を記録する。これらのデータは、どのプレートに活性クローンがあるかを示す。
【０３４３】
基質に関する鏡像選択性の値、Ｅは、次の式に従って決定される。
【０３４４】
【数】

この式で、ｅｅ_ｐ＝加水分解産物の鏡像体過剰率であり、ｃ＝反応の変換パーセントである。ウォン及びホワイトサイズ，Ｅｎｚｙｍｅｓ ｉｎ Ｓｙｎｔｈｅｔｉｃ Ｏｒｇａｎｉｃ Ｃｈｅｍｉｓｔｒｙ， １９９４， Ｅｌｓｅｖｉｅｒ， Ｔａｒｒｙｔｏｗｎ， Ｎ．Ｙ．， ｐｐ． ９−１２を参照せよ。
【０３４５】
鏡像体過剰率は、キラル高性能液体クロマトグラフィ（ＨＰＬＣ）又はキラルキャピラリ電気永動（ＣＥ）により決定される。検定は、以下のように実行される：２００μｌの適切なバッファを、９６ウェル白色マイクロタイタプレートの各ウェルに追加し、続いて５０μｌの部分又は完全精製酵素を追加し、５０μｌ基質を追加して、モニタされた蛍光の増加と時間とを、基質の５０％の消費又は反応の停止のいずれか早いほうが発生するまで比較する。
【０３４６】
実施例４
陽性酵素活性クローンの定方向突然変異生成
定方向突然変異生成は、野生型酵素より高い度合いの活性を示す新しい酵素を生成するために、二種類の酵素（アルカリホスファターゼ及びβ−グリコシダーゼ）で実行した。
【０３４７】
アルカリホスファターゼ
ＸＬ１−Ｒｅｄ株（Ｓｔｒａｔａｇｅｎｅ）を、製造業者のプロトコルに従ってクジラの骨表面から分離した有機体である有機体ＯＣ９ａからのアルカリホスファターゼ遺伝子を符号化するゲノムクローン２７ａ３ａ（プラスミドｐＢｌｕｅｓｃｒｉｐｔ内）により形質転換させた。ＬＢ＋０．１ｍｇ／ｍｌアンピシリンの５ｍｌ培地を、２００μｌの形質転換体により接種し、この培地を３７℃で３０時間に渡って成長させた。その後、この培地でミニプレップを実行し、製造業者のプロトコルに従い、下で概要を説明する検定手順に従って、結果として生じたＤＮＡ２μｌをＸＬ−１ Ｂｌｕｅ細胞（Ｓｔｒａｔａｇｅｎｅ）で形質転換することで分離ＤＮＡをスクリーニングした。スクリーニング検定において、突然変異したＯＣ９ａホスファターゼは、発色までに１０分を要し、野生型酵素は、発色までに３０分を要した。
【０３４８】
標準のアルカリホスファターゼスクリーニング検定
形質転換したＸＬ１ Ｂｌｕｅ細胞は、ＬＢ／ａｍｐプレートで平板培養した。結果として生じたコロニは、Ｄｕｒａｌｏｎ ＵＶ（Ｓｔｒａｔａｇｅｎｅ）又はＨＡＴＦ（Ｍｉｌｌｉｐｏｒｅ）薄膜によりリフトし、クロロホルム蒸気内で３０秒間溶解させた。３０分間８５℃で培養し、細胞を熱で殺した。フィルタを、ＢＣＩＰバッファにおいて室温で構築し、最も早く発展したコロニ（「陽性物」）を選択し、この「陽性物」をＢＣＩＰプレート（ＢＣＩＰバッファ。２０ｍｍ ＣＡＰＳ ｐＨ９．０、１ｍｍ ＭｇＣｌ_２、０．０１ｍｍ ＺｎＣｌ_２、０．１ｍｇ／ｍｌ ＢＣＩＰ）上で再び線条接種した。
【０３４９】
β−グリコシダーゼ
このプロトコルは、Ｔｈｅｒｍｏｃｏｃｃｕｓ ９Ｎ２ β−グリコシダーゼを突然変異生成させるために使用した。
【０３５０】
２マイクロリットルのｄＮＴＰ（１０ｍＭストック）と、１０マイクロリットルの１０×ＰＣＲバッファと、０．５マイクロリットルのベクタＤＮＡ−３１Ｇ１Ａ−１００ナノグラムと、２０マイクロリットルの３’プライマ（１００ｐｍｏｌ）と２０マイクロリットルの５’プライマ（１００ｐｍｏｌ）と、１６マイクロリットルのＭｎＣｌ ４Ｈ_２Ｏ（１．２５ｍＭストック）と、２４．５マイクロリットルのＨ_２Ｏと、１マイクロリットルのＴａｑポリメラーゼ（５．０ユニット）とを、合計１００マイクロリットルとして培養することにより、ＰＣＲを実行した。ＰＣＲサイクルは、９５℃１５秒、５８℃３０秒、７２℃９０秒、２５サイクルである（７２℃乃至４℃の培養で１０分延長）。
【０３５１】
５マイクロリットルのＰＣＲ産物を、１％アガロースゲルに流し、反応をチェックした。ＱＩＡＱＵＩＣＫカラム（Ｑｉａｇｅｎ）で精製した。５０マイクロリットルのＨ_２Ｏで再懸濁させた。
【０３５２】
２５マイクロリットルの精製ＰＣＲ産物と、１０マイクロリットルのＮＥＢバッファ＃２と、３マイクロリットルのＫｐｎ Ｉ（１ＯＵ／マイクロリットル）と、３マイクロリットルのＥｃｏＲ１（２０Ｕ／マイクロリットル）と、５９マイクロリットルのＨ_２Ｏとを、２時間３７℃で培養し、ＰＣＲ産物を消化させ、ＱＩＡＱＵＩＣＫカラム（Ｑｉａｇｅｎ）で精製した。３５マイクロリットルのＨ_２Ｏで溶出させた。
【０３５３】
１０マイクロリットルの消化済みＰＣＲ産物と、５マイクロリットルのベクタ（ＥｃｏＲＩ／ＫｐｎＩによる切断、及びエビアルカリホスファターゼによるホスファターゼ処理済み）と、４マイクロリットルの５×ライゲーションバッファと、１マイクロリットルのＴ４ ＤＮＡリガーゼ（ＢＲＬ）とを一晩培養し、ＰＣＲ産物をベクタ内に連結させた。
【０３５４】
結果として生じたベクタは、エレクトロポレーションを使用して、Ｍ１５ｐＲＥＰ４細胞で形質転換させた。１００又は２００マイクロリットルの細胞を、ＬＢ ａｍｐ ｍｅｔｈ ｋａｎプレート上で平板培養し、３７℃で一晩成長させた。
【０３５５】
β−ガラクトシダーゼの検定は、（１）Ｍｉｌｌｉｐｏｒｅ ＨＡＦＴ薄膜フィルタを使用してコロニリフトを実行し、（２）１５０ｍｍガラスペトリ皿において、クロロホルム蒸気でコロニを溶解させ、（３）１ｍｇ／ｍｌ ＸＧＬＵを含むＺバッファを染み込ませたＷｈａｔｍａｎ ３ＭＭ濾過紙１枚を含む１００ｍｍガラスペトリ皿にフィルタを移動させ（溶解コロニを有するフィルタをガラスペトリ皿に移動させた後、皿は室温に保つ）、（４）フィルタ薄膜上の青いスポットとして「陽性物」を観察すること（「陽性物」は早期に現れるスポット）により実行した。パスツールピペット（又はガラスキャピラリ管）を使用して、フィルタ薄膜上の青いスポットの中心を取った。小さなフィルタディスクを、２０μｌの水を含むエッペンドルフ管内に配置した。エッペンドルフ管を、７５℃で５分間培養し、続いて、渦動させて、プラスミドＤＮＡをフィルタから溶出させた。このＤＮＡを電気反応性Ｅ．ｃｏｌｉ細胞で形質転換させ、形質転換プレートでフィルタリフト検定を繰り返し、「陽性物」を同定した。形質転換プレートは、フィルタリフト後、３７℃の培養器に戻し、コロニを再生させた。３ｍｌのＬＢａｍｐ液に再精製した陽性物を接種し、３７℃で一晩培養した。こうした培地からプラスミドＤＮＡを分離し、プラスミド挿入の配列を決定した。このフィルタ検定では、１ｍｇの基質５−ブロモ−４−クロロ−３−インドリル−β−ｏ−グルコピラノシド（ＸＧＬＵ）（Ｄｉａｇｎｏｓｔｉｃ Ｃｈｅｍｉｃａｌｓ Ｌｉｍｉｔｅｄ又はＳｉｇｍａ）を含むバッファＺ（下の配合表参照）を使用する。
【０３５６】
Ｚバッファ（Ｊ・Ｈ・ミラー（１９９２）、Ａ Ｓｈｏｒｔ Ｃｏｕｒｓｅ ｉｎ Ｂａｃｔｅｒｉａｌ Ｇｅｎｅｔｉｃｓ， ｐ．４４５を引用）１リットル当たり
Ｎａ_２ＨＰＯ_４−７Ｈ_２Ｏ １６．１ｇ
Ｎａ_２ＨＰＯ_４−４Ｈ_２Ｏ ５．５ｇ
ＫＣｌ ０．７５ｇ
Ｎａ_２ＨＰＯ_４−７Ｈ_２Ｏ ０．２４６ｇ
６−メルカプトエタノール ２．７ｍｌ
ｐＨを７．０に調整
【０３５７】
実施例５
ピコプランクトンゲノムＤＮＡの安定した大きな挿入ＤＮＡライブラリの構築 細胞収集及びＤＮＡの準備。濃縮ピコプランクトン細胞を含むアガロースプラグは、オレゴン州ニューポート乃至ハワイ州ホノルルの海洋クルーズで採集した試料より準備した。海水（３０リットル）をニスキンボトルに収集し、１０μｍ Ｎｉｔｅｘによりスクリーニングし、３０，０００ＭＷカットオフポリフルフォンフィルタを通じた中空糸濾過（Ａｍｉｃｏｎ ＤＣ１０）により濃縮した。濃縮したバクテリアプランクトン細胞は、０．２２μｍ、４７ｍｍ Ｄｕｒａｐｏｒｅフィルタで収集し、１ｍｌの２×ＳＴＥバッファ（１Ｍ ＮａＣｌ、０．１Ｍ ＥＤＴＡ、１０ｍＭトリス、ｐＨ８．０）に再懸濁し、最終濃度を１ｍｌ当たり約１×１０^１０細胞とした。細胞懸濁液は、４０℃に冷却した一体積の１％溶融Ｓｅａｐｌａｑｕｅ ＬＭＰアガロース（ＦＭＣ）と混合し、直後に１ｍｌ注射器に引き込んだ。この注射器は、パラフィルムで密封し、氷上に１０分間置いた。細胞含有アガロースプラグを、１０ｍｌの溶解バッファ（１０ｍＭトリス、ｐＨ８．０、５０ｍＭ ＮａＣｌ、０．１Ｍ ＥＤＴＡ、１％サルコシル、０．２％デオキシコール酸ナトリウム、１ｍｇ／ｍｌリゾチーム）に押し出し、３７℃で１時間培養した。このアガロースプラグは、その後、４０ｍｌのＥＳＰバッファ（１％サルコシル、１ｍｇ／ｍｌプロテイナーゼＫ、０．５Ｍ ＥＤＴＡ中）に移動させ、５５℃で１６時間培養した。この溶液をデカントし、新鮮なＥＳＰバッファに取り替え、５５℃で更に一時間培養した。このアガロースプラグを、次に、５０ｍＭ ＥＤＴＡ中に入れ、海洋クルーズの期間中、船上において４℃で保存した。
【０３５８】
オレゴン州沿岸で採集した試料から準備したアガロースプラグ一切片（７２μｌ）を、２ｍＬマイクロ遠心分離管において、１ｍＬのバッファＡ（１００ｍＭ ＮａＣｌ、１０ｍＭビストリスプロパン−ＨＣｌ、１００μｇ／ｍｌアセチル化ＢＳＡ、２５℃でｐＨ７．０）に対して、４℃で一晩透析した。この溶液を、１０ｍＭ ＭｇＣｌ_２及び１ｍＭ ＤＴＴを含む２５０μｌの新鮮なバッファＡと取り替え、ロッキングプラットフォーム上において、室温で１時間培養した。次に、この溶液を、４ＵのＳａｕ３Ａ１（ＮＥＢ）を含む２５０μｌの同じバッファに変え、水槽内において３７℃で平衡化し、その後、３７℃の培養器内のロッキングプラットフォーム上で４５分間培養した。プラグは、１．５ｍｌマイクロ遠心分離管に移動させ、６８℃で３０分間培養し、酵素を不活性化し、アガロースを溶かした。Ｇｅｌａｓｅ及びＨＫ−ホスファターゼ（Ｅｐｉｃｅｎｔｕｒｅ）をそれぞれ使用し、製造業者の推奨に従って、アガロースを消化し、ＤＮＡを脱ホスホリル化した。タンパク質は、穏やかなフェノール／クロロホルム抽出により取り除き、ＤＮＡは、エタノール沈殿させ、ペレット化し、その後、７０％エタノールで洗浄した。この部分消化ＤＮＡを、無菌Ｈ_２Ｏに再懸濁させ、ｐＦＯＳ１ベクタとのライゲーションのために濃度を２．５ｎｇ／μｌにした。
【０３５９】
いくつかのアガロースプラグからのＰＣＲ増幅の成果は、多量の古細菌ＤＮＡの存在を示した。オレゴンコースト沖、深さ２００ｍで採集した一試料から抽出したｒＲＮＡを使用する定量的ハイブリッド形成実験は、この集合のプランクトン古細菌が全ピコプランクトンのバイオマスの約４．７％を占めることを示した（この試料は、デロングら，Ｎａｔｕｒｅ， ３７１：６９５−６９８， １９９４のＴａｂｌｅ １の”ＰＡＣＩ”−２００ｍに対応する）。アガロースプラグ溶解物で実行された古細胞バイアス済みｒＤＮＡ ＰＣＲ増幅の結果により、この試料における比較的多量の古細菌ＤＮＡの存在が確認された。このピコプランクトン試料から準備されたアガロースプラグを、その後のフォスミドライブラリ作成のために選択した。このサイトからの各１ｍｌアガロースプラグは、約７．５×１０^５の細胞を含んでいたため、部分消化ＤＮＡの作成に使用された７２μｌスライスには、約５．４×１０^５の細胞が存在した。
【０３６０】
ベクタ腕部は、説明の通り、ｐＦＯＳ１から作成した（キムら、Ｓｔａｂｌｅ ｐｒｏｐａｇａｔｉｏｎ ｏｆ ｃｏｎｍｉｄ ｓｉｚｅｄ ｈｕｍａｎ ＤＮＡ ｉｎｓｅｒｔｓ ｉｎ ａｎ Ｆ ｆａｃｔｏｒ ｂａｓｅｄ ｖｅｃｔｏｒ， Ｎｕｃｌ． Ａｃｉｄｓ Ｒｅｓ．， ２０：１０８３２−１０８３５， １９９２）。簡単に言うと、プラスミドを、ＡｓｔＩＩにより完全に消化し、ＨＫホスファターゼにより脱ホスホリル化し、その後、ＢａｍＨＩにより消化して二つの腕部を生成し、そのそれぞれが、３５乃至４５ｋｂｐの連結ＤＮＡをクローニング及びパッケージングするのに適切な方向性であるｃｏｓ部位を含む。部分消化ピコプランクトンＤＮＡは、それぞれ２５ｎｇのベクタ及び挿入物と１ＵのＴ４ ＤＮＡリガーゼ（Ｂｏｅｈｒｉｎｇｅｒ−Ｍａｎｎｈｅｉｍ）とを含む１５μｌのライゲーション反応物において、ＰＦＯＳ１腕部に一晩で連結させた。この反応物４マイクロリットルに含まれる連結ＤＮＡは、Ｇｉｇａｐａｃｋ ＸＬパッケージングシステム（Ｓｔｒａｔａｇｅｎｅ）を使用してｉｎ ｖｉｔｒｏでパッケージし、フォスミド粒子をＥ．ｃｏｌｉ株ＤＨ１０Ｂ（ＢＲＬ）にトランスフェクションし、この細胞をＬＢ_ｃｍ１５プレート状に塗り広げた。結果として生じたフォスミドクローンを、７％グリセロールを補ったＬＢ_ｃｍ１５を含む９６ウェルマイクロリットル皿に採取して入れた。約４０ｋｂのピコプランクトンＤＮＡ挿入物を含む組み換えフォスミドにより、３．５５２フォスミドクローンのライブラリが生じ、これは約１．４×１０^８塩基対のクローンＤＮＡを含む。検査したすべてのクローンは、３８乃至４２ｋｂの範囲の挿入物を含んでいた。このライブラリは、その後の検定のために−８０℃で冷凍保存した。
【０３６１】
実施例６
紫外線誘導光産物を使用したランダムサイズポリヌクレオチドの生成
鋳型ＤＮＡの１マイクログラム試料は、ＴＴ二量体、特にプリン二量体を含む二量体を形成させるために、取得し、紫外線により処理する。紫外線による露光は、鋳型ＤＮＡ試料で遺伝子ごとにほんの僅かな光産物が生成されるように制限される。多数の試料を、紫外線により、時間の長さを変化させて処理し、紫外線露光による二量体の数が異なる鋳型ＤＮＡ試料を取得する。
【０３６２】
非プルーフリーディングポリメラーゼを利用するランダム初回免疫キット（例えば、Ｓｔｒａｔａｇｅｎｅ Ｃｌｏｎｉｎｇ ＳｙｓｔｅｍｓのＰｒｉｍｅ−Ｉｔ ＩＩ Ｒａｎｄｏｍ Ｐｒｉｍｅｒ Ｌａｂｅｌｉｎｇキット）を利用し、紫外線によって（上記の通り）作成された鋳型のランダムな部位で初回免疫を行い、鋳型に沿って伸長することで、様々なサイズのポリヌクレオチドが生成される。Ｐｒｉｍｅ−Ｉｔ ＩＩ Ｒａｎｄｏｍ Ｐｒｉｍｅｒ Ｌａｂｅｌｉｎｇキットにおいて説明されるような初回免疫プロトコルは、プライマを伸長するのに利用することができる。紫外線露光によって形成される二量体は、非プルーフリーディングポリメラーゼによる伸長の障害物としての役割を果たす。したがって、ランダムなプライマによる伸長が終了した後には、ランダムなサイズのポリヌクレオチドのプールが存在する。
【０３６３】
実施例７
ランダムサイズのポリヌクレオチドの分離
例１に従って生成された所定のポリヌクレオチドは、１．５％アガロースゲル状でゲル分離される。１００乃至３００ｂｐの範囲のポリヌクレオチドを、ゲルから切り取り、三体積の６Ｍ Ｎａｌをゲル切片に追加する。この混合物を、５０℃で１０分間培養し、１０μｌのグラスミルク（Ｂｉｏ １０１）を追加する。この混合物を、一分間回転させ、上澄みをデカントする。ペレットを５００μｌのＣｏｌｕｍｎ Ｗａｓｈで洗浄し（Ｃｏｌｕｍｎ Ｗａｓｈは、５０％エタノール、１０ｍｌトリス−ＨＣｌ、ｐＨ７．５、１００ｍＭ ＮａＣｌ、及び２．５ｍＭ ＥＤＴＡ）、１分間回転させ、その後、上澄みをデカントする。その後、洗浄、回転、及びデカントのステップを繰り返す。グラスミルクペレットを、２０μｌのＨ_２Ｏに再懸濁し、一分間回転させる。ＤＮＡは、水相を維持する。
【０３６４】
実施例８
分離したランダムサイズの１００乃至３００ｂｐのポリヌクレオチドのシャッフリング
実施例２において取得された１００乃至３００ｂｐのポリヌクレオチドは、プライマを追加することなく、アニーリング混合物（０．２ｍＭの各ｄＮＴＰ、２．２ｍＭ ＭｇＣｌ_２、５０ｍＭ ＫＣｌ、１０ｍＭトリスＨＣｌ、ｐＨ８．８，０．１％ Ｔｒｉｔｏｎ Ｘ−１００、０．３μ、Ｔａｑ ＤＮＡポリメラーゼ、合計体積５０μｌ）内で組み換える。以下のプログラムによるアニーリングステップに、ＳｔｒａｔａｇｅｎｅのＲｏｂｏｃｙｃｌｅｒを使用した：９５℃３０秒、［９５℃３０秒、５０乃至５０℃（好ましくは５８℃）３０秒、及び７２℃３０秒］を２５乃至５０サイクル、及び７２℃５分間。これにより、１００乃至３００ｂｐのポリヌクレオチドは、結合し、長い配列を有する二本鎖ポリヌクレオチドを発生させる。再構成した二本鎖ポリヌクレオチドを分離し、変性させて、一本鎖ポリヌクレオチドを形成した後、随意的にサイクリングを再び繰り返し、一部の試料では鋳型としての一本鎖とプライマＤＮＡとを利用し、その他の試料では一本鎖に加えてランダムなプライマを利用する。
【０３６５】
実施例９
シャッフル済みポリヌクレオチドからのポリヌクレオチドのスクリーニング
実施例３のポリヌクレオチドを分離し、ここからポリヌクレオチドを発現させる。実施例３のシャッフル済みポリヌクレオチドから取得したポリヌクレオチドは、オリジナルの鋳型から得られたポリペプチドの活性に関してスクリーニングし、活性レベルの制御を比較する。スクリーニング中に発見された関心のあるポリヌクレオチドをコーディングするシャッフル済みポリヌクレオチドは、更に、二次的な望ましい形質に関して比較される。関心の少ないスクリーン済みポリヌクレオチドに対応する一部のシャッフル済みポリヌクレオチドには、再びシャッフリングを施す。
【０３６６】
実施例１０
飽和突然変異生成による定方向進化
部位飽和突然変異生成：部位飽和突然変異生成を達成するために、デハロゲナーゼ酵素のすべての残基を、下のように、３２重縮退オリゴヌクレオチドプライマを使用した部位指定突然変異生成により、全２０種類のアミノ酸に変換した。
【０３６７】
１． デハロゲナーゼ発現構造の培地を成長させ、プラスミドの作成を行った。
【０３６８】
プライマは、各コドンをランダム化させ、これらは共通の構造Ｘ_２０ＮＮ（Ｇ／Ｔ）Ｘ_２０を有し、このＸ_２０は鋳型と結合する十分な相同性を備えた２０塩基配列を表し、ＮＮ（Ｇ／Ｔ）は全２０種類のアミノ酸を符号化する３２重縮退３塩基配列である。代替の態様において、本発明は、Ｎ，Ｎ，Ｇ／Ｔ又は同様なＮＮ（Ｇ／Ｔ）カセットに加え、全１９種類のアミノ酸置換を導入するのに実用的なコドンの任意のセットの使用を提供し、これは（その一部として）３２重縮退ＮＮ（Ｇ／Ｃ）カセット、６４重縮退ＮＮＮカセット、４８重ＮＮ（Ａ／Ｃ／Ｇ）カセット、４８重ＮＮ（Ａ／Ｇ／Ｔ）カセット、全１９アミノ酸置換を導入するのに実用的な１９コドンの任意の非縮退セット、及び全１９アミノ酸置換を導入するのに実用的な２０コドンの任意の非縮退セットを含む。更に別の代替の態様において、本発明は、２０種類のアミノ酸のサブセットがコドン位置のセットのそれぞれに導入されることを提供し、こうしたサブセットの例は、グループ化体系に基づくサブセットから選択された（複数の）アミノ酸を含むことが可能であり、このグループ化体系は、非制限的な例証として、以下の８種類のサブセットを有するグループ化体系にすることができる。
【０３６９】
・芳香族（Ｐｈｅ、Ｔｒｐ、Ｔｙｒ）
・脂肪族（Ｇｌｙ、Ａｌａ、Ｖａｌ、Ｌｅｕ、Ｉｌｅ）
・ＯＨ含有（Ｓｅｒ、Ｔｙｒ、Ｔｈｒ）
・酸性（Ａｓｐ、Ｇｌｕ、Ａｓｎ、Ｇｉｎ）
・塩基性（Ｌｙｓ、Ａｒｇ、Ｈｉｓ）
・硫黄含有（Ｍｅｔ、Ｃｙｓ）
・極性（Ｓｅｒ、Ｔｈｒ、Ｃｙｓ、Ａｓｎ、Ｇｉｎ、Ｔｙｒ）
・無極性（Ｇｌｙ、Ａｌａ、Ｖａｉ、Ｌｅｕ、Ｉｌｅ、Ｍｅｔ、Ｐｈｅ、Ｔｒｐ、Ｐｒｏ）
【０３７０】
２． ５０ｎｇまでのプラスミド鋳型と、１２５ｎｇの各プライマと、１×ネイティブＰｆｕバッファと、２０ｕｍの各ｄＮＴＰと、２．５ＵネイティブＰｆｕ ＤＮＡポリメラーゼとを含む２５μｌの反応混合物を作成した。
【０３７１】
３． Ｒｏｂｏ９６ Ｇｒａｄｉｅｎｔ Ｃｙｃｌｅｒにおいて、次のように反応をサイクルさせた。
【０３７２】
９５℃１時間の初期変性
９５℃４５秒間、５３℃１分、７２℃１１分を２０サイクル
７２℃１０分の最終延長ステップ
４． この反応混合物を、１０ＵのＤｐｎＩにより３７℃で１時間消化し、メチル化鋳型ＤＮＡを消化した。
【０３７３】
５． ２ｕｌの反応混合物を使用して、５０ｕｌのＸＬ１−Ｂｌｕｅ ＭＲＦ細胞を形質転換し、形質転換混合物全体を大きなＬＢ−Ａｍｐ−Ｍｅｔプレート上で平板培養し、２００乃至１０００のコロニを作成した。
【０３７４】
６． 個別のコロニを、ＬＢ−Ａｍｐ−ＩＰＴＧを含む９６ウェルマイクロタイタプレートのウェルに楊枝で入れ、一晩成長させた。
【０３７５】
７． 翌日、これらのプレートのクローンを検定した。
【０３７６】
スクリーニング：各位置の突然変異体の約２００クローンを、液体培地（３８４ウェルマイクロタイタプレート）で成長させ、次のようにスクリーニングした。
【０３７７】
１． ３８４ウェルプレートにおいて一晩培養したものを、遠心分離し、培地を除去した。各ウェルに０．０６ｍＬ １ｍＭトリス／ＳＯ_４ ^２−、ｐＨ７．８を追加した。
【０３７８】
２． 各親成長プレートからの二枚の検定プレートが、０．０２ｍＬ細胞懸濁で構成されるようにした。
【０３７９】
３． 一方の検定プレートを室温で配置し、他方を一定期間（最初は３０分間）に渡って高温にした（初期スクリーニングでは５５℃を使用）。
【０３８０】
４． 規定の時間後、０．０８ｍＬの室温の基質（１．５ｍＭ ＮａＮ_３及び０．１ｍＭブロモチモールブルーを含むＴＣＰ飽和１ｍＭトリス／ＳＯ_４ ^２−、ｐＨ７．８）を各ウェルに追加した。
【０３８１】
５． ６２０ｎｍでの測定値を様々な時点で取り、各ウェルのプログレス曲線を生成した。
【０３８２】
６． データを分析し、加熱した細胞と加熱していない細胞のキネティクスを比較した。各プレートは、１乃至２カラム（２４ウェル）の非突然変異２０Ｆ１２制御を含んでいた。
【０３８３】
７． 安定性が向上したと思われるウェルは、再び成長させ、同じ条件でテストした。
【０３８４】
この手順の後、九種類の単一部位突然変異が酵素に熱安定性の増加を与えたと思われた。この改善を本質的に引き起こした各位置での正確なアミノ酸の変化を判断するために、配列分析を実行した。簡単に言うと、この改善は、七つの部位では一種類のアミノ酸の変化、第八の部位では二種類のアミノ酸の変化それぞれ、第九の部位では三種類のアミノ酸の変化それぞれによって与えられた。その後、いくつかの突然変異を作成し、この突然変異はそれぞれ、これら九種類の有益な部位突然変異のうち複数を組み合わせて有し、そのうち二種類の突然変異は、単一ポイント突然変異のものを含め、他のすべての突然変異よりも優れていることが分かった。
【０３８５】
実施例１１
末端選択を使用したダイレクト発現クローニング
エステラーゼ遺伝子を、標準のＰＣＲ反応において、５’リン酸化プライマを使用して増幅した（１０ｎｇの鋳型、ＰＣＲ条件：３分９４℃、［１分９４℃、１分５０℃、１分３０秒６８℃］×３０、１０分６８℃）。
【０３８６】
フォワードプライマ＝９５１１ＴｏｐＦ（ＣＴＡＧＡＡＧＧＧＡＧＧＡＧＡＡＴＴＡＣＡＴＧＡＡＧＣＧＧＣＴＴＴＴＡＧＣＣＣ）
リバースプライマ＝９５１１ＴｏｐＲ（ＡＧＣＴＡＡＧＧＧＴＣＡＡＧＧＣＣＧＣＡＣＣＣＧＡＧＧ）
結果として生じたＰＣＲ産物（約１０００ｂｐ）は、ゲル精製及び定量化した。
【０３８７】
発現クローニングのベクタ、ｐＡＳＫ３（ドイツ、ゲッチンゲンのＩｎｓｔｉｔｕｔ ｆｕｅｒ Ｂｉｏａｎａｌｙｔｉｋ）は、Ｘｂａ Ｉ及びＢｇｌ ＩＩにより切断し、ＣＩＰにより脱ホスホリル化した。
【０３８８】
０．５ｐｍｏｌｅのＶａｃｃｉｎａ Ｔｏｐｏｉｓｏｍｅｒａｓｅ（カリフォルニア州カールズバッドのＩｎｖｉｔｒｏｇｅｎ）を、バッファＮＥＢ Ｉ（マサチューセッツ州ベバリのＮｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ）内の６０ｎｇ（約０．１ｐｍｏｌｅ）の精製ＰＣＲ産物に追加し、合計体積５μｌとし、５分間３７℃にした。このトポゲートしたＰＣＲ産物を、ベクタｐＡＳＫ３（５μｌ、ＮＥＢＩ内で約２００ｎｇ）でクローニングし、５分間室温にした。この混合物を、Ｈ_２Ｏに対して３０分間透析した。ＤＨ１０Ｂ細胞（メリーランド州ゲーサーズバーグのＧｉｂｃｏ ＢＲＬ）のエレクトロポレーションに、２μｌを使用した。
【０３８９】
効率性：実際のクローン数に基づき、この方法は、１μｇのベクタ当たり２×１０^６クローンを生成可能である。
【０３９０】
実施例１２
デハロゲナーゼの熱安定性
本発明では、定方向進化によって生成される望ましい特性は、厳しい環境と考えられるものを含め、変化した環境に指定の時間に渡って晒された分子の改善された残基活性（例えば、酵素活性、免疫反応性、抗生物質活性、その他）により、限定的な形で例証される。こうした厳しい環境は、以下の任意の組み合わせ（反復性の有無を問わず、及び任意の順序又は順列）を含む場合がある：高温（動作する酵素の変性を引き起こす温度を含む）、低温、高塩度、低塩度、高ｐＨ、低ｐＨ、高圧、低圧、及び放射源への露出の変化（紫外線放射、可視光線、及び電磁スペクトル全体を含む）。
【０３９１】
本発明によれば、改善された前記活性等の望ましい特性は、厳しい環境（例えば、高温、低温、高ｐＨ、低ｐＨ、高塩度、低塩度、有機溶媒の存在の増加、有機溶媒の存在の減少、その他）で特定の期間維持した後の改善された活性（高い活性等）にすることが可能で、定方向進化ステップの適用に続くスクリーニング又は選択ステップに関する選択基準を含むことが可能であり、その一部として、末端選択ベースのステップ、エクソヌクレアーゼ処理ベースのステップ、部位飽和突然変異生成ベースのステップ、又は合成ライゲーション再構成ベースのステップ等がある。
【０３９２】
本発明の別の態様によれば、改善された前記活性等の望ましい特性は、厳しい環境（例えば、高温、低温、高ｐＨ、低ｐＨ、高塩度、低塩度、有機溶媒の存在の増加、有機溶媒の存在の減少、その他）に晒し、ここから移動させた後の改善された活性（高い活性等）にすることが可能で、定方向進化ステップの適用に続くスクリーニング又は選択ステップに関する選択基準を含むことが可能であり、その一部として、末端選択ベースのステップ、エクソヌクレアーゼ処理ベースのステップ、部位飽和突然変異生成ベースのステップ、又は合成ライゲーション再構成ベースのステップ等がある。
【０３９３】
好適な実施形態において、残基活性は、変性条件に晒された後、活性のある三次元形態（オリジナルの三次元形態、オリジナルの三次元形態に類似する三次元形態、或いはオリジナルの三次元形態とは異なる三次元形態を含む）を回復する分子の能力増加から生じる。こうした再び折り重なるための能力は、「変性の可逆性」又は「再生能力」又は「変性−再生の可逆性」と呼ばれる場合があり、本発明による選択パラメータである。
【０３９４】
次の例は、高温に晒された時に酵素が活性を再生及び又は維持する能力を進化させる定方向進化の適用を示している。デハロゲナーゼ酵素のすべての残基（３１６）は、３２重縮退オリゴヌクレオチドプライマを使用した部位指定突然変異生成により、全２０種類のアミノ酸に変換した。この突然変異は、既に比率を改善した変異体Ｄｈｌａ ２０Ｆ１２に導入した。各位置の約２００クローンを、スクリーニングするために液体培地（３８４ウェルマイクロタイタプレート）で成長させた。スクリーニング手順は、次の通りである。
【０３９５】
１． ３８４ウェルプレートにおいて一晩培養したものを、遠心分離し、培地を除去した。各ウェルに０．０６ｍＬ １ｍＭトリス／ＳＯ_４ ^２−、ｐＨ７．８を追加した。
【０３９６】
２． ロボットにより、各親成長プレートからの二枚の検定プレートが、０．０２ｍＬ細胞懸濁で構成されるようにした。
【０３９７】
３． 一方の検定プレートを室温で配置し、他方を一定期間（最初は３０分間）に渡って高温にした（初期スクリーニングでは５５℃を使用）。
【０３９８】
４． 規定の時間後、０．０８ｍＬの室温の基質（１．５ｍＭ ＮａＮ_３及び０．１ｍＭブロモチモールブルーを含むＴＣＰ飽和１ｍＭトリス／ＳＯ_４ ^２−、ｐＨ７．８）を各ウェルに追加した。ＴＣＰ＝トリクロロプロパン。
【０３９９】
５． ６２０ｎｍでの測定値を様々な時点で取り、各ウェルのプログレス曲線を生成した。
【０４００】
６． データを分析し、加熱した細胞と加熱していない細胞のキネティクスを比較した。各プレートは、１乃至２カラム（２４ウェル）の非突然変異２０Ｆ１２制御を含んでいた。
【０４０１】
７． 安定性が向上したと思われるウェルは、再び成長させ、同じ条件でテストした。
【０４０２】
この手順の後、九種類の単一部位突然変異がＤｈｌａ−２０Ｆ１２に熱安定性の増加を与えたと思われた。配列分析では、以下の変化が有益であることが明らかになった。
【０４０３】
Ｄ８９Ｇ
Ｆ９１Ｓ
Ｔ１５９Ｌ
Ｇ１８９Ｑ、Ｇ１８９Ｖ
Ｉ２２０Ｌ
Ｎ２３８Ｔ
Ｗ２５１Ｙ
Ｐ３０２Ａ、Ｐ３０２Ｌ、Ｐ３０２Ｓ、Ｐ３０２Ｋ
Ｐ３０２Ｒ／Ｓ３０６Ｒ
二つの部位（１８９及び３０２）のみが、二つ以上の置換を有した。リストの最初の五つは、（Ｇ１８９Ｑを使用して）単一の遺伝子へと結合された（この突然変異は「Ｄｈｌａ５」と呼ばれる）。Ｓ３０６Ｒを除くすべての変化は、Ｄｈｌａ８と呼ばれる別の変異体に組み込まれた。
【０４０４】
熱安定性は、酵素を一定期間高温（５５℃及び８０℃）で培養し、３０℃で活性検定することで評価した。初速度は、高温での時間と対比させてプロットした。酵素は、培養及び検定の両方で、５０ｍＭトリスＳＯ_４、ｐＨ７．８に入れた。産物（Ｃｌ⁻）は、Ｆｅ（ＮＯ_３）_３及びＨｇＳＣＮを使用した標準的な方法で検出した。Ｄｈｌａ ２０Ｆ１２を、事実上の野生型として使用した。見かけの半減期（Ｔ_１／２）は、データを指数減衰関数に適合させることで計算した。
【０４０５】
５５℃でのＤｈｌａ５デハロゲナーゼの熱安定性。５０ｍＭトリスＳＯ_４ ^２−、ｐＨ７．８内の精製タンパク質を、５５℃で、様々な期間にわたって培養し、アリコートを取り出し、トリクロロプロパン（ＴＣＰ）加水分解に関して検定した。この検定は、５０ｍＭトリスＳＯ_４ ^２−、ｐＨ７．８において、３０℃で、１０ｍＭ ＴＣＰにより実行した。産物（Ｃｌ⁻）の放出は、標準のＣｌ⁻検定により分析した。初速度（Ａ４５０／分）は、酵素プログレス曲線から導き、培養時間と対比させてプロットした。このデータは、指数減衰（Ｃ^＊ｅｘｐ^−ｋｔ）に適合し、ここでｔ_１／２＝ｌｎ２／ｋである（図１３参照）。
【０４０６】
Ｄｈｌａ及び２０Ｆ１２の３０℃及び５５℃での酵素プログレス曲線（図１４参照）。この反応は、精製タンパク質を使用して、５０ｍＭトリスＳＯ_４ ^２−、ｐＨ７．８において、１０ｍＭ ＴＣＰにより実行した。各検定において、等量のタンパク質を使用した。産物（Ｃｌ⁻）の濃度は、標準のＣｌ⁻検定を使用して、様々な時点で判定した。Ｄｈｌａ ５は、３０℃及び５５℃の両方で、２０Ｆ１２と同じ特異活性を有すると思われるが、この酵素は、２０Ｆ１２が熱的に不活性となった後も、生成を継続する。
【０４０７】
８０℃でのＤｈｌａ５デハロゲナーゼの熱安定性。５０ｍＭトリスＳＯ_４ ^２−、ｐＨ７．８内の精製タンパク質を、８０℃で、様々な期間にわたって培養し、アリコートを取り出し、トリクロロプロパン（ＴＣＰ）加水分解に関して検定した。この検定は、５０ｍＭトリスＳＯ_４ ^２−、ｐＨ７．８において、３０℃で、１０ｍＭ ＴＣＰにより実行した。産物（Ｃｌ⁻）の放出は、標準のＣｌ⁻検定により分析した。初速度（Ａ４５０／分）は、酵素プログレス曲線から導き、培養時間と対比させてプロットした。このデータは、指数減衰（Ｃ^＊ｅｘｐ^−ｋｔ）に適合し、ここでｔ_１／２＝ｌｎ２／ｋである（図１５参照）。
【０４０８】
８０℃でのＤｈｌａ５及びＤｈｌａ８の熱安定性。５０ｍＭトリスＳＯ_４ ^２−、ｐＨ７．８内の精製タンパク質を、８０℃で、様々な期間にわたって培養し、アリコートを取り出し、トリクロロプロパン（ＴＣＰ）加水分解に関して検定した。この検定は、５０ｍＭトリスＳＯ_４ ^２−、ｐＨ７．８において、３０℃で、１０ｍＭ ＴＣＰにより実行した。産物（Ｃｌ⁻）の放出は、標準のＣｌ⁻検定により分析した。初速度（Ａ４５０／分）は、酵素プログレス曲線から導き、相対速度を培養時間と対比させてプロットした。このデータは、指数減衰（Ｃ^＊ｅｘｐ^−ｋｔ）に適合し、ここでｔ_１／２＝ｌｎ２／ｋであり、Ｄｈｌａ５及びＤｈｌａ８に関して、それぞれ１３及び１３８分の値を与えた（図１６参照）。
【０４０９】
こうしたデハロゲナーゼ変異体の示唆走査熱量測定は、図１７Ａ、Ｂ、及びＣに表示されている。このデータは、Ｐｅｒｋｉｎ ＥｌｍｅｒのＰｙｒｉｓ Ｉを使用し、走査速度１０℃／分で記録した。タンパク質濃度は、４０μｌにおいて１０ｍｇ／ｍｌである（合計０．４ｍｇのタンパク質）。バッファは、１０ｍＭ ＮａＰ_ｉ、ｐＨ７．５である。データから導かれた溶解温度は、６８℃（２０Ｆ１２）、７３℃（Ｄｈｌａ５）、及び７３℃（Ｄｈｌａ８）。このデータは、更に、２０Ｆ１２が不可逆変性を受けており、Ｄｈｌａ５が部分的に可逆であり、Ｄｈｌａ８が完全に可逆であることを示している（図１７参照）。
【０４１０】
５５℃での野生型（２０Ｆ１２）及び突然変異体（Ｄｈｌａ５）デハロゲナーゼの熱安定性。５０ｍＭトリスＳＯ_４ ^２−、ｐＨ７．８内の精製タンパク質を、５５℃で、様々な期間にわたって培養し、アリコートを取り出し、トリクロロプロパン（ＴＣＰ）加水分解に関して検定した。この検定は、５０ｍＭトリスＳＯ_４ ^２−、ｐＨ７．８において、３０℃で、１０ｍＭ ＴＣＰにより実行した。産物（Ｃｌ⁻）の放出は、標準のＣｌ⁻検定により分析した。初速度（Ａ４５０／分）は、酵素プログレス曲線から導き、培養時間と対比させてプロットした。このデータは、指数減衰（Ｃ^＊ｅｘｐ^−ｋｔ）に適合し、ここでｔ_１／２＝ｌｎ２／ｋである（図１２参照）。
【０４１１】
上述した教示を踏まえて、本発明の多数の修正及び変形が可能であり、そのため、特許請求の範囲内で、本発明は、詳述したもの以外の態様で実施することができる。以上、本発明をその特定の好適な実施形態に基づき詳細に説明してきたが、修正及び変形は、説明され且つ請求されている発明の趣旨及び範囲内に入るものと理解されよう。
【図面の簡単な説明】
【図１】部位飽和突然変異生成の好適な実施形態を示す図である。
【図２】合成ライゲーション再構成によるキメラ結合の網羅的なセットの生成を示す図である。
【図３Ａ及び図３Ｂ】（例えば、合成ライゲーション再構成による）ポリヌクレオチド再構成によって生成された望ましいポリヌクレオチドを選択する（例えば、全長分子の選択を行う）トポイソメラーゼ誘導選択ステップを使用した末端選択の応用を示す図である。
【図４】末端選択技術の様々な態様を示す図である。
【図５】オリジナルの平滑末端ＤＮＡの定方向発現クローニングを達成する目的で、当初の平滑末端ＤＮＡに「付着末端」を導入するために、酵素Ｎ．ＢｓｔＮＢｌを使用して、「ｃ）定方向発現クローニングを実行すること」のステップが達成される、末端選択技術の特定の態様を示す図である。
【図６】酵素エクソヌクレアーゼＩＩＩの活性を示す図である。アスタリスクは、この酵素がポリヌクレオチド基質の３’方向から５’方向に向けて働くことを示す。
【図７】エクソヌクレアーゼ仲介ポリヌクレオチド再構成における酵素エクソヌクレアーゼＩＩＩの例示的な応用を示す図である。
【図８】多結合核酸鎖の生成を示す図である。
【図９】ヘテロメリック核酸複合体において、アニーリングした核酸鎖の未ハイブリッド形成一本鎖末端から、３’及び５’末端ヌクレオチドを遊離させ、短縮されているがハイブリッド形成された末端を残し、処理済み末端のポリメラーゼベース伸長及び又はリガーゼ仲介ライゲーションを促進する手段としてのエクソヌクレアーゼ処理の使用を示す図である。
【図１０】一本のメチル化多結合核酸鎖をアニーリングし、いくつかの非メチル化単結合核酸鎖にする例における、エクソヌクレアーゼ仲介核酸再構成の使用を示す図である。
【図１１】複数のメチル化多結合核酸鎖をアニーリングし、いくつかの非メチル化単結合核酸鎖にする例における、エクソヌクレアーゼ仲介核酸再構成の使用を示す図である。
【図１２】５５℃での野生型（２０Ｆ１２）及び突然変異型（Ｄｈｌａ ５）デハロゲナーゼの熱安定性のグラフを示す図である。
【図１３】５５℃でのＤｈｌａ ５デハロゲナーゼの熱安定性のグラフを示す図である。
【図１４】３０℃及び５５℃でのＤｈｌａ ５及び２０Ｆ１２の酵素プログレス曲線を示す図である。
【図１５】８０℃でのＤｈｌａ ５デハロゲナーゼの熱安定性を示す図である。このデータは、ｔ_１／２＝ｌｎ２／ｋである指数減衰（Ｃ^＊ｅｘｐ^−ｋｔ）に適合する。
【図１６】８０℃でのＤｈｌａ ５及びＤｈｌａ ８デハロゲナーゼの熱安定性を示す図である。このデータは、Ｄｈｌａ ５及びＤｈｌａ ８それぞれに関して、１３及び１３８分の値を与えたｔ_１／２＝ｌｎ２／ｋである指数減衰（Ｃ^＊ｅｘｐ^−ｋｔ）に適合する。
【図１７】（図１７Ａ、図１７Ｂ、及び図１７Ｃ）三種類のデハロゲナーゼ変種の示差走査熱量測定を示す図である。このデータは、Ｐｅｒｋｉｎ Ｅｌｍｅｒ Ｐｙｒｉｓ １を使用して１０℃／分の走査速度で記録された。
【配列表】

[0001]
FIELD OF THE INVENTION
The present invention relates to the screening and identification of biologically active molecules, and in particular, to the generation and screening of gene libraries generated from nucleic acids isolated from two or more organisms for biologically active molecules or biological activities.
[0002]
【background】
Most of the currently used bioactive compounds are derived from soil microorganisms. Many microorganisms that live in soil and other complex ecological communities produce various compounds that enhance their ability to survive and reproduce. These compounds are generally considered not essential for the growth of organisms and are synthesized with the help of genes involved in intermediate metabolism. These secondary metabolites, which affect the growth or survival of other organisms, are known as "bioactive" compounds and are a major component of the chemical defense reserve for both microbial and non-microbial organisms. Plays a role. Humans have exploited these compounds and used them as antibiotics, anti-infectives, and other bioactive compounds with activity against a wide range of prokaryotic and eukaryotic pathogens (Burns et al., Proc. Nat. Acad. Sci. USA, 91, 1994).
[0003]
Although there appears to be a large number of available bioactive compounds, modern biomedicine is facing the growth of antibiotic resistant pathogens as one of the biggest challenges. Due to short generation times and the ability to easily exchange genetic information, pathogenic microorganisms are evolving rapidly, spreading resistance mechanisms against virtually any class of complex antibiotics. For example, the virulent strains of the human pathogens Staphylococcus and Streptococcus are currently only treatable with a single antibiotic, vancomycin, and developing resistance to this compound requires only that the resistant Enterococcus species It only transcribes a single gene, vanA (Bateson et al., System. Appl. Microbiol., 12, 1989). Combining the critical need for new antimicrobial compounds with the increasing demand for enzyme inhibitors, immunosuppressants, and anticancer agents, it is easy to see why pharmaceutical companies have stepped up screening microbial samples for bioactive compounds. It becomes clear.
[0004]
The approach currently used to screen microorganisms for new bioactive compounds has largely changed since the field began. Fresh isolates of bacteria, especially Gram-positive strains from the soil environment, are collected and their metabolites are tested for pharmacological activity.
[0005]
There is still a huge amount of biological diversity that has not yet been used as a source of lead compounds. However, currently available methods for screening and generating lead compounds cannot be efficiently applied to such sparse resources. For example, it has been estimated that at least 99% of marine bacterial species cannot survive on laboratory media, and commercial fermentation equipment is not suitable for use in conditions in which such species grow, so such organisms are: It is difficult or impossible to culture for screening or resupply. Reharvesting, growing, improving strains, improving media, and expanding production of drug producing organisms often creates problems in the synthesis and development of lead compounds. Furthermore, the need to interact with certain organisms to synthesize some compounds makes their use extremely difficult in the discovery process. The use of new methods in drug discovery to take advantage of the genetic resources and chemical diversity of the source of compounds that have not yet been used would be highly beneficial.
[0006]
At the heart of modern biology is the presence of genetic information in nucleic acid genomes, and the information embodied in these genomes (ie, genotypes) directs cellular function. This occurs through the expression of various genes in the genome of the organism and the regulation of the expression of these genes. The expression of a gene in a cell or organism determines the physical properties (ie, phenotype) of the cell or organism. This is achieved through the translation of genes into proteins. Determining the biological activity of a protein obtained from an environmental sample can provide useful information about the role of the protein in the environment. In addition, such information will aid in the development of biology, diagnostics, therapeutics, and industrial applications.
[0007]
Thus, the present invention provides a method for accessing this unused biological diversity and rapidly screening for predetermined sequences and activities utilizing recombinant DNA technology. The present invention combines the advantages associated with the ability to rapidly screen for natural compounds with the flexibility and reproducibility provided in working with genetic material of an organism.
[0008]
Summary of the Invention
The present invention provides for rapid screening of samples for a given biological activity or biomolecule. Samples can be derived from a wide range of sources, including, for example, environmental libraries, samples containing more than one organism (such as a mixed population of organisms), and samples from non-cultureable organisms. Includes deep sea vents and others. As described herein, such samples provide a rich source of virgin molecules useful in biology, therapeutics, and industrial applications, which have been characterized prior to the present invention. And difficult and time consuming methods to identify, or have been impossible to identify or characterize.
[0009]
In one embodiment of the invention, the invention comprises screening a library of clones generated by nucleic acids from a mixed population of cells for a particular bioactivity or biomolecule, and having the particular bioactivity or biomolecule. Altering the nucleic acid sequence contained in the clone and comparing the altered biological activity or biomolecule with a specific biological activity or biomolecule, wherein the difference in the biological activity or biomolecule is the effect of the sequence change. And thereby providing a predetermined biological activity or biomolecule, thereby providing a method for obtaining the predetermined biological activity or biomolecule.
[0010]
In another embodiment, the invention comprises screening a library of clones produced by pooled nucleic acids obtained from multiple isolates for a particular biological activity or biomolecule, and comprising the particular biological activity or biomolecule. Identifying the clone provides a method for identifying a given biological activity or biomolecule.
[0011]
In yet another embodiment, the invention provides a method for identifying a given biological activity or biomolecule. The method comprises the steps of screening a library of clones generated by pooled nucleic acids obtained from the plurality of isolates for a particular biological activity or biomolecule, and the nucleic acid sequence contained in the clone having the particular biological activity or biomolecule. And comparing the altered biological activity or biomolecule to a particular biological activity or biomolecule, wherein in the comparison the difference in the biological activity or biomolecules has introduced at least one sequence change. Indicating an effect, thereby providing a predetermined biological activity or biomolecule.
[0012]
In another embodiment, the invention provides a method of identifying a given biological activity or biomolecule, the method comprising, for a particular biological activity or biomolecule, a nucleic acid obtained from each of the plurality of isolates. Screening a library of clones generated by pooling the generated individual gene libraries, and identifying clones containing a particular biological activity or biomolecule.
[0013]
In another embodiment, the invention provides for screening a library for a particular biological activity or biomolecule, wherein the library pools individual gene libraries produced by nucleic acids obtained from each of a plurality of isolates. And the step of causing a change in the nucleic acid sequence contained in the clone having the specific biological activity or biomolecule, comparing the altered biological activity or biomolecule with the specific biological activity or biomolecule, In this comparison, the difference in bioactivity or biomolecule indicates the effect of introducing at least one sequence change, thereby providing a given bioactivity or biomolecule, thereby providing a given bioactivity or biomolecule. The present invention provides a method for identifying
[0014]
In yet another embodiment, the present invention provides a method of identifying a given biological activity or biomolecule, wherein the method is provided by a nucleic acid from an enriched population of organisms for a particular biological activity and biomolecule. Screening a library of clones and identifying clones containing a particular biological activity or biomolecule.
[0015]
In yet another embodiment, the invention includes screening a library of clones generated by nucleic acids from an enriched population of organisms for a particular biological activity or biomolecule; Altering the nucleic acid sequence comprising the clone and comparing the altered biological activity or biomolecule to a particular biological activity or biomolecule, wherein the difference in the biological activity or biomolecule is at least one sequence change. Providing a predetermined biological activity or biomolecule, thereby providing a method of identifying the predetermined biological activity or biomolecule.
[0016]
In another embodiment, the invention provides a method for identifying a given biological activity or molecule. The predetermined biological activity or biomolecule comprises a complementary sequence of a nucleic acid from a mixed population of organisms, together with at least one oligonucleotide probe having a detectable molecule and at least a portion of the nucleic acid sequence encoding the predetermined molecule. Culturing under conditions that permit interaction and identifying a nucleic acid sequence that has a complement to the oligonucleotide probe using an analyzer that detects the detectable molecule. Thereafter, a library is generated from the identified nucleic acid sequences, and the library is screened for a particular biological activity or biomolecule. A nucleic acid sequence contained in a clone having a specific biological activity or biomolecule is altered, and the altered biological activity or biomolecule is compared with the specific biological activity or biomolecule. A molecular difference indicates the effect of introducing at least one sequence change, which provides a given biological activity or biomolecule.
[0017]
In another embodiment, the present invention relates to the use of a nucleic acid obtained from a mixed population of organisms in a microenvironment with at least one oligonucleotide probe having a detectable molecule and at least one nucleic acid sequence encoding a given molecule. Co-encapsulating over such a time period and separating the encapsulated nucleic acid with an analyzer that detects detectable molecules, under conditions that allow interaction of the complementary sequences, in part. In identifying an encapsulated nucleic acid containing a complement to an oligonucleotide probe encoding a predetermined molecule, generating a library from the separated encapsulated nucleic acid, and converting the library to a specific biological activity or biomolecule. Screening for clones having a particular biological activity or biomolecule. Altering the nucleic acid sequence and comparing the altered biological activity or biomolecule with a particular biological activity or biomolecule, wherein the difference in the biological activity or biomolecule results in at least one sequence change. Indicating the effect of the introduction and thereby providing a predetermined biological activity or biomolecule, thereby providing a method of identifying the predetermined biological activity or biomolecule.
[0018]
In yet another embodiment, the present invention relates to a method for detecting, in a microenvironment, a nucleic acid obtained from a segregate of a mixed population of organisms with at least one oligonucleotide probe having a detectable marker and a molecule having a predetermined biological activity. Co-encapsulating over such a time period and detecting a detectable marker under conditions that allow interaction of the complementary sequence with at least a portion of the polynucleotide sequence encoding Separating the encapsulated nucleic acid by an analyzer to identify an encapsulated nucleic acid containing a complement to an oligonucleotide probe encoding a predetermined molecule, and generating a library from the separated encapsulated nucleic acid, Screening the library for a particular biological activity or biomolecule; Altering a nucleic acid sequence contained in a clone having a defined biological activity or biomolecule; comparing the altered biological activity or biomolecule with a specific biological activity or biomolecule; Indicating that the difference in the biomolecules has introduced at least one sequence change, whereby a given biological activity or biomolecule is provided.
[0019]
In another embodiment, the present invention provides a method for detecting, in a microenvironment, nucleic acids obtained from one or more isolates of a mixed population of organisms with at least one oligonucleotide probe having a detectable marker and a predetermined biological activity. Co-encapsulating with a polynucleotide sequence encoding a molecule having at least a portion thereof over such a time under conditions that allow interaction of the complementary sequence, and a detectable marker Isolating the encapsulated nucleic acid by an analyzer that detects the nucleic acid, thereby identifying an encapsulated nucleic acid containing a complement to an oligonucleotide probe encoding a predetermined molecule, and generating a library from the separated encapsulated nucleic acid. And screening the library for specific biological activities or biomolecules. And altering the nucleic acid sequence contained in the clone having the specific biological activity or biomolecule; and comparing the altered biological activity or biomolecule with the specific biological activity or biomolecule; A step of obtaining a given biological activity or biomolecule by the step of providing a given biological activity or biomolecule, wherein the difference in activity or biomolecule indicates the effect of introducing at least one sequence change. provide.
[0020]
In yet another embodiment, the invention provides a method for identifying a given biological activity or biomolecule. The method comprises, in a microenvironment, converting a nucleic acid obtained from a mixture of isolates of a mixed population of organisms into at least one oligonucleotide probe having a detectable marker and a polynucleotide encoding a molecule having a predetermined biological activity. Co-encapsulating over such a time period under such conditions that the interaction of the complementary sequence with at least a portion of the nucleotide sequence is possible, and the nucleic acid encapsulated by the analyzer to detect a detectable marker Identifying an encapsulated nucleic acid containing a complement to an oligonucleotide probe encoding a predetermined molecule, generating a library from the separated encapsulated nucleic acid, and converting the library to a specific organism. Screening for activity or biomolecules; and Altering a nucleic acid sequence contained in a clone having a biomolecule, and comparing the altered bioactivity or biomolecule with a particular bioactivity or biomolecule; Indicating the effect of introducing at least one sequence change, thereby providing a predetermined biological activity or biomolecule.
[0021]
These and other aspects of the invention will be apparent to those skilled in the art from the teachings herein.
[0022]
The present invention provides for rapid screening of libraries derived from two or more organisms, such as, for example, an environmental sample or a non-cultured population of organisms or a mixed population of organisms from a cultured population of organisms.
[0023]
In one embodiment, the gene library is obtained by obtaining the nucleic acid from a mixed population of organisms and cloning the nucleic acid into a suitable vector to transform a plurality of clones to generate the gene library. Generated. Thus, a gene library contains genes or gene fragments present in a mixed population of organisms. The gene library can be an expression library, in which case the library can be screened for expressed polypeptides having the desired activity. Alternatively, the gene library can be screened for a given sequence, for example, by PCR or hybridization screening.
[0024]
In one embodiment, nucleic acids from a sample isolate comprising a mixed population of organisms are pooled, and the pooled nucleic acids are used to generate a gene library.
[0025]
"Isolate" means that a specific species, genus, family, order, or class of an organism has been obtained or withdrawn from a sample having more than one organism or from a mixed population of organisms Means that. Nucleic acids from such segregated populations can then be used to generate gene libraries. Isolates can be obtained from samples containing two or more organisms or a mixed population of organisms by selective filtering or culturing. For example, bacterial isolates can be used to filter a sample with a filter that excludes organisms based on size, or to cultivate the sample on a medium that allows for selective growth or selective inhibition of a particular population of organisms. Can be obtained.
[0026]
An “enriched population” is an organism population in which the percentage of organisms belonging to a particular species, genus, family, order, or class of organisms relative to the entire population is increasing. For example, selective growth or inhibition media can increase the overall number of organisms. Prokaryotes can be enriched for the total number of organisms in the population. Similarly, growing a mixed population on a selective medium that inhibits or promotes the growth of a small population within the mixed population can enrich a particular species, genus, family, order, or class of organisms. .
[0027]
In another embodiment, nucleic acids from multiple (eg, two or more) isolates from a mixed population of organisms are used to generate a plurality of gene libraries, including a plurality of clones, wherein the at least two The gene libraries from the isolates are then pooled to obtain a “pooled isolate library”.
[0028]
After the gene library has been generated, the clones can be of a defined biological activity (eg, enzymatic activity, second messenger activity, binding activity, transcriptional activity, etc.) or biomolecule (eg, nucleic acid sequence, peptide, polypeptide, lipid or Other small molecules, etc.). Such screening techniques can be used, for example, to convert a clone, population of clones, or population of nucleic acid sequences to a substrate or substrates having a detectable molecule that provides a detectable signal upon interaction with a predetermined biological activity or biomolecule. Including contacting. The substrate can be an enzyme substrate, a bioactive molecule, an oligonucleotide, and others.
[0029]
In one embodiment, a gene library is generated and the clone is exposed to a predetermined chromogenic or fluorescent substrate or substrates, or a labeled probe having a sequence corresponding to the predetermined sequence (oligo with detectable molecule). A positive clone is identified by a detectable signal (such as fluorescence).
[0030]
In one embodiment, an expression library generated from a mixed population of organisms is screened for a predetermined activity. Specifically, an expression library is generated, the clones are exposed to a predetermined substrate or substrates, and positive clones are identified and separated. In the present invention, cells need not survive. The cells must survive long enough to produce the molecule to be detected, and then either live or non-viable cells, as long as the expressed biomolecules (such as enzymes) remain active. There may be.
[0031]
In certain embodiments, the present invention relates to the direct cloning of genes encoding new or desired biological activities from environmental samples into high-throughput screening systems designed to rapidly discover new molecules, such as enzymes. Provide a combined approach. This approach is described in E. based on the construction of an environmental "expression library" capable of representing the population genome of a large number of naturally occurring microorganisms stored in cloning vectors that can be propagated in E. coli or other suitable host cells. This library is not limited to the small fraction of prokaryotes that can be grown in pure culture, since cloned DNA can be initially extracted directly from environmental samples or from isolates of environmental samples. In addition, by averaging the environmental DNA present in such a sample, it is possible to more evenly represent DNA from all species present in the sample. The averaging technique (described below) dramatically increases the efficiency of finding interesting genes from trace components of a sample, which may have orders of magnitude less than the dominant species in the sample. Averaging can be performed subsequent to obtaining the nucleic acid from the sample or substrate (s) in any of the above embodiments.
[0032]
In another embodiment, the present invention evaluates a large number of clones and identifies and recovers cells encoding useful enzymes and other biomolecules (such as ligands). Provide a throughput capillary array system. In particular, the capillary array-based approach described herein can be used to screen, identify, and recover proteins with the desired biological activity or other ligands with the desired binding affinity. For example, a binding assay can be performed using a suitable substrate or other marker that generates a detectable signal upon the occurrence of a desired binding event.
[0033]
In addition, fluorescently active cell sorting can be used to screen and isolate clones having a given activity or sequence. Heretofore, FACS devices have been used in research centered on the analysis of eukaryotic and prokaryotic cell lines and cell culture processes. FACS has also been used to monitor the production of foreign proteins in both eukaryotes and prokaryotes to be studied, eg, differential gene expression and the like. The detection and counting functions of the FACS system have been applied in such instances. However, FACS has not been used in discovery processes to screen and recover biological activity in prokaryotes. Furthermore, in the present invention, the desired nucleic acid (recombinant clone) can be obtained from live or dead cells, so that the cells do not need to survive as in the previously described techniques. The cells need only survive long enough to produce the compound to be detected, and thereafter, either live or non-viable cells, as long as the expressed biomolecule remains active. May be. The present invention further relates to E.C. It solves the problems that would be associated with the detection and selection of E. coli expressed recombinant enzymes and the recovery of the encoding nucleic acid. In addition, the present invention includes in its embodiment any device capable of detecting a fluorescent wavelength associated with a biological material, such a device being defined herein as a fluorescence analyzer (FACS device as an example).
[0034]
It is also desirable to identify nucleic acid sequences from a mixed population, segregant, or enriched population of organisms. In this embodiment, there is no need to express the gene product. A given nucleic acid sequence can be identified or "biopanned" by contacting a clone, device (such as a gene chip), filter, or nucleic acid sample with a probe labeled with a detectable molecule. Probes usually have a sequence that is substantially identical to the given nucleic acid sequence. Alternatively, the probe will be a fragment-length or full-length nucleic acid sequence encoding a given polypeptide. The probe and the nucleic acid are cultured for such a time under conditions that allow hybridization of the probe to a substantially complementary sequence. The stringency of hybridization varies, for example, with probe length and GC content. These factors can be determined empirically (e.g., Sambrook et al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, and Current Protocols, Inc., Columbia, Canada). (See Current Protocols, Green Publishing Associates, Inc., Joint Venture with John Wiley & Sons, Inc, Latest Supplement). After hybridization, the complementary sequence can be PCR amplified, identified by hybridization techniques (eg, exposing the probe and nucleic acid mixture to a film), or the nucleic acid can be detected using a chip.
[0035]
Prior to the present invention, the evaluation of complex gene or environmental expression libraries was rate-limited. The present invention allows for the rapid screening of complex environmental libraries, including, for example, genomic sequences from thousands of organisms or subsets and isolates. The advantages of the present invention can be seen, for example, in screening complex environmental samples. Screening complex samples has previously required the use of labor intensive methods to screen millions of clones to cover genomic diversity. The present invention means an extremely high-throughput screening method capable of evaluating such a huge number of clones. The disclosed method allows for the optional screening of about 30 million to about 200 million clones per hour for a given desired nucleic acid sequence, biological activity, or biomolecule. This allows full screening of environmental libraries for new biological activities or biomolecules of clonal expression.
[0036]
After identifying a given sequence or biological activity (eg, a given enzyme), modifying the amino acid sequence by developing, mutating, or inducing a sequence or polynucleotide encoding the given biological activity; For example, a modifying activity such as increased thermostability, specificity, or activity can be provided.
[0037]
“Amino acid” includes a hydrogen atom at the central carbon atom (α-carbon atom), a carboxylic acid group (the carbon atom therein is referred to as a “carboxyl carbon atom”), and an amino group (nitrogen atom therein). Atoms are referred to as “amino nitrogen atom” in the present specification) and a side chain group R. When incorporated into a peptide, polypeptide, or protein, an amino acid loses one or more atoms at the carboxyl group of the amino acid in a dehydration reaction linking one amino acid to another. As a result, when incorporated into a protein, the amino acids will be referred to as "amino acid residues."
[0038]
“Protein” or “polypeptide” refers to any polymer of two or more individual amino acids, whether or not naturally occurring, linked via peptide bonds, and one amino acid (or amino acid residue) This occurs when the carboxyl carbon atom of the carboxylic acid group bonded to the α-carbon atom of the above) is covalently bonded to the amino nitrogen atom of the amino group bonded to the α-carbon atom of the adjacent amino acid. The term "protein" is understood to include within its meaning the terms "polypeptide" and "peptide", which may be used interchangeably herein. In addition, proteins containing multiple polypeptide subunits (eg, DNA polymerase III, RNA polymerase II) or other components (eg, RNA molecules as occurs in telomerase) are also used herein as “proteins”. It is understood to be included in the meaning of "." Similarly, fragments of proteins and polypeptides fall within the scope of the invention and may be referred to herein as "proteins."
[0039]
The specific amino acid sequence of a given protein (ie, the "primary structure" of a polypeptide when writing the amino to carboxy termini) is determined by the nucleotide sequence of the encoded portion of the mRNA, which in turn comprises the genetic information, Usually specified by genomic DNA (including organelle DNA such as mitochondrial or chloroplast DNA). Thus, determining the sequence of a gene helps predict the primary structure of the corresponding polypeptide, and more particularly, the role or activity of the polypeptide or protein encoded by the gene or polynucleotide sequence.
[0040]
The terms "isolated" or "purified" when referring to a nucleic acid sequence or a polypeptide sequence, respectively, mean that they have been altered from their natural states "by the hand of man"; If it does, it has changed or moved from the original environment, or both. For example, naturally occurring polynucleotides or polypeptides that are naturally present in existing extant animals, biological samples, or environmental samples in their natural state are not "isolated" or "purified," The same polynucleotide or polypeptide that has been cleaved from is "separated" or "purified" as the term is used herein. Such polynucleotides, when introduced into a culture or whole intact host cell, are different from the naturally occurring form or environment and, as a term used herein, will still be isolated. . Similarly, when polynucleotides and polypeptides are generated in a composition, such as a medium composition (eg, a solution for introducing a polynucleotide or polypeptide into a cell or composition, or a solution for a chemical or enzymatic reaction). There is.
[0041]
"Polynucleotide" or "nucleic acid sequence" refers to a polymerized form of a nucleotide. Polynucleotide also refers to a sequence that is not directly contiguous with any of the immediately contiguous coding sequences (one at the 5 'end and one at the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, the term is used to describe, for example, a recombinant DNA incorporated into a vector, a self-replicating plasmid or virus, or a procaryotic or eukaryotic genomic DNA, or a separate molecule independent of other sequences (such as a cDNA). As well as recombinant DNA that exists as The nucleotides of the present invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. Polynucleotides, as used herein, include, in particular, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, Includes RNA, which is a mixture of single-stranded and double-stranded regions, and DNA and RNA, which may be single-stranded, or more generally, double-stranded, or a mixture of single- and double-stranded regions Hybrid molecule.
[0042]
In addition, polynucleotide, as used herein, refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The chains in these regions can be from the same molecule or from different molecules. This region can include all of one or more of the molecules, but more generally includes only one region of some of the molecules. One of the molecules of the triple helix region is often an oligonucleotide. The term polynucleotide encompasses genomic DNA or RNA (varies depending on the organism, ie the viral RNA genome), as well as mRNA and cDNA encoded by the genomic DNA.
[0043]
As noted above, there is currently a need in the biology and chemical industry for molecules (such as enzymes) that can optimally perform biological or chemical processes. For example, molecules and compounds utilized in conventional and emerging chemical, pharmaceutical, fiber, food and sample, and potential markets must meet stringent economic and environmental standards. The synthesis of polymers, pharmaceuticals, natural products, and pesticides creates harmful by-products and is often hampered by expensive processes where poor quality or inefficient catalysis is problematic. For example, enzymes have a number of remarkable advantages that can overcome these catalysis problems, acting on a single functional group, identifying similar functional groups on a single molecule, and enantiomers. Identify. In addition, enzymes are biodegradable and function in very small mole fractions in the reaction mixture. Due to their chemical, region and stereospecificity, enzymes offer a unique opportunity to properly achieve the desired selective transformation. Enzymes are often very difficult to replicate chemically, especially in single-step reactions. The elimination of the need for protecting groups, selectivity, and the ability to perform multi-step transformations in a single reaction vessel have led to an increased demand for enzymes in the chemical and pharmaceutical industries, with the attendant reduced environmental burden. Enzymatic processes are gradually replacing many conventional chemical methods. A major cause of the current limited use in a wider industry is the relatively small number of commercially available enzymes. Of the over 3000 non-DNA amplifying enzyme activities described so far, only up to 300 enzymes (excluding DNA amplifying enzymes) are currently commercially available.
[0044]
The use of enzymes in technical applications also requires performance under demanding industrial conditions. This includes activities in environments or on substrates where the stock of currently known enzymes is not evolutionarily selected. However, the natural environment offers extreme conditions, including, for example, extreme temperatures and pH. Many organisms have adapted to such conditions, such as by selecting polypeptides to be able to withstand such extreme conditions.
[0045]
Enzymes have evolved under the conditions of temperature, pH, and salt concentration due to selective pressures that perform very specific functions in the environment of living organisms. Most of the non-DNA modifying enzyme activities identified to date have been isolated from mesophilic organisms, representing a very small part of the available phylogenetic diversity. The dynamic field of biocatalysis is entering a new dimension with the help of enzymes isolated from microorganisms that thrive in extreme environments. For example, such enzymes may be in excess of 100 ° C. in hot springs and hot water vents on land and below 0 ° C. in polar waters, in the Dead Sea's saturated salt environment, in coal reserves and in geothermal sulfur. It must function at pH values near zero in spring water or greater than 11 in sewage sludge. For example, environmental samples obtained from extreme conditions including organisms, polynucleotides, or polypeptides (such as enzymes) open up a new field of biocatalysis. By rapidly screening for polynucleotides encoding a given polypeptide, the present invention provides a source of materials that will develop enzymes for biology, therapeutics, and industrial use, as well as specific activities, specificities, Or to provide new substances for further processing, for example by directed evolution and mutagenesis, in order to develop molecules or polypeptides that modify for the conditions.
[0046]
In addition to the need for new enzymes for use in the industry, the need for bioactive compounds with new activities has increased dramatically. This demand stems primarily from changes in global demographics coupled with a clear and growing trend in a number of pathogens that are resistant to currently available antibiotics. For example, in emerging nations with a young population, demand for antimicrobial drugs has surged, while in aging countries, such as the United States, the need to increase the repertoire of drugs for cancer, diabetes, arthritis, and other debilitating conditions There is. Infectious disease mortality has increased 58% between 1980 and 1992, and the emergence of antibiotic-resistant bacteria is estimated to add more than $ 30 billion annually to health care costs in the United States alone ( Adams et al., Chemical and Engineering News, 1995; Amman et al., Microbiological Reviews, 59, 1995). In response to this trend, pharmaceutical companies have greatly increased the screening of bacterial diversity for compounds with unique activities or specificities. Accordingly, the present invention can be used to obtain and identify information unique to polynucleotides and related sequences, for example, from infectious microorganisms present in the environment such as the digestive tract of various microorganisms.
[0047]
Identifying new enzymes in environmental samples is one solution to these problems. By rapidly identifying polypeptides having a given activity and polynucleotides encoding a given polypeptide, the present invention helps develop compositions for biology, diagnostics, therapeutics, and industrial applications. Methods, compositions, and sauces are provided.
[0048]
The methods and compositions of the present invention provide for the identification of a lead compound of a drug present in an environmental sample. The methods of the invention provide the ability to delve into the environment for new drugs or to identify related drugs contained in different microorganisms. There are several common sources of lead compounds (drug candidates), including natural products, such as nucleotides, peptides, or other polymeric molecules that have been identified or developed as a result of delving into the environment. A set, a set of synthetic chemicals, and a synthetic combined chemicals library are included. Each of these sources has advantages and disadvantages. The success of these candidate screening programs is largely determined by the number of compounds in the program, and pharmaceutical companies are looking to pioneer the search for a vast number of synthetic and natural compounds that have been screened out of date. There is a need to. Unfortunately, the ratio of new compounds to compounds discovered in the past has decreased over time. The discovery rate of new lead compounds has not kept up with demand, despite the best efforts of pharmaceutical companies. There is a strong need to access new sources of potential drug candidates. Thus, the present invention provides a rapid and efficient method for identifying and characterizing environmental samples containing new drug compounds.
[0049]
Most of the currently used bioactive compounds are derived from soil microorganisms. These compounds are generally considered not essential for microbial growth and are called "secondary metabolites" because they are synthesized with the help of genes involved in intermediate metabolism. Secondary metabolites that affect the growth or survival of other organisms are known as "bioactive" compounds and play a role as a major component of chemical defense reserves for both microbial and non-microscopic organisms. Fulfill. About 6,000 bioactive compounds of microbial origin have been characterized, of which more than 60% are produced by Gram-positive soil bacteria of the genus Streptomyces (Barnes et al., Proc. Nat. Acad. Sci. U.S.A.). SA, 91, 1994). Of these, at least 70 are currently used in biomedical and agricultural applications. Polyketides, the largest class of bioactive compounds, include a wide range of antibiotics, immunosuppressants, and anticancer agents, for a combined turnover of $ 5 billion annually.
[0050]
The present invention provides a method for identifying a nucleic acid sequence encoding a polypeptide having a known or unknown function. For example, much of the diversity in microbial genomes results from rearrangements of genes in the microbial genome. These genes may exist across species or may be systematically related to other organisms.
[0051]
For example, bacteria and many eukaryotes have a coordinated mechanism for regulatory genes whose products are involved in related processes. These genes assemble on a single chromosome, form a structure called "genes," and are transcribed together under the control of a single regulatory sequence that contains a single promoter that initiates transcription of the entire cluster. . Genes, promoters, and auxiliary sequences that function in regulation are collectively referred to as "operons" and can include 20 or more genes, and typically include 2 to 6 genes. Thus, a group of genes is a group of adjacent genes that are usually identical or related in terms of their function.
[0052]
Some gene families are composed of identical components. Although the genes to be grouped need not necessarily be identical, grouping is a prerequisite for maintaining identity between the genes. Genes range from extremes, where duplications to adjacent related genes are generated, to cases where hundreds of identical genes are present in tandem sequence. In some cases, no meaning can be found for a particular gene repeat. A prime example of this is the expressed replicative insulin gene in some species, while a single insulin gene is sufficient in other mammalian species.
[0053]
In addition, the ability of genes to form heterogeneous libraries of genes, for example, from bacteria and other prokaryotic sources, e.g., from bacterial and other prokaryotic sources, can result in the synthesis of polyketides with a vast number of active activities, for example. Is of value in determining the source of new proteins, particularly including enzymes such as polyketide synthases that cause For example, polyketides are molecules that are extremely abundant sources of biological activity, including antibiotics (such as tetracycline and erythromycin), anticancer drugs (daunomycin), immunosuppressants (FK506 and rapamycin), and veterinary products (monensin). And Many polyketides (produced by polyketide synthases) are valuable as therapeutic agents. Polyketide synthases are multifunctional enzymes that catalyze the biosynthesis of various carbon chains that differ in length, function, and pattern of reduction. Polyketide synthase genes are categorized into genes, and at least one type of polyketide synthase (designated as Type I) has large size genes and enzymes, genetic manipulation and the incorporation of such genes / proteins. In vitro research is complicated. Other types of proteins that are the product (s) of a group of genes are also contemplated, including, for example, regulatory proteins such as antibiotics, antivirals, antitumor agents, and insulin.
[0054]
The ability to select and combine desired components from a library of polyketide and post-polyketide biosynthetic genes to generate new polyketides for research is attractive. The method (s) of the present invention make this possible, allowing the generation of gene banks with clones containing large insertions (especially when using factor F-based vectors), which is the cloning of genes. To facilitate the cloning of new polyketide synthases and other genes.
[0055]
For example, the genes can be linked to a vector containing an expression control sequence capable of controlling and regulating the production of a detectable protein or protein-related sequence activity from the linked genes. The use of vectors having a very high capacity for the introduction of exogenous nucleic acids is particularly suitable for use with such a group of genes; E. coli F factor (or fertility factor) is explained. This E. The E. coli F factor is a plasmid that affects its own high frequency transmission during conjugation and is ideal for completing large nucleic acid fragments, such as genes from mixed microbial samples, and performing stable propagation.
[0056]
Nucleic acids isolated or extracted from such a sample (such as a mixed population of microorganisms) or isolates thereof can be inserted into a vector or plasmid prior to polynucleotide screening. Such vectors or plasmids usually contain expression control sequences, including promoters, enhancers, and others.
[0057]
Accordingly, the present invention provides a new system for cloning and screening a mixed population of microbial enriched samples or isolates thereof in vitro for a polynucleotide encoding a molecule having a predetermined activity, ie, a predetermined enzymatic and biological activity. I will provide a. The method (s) of the present invention allow for the cloning and discovery of new bioactive molecules in vitro, especially new bioactive molecules derived from uncultured or cultured samples. The method (s) of the invention can be used to clone, sequence, and screen large groups of genes, genes, and gene fragments. Unlike previous approaches, the method (s) of the present invention clone, screen, and identify polynucleotides and polypeptides encoded by such polynucleotides in vitro from a wide range of environmental samples. be able to.
[0058]
In the present invention, a polynucleotide sequence can be screened and identified from a complex environmental sample, its concentrated sample, or its isolate. A gene library can be generated from a cell-free sample, as long as the sample contains a nucleic acid sequence, or can be generated from a sample that contains cells, cellular materials, or viral particles. Organisms from which libraries can be created include prokaryotic microorganisms such as Eubacteria and Archaebacteria, and lower eukaryotic microorganisms such as fungi, algae, and protozoa, as well as mixed populations of plants, plant spores, and pollen. . Such organisms can be cultured organisms or uncultured organisms obtained from environmental samples, and can be used in extreme environmental microorganisms such as thermophilic, hyperthermophilic, cold and psychrotrophic organisms. including.
[0059]
Sources of nucleic acids used to generate DNA libraries can be obtained from environmental samples, including, in part, Arctic and Antarctic ice, sea and permafrost sources, Microbial samples obtained from materials of volcanic origin, materials from tropical soil or plant sources, and feces of various organisms, including mammals and invertebrates, as well as microbial samples obtained from minerals, spoils, etc. Is included. The nucleic acids used to generate the gene library can be obtained, for example, from a concentrated subpopulation or isolate of a sample. In another embodiment, DNA from multiple isolates can be pooled to form a source of nucleic acids for generating a library. Alternatively, the nucleic acid can be obtained from multiple isolates and multiple gene libraries generated from multiple isolates to include multiple gene libraries. A pool separation library can be obtained by pooling or combining two or more gene libraries. Thus, for example, nucleic acids can be recovered from cultured or uncultured organisms, generate appropriate gene libraries (such as recombinant expression libraries), and determine the identity of certain biomolecules (polynucleotides). Sequence, etc.) can then be used to determine or be screened for a given biological activity (such as an enzyme or biological activity).
[0060]
The following outlines a general procedure for generating a library from both cultured and uncultured organisms, enriched populations, and mixed populations of organisms and their isolates, By performing a survey, sequence, or screening, a nucleic acid sequence having a confirmed, desired, or predicted biological activity (such as an enzymatic activity) can be selected therefrom. Further evolution, mutagenesis, or induction is possible.
[0061]
An environmental sample, as used herein, is any sample that contains an organism or polynucleotide or a combination thereof. Thus, environmental samples can be obtained from any number of sources (as described above), including, for example, insect dung, hot springs, soil, and others. Any source of nucleic acid, in purified or unpurified form, can be utilized as starting material. Thus, nucleic acids can be obtained from any source contaminated by the organism or from any sample, including cells. The environmental sample can be an extract from any biological sample, such as blood, urine, spinal fluid, tissue, vaginal swabs, stool, amniotic fluid, or buccal mouthwash of any mammalian organism. For a non-mammalian (eg, invertebrate) organism, the sample can be a tissue sample, a saliva sample, a stool sample, or a substance in the gastrointestinal tract of the organism. Environmental samples further include samples obtained from extreme environments, including, for example, hot sulfur pools, conduits, and frozen tundra. Samples can be obtained from various sources. For example, in horticultural and agricultural tests, the sample can be a plant, fertilizer, soil, liquid, or other horticultural and agricultural products, and in a food test, the sample can be a fresh or processed food (eg, Specialty milk powder, marine products, fresh agricultural products, and packaged foods), and in environmental tests, the samples are judged or assumed to include liquids, soil, sewage treatment, sludge, and organisms or polynucleotides. It can be any other sample in the environment.
[0062]
If the sample is a mixture of substances comprising a mixed population of organisms, e.g., blood, soil, or sludge, treating with a suitable reagent that is effective to open cells and expose or separate nucleic acid strands Can be. Although not required, this lysis and nucleic acid denaturation step results in cloning, amplification, or sequence occurring more rapidly. In addition, if desired, a particular population or desired isolate (eg, a isolate of a particular species, genus, or family of organisms) may be purified or concentrated, and thus mixed to obtain a low-impurity sample. The population can be cultured prior to analysis. However, this is not required. For example, culturing the organism in the sample includes culturing the organism in the microdroplet, and separating the cultured microdroplet into individual wells of a multi-well tissue culture plate by using a cell sorter. May be included. Alternatively, the sample can be cultured in any number of selective media compositions designed to inhibit or promote the growth of a particular small population of organisms.
[0063]
If the isolate is from a sample that contains a mixed population of organisms, the nucleic acid can be obtained from the isolate as described below. The nucleic acids obtained from this isolate can be used to generate a gene library or, alternatively, can be pooled with other separated fragments of the sample, in which case the pooled nucleic acids Is used to generate a gene library. The isolate can be cultured prior to nucleic acid extraction or can be uncultivated. The method of separating a particular population of organisms is performed in a mixed population.
[0064]
Thus, the sample includes, for example, nucleic acids from a diverse mixed population of organisms (such as microorganisms present in the digestive tract of insects). Nucleic acids are separated from a sample using any number of methods that separate DNA and RNA. Such nucleic acid separation methods are commonly practiced in the art. If the nucleic acid is RNA, the RNA can be reverse transcribed into DNA using primers known in the art. If the DNA is genomic DNA, the DNA can be sheared using, for example, a 25 gauge needle.
[0065]
The nucleic acid can be cloned in a suitable vector. The vector used will be determined by whether to express, amplify, or manipulate the DNA in any number of ways known in the art (eg, US Pat. No. 6,022,716). Cloning techniques are known in the art and can be constructed by one of ordinary skill in the art without undue explanation. The choice of vector will also depend on the size of the host cell and the polynucleotide sequence utilized in the methods of the invention. Thus, the vectors used in the present invention can be plasmids, phages, cosmids, phagemids, viruses (eg, retroviruses, parainfluenza viruses, herpes viruses, reoviruses, paramyxoviruses, and others), or selected portions thereof (eg, , Coat protein, spike glycoprotein, capsid protein). For example, cosmids and phagemids are commonly used when the particular nucleic acid sequence to be analyzed or modified is large, since such vectors can stably propagate large polynucleotides.
[0066]
Vectors containing the cloned nucleic acid sequences can then be amplified by plating (ie, cloning) or transfecting appropriate host cells with the vector (eg, a phage on an E. coli host). The cloned nucleic acid sequence can be used to transform a suitable organism to create a library for screening (eg, expression screening, PCR screening, hybridization screening, or the like). Hosts known in the art are transformed by artificially introducing a vector containing the nucleic acid sequence by inoculation under conditions conducive to such transformations. Transformation with double-stranded circular or linear nucleic acids is possible, and in some cases, with single-stranded circular or linear nucleic acid sequences. Transformation or transformation refers to permanent or transient changes in a gene induced within a cell following the incorporation of new DNA (such as DNA external to the cell). When the cell is a mammalian cell, permanent genetic changes are usually achieved by introducing DNA into the genome of the cell. A transformed cell or host cell is generally a cell (eg, prokaryotic or eukaryotic) into which DNA molecules not normally present in the host organism have been introduced (or introduced into their ancestors) using recombinant DNA techniques. Point to.
[0067]
Certain types of vectors used in the present invention contain a copy of the F factor origin. E. FIG. E. coli F factor (or fertility factor) is a plasmid that affects its own high frequency transmission during conjugation and the low frequency transmission of the bacterial chromosome itself. In certain embodiments, a cloning vector called a "fosmid" or bacterial artificial chromosome (BAC) vector is used. These are E. coli capable of stably integrating large segments of DNA. E. coli F factor. When integrated with DNA from mixed uncultured environmental samples, this allows large genomic fragments to be achieved in the form of a stable environmental gene library.
[0068]
Nucleic acids from a mixed population or sample can be inserted into a vector by various procedures. Generally, the nucleic acid sequence is inserted into the appropriate restriction endonuclease site (s) by procedures known in the art. Such procedures and others will be within the purview of those skilled in the art. In a typical cloning scenario, DNA that has been "blunted" with an appropriate nuclease (such as mung bean nuclease) is methylated, for example, with EcoR I methylase, and ligated to the EcoR I linker GGAATTCC (SEQ ID NO: 1). May be included. The linker is then digested with EcoRI restriction endonuclease and the size of the DNA is fragmented (eg, using a sucrose gradient). The resulting size-fragmented DNA is then ligated into a suitable vector for sequencing, screening, or expression (eg, packaging using a λ vector and in vitro λ packaging extraction).
[0069]
Transformation of a host cell with the recombinant DNA can be performed by conventional techniques widely known to those skilled in the art. If the host is E. coli. In the case of prokaryotes such as E. coli, competent cells capable of taking up DNA are harvested after the exponential growth phase, and then purified by procedures well known in the art.₂It can be made from cells treated by the method. Instead, MgCl₂Alternatively, RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
[0070]
When the host is eukaryotic, methods for transfection or transformation with DNA include conventional mechanical procedures such as calcium phosphate co-precipitation, microinjection, electroporation, insertion of liposome-encapsulated plasmids, or viral Vectors may be included and other methods known in the art may be used. Eukaryotic cells can be further co-transfected with a second heterologous DNA molecule encoding a selectable marker, such as the herpes simplex thymidine kinase gene. Another method uses eukaryotic viral vectors, such as Simian Virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express proteins (Eukaryotic Viral Vector, Cold Spring). Laboratory, Glutzmann ed., 1982). The eukaryotic cells can be yeast cells (such as Saccharomyces cerevisiae), insect cells (such as Drosophila species), or mammalian cells, including human cells.
[0071]
Eukaryotic and mammalian expression systems allow for post-translational modifications of expressed mammalian proteins. Eukaryotic cells should be used that have a cellular machine that handles primary transcription, glycosylation, phosphorylation, or secretion of the gene product. Such cell lines can include CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and WI38 as part thereof.
[0072]
In one embodiment, after a library of clones has been created using any number of methods, including those described above, the clones are transformed into, for example, nutrient-rich broth or other growth media known in the art. It is resuspended in a liquid medium such as a medium. Typically, the medium is a liquid medium that can be quickly pipetted. Then, one or more media types, including at least one clone of the library, are introduced individually or together as a mixture (all or part thereof) into the capillaries of the capillary array.
[0073]
In another embodiment, the library is first biopanned prior to introduction or delivery to a capillary device or other screening technique. Such biopanning methods enrich the library for a given sequence or activity. Examples of biopanning methods are described below.
[0074]
In one embodiment, a library can be screened or sorted for clones containing a given sequence or activity based on the polynucleotide sequences present in the library or clone. Thus, the present invention is useful for screening organisms for desirable biological activities or sequences, and furthermore, can be used for directed evolution, molecular biology, biotechnology, and industrial applications. Methods and compositions are provided that assist in obtaining a sequence.
[0075]
Accordingly, the present invention provides a method for rapidly screening, enriching, and / or identifying sequences in a sample by screening and identifying nucleic acid sequences present in the sample. As such, the present invention increases the repertoire of available sequences that can be used to develop molecules for diagnostics, therapeutics, or industrial applications. Thus, the methods of the present invention can identify new nucleic acid sequences that encode proteins or polypeptides having a desired biological activity.
[0076]
After a gene library (such as an expression library) has been generated and prior to expression screening, an additional step of "biopanning" such a library may be included. This “biopanning” procedure refers to the process of screening clones for sequence homology in a library of clones to identify clones with designated biological activities.
[0077]
The probe sequence used to selectively interact with a given target sequence in the library can be a full-length or partial coding region sequence for a known biological activity. Libraries can be probed using a mixture of probes that include at least a portion of a sequence that encodes a known biological activity or that has a desired biological activity. Such a plurality of probes or probe libraries are preferably single-stranded. In one embodiment, the library is preferably converted to single-stranded form. Particularly suitable probes are those derived from DNA encoding an activity similar to, or having the same activity as, the specified biological activity being screened. Probes can be used for PCR amplification and, therefore, target sequences can be selected. Alternatively, the probe sequence can be used as a hybridization probe that can be used to identify sequences with substantial or desired homology.
[0078]
In another embodiment, FACS-based devices can be utilized to perform in vivo biopanning. Gene or expression libraries are constructed with vectors containing elements that stabilize the transcribed RNA. For example, including sequences that generate secondary structures, such as hairpins, designed to flanking the transcribed region of RNA can help enhance stability and therefore increase half-life in cells Let it. The probe molecules used in the biopanning process provide an detectable signal upon interaction with the target sequence (eg, an oligonucleotide labeled with a detectable molecule that fluoresces only when the probe binds to the target molecule). It is composed of nucleotides. For example, Howard M. Shapiro M. D. "Practical Flow Cytometry", 1995, Wiley-Liss, Inc. Various dyes or stains known in the art, such as those described in, can be used to intercalate or associate with nucleic acids to "label" oligonucleotides. Such probes are introduced into recombinant cells of the library using one of several transformation methods. The probe molecule interacts or hybridizes with the transcribed target mRNA or DNA, generating a DNA / RNA heteroduplex or DNA / RNA double-stranded molecule. Binding of the probe to the target will produce a detectable signal (such as a fluorescent signal) that is detected and sorted by the FACS device or otherwise during the screening process.
[0079]
The probe DNA should be at least about 10 bases, preferably at least 15 bases. In one embodiment, the entire coding region of a portion of the pathway can be utilized as a probe. When the probe hybridizes to the target DNA in an in vitro system, the conditions for hybridization in which the target DNA is separated by using at least one DNA probe are stringent hybridization conditions that result in at least about 50% sequence identity. Designed to provide stringency, and more particularly, to provide stringency with at least about 70% sequence identity.
[0080]
Hybridization techniques for probing microbial DNA libraries to isolate potential target DNA are well known in the art and include any of the techniques described in the literature for use herein. Are suitable, including, for example, chip-based assays, membrane-based assays, and others.
[0081]
The resulting library of transformed clones can then be further screened for clones exhibiting the desired activity. Clones can be transferred to an alternative host for expression of the active compound, or can be screened using the methods described herein.
[0082]
An alternative to the in vitro biopanning described above is the encapsulation technique, for example, gel microdroplets, which localizes a large number of clones to one location and uses it for screening on a FACS device. Can be. Clones can then be rescreened on a FACS machine and split into individual clones to identify individual clones that are positive. Screening using such a FACS device is fully described in patent application Ser. No. 08 / 876,276, filed Jun. 16, 1997. Thus, for example, if the clone mixture has the desired activity, it may be necessary to collect individual clones and rescreen using a FACS machine to determine among these clones those having the specified desired activity. it can.
[0083]
Various types of encapsulation methods and compounds or polymers can be used in the present invention. For example, high temperature agarose can be used to create high temperature stable microdroplets, which can be used to remove all background activity when screening for thermostable biological activity. After the heat sterilization step, stable cell encapsulation can be obtained. Encapsulation can be by beads, hot agarose, gel microdroplets, cells such as ghost red blood cells or macrophages, liposomes, or any other means of encapsulating and localizing molecules.
[0084]
For example, methods for making liposomes have been described (eg, US Pat. Nos. 5,653,996, 5,393,530, and 5,651,981) to encapsulate various molecules. Also described are methods of using liposomes (eg, US Pat. Nos. 5,595,756, 5,605,703, 5,627,159, 5,652,225, 5). No., 567,433, 4,235,871, and 5,227,170). The inclusion of proteins, viruses, bacteria, and DNA during endocytosis has been similarly described (see, for example, Journal of Applied Biochemistry 4, 418-435 (1982)). Erythrocytes used as carriers in vitro or in vivo for substances involved during hypotonic lysis or breakdown of membranes have also been described (Their, GM (1983) J. Pharm. Ther. Has been considered in). These techniques are useful in the present invention to encapsulate a sample in a microenvironment for screening.
[0085]
"Microenvironment" refers to any molecular environment that, in use with the present invention, provides an environment suitable for promoting the interactions required for the methods of the present invention. Suitable environments for promoting the interaction of molecules include, for example, liposomes. Liposomes can be made from various lipids including phospholipids, glycolipids, steroids, long chain alkyl esters such as alkyl phosphates, fatty acid esters such as lecithin, aliphatic amines, and others. it can. Fat mixtures may be utilized in combinations such as neutral steroids, charged amphiphiles, and phospholipids. Illustrative examples of phospholipids include lecithin, sphingomyelin, and dipalmitoyl phosphatidylcholine. Representative steroids include cholesterol, cholestanol, and lanosterol. Representative charged amphiphile compounds generally contain 12 to 30 carbon atoms. This is a monoalkyl or dialkyl phosphate, or an alkylamine, such as dicetyl phosphate, stearylamine, hexadecylamine, dilauryl phosphate, and others.
[0086]
Further, some or all of the above-described embodiments may be combined, performing an averaging step prior to generating the expression library, and then generating the expression library, and then bioexpressing the thus generated expression library. Panned and then biopanned expression libraries can be screened using high-throughput cell sorting and screening equipment. Thus, there are various options, including (i) generating a library and then screening, (ii) averaging the target DNA, generating a library and then screening, (ii) Averaging, generating a library, biopanning and screening, or (iv) generating, biopanning and screening a library. The nucleic acid used to generate the library can be obtained, for example, from an environmental sample, a mixed population of organisms (cultured or uncultured, etc.), an enriched population, and a isolate thereof. In addition, screening techniques include, for example, hybridization screening, PCR screening, expression screening, and others.
[0087]
Gel microdroplet technology has had significance in amplifying the signals available for flow cytometric analysis and in enabling microbial strain screening with strain improvement programs related to biotechnology. (Biotechnolo. Bioeng. (1993) 42: 351-356) reported that Aspergillus awamori glucoamylase has a 10-fold increase in secretion.⁶We have developed a microencapsulation selection method that enables rapid quantitative screening of yeasts exceeding 100%. This method provides a 400-fold single pass enrichment for high secretion mutants.
[0088]
Gel microdroplets or other related techniques can be used in the present invention to perform localization, sorting, and signal amplification in high-throughput screening of recombinant libraries. Because nucleic acids can be recovered from the microdroplets, the viability of the cells during screening is not an issue or is not critical.
[0089]
Following any number of biopanning procedures that can enrich the library for clones containing a given sequence, enriched clones are suspended in a liquid medium such as broth or other growth medium. Thus, an enriched clone includes a plurality of host cells that have been transformed with a construct that includes a vector that has incorporated nucleic acid sequences from a sample (eg, a mixed population of organisms, isolates thereof, and the like). A liquid medium containing a subset of the clones and one or more substrates having detectable molecules (such as enzyme substrates) is then introduced or contacted individually or as a mixture with the enriched clones (capillary array capillary). Etc.). The interaction (including the reaction) of the substrate with a clone that expresses an enzyme having the desired enzymatic activity produces a product or detectable signal, which can include one or more signal-generating clones, including at least one signal-generating clone. Spatial detection can be used to identify clones or capillaries. This signal producing clone or the nucleic acid contained in the signal producing clone can then be recovered using any number of techniques.
[0090]
"Substrate" as used herein includes, for example, a substrate for detecting a biological activity or a biomolecule (eg, an enzyme and its specific enzymatic activity). Such substrates are widely known in the art. For example, various enzymes, and suitable substrates specific to such enzymes, are disclosed in Molecular Probes, Handbook of Fluorescent Probes and Research Chemical (Molecular Probes, Inc., Eugene, Oreg.), The disclosure of which is incorporated herein by reference. Part of the specification of the present application is explicitly stated. Substrates can have an associated detectable molecule, including, for example, a chromogenic or fluorescent molecule. Substrates suitable for use in the invention are those that produce an optically detectable signal upon interaction (eg, a reaction) with certain enzymes having the desired activity, or with certain clones encoding such enzymes. Any substrate that does.
[0091]
One skilled in the art can, for example, select an appropriate substrate based on the desired enzyme activity. Examples of desirable enzymes / enzyme activities include those listed herein. Desirable enzymatic activity can further include a group of enzymes in the enzymatic pathway where the optical signal substrate is present. An example of this is the series of carotenoid synthases.
[0092]
Substrates are known and / or commercially available, especially for glucosidases, proteases, phosphatases, and monooxygenases. Proteases with a detectable (such as optical) signal substrate include serine proteases—trypsin and chymotrypsin. Glucosidases include mannosidase, amyloglucosidase, cellulase, neuraminidase, β-galactosidase, and alpha amylase.
[0093]
When the desired activity is of the same class as that of other biomolecules or enzymes with multiple substrates, the activity can be tested using a cocktail of known substrates. For example, substrates are known for about 20 commercially available esterases, and combinations of such known substrates provide detectable, if not optical, signal generation.
[0094]
The optical signal substrate can be a chromogenic substrate, a fluorescent substrate, a bioluminescent or chemiluminescent substrate, or a fluorescence resonance energy transfer (FRET) substrate. The detectable species can be derived from a secondary molecule that is affected by cleavage or other substrate / biomolecule interactions, such that the cleavage of the substrate or undergoes a detectable change. Numerous examples of detectable assay formats are known from diagnostic techniques using immunoassays, chromogenic assays, and labeled probe methods.
[0095]
In one embodiment, the optical signal substrate can be a bioluminescent or chemiluminescent substrate. Chemiluminescent substrates for some enzymes are available from Tropix (Bedford, Mass.). Enzymes with known chemiluminescent substrates include alkaline phosphatase, β-galactosidase, β-glucoronidase, and β-glucosidase.
[0096]
In another embodiment, a chromogenic substrate can be used, particularly for certain enzymes such as hydrolases. For example, the optical signal substrate can be an indolyl derivative that is enzymatically cleaved to generate a chromogenic product. If a chromogenic substrate is used, the optically detectable signal is light absorbance (including changes in absorbance). In this embodiment, the signal can be detected by absorbance measurement using a spectrophotometer or other.
[0097]
In another embodiment, a fluorescent substrate is used and the optically detectable signal is fluorescent. Fluorescent substrates provide high sensitivity to enhance detectability and alternative modes of detection. Hydroxyl- and amino-substituted coumarins are most widely used as fluorescent materials used to make fluorescent substrates. A common coumarin-based fluorescent substrate is 7-hydroxycoumarin, commonly known as umbelliferone (Umb). Derivatives and analogs of umbelliferone are also used. Derivatives of fluorescein (such as FDG or C12-FDG) and rhodamine and analogous substrates are also used. Resorufin (eg, resorufin β-D-galactopyranoside or resorufin β-D-glucuronide) is particularly useful in the present invention. Resorufin-based substrates are useful, for example, in screening for glycosidases, hydrolases, and dealkylases. Lipophilic derivatives of the above substrates (such as alkylated derivatives) may generally be useful in certain embodiments because they may be readily taken up by cells and have a tendency to associate with lipid regions of the cell. Fluorescein and resorufin are commercially available as alkylated derivatives that form relatively water-insoluble (ie, lipophilic) products. For example, fluorescence imaging can be performed using C12-resorphin galactoside manufactured by Molecular Probes (Eugene, Oreg.) As a substrate.
[0098]
The particular fluorescent substrate used can be selected based on the enzyme activity to be screened. The following is an example.
[0099]
Lipase / esterase. When screening for enzymes having lipase or esterase activity, acylated derivatives of fluorescein are used. The fluorescent substance is hydrolyzed from this derivative to generate a signal.
[0100]
Protease. Enzymes with protease activity are screened in the same way as esterases, where the amide bond is cleaved instead of the ester. Currently, significantly more than 100 protease substrates are available at the cleavage site of an acylated fluorophore.
[0101]
Monooxygenase (dealkylase). Several coumarin derivatives suitable as monooxygenase substrates are commercially available. Typically, in such substrates, hydroxylation of the ethyl group of the compound results in release of the resorufin phosphor.
[0102]
Usually, the substrate can enter the cell and maintain its presence in the cell for a period of time sufficient to perform the analysis (e.g., the substrate produces a detectable reaction upon entry into the cell). Do not "leak" again before reacting with the enzyme being screened for a sufficient period of time). Substrate retention in cells can be enhanced by a variety of techniques. In one method, the substrate compound is structurally modified by adding a hydrophobic (eg, alkyl) tail. In another embodiment, a solvent such as DMSO or glycerol can be used to cover the outside of the cells. Further, the substrate can be provided to the cells at reduced temperatures that have been observed to slow the escape of the substrate from the cells. However, entry of the substrate into the cell may occur, for example, if the enzyme or polypeptide is secreted and present in a lysed cell sample or the like, or if the substrate is capable of acting externally on the cell (extracellular receptor). Ligand complex, etc.) is not essential.
[0103]
The optical signal substrate can, in some embodiments, be a FRET substrate. FRET is a spectroscopic method that can monitor the proximity and relative angular orientation of fluorescent materials. Fluorescent indicator systems that measure the concentration of a substrate or product using FRET include two fluorescent moieties with emission and excitation spectra, one as a "donor" fluorescent moiety and the other as an "acceptor" fluorescent moiety. . The two fluorescent moieties are selected such that the excitation spectrum of the acceptor fluorescent moiety overlaps the emission spectrum of the excited moiety (donor fluorescent moiety). The donor moiety is excited by light of the appropriate intensity in the excitation spectrum of the donor moiety and emits absorbed energy as fluorescence. When the acceptor fluorescent protein moiety is positioned to suppress the excited donor moiety, the fluorescent energy is transferred to the acceptor moiety, which can emit a second photon. Since the emission spectra of the donor and acceptor moieties have minimal overlap, it is possible to distinguish between the two emissions. Thus, when the acceptor fluoresces at a longer wavelength than the donor, as a result of the final steady state, the emission of the donor is suppressed and the acceptor emits when excited at the absorption maximum of the donor.
[0104]
The detectable or optical signal can be measured, for example, using a fluorimeter (or the like) that detects fluorescence, including fluorescence deflection, time-resolved fluorescence, or FRET. Generally, excitation radiation from an excitation source having a first wavelength generates excitation radiation that excites the sample. In response, the fluorescent compound in the sample emits radiation having a wavelength different from the excitation wavelength. Methods for performing assays on fluorescent materials are widely known in the art, and include, for example, Racowitz (Principles of Fluorescence Spectroscopy, New York, Plenum Press, 1983) and Herman ("Resonance energy profession system, France"). Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor & Wang, San Diego, Academic Press, 1989, pp. 219-2. Examples of fluorescence detection techniques are described in further detail below.
[0105]
In addition, several methods for measuring gene expression using reporter genes have been described in the literature. Nolan et al. Describe a technique for analyzing β-galactosidase expression in mammalian cells. This approach utilizes fluorescein-di-β-D-galactopyranoside (FDG) as a substrate for β-galactosidase, which upon hydrolysis releases fluorescein, a product detectable by fluorescence. (Nolan et al., 1991). Other fluorescent substrates have also been developed, for example, 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12-FDG) (Molecular Probes), which under physiological culture conditions, It differs from FDG in that it is an easily traversable lipophilic fluorescein derivative.
[0106]
The β-galactosidase assay described above can be used, for example, to express a single E. coli expressing recombinant β-D-galactosidase isolated from hyperthermophilic archae such as Sulfolobus solfataricus. It can be used to screen E. coli cells. Other reporter genes may also be useful as substrates and are known for β-glucoronidase, alkaline phosphatase, chloramphenicol acetyltransferase (CAT), and luciferase.
[0107]
Libraries can be screened, for example, for specific enzyme activities. For example, the enzymatic activity screened can be one or more of the six IUB classes of oxidoreductase, transferase, hydrolase, lyase, isomerase, and ligase. Recombinant enzymes that test positive for one or more of the IUB classes can then be rescreened for more specific enzyme activity.
[0108]
Alternatively, the library can be screened for more specialized enzyme activity. For example, instead of screening the library in general for hydrolase activity, the library can be screened for more specialized activity, ie, the type of binding on which the hydrolase acts. Thus, for example, the library may include one or more specific compounds such as (a) amides (peptide bonds), ie, proteases, (b) ester bonds, ie, esterases and lipases, and (c) acetals, ie, glycosidases and others. Can be screened to identify those hydrolases that affect the chemical functionality of the enzyme.
[0109]
As described with respect to one of the above embodiments, the present invention provides a process for screening the activity of clones containing selected DNA from a microorganism, the method comprising:
Screening a library for a given biomolecule or a given biological activity, wherein the library comprises a plurality of clones, which clones recover nucleic acids (such as genomic DNA) from a mixed population of organisms, enriched populations, or isolates thereof And transforming the host with the nucleic acid to generate a clone to be screened for a given biomolecule or biological activity.
[0110]
In another embodiment, an enrichment step can be used prior to activity-based screening. This concentration step can be, for example, a biopanning method. Such a "biopanning" procedure is described and exemplified in U.S. Patent No. 6,054,002, issued April 25, 2000, the disclosure of which is hereby incorporated by reference. Partly.
[0111]
In another embodiment, the polynucleotide is included in a clone, the clone being created from a nucleic acid sequence of a mixed population of organisms, which nucleic acid sequence is used to create a gene library of the mixed population of organisms. Is done. The gene library can be used to transfect a host cell containing the library with at least one nucleic acid sequence having a detectable molecule that is all or part of a DNA sequence encoding a biological activity having the desired activity, e.g., using a fluorescent system. By screening, library clones containing the desired sequence are isolated and screened for a predetermined sequence.
[0112]
The present invention provides the ability to screen for many types of biological activity. For example, the ability to select and combine desirable components from a library of polyketide and post-polyketide biosynthetic genes to produce new polyketides for research can be attractive. The methods of the present invention make this possible, particularly in gene libraries with clones containing large inserts (especially when using vectors that can accept large inserts, such as factor F-based vectors). A new polyketide synthase can be cloned with other relevant pathways or genes encoding secondary metabolites that are commercially relevant, since production is possible, which facilitates the cloning of genes.
[0113]
The biopanning approach described above can be used to generate libraries enriched in clones carrying sequences homologous to certain probe sequences. Using this approach, libraries containing clones with up to 40 kbp of insert can be enriched approximately 1,000-fold after each round of panning. This can reduce the number of clones screened after one round of biopanning enrichment. This approach can be applied, for example, to generate enriched libraries for clones that carry a given sequence associated with a given biological activity, such as a polyketide sequence.
[0114]
Hybridization screening using high density filters or biopanning has proven to be an efficient approach for detecting homologues of pathways involving conserved genes. However, another approach is needed to discover new bioactive molecules for which there may be no known counterparts. In another approach of the invention, the expression of a small molecule ring structure or "backbone" is described in E. coli. Perform screening in E. coli. The gene encoding such a polycyclic structure is E. coli. Because they are often expressed in E. coli, small molecule backbones can be produced, even in an inactive form. Biological activity is conferred upon transfer of the molecule or pathway to an appropriate host that expresses the necessary glycosylation and methylation genes that can modify or "decorate" the structure into its active form. Therefore, the E. coli was recombined. Inactive ring compounds expressed in E. coli are identified to identify the clone and then transport it to a metabolically enriched host, such as Streptomyces, after which it is detected to produce a bioactive molecule. The use of high-throughput robotic systems makes it possible to screen vast numbers of clones in multiple arrays of microtiter dishes.
[0115]
One approach to detecting and enriching clones carrying such a structure, using capillary screening or FACS screening, is described in US application Ser. No. 08 / 876,276, filed Jun. 16, 1997. Explained and illustrated. Polycyclic ring compounds typically have a characteristic fluorescence spectrum when excited by ultraviolet light. Thus, clones expressing these structures can be distinguished from the background using detection methods with sufficient sensitivity. For example, high-throughput FACS screening is described in E.I. It can be used for screening of the E. coli library for the small molecule backbone. Commercially available FACS machines have the ability to screen up to 100,000 clones per second for UV active molecules. Such clones can be further screened by FACS screening, or the stationary plasmid can be extracted and transported to Streptomyces for activity screening.
[0116]
In an alternative screening approach, after transfer to a Streptomyces host, organic extracts from candidate clones can be tested for biological activity by susceptibility screening against a test organism such as Staphylococcus aureus or Saccharomyces cerevisiae.
[0117]
An alternative to the above-described screening method provided by the present invention is an approach called "mixed extraction" screening. The “mixed extraction” screening approach relies on the fact that accessory genes are required to confer activity when the polycyclic backbone is expressed in metabolically rich hosts such as Streptomyces, and the in vitro bioactive compound The enzyme is extracted to produce E. It takes advantage of the fact that it can be combined with a backbone extracted from E. coli clones. At various stages of growth, enzyme-extracted specimens from metabolically-rich hosts such as Streptomyces strains were obtained from E. coli. A pool of organic extracts from the E. coli library is combined and then evaluated for biological activity.
[0118]
E. FIG. Another approach to detecting activity in E. coli clones involves screening for genes that can convert bioactive compounds to different forms. For example, recently, a recombinant enzyme capable of converting low-value daunomycin to high-value doxorubicin has been discovered. A similar enzymatic pathway has been explored to convert penicillin to cephalosporins.
[0119]
Capillary screening can be used, for example, to detect the expression of UV fluorescent molecules in metabolically rich hosts such as Streptomyces. Recombinant oxytetracycline is available from S.A. When produced heterologously in C. lividans TK24, it retains diagnostic red fluorescence. Pathway clones identifiable by the methods and systems of the present invention can therefore be screened for polycyclic molecules in a high-throughput manner.
[0120]
Recombinant bioactive compounds can be screened in vivo using a "two-hybrid" system, which involves the interaction between a transcription factor and its active substance or between a receptor and its cognate target. Certain protein-protein or other interaction enhancers and inhibitors can be detected. In this embodiment, both the small molecule pathway and the GFP reporter construct are co-expressed. Clones that have altered in GFP expression can then be identified, and the clones are isolated for characterization
As noted, a common approach to drug discovery involves screening assays that expose disease targets (macromolecules involved in disease development) to potential drug candidates to be tested for therapeutic activity. In another approach, whole cells or organisms, such as bacteria or tumor cell lines, that are the causative agents of the disease are exposed to potential candidates for screening purposes. Any of these approaches can be utilized in the present invention.
[0121]
The present invention further provides for the clonal pathway from uncultured samples to be metabolically metabolized for heterologous expression and downstream screening for a given bioactive compound using the various screening approaches described briefly above. Can be transferred to a rich host.
[0122]
After screening live or non-viable cells, each containing a different expression clone from the gene library, and collecting the positive clones, DNA can be isolated from the positive clones using techniques well known in the art. The DNA can then be amplified either in vivo or in vitro, using any of the various amplification techniques known in the art. In vivo amplification involves the conversion of the clone (s) or subclone (s) into a host suitable for survival and subsequent growth of the host. In vivo amplification can be performed using techniques such as the polymerase chain reaction. After amplification, the identified sequences can be "evolved" or sequenced.
[0123]
One of the advantages provided by the present invention is the ability to manipulate the identified biomolecule or biological activity to generate and select encoded variants with altered sequence, activity, or specificity. .
[0124]
The screened biomolecules or clones determined to have bioactivity can be subjected to directed mutagenesis to develop new biomolecules or bioactivity with desired properties, or Modified biomolecules or biological activities can be developed that have particularly desirable properties that are absent or not significant in their natural state (such as wild-type activity), such as stability. Any known technique for directed mutagenesis can be applied to the present invention. For example, particularly suitable mutagenesis techniques for use in accordance with the present invention include those described below.
[0125]
Alternatively, when it is desirable to alter the biological molecule (eg, peptide, protein, or polynucleotide sequence) or biological activity (eg, enzymatic activity) obtained, identified, or cloned as described herein. There is. Such changes can modify a biomolecule or biological activity, for example, to increase or decrease the activity, specificity, affinity, function, etc., of the polypeptide. DNA shuffling refers to recombination between sequences that are substantially homologous but not identical, and in some embodiments, DNA shuffling involves non-sequencing, such as through the cer / lox and / or flp / frt systems. It may be accompanied by transfer via homologous recombination (for example, issued to Dr. Jay Short on August 17, 1997, the disclosure of which is incorporated herein by reference, and See U.S. Patent No. 5,939,250 assigned to Corporation). Various methods relating to shuffling, mutating, or altering polynucleotide or polypeptide sequences are described below.
[0126]
Nucleic acid shuffling is a method of homologous recombination of a pool of short or small polynucleotides in vitro or in vivo to produce a polynucleotide or polynucleotides. A mixture of related nucleic acid sequences or polynucleotides is subjected to sexual PCR to provide a random polynucleotide, which is reconstituted to generate a library or mixed population of recombinant hybrid nucleic acid molecules or polynucleotides. In contrast to cassette mutagenesis, shuffling and error prone PCR alone can blindly mutate a sequence pool (without sequence information other than the primers).
[0127]
The advantages of the mutagenized shuffling of the present invention over error-prone PCR alone for repeated selection can best be understood by describing as follows. Consider DNA shuffling compared to error-prone PCR (as opposed to sexual PCR). The initial library of selected or pooled sequences may consist of related sequences of various origins, or may be derived from any type of mutagenesis (including shuffling) of a single gene There is. The set of selected sequences is obtained after the first round of activity selection. Shuffling, for example, allows association of all related sequences in any combination.
[0128]
This method differs from error-prone PCR in that it is a reverse chain reaction. In error-prone PCR, the number of polymerase start sites and the number of molecules increase exponentially. However, the sequence of the polymerase start site and the sequence of the molecule remain essentially the same. In contrast, in rearrangement of nucleic acids or shuffling of random polynucleotides, the number of start sites and the number (but not size) of random polynucleotides decrease over time. For polynucleotides derived from whole plasmids, the theoretical endpoint is a single large concatemer molecule.
[0129]
Recombination mainly occurs between members of the same sequence family because crossovers occur in regions of homology. This prevents combinations of significantly mismatched sequences (eg, having different activities or specificities). It is believed that multiple families of sequences can be shuffled in the same reaction. Furthermore, shuffling generally preserves the relative order.
[0130]
Those that are shuffled will rarely contain a large number of the best molecules (eg, the highest activity or specificity), and those that are rare may be selected based on their superior activity or specificity. it can.
[0131]
The pool of 100 polypeptide sequences is 10³You can sort in different ways. These multiple permutations cannot be represented in a single library of DNA sequences. Therefore, it is contemplated that multiple cycles of DNA shuffling and selection may be required, depending on the desired sequence length and sequence diversity. Error-prone PCR, in contrast, maintains all selected sequences in the same relative orientation, producing a much smaller cluster of mutants.
[0132]
The template polynucleotide that can be used in the method of the present invention can be DNA or RNA. This can be of various lengths depending on the size of the gene or the short or small polynucleotide to be recombined or reconstituted. Preferably, the template polynucleotide is between 50 bp and 50 kb. It is believed that the entire vector containing the nucleic acid encoding a given protein can be used in the methods of the invention and has been successfully used.
[0133]
The template polynucleotide can be obtained by PCR reaction (US Pat. Nos. 4,683,202 and 4,683,195) or amplification using other amplification or cloning methods. However, by removing free primers from the PCR products before pooling the PCR products, sexual PCR can provide more efficient results. If primers are not sufficiently removed from the original pool prior to sexual PCR, the frequency of transfer clones may be low.
[0134]
The template polynucleotide is often double-stranded. Double-stranded nucleic acid molecules are recommended to ensure that the resulting single-stranded polynucleotides are complementary to each other and thus can form double-stranded molecules upon hybridization.
[0135]
It is contemplated that a single or double stranded nucleic acid polynucleotide having a region of identity with the template polynucleotide and a region of heterogeneity with the template template can be added to the template polynucleotide at this step. It is further contemplated that the two related polynucleotide templates can be mixed at this step.
[0136]
The double-stranded polynucleotide template and any additional double- or single-stranded polynucleotides undergo sexual PCR, including slowing or stopping to provide a mixture of about 5 bp to 5 kb or more. Preferably, the size of the random polynucleotide is between about 10 bp and 1000 bp, and more preferably, the size of the polynucleotide is between about 20 bp and 500 bp.
[0137]
Alternatively, it is contemplated that double-stranded nucleic acids having multiple nicks can be used in the methods of the invention. A nick is a break in one strand of a double-stranded nucleic acid. The distance between such nicks is preferably between 5 bp and 5 kb, more preferably between 10 bp and 1000 bp. This can provide, for example, a self-sufficient area that produces short or small polynucleotides included with the polynucleotides generated from random primers.
[0138]
The concentration of any one particular polynucleotide does not exceed 1% by weight of the total polynucleotide, more preferably the concentration of any one particular nucleic acid sequence is 0.1% by weight of the total nucleic acid. % Will not be exceeded.
[0139]
The number of different types of specific polynucleotides in the mixture is at least about 100, preferably at least about 500, and more preferably at least about 1000.
[0140]
In this step, single or double stranded polynucleotides, synthetic or naturally occurring, can be added to random double stranded short or small polynucleotides to increase the heterogeneity of the mixture of polynucleotides. .
[0141]
Furthermore, the population of double-stranded, randomly cut polynucleotides can be mixed or combined with the polynucleotides from the sexual PCR process in this step, optionally involving one or more sexual PCR cycles. It is believed that it can be applied.
[0142]
If insertion of a mutant into the template polynucleotide is desired, a single- or double-stranded polynucleotide having a region of identity with the template polynucleotide and a region of heterogeneity with the template polynucleotide is compared to the total nucleic acid. It is possible to add more than 20 times the weight of the single-stranded polynucleotide, more preferably more than 10 times the weight of the total nucleic acid.
[0143]
If a mixture of different but related template polynucleotides is desired, the populations of polynucleotides from each of the templates are combined in a ratio of less than about 1: 100, and more preferably in a ratio of less than about 1:40. be able to. For example, when it is necessary to backcross a wild-type polynucleotide with a population of mutated polynucleotides to eliminate neutral mutations (mutations that result in intangible alterations in the selected phenotypic trait). There is. In such instances, the ratio of randomly provided wild-type polynucleotide that can be added to the randomly provided sexual PCR cycle hybrid polynucleotide is from about 1: 1 to about 100: 1, and more preferably. Is 1: 1 to 40: 1.
[0144]
The mixed population of random polynucleotides is denatured to form single-stranded polynucleotides and then re-annealed. Only single-stranded polynucleotides having regions of homology to other single-stranded polynucleotides will be reannealed.
[0145]
Random polynucleotides can be denatured by heating. Conditions necessary for completely denaturing the double-stranded nucleic acid can be determined by those skilled in the art. Preferably, this temperature is between 80 ° C and 100 ° C, more preferably, this temperature is between 90 ° C and 96 ° C. Other methods that can be used to denature a polynucleotide include pressure and pH.
[0146]
The polynucleotide can be re-annealed by cooling. Preferably, this temperature is between 20 ° C and 75 ° C, more preferably, this temperature is between 40 ° C and 65 ° C. If frequent transfers are required, based on an average of only four consecutive bases of homology, a lower annealing temperature can be used to force recombination, but this process is more difficult. The degree of regeneration that occurs varies depending on the degree of homology in the population of single-stranded polynucleotides.
[0147]
Regeneration can be accelerated by adding polyethylene glycol ("PEG") or a salt. The salt concentration is preferably 0 mM to 200 mM, and more preferably the salt concentration is 10 mM to 100 mM. This salt can be KCl or NaCl. The concentration of PEG is preferably 0% to 20%, more preferably 5% to 10%.
[0148]
The annealed polynucleotide is then cultured in the presence of a nucleic acid polymerase and dNTPs (ie, dATP, dCTP, dGTP, and dTTP). The nucleic acid polymerase can be Klenow fragment, Taq polymerase, or any other DNA polymerase known in the art.
[0149]
The approach used for this assembly will vary depending on the minimum degree of homology that will cause further transfers. If the same area is large, Taq polymerase can be used at an annealing temperature of 45-65 ° C. If the same area is small, Klenow polymerase can be used at an annealing temperature of 20-30 ° C. One skilled in the art can vary the temperature of the annealing to increase the number of transfers achieved.
[0150]
The polymerase can be added to the random polynucleotide prior to, simultaneously with, or after annealing.
[0151]
The cycle of denaturation, regeneration, and culture in the presence of polymerase is referred to herein as nucleic acid shuffling or reconstitution. This cycle is repeated a desired number of times. Preferably, this cycle is repeated 2 to 50 times, and more preferably, the sequence is repeated 10 to 40 times.
[0152]
The resulting nucleic acid is a large double-stranded polynucleotide of about 50 bp to about 100 kb, preferably, the large polynucleotide is 500 bp to 50 kb.
[0153]
The large polynucleotide can include multiple copies of the polynucleotide having the same size as the template polynucleotide in tandem. The concatemer polynucleotide is then denatured into a single copy of the template polynucleotide. The result is a population of polynucleotides about the same size as the template polynucleotide. This population is a mixed population in which single- or double-stranded polynucleotides having the same area and different areas are added to the template polynucleotide before shuffling. Such polynucleotides are then cloned into the appropriate vector and ligation mixture used to transform bacteria.
[0154]
It is believed that single polynucleotides can be obtained from large concatemer polynucleotides by amplifying the single polynucleotide prior to cloning by various methods, including PCR, rather than by digestion of the concatemer (US Patent No. 4,683,195 and 4,683,202).
[0155]
The vector used for cloning is not critical if it accepts the desired size polynucleotide. If expression of a particular polynucleotide is desired, the cloning vehicle will further include transcription and translation signals adjacent to the site of insertion of the polynucleotide to allow expression of the polynucleotide in a host cell.
[0156]
The resulting bacterial population contains a large number of recombinant polynucleotides with random mutations. This mixed population can be tested to identify the desired recombinant polynucleotide. The method of selection will vary depending on the desired polynucleotide.
[0157]
For example, if a polynucleotide identified by the methods described herein encodes a protein with a first binding affinity, then a subsequently mutated (eg, shuffled) sequence will have increased binding efficiency for the ligand. It is desirable to have. In such cases, the protein expressed by each portion of the polynucleotides in the population or library can be tested for its ability to bind the ligand by methods known in the art (panning, affinity chromatography, etc.). If a polynucleotide encoding a protein with increased drug resistance is desired, the protein expressed by each portion of the polynucleotide in the population or library can be tested for its ability to confer drug resistance to the host organism. Given the knowledge of a desired protein, one of skill in the art can easily test the population to identify polynucleotides that confer the desired properties on the protein.
[0158]
One of skill in the art could use a phage display system that expresses a fragment of the protein as a fusion protein on the phage surface (Pharmacia, Milwaukee, Wis.). The recombinant DNA molecule is cloned in the phage DNA at the site where transcription of the fusion protein occurs, and a portion of the fusion protein is encoded by the recombinant DNA molecule. Phage containing the recombinant nucleic acid molecule are replicated and transcribed within the cell. The fusion protein leader sequence directs delivery of the fusion protein to the tip of the phage particle. Thus, the fusion protein partially encoded by the recombinant DNA molecule is displayed on phage particles for detection and selection by the methods described above.
[0159]
Further, it is contemplated that polypeptides from a sub-population of the first population can perform multiple cycles of nucleic acid shuffling, the sub-population comprising DNA encoding the desired recombinant protein. Thereby, a protein having higher binding affinity or enzymatic activity can be achieved.
[0160]
Additionally, multiple cycles of nucleic acid shuffling may be performed with a mixture of wild-type polynucleotide and a small population of nucleic acids from the first or subsequent rounds of nucleic acid shuffling to remove any silent mutations from the small population. It can be done.
[0161]
Any source of nucleic acid in purified form can be used as starting nucleic acid. Thus, the process can utilize DNA or RNA, including messenger RNA, which can be single-stranded or double-stranded. In addition, DNA-RNA hybrids containing each single strand can be utilized. Nucleic acid sequences can be of various lengths, depending on the size of the nucleic acid sequence to be mutated. Preferably, a particular nucleic acid sequence is between 50 and 50,000 base pairs. It is contemplated that the entire vector containing the nucleic acid encoding the given protein can be used in the methods of the invention.
[0162]
Any particular nucleic acid sequence can be used to generate a population of hybrids by this process. All that is needed is a small population of hybrid sequences of a particular nucleic acid sequence, either present or available in the process.
[0163]
A particular population of nucleic acid sequences having a mutation can be formed by a number of different methods. Mutations can be made by error-prone PCR. Error-prone PCR uses low-fidelity polymerization conditions to randomly introduce low levels of point mutations throughout long sequences. Alternatively, mutations can be introduced into the template polynucleotide by oligonucleotide directed mutagenesis. In oligonucleotide directed mutagenesis, a short sequence of a polynucleotide is removed from the polynucleotide using restriction enzyme digestion, replacing various bases with a synthetic polynucleotide that has been altered from the original sequence. The polynucleotide sequence can be altered by chemical mutagenesis. Chemical mutagens include, for example, sodium bisulfite, nitrite, hydroxylamine, hydrazine, or formic acid. Other agents that are analogs of nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Generally, such agents are added to the PCR reaction at the nucleotide precursor, thereby mutating the sequence. Inserting agents such as proflavine, acriflavine, quinacrine, and others can also be used. Random mutagenesis of the polynucleotide sequence can also be achieved by X-ray or UV irradiation. Generally, the polynucleotide of the plasmid so mutated is E. coli. coli and grown as a pool or library of hybrid plasmids.
[0164]
Alternatively, a small mixed population of a particular nucleic acid can be found in nature, in that it is made up of different alleles of the same gene, or the same gene from different related species (ie, cognate genes). . Alternatively, they can be related DNA sequences found in one species, for example, immunoglobulin genes.
[0165]
After a mixed population of specific nucleic acid sequences has been generated, the polynucleotides can be used directly or inserted into an appropriate cloning vector using techniques well known in the art.
[0166]
The choice of vector will vary depending on the polynucleotide sequence utilized in the method of the invention and the size of the host cell. The template of the present invention may be a plasmid, phage, cosmid, phagemid, virus (eg, retrovirus, parainfluenza virus, herpes virus, reovirus, paramyxovirus, and others), or a selected portion thereof (eg, coat protein, spike Glycoprotein, capsid protein). For example, cosmids and phagemids are preferred when the particular nucleic acid sequence to be mutated is large, since such vectors can stably propagate large polynucleotides.
[0167]
A mixed population of specific nucleic acid sequences can be clonally amplified when cloned in a vector. Utility can be readily determined by screening the expressed polypeptide.
[0168]
The DNA shuffling method of the present invention can be performed blindly on pools of unknown sequences. By adding to the oligonucleotides of the reconstituted mixture (having ends homologous to the sequence to be reconstituted), any sequence mixture can be incorporated into another sequence mixture at any particular position. Thus, a mixture of synthetic oligonucleotides, PCR polynucleotides or even complete genes can be mixed into another sequence library at defined locations. Insertion of one sequence (mixture) is independent of insertion of the sequence in another part of the template. Thus, the degree of recombination, the required homology, and the diversity of the library can be varied independently and simultaneously, along with the length of the DNA to be reconstituted.
[0169]
Shuffling requires the presence of regions of homology that separate regions of diversity. Scaffold-like protein structures may be particularly suitable for shuffling. Conserved scaffolds determine the overall fold by self-association, while exhibiting relatively unrestricted loops that mediate specific binding. Examples of such scaffolds are immunoglobulin β-barrels and four-helix bundles well known in the art. This shuffling can be used to create scaffold-like proteins by combining various combinations of mutated sequences.
[0170]
Equivalents of some standard genetic crosses can also be performed by shuffling in vitro. For example, "molecular backcrossing" can be performed by repeatedly mixing hybrid nucleic acid with wild-type nucleic acid while selecting a given mutation. As with conventional breeding, this approach can be used to combine phenotypes from various sources into a selected background. This is useful, for example, for the removal of neutral mutations affecting unselected characteristics (such as immunogenicity). Thus, this may be useful in determining which mutations in a protein are involved and which are not involved in the enhanced biological activity, and this advantage may be attributed to the error prone It cannot be achieved by mutagenesis or cassette mutagenesis.
[0171]
Large functional genes can be assembled accurately from a mixture of small, random polynucleotides. This reaction can be used to reconstruct genes from highly fragmented fossil DNA. In addition, random nucleic acid sequences from fossils can be combined with polynucleotides from similar genes of related species.
[0172]
Further, it is contemplated that the methods of the present invention can be used for in vitro amplification of the complete genome from a single cell, as required for various research and diagnostic applications. DNA amplification by PCR usually involves a sequence of about 40 kb. E. PCR by PCR. Amplification of a complete genome, such as E. coli (5,000 kb), requires approximately 250 primers that generate 125 40 kb polynucleotides. On the other hand, random production of genomic polynucleotides by sexual PCR cycles, followed by gel purification of small polynucleotides, provides a large number of potential primers. If this random mixture of small polynucleotides is used alone or with the entire genome as a template in a PCR reaction, a single concatemer containing many copies of the genome will be the theoretical endpoint. A reverse chain reaction occurs.
[0173]
When only random nucleotides are used, an amplification of 100 times the copy number and an average polynucleotide size of greater than 50 kb is obtained. Large concatemers are thought to be generated by the duplication of many small polynucleotides. The quality of certain PCR products obtained using synthetic primers is indistinguishable from products obtained from unamplified DNA. This approach is expected to be useful for genome mapping.
[0174]
The shuffled polynucleotides can be generated as random or non-random polynucleotides, at the discretion of the practitioner. In addition, the present invention provides methods of shuffling applicable to a wide range of polynucleotide sizes and types, including the steps of producing polynucleotide monomers used as building blocks in large polynucleotide reconstitution. included. For example, the basic unit can be a fragment of a gene, or can include an entire gene or a gene pathway, or any combination thereof.
[0175]
In an in vivo shuffling embodiment, a mixed population of specific nucleic acid sequences is introduced into a bacterial or eukaryotic cell under conditions such that at least two nucleic acid sequences are present in each host cell. The polynucleotide can be introduced into the host cell in various ways. Host cells can be transformed with small polynucleotides using methods known in the art, for example, treatment with calcium chloride. If the polynucleotide is inserted into the phage genome, the host cell can be transfected with the recombinant phage genome having the particular nucleic acid sequence. Alternatively, the nucleic acid sequence can be introduced into a host cell using electroporation, transfection, lipofection, biolistics, conjugation, and the like.
[0176]
Generally, in this embodiment, the particular nucleic acid sequence will be present in a vector that is capable of stable replication of this sequence in the host cell. In addition, the vector will encode a marker gene so that a host cell having the vector can be selected. This ensures that the particular mutated nucleic acid sequence can be recovered after introduction into the host cell. However, it is not believed that the entire mixed population of a particular nucleic acid sequence need be present on the vector sequence. Rather, after introduction of the polynucleotide into the host cells, it is necessary that each host cell be cloned into a vector in order to ensure that it contains a single vector within which at least one particular nucleic acid sequence is present. , Only a sufficient number of sequences. Furthermore, rather than cloning a subset of a particular population of nucleic acid sequences into a vector, it is likely that this subset may already be stably integrated into the host cell.
[0177]
It has been found that when two polynucleotides having regions of identity are inserted into a host cell, homologous recombination occurs between the two polynucleotides. Such recombination between two particular mutated nucleic acid sequences will, in some circumstances, result in the formation of a double or triple hybrid.
[0178]
Furthermore, it has been found that the recombination frequency increases when a portion of a particular mutated nucleic acid sequence is present on a linear nucleic acid molecule. Thus, in one embodiment, a portion of a particular nucleic acid sequence is on a linear polynucleotide.
[0179]
After transformation, transformants of the host cell are selected for identification of transformants of the host cell that contain the particular mutated nucleic acid sequence having the desired quality. For example, if increased resistance to a particular drug is desired, transformed host cells can be affected by increased concentrations of the particular drug, and mutations that can confer increased drug resistance. The transformant that produced the protein will be selected. If it is desired to increase the ability of a particular protein to bind to the receptor, it is possible to induce expression of the protein from the transformant, and the resulting protein can be converted to a ligand by methods known in the art. Evaluate in a binding assay to identify a subset of the mutant population that shows enhanced binding to the ligand. Alternatively, the protein can be expressed in another system to ensure proper processing.
[0180]
Once identified, a subset of the first recombined specific nucleic acid sequence (daughter sequence) having the desired characteristics is subjected to a second round of recombination. In the second cycle of recombination, the particular recombined nucleic acid sequence can be mixed with the original mutated particular nucleic acid sequence (the parent sequence), and this cycle is performed as described above. Is repeated. In this way, a second set of recombined specific nucleic acid sequences can be identified that have enhanced characteristics or encode proteins with enhanced characteristics. This cycle can be repeated as many times as desired.
[0181]
It is further contemplated that backcrossing can be performed in a second or subsequent recombination cycle. Molecular backcrossing involves mixing a particular nucleic acid sequence with a number of wild-type sequences so that at least one wild-type nucleic acid sequence and the mutated nucleic acid sequence are present in the same host cell after transformation. Can be run with Recombination with a particular nucleic acid sequence of the wild type will eliminate neutral mutations that affect unselected features, such as immunogenicity, but may not affect the selected features.
[0182]
In another embodiment of the present invention, slowing or stopping PCR amplification prior to introduction into a host cell produces a subset of a particular nucleic acid sequence as a small polynucleotide during a first round. It is thought that it is possible. The size of the polynucleotide must be large enough to include some region of identity with the other sequence so that it can recombine homologously with the other sequence. The size of the polynucleotide will range from 0.03 kb to 100 kb, more preferably from 0.2 kb to 10 kb. Further, in subsequent rounds, all of the specific nucleic acid sequences other than those selected from the previous round can be utilized to generate a PCR polynucleotide before introduction into a host cell.
[0183]
Short polynucleotide sequences can be single-stranded or double-stranded. Suitable reaction conditions for separating nucleic acid strands are widely known in the art.
[0184]
The steps of this process can be repeated indefinitely and are limited only by the number of hybrids that can be achieved.
[0185]
Therefore, the first pool or population of mutated template nucleic acids is E. coli. E. coli or the like, and cloned into a vector capable of replicating in bacteria. E. FIG. No particular vector is essential as long as autonomous replication in E. coli is possible. In one embodiment, the vector is designed to allow expression and production of any protein encoded by the particular mutated nucleic acid associated with the vector. Further, preferably, the vector contains a gene encoding a selectable marker.
[0186]
A population of vectors containing a pool of mutated nucleic acid sequences is E. coli. E. coli host cells. The nucleic acid sequence of the vector can be introduced by transformation, transfection, or infection in the case of phage. The concentration of the vector used to transform the bacteria is such that multiple vectors are introduced into each cell. After entering the cell, the efficiency of homologous recombination is such that homologous recombination occurs between the various vectors. This results in a hybrid (daughter) having a different combination of mutations than the original parent mutant sequence. Host cells are then clonally replicated and selected for the marker gene present in the vector. Only cells that have the plasmid will grow for selection. The host cell containing the vector is then tested for the presence of the preferred mutation.
[0187]
After the identification of a particular daughter mutant nucleic acid that confers the desired characteristics, the nucleic acid is isolated either already in association with the vector or away from the vector. The nucleic acid is then mixed with the first or parent population of nucleic acids and the cycle is repeated.
[0188]
The parental mutated population of specific nucleic acids is cloned as a polynucleotide or in the same vector and introduced into a host cell that already contains the daughter nucleic acid. In vivo, recombination is allowed to occur, and next generation recombinants, or granddaughters, are selected by the methods described above. This cycle can be repeated many times until a nucleic acid or peptide with the desired characteristics is obtained. In subsequent cycles, the population of mutated sequences that add to the hybrid may be from the parent hybrid or any sequence generation.
[0189]
In an alternative embodiment, the present invention provides a method of performing a "molecular" backcross of the specific, recombined nucleic acid obtained to eliminate any neutral mutations. Neutral mutations are mutations that do not confer the desired properties on the nucleic acid or peptide. However, such mutations impart undesired characteristics to the nucleic acid or peptide. Therefore, it is desirable to eliminate such neutral mutations. The method of the present invention provides a means to do this.
[0190]
In this embodiment, the nucleic acid, the vector containing the nucleic acid, or the host cell containing the vector and the nucleic acid is isolated after obtaining the hybrid nucleic acid having the desired characteristics by the method of the embodiment.
[0191]
The nucleic acid or vector is then introduced into a host cell that has significantly exceeded wild-type nucleic acid. The hybrid nucleic acid and the wild-type sequence nucleic acid can be recombined. The resulting recombinant is subject to selection, as is a hybrid nucleic acid. Only those recombinants that retain the desired characteristics will be selected. Any silent mutations that do not provide the desired characteristics will be lost through recombination with wild-type DNA. This cycle can be repeated many times until all silent mutations have been eliminated.
[0192]
In another embodiment, the invention provides for shuffling, assembling, and reassembling at least two polynucleotides to form a progeny polynucleotide (eg, a chimeric progeny nucleotide that can be expressed to produce a polypeptide or gene pathway). , Recombination, and / or linearization methods. In certain embodiments, a double-stranded polynucleotide (eg, two single-stranded sequences hybridized to each other as a partner for hybridization) is treated with an exonuclease to release nucleotides from one of the two strands. And leave the remaining strand away from the original partner so that, if desired, the remaining strand can be used to achieve hybridization with another partner.
[0193]
In certain embodiments, the double-stranded polynucleotide terminus (which can be part of or attached to a polynucleotide or non-polynucleotide sequence) is affected by the source of exonuclease activity. An enzyme having 3 'exonuclease activity, an enzyme having 5' exonuclease activity, an enzyme having both 3 'exonuclease activity and 5' exonuclease activity, and any combination thereof are used in the present invention. be able to. Exonucleases can be used to release nucleotides from one or both ends of a linear double-stranded polynucleotide, or from one to all ends of a branched polynucleotide having three or more ends. .
[0194]
In contrast, non-enzymatic steps can be used to shuffle, assemble, reassemble, recombine, and / or linearize the polynucleotide building blocks, which is a process in which a working double-stranded poly Denaturing (or "lysing") conditions are applied to the working sample (e.g., by changing temperature, pH, and / or salinity conditions) so that the set of nucleotides can be dissolved into a single polynucleotide chain. Including giving. With respect to shuffling, it is desirable to involve a single polynucleotide strand in annealing with different hybridization partners to some extent (i.e., simply to have exclusive re-annealing with the original partner before the denaturation step). Not back). However, due to the presence of the original hybridization partner in the reaction vessel, re-annealing of the single-stranded polynucleotide to the original partner does not preclude, and in some cases, re-formation of the original double-stranded nucleotide. It may work in some cases.
[0195]
In contrast to the non-enzymatic shuffling step, which involves denaturing the double-chain polypeptide building blocks and then annealing, the present invention further provides an exonuclease-based approach, which requires denaturation. Conversely, avoiding denaturing conditions and maintaining the double-stranded polynucleotide substrate in annealing (ie, non-denaturing) conditions is critical to the activity of exonucleases (eg, exonuclease III and red alpha gene products). It is a necessary condition. In further contrast, the generation of single-stranded polynucleotide sequences capable of hybridizing with other single-stranded polynucleotide sequences is a consequence of covalent cleavage and consequent disruption of one of the hybridization partners. is there. For example, the exonuclease III enzyme can be used to enzymatically release the 3 ′ terminal nucleotide of one hybridizing strand (to achieve covalent hydrolysis in that polynucleotide strand), Helps the strand hybridize with the new partner (since the original partner has undergone a covalent cleavage).
[0196]
It is particularly understood that enzymes having more optimal rates and / or higher specific activities and / or less unnecessary activities are specifically discovered and optimized for the approach disclosed immediately above. (Eg, created by directed evolution), or both discovery and optimization. Indeed, it is expected that the present invention may facilitate the discovery and / or development of such designer enzymes.
[0197]
It is further understood that the ends of the double-stranded polynucleotide can be protected, if desired, or made susceptible to the desired enzymatic activity of exonucleases. For example, ends of a double-stranded polynucleotide having a 3 'overhang are not affected by exonuclease activity of exonuclease III. However, by various means, it can be made susceptible to exonuclease activity of exonuclease III, for example, blunting by treatment with a polymerase, cleavage to provide blunt ends or 5 'overhangs, Ligating (ligating or hybridizing) to another double stranded polynucleotide to provide a blunt end or 5 'overhang, hybridizing to a single stranded polynucleotide to provide a blunt end or 5' overhang, or various It can be modified by any of the various means.
[0198]
According to one aspect, the exonuclease can act on one or both termini of the linear double-stranded polynucleotide and can proceed to completion, near completion, or partial completion. When exonuclease activity can proceed to completion, the result is that the length of each 5 'overhang increases toward the central region of the polynucleotide by a "rendezvous point" (somewhere near the central point of the polynucleotide). (May be). Ultimately, this produces single-stranded polynucleotides, each of which is about half as long (separable) as the original double-stranded polynucleotide.
[0199]
Thus, exonuclease-mediated approaches are useful for shuffling, assembling and / or rearranging, recombination, and linearizing polynucleotide building blocks. This polynucleotide basic unit is up to ten bases in length, or tens of bases in length, or hundreds of bases in length, or thousands of bases in length, or tens of thousands of bases in length, or hundreds of thousands of bases in length, or It can be millions of bases in length, or longer.
[0200]
Exonuclease substrates can be generated by fragmenting double-stranded polynucleases. Fragmentation can be achieved by mechanical means (eg, shearing, sonication, and others), enzymatic means (eg, using restriction enzymes), and any combination thereof. Large polynucleotide fragments can also be produced by polymerase-mediated synthesis.
[0201]
Additional examples of enzymes having exonuclease activity include red alpha and snake venom phosphodiesterase. The red alpha (redα) gene product (also called λ-exonuclease) is of bacteriophage λ origin. The red alpha gene product acts progressively from the 5'-phosphate end, releasing mononucleotides from double-stranded DNA (Takahashi and Kobayashi, 1990). Snake venom phosphodiesterase (Lascousky, 1980) can rapidly open supercoiled DNA.
[0202]
In one aspect, the invention provides a non-stochastic method called synthetic ligation rearrangement (SLR), which is somewhat related to stochastic shuffling, except that nucleic acid building blocks are randomly shuffled, or Assemble non-stochastically instead of chaining or chimerizing
The SLR method does not rely on the presence of high levels of homology between shuffling polynucleotides. The present invention relates to 10¹⁰⁰It can be used to non-stochastically generate libraries (or sets) of progeny molecules containing more than one type of chimera. Perhaps the SLR is 10¹⁰⁰⁰It can even be used to generate libraries containing more than one progeny chimera.
[0203]
Thus, in one aspect, the present invention provides a non-stochastic method for generating a set of completed chimeric nucleic acid molecules having a deliberately selected overall assembly order, which method is practical and compatible. Deliberately generating a plurality of specific nucleic acid building blocks with ligable ends and assembling such nucleic acid building blocks such that a planned overall assembly sequence is achieved.
[0204]
The interchangeable linkable ends of the nucleic acid building blocks to be assembled are said to be "practical" for this type of ordered assembly if the building blocks can be joined in a predetermined order. You. Thus, in one aspect, the overall assembly order in which the nucleic acid building blocks can be joined is dictated by the design of the ligatable ends, and if more than one assembly step is used, the nucleic acid building blocks are joined together. The overall assembly sequence that can be combined is further specified by the sequential order of the assembly step (s). In one embodiment of the present invention, the annealed building blocks are treated with an enzyme such as a ligase (eg, T4 DNA ligase) to achieve the covalent attachment of the building blocks.
[0205]
In another embodiment, the design of a nucleic acid building block is obtained by analyzing the sequence of a set of precursor nucleic acid templates that serve as a basis for generating a set of progeny of the completed chimeric nucleic acid molecule. Thus, such precursor nucleic acid templates serve as a source of sequence information to assist in mutagenesis, ie, the design of chimeric or shuffled nucleic acid building blocks.
[0206]
In one illustrative form, the invention provides for chimerization of a family of related genes and a coding family of related genes. In certain illustrative forms, the encoded product is an enzyme. As a representative list of a family of enzymes capable of mutagenesis according to aspects of the present invention, the enzymes and their functions that may be mentioned are lipase / esterase, protease, glycosidase / glycosyl, transferase, phosphatase / Kinases, mono / dioxygenases, haloperoxidases, lignins, peroxidases / diallylpropaneperoxidases, epoxide hydrolases, nitrile hydratases / nitrilases, transaminases, amidases / acylases. These illustrative forms exemplify some specific aspects of the invention, but do not represent limitations or limit the scope of the disclosed invention.
[0207]
Thus, according to one aspect of the present invention, the sequences of the plurality of precursor nucleic acid templates identified using the methods of the present invention are aligned to select one or more demarcation points, wherein the demarcation points are: It can be located in the homology area. Boundary points can be used to delineate the resulting nucleic acid building blocks. Thus, the demarcation points identified and selected in the precursor molecule serve as potential chimerization points in the assembly of progeny molecules.
[0208]
Usually, the demarcation point is an area of homology (comprising at least one homologous nucleotide base) shared by at least two pre-templates, but this demarcation point comprises at least half of the pre-template and at least two-thirds of the pre-template And a homology area shared by at least three-quarters of the pre-template and, preferably, almost all of the pre-template. More preferably, the demarcation points are homology areas shared by all precursor templates.
[0209]
In another embodiment, the ligation reconstruction process is performed exhaustively to generate a comprehensive library. In other words, all possible order combinations of the nucleic acid building blocks are represented in the set of completed chimeric nucleic acid molecules. At the same time, the order of assembly in each combination (i.e., the order of assembly of each building block in the 5 'to 3 sequences of each completed chimeric nucleic acid) is deliberate (or non-stochastic). Due to the non-stochastic nature of the present invention, the potential for unwanted by-products is greatly reduced.
[0210]
In yet another embodiment, in the present invention, the ligation reconstruction process is performed systematically, for example, to generate a systematically compartmentalized library, wherein the compartments are, for example, one by one, etc. Can be screened systematically. In other words, the present invention combines the selective and judicious use of a particular nucleic acid building block with the selective and judicious use of successive step-by-step assembly reactions so that a particular set of progeny products can Can achieve the experimental design created by each of the. This makes it possible to carry out a systematic survey and screening procedure. Thus, it is possible to systematically examine a potentially very large number of progeny molecules within a small group.
[0211]
Due to the ability to perform chimerization in a very flexible yet comprehensive and systematic way, especially when the homology between the precursor molecules is at a low level, the present invention provides a library (or Set) generation. Due to the non-stochastic nature of the invention of this ligation rearrangement, the resulting progeny molecules will preferably be part of a library of completed chimeric nucleic acid molecules having a deliberately selected overall assembly order. In certain embodiments, such a generated library comprises 10³More than kind to 10¹⁰⁰⁰Includes more than one progeny molecular species.
[0212]
In one aspect, the set of completed chimeric nucleic acid molecules generated according to the description comprises a polynucleotide encoding a polypeptide. According to one embodiment, the polynucleotide is a gene, which can be an artificial gene. According to another embodiment, the polynucleotide is a genetic pathway, which can be an artificial gene pathway. In the present invention, one or more artificial genes generated according to the present invention can be incorporated into artificial gene pathways such as those that function in eukaryotic organisms (including plants).
[0213]
In another illustrative form, the synthetic nature of the generation of the building blocks makes use of the gene splicing capacity of the host organism (eg, by mutagenesis) or in vivo (eg, by mutagenesis). Then, the design and introduction of nucleotides that can optionally be removed at a later time (eg, one or more nucleotides can be codons or introns or regulatory sequences, etc.) becomes possible. It will be appreciated that in many cases, the introduction of such nucleotides may be desirable for many other reasons, in addition to the potential benefits of forming a demarcation point.
[0214]
Thus, according to another embodiment, the invention allows the use of nucleic acid building blocks to introduce introns. Therefore, in the present invention, a functional intron can be introduced into the artificial gene of the present invention. In the present invention, a functional intron can be further introduced into the artificial gene pathway of the present invention. Accordingly, the invention provides for the generation of a chimeric polynucleotide that is an artificial gene containing one (or more) artificially introduced intron (s).
[0215]
Accordingly, the present invention further provides for the generation of chimeric polynucleotides that are artificial gene pathways containing one (or more) artificially introduced intron (s). Preferably, the artificially introduced intron (s) is capable of functioning in one or more host cells for gene splicing in a manner similar to that of naturally occurring introns in gene splicing. The present invention provides a process for producing an artificial intron-containing polynucleotide that is introduced into a host organism for recombination and / or splicing.
[0216]
An artificial gene generated using the present invention can also serve as a substrate for recombination with another nucleic acid. Similarly, an artificial gene pathway generated using the present invention can also serve as a substrate for recombination with another nucleic acid. In a preferred case, the recombination is facilitated by, and occurs in, an area of homology between the artificial intron containing the gene and the nucleic acid that acts as a recombination partner. In certain preferred cases, the recombinant partner can be a nucleic acid produced according to the present invention, including an artificial gene or an artificial gene pathway. Recombination may be facilitated by and may occur in homologous areas present in one (or more) artificially introduced intron (s) in the artificial gene.
[0217]
The synthetic ligation rearrangement method of the present invention utilizes a plurality of nucleic acid building blocks, each of which preferably has two ligatable ends. The two linkable ends of each nucleic acid building block may be two blunt ends (ie, each having no nucleotide overhang), or, preferably, one blunt end and one overhang, or more preferably, two blunt ends. There can be overhangs.
[0218]
The overhang for this purpose can be a 3 'overhang or a 5' overhang. Thus, a nucleic acid building block can have a 3 'overhang, or alternatively a 5' overhang, or alternatively two 3 'overhangs, or alternatively two 5' overhangs. The overall order in which the nucleic acid building blocks are assembled to form the completed chimeric nucleic acid molecule is not random, but is determined by the desired experimental design.
[0219]
According to one preferred embodiment, the nucleic acid building blocks are contacted such that two single-stranded nucleic acids (also called single-stranded oligos) are chemically synthesized and annealed to form double-stranded nucleic acid building blocks. And causing
[0220]
Double-stranded nucleic acid building blocks can be of various sizes. The size of such basic units can be small or large. Suitable sizes for the basic units range from 1 base pair (not including any overhangs) to 100,000 base pairs (not including any overhangs). Other suitable size ranges are also provided, with a lower limit of 1 bp to 10,000 bp (including all integer values between) and an upper limit of 2 bp to 100,000 bp (including all integer values between). Have.
[0221]
FIG. 6 shows the activity of the enzyme exonuclease III. This is an exemplary enzyme that can be used for shuffling, assembling, reassembling, recombining, and / or linearizing polynucleotide building blocks. The asterisk indicates that the enzyme acts from the 3 'direction to the 5' direction of the polynucleotide substrate. FIG. 7 shows an exemplary application of the enzyme exonuclease III in exonuclease-mediated polynucleotide reconstitution. In addition, transforming a suitable host (Escherichia, Pseudomonas, Streptomyces, or Bacillus) and transforming the clonal mutagenic progeny nucleic acids (and preferably the polypeptides expressed by such nucleic acids) that can be analyzed by expression screening. Figure 4 illustrates the combined use of in vivo "repair" by utilizing host repair mechanisms to generate libraries and provide additional diversity.
[0222]
FIG. 8 shows the generation of a multilinked nucleic acid strand. In this case, the resulting strand is the same length as the parent template, but unmethylated. Thus, the resulting strand can be selected using the Dpn I treatment. Although not shown, the template and the product can be part of a vector (linear or circular). FIG. 9 shows that in the heteromeric nucleic acid complex, the 3 ′ and 5 ′ terminal nucleotides were released from the unhybridized single-stranded ends of the annealed nucleic acid strands, leaving a shortened but hybridized end. 9 illustrates the use of exonuclease treatment as a means of promoting polymerase end extension and / or ligase-mediated ligation of the terminated ends. In addition, transforming a suitable host (Escherichia, Pseudomonas, Streptomyces, or Bacillus) and transforming the clonal mutagenized progeny nucleic acids (and preferably the polypeptides expressed by such nucleic acids) that can be analyzed by expression screening. Figure 4 illustrates the combined use of in vivo "repair" by utilizing host repair mechanisms to generate libraries and provide additional diversity.
[0223]
FIG. 10 shows the use of exonuclease-mediated nucleic acid rearrangement in an example of annealing one methylated multi-linked nucleic acid strand into several unmethylated single-linked nucleic acid strands. The annealed nucleic acid strands form a heteromeric nucleic acid complex, which releases the 3 ′ and 5 ′ terminal nucleotides from the unhybridized single-stranded ends of the multiple annealed nucleic acid strands in the heteromeric nucleic acid complex. However, the treated ends are subjected to exonuclease treatment as a means of promoting polymerase extension and / or ligase-mediated ligation, leaving the hybridized ends. The treatment with Dpn I then becomes practical for the selection of the generated annealed single-bonded nucleic acid strands that are unmethylated and chimeric in nature.
[0224]
FIG. 11 illustrates the use of exonuclease-mediated nucleic acid rearrangement in an example of annealing multiple methylated multi-linked nucleic acid strands into several unmethylated single-linked nucleic acid strands. The annealed nucleic acid strands form a heteromeric nucleic acid complex, which releases the 3 ′ and 5 ′ terminal nucleotides from the unhybridized single-stranded ends of the multiple annealed nucleic acid strands in the heteromeric nucleic acid complex. However, the treated ends are subjected to exonuclease treatment as a means of promoting polymerase extension and / or ligase-mediated ligation, leaving the hybridized ends. The treatment with Dpn I then becomes practical for the selection of the generated annealed single-bonded nucleic acid strands that are unmethylated and chimeric in nature.
[0225]
There are a number of methods by which double-stranded nucleic acid building blocks can be produced that are useful in the present invention and are known in the art and can be readily implemented by those skilled in the art.
[0226]
According to one embodiment, the double-stranded nucleic acid building blocks are generated by first generating two single-stranded nucleic acids and annealing them so that double-stranded nucleic acid building blocks can be generated. Is done. The two strands of the double-stranded nucleic acid building block can be complementary at all nucleotides except for any that form an overhang, thus eliminating mismatches except for any overhang (s). Not included. According to another embodiment, the two strands of the double-stranded nucleic acid building block are complementary in all fewer nucleotides, except for any that form an overhang. Thus, according to this embodiment, double-stranded nucleic acid building blocks can be used to introduce codon degeneracy. Preferably, codon degeneracy is introduced using site saturation mutagenesis as described herein, which uses one or more N, N, G / T cassettes, or alternatively, One or more N, N, N cassettes are used.
[0227]
FIG. 1 illustrates a preferred embodiment of the site saturation mutagenesis described herein. Nucleic acid sequences that can be mutated by non-stochastic site saturation mutagenesis include any polynucleotide between 15 and 100,000 bases in length (eg, any coding region and any non-coding region). Including. For example, such nucleic acid sequences may include, for example, promoters, enhancers, introns, whole genes, cDNAs, gene segments, operators, polynucleotide functional groups, expression control sequences, expression sequence tags (ESTs), open reading frames (ORFs), repressors / Trans-acting factor, origin of replication, or genetic pathway.
[0228]
Mutagenic cassettes that can be mutagenized by non-stochastic abrupt mutagenesis include any polynucleotide cassette from 1 to 500 bases in length. Site saturation mutagenesis is practical for mutagenizing the complete set of cassettes contained within the polynucleotide sequence to be mutagenized. As shown in FIG. 1, the cassettes can be variously spaced (ie, immediately adjacent, evenly or unevenly spaced) along each polynucleotide and can be of any size Can be. In a preferred, non-limiting illustrative form, the set of mutagenic cassettes is a set of contiguous codons within a sequence of defined length. Alternatively, in another preferred, non-limiting example, the set of mutagenic cassettes is a set of nucleotide cassettes that are not shared by the related polynucleotides in alignment.
[0229]
The type of mutation introduced into the set of mutagenic cassettes can be of the same or different types in each round of polynucleotide site saturation mutagenesis. Each mutagenic cassette (within the nucleic acid sequence to be mutagenized) is preferably usually mutagenized (including with degenerate oligos) by using the corresponding oligo. Examples of degenerate mutations provided by the present invention include:
[0230]
Codons for all 20 amino acids (eg, N, N, N or N, N, G / T or N, N, G / C)
All degenerate codons that do not change the amino acid sequence of the parent template (ie, codons for the same amino acid present in the parent template)
Selected amino acid grouping system^*(All or selected) codons for amino acids in the same group according to
Each amino acid group^*Codon for at least one amino acid in
1. Aromatic (Phe, Trp, Tyr)
2. Aliphatic (Gly, Ala, Val, Leu, Ile)
3. OH content (Ser, Tyr, Thr)
4. Acidic (Asp, Glu, Asn, Gin)
5. Basic (Lys, Arg, His)
6. Sulfur-containing (Met, Cys)
7. Polarity (Ser, Thr, Cys, Asn, Gin, Tyr)
8. Non-polar (Gly, Ala, Vai, Leu, Ile, Met, Phe, Trp, Pro)
[0231]
FIG. 2 illustrates the power of the present invention in its ability to generate all possible combinations of nucleic acid building blocks designed in this example in an exhaustive and systematic manner. In particular, large sets (or libraries) of progeny chimeric cells can be generated. This method can be performed comprehensively and systematically, so that iterative methods can be applied by selecting new boundary points, and correspondingly newly designed nucleic acid building blocks have previously been investigated and The burden of regenerating and rescreening rejected molecular species is avoided. It is understood that codon fluctuations can be used to advantage in increasing the frequency of boundary points. In other words, a particular base can often be replaced in a nucleic acid building block without changing the amino acid encoded by the precursor codon (altered codon) due to codon degeneracy. As illustrated, the boundary points are selected with the eight precursor templates aligned. Thereafter, nucleic acid building blocks containing overhangs (practical for the generation of ordered bonds) are designed and synthesized. In this case, 18 nucleic acid building blocks are generated based on the sequence of each of the eight precursor templates, for a total of 144 nucleic acid building blocks (or double-stranded oligos). Then, by performing the ligation reconstitution procedure, a library of progeny molecules containing 818 (or more than 1.8 x 1016) chimera production will be generated.
[0232]
The in vitro recombination method of the invention can be performed blindly on a pool of unknown hybrids or alleles of a particular polynucleotide or sequence. However, it is necessary that the actual DNA or RNA sequence of a particular polynucleotide be known.
[0233]
An approach that uses recombination within a mixed population of genes can help generate any useful protein, for example, interleukin I, antibodies, tPA, and growth hormone. This approach can be used to generate proteins with altered specificity or activity. This approach can also be useful in generating hybrid nucleic acid sequences, for example, promoter regions, introns, exons, enhancer sequences, 31 untranslated regions, or 51 untranslated regions of a gene. Thus, this approach can be used to generate genes with increased rates of expression. This approach can also be useful in the study of repetitive DNA sequences. Finally, this approach can help mutate ribozymes or aptamers.
[0234]
The present invention provides a method for selecting a subset of polynucleotides from a starting set of polynucleotides, the method comprising selecting for each selectable polynucleotide (positive selection) and / or selecting against it (negative selection). Is performed based on the ability to identify one or more selectable features (or selectable markers) present somewhere in the working polynucleotide. In one aspect, the provided method is referred to as end selection, which is based on the use of selectable markers located in part or all in the terminal region of the selectable polynucleotide, such selectable markers comprising "terminal Sometimes referred to as a "selection marker."
[0235]
End selection can be based on the detection of naturally occurring sequences, or based on the detection of experimentally introduced sequences (including by any mutagenesis described herein and not described). Or even within the same polynucleotide, based on both of these. The end selection marker can be a structural or functional selection marker, or a structural and functional selection marker. The terminal selectable marker can include a polynucleotide sequence, or polypeptide sequence, or any chemical structure, or any biological or biochemical tag, including radioactivity, enzymatic activity, fluorescence, Includes optional optical features, magnetic properties (such as the use of magnetic beads), immunoreactivity, and markers selectable using methods based on detection of hybridization.
[0236]
End selection can be applied in combination with any method that performs mutagenesis. Such mutagenesis methods include, in part, the methods described herein (above and below). Such methods include, by way of non-limiting illustration, any method that may be referred to herein or by one of skill in the art by any of the following terms: “saturation mutation”, “shuffling”, “recombination” "Reconstruction", "error-prone PCR", "assembly PCR", "sexual PCR", "crossover PCR", "oligonucleotide primer-directed mutagenesis", "recursive (and / or exponential) ensemble mutation" Production (see Arkin and Uban, 1992) "," Cassette Mutagenesis "," In Vivo Mutagenesis ", and" In Vitro Mutagenesis ". In addition, end selection can be performed on molecules generated by any mutagenesis and / or amplification method (see Arnold, 1993, Caldwell and Joyce, 1992, Stema, 1994, etc.) Following, it is desirable to select the desired progeny molecule (including screening for its presence).
[0237]
In addition, end selection can be applied to polynucleotides unrelated to any mutagenesis method. In one embodiment, end selection can be used to facilitate a cloning step, such as a step of ligation with another polynucleotide (including ligation with a vector), as defined herein. Thus, the present invention provides end selection as a means to facilitate library construction, selection, and / or enrichment, and general cloning, of desired polynucleotides.
[0238]
In another embodiment, the end selection can be based on a (positive) selection for the polynucleotide; alternatively, the end selection can be based on a (negative) selection for the polynucleotide. Alternatively, the terminal selection can be based on both (positive) and (negative) selection for the polynucleotide. End selection can be performed in an iterative manner, along with other selection and / or screening methods, in which similar or dissimilar selection and / or screening methods and mutagenesis or directed evolution methods are optional. , All of which can be performed in an iterative manner, in any order, combination, and permutation. In addition, end selection can be circular (eg, a plasmid or any other circular vector, or any other polynucleotide that is partially circular), and / or branched, and / or modified or replaced by any chemical group or moiety. It can also be used to select polynucleotides in a state.
[0239]
In one non-limiting embodiment, the end-selection of the linear polynucleotide is performed by using at least one end-selection marker located at or near the end of the polynucleotide (which can be 5 'or 3'). Is performed using a general approach based on the presence of In one specific, non-limiting, illustrative form, end selection is based on the selection of a particular sequence at or near the end, including those recognized by enzymes that recognize polynucleotide sequences. Enzymes that recognize and catalyze chemical modifications of polynucleotides are referred to herein as polynucleotide acting enzymes. In a preferred embodiment, the polynucleotide acting enzyme is an enzyme having a polynucleotide cleavage activity, an enzyme having a polynucleotide methylation activity, an enzyme having a polynucleotide ligation activity, and a plurality of distinguishable enzyme activities (for example, (Exclusively including polynucleotide cleavage activity and polynucleotide ligation activity).
[0240]
Related polynucleotide acting enzymes include any enzymes known to those of skill in the art (e.g., commercially available), or are currently not available to help generate ligation compatible ends, preferably cohesive ends, in the polynucleotide. Is understood to include those that may be developed in the future. Use a restriction site that is not, is not expected to be, or is unlikely to be contained within the polynucleotide to be end-selected (eg, when the sequence information for the working polynucleotide is incomplete). May be preferred. It is recognized that methods (eg, mutagenesis methods) can be used to remove unnecessary internal restriction sites. In addition, partial digestion reactions (i.e., digestion reactions that go to partial completion) are used to achieve digestion at the recognition site in the terminal region to produce susceptibility generated within the polynucleotide and recognized by the same enzyme. It is understood that it is possible to leave certain restriction sites. In one aspect, partial digestion is useful because, depending on the location and environment in which the recognition sequence occurs, it is understood that certain enzymes will exhibit preferential cleavage of the same recognition sequence.
[0241]
In addition, the use of protection methods to selectively protect certain restriction sites (such as internal sites) from unnecessary digestion by enzymes that cut the working polypeptide in response to the presence of such sites. Such protection methods include modifications such as methylation and base substitution (eg, replacing T with U) to suppress unwanted enzyme activity.
[0242]
In another embodiment of the invention, a useful end selectable marker is an end sequence recognized by a polynucleotide acting enzyme that recognizes a particular polynucleotide sequence. In one aspect of the invention, useful polynucleotide acting enzymes further include other enzymes in addition to the standard type II restriction enzymes. According to this preferred aspect of the invention, useful polynucleotide acting enzymes further include gyrase (such as topoisomerase), helicase, recombinant enzymes, relaxase, and any enzymes related thereto.
[0243]
It is understood that end selection can be used to distinguish and separate parent template molecules (such as those that have undergone mutagenesis) from progeny molecules (such as those generated by mutagenesis). For example, a first set of primers lacking a topoisomerase I recognition site can be used to modify the terminal region of the parent molecule (eg, in a polymerase-based amplification). A second set of different primers (such as those having a topoisomerase I recognition site) can then be used to generate mutated progeny molecules from the amplified parent molecule (eg, intermittent synthesis, template switching polymerase). Using any polynucleotide chimerization method, such as base amplification, or intermittent synthesis, or using a saturation mutation, or any other method that introduces a topoisomerase I recognition site into the mutated progeny molecule. By using.) The use of topoisomerase I-based end selection then facilitates selective topoisomerase I-based ligation as well as identification of the desired progeny molecule.
[0244]
It is understood that the end selection approach using topoisomerase-based nicking and ligation has several advantages over previously available selection methods. In short, with this approach, directional cloning (including expression cloning) can be achieved.
[0245]
FIG. 3 illustrates the application of end-selection using a topoisomerase-induced selection step to select a desired polynucleotide (eg, to select a full-length molecule) generated by polynucleotide rearrangement (eg, by synthetic ligation rearrangement). FIG. FIG. 4 illustrates various aspects of the end selection technique. This includes a method of end selection in in vitro rearranged genes, which includes the following steps: a) amplifying a given gene with a gene-specific primer template: chromosomal DNA / λ clone / genome Clone / optional subclone DNA, b) Performing recombinant PCR: add general topo cloning primer to mixed PCR product from homologous gene, PCR can be performed, for example, at 5 min 94 ° C, 80 x (30 sec 94 C) performing directed expression cloning: gel-purifying the reconstituted PCR product and binding topoisomerase to vaccinia virus (optimal molar ratio is DNA: enzyme). , 1: 5), add vector (double the amount of insertion), and transformants are screened. FIG. 5 illustrates the use of the enzyme N.I. to introduce "sticky ends" into the original blunt-ended DNA in order to achieve directed expression cloning of the original blunt-ended DNA. 9 illustrates a particular embodiment of the end-selection technique in which the step of "c) performing directed expression cloning" is accomplished using BstNBl.
[0246]
The method can be used to shuffle a polynucleotide sequence selected by a peptide display method by in vitro and / or in vivo recombination with any disclosed method and any combination thereof, wherein The relevant polynucleotide encodes a display peptide that has been screened for a phenotype (such as affinity for a given receptor (ligand)).
[0247]
An aspect of increasing importance in biopharmaceutical drug development and molecular biology is the identification of peptide structures that include the primary amino acid sequence of peptides or peptidomimetics that interact with biological macromolecules. One method of identifying a peptide that possesses a desired structural or functional property, such as binding to a defined biological macromolecule (such as a receptor), involves individualized possession of the desired structural or functional property provided by the amino acid sequence of the peptide. Screening large libraries or peptides for library components is involved.
[0248]
In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have also been reported. One type involves the display of peptide sequences, antibodies, or other proteins on the surface of bacteriophage particles or cells. In general, in such methods, each bacteriophage particle or cell serves as a separate library member that displays a single species of display peptide in addition to the native bacteriophage or cellular protein sequence. Each bacteriophage or cell contains nucleotide sequence information that encodes a particular display peptide sequence, and thus the display peptide sequence can be ascertained by determining the nucleic acid sequence of the separated library components.
[0249]
Widely known peptide display methods involve the display of peptide sequences on the surface of filamentous bacteriophage, usually as a fusion with a bacteriophage coat protein. This bacteriophage library can be cultured with a fixed macromolecule or a small molecule (such as a receptor) immobilized, and bacteriophage particles displaying a peptide sequence that binds to the immobilized macromolecule can be cultured with the predetermined macromolecule. Can be distinguished from those that do not display peptide sequences that bind to The bacteriophage particles (ie, library components) that bind to the immobilized macromolecule are then recovered and replicated, amplifying the selected bacteriophage subpopulation for subsequent rounds of affinity enrichment and phage replication. can do. After several rounds of affinity enrichment and phage replication, the bacteriophage library components selected in this way are separated and the nucleotide sequence encoding the display peptide sequence is determined, whereby the defined macromolecules (such as receptors) )) Are identified. Such methods are further described in PCT Patent Publications WO 91/17271, WO 91/18980, WO 91/19818, and WO 93/08278.
[0250]
The present invention further provides peptide libraries of random, pseudorandom, and defined sequence frameworks and useful compounds (single-chain antibodies) that bind to a given receptor molecule or epitope or a gene product that modifies a peptide or RNA in a desired manner. And methods for generating and screening such libraries to identify peptides comprising Random, pseudorandom, and predefined sequence framework peptides are generated from a library of peptide library components, including display peptides or display single-chain antibodies, that attach the display peptide to the synthesized polynucleotide template. The form of attachment can vary depending on the particular embodiment of the invention chosen, and can include encapsulation within phage particles or uptake within cells.
[0251]
A great advantage of the present invention is that no prior information about the expected ligand structure is required to separate a given peptide ligand or antibody. The peptide identified can have biological activity, which means that it contains at least a specific binding affinity for the selected receptor molecule, and the ability to block the binding of other compounds And the ability to stimulate or inhibit metabolic pathways, the ability to act as a signal or messenger, the ability to stimulate or inhibit cell activity, and others.
[0252]
The invention further relates to heterodimers identified by the methods of the invention and binding to defined receptors (such as mammalian proteinaceous receptors, such as peptide hormone receptors, cell surface receptors, other protein (s)). Nascent peptides (single-chain antibodies) for library components that bind to intracellular proteins that form intracellular protein complexes, such as, etc., or epitopes (immobilized proteins, glycoproteins, oligosaccharides, etc.) Methods of Shuffling a Pool of Selected Polynucleotide Sequences by Affinity Screening a Library of Polysomes That Display
[0253]
The polynucleotide sequences selected in the first round of selection (usually by affinity selection for binding to the receptor (ligand)) by any of these methods are pooled, and the pool (s) are pooled in vitro and Alternatively, shuffled by in vivo recombination to produce a shuffled pool containing a population of recombinantly selected polynucleotide sequences. The recombined selection polynucleotide sequence is subjected to at least one post selection round. Polynucleotide sequences selected in the round (s) of post-selection can be used directly for the sequence, and / or can be subjected to shuffling and post-selection in one or more additional rounds. The selected sequence can also be backcrossed with a polynucleotide sequence that encodes a native sequence (ie, one that has no substantial functional effect on binding), for example, a native sequence that may be less immunogenic. Can be backcrossed with a wild-type or naturally occurring sequence that is substantially identical to the selected sequence. Generally, during backcrossing, post-selection is applied to maintain the binding properties with a given receptor (ligand).
[0254]
The sequence can be mutagenized before or simultaneously with shuffling of the selected sequence. In one embodiment, the selected library components are cloned in a prokaryotic vector (eg, a plasmid, phagemid, or bacteriophage), which produces a collection of individual colonies (or plaques) that represent the individual library components. Is done. The individual selected library components can then be manipulated to generate a set of library components that represent a core of sequence diversity based on the sequence of the selected library components (eg, site-directed mutations). Generation, cassette mutagenesis, chemical mutagenesis, PCR mutagenesis, and others). The sequences of the individual selected library members or pools can be random mutations, pseudo-random mutations, and defined core mutations (ie, including mutation and invariant residue positions, and / or defined amino acid residues). Segmented or over the entire length of the individual selected library component sequence to incorporate the variant residue positions including residues selected from the subset), codon-based mutations, and others. , Can be operated. The mutagenized selected library components are then shuffled by the in vitro and / or in vivo recombinant shuffling disclosed herein.
[0255]
The invention further provides a peptide library comprising a plurality of individual library components of the invention, wherein (1) each individual library component in said plurality shuffles a pool of selected sequences. (2) each individual library component is a variable peptide segment sequence or a variable peptide segment sequence or a single chain antibody segment sequence that is distinguishable from the variable peptide segment sequence or single chain antibody segment sequence of the other individual library components in the plurality. Contains single-chain antibody segment sequences (although some library components may be present in more than one copy per library due to unequal amplification, stochastic potential, or otherwise) .
[0256]
The present invention further provides a product-by-process, wherein a selected polynucleotide sequence having a defined binding specificity (or encoding a peptide having the same) is formed by the following process. : (1) To screen a display peptide or display single-chain antibody library for a given receptor (eg, ligand) or epitope (eg, antigenic macromolecule), and to generate a pool of selected library components, Identifying and / or enriching a library component that binds to a receptor or epitope of (2) a selected library component that binds to a predetermined epitope, thereby being separated and / or enriched from the library (or amplified or cloned thereof) Copy), shuffled library Shuffling recombinantly to produce; (3) shuffling the shuffled library against a given receptor (eg, ligand) or epitope (eg, antigenic macromolecule); and selecting a pool of shuffled library components. Identifying and / or enriching shuffled library components that bind to a given receptor or epitope to generate.
[0257]
The method can be used to shuffle a polynucleotide sequence selected by an antibody display method by in vitro and / or in vivo recombination with any disclosed method and any combination thereof, wherein Related polynucleotides encode display antibodies that have been screened for phenotype, such as affinity for binding to a given antigen (ligand).
[0258]
Various molecular genetic approaches have been devised to capture the vast immunological repertoire represented by the large number of distinct variable regions that may be present in the immunoglobulin chains. The naturally occurring germline immunoglobulin heavy chain locus contains an isolated tandem sequence of the variable segment gene, which is located upstream of the tandem sequence of the diversity segment gene, and is itself a tandem sequence of the junctional (i) region gene. Which is located upstream of the constant region gene. During B lymphocyte development, a VDJ rearrangement occurs in which rearrangements forming the fusion D segment form the heavy chain variable region gene (VH), followed by rearrangement of the V segment. Form a VDJ conjugation product gene, which, when productively rearranged, encodes a heavy chain functional variable region (VH). Similarly, the light chain locus rearranges one of several V segments with one of several J segments to form a gene encoding the variable region (LV) of the light chain.
[0259]
The vast repertoire of variable regions possible in immunoglobulins is due, in part, to the myriad possible combinations of joining V and i segments (and, in the case of heavy chain loci, D segments) during rearrangement in B cell development. Derived from sex. Additional sequence diversity in the heavy chain variable region results from heterogeneous rearrangement of the D segments during the VDJ junction and the addition of N regions. In addition, antigen selection of identified B cell clones selects for high affinity variants with non-germline mutations in one or both of the heavy and light chain variant regions, a phenomenon which is referred to as "affinity mutations". Or, it is called “affinity sharpening”. Usually, such "affinity sharpening" mutations are concentrated in specific areas of the variable region, most commonly in the complementarity determining regions (CDRs).
[0260]
To overcome a number of limitations in producing and identifying high affinity immunoglobulins through the generation of antigen-stimulated β cells (ie, immunization), various prokaryotic expression systems have been developed, It can be manipulated to generate a combinatorial antibody library that can screen for high affinity antibodies to the antigen. With recent advances in the expression of antibodies in Escherichia coli and bacteriophage systems (see "Alternate Peptide Display Methods" below), cloning antibody genes from characterized hybridomas, or using antibody gene libraries (eg, from Ig cDNAs) ) Increased the possibility of obtaining virtually any specificity.
[0261]
Combinatorial libraries of antibodies have been generated in bacteriophage lambda expression systems that can be screened as bacteriophage plaques or as lysogen colonies (Hughes et al., 1989, Caton and Koplowski, 1990, Marinax et al., 1990, Person et al. 1991). Various embodiments of bacteriophage antibody display libraries and phage lambda expression libraries have been described (Kang et al., 1991; Crackson et al., 1991; McCaferty et al., 1990; Burton et al., 1991; Hugenboom et al., 1991; 1991, Breitling et al., 1991, Marks et al., 1991, p581, Barbus et al., 1992, Hawkins and Winter, 1992, Marks et al., 1992, p779, Marks et al., 1992, p16007, and Roman et al., 1991, Lana et al. 1992, all of which are hereby incorporated by reference. Typically, bacteriophage antibody display libraries contain immobilized (eg, by covalent attachment to a chromatography resin for enriching reactive phage by affinity chromatography) and / or labeled (screening for plaque or colony lift). (Polypeptides, carbohydrates, glycoproteins, nucleic acids, etc.).
[0262]
One particular advantageous approach has been the use of so-called single-chain variable fragment (scfv) libraries (Marks et al., 1992, p779, Winter and Milstein, 1991, Cracson et al., 1991, Marks et al., 1991, p581. Chodoli et al., 1990; Chiswell et al., 1992; McCaferty et al., 1990; and Houston et al., 1998). Various embodiments of a scfv library displayed with bacteriophage coat proteins have been described.
[0263]
Since 1988, single-chain analogs and their fusion proteins have been reliably produced by antibody engineering methods. The first step usually involves obtaining genes encoding VH and VL domains with the desired binding properties, such V genes being selected from a combinatorial V gene library or generated by V gene synthesis. Can be isolated from certain hybridoma cell lines. The single-chain Fv encodes the component V gene with a properly designed linker peptide such as (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3)) or equivalent linker peptide (s). It is formed by connecting to an oligonucleotide to be converted. The linker bridges the C-terminus of the first V region and the N-terminus of the second, in the order of VH-linker-VL or VL-linker-VH '. In principle, the scfv binding site can faithfully replicate both the affinity and specificity of the parent antibody binding site.
[0264]
Thus, scfv fragments contain VH and VL domains linked by a flexible linker peptide to a single polypeptide chain. After the scfv genes have been assembled, they are cloned on a phagemid and expressed at the tip of M13 phage (or similar filamentous bacteriophage) as a fusion protein with a bacteriophage PIII (gene 3) coat protein. Enrichment of phage expressing a given antibody is achieved by panning recombinant phage displaying the scfv of a population for binding to a defined epitope (eg, target antigen, receptor).
[0265]
The linked polynucleotides of the library components provide a basis for replicating the library components after the screening or selection procedure, and also for determining the identity of the display peptide sequence or VH or VL amino acid sequence by nucleotide sequence. Provide the foundation. The display peptide (s) or single chain antibodies (such as scfv), and / or their VH and VL domains or their CDRs can be cloned and expressed in a suitable expression system. Polynucleotides encoding the separated VH and VL domains are linked to polynucleotides encoding the constant regions (CH and CL), and are used to encode whole antibodies (eg, chimeric or fully human), antibody fragments, and the like. Often, a polynucleotide to be encoded is formed. The polynucleotide encoding the separated CDRs can be grafted onto a polynucleotide encoding the appropriate variable region framework (and, optionally, the constant region), and a fully antibody (eg, humanized or fully human), Often, polynucleotides encoding antibody fragments, and others, are formed. Antibodies can be used to separate antigen reserves by immunoaffinity chromatography. Various uses for such antibodies include diagnosis and / or staging of diseases (such as tumorigenesis) and treatment of diseases such as, for example, tumorigenesis, autoimmune diseases, AIDS, heart disease, infections, and others. There are therapeutic uses for
[0266]
Various reports have been reported to increase the combinatorial diversity of scfv libraries and broaden the repertoire of binding species (idiotypic spectra). The use of PCR allows for the rapid cloning of variable regions from specific hybridoma sources or as a gene library from non-immunized cells, providing a combinatorial diversity for assortment of combinatorial VH and VL cassettes. Furthermore, VH and VL cassettes can diversify themselves by random, pseudo-random, or defined mutagenesis. Usually, the VH and VL cassettes are diversified at or near the complementarity determining region (CDRs), which is often the third CDR, CDR3. Enzymatic inverse PCR mutagenesis, like error-prone PCR and chemical mutagenesis (Den et al., 1994), should be a simple and reliable method of constructing a relatively large library of scfv site-directed hybrids Is shown (Stema et al., 1993). Leachman (Leachman et al., 1993) showed a semi-rational design of antibody scfv fragments using site-directed randomization by degenerate oligonucleotide PCR followed by phage display of the resulting scfv hybrid. Barbas (Barbas et al., 1992) randomized the sequence in the synthetic CDR regions of the human tetanus toxoid-binding Fab to avoid the problem of limited repertoire resizing resulting from the use of biased variable region sequences. Tried.
[0267]
CDR randomization is approximately 1 × 10 for heavy chain CDR3 only.²⁰It has the potential to form CDRs, approximately the same number of variants of heavy chain CDR1 and CDR2, and light chain CDR1 to 3 variants. When considered individually or together, the combinatorial possibility of CDR randomization of heavy and / or light chains generates an inordinate number of bacteriophage clones to generate a clone library that represents all possible combinations And most of the combinations are non-binding. The generation of such a large number of primary transformants is not feasible with current transformation techniques and bacteriophage display systems. For example, Barbas (Barbas et al., 1992) is 5 × 10⁷, Which represents only a small fraction of the potential diversity of a library of fully randomized CDRs.
[0268]
Despite these major limitations, bacteriophage display of scfv has already produced a variety of useful antibodies and antibody fusion proteins. Bispecific single-chain antibodies have been shown to mediate efficient tumor cell lysis (Gruba et al., 1994). Intracellular expression of anti-Rev scfv has been shown to suppress HIV-1 virus replication in vitro (Duan et al., 1994), and intracellular expression of anti-p2llar scfv was reduced in Xenopus oocytes. It has been shown to inhibit mitotic maturation (Biokka et al., 1993). Recombinant scfv that can be used to diagnose HIV infection has also been reported, demonstrating its importance in the diagnosis of scfv (Lilly et al., 1994). Fusion proteins where the scfv is linked to a second polypeptide, such as a toxin or fibrinolytic activator protein, have also been reported (Horbost et al., 1992; Nicholas et al., 1993).
[0269]
Given that it is possible to generate scfv libraries that have a wide range of antibody diversity and overcome many of the limitations of conventional CDR mutations and randomization that can cover only a very small fraction of potential sequence combinations The number and quality of scfv antibodies suitable for therapeutic and diagnostic applications is greatly improved. To address this, the CDRs obtained (usually via PCR amplification or cloning) from nucleic acids obtained from a selected display antibody using the in vitro and in vivo shuffling methods of the present invention are recombined. You. Such display antibodies can be displayed on cells, on bacteriophage particles, on polysomes, or in any suitable antibody display system in which the antibody relates to the encoding nucleic acid (s). As a mutation, the CDRs may be derived from plasma from immunized wild-type mice, humans, or transgenic mice capable of forming human antibodies, such as those found in antibody producing cells (eg, WO 92/03918, WO 93/12227, and WO 94/25585). Cells / splenocytes) from the mRNA (or cDNA), including the hybridomas derived therefrom.
[0270]
Polynucleotide sequences selected in a first round of selection (usually by affinity selection for display antibodies that bind to an antigen (such as a ligand)) by any of these methods are pooled, and the pool (s) are pooled. , In vitro and / or in vivo recombination, especially shuffling CDRs (usually shuffling heavy chain CDRs with other heavy chain CDRs and shuffling light chain CDRs with other light chain CDRs) and recombining A shuffled pool containing a population of the selected polynucleotide sequences is generated. The recombinant selection polynucleotide sequence is expressed in a selection format as a display antibody and subjected to at least one post-selection land. Polynucleotides selected in the post-selection round (s) can be sequenced directly and / or undergo one or more additional rounds of shuffling and post-selection until the desired binding affinity is obtained. be able to. The selected sequence can also be backcrossed with a polynucleotide sequence encoding a neutral antibody framework sequence (ie, one that has no substantial effect on antigen binding), for example, a human-like sequence antibody. It can be backcrossed with human variable regions to produce. Generally, during backcrossing, post-selection is applied to maintain the binding properties to the defined antigen.
[0271]
Alternatively or in combination with the described mutations, the valency of the target epitope can be altered to control the average binding affinity of the selected scfv library component. The target epitope can be bound to the surface or substrate at various concentrations, such as by including, diluting, or other methods known to those of skill in the art. High concentrations (valency) of a given epitope can be used to enrich scfv library components with relatively low affinity, whereas low concentrations (valency) can be used for high affinity scfv library The components can be preferentially concentrated.
[0272]
Insertion of a set of synthetic oligonucleotides encoding random, pseudo-random, or defined sequence core sets of peptide sequences into defined sites (such as CDRs) by ligation to generate diverse variable segments Can be. Similarly, extending the sequence diversity of one or more CDR (s) of the single chain antibody cassette (s) by site-directed mutagenesis, CDR substitution, and mutating the CDR (s) by other means. Can be. The resulting DNA molecule can be propagated in a host for cloning and amplification prior to shuffling, or can be used directly (i.e., the potential for occurring upon growth in a host cell). The selected library components are then shuffled (a certain loss of diversity can be avoided).
[0273]
Display peptide / polynucleotide complexes (library components) encoding predetermined various segmented peptide sequences or predetermined single chain antibodies are selected from the library by affinity enrichment techniques. This is achieved by means of immobilizing macromolecules or epitopes specific to a given peptide sequence, other macromolecules, or epitope species, such as receptors. Repeating the affinity selection procedure provides enrichment of library components that encode the desired sequence, which can then be pooled and shuffled, sequenced, and / or separated for further growth and affinity enrichment. it can.
[0274]
Library components without the desired specificity are removed by washing. The degree and intensity of washing required is determined for each peptide sequence or given single-chain antibody and the immobilized predefined macromolecule or epitope. By adjusting the conditions of the binding culture and subsequent washing, some control can be exerted on the binding properties of the recovered nascent peptide / DNA complex. Temperature, pH, ionic strength, divalent cation concentration, and amount and duration of washing are selected for nascent peptide / DNA complexes that exhibit an affinity for immobilized macromolecules within a specified range. be able to. Usually, selection based on low off-rates, where high affinity is expected, is often the most practical route. This can be accomplished by continuous culturing in the presence of a predetermined amount of macromolecule, free of saturation, or by increasing the amount, number and length of washes. In each case, reassociation of dissociated nascent peptide / DNA or peptide / RNA complexes is prevented and over time, higher affinity nascent peptide / DNA or peptide / RNA complexes are recovered. You.
[0275]
Additional modifications of the coupling and washing procedures can be applied to find peptides with particular characteristics. The affinity of some peptides changes depending on ionic strength or cation concentration. This is a useful feature for peptides used in affinity purification of various proteins when mild conditions are required to remove the protein from the peptide.
[0276]
One variation involves using multiple binding targets (multiple epitope species, multiple receptor species) and allowing the scfv library to be screened simultaneously for multiple scfv with different binding specificities. It is. Since the size of the scfv library often limits the diversity of possible scfv sequences, it is usually desirable to use the largest possible size of the scfv library. The time and economic considerations for generating a number of very large polysomal scFv display libraries can be quite large. To avoid these major problems, multiple defined epitope species (receptor species) can be screened together in a single library, or sequential screening for multiple epitope species can be used. . In one variation, multiple target epitope species, each encoded on a separate bead (or a subset of beads), can be mixed and cultured with a polysome-displayed scfv library under appropriate binding conditions. This set of beads contains multiple epitope species, which can then be used to separate scfv library components by affinity selection. In general, subsequent affinity screening rounds can include the same mixture of beads, a subset thereof, or beads containing only one or two individual epitope species. This approach provides efficient screening and is compatible with laboratory automation, batch processing, and high-throughput screening methods.
[0277]
In the present invention, various techniques are used to diversify a peptide library or a single-chain antibody library, or the variable regions discovered in an earlier round of panning before or in conjunction with shuffling. Repair peptides can be diversified to obtain sufficient binding activity for a given macromolecule or epitope. In one approach, a positive selection peptide / polynucleotide complex (identified in an early round of affinity enrichment) is sequenced to determine the identity of the active peptide. Oligonucleotides are then synthesized based on these active peptide sequences, utilizing low levels of all bases incorporated at each step to generate subtle changes in the primary oligonucleotide sequence. This mixture of (slightly) degenerate oligonucleotides is then cloned at appropriate positions in the various segment sequences. This method results in a systematic and controlled mutation of the starting peptide sequence, which can then be shuffled. However, individual positive nascent peptide / polynucleotide complexes need to be sequenced before mutagenesis, thus extending the diversity of the small number of recovered complexes, increasing affinity and / or higher binding. It is useful for selecting mutants having specificity. As a mutation, in mutagenic PCR amplification of a positive selection peptide / polynucleotide complex (especially of the variable region sequence), the amplification product is shuffled in vitro and or in vivo, and prior to sequencing, One or more additional rounds of screening are performed. The same general approach is to extend diversity and enhance binding affinity / specificity by diversifying CDRs or adjacent framework regions, usually before or in conjunction with shuffling. It can be used in single chain antibodies. If desired, the shuffling reaction can include mutagenic oligonucleotides that can be recombined in vitro with selected library components that can be included. Thus, a mixture of synthetic oligonucleotides and PCR-generated polynucleotides (synthesized by error prone or high fidelity methods) can be added to an in vitro shuffling mixture, resulting in a shuffled library construct Can be incorporated into elements (shuffled).
[0278]
The shuffling of the present invention can generate a vast library of CDR-mutated single-chain antibodies. One way to generate such antibodies is to insert the synthetic CDRs into a single-chain antibody and / or CDR randomization before or in conjunction with shuffling. The sequence of the synthetic CDR cassette is selected by reference to the known sequence data of the human CDRs and selected at the practitioner's discretion according to the following guidelines: It will have 40% positional sequence identity, and will preferably have at least 50-70% positional sequence identity to a known CDR sequence. For example, a set of synthetic CDR sequences can be generated by synthesizing a set of oligonucleotide sequences based on the naturally occurring human CDR sequences listed in Kabat (Kabat et al., 1991), and (multiple) of the synthetic CDR sequences. 2.) The pool is calculated to encode CDR peptide sequences having at least 40 percent sequence identity to at least one known naturally occurring human CDR sequence. Instead, the set of naturally occurring CDR sequences is such that frequently used amino acids at residue positions (ie, in at least 5% of the known CDR sequences) are incorporated at corresponding position (s) in the synthetic CDR. The comparisons can be made to generate a consensus sequence. Typically, several (eg, 3 to about 50) known CDR sequences are compared, and the natural sequence variation observed between known CDRs is tabulated, and all or most of the observed natural sequence variation is tabulated. A set of oligonucleotides encoding the CDR peptide sequence encompassing the permutation is synthesized. As a non-limiting example, if the collection of human VH CDR sequences has a carboxy-terminal amino acid that is either Tyr, Val, Phe, or Asp, the pool (s) of synthetic CDR oligonucleotide sequences will have a carboxy-terminal CDR residue. The group is designed to be able to be any of these amino acids. In some embodiments, residues other than those that occur naturally at residue positions in the set of CDR sequences are incorporated, in which conservative amino acid substitutions are incorporated at a high frequency, and the known naturally occurring CDR sequences have By comparison, up to five residue positions can be changed to incorporate non-conservative amino acid substitutions. Such CDR sequences can be used in the primary library component (prior to the first round of screening) and / or can be used to add to the in vitro shuffling reaction of the selected library component sequence. it can. Construction of such a pool of defined and / or degenerate sequences is readily achievable by those skilled in the art.
[0279]
The set of synthetic CDR sequences includes at least one component that is not known to be a naturally occurring CDR sequence. Whether or not to include a portion of the random or pseudo-random sequence corresponding to the N-region addition in the heavy chain CDRs is included in the judgment of the practitioner, and the N-region sequence may be one nucleotide to It is in the range of about 4 nucleotides. The set of synthetic heavy chain CDR sequences comprises at least about 100 unique CDR sequences, usually comprises at least about 1,000 unique CDR sequences, and preferably comprises at least about 10,000 unique CDR sequences. And frequently contains more than 50,000 unique CDR sequences; however, typically about 1 × 10⁶The following unique CDR sequences are included in the set, but in some cases, especially at positions where conservative amino acid substitutions are absent or rare (ie, less than 0.1 percent) at the same position in naturally occurring human CDRs. 1 × 10 if conservative amino acid substitutions are possible⁷~ 1 × 10⁸There are unique CDR sequences. In general, the number of unique CDR sequences contained in a library should not exceed 10 times the expected number of primary transformants in the library. Such single chain antibodies generally bind at least 1 × 10 m −, preferably at least about 5 × 10⁷M-1 affinity, more preferably at least about 1 × 10⁸M-1 to 1 × 10⁹M-1 or higher affinity, sometimes 1 × 10¹⁰M-1 or more. Predetermined antigens are frequently human proteins, eg, human cell surface antigens (eg, CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor), and other human biological macromolecules (eg, Thrombomodulin, protein C, sugar chain antigen, sialyl Lewis antigen, L-selectin) or non-human disease-related macromolecules (eg, bacterial LPS, virion capsid protein, or envelope glycoprotein), and others.
[0280]
High affinity single chain antibodies of the desired specificity can be engineered and expressed in a variety of systems. For example, scfv has been produced in plants (Philek et al., 1993) and can easily be made in prokaryotic systems (Owen and Young, 1994; Johnson and Bird, 1991). In addition, single-chain antibodies can be used as a basis for constructing whole antibodies or various fragments thereof (Kettlebarlow et al., 1994). The variable regions encoding the sequences may be separated (eg, by PCR amplification or subcloning) and encoded using the desired human constant to encode a human sequence antibody suitable for human therapeutic use, preferably minimizing immunogenicity. The region can be spliced into a coding sequence. The resulting polynucleotide (s) having the fully human coding sequence (s) can be expressed in a host cell (eg, from a mammalian cell expression vector) and purified for drug formulation. .
[0281]
After expression, the antibodies, individual mutant immunoglobulin chains, mutant antibody fragments, and other immunoglobulin polypeptides of the invention can be purified according to standard procedures in the art, This includes ammonium sulfate precipitation, fraction column chromatography, gel electrophoresis, and others (see generally, Scopes, 1982). After purification, partially or to the desired homogeneity, the polypeptide can be used therapeutically, or used in the development and performance of assay procedures, immunofluorescent staining, and the like (generally , Lefkovitz and Parnis, 1979 and 1981; Lefkovitz, 1997).
[0282]
Antibodies generated by the methods of the present invention can be used for diagnosis and therapy. By way of example and not limitation, they can be used to treat cancer, autoimmune diseases, or viral infections. For the treatment of cancer, the antibody will normally bind to an antigen that is preferentially expressed on cancer cells, including erB-2, CEA, CD33, and other antigens widely known to those of skill in the art. Many antigens and binding elements are included.
[0283]
Shuffling can also be used to recombine and diversify the pool of library components obtained by the two-hybrid screening system and identify library components that bind to a given polypeptide sequence. The selected library components are pooled and shuffled by in vitro and / or in vivo recombination. The shuffled pool is then screened in an yeast-to-hybrid system, and library components that bind to the predetermined polypeptide sequence (eg, SH2 domain), or alternative predetermined polypeptide sequences (eg, another protein species). Library component that binds to the SH2 domain from SH2).
[0284]
Approaches to identify polypeptide sequences that bind to a given polypeptide sequence have used a so-called "two-hybrid" system in which the given polypeptide sequence is present in the fusion protein (Shang et al., 1991). This approach identifies protein-protein interactions in vivo through rearrangement of the transcriptional activator, yeast Gal4 transcribed protein (Fields and Song, 1989). Usually, this method is based on the properties of the yeast Gal4 protein, which is composed of separable domains that cause DNA binding and transcriptional activation. A polynucleotide encoding a two-hybrid protein, one composed of a yeast Gal4 DNA binding domain fused to a polypeptide sequence of a known protein and the other a Gal4 activation domain fused to a polypeptide sequence of a second protein The construct is constructed and introduced in a yeast host cell. The intermolecular binding between the two fusion proteins reconstitutes the Gal4 DNA binding domain with the Gal4 activation domain, which is the transcriptional activity of a receptor gene (eg, lacZ, HIS3) that is operatively linked to the Gal4 binding site. Will lead to Usually, the two-hybrid method is used to identify new polypeptide sequences that interact with known proteins (Silva & Hunt, 1993, Darphy et al., 1993, Yang et al., 1992, Luban et al., 1993, Hardy et al. , 1992, Bartel et al., 1993, and Boyek et al., 1993). However, two-hybrid mutations have been used to identify mutations in known proteins that affect binding to a second known protein (Lee and Fields, 1993; Lalo et al., 1993; Jackson et al. 1993, and Madou et al., 1993). The two-hybrid system can be used to interact the structural domains of two known proteins (Birdwell et al., 1993, Chakraberti et al., 1992, Staudinger et al., 1993, and Milne and Weaver, 1993), or the oligos of a single protein It has also been used to identify regions that cause lysis (Iwabuchi et al., 1993, Bogard et al., 1993). Mutations in the two-hybrid system have been used to study the in vivo activity of proteolytic enzymes (Dasmasha Patra et al., 1992). Instead, E. The E. coli / BCCP two-way screening system (Germino et al., 1993, Guarente, 1993) can be used to identify interacting protein sequences (ie, protein sequences that heterodimerize or form the upper heteromultimers). Sequences selected by the two-hybrid system are pooled and shuffled and introduced into the two-hybrid system for one or more subsequent rounds of screening to identify polypeptide sequences that bind to the hybrid, including the defined binding sequence can do. The sequences thus identified can be compared to identify the consensus sequence (s) and the core of the consensus sequence.
[0285]
One microgram sample of the template DNA is obtained and treated with ultraviolet light to form a dimer containing a TT dimer, especially a purine dimer. Ultraviolet light exposure is limited so that only a small amount of photoproduct is generated for each gene in the template DNA sample. A large number of samples are treated with ultraviolet light for varying lengths of time to obtain template DNA samples with different numbers of dimers due to ultraviolet light exposure.
[0286]
Using a random priming kit utilizing a non-proofreading polymerase (e.g., the Stratagene Cloning Systems Prime-It II Random Primer Labeling kit), the primary immunization is performed at random sites on the template created by ultraviolet light (as described above). Performing and extending along the template produces polynucleotides of various sizes. A prime immunization protocol as described in the Prime-It II Random Primer Labeling kit can be used to extend primers. Dimers formed by UV exposure serve as obstacles to extension by non-proofreading polymerases. Thus, after completion of the random primer extension, there is a pool of polynucleotides of random size.
[0287]
The present invention is further directed to a method of producing a selection mutant polynucleotide sequence (or a population of selection polynucleotide sequences), usually in the form of an amplified and / or cloned polynucleotide, The selected polynucleotide sequence (s) can be selected from at least one desired phenotypic characteristic (e.g., encoding a polypeptide, promoting transcription of a linked polynucleotide, binding a protein, and Other). One method of identifying hybrid polypeptides that possess desirable structural or functional properties, such as binding to a defined biological macromolecule (such as a receptor), possesses the desirable structural or functional properties conferred by the amino acid sequence of the polypeptide. Screening large libraries of polypeptides for individual library components.
[0288]
In one embodiment, the invention provides a method of generating a library of display polypeptides or display antibodies suitable for affinity interaction screening or phenotypic screening. The method comprises: (1) obtaining a first plurality of selected library components comprising a display polypeptide or display antibody and a related polynucleotide encoding the display polypeptide or display antibody; and Obtaining a copy thereof, wherein said related polynucleotide comprises a region of sequence that is substantially identical, appropriately introducing a mutation into said polynucleotide or copy, and (2) pooling said polynucleotide or copy. (3) generating small or short polynucleotides by interrupting the random or specialized priming and synthesis or amplification processes; and (4) homologating newly synthesized polynucleotides. Amplification, preferably PCR amplification, for recombination And and performing an optional mutagenesis.
[0289]
It is an object of the present invention to provide a process for producing a hybrid polynucleotide that expresses a useful hybrid polypeptide by a series of steps including:
[0290]
(A) interrupting the polynucleotide amplification or synthesis process by means of inhibiting or interrupting the amplification or synthesis process to provide a plurality of small or short polynucleotides by replication of the polynucleotide at various completion stages; Producing nucleotides,
(B) adding one or more single-stranded or double-stranded oligonucleotides to the resulting single-stranded or double-stranded polynucleotide, wherein said additional oligonucleotides are one or more single-stranded or Including an area of identity in the heterogeneous structural area with the double-stranded polynucleotide,
(C) denaturing the resulting single- or double-stranded oligonucleotides, optionally separating short or small polynucleotides into pools of polynucleotides of various lengths, and optionally, at least Subjecting said polynucleotide to a PCR procedure to amplify one or more oligonucleotides contained in one said polynucleotide pool;
(D) annealing of the single-stranded polynucleotide occurs in a region of identity between the single-stranded polynucleotides, and under the condition that a double-stranded polynucleotide chain due to mutation is formed, a plurality of the polynucleotides or the polynucleotides Culturing at least one pool of nucleotides with a polymerase;
(E) optionally repeating steps (c) and (d);
(F) expressing at least one hybrid polypeptide from the polynucleotide chain or a plurality of chains;
(G) screening said at least one hybrid polypeptide for useful activity.
[0291]
In one aspect of the invention, the means of inhibiting or interrupting the amplification or synthesis process is through the use of ultraviolet light, DNA adducts, and DNA binding proteins.
[0292]
In one embodiment of the present invention, the DNA adduct or polynucleotide containing the DNA adduct is separated from the polynucleotide or polynucleotide pool, such as by a process comprising heating the solution containing the DNA fragment prior to further processing. Removed.
[0293]
In another embodiment, a clone identified as having a given biomolecule or biological activity also includes, for example, a DNA sequence encoding a polypeptide (such as an enzyme), or the polypeptide sequence itself having a particular activity. Can be sequenced to Therefore, according to the present invention, (i) a DNA encoding a predetermined biological activity (for example, an enzyme having a specific enzymatic activity) and (ii) a biomolecule (for example, a polypeptide or an enzyme having such an activity ( (Including its amino acid sequence)) and (iii) the resulting recombinant biomolecule or biological activity.
[0294]
Suitable clones from the library (eg, 1 to 1000 or more clones) are identified by the methods of the invention and sequenced, for example, using high-throughput sequencing techniques. The exact method of sequencing is not a limiting factor of the present invention. Any method useful for identifying the sequence of a particular cloned DNA sequence can be used. Generally, sequencing is an adaptation of the natural process of DNA replication. For this purpose, a template (such as a vector) and a primer sequence are used. One of the common template generation and sequencing protocols begins with automated collection of bacterial colonies, each of which contains a separate DNA clone that serves as a template for a sequencing reaction. Selected clones are placed in culture medium and grown overnight. The DNA template is then purified from the cells and suspended in water. After quantification of the DNA, Applied Biosystems, Inc. High-throughput sequencing is performed using a sequencer such as the Prism 377 DNA Sequencer. The resulting sequence data can then be used in additional ways, including searching a database or multiple databases.
[0295]
A number of source databases containing nucleic acid sequences and / or deduced amino acid sequences for use in the present invention in identifying and determining the activity encoded by a particular polynucleotide sequence are available. All or a representative portion of the sequences to be tested (eg, 100 individual clones) are used simultaneously or individually to search a sequence database (eg, GenBank, PFAM, or ProDom). Many different ways to perform such a sequence search are known in the art. This database may be limited to a particular organism or set of organisms. For example, C.I. elegans, Arabadopsis. sp, M.P. genitalium, M.P. Jannaschii, E .; coli, H.E. influenzae, S.P. There is a database for cerevisiae and others. The sequence data of the clones is then aligned to the sequence of the database or databases using an algorithm designed to determine the homology of two or more sequences.
[0296]
Such sequence alignment methods include, for example, BLAST (Altschul et al., 1990), BLITZ (MPsrch) (Starlock and Collins, 1993), and FASTA (Person and Lippman, 1988). Probe sequences (eg, sequence data from clones) can be of any length and are recognized as homologous based on a homology threshold. This threshold can be a predefined one, but this is not required. This threshold can be based on the length of a particular polynucleotide. A number of different procedures can be used for sequence alignment. Usually, the Smith-Waterman or Needleman-Bunsch algorithm is used. However, as described, fast procedures such as BLAST, FASTA, and PSI-BLAST may be used.
[0297]
For example, the proper alignment of the sequences that line up the comparison window can be found in Smith's local homology algorithm (Smith and Waterman, Adv Appl. Math., 1981, Smith and Waterman, J. Teor Biol, 1981, Smith and Waterman, J Mol Biol, 1981, Smith Et al., J Mol Evol, 1981), Needleman's homology alignment algorithm (Needleman and Bunsch, 1970), Pearson's similarity search method (Pearson and Lippman, 1988), and computer implementation of such an algorithm (Madison, Wisconsin, 575). Science Dr., Wisconsin Genetics Software Package of Genetics Computer Group Release 7.0, or GAP, BESTFIT, FASTA, and TFASTA in the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin, Madison, Wisconsin), or by any method that can be performed by inspection. The best alignment (ie, the one that produces the highest percentage homology over the comparison window) is selected. The similarity of the two sequences (ie, the probe sequence and the database sequence) can then be predicted.
[0298]
Such software matches similar sequences by assigning degrees of homology to various deletions, substitutions, and other modifications. The terms "homologous" and "identical" in the context of two or more nucleic acid or polypeptide sequences may refer to a comparison window or a sequence as determined using any number of sequence comparison algorithms or by manual alignment and visual inspection. Refers to two or more sequences or subsequences that have the same or specific percentage of amino acid residues or nucleotides that are the same or the same when compared and aligned with maximum correspondence across the designated regions.
[0299]
In sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using the sequence comparison algorithm, the test and reference sequences are loaded into a computer, where necessary specify small sequence coordinates, and specify sequence algorithm program parameters. Default program parameters can be used, or alternative parameters can be specified. Thereafter, the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence, based on the program parameters.
[0300]
A “comparison window”, as used herein, is any one of a series of positions selected from the group consisting of 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150. It includes a reference to a number of segments in which, after properly aligning the two sequences, it is possible to compare the sequence to the same number of reference sequences at consecutive positions.
[0301]
One example of a useful algorithm is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25: 3389-3402 (1977) and Altshull et al. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.gov/). The algorithm involves first identifying high-scoring sequence pairs (HSPs) in the query sequence by identifying short words of length W, which are matched to words of the same length in the database sequence. When they are arranged side by side, they match or satisfy a threshold score T of some positive value. T is called the adjacent word score threshold (Altschul et al., Supra). These first adjacent word hits play a role in initiating a search to find long HSPs containing them. Word hits are extended in both directions, along each sequence, as long as the cumulative alignment score can be increased. Cumulative scores are calculated for nucleotide sequences using the parameter M (reward score for a pair of matching residues, always greater than 0). The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M = 5, N = 4, and a comparison of both strands.
[0302]
The BLAST algorithm also performs a statistical analysis of the similarity between the two sequences (see, eg, Carlin & Altshull, Proc. Natl. Acad. Sci. USA 90: 5873 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)), which provides an indication of the probability that a match between two nucleotide sequences will occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the minimum sum probability in comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably, less than about 0.01, and most preferably, less than about 0.001. It is.
[0303]
Sequence homology means that the two polynucleotide sequences are homologous (compare nucleotide by nucleotide) over the window of comparison. The percentage of sequence identity or homology is determined by comparing two properly aligned sequences over the entire window of comparison, and by comparing nucleobases identical in both sequences (eg, A, T, Determine the number of positions where C, G, U, or I) occur, divide the number of matched positions by the total number of positions in the comparison window (ie, window size) to obtain the percentage of sequence homology. It is calculated by multiplying the result by 100. Such substantial homology is characteristic of a polynucleotide sequence in which the polynucleotide has at least 60% sequence homology, usually at least 60%, compared to a reference sequence in a comparison window of at least 25 to 50 nucleotides. It includes 70% homology, often 80-90% sequence homology, most commonly at least 99% sequence homology, wherein the percentage of sequence homology is determined by comparing the reference sequence to the comparison window. In total, it is calculated by comparison to polynucleotide sequences that may contain up to 20 percent of the deletion or addition of the reference sequence.
[0304]
Sequences with sufficient homology can then be further identified by any annotation included in the database, including, for example, species and activity information. Thus, in a typical environmental sample, multiple nucleic acid sequences will be obtained, cloned, sequenced, and correspond to homologous sequences from the identified database. This information provides a profile of the polynucleotides present in the sample, which may include the sequence or any polynucleotide associated with any polypeptide encoded by the sequence based on the database information and the activity of the polynucleotide. Contains one or more related functions. As used herein, "fingerprint" or "profile" refers to the fact that each sample will be associated with the set of polynucleotide properties of the sample and the environment from which it is derived. Such a profile may include the amount and type of sequence present in the sample, and the potential activity encoded by the polynucleotide and information about the organism from which the polynucleotide is derived. These unique patterns are the profile or fingerprint of each document.
[0305]
It may also be desirable to express a particular cloned polynucleotide sequence when the identity or activity has been determined, or when the suggested identity or activity is associated with the polynucleotide. In such cases, the desired clone, if not already cloned in the expression vector, is ligated downstream of regulatory control elements (eg, a promoter or enhancer) and cloned in a suitable host cell. Expression vectors are commercially available with corresponding host cells used in the present invention.
[0306]
Representative examples of expression vectors that can be used include viral particles, baculoviruses, phages, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral nucleic acids (eg, vaccinia, adenovirus). , Fowl pox virus, pseudorabies, and derivatives of SV40), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vector specific to a given particular host (Bacillus, Aspergillus, Yeast, and others). Thus, for example, DNA may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, non-chromosomal, and synthetic DNA sequences. Many suitable vectors are known to those of skill in the art and are commercially available. The following vectors are provided as examples. Bacterial: pQE70, pQE60, pQE-9 (Quiagen), psiX174, pBluescript SK, pBluescript KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), pTRC2apR, pTRC2APKp, KTR2Kp Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector can be used, so long as it can replicate and survive in the host.
[0307]
The nucleic acid sequence in the expression vector is operatively linked to the appropriate expression control sequence (s) to direct mRNA synthesis. Particular designated bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL, and trp. Prokaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector further includes a ribosome binding site for translation initiation and a transcription terminator. The vector may further include appropriate sequences for amplifying expression. The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) vector or other vector with a selectable marker.
[0308]
In addition, expression vectors may be resistant to dihydrofolate reductase or neomycin for eukaryotic cell culture, or E. coli. One or more selectable marker genes are usually included to provide a phenotypic trait for selection of transformed host cells, such as tetracycline or ampicillin resistance in E. coli.
[0309]
The nucleic acid sequence (s) that are selected, cloned, and sequenced as described above, in addition, can be introduced into an appropriate host to prepare a library that is screened for the desired biomolecule or biological activity. . The selection nucleic acid is preferably already in a vector containing the appropriate control sequences, which allows the expression of the biomolecule or the selection nucleic acid encoding the biological activity to detect the desired activity. . The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast, or the host cell can be a prokaryotic cell, such as a bacterial cell. The selection of an appropriate host will be within the skill of the art in light of the present description.
[0310]
It may also be desirable to perform amplification of a nucleic acid sequence present in a sample or of a particular isolated clone. In this embodiment, the nucleic acid sequence is amplified by a PCR reaction or a similar reaction known to those skilled in the art. To perform such an amplification reaction, a commercially available amplification kit can be used.
[0311]
In addition, it is important to recognize that alignment algorithms and searchable databases can be implemented by computer hardware, software, or a combination thereof. Thus, separation, processing, and identification of nucleic acid or polypeptide sequences can be performed in an automated system.
[0312]
In addition to the sequence-based approaches described above, there are a number of traditional assay systems for measuring enzyme activity using multi-well plates. For example, existing screening techniques typically rely on two-dimensional wells (eg, 96, 384, 1536 wells). The present invention further provides a capillary array-based approach that has a number of advantages over well-based screening techniques, including the need for a fluid dispenser to dispense fluids (such as reactants) to individual well reservoirs. And reduced cost per array (e.g., glass capillaries are reusable) (e.g., November 22, 1999, the disclosure of which is hereby incorporated by reference). (See U.S. patent application Ser.
[0313]
Accordingly, the capillaries, capillary arrays, and systems of the present invention are particularly suitable for screening libraries for a given activity or biomolecule, including polynucleotides. Screening for activity can be performed on individual expression clones, or, on a mixture of expression clones, first to determine whether the mixture has one or more specified activities. can do. If the mixture has the specified activity, after collection from the capillary array, individual clones can be screened again for such activity, or for more specified activity.
[0314]
Any headings and subheadings used herein are presented for the convenience of the reader and should not be construed as limiting the invention.
[0315]
As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a clone" includes a plurality of clones, and reference to "the nucleic acid sequence" generally refers to one or more nucleic acid sequences and those skilled in the art. And the like, including references to its equivalents, as well as others.
[0316]
Unless defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are described. ing.
[0317]
All printed materials mentioned herein are sourced for the purpose of explaining and disclosing the databases, proteins, and methods described in publications that may be used in connection with the described invention. The disclosure is incorporated herein by reference. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing in this specification should be construed as an admission that the inventors have not been entitled to antedate such disclosure by virtue of prior invention.
[0318]
The invention will now be described in more detail with reference to the following non-limiting examples.
[0319]
Example 1
DNA separation. DNA is isolated using the IsoQuick Procedure according to the manufacturer's instructions (Orca Research Inc., Bothell, WA). The separated DNA can optionally be averaged according to Example 2 (below). Upon separation, the DNA is sheared by pushing and pulling the DNA about 500 times with a 25 gauge double hub needle and a 1 cc syringe. Run a small amount on a 0.8% agarose gel to ensure that the majority of the DNA is in the desired size range (about 3-6 kb).
[0320]
DNA blunting. The DNA is blunt-ended by mixing 45 μl of 10 × mung bean buffer, 2.0 μl of mung bean nuclease (1050 u / μl) and water to a final volume of 405 μl. This mixture is incubated at 37 ° C. for 15 minutes. The mixture is extracted with phenol / chloroform and then further with chloroform. Add 1 ml of ice-cold ethanol to the final extract and precipitate the DNA. DNA is precipitated on ice for 10 minutes. DNA is removed by centrifugation in a microcentrifuge for 30 minutes. The pellet is washed with 1 ml of 70% ethanol and pelleted again in a microcentrifuge. Following centrifugation, the DNA is dried and gently resuspended in 26 μl of TE buffer.
[0321]
DNA methylation. The DNA is methylated by mixing 4 μl of 10 × EcoRI methylase buffer, 0.5 μl of SAM (32 mM) and 5.0 μl of EcoRI methylase (40 u / μl), and culturing at 37 ° C. for 1 hour. . To ensure blunt ends, the following can be added to the methylation reaction: 5.0 μl of 100 mM MgCl₂, 8.0 μl dNTP mixture (dGTP, dATP, dTTP, dCTP, 2.5 mM each), 4.0 μl Klenow (5 u / μl). This mixture is then incubated at 12 ° C. for 30 minutes.
[0322]
After incubation for 30 minutes, add 450 μl of 1 × STE. The mixture is extracted once with phenol / chloroform, followed by another chloroform extraction. Add 1 ml of ice-cold ethanol to the final extract and precipitate the DNA. DNA is precipitated on ice for 10 minutes. DNA is removed by centrifugation in a microcentrifuge for 30 minutes. The pellet is washed with 1 ml of 70% ethanol, pelleted again in a microcentrifuge and dried for 10 minutes.
[0323]
Ligation. The DNA was prepared by adding the DNA to 8 μl of EcoRI adapter (from Stragene's cDNA Synthesis Kit), 1.0 μl of 10 × ligation buffer, 1.0 μl of 10 mM rATP, and 1.0 μl of T4 DNA ligase (4 Wu / μl). And ligated by culturing at 4 ° C. for 2 days. The ligation reaction is terminated by heating at 70 ° C. for 30 minutes.
[0324]
Phosphorylation of the adapter. Adapter ends were used for ligation reaction with 1.0 μl of 10 × ligation buffer, 2.0 μl of 10 mM rATP, and 6.0 μl of H₂O is mixed with 1.0 μl of polynucleotide kinase (PNK) and cultured at 37 ° C. for 30 minutes to be methylated. After 30 minutes of culture, 30 μl of H₂Add O and 5 ml of 10 × STE and size fragment the sample with a Sephacryl S-500 spin column. The pooled fragments (1-3) are extracted once with phenol / chloroform, followed by a further chloroform extraction. DNA is precipitated on ice for 10 minutes by adding ice-cold ethanol. The precipitate is pelleted by centrifugation in a microcentrifuge for 30 minutes. The resulting pellet is washed with 1 ml of 70% ethanol, pelleted again by centrifugation and dried for 10 minutes. This sample is resuspended in 10.5 μl of TE buffer. This sample is not plated, but is ligated directly to the lambda arm as described above, except that 2.5 μl of DNA is used and no water is used.
[0325]
Sucrose gradient (2.2 ml) size fragmentation. Ligation is stopped by heating the sample at 65 ° C. for 10 minutes. The sample is placed in a 2.2 ml sucrose gradient and centrifuged for 4 hours at 20 ° C. in a 45000 rpm mini ultracentrifuge (without brake). The fragments are collected by piercing the bottom of the gradient tube with a 20 gauge needle and allowing sucrose to flow through the needle. The first 20 droplets are collected in a Falcon 2059 tube, after which ten 1-drop fragments (labeled 1-10) are collected. Each droplet has a volume of about 60 μl. Run 5 μl of each fragment on a 0.8% agarose gel and check the size. Fragments 1-4 (about 10-1.5 kb) are pooled, and fragments 5-7 (about 5-0.5 kb) are pooled in separate tubes. Add 1 ml of ice-cold ethanol to precipitate the DNA, then place on ice for 10 minutes. The precipitate is pelleted by centrifugation in a microcentrifuge for 30 minutes. The pellet is washed by resuspension in 70% ethanol, re-pelleted by centrifugation at high speed for 10 minutes in a microcentrifuge, and then dried. Each pellet is then resuspended in 10 μl of TE buffer.
[0326]
Test ligation with lambda arms. Analytes are plated by placing 0.5 μl of sample on agarose containing ethidium bromide with standards (DNA samples of known concentration) and obtaining approximate concentrations. The sample is then observed using ultraviolet light and the estimated concentration is compared to a reference.
[0327]
As shown in Table 1 below, prepare the following ligation reaction (5 μl reaction) and incubate at 4 ° C. overnight.
[0328]
Table 1

Test package and plate. The ligation reaction is packaged according to the manufacturer's protocol. The packaging reaction is stopped with 500 μl SM buffer and pooled with packaging from the same ligation. Titrate 1 μl of each pooled reaction on the appropriate host (OD₆₀₀= 1.0) (XLl-Blue MRF). 200 μl of host (MgSO 4₄) Is added to a Falcon 2059 tube, inoculated with 1 μl of packaged phage, and cultured at 37 ° C. for 15 minutes. Add about 3 ml of 48 ° C. top agar (50 ml stock containing 150 μl IPTG (0.5 M) and 300 μl X-GAL (350 mg / ml)) and plate on a 100 mm plate. Plates are incubated overnight at 37 ° C.
[0329]
Library amplification (5.0 × 10 from each library)⁵Recombination). About 3.0 ml of host cells (OD₆₀₀= 1.0) was added to two 50 ml conical tubes and 2.5 × 10⁵The pfu phage is inoculated, and then cultured at 37 ° C. for 20 minutes. Add top agar to each tube to bring the final volume to 45 ml. Each tube is plated on five 150 mm plates. Plates are incubated at 37 ° C. for 6-8 hours or until the plaque has reached the size of the pinhead. Plates are covered with 8-10 ml of SM buffer and placed at 4 ° C. overnight (gently rock if possible).
[0330]
Phage recovery. Phage suspension is recovered by pouring SM buffer from each plate into a 50 ml conical tube. Add about 3 ml of chloroform, shake vigorously, and incubate at room temperature for 15 minutes. The tube is centrifuged at 2000 rpm for 10 minutes to remove cell debris, the supernatant is poured into a sterile flask, added with 500 μl chloroform and stored at 4 ° C.
[0331]
Titration amplification library. The collected phages were serially diluted (for example, 10^-5= 1 μl amplified phage, 10 ml in 1 ml SM buffer^-6= 1 ml of 1 ml SM buffer 10^-3200 μl of host (10 mM MgSO)₄) Is added to the two tubes. In one tube, 10 μl of 10^-6Diluted (10^-5). In the other tube, 1 μl of 10^-6Diluted (10^-6) And incubate at 37 ° C. for 15 minutes.
[0332]
Add about 3 ml of 48 ° C top agar (50 ml stock containing 150 μl IPTG (0.5 M) and 37 μl X-GAL (350 mg / ml)) to each tube and plate on a 100 mm plate. Plates are incubated overnight at 37 ° C.
[0333]
The ZAP II library is excised to form a pBLUESCRIPT library according to the manufacturer's protocol (Stratagene).
[0334]
The DNA library can be transformed with a host cell (such as E. coli) to generate a clone expression library.
[0335]
Example 2
Averaging
Prior to library generation, the purified DNA can be averaged. DNA is fragmented according to the following protocol. Samples containing genomic DNA are purified on a cesium chloride gradient. The cesium chloride (Rf = 1.3980) solution is filtered through a 0.2 μl filter and 15 ml is placed in a 35 ml OptiSeal tube (Beckman). Add DNA and mix thoroughly. Add 10 micrograms of bisbenzimide (Sigma, Hoechst 33258) and mix thoroughly. The tube is then filled with the filtered cesium chloride solution and spun at 33000 rpm for 72 hours in a Beckman L8-70 Ultracentrifuge Bti50 rotor. Following centrifugation, the gradient is passed through an ISCO UA-5 UV absorbance detector set at 280 nm using a syringe pump and a rectification column (Brandel Model 186). Peaks representing DNA from organisms present in the environmental sample are obtained. Eubacteria sequences were obtained using the following primers for amplification. DNA encoding rRNA from a 10-fold dilution of the E. coli peak can be detected by PCR amplification.
[0336]
Forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 4)
Reverse primer: 5'-GGTTACCTTGTTTACGACTT-3 '(SEQ ID NO: 5)
The recovered DNA is sheared or enzymatically digested into 3-6 kb fragments. The lone linker primer is ligated and the DNA is size selected. The size selection DNA is amplified, if necessary, by PCR.
[0337]
Averaging was then performed by combining the double-stranded DNA sample with hybridization buffer (0.12 M NaH₂PO₄6.8 / 0.82 M NaCl / 1 mM EDTA / 0.1% SDS). The sample is denatured by overlaying with mineral oil and boiling for 10 minutes. This sample is incubated at 68 ° C. for 12 to 36 hours. Double-stranded DNA is separated from single-stranded DNA in hydroxyapatite at 60 ° C. according to standard protocols (Sambrook, 1989). The single-stranded DNA fragment is desalted and amplified by PCR. This process is repeated several more rounds (up to five or more).
[0338]
Example 3
Enzyme activity assay
The following is a representative procedure for screening an expression library prepared according to Example 1 for hydrolase activity.
[0339]
The library plates prepared as described in Example 1 are used to multi-inoculate a single plate containing 200 μl LB Amp / Meth glycerol in each well. This step is performed using Beckman BIOMEK RTM's High Density Replicating Tool (HDRT) with 1% bleach, water, isopropanol, and air-dry sterilization between each inoculation. This single plate is grown for 2 hours at 37 ° C. and then used to inoculate two white 96-well Dynatech microtiter daughter plates containing 250 μl LB Amp / Meth glycerol in each well. The original single plate is incubated at 37 ° C. for 18 hours and then stored at −80 ° C. The two condensed daughter plates are also incubated at 37 ° C. for 18 hours. The condensing daughter plate was then heated at 70 ° C. for 45 minutes to kill the cells and the host E. coli. inactivate the E. coli enzyme. A stock solution of 5 mg / mL morphourea phenylalanyl-7-amino-4-trifluoromethyl coumarin (MuPheAFC, “substrate”) in DMSO is 50 mM pH 7.0 containing 0.6 mg / mL surfactant dodecyl maltoside. Dilute to 600 μM with 5 Hepes buffer. Add 50 μl of a 600 μM MuPheAFC solution to each of the wells of the white condensing plate using BIOMEK in a 100 μl mixing cycle to bring the final substrate concentration to about 100 μM. Fluorescence value immediately after addition of substrate
[0340]
[Formula]

At (t = 0), record by plate reading fluorometer. The plate is incubated at 70 ° C. for 100 minutes and then allowed to cool at ambient temperature for another 15 minutes. The fluorescence value is recorded again (t = 100). The value of t = 0 is subtracted from the value of t = 100 to determine whether an active clone exists.
[0341]
MuPheAFC
This data will indicate whether one of the clones in a particular well has hydrolyzed the substrate. To determine individual clones with activity, the source library is thawed and individual clones are used to inoculate a new plate containing glycerol of LB Amp / Meth alone. As described above, the plate is cultured at 37 ° C. to grow the cells, heated at 70 ° C. to inactivate the host enzymes, and added 50 μl of 600 μM MuPheAFC using Biomek.
[0342]
After addition of the substrate, the fluorescence value at t = 0 is recorded as above, the plate is incubated at 70 ° C., and the value at t = 100 is recorded. These data indicate which plates have active clones.
[0343]
The value of the enantioselectivity for the substrate, E, is determined according to the following equation:
[0344]
【number】

In this equation, ee_p= Enantiomeric excess of hydrolyzate, c = percent conversion of reaction. Won and White Size, Enzymes in Synthetic Organic Chemistry, 1994, Elsevier, Tarrytown, N.W. Y. Pp. See 9-12.
[0345]
Enantiomeric excess is determined by chiral high performance liquid chromatography (HPLC) or chiral capillary electrophoresis (CE). The assay is performed as follows: 200 μl of the appropriate buffer is added to each well of a 96-well white microtiter plate, followed by the addition of 50 μl partially or fully purified enzyme and the addition of 50 μl substrate. The increase in monitored fluorescence is compared to the time until 50% consumption of substrate or termination of the reaction, whichever occurs first.
[0346]
Example 4
Directed mutagenesis of positive enzyme-active clones
Directed mutagenesis was performed with two enzymes (alkaline phosphatase and β-glycosidase) to produce a new enzyme that showed a higher degree of activity than the wild-type enzyme.
[0347]
Alkaline phosphatase
The XL1-Red strain (Stratagene) was transformed with a genomic clone 27a3a (in plasmid pBluescript) encoding the alkaline phosphatase gene from organism OC9a, an organism isolated from the surface of whale bone according to the manufacturer's protocol. . 5 ml medium of LB + 0.1 mg / ml ampicillin was inoculated with 200 μl of the transformants and the medium was grown at 37 ° C. for 30 hours. The isolated DNA was then performed by performing a miniprep on this medium and transforming 2 μl of the resulting DNA into XL-1 Blue cells (Stratagene) according to the manufacturer's protocol and according to the assay procedure outlined below. Screened. In the screening assay, the mutated OC9a phosphatase required 10 minutes to develop color and the wild-type enzyme required 30 minutes to develop color.
[0348]
Standard alkaline phosphatase screening assay
The transformed XL1 Blue cells were plated on LB / amp plates. The resulting colonies were lifted by Duralon UV (Stratagene) or HATF (Millipore) thin films and dissolved in chloroform vapor for 30 seconds. The cells were cultured for 30 minutes at 85 ° C., and the cells were killed by heat. Filters were constructed at room temperature in BCIP buffer and the fastest growing colonies ("positives") were selected and the "positives" were transferred to a BCIP plate (BCIP buffer; 20 mm CAPS pH 9.0, 1 mm MgCl2).₂, 0.01mm ZnCl₂, 0.1 mg / ml BCIP).
[0349]
β-glycosidase
This protocol was used to mutate Thermococcus 9N2 β-glycosidase.
[0350]
2 microliters of dNTP (10 mM stock), 10 microliters of 10 × PCR buffer, 0.5 microliters of vector DNA-31G1A-100 nanograms, 20 microliters of 3 ′ primer (100 pmol) and 20 microliters 5 'primer (100 pmol) and 16 microliters of MnCl 4H₂O (1.25 mM stock) and 24.5 microliters of H₂PCR was performed by culturing O and 1 microliter of Taq polymerase (5.0 units) for a total of 100 microliters. The PCR cycle is 95 ° C. for 15 seconds, 58 ° C. for 30 seconds, 72 ° C. for 90 seconds, and 25 cycles (culture at 72 ° C. to 4 ° C. for 10 minutes).
[0351]
Five microliters of the PCR product was run on a 1% agarose gel and the reaction was checked. Purified on a QIAQUICK column (Qiagen). 50 microliters of H₂Resuspended with O.
[0352]
25 microliters of purified PCR product, 10 microliters of NEB buffer # 2, 3 microliters of Kpn I (1OU / microliter), 3 microliters of EcoR1 (20U / microliter), and 59 microliters of H₂O was incubated for 2 hours at 37 ° C. to digest the PCR product and purified on a QIAQUICK column (Qiagen). 35 microliters of H₂Eluted with O.
[0353]
10 microliters of digested PCR product, 5 microliters of vector (cut with EcoRI / KpnI and phosphatase with shrimp alkaline phosphatase), 4 microliters of 5 × ligation buffer and 1 microliter of T4 DNA ligase (BRL) was cultured overnight, and the PCR product was ligated into the vector.
[0354]
The resulting vector was transformed in M15pREP4 cells using electroporation. 100 or 200 microliters of cells were plated on LB amp meth kan plates and grown overnight at 37 ° C.
[0355]
Assays for β-galactosidase consisted of (1) performing a colony lift using a Millipore HAFT membrane filter, (2) dissolving the colonies with chloroform vapor in a 150 mm glass Petri dish, and (3) Z containing 1 mg / ml XGLU. Move the filter to a 100 mm glass Petri dish containing one Whatman 3MM filter paper soaked with buffer (after moving the filter with dissolving colonies to the glass Petri dish, keep the dish at room temperature); (4) Filter membrane This was done by observing "positives" as blue spots above ("positives" are spots that appear early). The center of the blue spot on the filter membrane was centered using a Pasteur pipette (or glass capillary tube). A small filter disc was placed in an Eppendorf tube containing 20 μl of water. The Eppendorf tube was incubated at 75 ° C. for 5 minutes, followed by vortexing to elute the plasmid DNA from the filter. This DNA was converted to electroreactive E. coli. E. coli cells were transformed and the filter lift assay was repeated on the transformation plates to identify "positives". After the filter lift, the transformed plate was returned to the incubator at 37 ° C. to regenerate the colonies. Repurified positives were inoculated into 3 ml of LBamp solution, and cultured at 37 ° C. overnight. Plasmid DNA was isolated from these media and the sequence of the plasmid insert was determined. This filter assay uses Buffer Z (see recipe below) containing 1 mg of the substrate 5-bromo-4-chloro-3-indolyl-β-o-glucopyranoside (XGLU) (Diagnostic Chemicals Limited or Sigma).
[0356]
Per liter of Z buffer (cited from JH Miller (1992), A Short Course in Bacterial Genetics, p. 445)
Na₂HPO₄-7H₂O 16.1 g
Na₂HPO₄-4H₂O 5.5g
0.75 g of KCl
Na₂HPO₄-7H₂O 0.246g
2.7 ml of 6-mercaptoethanol
Adjust pH to 7.0
[0357]
Example 5
Construction of a stable large insert DNA library of picoplankton genomic DNA. Cell collection and DNA preparation. Agarose plugs containing concentrated picoplankton cells were prepared from samples collected on a marine cruise from Newport, OR to Honolulu, Hawaii. Seawater (30 liters) was collected in a Niskin bottle, screened with a 10 μm Nitex, and concentrated by hollow fiber filtration (Amicon DC10) through a 30,000 MW cutoff polyfluphon filter. The concentrated bacterial plankton cells were collected with a 0.22 μm, 47 mm Durapore filter, resuspended in 1 ml of 2 × STE buffer (1 M NaCl, 0.1 M EDTA, 10 mM Tris, pH 8.0) to a final concentration of 1 ml / ml. About 1 × 10¹⁰Cells. The cell suspension was mixed with one volume of 1% molten Seaplaque LMP agarose (FMC) cooled to 40 ° C. and immediately drawn into a 1 ml syringe. The syringe was sealed with parafilm and placed on ice for 10 minutes. The cell-containing agarose plug was extruded into 10 ml of lysis buffer (10 mM Tris, pH 8.0, 50 mM NaCl, 0.1 M EDTA, 1% sarkosyl, 0.2% sodium deoxycholate, 1 mg / ml lysozyme) at 37 ° C. Cultured for 1 hour. The agarose plug was then transferred to 40 ml of ESP buffer (1% sarkosyl, 1 mg / ml proteinase K, in 0.5 M EDTA) and cultured at 55 ° C. for 16 hours. The solution was decanted, replaced with fresh ESP buffer and incubated at 55 ° C. for another hour. The agarose plug was then placed in 50 mM EDTA and stored at 4 ° C. on board during the ocean cruise.
[0358]
An agarose plug piece (72 μl) prepared from a sample collected along the Oregon coast was placed in a 2 mL microcentrifuge tube with 1 mL of buffer A (100 mM NaCl, 10 mM bistrispropane-HCl, 100 μg / ml acetylated BSA, 25 ° C. (PH 7.0) at 4 ° C. overnight. This solution was added to 10 mM MgCl₂And 250 μl of fresh buffer A containing 1 mM DTT and incubated on a rocking platform for 1 hour at room temperature. This solution was then changed to 250 μl of the same buffer containing 4 U of Sau3A1 (NEB), equilibrated in a water bath at 37 ° C., and then cultured for 45 minutes on a rocking platform in a 37 ° C. incubator. The plug was transferred to a 1.5 ml microcentrifuge tube and incubated at 68 ° C. for 30 minutes to inactivate the enzyme and dissolve the agarose. Agarose was digested and DNA was dephosphorylated using Gelase and HK-phosphatase (Epicenture), respectively, according to the manufacturer's recommendations. Protein was removed by gentle phenol / chloroform extraction and DNA was ethanol precipitated, pelleted, and then washed with 70% ethanol. This partially digested DNA is converted into sterile H₂Resuspended in O and brought to a concentration of 2.5 ng / μl for ligation with the pFOS1 vector.
[0359]
Results of PCR amplification from several agarose plugs indicated the presence of large amounts of archaeal DNA. Quantitative hybridization experiments using rRNA extracted from a single sample offshore the Oregon Coast at a depth of 200 m showed that the plankton archaea in this assembly accounted for approximately 4.7% of the total picoprankton biomass. (This sample corresponds to Table 1, "PACI" -200 m of Delong et al., Nature, 371: 695-698, 1994). Results of archaeal biased rDNA PCR amplification performed on agarose plug lysates confirmed the presence of relatively large amounts of archaeal DNA in this sample. Agarose plugs prepared from this picoplankton sample were selected for subsequent fosmid library construction. Each 1ml agarose plug from this site is about 7.5x10⁵Of the cells used for the preparation of the partially digested DNA, about 5.4 × 10⁵Cells were present.
[0360]
The vector arm was prepared from pFOS1 as described (Kim et al., Stable propagation of common sized human DNA inserts in an Factor based vector, Nucl. Res. Briefly, plasmids were completely digested with AstII, dephosphorylated with HK phosphatase, and then digested with BamHI to generate two arms, each of which cloned and ligated 35-45 kbp of ligated DNA. Includes a cos site that is in the proper orientation for packaging. The partially digested picoplankton DNA was ligated overnight to the PFOS1 arm in a 15 μl ligation reaction containing 25 ng each of the vector and insert and 1 U of T4 DNA ligase (Boehringer-Mannheim). The ligated DNA contained in 4 microliters of this reaction was packaged in vitro using a Gigapack XL packaging system (Stratagene) and the fosmid particles were transformed into E. coli. E. coli strain DH10B (BRL), and the cells were_cm15It was spread on a plate. The resulting fosmid clone was replaced with LB supplemented with 7% glycerol._cm15And placed in a 96-well microliter dish containing Recombinant fosmid containing the approximately 40 kb picoplankton DNA insert yielded a library of 3.552 fosmid clones, which was approximately 1.4 × 10 5⁸Includes base pair cloned DNA. All clones tested contained inserts ranging from 38 to 42 kb. This library was stored frozen at -80 ° C for subsequent assays.
[0361]
Example 6
Generation of random-sized polynucleotides using UV-induced photoproducts
One microgram sample of the template DNA is obtained and treated with ultraviolet light to form a dimer containing a TT dimer, especially a purine dimer. Ultraviolet light exposure is limited so that only a small amount of photoproduct is generated for each gene in the template DNA sample. A large number of samples are treated with ultraviolet light for varying lengths of time to obtain template DNA samples with different numbers of dimers due to ultraviolet light exposure.
[0362]
Using a random priming kit utilizing a non-proofreading polymerase (e.g., the Stratagene Cloning Systems Prime-It II Random Primer Labeling kit), the primary immunization is performed at random sites on the template created by ultraviolet light (as described above). Performing and extending along the template produces polynucleotides of various sizes. A prime immunization protocol as described in the Prime-It II Random Primer Labeling kit can be used to extend primers. Dimers formed by UV exposure serve as obstacles to extension by non-proofreading polymerases. Thus, after completion of the random primer extension, there is a pool of polynucleotides of random size.
[0363]
Example 7
Separation of randomly sized polynucleotides
Certain polynucleotides produced according to Example 1 are gel separated on a 1.5% agarose gel. Polynucleotides ranging from 100 to 300 bp are excised from the gel and three volumes of 6M NaI are added to the gel slice. The mixture is incubated at 50 ° C. for 10 minutes and 10 μl of glass milk (Bio 101) is added. The mixture is spun for 1 minute and the supernatant decanted. Wash the pellet with 500 μl Column Wash (Column Wash is 50% ethanol, 10 ml Tris-HCl, pH 7.5, 100 mM NaCl, and 2.5 mM EDTA), spin for 1 minute, and then decant the supernatant. Thereafter, the steps of washing, spinning, and decanting are repeated. Glass milk pellet is added to 20 μl of H₂Resuspend in O and spin for 1 minute. DNA maintains an aqueous phase.
[0364]
Example 8
Shuffling of isolated random-sized 100 to 300 bp polynucleotides
The 100-300 bp polynucleotide obtained in Example 2 was added to the annealing mixture (0.2 mM of each dNTP, 2.2 mM MgCl 2) without adding a primer.₂, 50 mM KCl, 10 mM Tris HCl, pH 8.8, 0.1% Triton X-100, 0.3 μ, Taq DNA polymerase, total volume 50 μl). Stratagene Robocycler was used for the annealing step according to the following program: 95 ° C. for 30 seconds, [95 ° C. for 30 seconds, 50 to 50 ° C. (preferably 58 ° C.) for 30 seconds, and 72 ° C. for 30 seconds] for 25 to 50 cycles. And 72 ° C for 5 minutes. Thus, the 100 to 300 bp polynucleotides combine to generate a double-stranded polynucleotide having a long sequence. After the reconstituted double-stranded polynucleotide is separated and denatured to form a single-stranded polynucleotide, cycling is optionally repeated again, and in some samples, the single-stranded as a template and the primer DNA are used. Other samples use random primers in addition to single strands.
[0365]
Example 9
Screening polynucleotides from shuffled polynucleotides
The polynucleotide of Example 3 is separated, and the polynucleotide is expressed therefrom. Polynucleotides obtained from the shuffled polynucleotides of Example 3 are screened for the activity of the polypeptide obtained from the original template, and control of the level of activity is compared. Shuffled polynucleotides encoding polynucleotides of interest discovered during the screening are further compared for secondary desirable traits. Some shuffled polynucleotides corresponding to the screened polynucleotides of less interest are shuffled again.
[0366]
Example 10
Directed evolution by saturation mutagenesis
Site Saturation Mutagenesis: To achieve site saturation mutagenesis, all residues of the dehalogenase enzyme were subjected to site directed mutagenesis using 32-fold degenerate oligonucleotide primers, as shown below, for a total of 20 variants. Amino acid.
[0367]
1. A medium having a dehalogenase expression structure was grown to prepare a plasmid.
[0368]
Primers randomize each codon so that they have a common structure X₂₀NN (G / T) X₂₀And this X₂₀Represents a 20-base sequence with sufficient homology to bind to the template, and NN (G / T) is a 32-fold degenerate 3-base sequence encoding all 20 amino acids. In an alternative embodiment, the invention provides for the use of any set of codons that are practical to introduce all 19 amino acid substitutions in addition to the N, N, G / T or similar NN (G / T) cassette. Which include (as part of) a 32-fold degenerate NN (G / C) cassette, a 64-fold degenerate NNN cassette, a 48-fold NN (A / C / G) cassette, a 48-fold NN (A / G / T) ) Cassette, including any non-degenerate set of 19 codons that are practical to introduce all 19 amino acid substitutions, and any non-degenerate set of 20 codons that are practical to introduce all 19 amino acid substitutions. In yet another alternative embodiment, the invention provides that a subset of 20 amino acids is introduced into each of the set of codon positions, examples of such subsets being selected from subsets based on a grouping scheme. It can include amino acid (s), and this grouping system can be, by way of non-limiting example, a grouping system having the following eight subsets.
[0369]
・ Aromatic (Phe, Trp, Tyr)
・ Aliphatic (Gly, Ala, Val, Leu, Ile)
-Contains OH (Ser, Tyr, Thr)
・ Acid (Asp, Glu, Asn, Gin)
・ Basic (Lys, Arg, His)
・ Sulfur content (Met, Cys)
-Polarity (Ser, Thr, Cys, Asn, Gin, Tyr)
-Non-polar (Gly, Ala, Vai, Leu, Ile, Met, Phe, Trp, Pro)
[0370]
2. A 25 μl reaction mixture was made containing up to 50 ng of plasmid template, 125 ng of each primer, 1 × native Pfu buffer, 20 μm of each dNTP, and 2.5 U native Pfu DNA polymerase.
[0371]
3. The reaction was cycled on a Robo96 Gradient Cycler as follows.
[0372]
Initial denaturation at 95 ° C for 1 hour
20 cycles of 95 ° C for 45 seconds, 53 ° C for 1 minute, and 72 ° C for 11 minutes
Final extension step at 10 minutes at 72 ° C
4. This reaction mixture was digested with 10 U of DpnI at 37 ° C. for 1 hour to digest methylated template DNA.
[0373]
5. Two ul of the reaction mixture was used to transform 50 ul of XL1-Blue MRF cells, and the entire transformation mixture was plated on large LB-Amp-Met plates to make 200-1000 colonies.
[0374]
6. Individual colonies were picked into wells of a 96-well microtiter plate containing LB-Amp-IPTG and grown overnight.
[0375]
7. The next day, clones from these plates were assayed.
[0376]
Screening: Approximately 200 clones of the mutant at each position were grown in liquid medium (384-well microtiter plates) and screened as follows.
[0377]
1. The overnight culture in a 384-well plate was centrifuged to remove the medium. 0.06 mL 1 mM Tris / SO in each well₄ ^2-, PH 7.8 was added.
[0378]
2. Two assay plates from each parent growth plate were made up with 0.02 mL cell suspension.
[0379]
3. One assay plate was placed at room temperature and the other was heated for a period of time (30 minutes initially) (55 ° C. used for initial screening).
[0380]
4. After the specified time, 0.08 mL of room temperature substrate (1.5 mM NaN₃Saturated with 1 mM Tris / SO containing 0.1 mM and 0.1 mM bromothymol blue₄ ^2-, PH 7.8) was added to each well.
[0381]
5. Measurements at 620 nm were taken at various time points to generate a progress curve for each well.
[0382]
6. The data was analyzed to compare the kinetics of heated and unheated cells. Each plate contained 1-2 columns (24 wells) of non-mutated 20F12 controls.
[0383]
7. Wells that appeared to have improved stability were grown again and tested under the same conditions.
[0384]
After this procedure, nine single-site mutations appeared to have given the enzyme increased thermostability. Sequence analysis was performed to determine the exact amino acid change at each position that essentially caused this improvement. Briefly, the improvement was provided by one amino acid change at the seven sites, two amino acid changes at the eighth site, and three amino acid changes at the ninth site, respectively. We then made several mutations, each of which had some combination of these nine beneficial site mutations, two of which were single-point mutations. Was found to be superior to all other mutations, including
[0385]
Example 11
Direct expression cloning using end selection
The esterase gene was amplified using a 5 ′ phosphorylated primer in a standard PCR reaction (10 ng template, PCR conditions: 3 min 94 ° C., [1 min 94 ° C., 1 min 50 ° C., 1 min 30 sec 68 ° C] x 30, 10 minutes 68 ° C).
[0386]
Forward primer = 9511TopF (CTAGAAGGGAGGAGAATTACATGAAGCGGCTTTTTAGCCC)
Reverse primer = 9511TopR (AGCTAAGGGTCAAGGCCGCACCCGGAGG)
The resulting PCR product (about 1000 bp) was gel purified and quantified.
[0387]
The vector for expression cloning, pASK3 (Institut fuel Bioanalytik, Göttingen, Germany) was cut with Xba I and Bgl II and dephosphorylated with CIP.
[0388]
0.5 pmole Vaccina Topoisomerase (Invitrogen, Carlsbad, CA) was added to 60 ng (approximately 0.1 pmole) of the purified PCR product in buffer NEB I (New England Biolabs, Beverly, MA), for a total volume of 5 μl. 37 ° C. for minutes. The topogated PCR product was cloned with the vector pASK3 (5 μl, about 200 ng in NEBI) and brought to room temperature for 5 minutes. This mixture is₂Dialyzed against O for 30 minutes. 2 μl was used for electroporation of DH10B cells (Gibco BRL, Gaithersburg, MD).
[0389]
Efficiency: Based on the actual number of clones, the method was 2x10 / μg vector⁶Clones can be generated.
[0390]
Example 12
Thermostability of dehalogenase
In the present invention, desirable properties produced by directed evolution include improved residue activity (eg, enzymatic activity) of molecules exposed to a changed environment for a specified period of time, including those that are considered harsh environments. , Immunoreactivity, antibiotic activity, etc.). Such harsh environments may include any combination of the following (with or without repetition and in any order or permutation): high temperature (including the temperature that causes denaturation of the working enzyme), low temperature, high salt Degree, low salinity, high pH, low pH, high pressure, low pressure, and changes in exposure to radiation sources (including ultraviolet radiation, visible light, and the entire electromagnetic spectrum).
[0391]
According to the present invention, the desired properties such as improved activity can be achieved in harsh environments (eg, high temperature, low temperature, high pH, low pH, high salinity, low salinity, increased organic solvent presence, increased organic solvent content). (E.g., reduced presence, etc.) and can have improved activity (e.g., higher activity) after maintaining for a specified period of time, and can include selection criteria for screening or selection steps following the application of directed evolution steps Some of which include end selection-based steps, exonuclease treatment-based steps, site saturation mutagenesis-based steps, or synthetic ligation reconstruction-based steps.
[0392]
According to another aspect of the present invention, the desired properties such as improved activity may be in harsh environments (eg, high temperature, low temperature, high pH, low pH, high salinity, low salinity, increased presence of organic solvents). , Reduction of the presence of organic solvents, etc.) and improved mobility (such as high activity) after removal therefrom, and the choice of a screening or selection step following the application of a directed evolution step. Criteria can be included, some of which include end selection based steps, exonuclease treatment based steps, site saturation mutagenesis based steps, or synthetic ligation reconstruction based steps.
[0393]
In a preferred embodiment, the residue activity is such that after exposure to denaturing conditions, the active three-dimensional form (the original three-dimensional form, a three-dimensional form similar to the original three-dimensional form, or the original three-dimensional form) (Including a different three-dimensional form). Such ability to fold again, sometimes referred to as "denaturation reversibility" or "regeneration ability" or "denaturation-regeneration reversibility", is a selection parameter according to the present invention.
[0394]
The following example illustrates the application of directed evolution to evolve the ability of enzymes to regenerate and / or maintain activity when exposed to high temperatures. All residues (316) of the dehalogenase enzyme were converted to a total of 20 amino acids by site-directed mutagenesis using a 32-fold degenerate oligonucleotide primer. This mutation was introduced into mutant Dhla 20F12, which already had an improved ratio. About 200 clones at each location were grown in liquid medium (384-well microtiter plates) for screening. The screening procedure is as follows.
[0395]
1. The overnight culture in a 384-well plate was centrifuged to remove the medium. 0.06 mL 1 mM Tris / SO in each well₄ ^2-, PH 7.8 was added.
[0396]
2. The robot made sure that two assay plates from each parent growth plate consisted of 0.02 mL cell suspension.
[0397]
3. One assay plate was placed at room temperature and the other was heated for a period of time (30 minutes initially) (55 ° C. used for initial screening).
[0398]
4. After the specified time, 0.08 mL of room temperature substrate (1.5 mM NaN₃Saturated with 1 mM Tris / SO containing 0.1 mM and 0.1 mM bromothymol blue₄ ^2-, PH 7.8) was added to each well. TCP = trichloropropane.
[0399]
5. Measurements at 620 nm were taken at various time points to generate a progress curve for each well.
[0400]
6. The data was analyzed to compare the kinetics of heated and unheated cells. Each plate contained 1-2 columns (24 wells) of non-mutated 20F12 controls.
[0401]
7. Wells that appeared to have improved stability were grown again and tested under the same conditions.
[0402]
Following this procedure, nine single-site mutations appeared to have conferred increased thermal stability on Dhla-20F12. Sequence analysis revealed that the following changes were beneficial.
[0403]
D89G
F91S
T159L
G189Q, G189V
I220L
N238T
W251Y
P302A, P302L, P302S, P302K
P302R / S306R
Only two sites (189 and 302) had more than one substitution. The first five in the list were linked to a single gene (using G189Q) (this mutation is called "Dhla5"). All changes except S306R have been incorporated into another mutant called Dhla8.
[0404]
The thermostability was evaluated by culturing the enzyme at a high temperature (55 ° C. and 80 ° C.) for a certain period and assaying the activity at 30 ° C. Initial velocity was plotted against time at elevated temperature. The enzyme was used at 50 mM Tris SO in both culture and assay.₄, PH 7.8. Product (Cl⁻) Is Fe (NO)₃)₃And HgSCN using standard methods. Dhla 20F12 was used as the de facto wild type. Apparent half-life (T_1/2) Was calculated by fitting the data to an exponential decay function.
[0405]
Thermostability of Dhla5 dehalogenase at 55 ° C. 50 mM Tris SO₄ ^2-The purified protein in pH 7.8 was incubated at 55 ° C. for various periods of time, aliquots were removed and assayed for trichloropropane (TCP) hydrolysis. This assay was performed using 50 mM Tris SO₄ ^2-, PH 7.8 at 30 ° C with 10 mM TCP. Product (Cl⁻) Is released from standard Cl⁻Analyzed by a test. The initial rate (A450 / min) was derived from an enzyme progress curve and plotted against incubation time. This data has an exponential decay (C^*exp^-Kt) Where t_1/2= Ln2 / k (see FIG. 13).
[0406]
Enzyme progress curves of Dhla and 20F12 at 30 ° C. and 55 ° C. (see FIG. 14). This reaction is performed using purified protein at 50 mM Tris SO₄ ^2-, PH 7.8, performed with 10 mM TCP. Equal amounts of protein were used in each assay. Product (Cl⁻) Is the standard Cl⁻The test was used to determine at various time points. Dhla 5 appears to have the same specific activity as 20F12 at both 30 ° C. and 55 ° C., but the enzyme continues to form after 20F12 becomes thermally inactive.
[0407]
Thermostability of Dhla5 dehalogenase at 80 ° C. 50 mM Tris SO₄ ^2-, PH 7.8, were incubated at 80 ° C. for various periods of time, aliquots were removed and assayed for trichloropropane (TCP) hydrolysis. This assay was performed using 50 mM Tris SO₄ ^2-, PH 7.8 at 30 ° C with 10 mM TCP. Product (Cl⁻) Is released from standard Cl⁻Analyzed by a test. The initial rate (A450 / min) was derived from an enzyme progress curve and plotted against incubation time. This data has an exponential decay (C^*exp^-Kt) Where t_1/2= Ln2 / k (see FIG. 15).
[0408]
Thermal stability of Dhla5 and Dhla8 at 80 ° C. 50 mM Tris SO₄ ^2-The purified protein in pH 7.8 was incubated at 80 ° C. for various periods of time, aliquots were removed and assayed for trichloropropane (TCP) hydrolysis. This assay was performed using 50 mM Tris SO₄ ^2-, PH 7.8 at 30 ° C with 10 mM TCP. Product (Cl⁻) Is released from standard Cl⁻Analyzed by a test. The initial rate (A450 / min) was derived from the enzyme progress curve and the relative rate was plotted against the incubation time. This data has an exponential decay (C^*exp^-Kt) Where t_1/2= In2 / k, giving values of 13 and 138 minutes for Dhla5 and Dhla8, respectively (see FIG. 16).
[0409]
Indicative scanning calorimetry of such dehalogenase variants is displayed in FIGS. 17A, B, and C. The data were recorded using a Perkin Elmer Pyris I at a scan rate of 10 ° C./min. The protein concentration is 10 mg / ml in 40 μl (0.4 mg total protein). Buffer is 10 mM NaP_i, PH 7.5. Dissolution temperatures derived from the data were 68 ° C (20F12), 73 ° C (Dhla5), and 73 ° C (Dhla8). The data further shows that 20F12 has undergone irreversible denaturation, Dhla5 is partially reversible, and Dhla8 is completely reversible (see FIG. 17).
[0410]
Thermostability of wild-type (20F12) and mutant (Dhla5) dehalogenase at 55 ° C. 50 mM Tris SO₄ ^2-The purified protein in pH 7.8 was incubated at 55 ° C. for various periods of time, aliquots were removed and assayed for trichloropropane (TCP) hydrolysis. This assay was performed using 50 mM Tris SO₄ ^2-, PH 7.8 at 30 ° C with 10 mM TCP. Product (Cl⁻) Is released from standard Cl⁻Analyzed by a test. The initial rate (A450 / min) was derived from an enzyme progress curve and plotted against incubation time. This data has an exponential decay (C^*exp^-Kt) Where t_1/2= Ln2 / k (see FIG. 12).
[0411]
Many modifications and variations of the present invention are possible in light of the above teachings, so that, within the scope of the appended claims, the invention may be practiced other than as specifically described. While the present invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations fall within the spirit and scope of the described and claimed invention.
[Brief description of the drawings]
FIG. 1 shows a preferred embodiment of site saturation mutagenesis.
FIG. 2 shows the generation of an exhaustive set of chimeric linkages by synthetic ligation rearrangement.
3A and 3B. Select end-points using a topoisomerase-derived selection step to select the desired polynucleotide (eg, perform full-length molecule selection) produced by polynucleotide rearrangement (eg, by synthetic ligation rearrangement). It is a figure showing an application.
FIG. 4 illustrates various aspects of the end selection technique.
FIG. 5. In order to achieve directed expression cloning of the original blunt-ended DNA, the enzyme N.A. FIG. 3 illustrates a particular embodiment of the end-selection technique in which the step of “c) performing directed expression cloning” is accomplished using BstNB1.
FIG. 6 shows the activity of the enzyme exonuclease III. The asterisk indicates that the enzyme works from the 3 'direction to the 5' direction of the polynucleotide substrate.
FIG. 7 illustrates an exemplary application of the enzyme exonuclease III in exonuclease-mediated polynucleotide reconstitution.
FIG. 8 is a diagram showing generation of a multi-bond nucleic acid chain.
FIG. 9: In the heteromeric nucleic acid complex, the 3 ′ and 5 ′ terminal nucleotides are released from the unhybridized single-stranded end of the annealed nucleic acid strand, leaving the shortened but hybridized end. FIG. 3 illustrates the use of exonuclease treatment as a means to promote polymerase-based extension of the terminated ends and / or ligase-mediated ligation.
FIG. 10 illustrates the use of exonuclease-mediated nucleic acid rearrangement in an example of annealing a single methylated multi-linked nucleic acid strand into several unmethylated single-linked nucleic acid strands.
FIG. 11 illustrates the use of exonuclease-mediated nucleic acid rearrangement in an example of annealing a plurality of methylated multi-binding nucleic acid strands into several unmethylated single-binding nucleic acid strands.
FIG. 12 is a graph showing the thermostability of wild-type (20F12) and mutant (Dhla 5) dehalogenase at 55 ° C.
FIG. 13 shows a graph of the thermostability of Dhla 5 dehalogenase at 55 ° C.
FIG. 14 shows enzyme progress curves of Dhla 5 and 20F12 at 30 ° C. and 55 ° C.
FIG. 15 shows the thermostability of Dhla 5 dehalogenase at 80 ° C. This data is t_1/2= Ln2 / k exponential decay (C^*exp^-Kt).
FIG. 16 shows the thermostability of Dhla 5 and Dhla 8 dehalogenase at 80 ° C. This data gives values of 13 and 138 minutes for Dhla 5 and Dhla 8, respectively._1/2= Ln2 / k exponential decay (C^*exp^-Kt).
FIG. 17 (FIGS. 17A, 17B, and 17C) shows differential scanning calorimetry of three dehalogenase variants. This data was recorded using a Perkin Elmer Pyris 1 at a scan rate of 10 ° C./min.
[Sequence list]

Claims (62)

A method for obtaining a predetermined biological activity or biomolecule,
a) screening a library of clones produced by nucleic acids from a mixed population of cells for a particular biological activity or biomolecule;
b) altering a nucleic acid sequence contained in a clone having a particular biological activity or biomolecule;
c) comparing the biological activity or biomolecule from step b) to a specific biological activity or biomolecule, wherein the difference in the biological activity or biomolecule indicates the effect of the sequence change, whereby Providing a molecule;
A method comprising: The method according to claim 1, wherein the biomolecule is a nucleic acid sequence. 3. The method according to claim 2, wherein the nucleic acid sequence is a DNA or RNA sequence. The nucleic acid sequence is
Contacting the nucleic acid contained in the clone with at least one oligonucleotide probe containing a detectable molecule and at least a part of a predetermined nucleic acid sequence,
Identifying a nucleic acid sequence comprising complement to at least one oligonucleotide probe by an analyzer that detects a detectable signal from the detectable molecule;
3. The method of claim 2, which is screened by: 5. The method according to claim 4, wherein the detectable molecule is a chromogenic or fluorescent substrate. The method of claim 4, wherein the detectable signal is fluorescence. The method according to claim 5, wherein the fluorescent substrate is umbelliferone or a derivative or analog thereof, resorufin or a derivative or analog thereof, fluorescein or a derivative or analog thereof, or rhodamine or a derivative or analog thereof. 5. The method of claim 4, wherein the detectable molecule is a detectably labeled oligonucleotide having a sequence encoding a given polynucleotide or fragment thereof. 9. The method of claim 8, wherein the detectably labeled oligonucleotide is labeled with a fluorescent molecule. 3. The method of claim 2, wherein the screening is by PCR amplification of the predetermined nucleic acid sequence using a primer having a detectable molecule that substantially complements the predetermined sequence or the sequence flanking the predetermined nucleic acid sequence. the method of. 3. The method of claim 2, wherein the screening is by hybridization of an oligonucleotide that substantially complements the predetermined nucleic acid sequence. 3. The method of claim 2, further comprising comparing the altered nucleic acid sequence with the unaltered nucleic acid sequence of step (c), thereby identifying an altered nucleotide sequence. 13. The method of claim 12, wherein the comparison is performed using a sequence comparison algorithm. 2. The method of claim 1, wherein the biological activity is provided by a polypeptide. 2. The method according to claim 1, wherein the biological activity is an enzymatic activity. Enzyme activity, lipase, esterase, protease, glycosidase, glycosyltransferase, phosphatase, kinase, mono- and dioxygenase, haloperoxidase, lignin peroxidase, diallyl propane peroxidase, epoxide hydrolase 16. The method of claim 15, wherein the method is provided by an enzyme selected from the group consisting of: nitrile hydratase, nitrilase, transaminase, amidase, and acylase. 2. The method according to claim 1, wherein the library is an expression library. 2. The method of claim 1, wherein the library comprises DNA obtained from an environmental sample. 2. The method of claim 1, wherein the library comprises DNA obtained from an extreme environment microorganism. 20. The method of claim 19, wherein the extreme environmental microorganism is a thermophilic organism. Extreme environmental microorganisms are selected from the group consisting of hyperthermophilic organisms, cold and warm organisms, halophilic organisms, psychrotrophs, alkalophilic organisms, and acidophilic organisms, The method of claim 20. The screening step comprises contacting the clone with a substrate labeled with a detectable molecule, wherein the interaction of the substrate with a biological activity or biomolecule contained in the clone produces a detectable signal. The method of claim 17. 23. The method of claim 22, wherein the substrate is a biologically active substrate. 23. The method of claim 22, wherein the bioactive substrate comprises C12FDCG. The substrate includes a first test protein linked to a DNA binding moiety and a second test protein linked to a transcriptionally active moiety, wherein the first test protein linked to the DNA binding moiety and the second test protein linked to the transcriptionally active moiety. 23. The method of claim 22, wherein modulating interaction with the second test protein results in a change in expression of the detectable protein. 23. The method of claim 22, wherein the screening is by expression of a nucleic acid. 2. The method of claim 1, further comprising, prior to step (d), obtaining nucleic acids from a clone containing the particular biological activity or biomolecule. Obtaining the nucleic acid contained in the clone comprises contacting the clone with a complementary nucleic acid or a fragment thereof, thereby allowing hybridization of the cloned nucleic acid with the complementary nucleic acid and its separation. Item 28. The method according to Item 27. 29. The method of claim 28, wherein the complementary nucleic acid or fragment thereof comprises a solid phase binding hybridization probe. Error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, and recursive ensemble mutation of nucleic acid sequences The method of claim 1, wherein the altering is provided in a manner selected from a group consisting of generation, exponential ensemble mutagenesis, site-directed mutagenesis, ligation rearrangement, GSSM, and any combination thereof. the method of. The method according to claim 1, wherein the nucleic acid sequence is changed by error-prone PCR. 2. The method of claim 1, wherein the nucleic acid sequence is altered by shuffling. 2. The method of claim 1, wherein the nucleic acid sequence is altered by oligonucleotide directed mutagenesis. 2. The method of claim 1, wherein the nucleic acid sequence is altered by assembly PCR. 2. The method of claim 1, wherein the nucleic acid sequence is altered by sexual PCR mutagenesis. 2. The method of claim 1, wherein the nucleic acid sequence is altered by in vivo mutagenesis. 2. The method of claim 1, wherein the nucleic acid sequence is altered by cassette mutagenesis. 2. The method of claim 1, wherein the nucleic acid sequence is altered by recursive ensemble mutagenesis. 2. The method of claim 1, wherein the nucleic acid sequence is altered by exponential ensemble mutagenesis. 2. The method of claim 1, wherein the nucleic acid sequence is altered by site-directed mutagenesis. 2. The method of claim 1, comprising screening the clone of step (c) for further specific protein or enzyme activity prior to the step of altering the nucleic acid. 2. The method of claim 1, wherein the library is produced in a prokaryotic cell. 2. The method of claim 1, wherein the library is generated in a Streptomyces species. 44. The method of claim 43, wherein the Streptomyces is Streptomyces venezuelae. 43. The method of claim 42, wherein the prokaryotic cells are Gram negative. 43. The method of claim 42, wherein the prokaryotic cell is a Bacillus species. 43. The method of claim 42, wherein the prokaryotic cell is a Pseudomonas species. Libraries are screened by contacting, or encapsulating, a clone of the library with a biologically active substrate so that the biological activity or biomolecules produced by the clones can be compared after contact with the substrate before contacting the clones. The method of claim 1, wherein the difference is detected by a compared difference. 2. The method of claim 1, wherein the libraries are averaged prior to the step of screening the libraries. The method according to claim 1, wherein the biological activity or biomolecule is a gene group or a fragment thereof. 2. The method of claim 1, wherein the biological activity or biomolecule is a polypeptide that is in a metabolic pathway. A method for identifying a given biological activity or biomolecule,
a) screening a library of clones generated by pooled nucleic acids obtained from the plurality of isolates for a particular biological activity or biomolecule;
b) identifying a clone containing a particular biological activity or biomolecule;
A method comprising: A method for identifying a given biological activity or biomolecule,
a) screening a library of clones generated by pooled nucleic acids obtained from the plurality of isolates for a particular biological activity or biomolecule;
b) altering a nucleic acid sequence contained in a clone having a particular biological activity or biomolecule;
c) comparing the biological activity or biomolecule from b) to a specific biological activity or biomolecule, wherein the difference in the biological activity or biomolecule indicates the effect of introducing at least one sequence change, Providing a predetermined biological activity or biomolecule;
A method comprising: A method for identifying a given biological activity or biomolecule,
a) screening a library of clones generated from pooling individual gene libraries generated by nucleic acids obtained from each of the plurality of isolates for a particular biological activity or biomolecule;
b) identifying a clone containing a particular biological activity or biomolecule;
A method comprising: A method for identifying a given biological activity or biomolecule,
a) screening the library for a particular biological activity or biomolecule, wherein the library is generated from pooling individual gene libraries generated by nucleic acids obtained from each of a plurality of isolates; ,
b) altering a nucleic acid sequence contained in a clone having a particular biological activity or biomolecule;
c) comparing the bioactivity or biomolecule from step c) to a particular bioactivity or biomolecule, indicating the effect of the difference in bioactivity or biomolecule introducing at least one sequence change, Providing a predetermined biological activity or biomolecule,
A method comprising: A method for identifying a given biological activity or biomolecule,
(A) screening a library of clones generated by nucleic acids from an enriched population of organisms for a particular biological activity or biomolecule;
(B) identifying a clone containing a particular biological activity or biomolecule;
A method comprising: A method for identifying a given biological activity or biomolecule,
(A) screening a library of clones generated by nucleic acids from an enriched population of organisms for a particular biological activity or biomolecule;
(B) altering a nucleic acid sequence contained in a clone having a specific biological activity or biomolecule;
(C) comparing the bioactivity or biomolecule from step b) to a particular bioactivity or biomolecule, indicating the effect of the difference in bioactivity or biomolecule introducing at least one sequence change; Thereby providing a predetermined biological activity or biomolecule;
A method comprising: A method for identifying a given biological activity or biomolecule,
(A) enabling the interaction of a complementary sequence of a nucleic acid from a mixed population of organisms with at least one oligonucleotide probe containing a detectable molecule and at least a portion of the nucleic acid sequence encoding a given molecule. Culturing under such conditions and for a time that allows the interaction of the complementary sequences,
(B) using an analyzer to detect the detectable molecule, identifying a nucleic acid sequence having a complement to the oligonucleotide probe;
(C) generating a library from the identified nucleic acid sequence;
(D) screening the library for a particular biological activity or biomolecule;
(E) altering a nucleic acid sequence contained in a clone having a particular biological activity or biomolecule;
(F) comparing the bioactivity or biomolecule from step (e) to a particular bioactivity or biomolecule, indicating the effect of the difference in bioactivity or biomolecule introducing at least one sequence change. Providing a predetermined biological activity or biomolecule,
A method comprising: A method for identifying a given biological activity or biomolecule,
(A) enabling the interaction of a complementary sequence with a nucleic acid obtained from a mixed population of organisms, together with at least one oligonucleotide probe containing a detectable molecule and at least a portion of a nucleic acid sequence encoding a given molecule. Co-encapsulating in a microenvironment under such conditions and for a time such that complementary sequence interactions are possible;
(B) identifying an encapsulated nucleic acid containing a complement to an oligonucleotide probe encoding a predetermined molecule by separating the encapsulated nucleic acid with an analyzer that detects a detectable molecule;
(C) generating a library from the separated encapsulated nucleic acids;
(D) screening the library for a particular biological activity or biomolecule;
(E) altering a nucleic acid sequence contained in a clone having a particular biological activity or biomolecule;
(F) comparing the bioactivity or biomolecule from step (e) to a particular bioactivity or biomolecule, indicating the effect of the difference in bioactivity or biomolecule introducing at least one sequence change. Providing a predetermined biological activity or biomolecule,
A method comprising: A method for identifying a given biological activity or biomolecule,
(A) combining a nucleic acid obtained from a segregate of a mixed population of organisms with at least one oligonucleotide probe comprising a detectable marker and at least a portion of a polynucleotide sequence encoding a molecule having a predetermined biological activity. Co-encapsulating in a microenvironment under conditions that allow interaction of the complementary sequences and for a time that allows interaction of the complementary sequences;
(B) identifying an encapsulated nucleic acid containing a complement to an oligonucleotide probe encoding a predetermined molecule by separating the encapsulated nucleic acid by an analyzer that detects a detectable marker;
(C) generating a library from the separated encapsulated nucleic acids;
(D) screening the library for a particular biological activity or biomolecule;
(E) altering a nucleic acid sequence contained in a clone having a particular biological activity or biomolecule;
(F) comparing the bioactivity or biomolecule from step (e) to a particular bioactivity or biomolecule, indicating the effect of the difference in bioactivity or biomolecule introducing at least one sequence change. Providing a predetermined biological activity or biomolecule,
A method comprising: A method for identifying a given biological activity or biomolecule,
(A) combining nucleic acid obtained from one or more isolates of a mixed population of organisms with at least one oligonucleotide probe containing a detectable marker and a polynucleotide sequence encoding a molecule having a predetermined biological activity. Co-encapsulating in a microenvironment, at least in part, under conditions such that complementary sequence interaction is possible and for a time such that complementary sequence interaction is possible;
(B) identifying the encapsulated nucleic acid containing a complement to an oligonucleotide probe encoding a predetermined molecule by separating the encapsulated nucleic acid with an analyzer that detects a detectable marker;
(C) generating a library from the separated encapsulated nucleic acids;
(D) screening the library for a particular biological activity or biomolecule;
(E) altering a nucleic acid sequence contained in a clone having a particular biological activity or biomolecule;
(F) comparing the bioactivity or biomolecule from step (e) to a particular bioactivity or biomolecule, indicating the effect of the difference in bioactivity or biomolecule introducing at least one sequence change. Providing a predetermined biological activity or biomolecule,
A method comprising: A method for identifying a given biological activity or biomolecule,
(A) combining a nucleic acid obtained from a mixture of isolates of a mixed population of organisms with at least one oligonucleotide probe containing a detectable marker and at least one polynucleotide sequence encoding a molecule having a predetermined biological activity. Co-encapsulating in a microenvironment under conditions that allow interaction of the complementary sequences and for a time that allows interaction of the complementary sequences,
(B) identifying the encapsulated nucleic acid containing a complement to an oligonucleotide probe encoding a predetermined molecule by separating the encapsulated nucleic acid with an analyzer that detects a detectable marker;
(C) generating a library from the separated encapsulated nucleic acids;
(D) screening the library for a particular biological activity or biomolecule;
(E) altering a nucleic acid sequence contained in a clone having a particular biological activity or biomolecule;
(F) comparing the bioactivity or biomolecule from step (e) to a particular bioactivity or biomolecule, indicating the effect of the difference in bioactivity or biomolecule introducing at least one sequence change. Providing a predetermined biological activity or biomolecule,
A method comprising:

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