JP2004500812A - Protease - Google Patents

Abstract

The present invention relates to human proteases (PRTS) and polynucleotides that identify and encode PRTS. The invention also provides expression vectors and host cells, antibodies, agonists, antagonists. Further, the present invention provides a method for diagnosing, treating, and preventing a disease associated with abnormal expression of PRTS.

Description

として定義される積スコアである。積スコアは、０〜１００の標準化された値であり、以下のように求める。ＢＬＡＳＴスコアにヌクレオチド配列の一致率を乗じ、その積を２つの配列の短い方の長さの５倍で除する。高スコアのセグメントの対（ＨＳＰ）において一致する各塩基に＋５のスコアを割り当て、各不適性塩基対に−４を割り当てることにより、ＢＬＡＳＴスコアを計算する。２つの配列は、２以上のＨＳＰを共有し得る（ギャップにより離隔される）。２以上のＨＳＰがある場合には、最高ＢＬＡＳＴスコアの塩基対を用いて積スコアを計算する。積スコアは、ＢＬＡＳＴアラインメントの断片的重複と質とのバランスを表す。例えば積スコア１００は、比較した２つの配列の短い方の長さ全体にわたって１００％一致する場合にのみ得られる。積スコア７０は、１００％の一致で一端が７０％重畳しているか、或いは８８％一致で他端が１００％重畳しているかの何れかの場合である。積スコア５０は、１００％の一致で一端が５０％重畳しているか、或いは７９％の一致で他端が１００％重畳しているかの何れかの場合である。
【０２５８】
或いは、ＰＲＴＳをコードするポリヌクレオチド配列は、由来する組織に対して分析する。例えば、ある完全長の配列は、少なくとも部分的にインサイトｃＤＮＡ配列をオーバーラップさせて組み立てられる（実施例３を参照）。各ｃＤＮＡ配列は、ヒト組織から作製されたｃＤＮＡライブラリに由来する。各ヒト組織は、心血管系、結合組織、消化系、胚構造、内分泌系、外分泌腺、女性生殖器、男性生殖器、生殖細胞、血液および免疫系、肺、筋骨格系、神経系、膵臓、呼吸器系、感覚器官、皮膚、顎口腔系、分類不能／混合、または尿管などの１つの生物／組織のカテゴリーに分類される。各カテゴリーにおけるライブラリの数をカウントし、その合計数を全カテゴリーのライブラリ数で除す。同様に、各ヒト組織は、癌、細胞系、発生、炎症、神経、外傷、心血管、プール（ｐｏｏｌｅｄ）などの１つの疾患／症状のカテゴリーに分類され、各カテゴリーにおけるライブラリの数をカウントし、その合計数を全カテゴリーのライブラリ数で除す。得られるパーセンテージは、ＰＲＴＳをコードするｃＤＮＡの疾患特異的な発現を反映する。ｃＤＮＡ配列およびｃＤＮＡライブラリ／組織の情報は、ＬＩＦＥＳＥＱ ＧＯＬＤ データベース（Ｉｎｃｙｔｅ Ｇｅｎｏｍｉｃｓ， Ｐａｌｏ Ａｌｔｏ ＣＡ）から得ることができる。
【０２５９】
８　 ＰＲＴＳ をコードするポリヌクレオチドの伸長
完全長のポリヌクレオチド配列は、完全長分子の好適な断片から設計したオリゴヌクレオチドプライマーを用いてその完全長分子の好適な断片を伸長して作製した。一方のプライマーは既知の断片の５’の伸長を開始するために合成し、他方のプライマーは既知の断片の３’の伸長を開始するために合成した。開始プライマーは、ＯＬＩＧＯ ４．０６ソフトウェア（Ｎａｔｉｏｎａｌ Ｂｉｏｓｃｉｅｎｃｅｓ）或いは他の適切なプログラムを用いて、約２２個から約３０個のヌクレオチドの長さで約５０％以上のＧＣ含量を有し、かつ約６８〜７２℃の温度で標的配列にアニールするように設計した。ヘアピン構造及びプライマー−プライマー二量体が生じないようにヌクレオチドを伸長した。
【０２６０】
選択されたヒトｃＤＮＡライブラリを用いてこの配列を伸長した。２段階以上の伸長が必要な場合、若しくは望ましい場合は、追加或いはネスト化プライマーの組を設計する。
【０２６１】
当分野で既知の方法を利用したＰＣＲ法で高い忠実度で増幅した。ＰＣＲはＰＴＣ−２００ ｔｈｅｒｍａｌ ｃｙｃｌｅｒ （ＭＪ Ｒｅｓｅａｒｃｈ， Ｉｎｃ．）用いて９６ウェルブロックプレートで行った。反応混合液は、鋳型ＤＮＡ及び２００ ｎｍｏｌの各プライマー、Ｍｇ^{２ ＋}と（ＮＨ_４）_２ＳＯ_４とβ−メルカプトエタノールを含むバッファー、Ｔａｑ ＤＮＡポリメラーゼ（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）、ＥＬＯＮＧＡＳＥ酵素（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）、Ｐｆｕ ＤＮＡポリメラーゼ（Ｓｔｒａｔａｇｅｎｅ）を含む。プライマーの組、ＰＣＩ ＡとＰＣＩ Ｂに対して以下のパラメーターで増幅を行った。
ステップ１　　９４℃で３分間
ステップ２　　９４℃で１５秒
ステップ３　　６０℃で１分間
ステップ４　　６８℃で２分間
ステップ５　　ステップ２、３、及び４を２０回繰り返す
ステップ６　　６８℃で５分間
ステップ７　　４℃で保管
別法では、プライマーの組、Ｔ７とＳＫ＋に対して以下のパラメーターで増幅を行った。
ステップ１　　９４℃で３分間
ステップ２　　９４℃で１５秒
ステップ３　　５７℃で１分間
ステップ４　　６８℃で２分間
ステップ５　　ステップ２、３、及び４を２０回繰り返す
ステップ６　　６８℃で５分間
ステップ７　　４℃で保管。
【０２６２】
各ウェルのＤＮＡ濃度は、１Ｘ ＴＥ及び０．５μｌの希釈していないＰＣＲ産物に溶解した１００μｌのＰＩＣＯＧＲＥＥＮ定量試薬（０．２５％ （ｖ／ｖ） ＰＩＣＯＧＲＥＥＮ； Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ， Ｅｕｇｅｎｅ ＯＲ）を不透明な蛍光光度計プレート（Ｃｏｍｉｎｇ Ｃｏｓｔａｒ， Ａｃｔｏｎ ＭＡ）の各ウェルに分配してＤＮＡが試薬と結合できるようにして測定する。このプレートをＦｌｕｏｒｏｓｋａｎ ＩＩ （Ｌａｂｓｙｓｔｅｍｓ Ｏｙ， Ｈｅｌｓｉｎｋｉ， Ｆｉｎｌａｎｄ）でスキャンして、サンプルの蛍光を計測してＤＮＡの濃度を定量化する。反応混合物の５〜１０μｌのアリコットを１％のアガロースミニゲル上での電気泳動によって解析し、何れの反応物が配列を伸長することに成功したかを決定する。
【０２６３】
伸長したヌクレオチドを脱塩及び濃縮してから３８４ウェルプレートに移し、ＣｖｉＪＩコレラウィルスエンドヌクレアーゼ（Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ Ｒｅｓｅａｒｃｈ， Ｍａｄｉｓｏｎ ＷＩ）で消化し、ｐＵＣ １８ベクター（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）に再連結する前に音波処理またはせん断を行った。ショットガンシークエンシングのために、消化したヌクレオチドを低濃度（０．６〜０．８％）のアガロースゲル上に分離して断片を切断し、寒天をＡｇａｒ ＡＣＥ （Ｐｒｏｍｅｇａ）で消化した。Ｔ４リガーゼ（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ， Ｂｅｖｅｒｌｙ ＭＡ）を用いて伸長したクローンをｐＵＣ １８ベクター（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）に再連結し、Ｐｆｕ ＤＮＡポリメラーゼ（Ｓｔｒａｔａｇｅｎｅ）で制限部位の延び出しを処理してコンピテント大腸菌細胞に形質移入した。形質移入した細胞を選択して抗生物質を含む培地に移し、それぞれのコロニーを切りとってＬＢ／２Ｘカルベニシリン培養液の３８４ウェルプレートに３７℃で一晩培養した。
【０２６４】
細胞を溶解して、Ｔａｑ ＤＮＡポリメラーゼ（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）及びＰｆｕ ＤＮＡポリメラーゼ（Ｓｔｒａｔａｇｅｎｅ）を用いて以下の手順でＤＮＡをＰＣＲ増幅した。
ステップ１　　９４℃で３分間
ステップ２　　９４℃で１５秒
ステップ３　　６０℃で１分間
ステップ４　　７２℃で２分間
ステップ５　　ステップ２、３、及び４を２９回繰り返す
ステップ６　　７２℃で５分間
ステップ７　　４℃で保管。
上記したようにＰＩＣＯＧＲＥＥＮ試薬（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ）でＤＮＡを定量化した。ＤＮＡ回収率の悪いサンプルは、上記した条件で再び増幅した。サンプルを２０％のジメチルサルホサイド（ｄｉｍｅｔｈｙｓｕｌｐｈｏｘｉｄｅ）（１：２， ｖ／ｖ）で希釈し、ＤＹＥＮＡＭＩＣ ＤＩＲＥＣＴキット（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）またはＡＢＩ ＰＲＩＳＭ ＢＩＧＤＹＥ Ｔｅｒｍｉｎａｔｏｒ ｃｙｃｌｅ ｓｅｑｕｅｎｃｉｎｇ ｒｅａｄｙ ｒｅａｃｔｉｏｎキット（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いてシークエンシングした。
【０２６５】
同様に上述の手順で、完全長のポリヌクレオチド配列を検査したり、或いは完全長のポリヌクレオチド配列を利用して、この伸長のために設計したオリゴヌクレオチドと好適なゲノムライブラリを用いて５′調節配列を得た。
【０２６６】
９　個々のハイブリダイゼーションプローブの標識化及び使用法
ＳＥＱ ＩＤ ＮＯ：１５−２８から導き出されたハイブリダイゼーションプローブを用いて、ｃＤＮＡ、ｍＲＮＡ、またはゲノムＤＮＡをスクリーニングする。約２０塩基対からなるオリゴヌクレオチドの標識について特に記すが、より大きなｃＤＮＡフラグメントの場合でも基本的に同じ手順を用いる。オリゴヌクレオチドを、ＯＬＩＧＯ４．０６ソフトウェア（Ｎａｔｉｏｎａｌ Ｂｉｏｓｃｉｅｎｃｅ）のような最新式のソフトウェアを用いてデザインし、５０ｐｍｏｌの各オリゴマーと、２５０μＣｉの［γ‐^３２Ｐ］アデノシン三リン酸（Ａｍｅｒｓｈａｍ， Ｃｈｉｃａｇｏ， ＩＬ）及びＴ４ポリヌクレオチドキナーゼ（ＤｕＰｏｎｔ ＮＥＮ、Ｂｏｓｔｏｎ ＭＡ）とを組み合わせて用いることにより標識する。標識されたオリゴヌクレオチドを、ＳＥＰＨＡＤＥＸ Ｇ−２５超精細排除デキストランビードカラム（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）を用いて実質的に精製する。毎分１０^７カウントの標識されたプローブを含むアリコットを、次のエンドヌクレアーゼ、Ａｓｅ Ｉ、Ｂｇｌ ＩＩ、Ｅｃｏ ＲＩ、Ｐｓｔ Ｉ、Ｘｂａ１或いはＰｖｕ ＩＩ（ＤｕＰｏｎｔ ＮＥＮ）の１つを用いて切断したヒトゲノムＤＮＡの典型的な膜ベースのハイブリダイゼーション解析において用いる。
【０２６７】
各切断物からのＤＮＡを、０．７％アガロースゲル上で分画して、ナイロン製メンブラン（Ｎｙｔｒａｎ Ｐｌｕｓ， Ｓｃｈｌｅｉｃｈｅｒ ＆ Ｓｃｈｕｅｌｌ， Ｄｕｒｈａｍ ＮＨ）に転写する。ハイブリダイゼーションは４０℃で１６時間かけて行う。非特異的シグナルを取り除くため、例えば、最大０．１ｘクエン酸ナトリウム食塩水及び０．５％ドデシル硫酸ナトリウムの条件の下、ブロットを順次室温にて洗浄する。ハイブリダイゼーションパターンをオートラジオグラフィー或いは別のイメージ化手段で視覚化して比較する。
【０２６８】
１０　マイクロアレイ
マイクロアレイ上のアレイエレメントの連結または合成は、フォトリソグラフィ、ピエゾプリント（インクジェットプリンター、前出のＢａｌｄｅｓｃｈｗｅｉｌｅｒ等を参照）、機械的マイクロスポッティング技術及びこれらから派生したものを用いて達成することが可能である。上記各技術において基板は、均一な非多孔性の固体とするべきである（Ｓｃｈｅｎａ （１９９９）．前出）。推奨する基板には、シリコン、シリカ、スライドガラス、ガラスチップ及びシリコンウエハがある。別法では、ドットブロット法またはスロットブロット法に類似のアレイを利用して、熱や紫外線、または化学的或いは機械的な結合手段で基板の表面にエレメントを配置して結合させることができる。通常のアレイは利用可能な方法や機械を用いて作製でき、任意の適正な数のエレメントを含めることができる（Ｓｃｈｅｎａ， Ｍ． 他 （１９９５） Ｓｃｉｅｎｃｅ ２７０：４６７−４７０、Ｓｈａｌｏｎ． Ｄ． 他 （１９９６） Ｇｅｎｏｍｅ Ｒｅｓ． ６：６３９−６４５、Ｍａｒｓｈａｌｌ， Ａ． ａｎｄ Ｊ． Ｈｏｄｇｓｏｎ （１９９８） Ｎａｔ． Ｂｉｏｔｅｃｈｎｏｌ． １６：２７−３１．を参照）。
【０２６９】
完全長ｃＤＮＡ、発現遺伝子配列断片（ＥＳＴ）、或いはそれらの断片やオリゴマーが、マイクロアレイのエレメントとなり得る。ハイブリダイゼーションに好適な断片やオリゴマーを、ＬＡＳＥＲＧＥＮＥソフトウェア（ＤＮＡＳＴＡＲ）などの当分野で周知のソフトウェアを用いて選択することが可能である。このアレイエレメントを、生体サンプル中のポリヌクレオチドとハイブリダイズさせる。生体サンプル中のポリヌクレオチドは、検出を容易にするために蛍光標識またはその他の分子タグに結合する。ハイブリダイゼーションの後、生体サンプルからハイブリダイズしなかったヌクレオチドを除去し、蛍光スキャナを用いて各アレイエレメントにおけるハイブリダイゼーションを検出する。別法では、レーザー脱離及び質量スペクトロメトリーを用いてもハイブリダイゼーションを検出し得る。マイクロアレイ上のエレメントにハイブリダイズする各ポリヌクレオチドの相補性の程度及び相対的存在量は、算定することができる。一実施例におけるマイクロアレイの調整及び使用について、以下に詳述する。
【０２７０】
組織または細胞サンプルの調製
グアニジウムチオシアネート法を用いて組織サンプルから全ＲＮＡを単離し、オリゴ（ｄＴ）セルロース法を用いてポリ（Ａ）^＋ＲＮＡを精製する。各ポリ（Ａ）^＋ＲＮＡサンプルは、ＭＭＬＶ逆転写酵素、０．０５ ｐｇ／μｌのオリゴ（ｄＴ）プライマー（２１ｍｅｒ）、１×第１鎖緩衝液、０．０３単位／μｌのＲＮアーゼインヒビター、５００μＭ ｄＡＴＰ、５００μＭ ｄＧＴＰ、５００μＭ ｄＴＴＰ、４０μＭ ｄＣＴＰ、４０μＭ ｄＣＴＰ−Ｃｙ３（ＢＤＳ）またはｄＣＴＰ−Ｃｙ５（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）を用いて逆転写する。この逆転写反応は、ＧＥＭＢＲＩＧＨＴキット（Ｉｎｃｙｔｅ）を用いて、２００ ｎｇのポリ（Ａ）^＋ＲＮＡを含む２５ ｍｌ容量で行う。特異的なコントロールポリ（Ａ）^＋ＲＮＡは、ｉｎ ｖｉｔｒｏ転写により非コーディング酵母ゲノムＤＮＡから合成する。３７０℃で２時間インキュベートした後、各反応サンプル（一方はＣｙ３標識、他方はＣｙ５標識）は、２．５ｍｌの０．５Ｍ 水酸化ナトリウムで処理し、８５０℃で２０分間インキュベートし、反応を停止させてＲＮＡを変性する。サンプルは、２つの連続するＣＨＲＯＭＡ ＳＰＩＮ ３０ゲル濾過スピンカラム（ＣＬＯＮＴＥＣＨ Ｌａｂｏｒａｔｏｒｉｅｓ， Ｉｎｃ． （ＣＬＯＮＴＥＣＨ）， Ｐａｌｏ Ａｌｔｏ ＣＡ）を用いて精製する。結合後、２つの反応サンプルを、１ｍｌのグリコーゲン（１ｍｇ／ｍｌ）、６０ｍｌの酢酸ナトリウム及び３００ｍｌの１００％エタノールを用いてエタノール沈殿させる。サンプルは次に、ＳｐｅｅｄＶＡＣ（Ｓａｖａｎｔ Ｉｎｓｔｒｕｍｅｎｔｓ Ｉｎｃ．， Ｈｏｌｂｒｏｏｋ ＮＹ）を用いて乾燥して仕上げ、１４μｌ ５×ＳＳＣ／０．２％ ＳＤＳ中で再懸濁する。
【０２７１】
マイクロアレイの準備
本発明の配列を用いて、アレイエレメントを作製する。各アレイエレメントは、クローン化ｃＤＮＡ挿入断片を含むベクターを含有する細菌性細胞から増幅する。ＰＣＲ増幅は、ｃＤＮＡ挿入断片に隣接するベクター配列に相補的なプライマーを用いる。３０サイクルのＰＣＲによって、１〜２ｎｇの初期量から５μｇを超える最終量までアレイエレメントを増幅する。増幅されたアレイエレメントは、ＳＥＰＨＡＣＲＹＬ−４００（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）を用いて精製する。
【０２７２】
精製したアレイエレメントを、ポリマーコートされたスライドガラス上に固定する。顕微鏡スライドガラス（Ｃｏｒｎｉｎｇ）は、処理中及び処理後に大量の蒸留水での洗浄と、０．１％のＳＤＳ及びアセトン中で超音波による洗浄を行う。スライドガラスは、４％フッ化水素酸（ＶＷＲ Ｓｃｉｅｎｔｉｆｉｃ Ｐｒｏｄｕｃｔｓ Ｃｏｒｐｏｒａｔｉｏｎ （ＶＷＲ）， Ｗｅｓｔ Ｃｈｅｓｔｅｒ ＰＡ）中でエッチングし、蒸留水中で広範囲にわたって洗浄し、９５％エタノール中の０．０５％アミノプロピルシラン（Ｓｉｇｍａ）でコーティングする。コーティングしたスライドガラスは、１１０℃の天火で硬化させる。
【０２７３】
米国特許第５，８０７，５２２号に記載されている方法を用いて、コーティングしたガラス基板にアレイエレメントを付加する。この特許に引用することを以って本明細書の一部とする。平均濃度が１００ｎｇ／μｌのアレイエレメントＤＮＡ１μｌを高速機械装置により開放型キャピラリープリンティングエレメント（ｏｐｅｎ ｃａｐｉｌｌａｒｙ ｐｒｉｎｔｉｎｇ ｅｌｅｍｅｎｔ）に充填する。次にこの装置が、スライド毎に約５ｎｌのアレイエレメントサンプルを分注する。
【０２７４】
マイクロアレイには、ＳＴＲＡＴＡＬＩＮＫＥＲ ＵＶクロスリンカー（Ｓｔｒａｔａｇｅｎｅ）を用いてＵＶ架橋する。マイクロアレイは、室温において０．２％ＳＤＳで１回洗浄し、蒸留水で３回洗浄する。非特異的な結合部位は、リン酸緩衝生理食塩水 （ＰＢＳ）（Ｔｒｏｐｉｘ， Ｉｎｃ．， Ｂｅｄｆｏｒｄ ＭＡ）における０．２％カゼイン中で６０℃で３０分間マイクロアレイをインキュベートし、その後上述したように０．２％ＳＤＳ及び蒸留水で洗浄することによってブロックする。
【０２７５】
ハイブリダイゼーション
ハイブリダイゼーション反応液は、５×ＳＳＣ、０．２％ＳＤＳハイブリダイゼーション緩衝液にＣｙ３及びＣｙ５標識したｃＤＮＡ合成産物を各０．２μｇ含む９μｌのサンプル混合体を含めたものである。サンプル混合液を、６５℃で５分間加熱し、マイクロアレイ表面上に一定量分注してから１．８ｃｍ^２ のカバーガラスで覆う。このアレイを、顕微鏡スライドより僅かに大きいキャビティを有する防水チェンバーに移す。チャンバーの角に１４０μｌの５×ＳＳＣを加えて、チャンバー内を湿度１００％に保持する。このアレイを含むチャンバーを、６０℃で約６．５時間インキュベートする。アレイは、第１洗浄緩衝液中（１×ＳＳＣ，０．１％ＳＤＳ）において４５℃で１０分間、第２洗浄緩衝液中（０．１×ＳＳＣ）において４５℃で１０分間それぞれ３回洗浄し、その後乾燥させる。
【０２７６】
検出
レポーター標識されたハイブリダイゼーション複合体は、Ｃｙ３を励起するための４８８ｎｍ、及びＣｙ３を励起するための６３２ｎｍのスペクトル線を生成し得るＩｎｎｏｖａ ７０混合ガス１０ Ｗレーザー（Ｃｏｈｅｒｅｎｔ， Ｉｎｃ．， Ｓａｎｔａ Ｃｌａｒａ ＣＡ）を備えた顕微鏡で検出する。２０倍の顕微鏡対物レンズ（Ｎｉｋｏｎ， Ｉｎｃ．， Ｍｅｌｖｉｌｌｅ ＮＹ）を用いて、アレイ上に励起レーザー光を集中させる。このアレイを含むスライドを顕微鏡のコンピュータ制御Ｘ−Ｙステージに置き、対物レンズを通してラスタスキャンする。本実施例で用いた１．８ｃｍ×１．８ｃｍのアレイは、２０μｍの解像度でスキャンする。
【０２７７】
２つの異なるスキャンにおいて、混合ガスマルチラインレーザーは２つの蛍光体を連続的に励起する。放射された光は、波長に基づいて２つの蛍光体に対応する２つの光電子増倍管検出器（ＰＭＴ Ｒ１４７７， Ｈａｍａｍａｔｓｕ Ｐｈｏｔｏｎｉｃｓ Ｓｙｓｔｅｍｓ， Ｂｒｉｄｇｅｗａｔｅｒ ＮＪ）に分割される。アレイと光電子増倍管との間に配設された好適なフィルターを用いて信号をフィルタリングする。用いる蛍光体の最大発光は、Ｃｙ３では５６５ｎｍ、Ｃｙ５では６５０ｎｍである。装置は両方の蛍光体からのスペクトルを同時に記録できるが、レーザー源に好適なフィルターを用いて、蛍光体１つにつき１回スキャンし、各アレイを通常２回スキャンする。
【０２７８】
スキャンの感度は通常、既知濃度のサンプル混合体に添加されるｃＤＮＡコントロール種により生成されるシグナル強度を用いて較正する。アレイ上の特定の位置には相補的ＤＮＡ配列を含め、その位置におけるシグナルの強度がハイブリダイズする種の重量比１：１００，０００に相関するようにする。異なる試料（例えば検査細胞及びコントロール細胞を代表する）からの２つのサンプルを、各々異なる蛍光体で標識し、他と異なって発現する遺伝子を同定するために単一のアレイにハイブリダイズさせる場合には、較正は２つの蛍光体を有する較正するｃＤＮＡのサンプルを標識して、ハイブリダイゼーション混合液に各々等量を加えて行う。
【０２７９】
光電子増倍管の出力は、ＩＢＭコンパチブルＰＣコンピュータにインストールされた１２ビットＲＴＩ−８３５Ｈアナログ−ディジタル（ＡＩＤ）変換ボード（Ａｎａｌｏｇ Ｄｅｖｉｃｅｓ， Ｉｎｃ．， Ｎｏｒｗｏｏｄ ＭＡ）を用いてディジタル化される。ディジタル化されたデータは、リニア２０色変換を用いてシグナル強度が青色（低シグナル）から赤色（高シグナル）までの擬似カラー範囲にマッピングされるイメージとして表示される。データはまた、定量的に分析される。２つの異なる蛍光体を同時に励起して測定する場合には、各蛍光体の発光スペクトルを用いて、先ずデータは蛍光体間の光学的漏話（重複発光スペクトルに起因する）に対して補正される。
【０２８０】
グリッドを蛍光シグナルイメージ上に重畳して、各スポットからのシグナルがグリッドの各エレメントに中央に位置するようにする。各エレメント内の蛍光シグナルを統合し、シグナルの平均強度に対応する数値を得る。シグナル分析に用いるソフトウェアは、ＧＥＭＴＯＯＬＳ遺伝子発現分析プログラム（Ｉｎｃｙｔｅ）である。
【０２８１】
１１　相補的ポリヌクレオチド
ＰＲＴＳをコードする配列或いはその任意の一部に対して相補的な配列は、天然のＰＲＴＳの発現を低下させるため即ち阻害するために用いられる。約１５〜約３０個の塩基対を含むオリゴヌクレオチドの使用について記すが、より小さな或いはより大きな配列の断片の場合でも本質的に同じ方法を用いることができる。Ｏｌｉｇｏ４．０６ソフトウェア（Ｎａｔｉｏｎａｌ Ｂｉｏｓｃｉｅｎｃｅｓ）及びＰＲＴＳのコーディング配列を用いて、適切なオリゴヌクレオチドを設計する。転写を阻害するためには、最も独特な５′配列から相補的なオリゴヌクレオチドを設計し、これを用いてプロモーターがコーディング配列に結合するのを阻害する。翻訳を阻害するためには、相補的なオリゴヌクレオチドを設計して、リボソームがＰＲＴＳをコードする転写物に結合するのを阻害する。
【０２８２】
１２　 ＰＲＴＳ の発現
ＰＲＴＳの発現及び精製は、細菌若しくはウイルスを基にした発現系を用いて行うことができる。細菌でＰＲＴＳが発現するために、抗生物質耐性及びｃＤＮＡの転写レベルを高める誘導性のプロモーターを含む好適なベクターにｃＤＮＡをサブクローニングする。このようなプロモーターには、ｌａｃオペレーター調節エレメントに関連するＴ５またはＴ７バクテリオファージプロモーター及びｔｒｐ−ｌａｃ（ｔａｃ）ハイブリッドプロモーターが含まれるが、これらに限定されるものではない。組換えベクターを、ＢＬ２１（ＤＥ３）などの好適な細菌宿主に形質転換する。抗生物質耐性をもつ細菌が、イソプロピルβ−Ｄチオガラクトピラノシド（ＩＰＴＧ）で誘発されるとＰＲＴＳを発現する。真核細胞でのＰＲＴＳの発現は、昆虫細胞株または哺乳動物細胞株に一般にバキュロウイスルスとして知られているＡｕｔｏｇｒａｐｈｉｃａ ｃａｌｉｆｏｒｎｉｃａ核多面性ウイルス（ＡｃＭＮＰＶ）を感染させて行う。バキュロウイルスの非必須ポリヘドリン遺伝子を、相同組換え或いは転移プラスミドの媒介を伴う細菌の媒介による遺伝子転移のどちらかによって、ＰＲＴＳをコードするｃＤＮＡと置換する。ウイルスの感染力は維持され、強いポリヘドリンプロモータによって高いレベルのｃＤＮＡの転写が行われる。組換えバキュロウイルスは、多くの場合はＳｐｏｄｏｐｔｅｒａ ｆｒｕｇｉｐｅｒｄａ （Ｓｆ９）昆虫細胞に感染に用いられるが、ヒト肝細胞の感染にも用いられることもある。後者の感染の場合は、バキュロウイルスの更なる遺伝的変更が必要になる。（例えば、Ｅｎｇｅｌｈａｒｄ． Ｅ． Ｋ．他 （１９９４） Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ９１：３２２４−３２２７； Ｓａｎｄｉｇ， Ｖ． 他 （１９９６） Ｈｕｍ． Ｇｅｎｅ Ｔｈｅｒ． ７：１９３７−１９４５．を参照）。
【０２８３】
殆どの発現系では、ＰＲＴＳが、例えばグルタチオンＳトランスフェラーゼ（ＧＳＴ）、またはＦＬＡＧや６−Ｈｉｓなどのペプチドエピトープ標識で合成された融合タンパク質となるため、未精製の細胞溶解物からの組換え融合タンパク質の親和性ベースの精製が素早く１回で行うことができる。Ｓｃｈｉｓｔｏｓｏｍａ ｊａｐｏｎｉｃｕｍからの２６キロダルトンの酵素ＧＳＴによって、タンパク質の活性及び抗原性を維持した状態で固定されたグルタチオンで融合タンパク質の精製が可能となる（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）。精製の後、ＧＳＴ部分を特定の操作部位でＰＲＴＳからタンパク分解的に切断できる。アミノ酸８個のペプチドであるＦＬＡＧで、市販のモノクローナル及びポリクローナル抗ＦＬＡＧ抗体（Ｅａｓｔｍａｎ Ｋｏｄａｋ）を用いた免疫親和性の精製が可能となる。６個の連続するヒスチジン残基のストレッチである６−Ｈｉｓによって、金属キレート樹脂（ＱＩＡＧＥＮ）で精製が可能となる。タンパク質の発現及び精製の方法は、Ａｕｓｕｂｅｌ （１９９５，前出， ｃｈ １０， １６）に記載されている。これらの方法で精製したＰＲＴＳを直接用いて以下の実施例１６、１７、１８、及び１９のアッセイを行うことができる。
【０２８４】
１３　機能のアッセイ
ＰＲＴＳの機能は、哺乳動物細胞培養系において生理学的に高められたレベルでのＰＲＴＳをコードする配列の発現によって評価する。ｃＤＮＡを、ｃＤＮＡを高いレベルで発現する強いプロモーターを含む哺乳動物発現ベクターにサブクローニングする。このようなベクターには、ｐＣＭＶ ＳＰＯＲＴ^ＴＭ （Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ．）及びｐＣＲ ３．１ （Ｉｎｖｉｔｒｏｇｅｎ， Ｃａｒｌｓｂａｄ， ＣＡ）が含まれ、どちらもサイトメガロウイルスプロモーターを含んでいる。５〜１０μｇの組換えベクターを、例えば内皮由来か造血由来のヒト細胞株にリポソーム製剤或いは電気穿孔法によって一時的に形質移入する。更に、標識タンパク質をコードする配列を含む１〜２μｇのプラスミドを同時に形質移入する。標識タンパク質の発現により、形質移入された細胞と形質移入されていない細胞とを区別できる。また、標識タンパク質の発現によって、ｃＤＮＡの組換えベクターからの発現を正確に予想できる。このような標識タンパク質には、緑色蛍光タンパク質（ＧＦＰ；Ｃｌｏｎｔｅｃｈ）、及びＣＤ６４またはＣＤ６４−ＧＦＰ融合タンパク質が含まれる。レーザー光学に基づいた技術を利用した自動流動細胞計測法（ＦＣＭ）を用いて、ＧＦＰまたはＣＤ６４−ＧＦＰを発現する形質移入された細胞を同定し、その細胞のアポトーシス状態や他の細胞特性を評価する。また、ＦＣＭで、先行した或いは同時の細胞死の現象を診断する蛍光分子の取り込みを検出して計量する。これらの現象には、プロピジウムヨウ化物でのＤＮＡの染色によって計測される核ＤＮＡ内容物の変化と、ブロモデオキシウリジンの取り込み量の低下によって計測されるＤＮＡ合成の下方調節と、特異的な抗体との反応性によって計測される細胞表面及び細胞内のタンパンク質の発現の変化と、蛍光複合アネキシンＶタンパク質の細胞表面への結合によって計測される原形質膜組成の変化とが含まれる。流動細胞計測法は、Ｏｒｍｅｒｏｄ， Ｍ． Ｇ．による （１９９４） Ｆｌｏｗ Ｃｙｔｏｍｅｔｒｙ Ｏｘｆｏｒｄ， Ｎｅｗ Ｙｏｒｋ， ＮＹ．に記載されている。
【０２８５】
遺伝子発現におけるＰＲＴＳの影響は、ＰＲＴＳをコードする配列とＣＤ６４またはＣＤ６４−ＧＦＰのどちらかが形質移入された高度に精製された細胞集団を用いて評価することができる。ＣＤ６４またはＣＤ６４−ＧＦＰは形質転換された細胞表面で発現し、ヒト免疫グロブリンＧ（ＩｇＧ）の保存された領域と結合する。形質転換された細胞と形質転換されない細胞とは、ヒトＩｇＧかＣＤ６４に対する抗体のどちらかで被覆された磁気ビードを用いて分離することができる（ＤＹＮＡＬ． Ｌａｋｅ Ｓｕｃｃｅｓｓ． ＮＹ）。ｍＲＮＡは、当分野で周知の方法で細胞から精製することができる。ＰＲＴＳ及び目的の他の遺伝子をコードするｍＲＮＡの発現は、ノーザン分析やマイクロアレイ技術で分析することができる。
【０２８６】
１４　 ＰＲＴＳ に特異的な抗体の作製
ポリアクリルアミドゲル電気泳動法（ＰＡＧＥ；例えば、Ｈａｒｒｉｎｇｔｏｎ， Ｍ．Ｇ． （１９９０） Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ． １８１６−３０８８−４９５を参照）または他の精製技術で実質的に精製されたＰＲＴＳを用いて、標準的なプロトコルでウサギを免疫化して抗体を作り出す。
【０２８７】
別法では、ＰＲＴＳアミノ酸配列をＬＡＳＥＲＧＥＮＥソフトウェア（ＤＮＡＳＴＡＲ）を用いて解析して免疫原性の高い領域を決定し、対応するオリゴペプチドを合成してこれを用いて当業者に周知の方法で抗体を生産する。Ｃ末端付近の、或いは隣接する親水性領域内のエピトープなどの適切なエピトープの選択については、当分野で周知である（例えば、前出のＡｕｓｕｂｅｌ， １９９５，１１章を参照）。
【０２８８】
通常、約１５残基の長さのオリゴペプチドを、Ａｐｐｌｉｅｄ ＢｉｏｓｙｓｔｅｍｓのＡＢＩ ４３１Ａペプチドシンセサイザー（ＰＥ Ｂｉｏｓｙｓｔｅｍｓ）を用いてｆｍｏｃ法のケミストリにより合成し、Ｎ−マレイミドベンゾイル−Ｎ−ヒドロキシスクシンイミドエステル（ＭＢＳ）を用いた反応によりＫＬＨ（Ｓｉｇｍａ−Ａｌｄｒｉｃｈ， Ｓｔ． Ｌｏｕｉｓ ＭＯ）に結合させて、免疫原性を高める（例えば、前出のＡｕｓｕｂｅｌ， １９９５を参照）。フロイントの完全アジュバントにおいてオリゴペプチド−ＫＬＨ複合体を用いてウサギを免疫化する。得られた抗血清の抗ペプチド活性及び抗ＰＲＴＳ活性を検査するには、ペプチドまたはＰＲＴＳを基板に結合し、１％ＢＳＡを用いてブロッキング処理し、ウサギ抗血清と反応させて洗浄し、さらに放射性ヨウ素標識されたヤギ抗ウサギＩｇＧと反応させる。
【０２８９】
１５　特異的抗体を用いる天然 ＰＲＴＳ の精製
天然ＰＲＴＳ或いは組換えＰＲＴＳを、ＰＲＴＳに特異的な抗体を用いるイムノアフィニティークロマトグラフィにより実質的に精製する。イムノアフィニティーカラムは、ＣＮＢｒ−活性化ＳＥＰＨＡＲＯＳＥ（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）のような活性化クロマトグラフィー用レジンと抗ＰＲＴＳ抗体とを共有結合させることにより形成する。結合の後、そのレジンを製造者の使用説明書に従ってブロッキング処理し洗浄する。
【０２９０】
ＰＲＴＳを含む培養液をイムノアフィニティーカラムに通し、ＰＲＴＳを優先的に吸着できる条件で（例えば、界面活性剤の存在下において高イオン強度のバッファーで）そのカラムを洗浄する。そのカラムを、抗体とＰＲＴＳとの結合を切るような条件で（例えば、ｐＨ２〜３のバッファー、或いは高濃度の尿素またはチオシアン酸塩イオンのようなカオトロピックイオンで）溶出させ、ＰＲＴＳを回収する。
【０２９１】
１６　 ＰＲＴＳ と相互作用する分子の同定
ＰＲＴＳまたは生物学的に活性なその断片を、^１２５Ｉボルトンハンター試薬（例えば、Ｂｏｌｔｏｎ Ａ．Ｅ．及びＷ．Ｍ． Ｈｕｎｔｅｒ （１９７３） Ｂｉｏｃｈｅｍ． Ｊ． １３３：５２９を参照）で標識する。マルチウェルプレートに予め配列しておいた候補の分子を、標識したＰＲＴＳと共にインキュベートし、洗浄して、標識したＰＲＴＳ複合体を有する全てのウェルをアッセイする。様々なＰＲＴＳ濃度で得られたデータを用いて、候補分子と結合したＰＲＴＳの数量及び親和性、会合についての値を計算する。
【０２９２】
別法では、ＰＲＴＳと相互作用する分子を、Ｆｉｅｌｄｓ， Ｓ．及びＯ． Ｓｏｎｇ（１９８９， Ｎａｔｕｒｅ ３４０：２４５−２４６）に記載の酵母２−ハイブリッドシステム（ｙｅａｓｔ ｔｗｏ−ｈｙｂｒｉｄ ｓｙｓｔｅｍ）やＭＡＴＣＨＭＡＫＥＲシステム（Ｃｌｏｎｔｅｃｈ）などの２−ハイブリッドシステムに基づいた市販のキットを用いて分析する。
【０２９３】
ＰＲＴＳはまた、ハイスループット型の酵母２ハイブリッドシステムを使用するＰＡＴＨＣＡＬＬＩＮＧプロセス（ＣｕｒａＧｅｎ Ｃｏｒｐ．， Ｎｅｗ Ｈａｖｅｎ ＣＴ）に用いて、遺伝子の２つの大きなライブラリによってコードされるタンパク質間の全ての相互作用を決定することができる（Ｎａｎｄａｂａｌａｎ， Ｋ． 他 （２０００） 米国特許第６，０５７，１０１号）。
【０２９４】
１７　 ＰＲＴＳ の活性の実証
プロテアーゼ活性は、様々な色素分子と共役された適切な合成ペプチド基質加水分解によって測定され、色素分子内部では、加水分解の程度が放出発色団の分光光度法（蛍光）吸収（Ｂｅｙｎｏｎ， Ｒ． Ｊ．及びＪ． Ｓ． Ｂｏｎｄ （１９９４）、Ｐｒｏｔｅｏｌｙｔｉｃ Ｅｎｚｙｍｅｓ ： Ａ Ｐｒａｃｔｉｃａｌ Ａｐｐｒｏａｃｈ， Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓ， Ｎｅｗ Ｙｏｒｋ ＮＹ， ｐｐ． ２５−５５）で定量される。ペプチド基質は、エンドペプチダーゼ（セリン、システイン、アスパラギン酸プロテアーゼ、若しくはメタロプロテアーゼ）、アミノペプチダーゼ（ロイシンアミノペプチダーゼ）、若しくはカルボキシペプチダーゼ（カルボキシペプチダーゼＡ及びＢ、プロコラーゲンＣ−プロテイナーゼ）としての、プロテアーゼ活性カテゴリーに従ってデザインされる。通常、用いられたクロモーゲンは、２−ナフチルアミン、４−ニトロアニリン、フリルアクリル酸である。アッセイは外界温度で実行され、好適なバッファ内の酵素及び適切な基質のアリコットを含んでいる。反応は蛍光キュベット内で実行され、ペプチド基質の加水分解の間に遊離された色素原の吸光度における増加／減少が測定される。吸光度に於ける変化は、アッセイ中の酵素活性に比例する。
【０２９５】
ユビキチン加水分解酵素活性のための代替アッセイは、ユビキチン前駆体の加水分解を測定する。アッセイは外界温度で実行され、適当なバッファ中に適切な基質及びＰＲＴＳのアリコットを含んでいる。化学的に合成されたヒトユビキチン−バリンは基質として用いられる。基質よりのＣ−末端バリン残基の切断は、毛細管電気泳動（Ｆｒａｎｋｌｉｎ， Ｋ．ら（１９９７） Ａｎａｌ． Ｂｉｏｃｈｅｍ． ２４７ ： ３０５−３０９）でモニターされる。
【０２９６】
別法では、プロテアーゼ活性のためのアッセイが蛍光共鳴エネルギー変換（ＦＲＥＴ）を利用する。ＦＥＲＴは、一つのドナー及び一つのアクセプターフルオロフォアが適切なスペクトルの重なりを有して近接している場合に発生する。ＰＲＴＳに特異的な切断部位を有するフレキシブルなペプチドリンカーは、緑蛍光タンパク質の、赤シフト（ｒｅｄ−ｓｈｉｆｔｅｄ ｖａｒｉａｎｔ）変異体（ＲＳＧＦＰ４）及び青変異体間で融着する。
【０２９７】
この融着タンパク質は、エネルギー移動をＢＦＰ５からＲＳＧＦＰ４へと発生させるスペクトル特性を有する。融着タンパク質がＰＲＴＳと共にインキューベートされる場合、基質が切断され二つの蛍光タンパク質が分離される。このことは、ＰＲＴＳの付加前後における発行スペクトルを比較すること（Ｍｉｔｒａ， Ｒ． Ｄら、（１９９６） Ｇｅｎｅ １７３：１３−１７）で定量される、エネルギー移動中の顕著な減少を伴う。アッセイは生細胞内でも行われ得る。このような場合は、蛍光基質タンパク質は細胞中で恒常的に発現され、またＰＲＴＳが誘導ベクター上に提供され、よってＦＲＥＴがＰＲＴＳの存在及び不在という形でモニターされうる（Ｓａｇｏｔ， Ｉら： （１９９９） ＦＥＢＳ Ｌｅｔｔ． ４４７ ： ５３−５７）。
【０２９８】
１８　 ＰＲＴＳ 基質の同定
ファージディスプレイライブラリが、ＰＲＴＳの光学基質配列を識別するのに用いられてもよい。リンカー及び既知の抗体エピトープに続くランダムな六量体は繊維状ファージライブラリ中の遺伝子ＩＩＩのＮ−末端延長としてクローン化される。遺伝子ＩＩＩコードが外殻蛋白質をコードし、またエピトープが各々のファージ分子表面上に表示されてもよい。ライブラリはタンパク分解性条件下で、ＰＲＴＳとインキュベートされ、エピトープはＰＲＴＳのための六量体コードが部位を切断する場合取り除かれてもよい。エピトープを認識する抗体が、固定化タンパク質Ａと共に添加される。エピトープを有している切断されていないファージは遠心分離によって取り除かれる。表面のファージは次に増幅され、スクリーニングのさらなる複数のラウンドが生じる。個々のファージクローンは次に単離され、配列決定される。これらペプチド基質に対する速度論は、実施例１７に於けるアッセイを用いて研究されてもよく、また最適切断配列が得られうる（Ｋｅ， Ｓ． Ｈ．ら（１９９７） Ｊ． Ｂｉｏｌ． Ｃｈｅｍ ２７２ ： １６６０３−１６６０９）。
【０２９９】
ｉｎ ｖｉｖｏＰＲＴＳ基質のスクリーンのため、この方法は、ファージ分子（Ｔ７ＳＥＬＥＣＴ^ＴＭ１０−３ ファージディスプレイベクター、 Ｎｏｖａｇｅｎ， Ｍａｄｉｓｏｎ， ＷＩ）、若しくは酵母菌（ｐＹＤ１ 酵母ディスプレイベクターキット、 Ｉｎｖｉｔｒｏｇｅｎ， Ｃａｒｌｓｂａｄ， ＣＡ）の表面上にディスプレイされたｃＤＮＡ発現ライブラリをスクリーンするべく拡張されてもよい。このような場合、全体のｃＤＮＡは、遺伝子ＩＩＩと適切なエピトープとの間で融着される。
【０３００】
１９　 ＰＲＴＳ 阻害剤の同定
試験される化合物は、実施例１７のアッセイで記述されているように、適切なバッファ及び基質に沿って濃度を変化させ、マルチウェルプレート上に配列される。ＰＲＴＳ活性は各々の穴で測定され、ＰＲＴＳ活性を抑制するために各化合物の各化合物の能力が決定されてもよい。用量‐応答の速度論も同様である。アッセイはＰＲＴＳの活性を向上させる分子を同定することに用いられてもよい。
【０３０１】
別法では、ファージディスプレイライブラリがペプチドＰＲＴＳ阻害剤をスクリーンするために用いられてもよい。候補はプロテアーゼに強く結合されたペプチド中に見られた。この場合、マルチウェルプレートの各ウェルは、ＰＲＴＳでコートされ、ランダムなペプチドファージディスプレイライブラリ若しくは周期的なペプチドライブラリでインキュベートされる（Ｋｏｉｖｕｎｅｎ， Ｅ．ら、（１９９９） Ｎａｔ． Ｂｉｏｔｅｃｈｎｏｌ． １７ ： ７６８−７７４）。結合していないファージは洗い流され、選択されたファージが複数のさらなるラウンドで増幅され、再スクリーニングされる。候補は、実施例１７に記載のアッセイを用いてＰＲＴＳの阻害活性のために試験される。
【０３０２】
当業者は、本発明の範囲及び精神から逸脱することなく本発明の記載した方法及びシステムの種々の改変を行うことができるであろう。特定の好適な実施例に基づいて本発明を説明したが、本発明の範囲が、そのような特定の実施例に不当に制限されるべきではないことを理解されたい。実際に、分子生物学或いは関連する分野の専門家には明らかな、本明細書に記載の本発明の実施例の様々な改変は、特許請求の範囲に含まれる。
【０３０３】
（表の簡単な説明）
表１は、本発明のポリヌクレオチド配列及びポリペプチド配列に対する系統的な名称を示す。
【０３０４】
表２は、本発明の各ポリペプチドに最も近いＧｅｎＢａｎｋの相同体のＧｅｎＢａｎｋの識別番号およびアノテーションを示す。各ポリペプチドとそのＧｅｎＢａｎｋの相同体との間の一致を表す確率値スコアも示す。
【０３０５】
表３は、推定上のモチーフおよびドメインを含む各ポリペプチド配列の構造的な特徴、並びに各ポリペプチドの分析に用いた方法、アルゴリズム、および検索可能なデータベースを示す。
【０３０６】
表４は、各ポリヌクレオチド配列の組み立てに用いたｃＤＮＡ断片およびゲノムＤＮＡ断片のリスト、並びにポリヌクレオチド配列の選択された断片のリストを示す。
【０３０７】
表５は、本発明の各ポリヌクレオチドの代表的なｃＤＮＡライブラリを示す。
【０３０８】
表６は、表５に示すｃＤＮＡライブラリの作製に用いた組織およびベクターを示す付録である。
【０３０９】
表７は、本発明のポリヌクレオチド及びポリペプチドの分析に用いたツール、プログラム、及びアルゴリズム、並びにその説明、引用文献、閾値パラメーターを示す。
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【表６】

【表７】

【表８】

【表９】

【表１０】

【表１１】

【表１２】

【表１３】

【表１４】

【表１５】

【表１６】

[0001]
The present invention relates to nucleic acid and amino acid sequences of proteases. The present invention also provides diagnosis, treatment, and prevention of gastrointestinal diseases, cardiovascular diseases, autoimmune / inflammatory diseases, abnormal cell proliferation, developmental disorders, epithelial diseases, neurological diseases, and reproductive disorders using these sequences. About. The invention further relates to the evaluation of the effects of foreign compounds on the expression of the nucleic acid and amino acid sequences of the protease.
[0002]
(Background of the Invention)
Proteases cleave proteins and peptides at the peptide backbone or peptide binding site forming a peptide chain. Proteolysis is one of the most important and frequently occurring enzymatic reactions occurring inside and outside cells. Proteolysis is responsible for the activation and maturation of nascent polypeptides, the degradation of misfolded and damaged proteins, and the controlled turnover of intracellular peptides. Proteases are involved in digestion, endocrine function, and tissue formation during embryonic development, and in wound healing and normal growth. Proteases play an important role in regulatory action by altering the half-life of regulatory proteins. Proteases are involved in the pathogenesis or progression of disease states such as inflammation, angiogenesis, tumor spread and metastasis, cardiovascular disease, neurological disease, bacterial, viral, and parasitic infections.
[0003]
Proteases can be classified by where to cut their substrate. Exopeptidases, including aminopeptidases, dipeptidyl peptidases, tripeptidases, carboxypeptidases, peptidyl dipeptidases, dipeptidases, and ω-peptidases cleave residues at the end of the substrate. Endopeptidases, including serine proteases, cysteine proteases, and metalloproteases, cleave residues within peptides. Mammalian proteases were divided into four main types by active site structure, mechanism of action, and overall three-dimensional structure (See Beynon, RJ and JS Bond (1994)).Proteolytic Enzymes : A Practical Approach, Oxford University Press, New York NY, pp. 1-5. ).
[0004]
Serine protease
Serine proteases (SPs) are a broad and large family of proteolytic enzymes, including the digestive enzymes trypsin and chymotrypsin, components of the complement and blood coagulation cascades, and the breakdown and turnover of macromolecules in the intracellular and extracellular matrix. Contains enzymes that regulate Most of the 20 or more subfamilies can be classified into six clans, each with a common ancestor. These six clans are presumed to be from at least four evolutionarily different ancestors. SP was named for the presence of a serine residue at the active catalytic site of most families. The active site is formed by three catalytic triads of a group of conserved aspartic, histidine, and serine residues important for catalysis. These residues form a charge relay network that facilitates substrate binding. Other residues outside the active site form oxyanion holes that stabilize the tetrahedral transition intermediate formed during catalysis. SP has a wide range of substrates and can be divided into subfamilies by their substrate specificity. The major subfamilies are named for the residues after cleavage, tryases (after arginine or lysine), asses (after aspartic acid), timase (after phenylalanine or leucine), metases (after methionine), and serases. (After Serine) (Rawlings, ND and AJ Barrett (1994) Meth. Enzymol. 244: 19-61).
[0005]
Most mammalian serine proteases are synthesized as pro-enzymes, which are inactive precursors that are activated by proteolysis. For example, trypsinogen is converted to its active form, trypsin, by enteropeptidase. Enteropeptidase is an intestinal protease that removes the N-terminal fragment from trypsinogen. The remaining active fragment is trypsin, which activates other precursors of pancreatic enzymes. Similarly, proteolysis of prothrombin, a precursor of thrombin, produces three different polypeptide fragments. The N-terminal fragment is cleaved when two fragments containing activated thrombin are linked by a disulfide bond.
[0006]
The two largest SP subfamilies are the chymotrypsin (S1) family and the subtilisin (S8) family. Some members of the chymotrypsin family contain two structural domains unique to this family. Kringle domains are triple loop disulfide bridging domains found in variable copy numbers. The kringle domain is thought to play a role in regulating membranes, other proteins and mediators such as phospholipids, as well as in regulating proteolysis (PROSITE PDOC00020). The Apple domain is a repetitive domain of 90 amino acids, each containing six conserved cysteines. The first cysteine and the sixth cysteine, the second cysteine and the fifth cysteine, and the third cysteine and the fourth cysteine are linked by three disulfide bonds (PROSITE PDOC00376). Apple domains are involved in protein-protein interactions. S1 family members include trypsin, chymotrypsin, coagulation factors IX-XII, complement factors B, C, and D, granzyme, kallikrein, and tissue and urokinase plasminoden activators. The subtilisin family includes members found in eubacteria, archaebacteria, eukaryotes, and viruses. Subtilisins include the protein-protein endopeptidases kexin and furin, pituitary prohormone convertases PC1, PC2, PC3, PC6, and PACE4 (Rawlings and Barrett, supra).
[0007]
Although SP acts in many normal processes, some SPs are involved in the etiology and treatment of disease. Enterokinase, an inhibitor of intestinal digestion, is found on the edge of the intestinal brush where it cleaves acidic propeptides from trypsinogen to produce active trypsin (Kitamoto, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91: 7588-7592). Prolyl carboxypeptidase, a lysosomal serine peptidase that cleaves peptides such as angiotensin II and III, and [des-Arg9] bradykinin, shares sequence homology with members of both the serine carboxypeptidase and prolyl endopeptidase families ( Tan, F. et al. (1993) J. Biol. Chem. 268: 16631-16638). The protease neuropsin can affect synapse formation and neuronal connections in the hippocampus in response to neural signaling (Chen, ZL-L. Et al. (1995) J Neurosci 15: 5088-5097). Tissue plasminogen activator has been identified in seizures (Zivin, JA (1999) Neurology 53: 14-19) and myocardial infarction (Ross, AM (1999) Clin. Cardiol. 22: 165-171). Useful for first aid. Certain receptors, which are highly expressed throughout the gastrointestinal tract (PARs: protease-activated receptors), are activated by proteolytic cleavage of the extracellular domain. Major agonists of PAR, thrombin, trypsin, and mast cell tryptase are released in allergic and inflammatory conditions. Regulation of PAR activity by proteases has been proposed as a promising therapeutic target (Vergnoll, N. (2000) Alignment. Pharmacol. Ther. 14: 257-266; Rice, KD et al. (1998) Curr. Pharm. Des. 4: 381-396). Prostate specific antigen (PSA) is a kallikrein-like serine protease that is synthesized and secreted only by prostate epithelial cells. Serum PSA is elevated in prostate cancer and is the most sensitive physiological marker for monitoring cancer progression and treatment response. PSA also allows the prostate to be identified as the primary cancer of metastatic cancer (Brawer, MK and PH Language (1989) Urology 33: 11-16).
[0008]
Signal peptidases are a class of SP found in all eukaryotic and prokaryotic cell types and affect the processing of signal peptides from certain proteins. A signal peptide is the amino-terminal domain of a protein that directs the protein from a ribosome assembly site to a predetermined intracellular or extracellular site. Once the protein is transported, its signal sequence is removed by signal peptidases and the protein is activated by post-translational processing such as glycosylation or phosphorylation. Signal peptidases exist as multi-subunit complexes in both yeast and mammals. The canine signal peptidase complex is composed of five subunits, all of which are associated with the microsomal membrane and contain a hydrophobic region that penetrates the membrane one or more times (Shelness, GS and G.S. Blobel (1990) J. Biol. Chem. 265: 9512-9519). Some of these subunits move the complex to the proper location on the membrane, others contain the actual catalytic activity.
[0009]
Another family of proteases that include a serine in the active site relies on ATP hydrolysis for its activity. These proteases contain a regulatory ATPase domain and a proteolytic core domain that can be identified by the presence of the ATP / GTP-binding motif P-loop (PROSITE PDOC008003). Members of this family include eukaryotic mitochondrial matrix proteases, C1p proteases, and proteasomes. C1p protease was first found in chloroplasts of plants, but is thought to be widely present in both prokaryotic and eukaryotic cells. The gene for premature torsion dystonia encodes a protein related to the C1p protease (Ozelius, LJ et al. (1998) Adv. Neurol. 78: 93-105).
[0010]
The proteasome is an intracellular protease complex found in certain bacteria and all eukaryotic cells and plays an important role in intracellular physiology. The proteasome is associated with the ubiquitin conjugation system (UCS), which is the main pathway for the degradation of all types of intracellular proteins. The intracellular proteins also include proteins that act to activate or suppress cellular processes such as transcription and cell cycle progression (Ciechanover, A. (1994) Cell 79: 13-21). In the UCS pathway, a small heat-resistant protein, ubiquitin, binds to a protein to be a degradation target. Next, the ubiquitin-bound protein is recognized and degraded by the proteasome. The resulting ubiquitin-peptide conjugate is hydrolyzed by ubiquitin carboxyl terminal hydrolase, releasing free ubiquitin for reuse by UCS. The ubiquitin proteasome system is involved in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes (p53), cell surface receptors involved in signal transduction, transcription factors, and mutated or damaged proteins (Ciechanover, supra). This pathway is involved in a variety of diseases including cystic fibrosis, Angelman's syndrome, and Riddle syndrome (Schwartz, AL and A. Ciechanover (1999) Annu. Rev. Med. 50: 57-74. See). The mouse proto-oncogene Unp encodes a nuclear ubiquitin protease whose overexpression causes oncogenic transformation of NIH3T3 cells. The human homolog of this gene has consistently high levels in small cell tumors of the lung and adenocarcinoma (Gray, DA (1995) Oncogene 10: 2179-2183). Ubiquitin carboxyl-terminal hydrolase is involved in the differentiation of lymphoblastic leukemia cell lines to a non-dividing maturation state (Maki, A. et al. (1996) Differentiation 60: 59-66). Ubiquitin carboxyl-terminal hydrolase (PGP9.5) is highly expressed in neurons in abnormal structures that occur in human neurodegenerative diseases. The proteasome contains a central catalytic core containing various proteases arranged in four 7-membered rings with the active site facing the central cavity, a terminal ATPase subunit covering the outer ports of the cavity and regulating substrate entry. (〜2000 kDa) (see Schmidt, M. et al. (1999) Curr. Opin. Chem. Biol. 3: 584591).
[0011]
Cysteine protease
Cysteine protease (CP) is involved in a variety of cellular processes, from precursor protein processing to intracellular degradation. About half of the known CPs are present in the virus only. CP has cysteine as the main catalytic residue in the active site. At the active site, catalysis proceeds through the diester intermediate and is facilitated by histidine and aspartic acid residues in close proximity. Glutamine residues are also important and aid in the formation of oxyanion holes. Two important CP families include papain-like enzymes (C1) and calpain (C2). Papain-like family members are synthesized from signal and propeptides, usually because they are lysosomal or secreted enzymes. Most members contain motifs conserved in the propeptide that may have structural significance (Karrer, KM et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3063-3067). The three-dimensional structure of papain family members is a bilobed molecule with a catalytic site located between two lobes. Papains include cathepsins B, C, H, L, and S, certain plant allergens and dipeptidyl peptidases (Rawlings, ND and AJ Barrett (1994) Meth. Enzymol. 244: 461-486).
[0012]
Some CPs are expressed everywhere while others are expressed only by cells of the immune system. Of note, CP is produced by monocytes, macrophages, and other cells that migrate to sites of inflammation and secrete molecules involved in tissue repair. Excess of these repair molecules plays a role in certain diseases. In autoimmune diseases such as rheumatoid arthritis, secretion of cysteine peptidase cathepsin C degrades collagen, laminin, elastin, and other structural proteins found in the extracellular matrix of bone. Bone weakened by such degradation is also susceptible to tumor invasion and metastasis. Cathepsin L expression may help the influx of mononuclear cells that promotes the destruction of rheumatoid synovial fluid (Keyszer, GM (1995) Arthritis Rheum. 38: 976-984).
[0013]
Calpain is a calcium-dependent cytoplasmic endopeptidase that contains an N-terminal catalytic domain and a C-terminal calcium binding domain. Calpain is expressed as a proenzyme heterodimer consisting of a catalytic subunit unique to each isoform and a regulatory subunit common to various isoforms. Each subunit has a calcium binding EF hand domain. The regulatory subunit also contains a hydrophobic high glycine domain that allows the enzyme to associate with the cell membrane. Calpain is activated by increasing intracellular calcium levels, causing changes in structure and limited autolysis. The resulting active molecule requires low calcium concentrations for its activity (Chan, SL and MP Mattson (1999) J. Neurosci. Res. 58: 167-190). Calpain is mainly expressed in neurons, but also in other tissues. Certain chronic neurodegenerative diseases, including ALS, Parkinson's disease, and Alzheimer's disease, are associated with elevated calpain expression (Chan and Mattson, supra). Calpain-mediated degradation of the cytoskeleton has been reported to be involved in brain injury due to head trauma (McCracken, E. et al. (1999) J. Neurotrauma 16: 749-761). Calpain 3 is mainly expressed in skeletal muscle and is associated with limb girdle muscular dystrophy type 2A (Minami, N. et al. (1999) J. Neurol. Sci. 171: 31-37).
[0014]
Caspases are another family of thiol proteases and are involved in the initiation and termination of apoptosis. Pro-apoptotic signals activate the initiator caspase, initiating the proteolytic caspase cascade, leading to target protein hydrolysis and classical apoptotic death of cells. Two active site residues, cysteine and histidine, are involved in the mechanism of catalysis. Caspases are one of the most specific endopeptidases and cleave after aspartic acid residues. Caspases are synthesized as inactive zymogens consisting of one large subunit (p20) and one small subunit (p10) separated by a small spacer region, and a variable N-terminal prodomain. This prodomain interacts with cofactors that positively or negatively affect apoptosis. Autoproteolysis by an activation signal cleaves certain aspartic acid residues (D297 of caspase 1 according to numbering rules), removes the spacer and prodomain, and leaves the p10 / p20 heterodimer. These two heterodimers interact via their small subunits to form catalytically active tetramers. The long prodomain of certain caspase family members was found to promote pro-caspase dimerization and self-processing. Certain caspases contain a "death effector domain" in their prodomain, whereby the caspase self-activates together with the FADD protein and other caspases associated with the death receptor or the TNF receptor complex. Can be recruited into the complex. In addition, two dimers from different caspase family members can bind and alter the substrate specificity of the resulting tetramer. There are also endogenous caspase inhibitors (inhibitors of apoptotic proteins, or IAPs). It is clear that all of these interactions influence the regulation of apoptosis (Chan and Mattson, supra; Salveson, GS and VM Dixit (1999) Proc. Natl. Acad. ScL USA 96: 10964-10967).
[0015]
Caspases are involved in certain diseases. Mice deficient in certain caspases have severe nervous system damage due to failure of sensory epithelium apoptosis and die early. Others show severe impairment in the inflammatory response. For example, caspases are involved in the processing of IL-1b and may also be involved in other inflammatory cytokines (Chan and Mattson, supra). Cowpox and baculoviruses target caspases to avoid death of their host cells and to succeed in infection. In addition, it has been reported that inappropriate apoptosis is increased in AIDS, neurodegenerative diseases, and ischemic trauma, and that reduced cell death is associated with cancer (Salveson and Dixit, supra: Thompson, CB. (1995) Science 267: 1456-1462).
[0016]
Aspartyl protease
Aspartyl proteases (AP) include the lysosomal proteases cathepsins D and E, chymosin, renin, and gastric pepsin. Most retroviruses are usuallypolEncodes AP as part of a polyprotein. AP, also called acidic protease, is a two-domain monomeric enzyme, each domain containing one-half active site with its own catalytic aspartic acid residue. AP is most active in the pH range 2-3, at which one aspartic acid residue is ionized and the others are neutral. The pepsin family of APs contains many secreted enzymes, all of which appear to be synthesized with signal and propeptides. Most family members contain three disulfide loops, with the first aspartic acid followed by a first loop of up to five residues, and the second aspartic acid by five to six residues. And the third largest loop is formed towards the C-terminus. On the other hand, retropepsins resemble one domain of pepsin and activate as a homodimer with each retropepsin monomer providing a 1/2 active site. Retropepsin is required for processing of viral polyprotein.
[0017]
APs act on various tissues, some have been implicated in disease. Renin mediates the first step in the processing of angiotensin, a hormone responsible for regulating electrolyte balance and blood pressure (Crews, DE and SR Williams (1999) Hum. Biol. 71: 475-503). See). It is clear that aberrant regulation and expression of cathepsin occurs in various inflammatory disease states. Cathepsin D expression is increased in synovial membrane tissue of patients with rheumatoid arthritis and osteoarthritis. Elevated cathepsin expression and differential regulation are associated with the metastatic potential of various cancers (Chambers, AF et al. (1993) Crit. Rev. Oncol. 4: 95-114).
[0018]
Metalloprotease
Most zinc-dependent metalloproteases share a common sequence in the zinc binding domain. This active site consists of two histidines acting as zinc ligands and the catalytic glutamic acid C-terminus of the first histidine. Proteins containing this signature sequence are known as metzincins and include aminopeptidase N, angiotensin converting enzyme, neurolysin, matrix metalloproteases, and adamalysins (ADAMS). Another sequence was found for zinc carboxypeptidase, in which three conserved residues, two histidine residues and one glutamic acid residue, are involved in zinc binding.
[0019]
Several neutral metalloendopeptidases, including angiotensin converting enzyme and aminopeptidase, are involved in the metabolism of peptide hormones. For example, high activity of aminopeptidase B is found in the thyroid and pituitary gland of hypertensive rats (Prieto, I. et al. (1998) Horm. Metab. Res. 30: 246-248). Oligopeptidase M / neurolysin is capable of hydrolyzing bradykinin and neurotensin (Serizawa, A. et al. (1995) J. Biol. Chem 270: 2092-2098). Neurotensin is a vasoactive peptide that can act as a neurotransmitter in the brain and appears to be involved in limiting dietary intake (Tritos, NA et al. (1999) Neuropeptides 33: 339-349).
[0020]
Matrix metalloproteases (MMPs) are a family of at least 23 enzymes that can degrade components of the extracellular matrix (ECM). MMP is Zn with N-terminal catalytic domain⁺²Endopeptidase. Almost all members of this family have a C-terminal domain and a hinge peptide capable of binding to inhibitors produced by the tissue or substrate molecules of the ECM (TIMP: tissue inhibitor of metalloproteases; Campbell, IL). (1999) Trends Neurosci. 22: 285). Utilizing the presence of fibruronectin-like repeats, transmembrane domains, or C-terminal hemopexinase-like domains, MMPs can be divided into collagenases, gelatinases, stromelysins, and membrane-type MMP subfamilies. In the active form, Zn at the active site⁺²Ions interact with cysteines in the prosequence. Activator is Zn that exposes the active site⁺²Inhibits cysteine interactions or "cysteine switches". This partially activates the enzyme, which then cleaves the propeptide to fully activate it. MMPs are often activated by the serine proteases plasmin and furin. MMPs are often regulated by stoichiometric non-covalent interactions with inhibitors, ie, the balance between proteases and inhibitors, and are critical in tissue homeostasis. (See Young, VW, et al. (1998) Trends Neurosci. 21:75).
[0021]
MMPs are involved in various diseases, among them osteoarthritis (Mitchell, P. et al. (1996) J. Clin. Invest. 97: 761), atheroma plaque rupture (Sukhova, GK et al. (1999) Circulation). 99: 2503), aortic aneurysm (Schneiderman, J. et al. (1998) Am. J. Path. 152: 703), non-wound healing (Sarialho-Kere, UK, et al. (1994) J. Clin. Invest. 94. : 79), bone resorption (Blavier, L. and JM Delaise (1995) J. Cell Sci. 108: 3649), age-related macular degeneration (Sten, B. et al. (1998) Invest. Opthalmol. Vis. S 39: 2194), emphysema (inlay, GA et al. (1997) Thorax 52: 502), myocardial infarction (Rohde, LE et al. (1999) Circulation 99: 3063), and dilated cardiomyopathy ( Thomas, CV, et al. (1998) Circulation 97: 1708). MMP inhibitors inhibit the metastasis of breast and experimental tumors in rats and the metastasis of Lewis lung carcinoma, hemangiomas, and human ovarian cancer xenografts in mice (Eccles, SA et al. (1996) Cancer Res. 56: 2815; Anderson et al. (1996) Cancer Res. 56: 715-718; Volpert, OV et al. (1996) J. Clin. Invest. 98: 671; Taraboletti, G. et al. (1995) J. NCI. Davies, B. et al. (1993) Cancer Res. 53: 2087). MMPs may be activated in Alzheimer's disease. Certain MMPs are involved in multiple sclerosis, and administration of MMP inhibitors can alleviate some of the symptoms (see Young, supra).
[0022]
Another family of metalloproteases is the ADAM (disintegrin domain and metalloprotease domain), which shares an analog of the adamalysins, a snake venom metalloprotease (SVMP). ADAM combines the structures of both cell surface adhesion molecules and proteases and includes a pro domain, a protease domain, a disintegrin domain, a high cysteine domain, an epidermal growth factor repeat, a transmembrane domain, and a cytoplasmic tail. These first three domains are also found in SVMP. ADAM has four potential functions: proteolysis, adhesion, signal transduction, and fusion. ADAM shares the metzincin zinc binding sequence and is inhibited by certain MMP antagonists such as TIMP-1.
[0023]
ADAMs are involved in processes such as sperm-egg binding and fusion, myoblast fusion, and processing or disruption of the protein ectodomain of cytokines, cytokine receptors, adhesion proteins and other extracellular protein domains (Schlondorff, J And CP Blobel (1999) J. Cell. Sci. 112: 3603-3617). The Kuzbania protein cleaves substrates of the NOTCH pathway (NOTCH itself is possible) and activates a program for lateral inhibition of Drosophila neural development. Two ADAMs, TACE (ADAM17) and ADAM10, have been proposed to perform similar functions in processing amyloid precursor protein in the brain (Schlondorff and Blobel, supra). TACE has also been identified as a TNF activating enzyme (Black, RA, et al. (1997) Nature 385: 729). TNF is a pleiotropic cytokine that is important in mobilizing host defenses in response to infection or trauma, but overexpression causes severe damage and is often overexpressed in autoimmune diseases. TACE cleaves membrane-bound pro-TNF, releasing a soluble form. Other ADAMs appear to be involved in similar processing of other membrane-bound molecules.
[0024]
The ADAMTS subfamily has all the features of the ADAM family metalloproteases and additionally contains a thrombospondin domain (TS). A prototype ADAMTS has been identified in mice, expressed in the heart and kidney, and up-regulated by proinflammatory stimuli (Kuno, K. et al. (1997) J. Biol. Chem. 272: 556). To date, 11 members have been identified by Human Genome Organization (HUGO; http://www.gene.ucl.ac.uk/users/hester/adamts.html#Approved). Members of this family have the ability to degrade aggrecan, a high molecular weight proteoglycan that provides cartilage with important mechanical functions, including the ability to contract, which is lost by the development of arthritis. Thus, enzymes that degrade aggrecan are considered attractive targets for preventing or reducing indirect cartilage degradation (eg, Tortorella, MD (1999) Science 284: 1664; Abbaszade, I.). 1999) J. Biol. Chem. 274: 23443). Other members have been reported to be involved in anti-angiogenic potential (Kuno et al., Supra) and / or in the processing of procollagen (Colige, A. et al. (1997) Proc. Natl. Acad. Sci. USA 94). : 2374).
[0025]
Protease inhibitor
Protease inhibitors and other modulators of protease activity modulate the activity and effects of proteases. Protease inhibitors have been reported to modulate the pathogenesis in animal models of proteolysis-related diseases (Murphy, G. (1991) Agents Actions Suppl. 35: 69-76). Decrease in cystatin, a low molecular weight inhibitor of cysteine protease, correlates with malignant tumor progression (Calcins, C. et al. (1995) Biol. Biochem. Hoppe Seyler 376: 71-80). Serpins are inhibitors of mammalian plasma serine proteases. Many serpins affect the regulation of the blood coagulation cascade and / or the complement cascade in mammals. SP32 is a positive regulator of the mammalian acrosomal protease acrosin, which binds to the proenzyme proacrosin and helps to fold that enzyme into the acrosome matrix (Baba, T. et al. (1994) J. Biol. Chem. 269: 10133-10140). The Kunitz family of serine protease inhibitors is characterized by one or more "Kunitz domains." The Kunitz domain contains a series of cysteine residues that are regularly spaced at about 50 amino acid residue intervals and form three intrachain disulfide bonds. Members of this family include aprotinin, tissue factor pathway inhibitors (TFPI-1 and TFPI-2), inter-α-trypsin inhibitor, and bikunin (Marlor, CW et al. (1997) J. Biol. Chem. 272: 12220-12208.). Members of this family are potent inhibitors (in the nanomolar range) for serine proteases such as kallikrein and plasmin. Aprotinin is useful in controlling blood loss during surgery.
[0026]
The discovery of novel proteases and polynucleotides encoding them can meet the needs in the art by providing new compositions. The novel compositions are useful in the diagnosis, treatment, and prevention of gastrointestinal, cardiovascular, autoimmune / inflammatory, cell proliferation abnormalities, developmental disorders, epithelial disorders, neurological disorders, and reproductive disorders. It is also useful for evaluating the effects of foreign compounds on the expression of protease nucleic acid and amino acid sequences.
[0027]
(Summary of the Invention)
The present invention is generally referred to as "PRTS", and individually referred to as "PRTS-1," "PRTS-2," "PRTS-3," "PRTS-4," "PRTS-5," and "PRTS-6." , "PRTS-7", "PRTS-8", "PRTS-9", "PRTS-10", "PRTS-11", "PRTS-12", "PRTS-13", and "PRTS-14". A purified polypeptide is provided, which is a protease called. In one embodiment of the present invention, (a) an amino acid sequence selected from a group consisting of SEQ ID NOs: 1 to 14 (SEQ ID NOs: 1 to 14) and (b) SEQ ID NOs: 1 to 14 A natural amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group; and (c) a biologically active fragment of the amino acid sequence selected from the group consisting of SEQ ID NO: 1-14. And (d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14. . Alternatively, provided is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1-14.
[0028]
Further, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14 and (b) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14 by 90% or more. A natural amino acid sequence having sequence identity, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, and (d) SEQ ID NOs: 1-14. And an immunogenic fragment of an amino acid sequence selected from the group consisting of: an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: Alternatively, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOs: 1-14. Alternatively, the polynucleotide is selected from the group consisting of SEQ ID NO: 15-28.
[0029]
Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14 and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14 by 90% or more. A natural amino acid sequence having the sequence identity of: (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14; and (d) a SEQ ID NO: 1- And a recombinant polypeptide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of an immunogenic fragment of the amino acid sequence selected from the group consisting of: Provide nucleotides. Alternatively, the invention provides a cell transformed with the recombinant polynucleotide. In yet another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.
[0030]
Further, the present invention relates to (a) an amino acid sequence selected from a group consisting of SEQ ID NO: 1-14 and (b) an amino acid sequence selected from a group consisting of SEQ ID NO: 1-14 by 90% or more. A natural amino acid sequence having the sequence identity of: (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14; and (d) a SEQ ID NO: 1- 14. A method for producing a polypeptide comprising an amino acid sequence selected from the group consisting of: an immunogenic fragment of the amino acid sequence selected from the group consisting of: The method comprises the steps of (a) culturing cells transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide under conditions suitable for expression of the polypeptide. And (b) recovering the polypeptide thus expressed.
[0031]
Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14 and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14 by 90% or more. A natural amino acid sequence having the sequence identity of: (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14; and (d) a SEQ ID NO: 1- 14. An isolated antibody that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of an immunogenic fragment of the amino acid sequence selected from the group consisting of:
[0032]
Further, the present invention relates to a method comprising: (a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 15-28; and (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 15-28. (E) a polynucleotide sequence complementary to (a), (d) a polynucleotide sequence complementary to (b), and (e) a polynucleotide sequence complementary to (a). The present invention provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of the RNA equivalents (a) to (d). Alternatively, the polynucleotide comprises at least 60 contiguous nucleotides.
[0033]
Further, the present invention relates to (a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 15-28 and (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 15-28 by 90% A natural polynucleotide sequence having the above sequence identity, (c) a polynucleotide sequence complementary to (a), (d) a polynucleotide sequence complementary to (b), (e) Provided is a method for detecting in a sample a target polynucleotide having a polynucleotide sequence that includes a polynucleotide sequence selected from the group consisting of the RNA equivalents of (a) to (d). The method comprises the steps of: (a) hybridizing a sample containing at least 20 contiguous nucleotides constituting a sequence complementary to a target polynucleotide in the sample with the sample, the method comprising: Said step of specifically hybridizing said probe to said target polynucleotide under conditions in which a hybridization complex is formed with nucleotides or fragments thereof, and (b) whether said hybridization complex is present And optionally measuring its yield, if present. Alternatively, the probe comprises at least 60 contiguous nucleotides.
[0034]
Further, the present invention relates to (a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 15-28 and (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 15-28 by 90% A natural polynucleotide sequence having the above sequence identity, (c) a polynucleotide sequence complementary to (a), (d) a polynucleotide sequence complementary to (b), (e) Provided is a method for detecting in a sample a target polynucleotide having a polynucleotide sequence that includes a polynucleotide sequence selected from the group consisting of the RNA equivalents of (a) to (d). The method comprises: (a) amplifying the target polynucleotide or a fragment thereof using polymerase chain reaction amplification; and (b) detecting whether the amplified target polynucleotide or a fragment thereof is present. Optionally measuring the yield, if present.
[0035]
Further, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14 and (b) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14 by 90% or more. A natural amino acid sequence having sequence identity, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, and (d) SEQ ID NOs: 1-14. A composition comprising an effective amount of a polypeptide comprising an amino acid sequence selected from a group consisting of an immunogenic fragment of an amino acid sequence selected from the group consisting of: and a suitable pharmaceutical excipient. I do. In one embodiment, there is provided a composition comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14. Further, the present invention provides a method for treating a disease or a symptom thereof associated with reduced expression of functional PRTS, comprising administering the composition to a patient.
[0036]
Further, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14 and (b) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14 by 90% or more. A natural amino acid sequence having sequence identity, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, and (d) SEQ ID NOs: 1-14. And an immunogenic fragment of an amino acid sequence selected from the group consisting of: and a method for screening a compound effective as an agonist of a polypeptide containing an amino acid sequence selected from the group consisting of: The method comprises: (a) exposing a sample comprising the polypeptide to a compound; and (b) detecting agonist activity of the sample. Alternatively, the invention provides a composition comprising an agonist compound identified by this method and a suitable pharmaceutical excipient. In a further alternative, the invention provides a method of treating a disease or condition associated with reduced expression of functional PRTS, comprising administering the composition to a patient.
[0037]
Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14 and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14 by 90% or more. A natural amino acid sequence having the sequence identity of: (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14; and (d) a SEQ ID NO: 1- 14. A method for screening a compound effective as an antagonist of a polypeptide comprising an amino acid sequence selected from the group consisting of an immunogenic fragment of an amino acid sequence selected from the group consisting of: The method includes: (a) exposing a sample containing the polypeptide to a compound; and (b) detecting the antagonist activity of the sample. Alternatively, the invention provides a composition comprising an antagonist compound identified by this method and a suitable pharmaceutical excipient. In yet another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional PRTS, comprising administering the composition to a patient.
[0038]
Further, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14 and (b) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14 by 90% or more. A natural amino acid sequence having sequence identity, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, and (d) SEQ ID NOs: 1-14. A method of screening for a compound that specifically binds to a polypeptide containing an amino acid sequence selected from the group consisting of: an immunogenic fragment of an amino acid sequence selected from the group consisting of: The method comprises the steps of: (a) binding the polypeptide to at least one compound under suitable conditions; and (b) detecting binding of the polypeptide to the test compound to specifically bind the polypeptide to the test compound. Identifying a compound that binds to.
[0039]
Further, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14 and (b) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14 by 90% or more. A natural amino acid sequence having sequence identity, (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, and (d) SEQ ID NOs: 1-14. A method of screening for a compound that modulates the activity of a polypeptide comprising an amino acid sequence selected from the group consisting of an immunogenic fragment of an amino acid sequence selected from the group consisting of: The screening method comprises the steps of (a) binding the polypeptide to at least one compound under conditions permitting its activity; and (b) assessing the activity of the polypeptide in the presence of the test compound. (C) comparing the activity of the polypeptide in the presence of the test compound to the activity of the polypeptide in the absence of the test compound, Is characterized by the fact that a change in the activity of the polypeptide in E. coli indicates the presence of a compound that modulates the activity of the polypeptide.
[0040]
The invention further provides a method of screening for a compound effective to alter the expression of a target polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 15-28, comprising the steps of: The present invention provides the screening method, comprising: exposing a sample containing nucleotides to a compound; and (b) detecting a change in expression of the target polynucleotide.
[0041]
The invention further provides a method for assessing the toxicity of a test compound. The method comprises the steps of (a) treating a biological sample containing a nucleic acid with the test compound, (b) hybridizing the nucleic acid of the treated biological sample with a probe, and (c) yield of a hybridization complex. And (d) comparing the yield of the hybridization complex in the treated biological sample with the yield of the hybridization complex in the untreated biological sample. Differences in the yield of hybridization complexes indicate toxicity of the test compound. The probe in this method comprises: (1) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 15-28; and (2) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 15-28. A natural polynucleotide sequence having at least 90% sequence identity, (3) a polynucleotide sequence complementary to (1), (4) a polynucleotide sequence complementary to (2), (5) And) at least 20 consecutive nucleotides of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of the RNA equivalents of (1) to (4). In addition, the hybridization is performed under a condition where a specific hybridization complex is formed between the probe and a target polynucleotide of the biological sample. Further, the target polynucleotide may be (1) a polynucleotide sequence selected from a group consisting of SEQ ID NO: 15-28, and (2) a polynucleotide sequence selected from a group consisting of SEQ ID NO: 15-28. (3) a polynucleotide sequence complementary to (1), (4) a polynucleotide sequence complementary to (2), and (3) a polynucleotide sequence complementary to (1). 5) RNA equivalents of the above (1) to (5). Alternatively, said target polynucleotide is a fragment of said polynucleotide sequence.
[0042]
(About the description of the present invention)
Before describing the protein and nucleic acid sequences and methods of the present invention, it is to be understood that this invention is not limited to particular devices, materials, and methods disclosed herein, which may vary in its embodiments. Further, the terms used herein are used only for the purpose of describing a specific embodiment, and are limited only by the claims described below, and are not intended to limit the scope of the present invention. It should be understood that.
[0043]
It should be noted that in the specification and claims, the terms "a", "an", and the like include "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, “a host cell” includes a plurality of host cells, and the “antibody” includes a plurality of antibodies, including equivalents well known to those skilled in the art.
[0044]
Unless defined otherwise, 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 all devices and materials and methods similar or equivalent to those described herein can be used in the practice and testing of the present invention, the preferred devices, materials and methods are described here. All references mentioned herein have been cited to describe and disclose the cell lines, protocols, reagents, and vectors described in the literature that may be used in connection with the present invention. Citing a conventional invention does not, however, be construed as impairing the novelty of the present invention.
[0045]
(Definition)
The term "PRTS" refers to substantially purified PRTS amino acids obtained from all species, particularly natural, synthetic, semi-synthetic or recombinant, including mammals, including cows, sheep, pigs, mice, horses and humans. Points to an array.
[0046]
The term “agonist” refers to a molecule that enhances or mimics the biological activity of PRTS. The agonist may interact directly with PRTS or with components of the biological pathways involving PRTS to regulate the activity of PRTS, including proteins, nucleic acids, carbohydrates, small molecules, any other compounds, It may include a composition.
[0047]
The term "allelic variant" refers to another form of the gene encoding PRTS. Allelic variant sequences result from at least one mutation in the nucleic acid sequence, resulting in a variant mRNA or variant polypeptide, whose structure and function may or may not change. Some genes have no natural allelic variant, one or a large number thereof. In general, mutations that result in allelic variants are due to natural deletions, additions, or substitutions of nucleotides. Each of these mutations may occur alone or concurrently with other mutations, and may occur one or more times within a given sequence.
[0048]
A “mutated” nucleic acid sequence that encodes PRTS refers to a polypeptide that retains the same polypeptide as PRTS or has at least one of the functional properties of PRTS despite the occurrence of various nucleotide deletions, insertions, or substitutions. This definition includes inappropriate or unexpected hybridization of the polynucleotide sequence encoding PRTS with an allelic variant at a position that is not a normal chromosomal locus, as well as specific oligonucleotide probes of the polynucleotide encoding PRTS. And polymorphisms that are easily detectable or difficult to detect using The encoded protein may also be mutated and may contain deletions, insertions or substitutions of amino acid residues which produce a silent change and are functionally equivalent to PRTS. Intentional amino acid substitutions are similar to residues in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity, as long as the activity of the PRTS is retained biologically or immunologically. It can be done based on For example, negatively charged amino acids can include aspartic acid and glutamic acid, and positively charged amino acids can include lysine and arginine. Amino acids with similar hydrophilicity values and polar uncharged side chains may include asparagine, glutamine, serine, threonine. Amino acids with similar hydrophilicity values and uncharged side chains may include leucine, isoleucine, valine, glycine, alanine, phenylalanine and tyrosine.
[0049]
The terms "amino acid" and "amino acid sequence" refer to oligopeptides, peptides, polypeptides, protein sequences, or any fragment thereof, and include natural and synthetic molecules. Where "amino acid sequence" refers to the sequence of a naturally occurring protein molecule, "amino acid sequence" and similar terms are not limited to the complete, intact amino acid sequence associated with the described protein molecule.
[0050]
The term "amplification" relates to making a copy of a nucleic acid sequence. Generally, amplification is performed by the polymerase chain reaction (PCR) technique well known in the art.
[0051]
The term "antagonist" is a molecule that inhibits or attenuates the biological activity of PRTS. Antagonists are antibodies, nucleic acids, carbohydrates, small molecules, any other compounds or compositions that directly interact with PRTS or interact with components of the biological pathway in which PRTS is involved to modulate the activity of PRTS. Can include proteins such as objects.
[0052]
The term "antibody" refers to Fab and F (ab ') that are capable of binding an antigenic determinant.₂, And their fragments, Fv fragments and the like. Antibodies that bind a PRTS polypeptide can be produced using intact molecules or fragments thereof, including small peptides that immunize the antibody. The polypeptide or oligopeptide used to immunize an animal (eg, a mouse, rat, or rabbit) can be synthesized from the translation of RNA or chemically, and can be coupled to a carrier protein if necessary. It is also possible. Commonly used carriers that are chemically coupled to the peptide include bovine serum albumin, thyroglobulin, and keyhole limpet haemonian (KLH). The animal is then immunized with the conjugated peptide.
[0053]
The term "antigenic determinant" refers to a region of a molecule (ie, an epitope) that contacts a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, various regions of the protein may be antibodies that specifically bind to an antigenic determinant (a specific region or three-dimensional structure on the protein). Can be induced. Antigenic determinants can compete with the intact antigen (ie, the immunogen used to elicit the immune response) to bind the antibody.
[0054]
As used herein, "antisense" refers to any composition capable of base-pairing with the sense (coding) strand of a particular nucleic acid sequence. Antisense components include DNA, RNA, peptide nucleic acids (PNA), oligonucleotides having a modified backbone linkage such as phosphorothionate, methylphosphonate, benzylphosphonate, and 2 ′. Oligonucleotides having a modified sugar, such as -methoxyethyl sugar or 2'-methoxyethoxy sugar, and oligos having a modified base, such as 5-methylcytosine or 2'-deoxyuracil, 7-deaza-2'-deoxyguanosine May contain nucleotides. Antisense molecules can be created by any method, including chemical synthesis and transcription. Once introduced into a cell, a complementary antisense molecule base pairs with a natural nucleic acid sequence created by the cell to form a duplex and inhibit transcription and translation. The expression "negative" or "minus" is the antisense strand, and the expression "positive" or "plus" is the sense strand.
[0055]
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a natural molecule. Similarly, the term "immunologically active" or "immunogenic" refers to the ability of natural or recombinant PRTS, synthetic PRTS or any of their oligopeptides to induce a specific immune response in a suitable animal or cell. Refers to the ability to induce and bind to a particular antibody.
[0056]
The term "complementary" refers to the relationship between two single-stranded nucleic acid sequences that anneal by base pairing. For example, the sequence "5'AGT3 '" pairs with the complementary sequence "3'TCA5'."
[0057]
"Composition comprising a given polynucleotide sequence" or "composition comprising a given amino acid sequence" refers in a broad sense to any composition comprising a given nucleotide or amino acid sequence. The composition may include a dry formulation or an aqueous solution. Compositions comprising a polynucleotide sequence encoding PRTS or a fragment of PRTS can be used as a hybridization probe. This probe can be stored in a lyophilized state, and can be bound to a stabilizer such as a carbohydrate. In hybridization, the probe is developed in an aqueous solution containing a salt (eg, NaCl), a surfactant (eg, SDS: sodium dodecyl sulfate), and other substances (eg, Denhardt's solution, dried milk, salmon sperm DNA, etc.). obtain.
[0058]
The “consensus sequence” is repeatedly subjected to DNA sequence analysis to separate unnecessary bases, and is extended in the 5 ′ and / or 3 ′ direction using an XL-PCR kit (PE Biosystems, Foster City CA), One or more overlapping cDNAs using a resequenced nucleic acid sequence or a computer program for fragment construction such as the GELVIEW fragment construction system (GCG, Madison, WI) or Phrap (University of Washington, Seattle WA). And ESTs, or nucleic acid sequences constructed from genomic DNA fragments. Some consensus sequences are constructed by both extension and duplication.
[0059]
The term "conservative amino acid substitution" refers to a substitution that does not significantly alter the properties of the original protein. That is, the structure and function of the protein are not significantly changed by the substitution, and the structure of the protein, particularly its function, is preserved. The following shows conservative amino acid substitutions in which the original amino acid of one protein is replaced by another amino acid.
Original residue conservative substitution
Ala @ Gly, Set
Arg @ His, Lys
Asn @ Asp, Gln, His
Asp @ Asn, Glu
Cys @ Ala, Ser
Gln @ Asn, Glu, His
Glu @ Asp, Gln, His
Gly @ Ala
His @ Asn, Arg, Gln, Glu
Ile @ Leu, Val
Leu @ Ile, Val
Lys @ Arg, Gln, Glu
Met @ Leu, Ile
Phe @ His, Met, Leu, Trp, Tyr
Ser @ Cys, Thr
Thr @ Ser, Val
Trp @ Phe, Tyr
Tyr @ His, Phe, Trp
Val @ Ile. Leu, Thr
In general, for conserved amino acid substitutions, a) the backbone structure of the polypeptide in the substituted region, for example, a β-sheet or α-helical conformation; b) the charge or hydrophobicity of the molecule at the substituted site; And / or c) the majority of the side chains are maintained.
[0060]
The term "deletion" refers to a change in the amino acid sequence that lacks one or more amino acid residues, or a change in the nucleic acid sequence that lacks one or more nucleotides.
[0061]
The term “derivative” refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide sequence include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide that retains at least one of the biological or immunological functions of the native molecule (the unmodified molecule). A derivative polypeptide is a polypeptide modified by glycosylation, polyethyleneglycolation, or any similar process that maintains at least one of the biological or immunological functions of the original polypeptide. That is.
[0062]
"Detectable label" refers to a reporter molecule or enzyme that is capable of producing a measurable signal, covalently or non-covalently linked to a polynucleotide or polypeptide.
[0063]
The term "fragment" refers to a unique portion of a PRTS or a polynucleotide encoding a PRTS that is identical to, but shorter in length than, its parent sequence. The maximum length of a "fragment" is the parent sequence minus one nucleotide / amino acid residue. For example, some fragments contain from 5 to 1000 contiguous nucleotide or amino acid residues. At least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250, or 500 probes, primers, antigens, therapeutic molecules, or fragments used for other purposes Is the length of consecutive nucleotides or amino acid residues. Fragments may be preferentially selected from particular regions of the molecule. For example, a polypeptide fragment can include a predetermined length of contiguous amino acids selected from the first 250 or 500 amino acids set forth in the predetermined sequence (or the first 25% or 50% of the polypeptide). . These lengths are examples, and any length described in the specification including the sequence listing and the tables and drawings can be included in the examples of the present invention.
[0064]
Fragments of SEQ ID NO: 15-28 include, for example, regions of the unique polynucleotide sequence that unambiguously identify SEQ ID NO: 15-28, different from other sequences in the genome from which the fragment was obtained. Certain fragments of SEQ ID NO: 15-28 are useful, for example, in hybridization and amplification techniques, or similar methods that distinguish SEQ ID NO: 15-28 from related polynucleotide sequences. The exact fragment length or region of SEQ ID NO: 15-28 that corresponds to a fragment can be routinely determined by techniques common in the art based on the purpose of the fragment.
[0065]
A fragment with SEQ ID NO: 1-14 is encoded by a fragment with SEQ ID NO: 15-28. Certain fragments of SEQ ID NOs: 1-14 include regions of unique amino acid sequences that specifically identify SEQ ID NOs: 1-14. For example, certain fragments of SEQ ID NO: 1-14 are useful as immunogenic peptides in the development of antibodies that specifically recognize SEQ ID NO: 1-14. The exact fragment length or region of SEQ ID NO: 1-14 that matches a fragment can be routinely determined by techniques common in the art based on the purpose of the fragment.
[0066]
A “full-length” polynucleotide sequence is a sequence that has at least one translation initiation codon (eg, methionine) followed by an open reading frame and a translation stop codon. A "full length" polynucleotide sequence encodes a "full length" polypeptide sequence.
[0067]
"Homology" is sequence similarity between two or more polynucleotide sequences or two or more polypeptide sequences. This sequence similarity can be translated into sequence identity.
[0068]
The terms "percent identity" or "% identity" with respect to polynucleotide sequences are the percentage of matching residues between two or more polynucleotide sequences that are aligned using a standardized algorithm. It is. Such an algorithm can insert gaps in the sequences to make a more meaningful comparison between the two sequences in a standardized and reproducible manner to optimize the alignment between the two sequences.
[0069]
The percent identity between polynucleotide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package and is a suite of molecular biology analysis programs (DNASTAR, Madison WI). This CLUSTAL V is available from Higgins, D.W. G. FIG. And P.A. M. Sharp (1989) CABIOS 5: 151-153; Higgins, D.W. G. FIG. Et al. (1992) CABIOS 8: 189-191. For alignment of pairs of polynucleotide sequences, the default parameters are set as Ktuple = 2, gap penalty = 5, window = 4, and “diagonals saved” = 4. A "weighted" residue weight table was selected as the default. Percent identity is reported by CLUSTAL V as "percent similarity" between the aligned polynucleotide sequences.
[0070]
Alternatively, National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, SF, et al. (1990) J. Mol. Biol. A complete set of free sequence comparison algorithms is available from several sources, including NCBI (Bethesda, MD) and the Internet (http://www.ncbi.nlm.nih.gov/BLAST/). The BLAST software suite includes various sequence analysis programs, including "blastn" used to align known polynucleotide sequences with other polynucleotide sequences in various databases. A tool called "BLAST 2 Sequences" is available and is used to directly compare pairs of two nucleotide sequences. “BLAST 2 Sequences” is available at http: // www. ncbi. nlm. nih. gov / gorf / b12. You can access html and use it interactively. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (described below). BLAST programs are typically used with gaps and other parameters to set defaults. For example, when comparing two nucleotide sequences, one would use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set to default parameters. Such default parameters are, for example, as follows.
[0071]
Matrix: BLOSUM62
Reward for match: 1
Penalty for Mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off: 50
Expect: 10
Word Size: 11
Filter: on
Percent identity may be measured, for example, for the entire length of a given sequence, as determined by a particular SEQ ID NO: or for a shorter length, for example, from a larger given sequence. It may be measured against the length of a given fragment, for example a fragment of at least 20 or 30, 40, 50, 70, 100, 200 nucleotides in a row. Such lengths are merely examples, and any length fragment of the sequences set forth in the specification, including the sequence listing and the tables and drawings, may be used to indicate the length at which percent identity is measured. it can.
[0072]
Nucleic acid sequences that do not show a high degree of identity may encode similar amino acid sequences due to the degeneracy of the genetic code. It should be understood that degeneracy can be used to alter nucleic acid sequences to produce various nucleic acid sequences, each encoding substantially the same protein.
[0073]
The terms "percent identity" or "% identity" as used in polypeptide sequences refer to the percentage of matching residues between two or more polypeptide sequences that are aligned using a standardized algorithm. It is. Methods for polypeptide sequence alignment are well known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, as described in detail above, generally preserve the charge and hydrophobicity of the substitution site and preserve the structure (and therefore function) of the polypeptide.
[0074]
The percent identity between polypeptide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated in the MEGALIGN version 3.12e sequence alignment program (described above). In the case of paired polypeptide sequence alignment using CLUSTAL V, the default parameters are set as Ktuple = 1, gap penalty = 3, window = 5, and “diagons saved” = 5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity of a pair of aligned polypeptide sequences is reported by CLUSTAL V as "percent similarity".
[0075]
Alternatively, the NCBI BLAST software suite is used. For example, when comparing two polypeptide sequences in pairs, one may use blastp with the "BLAST 2 Sequences" tool Version 2.0.12 (Apr-21-2000) set with default parameters. There will be. Such default parameters are, for example, as follows.
[0076]
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off: 50
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured, for example, over the entire length of a given polypeptide sequence, as determined by a particular SEQ ID NO: It may be measured against the length of fragments obtained from the peptide sequence, for example fragments of at least 15,20 or 30,40,50,70,150 contiguous residues. Such lengths are merely examples, and any length fragment of the sequences set forth in the specification, including the sequence listing and the tables and drawings, may be used to indicate the length at which percent identity is measured. it can.
[0077]
"Human artificial chromosomes (HACs)" are linear, small elements containing all the elements necessary for the separation and maintenance of stable mitotic chromosomes, which can include DNA sequences of about 6 kb (kilobases) to 10 Mb in size. It is a chromosome.
[0078]
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence of the non-antigen binding region has been changed to more closely resemble a human antibody while retaining its original binding ability.
[0079]
“Hybridization” is the process of annealing in which a single-stranded polynucleotide forms a base pair with a complementary single-strand under predetermined hybridization conditions. Specific hybridization means that two nucleic acid sequences have high homology. Under conditions that permit annealing, a specific hybridization complex is formed and remains hybridized after the washing step. The washing step is particularly important in determining the stringency or stringency of the hybridization process; in more stringent conditions, non-specific binding, ie, binding of pairs between nucleic acid strands that are not perfectly matched. Decrease. The conditions under which annealing between nucleic acid sequences is permissible are routinely determined by those skilled in the art, and are constant during hybridization, but the washing process is altered during the process to achieve the desired stringency. And hybridization specificity is obtained. Conditions that permit annealing include, for example, about 6 × SSC, about 1% (w / v) SDS at about 68 ° C., and about 100 μg / ml shear denatured salmon sperm DNA.
[0080]
In general, the stringency of hybridization also depends on the temperature at which the washing step is performed. This washing temperature is usually selected to be about 5-20C below the thermal melting point (Tm) of the particular arrangement at a given ionic strength and pH. This Tm is the temperature (under a given ionic strength and pH) at which 50% of the target sequence hybridizes with a perfectly matched probe. The equation for calculating the Tm and the conditions for nucleic acid hybridization are well known and are described in Sambrook, J. et al. By others, 1989,Molecular Cloning: A Laboratory Manual, Second Edition, Volume 1-3, Cold Spring Harbor Press, Plainview NY;
[0081]
High stringency hybridization between polynucleotides of the present invention involves a washing step at about 68 ° C. for 1 hour in the presence of about 0.2 × SSC and about 1% SDS. Alternatively, it is performed at a temperature of 65 ° C, 60 ° C, 55 ° C, 42 ° C. The concentration of SSC ranges from about 0.1 to 2x SSC in the presence of about 0.1% SDS. Usually, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for example, about 100-200 μg / ml of cleaved and denatured salmon sperm DNA. Organic solvents such as formamide at a concentration of about 35-50% v / v can be used in certain cases, such as, for example, hybridization of RNA and DNA. Useful modifications of these washing conditions are well known to those skilled in the art. Hybridization under particularly high stringency conditions can indicate evolutionary similarities between nucleotides. Such similarities strongly suggest that their nucleotides and encoded polypeptides play a similar role.
[0082]
The term "hybridization complex" refers to a complex of two nucleic acid sequences formed by hydrogen bonding between complementary bases. The hybridization complex is in solution (eg, C₀t or R₀t-analysis) or one nucleic acid sequence in solution and a solid support (eg, paper, membrane, filter, chip, pin, or glass slide, or any suitable fixation of cells and their nucleic acids) And another nucleic acid sequence immobilized on another substrate.
[0083]
The term "insertion" or "addition" refers to a change in the amino acid or nucleic acid sequence to which one or more amino acid residues or nucleotides are added, respectively.
[0084]
"Immune response" refers to symptoms associated with inflammatory diseases and trauma, immune disorders, infectious diseases, genetic diseases, and the like. These conditions are characterized by the expression of various factors, such as cytokines and chemokines, and other signaling molecules, that affect the cell line and the systemic defense system.
[0085]
The term “immunogenic fragment” refers to a polypeptide or oligopeptide fragment of GVRED that elicits an immune response when introduced into a living animal, eg, a mammal. The term "immunogenic fragment" also includes polypeptide or oligopeptide fragments of PRTS useful in any of the antibody production methods disclosed herein or known in the art.
[0086]
The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides or other compounds on a substrate.
[0087]
The term "element" or "array element" refers to a polynucleotide, polypeptide or other compound that has a unique, designated location on a microarray.
[0088]
The term "modulation" refers to a change in the activity of PRTS. For example, modulation results in a change in PRTS protein activity, or binding, or other biological, functional, or immunological properties.
[0089]
The terms "nucleic acid" and "nucleic acid sequence" refer to nucleotides, oligonucleotides, polynucleotides, or fragments thereof, single or double stranded, of genomic or synthetic origin that are sense or antisense strands DNA or RNA, peptide nucleic acid (PNA), any DNA-like substance, and RNA-like substance.
[0090]
"Operably linked" refers to the state in which a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship. For example, when a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Generally, operably linked DNA sequences are very close together or contiguous if, in the same reading frame, the regions encoding the two proteins need to be joined.
[0091]
“Peptide nucleic acid (PNA)” refers to an antisense molecule or an antigenic agent that comprises an oligonucleotide at least about 5 nucleotides in length attached to a peptide backbone of amino acid residues terminating in lysine. This terminal lysine renders the composition soluble. PNAs can preferentially bind to complementary single-stranded DNA or RNA, stop transcript elongation, and become polyethylene glycolated to extend cell life.
[0092]
"Post-translational modifications" of a PRTS may include lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage and other modifications known in the art. These processes can occur synthetically or biochemically. Biochemical modifications depend on the enzymatic environment of PRTS and can vary from cell type to cell type.
[0093]
The term "probe" refers to a nucleic acid sequence encoding PRTS, its complementary sequence, or a fragment thereof, which is used for detecting the same or allele nucleic acid sequence or a related nucleic acid sequence. A probe is an isolated oligonucleotide or polynucleotide to which a detectable label or reporter molecule has been attached. Typical labels include radioisotopes and ligands, chemiluminescent reagents, and enzymes. A “primer” is a short nucleic acid, usually a DNA oligonucleotide, capable of forming complementary base pairs and annealing to a target polynucleotide. After the primer anneals to the polynucleotide, it is extended along the target DNA single strand by a DNA polymerase enzyme. The set of primers can be used, for example, for amplification (and identification) of a nucleic acid sequence in a PCR method.
[0094]
Probes and primers used in the present invention comprise at least 15 contiguous nucleotides of a known sequence. Longer probes and primers can be used to increase specificity. For example, it comprises at least 20 or 25, 30, 40, 50, 60, 70, 80, 90, 100, 150 contiguous nucleotides of the disclosed nucleic acid sequences. It is to be understood that probes and primers can be used which are considerably longer than the examples described above, and that nucleotides of any length shown in the tables, drawings and sequence listings herein can be used.
[0095]
For the preparation and use of probes and primers, see, for example, Sambrook, J. et al. 1989, entitled "Molecular Cloning: A Laboratory Manual", Second Edition, Volume 1-3 (Cold Spring Harbor Press, Plainview NY), or Ausubel, F.C. M. In 1987, "Current Protocols in Molecular Biology" (Greene Pubi. Assoc. & Wiley-Intersciences, New York NY), and in 1990, by Innis et al. (Academic Press, San Diego CA.). A set of primers for PCR can be derived from a known sequence using a computer program for such purposes, such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA). Can be.
[0096]
Oligonucleotides to be used as primers are selected by a computer program for selecting primers well known in the art. For example, OLIGO 4.06 software can select primer pairs for PCR up to 100 nucleotides each, and large polynucleotides and oligonucleotides up to 5,000 nucleotides from input polynucleotide sequences of up to 32,000 bases. It is useful for analysis. Similar primer selection programs include additional features to expand capacity. For example, the PrimOU primer selection program (available from the Genome Center at University of Texas South West Medical Center, Dallas TX) allows the selection of specific primers from the megabase sequence, thus providing a primer for genome-wide design. Useful for The Primer3 primer selection program (available from Whitehead Institute / MIT Center for Genome Research, Cambridge MA1) allows the user to enter a "non-priming library" that allows the designation of sequences to be avoided as primer binding sites. In addition, Primer 3 is particularly useful for selecting oligonucleotides for microarrays (the source code of the latter two primer selection programs can be obtained from the respective sources and modified to meet the needs of the user. You can also). The PrimerGen program (available from the UK Human Genome Mapping Project Resource Center, Cambridge UK) designs the primers based on multiple sequence alignments, and therefore the most conserved or least conserved regions of the aligned nucleic acid sequences. Can be selected. Thus, the program is useful for identifying unique and conserved oligonucleotide and polynucleotide fragments. Oligonucleotide or polynucleotide fragments identified by any of the selection methods described above may be used, for example, to identify polynucleotides, sequencing primers, microarray elements, or polynucleotides that completely or partially complement the nucleic acids of the sample. Useful for hybridization techniques, such as a probe. The method for selecting the oligonucleotide is not limited to the method described above.
[0097]
As used herein, a “recombinant nucleic acid” is not a natural sequence, but a sequence in which two or more separate segments of a sequence are artificially combined. Although this artificial combination is often made by chemical synthesis, it is more common to artificially manipulate discrete segments of nucleic acids using genetic engineering techniques such as those described in Sambrook, supra. is there. The “recombinant nucleic acid” also includes a nucleic acid that has been changed simply by adding, replacing, or deleting a part of the nucleic acid. A recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid can be, for example, part of a vector used to transform a cell.
[0098]
Alternatively, such a recombinant nucleic acid may be one of vaccinia virus-based viral vectors that, when used for vaccination of a mammal expressing the recombinant nucleic acid, elicits a protective immune response in the mammal. Department.
[0099]
A "regulatory element" is a nucleic acid sequence usually derived from the untranslated region of a gene, and includes enhancers, promoters, introns, and 5 'and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins that regulate transcription or translation, or RNA stability.
[0100]
A “reporter molecule” is a chemical or biochemical moiety used to label nucleic acids, amino acids or antibodies. Reporter molecules include radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles, and other components known in the art.
[0101]
As used herein, the term "RNA equivalent" for a DNA sequence is composed of the same linear nucleic acid sequence as the reference DNA sequence, but the thymine of the nitrogenous base is replaced with uracil, and the backbone of the sugar chain is Consists of ribose, not deoxyribose.
[0102]
The term "sample" is used in its broadest sense. A sample presumed to contain PRTS, a nucleic acid encoding PRTS, or a fragment thereof is obtained by analyzing a body fluid, an extract from a cell or a chromosome or an organelle or a membrane isolated from a cell, a membrane, a cell, Genomic DNA, RNA, cDNA present in or immobilized on a substrate, tissue, tissue prints and the like.
[0103]
The terms "specific binding" and "specifically bind" refer to the interaction between a protein or peptide and an agonist, antibody, antagonist, small molecule, or any natural or synthetic binding composition. This interaction depends on the presence of specific structures of the protein, such as, for example, antigenic determinants or epitopes, recognized by the binding molecule. For example, if the antibody is specific for the epitope "A", the unbound labeled "A" and the reaction solution containing the antibody may contain the polypeptide containing epitope A or the unlabeled "A" ”Reduces the amount of label A that binds to the antibody.
[0104]
The term "substantially purified" refers to an isolated or isolated nucleic acid or amino acid sequence that has been removed from its natural environment and has at least about 60% of its naturally associated composition removed. Preferably at least about 75% removed, most preferably at least 90% removed.
[0105]
"Substitution" refers to the replacement of one or more amino acids or nucleotides with another amino acid or nucleotide, respectively.
[0106]
The term "substrate" refers to any suitable solid or semi-solid support, including membranes and filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubes, plates, polymers, microparticles, capillaries. Is included. The substrate has various surface features, such as walls or trenches, pins, channels, pores, etc., to which polynucleotides or polypeptides bind.
[0107]
"Transcription image" refers to the pattern of collective gene expression by a particular cell type or tissue at a given time under given conditions.
[0108]
"Transformation" refers to the process by which foreign DNA is introduced into recipient cells. Transformation can occur under natural or artificial conditions by a variety of methods known in the art, and can be performed by any known method of inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. . The method of transformation is selected according to the type of host cell to be transformed. The methods include, but are not limited to, bacteriophage or viral infection, electroporation, lipofection, and microparticle irradiation. "Transformed" cells include stably transformed cells in which the introduced DNA is capable of replicating autonomously as a plasmid or as part of the host chromosome. Furthermore, cells that express the introduced DNA or introduced RNA temporarily for a limited time are also included.
[0109]
As used herein, a "genetically modified organism" is any organism in which a human has introduced a heterologous nucleic acid into one or more cells of the organism, using genetically well-known techniques in the art, and the like. And plants, but is not limited thereto. Heterologous nucleic acids are introduced into cells directly or indirectly into their precursors by careful genetic manipulation, such as microinjection or infection with recombinant viruses. "Genetic manipulation" refers to typical cross breeding andin in vitroIs to introduce a recombinant DNA molecule instead of fertilization at Genetically modified organisms according to the present invention include bacteria and cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into a host by methods well known in the art, for example, infection, transfection, transformation, transconjugation, and the like. Techniques for introducing the DNA of the present invention into such organisms are well known and are described in Sambrook et al. (1989), supra.
[0110]
The “variant sequence” of a specific nucleic acid sequence is defined as a specific sequence for a certain nucleic acid sequence by blastn using a “BLAST 2 Sequences” tool Version 2.0.9 (May-07-1999) with default parameter settings. Is a nucleic acid sequence whose identity is determined to be at least 40%. Such pairs of nucleic acids may exhibit, for example, at least 50% or 60%, 70%, 80%, 85%, 90%, 95%, 98%, or more identity in certain lengths. Certain variant sequences can be referred to, for example, as "allelic" variant sequences (described above) or "splice" variant sequences, "species" variant sequences, "polymorphic" variant sequences. A splice variant may have very high identity to a reference molecule, but will have more or less polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may have additional functional domains present in the reference molecule, or may lack domains present in the reference molecule. Species variant sequences are polynucleotide sequences that vary from species to species. The resulting polypeptides have high amino acid identity with each other. Polymorphic variant sequences differ in the polynucleotide sequence of a particular gene in a given species. Polymorphic variant sequences can also include "single nucleotide polymorphisms" (SNPs) in which one nucleotide of the polynucleotide sequence differs. The presence of a SNP may, for example, indicate a certain population, condition, or propensity for the condition.
[0111]
A "variant" of a particular polypeptide sequence is defined as the blastp using a "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) with default parameter settings, for a given length of a nucleic acid sequence. A polypeptide sequence whose identity is determined to be at least 40% for a particular polypeptide sequence. Such pairs of polypeptides may exhibit, for example, at least 50% or 60%, 70%, 80%, 85%, 90%, 95%, 98% or more identity in a certain length. .
[0112]
(invention)
The present invention is based on the discovery of novel human proteases (PRTS) and polynucleotides encoding PRTS, and uses these compositions to treat gastrointestinal diseases, cardiovascular diseases, autoimmune / inflammatory diseases, abnormal growth, and developmental disorders. Diagnosing, treating, and preventing epithelial diseases, neurological diseases, and reproductive disorders.
[0113]
Table 1 shows the identification numbers of the polynucleotide and polypeptide sequences of the present invention. Each polynucleotide and its corresponding polypeptide correlate to one Incyte Project ID number (Incyte Project ID). Each polypeptide sequence is indicated by both a polypeptide sequence identification number (Polypeptide SEQ ID NO :) and an incyte polypeptide sequence number (Incyte Polypeptide ID) as described. Each polynucleotide sequence is indicated by both the polynucleotide sequence identification number (Polypeptide SEQ ID NO :) and the incyte polynucleotide consensus sequence number (Incyte Polypeptide ID) as described.
[0114]
Table 2 shows sequences having homology to the polypeptides of the invention identified by BLAST analysis in the GenBank protein (genept) database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO :) and the corresponding insite polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention. Column 3 shows the GenBank identification number (Genbank ID NO :) of the closest homolog of GenBank. Column 4 shows the probability score representing the match between each polypeptide and its GenBank homolog. Column 5 shows the annotation of the GwnBank homolog, with the appropriate citation in place. These are incorporated herein by reference.
[0115]
Table 3 shows various structural features of each polypeptide of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO :) and the corresponding insite polypeptide sequence number (Incyte Polypeptide ID) of each polypeptide of the invention. Column 3 shows the number of amino acid residues in each polypeptide. Rows 4 and 5 show the potential phosphorylation and glycosylation sites, respectively, as determined by the MOTIFS program (Genetics Computer Group, Madison WI) of the GCG sequence analysis software package. Column 6 shows the amino acid residues that include the signature sequence, domain, and motif. Column 7 shows the analysis method for analysis of the structure / function of the protein, and the relevant part shows a searchable database used for the analysis method.
[0116]
As shown in Table 4, the polynucleotide sequences of the present invention were assembled using cDNA sequences, or coding (exon) sequences derived from genomic DNA, or any combination of these two sequences. Columns 1 and 2 show the polynucleotide sequence identification number (Polynucleotide SEQ ID NO :) of each polynucleotide of the present invention and the corresponding insite polynucleotide consensus sequence number (Incyte Polynucleotide ID), respectively. Column 3 shows the length of each polynucleotide sequence in base pairs. Row 4 contains polynucleotide sequences useful for hybridization or amplification techniques, for example, to identify SEQ ID NO: 15-28 or to distinguish SEQ ID NO: 15-28 from related polynucleotide sequences. Shows a fragment. Column 5 indicates the cDNA sequence, the coding sequence (exons) deduced from genomic DNA, and / or the identification number corresponding to the group consisting of both cDNA and genomic DNA. These sequences were used to construct a polynucleotide sequence of the present invention. Columns 6 and 7 of Table 4 indicate the starting nucleotide (5 ') and ending nucleotide (3') positions of the cDNA and genomic sequences, respectively, corresponding to the sequence of column 5.
[0117]
Specifically, the identification numbers shown in column 5 of Table 4 indicate, for example, the identification numbers of in-site cDNAs and their corresponding cDNA libraries. For example, 7032724H1 is the identification number of the insite cDNA sequence, and BRAXTDR12 is the identification number of the cDNA library from which it was derived. Insite cDNAs for which no cDNA library is shown were derived from a pooled cDNA library (eg 70152356V1). The identification number in row 5 may be the identification number of the GenBank cDNA or EST (eg, g536364) used to assemble the full-length polynucleotide sequence. Alternatively, the identification number in column 5 may be the coding region of the genomic DNA deduced by Genscan analysis. For example, GNN. g6436155_002. edit is the identification number of the Genscan deduced coding sequence, and g6436155 is the identification number of the GenBank sequence obtained by Genscan analysis. This Genscan putative coding sequence may be edited before the sequence is assembled (Example 4See). Alternatively, the identification number in column 5 may be a group consisting of both cDNA and Genscan putative exons by the "exon-stitching" algorithm (Example 5See). Alternatively, the identification number in column 5 may be a group consisting of both cDNA and Genscan putative exons by the “exon-stretching” algorithm (Example 5See). In some cases, a range of in-site cDNAs that overlap the range of sequences shown in column 5 is obtained and the final consensus sequence is determined, but the corresponding in-site cDNA identification numbers are indicated. Absent.
[0118]
Table 5 shows a representative cDNA library from these polynucleotide sequences assembled using the insite cDNA sequence. A typical cDNA library is an insite cDNA library containing the largest amount of insite cDNA sequences used for assembling and determining the polynucleotide sequence. The tissues and vectors used to generate the cDNA libraries shown in Table 5 are shown in Table 6.
[0119]
The invention also includes variants of PRTS. Preferred variants of PRTS have at least one of functional and / or structural characteristics of PRTS and have at least about 80% amino acid sequence identity to the PRTS amino acid sequence, or at least about 90% amino acid sequence. Have identity, and even at least about 95% amino acid sequence identity.
[0120]
The present invention also provides a polynucleotide encoding PRTS. In certain embodiments, the present invention provides a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NOS: 15-28 encoding PRTS. The polynucleotide sequence of SEQ ID NO: 15-28 shown in the sequence listing includes an equivalent RNA sequence in which thymine, a nitrogenous base, is substituted with uracil, and the backbone of the sugar chain is composed of ribose instead of deoxyribose.
[0121]
The invention also includes mutant sequences of the polynucleotide sequence encoding PRTS. In particular, such a variant of the polynucleotide sequence will have at least 70% polynucleotide sequence identity to the polynucleotide sequence encoding PRTS, or at least 85% polynucleotide sequence identity, or even at least 95%. Has polynucleotide sequence identity. Certain embodiments of the present invention provide that at least 70% polynucleotide sequence identity, or at least 85% polynucleotide sequence identity, or even at least 85% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15-28. A variant sequence of the polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 15-28 having as much as 95% polynucleotide sequence identity is provided. Each of the above-described polynucleotide variants encodes an amino acid sequence having at least one of the functional or structural features of PRTS.
[0122]
Those skilled in the art will recognize that the various polynucleotide sequences encoding PRTS that can be created by the degeneracy of the genetic code include those that have minimal similarity to the polynucleotide sequences of any known naturally occurring genes. Will understand. Thus, the present invention may include all possible polynucleotide sequence variations that may be made by selecting combinations based on possible codon choices. These combinations are made based on the standard triplet genetic code as applied to the polynucleotide sequence of native PRTS, and all variations are considered to be expressly disclosed.
[0123]
The nucleotide sequence encoding PRTS and its variant sequences are generally capable of hybridizing to the nucleotide sequence of a native PRTS under suitably selected stringent conditions, but are substantially non-naturally occurring, such as including non-naturally occurring codons. It may be advantageous to generate nucleotide sequences encoding PRTS or derivatives thereof with different usage codons. Codons can be selected based on the frequency with which particular codons are utilized by the host to increase the rate at which expression of the peptide occurs in a particular eukaryotic or prokaryotic host. Another reason for substantially altering the nucleotide sequence encoding PRTS and its derivatives without altering the encoded amino acid sequence is that RNA transcripts have favorable properties, such as longer half-lives, than transcripts made from the native sequence. Is to make things.
[0124]
The present invention also includes the production of DNA sequences encoding PRTS and its derivatives, or fragments thereof, entirely by synthetic chemistry. After construction, the synthetic sequence can be inserted into any of a variety of available expression vectors and cell lines using reagents well known in the art. In addition, synthetic chemistry can be used to introduce mutations into the sequence encoding PRTS or any fragment thereof.
[0125]
The invention further includes polynucleotide sequences that are capable of hybridizing under various stringent conditions to the claimed polynucleotide sequences, particularly SEQ ID NO: 15-28 and fragments thereof ( See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152: 399-407; and Kimmel.AR (1987) Methods Enzymol. 152: 507-511.). Hybridization conditions, including annealing and washing conditions, are described in Definitions.
[0126]
Any of the embodiments of the present invention can be performed using DNA sequencing methods well known in the art. This method includes, for example, a Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham, Pharmacia Biotech or Pisca Nevada, Biotech, Pisca, NY, USA). Enzymes, such as a combination of a proofreading exonuclease and a polymerase, such as those found in Gaithersburg MD, are used. Preferably, a device such as MICROLAB2200 liquid transfer system (Hamilton, Reno, NV), PTC200 Thermal Cycler200 (MJ Research, Watertown MA) and ABI CATARYST 800 (PE Biosystems) are used to automatically prepare and arrange devices. Next, sequencing is performed using an ABI 373 or 377 DNA sequencing system (PE Biosystems), a MEGABACE 1000 DNA sequencing system (Molecular Dynamics. Sunnyvale CA), or another method known in the art. The resulting sequence is analyzed using various algorithms well known in the art (eg, Ausubel, FM (1997)).Short Protocols in Molecular BiologyMeyers, R., John Wiley & Sons, New York NY, unit 7.7; A. (1995)Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853. See).
[0127]
Various methods based on the PCR method well known in the art are used to extend the nucleic acid sequence encoding PRTS and detect upstream sequences such as promoters and regulatory elements using partial nucleotide sequences. For example, one method utilizing restriction site PCR involves amplifying an unknown sequence from genomic DNA in a cloning vector using common and nested primers (eg, Sarkar, G. (1993)). PCR Methods Appl. 2: 318-322). Another method using the inverse PCR method uses primers that amplify an unknown sequence from a circularized template that has been extended in a wide range of directions. This template is derived from restriction fragments containing known genomic loci and surrounding sequences (see, eg, Triglia, T. et al. (1988) Nucleic Acids Res 16: 8186). A third method using the capture PCR method involves PCR amplification of DNA fragments adjacent to known sequences of human and yeast artificial chromosomal DNA (eg, Lagerstrom, M. et al. (1991) PCR Methods Applic 1: 111-). 119). In this method, it is possible to insert the recombinant double-stranded sequence into a region of unknown sequence before performing PCR using digestion and ligation with a number of restriction enzymes. It is also possible to obtain unknown sequences using other methods well known in the art. (See, e.g., Parker, JD, et al. (1991) Nucleic Acids Res. 19: 3055-3060). Furthermore, the use of PCR, nested primers, and a PROMOTERFINDER library (Clontech, Palo Alto CA) allows walking within genomic DNA. This method eliminates the need to screen libraries and is useful for finding intron / exon junctions. In all PCR-based methods, the primers are 22-30 nucleotides in length using a commercially available OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth MN) or another suitable program. It is designed to anneal to the mold at a temperature of about 68 ° C to 72 ° C with a content of 50% or more.
[0128]
When screening for full-length cDNAs, it is preferable to use libraries whose size has been selected to include large cDNAs. In addition, if the oligo d (T) library cannot produce full-length cDNA, a randomly primed library, often containing a sequence having the 5 'region of the gene, is useful. Genomic libraries will be useful for extension of sequence into 5 'non-transcribed regulatory regions.
[0129]
Using a commercially available capillary electrophoresis system, it is possible to analyze or confirm the size of the nucleotide sequence of the sequencing or PCR product. Specifically, for capillary sequencing, a flowable polymer for electrophoretic separation, a laser-activated fluorescent dye specific for four different nucleotides, and a CCD camera for detecting the emitted wavelength It is possible to use The output / light intensity is converted to an electrical signal using appropriate software (eg, GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems), and the process from sample loading to computer analysis and electronic data display are computer controllable. Capillary electrophoresis is particularly suitable for sequencing small pieces of DNA that may be present in small amounts in a particular sample.
[0130]
In another embodiment of the present invention, a polynucleotide sequence encoding PRTS or a fragment thereof can be cloned into a recombinant DNA molecule to express the PRTS, fragment or functional equivalent in a suitable host cell. It is. Due to the inherent degeneracy of the genetic code, alternative DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence can be generated, and these sequences can be used for PRTS cloning and expression.
[0131]
The nucleotide sequence of the present invention can be recombined using methods generally known in the art to alter the sequence encoding PRTS for various purposes. This purpose includes, but is not limited to, cloning, processing and / or regulating expression of the gene product. Recombination of nucleotide sequences is possible using mixing of DNA with random fragments or PCR reassembly of gene fragments and synthetic oligonucleotides. For example, oligonucleotide-mediated directed mutagenesis can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, alter codon choices, create splice variants, and the like.
[0132]
The nucleotides of the present invention may be synthesized using MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; U.S. Pat. No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17: 793-797; Christians, F. C. et al. (1999) Nat. Biotechnol. 17: 259-264; Crameri, A. et al. (1996) Nat. Biotechnol. 14: 315-319). The biological properties of PRTS, such as its biological or enzymatic activity, or its ability to bind to other molecules or compounds, can be altered or improved. DNA shuffling is a process for preparing a library of gene variants by recombination of gene fragments by the PCR method. Next, the library is selected or screened to identify gene variants having the desired properties. These preferred variants are pooled and the DNA shuffling and selection / screening is repeated. Thus, diverse genes are created by artificial breeding and rapid molecular evolution. For example, recombination, screening, and shuffling of a single gene fragment having a mutation at a random position can be performed until the desired property is optimized. Alternatively, a given gene fragment may be recombined with a homologous gene fragment of the same gene family of the same or a different species, thereby controlling the genetic diversity of the large number of native genes in a controllable manner according to the protocol. Can be maximized.
[0133]
According to another embodiment, the sequence encoding PRTS can be synthesized, in whole or in part, using chemical methods well known in the art (eg, Caruthers. MH. Et al. (1980) Nucl. Acids Res. Symp. Ser 7: 215-223; and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232. Alternatively, PRTS itself or a fragment thereof can be synthesized using chemical methods. For example, peptide synthesis can be performed using a variety of solid-phase techniques (eg, Creighton, T. (1984)).Proteins. Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60; Roberte, J .; Y. (1995) Science 269: 202-204). Automation of the synthesis can also be achieved using, for example, an ABI 431A peptide synthesizer (PE Biosystems). In addition, the amino acid sequence of PRTS, or any portion thereof, may be altered by direct synthesis and / or in combination with sequences from other proteins or any portion thereof, using chemical methods, to produce the native sequence. It is possible to produce a polypeptide having a polypeptide sequence or a variant polypeptide.
[0134]
This peptide can be substantially purified using preparative high performance liquid chromatography (see, for example, Chiez, RM and FZ Regnier (1990) Methods Enzymol. 182: 392-421). The composition of the synthesized peptide can be confirmed by amino acid analysis or sequencing (see, eg, Creighton, supra, pp. 28-53).
[0135]
To express a biologically active PRTS, the nucleotide sequence encoding PRTS or a derivative thereof is inserted into a suitable expression vector. This expression vector contains the necessary elements for the regulation of transcription and translation of the coding sequence inserted in a suitable host. These elements include regulatory sequences such as enhancers, constitutive and inducible promoters, 5 'and 3' untranslated regions in the vector and polynucleotide sequences encoding PRTS. Such elements vary in their length and specificities. With certain initiation signals, it is possible to achieve more efficient translation of the sequence encoding PRTS. Such signals include the ATG start codon and nearby sequences such as the Kozak sequence. If the sequence encoding PRTS, its initiation codon, and upstream regulatory sequences are inserted into a suitable expression vector, no additional transcriptional or translational regulatory signals will be necessary. However, if only the coding sequence or a fragment thereof is inserted, an exogenous translational control signal including an in-frame ATG initiation codon must be included in the expression vector. Exogenous translational elements and initiation codons can be obtained from a variety of sources, both natural and synthetic. Inclusion of suitable enhancers for the particular host cell line used can increase the efficiency of expression. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 201-18-162.).
[0136]
Using methods well known to those skilled in the art, it is possible to create an expression vector containing a sequence encoding PRTS and suitable transcriptional and translational regulatory elements. These methods include:in in vitroRecombinant DNA technology, synthetic technology, andin VivoGenetic recombination techniques are included. (See, for example, Sambrook, J. et al. (1989)Molecular Cloning. A Laboratory ManualAusubel, F., Cold Spring Harbor Press, Plainview NY, Chapters 4 and 8, and 16-17; M. other. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, ch. (See Chapters 9 and 13 1-4).
[0137]
Various expression vector / host systems can be used to retain and express the sequence encoding PRTS. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, and viral expression vectors. (E.g., baculovirus) -infected insect cell lines, and plants transformed with a viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or a bacterial expression vector (e.g., Ti or pBR322 plasmid). Cell lines, animal cell lines, etc. (for example, Sambrook, supra, Ausubel, Van Heke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509; Enge Lhard, EK, et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3224-3227, Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945, Takamatsu, N. (1987). ) EMBOJ.6: 307-311;The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 146-64. 191-196, Logan, J.M. and T. Shenk (1984) Proc. Natl. Acad. Sci. ScL USA 81: 3655-3659, Harrington, J.M. J. Et al. (1997) Nat. Genet. 15: 345-355). Expression vectors derived from retroviruses, adenoviruses, herpesviruses or vaccinia viruses, or expression vectors derived from various bacterial plasmids can be used to transport nucleotide sequences to target organs, tissues or cell populations (Di Nicola, 5 (6): 350-356, Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90 (13): 6340-6344, Buller, R. M. et al. (1998) Cancer Gen. Ther. McGregor, DP et al. (1994) Mol. Immunol. 31 (3): 219-226, Verma, IM and N. Somia (1985) Nature 317 (6040): 813-815; 1997) Na See 239-242, etc.): ure 389. The present invention is not limited by the host cell used.
[0138]
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for the polynucleotide sequence encoding PRTS. For example, a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or pSPORT1 plasmid (GIBCO BRL) can be used for routine cloning, subcloning, and propagation of a polynucleotide sequence encoding PRTS. Ligation of the sequence encoding PRTS into the multiple cloning sites of the vector disrupts the lacZ gene, allowing a colorimetric screening method to identify transformed bacteria containing the recombinant molecule. In addition, using these vectors,in in vitroTranscription, dideoxin screening, single-stranded rescue by helper phage, and nested deletions can be created (eg, Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509.). For example, when a large amount of PRTS is required for production of an antibody or the like, a vector that induces PRTS expression at a high level can be used. For example, vectors containing a T5 or T7 bacteriophage promoter that strongly induces expression can be used.
[0139]
A yeast expression system can be used for PRTS expression. A variety of vectors containing constitutive or inducible promoters, such as α-factor, alcohol oxidase, and PGH promoter, have been used for yeast Saccharomyces cerevisiaePichia pastorisIt can be used for Furthermore, such vectors induce either the secretion of the expressed protein or its retention in cells, incorporating foreign sequences into the host genome for stable growth. (See, for example, Ausubel, 1995, supra, Bitter, GA, et al. (1987) Methods Enzymol. 153: 516-544, and Scorer. CA, et al. (1994) Bio / Technology 121-181-184. reference).
[0140]
Plant systems can also be used for expression of PRTS. Transcription of the sequence encoding PRTS can be performed, for example, by using a viral promoter such as CaMV-derived 35S and 19S promoters alone, or by using TMV (for example, Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie). , R. et al. (1984) Science 224: 838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17: 85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (For example,The McGraw Hill Yearbook of Science and Technology(1992) McGraw-Hill NY, pp. 191-196).
[0141]
In mammalian cells, a variety of virus-based expression systems may be utilized. When an adenovirus is used as an expression vector, a sequence encoding PRTS can be ligated to an adenovirus transcript / translation complex consisting of the late promoter and tripartite leader sequence. Insertion of the viral genome into a non-essential E1 or E3 region makes it possible to obtain a live virus expressing PRTS in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81: 3655-3659). In addition, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells. To express proteins at high levels, vectors based on SV40 or EBV can be used.
[0142]
Human artificial chromosomes (HACs) can be used to provide fragments of DNA larger than those expressed and contained in a plasmid. Approximately 6 kb to 10 Mb of HACs are made for treatment and supplied by conventional delivery methods (liposomes, polycationic amino polymers, or vesicles). (See, e.g., Harrington. JJ, et al. (1997) Nat Genet. 15: 345-355.).
[0143]
For long-term production of recombinant mammalian proteins, stable expression of PRTS in cell lines is desirable. For example, an expression vector can be used to transform a PRTS-encoding sequence into a cell line. Such expression vectors include replication and / or endogenous expression elements of viral origin and a selectable marker gene on the same or another vector. After the introduction of the vector, the cells are allowed to grow for about 1-2 days in an enriched medium before being transferred to a selective medium. The purpose of the selectable marker is to confer resistance to the selective mediator, and its presence allows the growth and recovery of cells that reliably express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type.
[0144]
The transformed cell lines can be recovered using any number of selection systems. Selection systems include, but are not limited to, the herpes simplex virus thymidine kinase gene and the adenine phosphoribosyltransferase gene, each with a tk⁻Or apr⁻Used in cells. (See, e.g., Wigler, M. et al. (1977) Cell 11: 223-232; and Lowy, I. et al. (1980) Cell 22: 817-823). Also, resistance to antimetabolites, antibiotics or herbicides can be used as a basis for selection. For example, dhfr confers resistance to methotrexate, neo confers resistance to aminoglycoside neomycin and G-418, als or pat confers resistance to chlorsulfuron (cPRTSsulfuron), phosphinothricin acetyltransferase (eg,). (1980) Proc. Natl. Acad. Sci. 77: 3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14). In addition, genes available for selection, such as trpB and hisD, which alter what cells need for metabolism, have been described in the literature (eg, Hartman, SC and RC Mulligan (1988) Proc. Natl. Acad. Sci. 85: 8047-51). Visible markers such as anitocyanin, green fluorescent protein (GFP; Clontech), β-glucuronidase and its substrate GUS, luciferase and its substrate luciferin are used. Green fluorescent protein (GFP) (Clontech, Palo Alto, CA) can also be used. These markers can be used to not only identify transformants, but also quantify transient or stable protein expression due to particular vector systems (see, eg, Rhodes, CA et al. 1995) Methods Mol. Biol. 55: 121-131).
[0145]
Even if the presence / absence of marker gene expression indicates the presence of the gene of interest, it may be necessary to confirm the presence and expression of that gene. For example, if the sequence encoding PRTS is inserted into a marker gene sequence, transformed cells containing the sequence encoding PRTS can be identified by a lack of marker gene function. Alternatively, the marker gene can be aligned with the sequence encoding PRTS under the control of one promoter. Expression of the marker gene in response to induction or selection usually also indicates expression of the tandem gene.
[0146]
Generally, host cells that contain the nucleic acid sequence encoding PRTS and that express PRTS can be identified using a variety of methods well known to those of skill in the art. These methods include DNA-DNA or DNA-RNA hybridization, PCR, and protein biology including membrane-based, solution-based, or chip-based techniques for the detection and / or quantification of nucleic acids or proteins. Test methods or immunological assays include, but are not limited to.
[0147]
Immunological methods for detecting and measuring expression of PRTS using either specific polyclonal or monoclonal antibodies are well known in the art. Such techniques include immunosorbent assays (ELISA), radioimmunoassays (RIAs), and fluorescence activated cell sorters (FACS) coupled to enzymes. A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on PRTS is preferred, but a competitive binding assay can also be used. These and other assays are well known in the art. (For example, Hampton. R. et al. (1990)Serological Methods, a Laboratory Manual. APS Press. St Paul. MN, Sect. IV; Coligan, J .; E. FIG. otherCurrent Protocols in Immunology, Green Pub. Associates and Wiley-Interscience, New York. NY; and Pound, J .; D. (1990)Immunochemical Protocols, Humans Press, Totowa NJ).
[0148]
Various labeling and conjugation techniques are well known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Methods for generating labeled hybridization or PCR probes for detecting sequences related to the polynucleotide encoding PRTS include oligo-labeling, nick translation, end-labeling, or labeled nucleotides. The PCR amplification used is included. Alternatively, the sequence encoding PRTS, or any fragment thereof, can be cloned into a vector for generating an mRNA probe. Such vectors, which are well known and commercially available in the art, can be prepared by adding a suitable RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.in in vitroCan be used for the synthesis of RNA probes. These methods are described, for example, in Amersham Pharmacia Biotech and Promega (Madison WI), U.S. Pat. S. Biochemical Corp (Cleveland OH) can use various kits commercially available. Suitable reporter molecules or labels that can be used for easy detection include substrates, cofactors, inhibitors, magnetic particles, and radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, and the like.
[0149]
Host cells transformed with the nucleotide sequence encoding PRTS are cultured under conditions suitable for the expression and recovery of the protein from cell culture. Whether a protein produced from a transformed cell is secreted or remains in the cell depends on the sequence used and / or the vector. Those skilled in the art will appreciate that expression vectors containing a polynucleotide encoding PRTS can be designed to include a signal sequence that directs secretion of PRTS across prokaryotic and eukaryotic cell membranes.
[0150]
In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves the "prepro" or "pro" form of the protein can be used to identify the target protein, folding and / or activity. A variety of host cells (eg, CHO, HeLa, MDCK, MEK293, WI38) with specific cellular devices and characteristic mechanisms for post-translational activity are available from the American Type Culture Collection (ATCC; Bethesda, MD). And selected to ensure correct modification and processing of the foreign protein.
[0151]
In another embodiment of the present invention, a natural or altered or recombinant nucleic acid sequence encoding PRTS is ligated to a heterologous sequence which translates into a fusion protein in any of the host systems described above. For example, a chimeric PRTS protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate screening of a peptide library for inhibitors of PRTS activity. Also, the heterologous protein portion and the heterologous peptide portion can facilitate purification of the fusion protein using commercially available affinity substrates. Such portions include glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-mc, hemagglutinin (HA). Including, but not limited to. GST and MBP, Trx, CBP, 6-His allow the purification of homologous fusion proteins with immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal chelating resins, respectively. FLAG, c-mc, and hemagglutinin (HA) allow immunoaffinity purification of the fusion protein using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. Alternatively, if the fusion protein is genetically engineered so that the fusion protein contains a proteolytic cleavage site between the sequence encoding PRTS and the heterologous protein sequence, PRTS may be cleaved from the heterologous portion after purification. Methods for expression and purification of the fusion protein are described in Ausubel. (1995, supra ch 10). It is described in. Various commercially available kits can be used to facilitate expression and purification of the fusion protein.
[0152]
In another embodiment of the invention, a TNT rabbit reticulocyte lysate or a wheat germ extract system (Promega) is used.in in vitroThe synthesis of PRTS radioactively labeled with is possible. These systems link the transcription and translation of sequences encoding proteins operably linked to the T7 or T3, SP6 promoter. Translation, for example,³⁵It occurs in the presence of a radiolabeled amino acid precursor that is S-methionine.
[0153]
A compound that specifically binds to PRTS can be screened using the PRTS of the present invention or a fragment thereof. At least one or more test compounds can be used to screen for specific binding to PRTS. Examples of test compounds include antibodies, oligonucleotides, proteins (eg, receptors) or small molecules.
[0154]
In one embodiment, the compound so identified is closely associated with a natural ligand of PRTS, eg, a ligand or fragment thereof, or a natural substrate, structural or functional mimetic or natural binding partner. (Coligan, JE, et al. (1991)Current Protocols in Immunology (See Chapter 5 of 1 (2)). Similarly, the compound may be closely related to the natural receptor to which the PRTS binds, or at least some fragment of the receptor, eg, a ligand binding site. In each case, the compound can be rationally designed using known techniques. In one embodiment, screening for such compounds involves producing suitable cells that express PRTS as either a secreted protein or a protein on the cell membrane. Suitable cells include cells from mammals, yeast, E. coli. Cells expressing PRTS or cell membrane fragments containing PRTS are contacted with a test compound and analyzed for binding, stimulation or inhibition of either PRTS or the compound.
[0155]
Some assays simply allow the test compound to be experimentally bound to the polypeptide and the binding detected by a fluorophore, radioisotope, enzyme conjugate or other detectable label. For example, the assay can include binding at least one test compound to PRTS, either in solution or immobilized on a solid support, and detecting binding of the compound to PRTS. Alternatively, detection and measurement of binding of a test compound in the presence of a labeled competitor can be performed. In addition, this assay can be performed using cell release agents, chemical libraries or natural product mixtures, wherein the test compound is released in solution or immobilized on a solid support.
[0156]
Using the PRTS of the present invention or a fragment thereof, it is possible to screen for a compound that regulates the activity of PRTS. Such compounds include agonists, antagonists, or partial or inverse agonists and the like. In one example, the assay is performed under conditions where the activity of the PRTS is such that the PRTS binds to at least one test compound, and the activity of the PRTS in the presence of the test compound is greater than the activity of the PRTS in the absence of the test compound. Compare with activity. A change in the activity of PRTS in the presence of the test compound indicates the presence of a compound that modulates the activity of PRTS. Alternatively, the test compound comprises PRTS under conditions suitable for PRTS activity.in in vitroAlternatively, the assay is performed in combination with a cell release system. In any of these assays, the test compound that modulates the activity of PRTS can bind indirectly and does not need to be in direct contact with the test compound. At least one and a plurality of test compounds can be screened.
[0157]
In another example, a polynucleotide encoding PRTS or a mammalian homolog thereof is "knocked out" in an animal model system using homologous recombination in embryonic stem cells (ES cells). Such techniques are well known in the art and are useful for generating animal models of human disease (see, eg, US Pat. Nos. 5,175,383 and 5,767,337). For example, mouse ES cells such as the 129 / SvJ cell line are derived from an early mouse embryo and can be grown in a medium. These ES cells are transformed with a vector containing a gene of interest disrupted with a marker gene such as the neomycin phosphotransferase gene (neo: Capecchi, MR (1989) Science 244: 1288-1292). This vector is integrated into the corresponding region of the host genome by homologous recombination. In another method, homologous recombination is performed using the Cre-loxP system to knock out the target gene in a tissue-specific or developmental stage-specific manner (Marth, JD (1996) Clin. Invest. 97: 1999-2002). Wagner, KU, et al. (1997) Nucleic Acids Res. 25: 4323-4330). The transformed ES cells are identified and microinjected into mouse cell blastocysts collected, for example, from the C57BL / 6 mouse line. The blastocysts are surgically introduced into pseudopregnant females, the genetic traits of the resulting chimeric progeny are determined, and they are bred to produce heterozygous or homozygous lines. The transgenic animals thus produced can be tested for potential therapeutic or toxic agents.
[0158]
A polynucleotide encoding PRTSin in vitroIt is possible to operate on ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight distinct cell lineages, including endoderm, mesoderm and ectoderm cell types. These cell lines differentiate into, for example, neurons, hematopoietic lineages and cardiomyocytes (Thomson, JA, et al. (1998) Science 282: 1145-1147).
[0159]
Using the polynucleotides encoding PRTS, it is possible to create "knock-in" humanized animals (pigs) or transgenic animals (mouse or rat) using human disease as a model. Using knock-in technology, a region of the polynucleotide encoding PRTS is injected into animal ES cells, and the injected sequence is integrated into the animal cell genome. The transformed cells are injected into a blastula and the blastula is implanted as described above. Study transgenic progeny or inbred lines, treat with potential medicines, and get information about the treatment of human disease. Alternatively, inbred mammalian strains that overexpress PRTS, eg, secrete PRTS into milk, may be a convenient source of proteins (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55). -74).
[0160]
(Treatment)
There is chemical and structural similarity between certain regions of the PRTS and certain regions of the protease, for example, in the context of sequence and motifs. In addition, expression of PRTS is closely related to gastrointestinal, epithelial, reproductive, cardiovascular, cancerous, and inflammatory tissues, and to healthy kidney and skin tissues. Thus, PRTS is believed to play a role in gastrointestinal, cardiovascular, autoimmune / inflammatory, proliferative, developmental, epithelial, neurological, and reproductive disorders. In treating diseases associated with increased PRTS expression or activity, it is desirable to reduce PRTS expression or activity. In the treatment of a disease associated with decreased expression or activity of PRTS, it is desirable to increase the expression or activity of PRTS.
[0161]
Thus, in one embodiment, it is possible to administer PRTS or a fragment or derivative thereof to a patient for the treatment or prevention of a disease associated with reduced expression or activity of PRTS. Such disorders include, but are not limited to, gastrointestinal disorders, cardiovascular disorders, autoimmune / inflammatory disorders, abnormal cell proliferation, developmental disorders, epithelial disorders, neurological disorders, and reproductive disorders, Some gastrointestinal disorders include dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal cancer, indigestion, digestive disorders, gastritis, stomach cancer, anorexia, nausea, vomiting, gastroparesis, sinus or pyloric edema , Abdominal angina, heartburn, gastroenteritis, ileus, intestinal infection, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic cancer, biliary disease, hepatitis, hyperbilirubinemia, cirrhosis, liver Passive congestion, hepatome, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple disease, Mallory-Weiss syndrome, colon cancer, colonic obstruction, irritable bowel syndrome, short small bowel syndrome, diarrhea, constipation , Gastrointestinal bleeding, and later Autoimmune deficiency syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatitis, hepatic steatosis, hemochromatosis, Wilson disease, α-1-antitrypsin deficiency, Reye syndrome, primary sclerosing cholangitis, Hepatic infarction, portal venous occlusion and thrombus, central lobular necrosis, hepatic purpura, hepatic vein thrombosis, hepatic vein obstruction, preeclampsia, eclampsia, gestational acute hepatic fat, gestational intrahepatic cholestasis, and nodularity Regeneration and adenomas, liver cancer including carcinoma, and cardiovascular diseases include arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, venous malformation, arterial dissection, varices, thrombophlebitis and Venous thrombosis, vascular tumor, thrombolysis complications, balloon angioplasty, vascular replacement, aortic coronary artery bypass graft surgery ary artery bypass graft), congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcified aortic stenosis, congenital bicuspid aortic valve, mitral Mitral annular calcification, mitral prolapse, rheumatic fever, rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, systemic lupus erythematosus endocarditis, carcinoid Heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, congenital lung anomalies, pulmonary insufficiency, pulmonary congestion and edema, pulmonary embolism, pulmonary bleeding, Pulmonary infarction, pulmonary hypertension, vascular sclerosis, obstructive pulmonary disease, restrictive pulmonary disease (restrictive pulmona) y disease, chronic obstructive pulmonary disease, emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral pneumonia and mycoplasma pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases ), Pneumoconiosis, sarcoidosis, idiopathic pulmonary fibrosis, exfoliative interstitial pneumonia, hypersensitivity pneumonia, hypereosinophilic obstructive bronchiolitis-organic pneumonia eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage (diffuse pulmonary hemorrhage) age syndromes), Goodpasture's syndrome, idiopathic pulmonary hematosis, pulmonary involvment in collagen-vascular disorders, alveolar proteinosis, lung tumors, inflammatory and non-inflammatory inflammatory effusions effusions, pneumothorax, pleural tumors, drug-induced lung disease, radiation-induced lung disease, and complications of lung transplantation, and autoimmune / inflammatory diseases. Among them are inflammation and actinic keratosis, acquired immunodeficiency syndrome (AIDS) and adrenal insufficiency, adult respiratory distress syndrome, allergyー, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, atherosclerotic plaque rupture, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyglandular endocrine candidal ectoderm dystrophy ( APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, pulmonary emphysema, lymphocotoxin temporary lymphopenia, erythroblastosis, erythema nodosum, atrophic Gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, Articular cartilage degradation, osteoporosis, pancreatitis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus Systemic sclerosis, primary thrombocythemia, thrombocytopenia, ulcerative colitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, viral infections, bacterial infections, fungal infections, parasitic infections, Protozoal infections, helminth infections, trauma, and cell proliferation abnormalities include actinic keratosis and atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), Myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically adrenal gland , Bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, testis, thymus, Includes thyroid and uterine cancers, including developmental disorders. Some include tubular acidosis, anemia, Cushing's syndrome, chondrodysplasia dwarfism, Duchenne-Becker muscular dystrophy, bone resorption, epilepsy, gonad dysplasia, WAGR syndrome (Wilms tumor, aniridia, urogenital abnormalities, Hereditary neuropathies such as mental retardation, Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucosal epithelial dysplasia, hereditary keratosis, Charcot-Marie-Tooth disease and neurofibromatosis , Hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spinal cord fission, anencephaly, cranial spondylolysis, congenital glaucoma, cataract, age-related macular degeneration Includes sensory nerve hearing loss, some epithelial diseases, eczema, eczema, alleles Germinal contact dermatitis, keratosis, melasma, vitiligo, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, seborrheic keratosis, folliculitis, herpes simplex, shingles, Chickenpox, candidiasis, dermatophytosis, scabies, insect sting, senile hemangioma, keloid, dermal fibroma, apical fibrous tenderness, urticaria, transient acantholytic dermatosis, xerosis, eczema , Atopic dermatitis, contact dermatitis, hand eczema, monetary eczema, lichen lichen, asthetic eczema, congestive dermatitis, congestive ulcer, seborrheic dermatitis, psoriasis, lichen planus, rose pity Rash, impetigo, acne, dermatophytosis, nausea, wart, acne vulgaris, red nose, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, bullous pemphigoid, gestational Herpes, herpes dermatitis, linear IgA disease, acquired epidermolysis bullosa, dermatomyositis, lupus erythematosus, scleroderma and Localized scleroderma, erythroderma, alopecia, fixed skin lesions, vasodilatation, hypopigmentation, hyperpigmentation, vesicle / blister, rash, skin drug reaction, papular nodule skin disorder (Papulonodular skin lesions), chronic non-healing wounds, photosensitive diseases, simple epidermolysis bullosa, exfoliative and non-epidermal palmoplantar keratoderma, ichthyosis bulosa of Siemens, exfoliative ichthyosis, palmar Plantar keratosis, palmatoplantar keratoderma (keratosis palmoplantaris or palmoplantar keratoderma), keratosis punctata, Meesmann's cornea dystrophy, congenital nail hyperplasia, white spongiform nevus Includes onset sebaceous cysts, epidermal nevus / epidermal keratinization, moniletbrix, trichothiodystrophy, chronic hepatitis / idiopathic cirrhosis, colorectal hyperplasia, and some neurological disorders include epilepsy, ischemic cerebrovascular disease Disorders, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neuromuscular atrophy, Retinitis pigmentosa, hereditary ataxia, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural pyometra, epidural abscess, purulent intracranial thrombotic vein Inflammation, myelitis and radiculitis, viral central nervous system disease, Kuru and Creutzfeldt-Jakob disease, Gerstmann syndrome, Gerstmann-Strau Prion diseases including sler-Scheinker syndrome, fatal familial insomnia, neurotrophic and metabolic diseases, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastoma, trigeminal neurovascular Syndrome, central nervous system mental retardation including Down syndrome and other developmental disorders, cerebral palsy, nervous skeletal disorders, autonomic nervous system disorders, peripheral neuropathy, dermatomyositis and polymyositis, and hereditary, metabolic, endocrine And toxic myopathy, myasthenia gravis, periodic limb paralysis, psychosis including disorders of mood and anxiety, schizophrenia, seasonal disorders (SAD), restlessness, amnesia, nervousness Disease, diabetic neuropathy, extrapyramidal terminal defect syndrome, dystonia, schizophrenic psychiatric disorder, postherpetic neuralgia, and Poulet's disease includes progressive supranuclear palsy, corticobasal degeneration and familial frontotemporal amnesia, among which reproductive disorders include abnormal production of prolactin, fallopian tube disease and ovulation Abnormalities, endometriosis, abnormal estrus, abnormal menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian cancer, uterine fibroids, autoimmune abnormalities, ectopic pregnancy, infertility including teratogenesis Disease, breast cancer, fibrocystic mastosis, mastolacia, spermatogenesis, physiological sperm abnormalities, testicular cancer, prostate cancer, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, male breast and female And breast cancer.
[0162]
In another embodiment, a vector capable of expressing PRTS or a fragment or derivative thereof for the treatment or prevention of a disease associated with reduced expression or activity of PRTS, including, but not limited to, the diseases listed above. It can also be administered to patients.
[0163]
In yet another embodiment, a composition comprising substantially purified PRTS for the treatment or prevention of a disease associated with reduced expression or activity of PRTS, including but not limited to the diseases listed above. Can be administered to a patient together with a suitable pharmaceutical carrier.
[0164]
In yet another embodiment, an agonist that modulates PRTS activity is administered to a patient for the treatment or prevention of a disease associated with reduced expression or activity of PRTS, including but not limited to the diseases listed above. It is also possible.
[0165]
In a further embodiment, an antagonist of PRTS can be administered to a patient for the treatment or prevention of a disease associated with increased expression or activity of PRTS. Examples of such diseases include, but are not limited to, gastrointestinal diseases, cardiovascular diseases, autoimmune / inflammatory diseases, abnormal growth, developmental disorders, epithelial disorders, neurological disorders, and reproductive disorders as described above. Is included. In one embodiment, an antibody that specifically binds to PRTS can be used directly as an antagonist or indirectly as a targeting or delivery mechanism for delivering an agent to cells or tissues that express PRTS.
[0166]
In another embodiment, the complement of a polynucleotide encoding PRTS is expressed for the treatment or prevention of a disease associated with increased expression or activity of PRTS, including but not limited to the diseases listed above. The vector can be administered to the patient.
[0167]
In another embodiment, any of the proteins, antagonists, antibodies, agonists, complementary sequences, and vectors of the invention may be administered in combination with another suitable therapeutic agent. One skilled in the art can select a suitable therapeutic agent to use in combination therapy according to conventional pharmaceutical principles. Combinations with therapeutic agents can have synergistic effects in the treatment or prevention of the various diseases listed above. Using this method, it is possible to achieve a medicinal effect with a small amount of each drug, and to reduce the possibility of a wide range of side effects.
[0168]
PRTS antagonists can be prepared using methods common in the art. Specifically, it is possible to produce antibodies using purified PRTS, or to screen a therapeutic drug library to identify those that specifically bind to PRTS. An antibody to PRTS can also be produced using a method common in the art. Such antibodies include polyclonal, monoclonal, chimeric, single chain, Fab fragments, and fragments produced by Fab expression libraries. However, it is not limited to these. For therapeutic use, neutralizing antibodies (ie, those that inhibit dimer formation) are particularly preferred.
[0169]
For the production of antibodies, various hosts, including goats, rabbits, rats, mice, humans, and others, are immunized by injection of PRTS or any fragment, or oligopeptide thereof with immunogenic properties. Can be done. Depending on the host species, various adjuvants can be used to enhance the immune response. Such adjuvants include surfactants such as Freund's adjuvant, mineral gel adjuvants such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oily emulsions, keyhole limpet hemocyanin, and dinitrophenol. However, the present invention is not limited to these. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvumIs particularly preferred.
[0170]
Oligopeptides, peptides, or fragments used to elicit antibodies to PRTS are composed of at least about 5 amino acids, and generally are preferably composed of about 10 or more amino acids. Desirably, these oligopeptides or peptides, or fragments thereof, are identical to a portion of the amino acid sequence of the native protein. Short stretches of PRTS amino acids can be fused to sequences of another protein, such as KLH, to produce antibodies against the chimeric molecule.
[0171]
Monoclonal antibodies to PRTS can be produced by continuous cell lines in culture using any technique that produces antibody molecules. These technologies include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology (eg, Kohler, G. et al. (1975) Nature 256: 495-495). (497) Kozbor, D. et al. (1985) J. Immunol. Methods 81-8-42; Cote, RJ. Et al. (1983) Proc. Natl. Acad. Sci. 80: 2026-2030; (See SP et al. (1984) Mol. Cell Biol. 62: 109-120).
[0172]
In addition, techniques such as splicing a mouse antibody gene to a human antibody gene developed for the production of a “chimeric antibody” are used to obtain molecules with suitable antigen specificity and biological activity (eg, Morrison (1984) Proc. Natl. Acad. Sci. 81-4855-14855; Neuberger, MS et al. (1984) Nature 312: 604-608; Takeda, S. et al. ) Nature 314: 452,454). Alternatively, PRTS-specific single-chain antibodies are generated using the techniques described for the production of single-chain antibodies, using methods well known in the art. Antibodies of related idiotype composition with related specificity can also be generated by chain mixing from random combinations of immunoglobulin libraries (see, eg, Burton DR (1991) Proc. Natl. Acad. Sci. 88: 11120-3).
[0173]
Antibodies in lymphocyte populationsin VivoIt can also be obtained by inducing production, or by screening immunoglobulin libraries or by screening a panel of highly specific binding reagents as indicated in the literature (eg, Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991) Nature 349: 293-299).
[0174]
Antibodies containing specific binding sites for PRTS can also be obtained. For example, such fragments include a fragment at F (ab ') generated by pepsin digestion of an antibody molecule and a Fab fragment at F (ab') generated by reducing the disulfide bridges of the fragment. However, the present invention is not limited to these. Alternatively, creating a Fab expression library allows for the rapid and easy identification of monoclonal Fab fragments with the desired specificity (eg, Huse, WD et al. (1989) Science 254: 1275-). 1281).
[0175]
Screening is performed using various immunoassays to identify antibodies with the desired specificity. Numerous protocols for competitive binding, using either polyclonal or monoclonal antibodies with sequestered specificity, or immunoradioactivity, are well known in the art. Usually, such immunoassays involve the measurement of complex modulation between the PRTS and its specific antibody. Two-site, monoclonal-based immunoassays using monoclonal antibodies reactive to two non-interfering PRTS epitopes are commonly used, but competitive binding assays can also be used (Pound, supra).
[0176]
Various methods, such as Scatchard analysis, are used in conjunction with radioimmunoassay techniques to assess the affinity of the antibody for PRTS. Affinity is represented by the binding constant Ka, which is a value obtained by dividing the molar concentration of the PRTS antibody complex by the molar concentration of free antibody and free antigen under equilibrium conditions. The Ka of a polyclonal antibody drug with heterogeneous affinity for multiple PRTS epitopes indicates the average affinity or avidity of the antibody for PRTS. The Ka of a monoclonal antibody drug monospecific for a particular PRTS epitope represents a true measure of affinity. Ka value is 10⁹-10¹²The L / mol high affinity antibody drug is preferably used for immunoassays in which the PRTS antibody complex must withstand severe manipulations. Ka value is 10⁶-10⁷The L / mol low-affinity antibody drug is preferably used for immunopurification and similar treatments where the PRTS must eventually dissociate in an activated state from the antibody. (Catty, D. (1988)Antibodies, Volume I: A Practical Approach. IRL Press, Washington, DC; E. FIG. and Cryer, A.C. (1991)A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
[0177]
To determine the quality and suitability of such drugs for certain downstream applications, the antibody titer and avidity of the polyclonal antibody drug are further evaluated. For example, polyclonal antibody medicaments containing at least 1-2 mg / ml specific antibody, preferably 5-10 mg / ml specific antibody, are generally used in processes where the PRTS antibody complex must be precipitated. Guidance on antibody specificity and titer, binding activity, antibody quality and usage in various applications is generally available. (See, eg, Catty, supra, and Colligan et al., Supra).
[0178]
In another embodiment of the invention, a polynucleotide encoding PRTS, or any fragment or complement thereof, can be used for therapeutic purposes. In one embodiment, sequences that are complementary to the coding and regulatory regions of the gene encoding PRTS and antisense molecules (DNA and RNA, modified nucleotides) can be designed to alter gene expression. Such techniques are well known in the art, and sense or antisense oligonucleotides or large fragments can be designed from the control region of the PRTS-encoding sequence or from various locations along the coding region.
[0179]
For use in therapy, any gene delivery system suitable for introducing the antisense sequences into suitable target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid that, upon transcription, expresses a sequence that is complementary to at least a portion of the cell sequence encoding the target protein (see, eg, Slater, JE. Et al. 1998) J. Allergy Clin. Immunol. 102 (3): 469-475; and Scanlon, KJ. Et al. (1995) 9 (13): 1288-1296. Antisense sequences can also be introduced into cells using, for example, viral vectors such as retroviruses and adeno-associated virus vectors (eg, Miller, AD (1990) Blood 76: 271; Ausubel, supra). Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63 (3): 323-347). Other genetic delivery mechanisms include liposome systems, artificial viral envelopes, and other systems well known in the art (Rossi, JJ (1995) Br. Med. Bull. 51 (1): 217-225; Boado, RJ, et al. (1998) J. Pharm. Sci. 87 (11): 1308-1315; and Morris, MC, et al. (1997) Nucleic Acids Res. 25 (14): 2730. -2736.).
[0180]
In another embodiment of the present invention, a polynucleotide encoding PRTS can be used for somatic or germ cell gene therapy. Gene therapy involves (i) a severe combined immunodeficiency (SCID) -X1 characterized by a genetic deficiency (eg, X chromosome linkage (Cavazzana-Calvo, M. et al. (2000) Science 288: 669-672)). ), A hereditary adenosine-deaminase (ADA) deficiency (Blaese, RM et al. (1995) Science 270: 475-480; Bordignon, C. et al. (1995) Science 270: 470-475). Complex immunodeficiency, cystic fibrosis (Zabner, J. et al. (1993) Cell 75: 207-216: Crystal, RG et al. (1995) Hum. Gene @ Therapy 6: 643-666; Crystal, RG Others (199 Hum. Gene Therapy 6: 667-703), thalassemia, familial hypercholesterolemia, hemophilia due to factor VIII or factor IX deficiency (Crystal, 35 ° RG (1995) Science 270: Verma, IM and Somia, N. (1997) Nature 389: 239-242), or (ii) a conditional lethal gene product (eg, in cancer due to uncontrolled cell proliferation). And (iii) intracellular parasites such as the human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335: 395-396; Poescbla, E. et al. (1996) Proc. Natl.Aca USA 93: 11395-11399), hepatitis B or C virus (HBV, HCV),Candida albicansas well asParacoccidioides brasiliensisFungal parasites, such asPlasmodium falciparumas well asTrypanosoma cruziEtc.) can be carried out by expressing a protein having a protective function against parasite parasites such as When a deficiency in a gene required for PRTS expression or regulation causes a disease, PRTS can be expressed from a suitable population of the introduced cells to mitigate the symptoms caused by the gene deficiency.
[0181]
In a further embodiment of the invention, diseases and disorders caused by PRTS deficiency are treated by creating mammalian expression vectors encoding PRTS and introducing these vectors into PRTS-deficient cells by mechanical means. .in VivoOrex in vitroMechanical transfection techniques used for cells of (i) include (i) microinjection of DNA into individual cells, (ii) bombardment of gold particles, (iii) liposome-mediated transfection, (iv) receptor-mediated Gene transfer and (v) DNA transposon (Morgan, RA and WF Anderson (1993) Annu. Rev. Biochem. 62: 191-217; Ivics, Z. (1997) Cell 91: 501-). 510; Boulay, JL and H. Recipon (1998) Curr. Opin. Biotechnol. 9: 445-450).
[0182]
Expression vectors that can affect PRTS expression include, but are not limited to, PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vector (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH / PERV (Stratagene, La Jolla CA), PTT-OFF, PTTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). To express PRTS, (i) a constitutively active promoter (eg, cytomegalovirus (CMV), rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin gene); (Ii) Inducible promoters (eg, a tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. Inc.) contained in a commercially available T-REX plasmid (Invitrogen). U.S.A. 89: 5547-5551; Gossen, M. et al. (1995) Science 268: 1766-1769; Rossi, FMV and HM Bluu (1998) Curr. Opin. Biotechn. 9: 451-456)), an ecdysone inducible promoter (included in commercially available plasmids PVGRXR and PIND: Invitrogen), an FK506 / rapamycin inducible promoter, or a RU486 / mifepristone inducible promoter ( Rossi, FMV and HM Blau, supra), or (iii) the natural or tissue-specific promoter of an endogenous gene encoding PRTS from a normal individual. It is.
[0183]
Using commercially available liposome transformation kits (eg, PERFECT LIPID and TRANSFECTION KIT sold by Invitrogen), one skilled in the art can introduce polynucleotides into target cells in culture without much recourse to experience. . Alternative methods include the calcium phosphate method (Graham. FL and AJ Eb (1973) Virology 52: 456-467) or the electroporation method (Neumann, B. et al. (1982) EMBO J. 1: 841-845. ). In order to introduce DNA into primary cells, these standard mammalian transfection protocols need to be modified.
[0184]
In another embodiment of the present invention, a disease or disorder caused by a gene deficiency associated with PRTS expression comprises: (i) a polynucleotide encoding PRTS under the control of a retroviral terminal repeat (LTR) promoter or an independent promoter; Nucleotides and (ii) a suitable RNA packaging signal and (iii) a Rev responsive element (RRE) with additional retroviral cis-acting RNA sequences and coding sequences necessary for efficient vector propagation. Retroviral vectors can be made and treated. Retroviral vectors (eg, PFB and PFBNEO) are available from Stratagene and published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6733-6737). ) Based on. The above data is incorporated herein by reference. This vector is VSVg (Armentano, D. et al. (1987) J. Virol. 61: 1647-1650; Bender, MA. Et al. (1987) J. Virol. 61: 1639-1646; Adam, MA. and AD Miller (1988) J. Virol. 62: 3802-3806; Dull, T. et al. (1998) J. Virol. 72: 8463-8471; Zufferey, R. et al. (1998) J. Virol. : 9873-9880) or a suitable vector producing cell line (VPCL) that expresses a promiscuous envelope protein or an envelope gene that has an affinity for the receptor on the target cell. U.S. Patent No. 5,910,434 to RIGG ("Method for observing retrovirus packaging cell lines producing high transducing effect"; a method for disseminating the virus in a retrospective system) Citation is incorporated herein by reference. Propagation of retroviral vectors, certain cell populations (eg, CD4⁺Methods for transducing T cells) and returning the transduced cells to the patient are well known in the field of gene therapy and are described in numerous references (Ranga, U. et al. (1997) J. Virol. 71: 7020-7029; Bauer, G. et al. (1997) Blood 89: 2259-2267; Bonyhadi, M. L. (1997) J. Virol. 71: 4707-4716; Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95: 1201-1206: Su, L. (1997) Blood 89: 2283- 2290).
[0185]
Alternatively, an adenovirus-based gene therapy delivery system is used to deliver a polynucleotide encoding PRTS to cells having one or more genetic abnormalities associated with PRTS expression. The production and packaging of adenovirus-based vectors is well known in the art. Replication-defective adenovirus vectors have been shown to be variable to introduce genes encoding immunomodulatory proteins into intact pancreatic islets of the pancreas (Csete, ME et al. (1995)). Transplantation 27: 263-268). Adenovirus vectors that may be used are described in US Pat. No. 5,707,618 (“Adenovirus vectors for gene therapy”), which is hereby incorporated by reference. For adenovirus vectors, see also Antinozzi, P. et al. A. Et al. (1999) Annu. Rev .. Nutr. 19: 511-544; and Verma, I .; M. and N.M. Somia (1997) Nature 18: 389: 239-242, which is hereby incorporated by reference.
[0186]
Alternatively, a herpes-based gene therapy delivery system is used to deliver a polynucleotide encoding PRTS to target cells having one or more genetic abnormalities associated with PRTS expression. Herpes simplex virus (HSV) -based vectors are particularly important in introducing PRTS into HSV-affected central nervous cells. The production and packaging of herpes-based vectors is well known in the art. Vectors of the replication-competent herpes simplex virus (HSV) type I system have been used to deliver reporter genes to primate eyes (Liu, X. et al. (1999) Exp. Eye Res. 169: 385). -395). The construction of the HSV-1 viral vector is described in U.S. Pat. No. 5,804,413 to DeLuca (Herpes simple viruses against genes transfer), which is incorporated herein by reference. . U.S. Patent No. 5,804,413 discloses a recombinant HSV comprising a genome containing at least one endogenous gene introduced into a cell under the control of a suitable promoter for purposes including human gene therapy. There is a description about d92. The patent also discloses methods for making and using recombinant HSV strains that are removed for ICP4, ICP27 and ICP22. For HSV vectors, see Goins, W. et al. F. Et al. (1999) J. Am. Virol. 73: 519-532 and Xu, H .; Et al. (1994) Dev. Biol. 163: 152-161, which are hereby incorporated by reference. Manipulation of cloned herpesvirus sequences, passage of recombinant virus after transfection of multiple plasmids containing different parts of the genome of the herpesvirus giant, growth and propagation of herpesvirus, and transfer to herpesvirus cells Infection is a technique well known in the art.
[0187]
Alternatively, a polynucleotide encoding PRTS is delivered to target cells using an alphavirus (positive single-stranded RNA virus) vector. Biological studies of the prototype alphavirus, Semliki Forest Virus (SFV), have been performed extensively and found that the gene transfer vector is based on the SFV genome (Garoff). , H. and K.-J. Li (1998) Cun. Opin. Biotech. 9: 464-469). During the replication of alpha viral RNA, subgenomic RNA is usually created that encodes the viral capsid protein. Because this subgenomic RNA is replicated at a higher level than full-length genomic RNA, capsid proteins are produced in excess of viral proteins having enzymatic activity (eg, proteases and polymerases). Similarly, by introducing the sequence encoding PRTS into the region encoding the capsid of the α virus genome, a large number of RNAs encoding PRTS are produced in the vector-transfected cells, and PRTS is synthesized at a high level. Alpha virus infection usually involves cell lysis within 2-3 days, but the ability to establish persistent infection of normal kidney cells of hamsters (BHK-21) with Sindbis virus (SIN) variants Suggest that the lytic replication of the α virus can be suitably altered to be applicable to gene therapy (Dryga, SA et al. (1997) Virology 228: 74-83). ). Since the α virus can be introduced into various hosts, PRTS can be introduced into various types of cells. Specific transduction of a subset of cells in a population may require sorting of the cells before transduction. The manipulation of infectious cDNA clones of α-virus, transfection of α-virus cDNA and RNA, and methods of infecting α-virus are well known in the art.
[0188]
For example, oligonucleotides derived from the transcription initiation site from about -10 to about +10 from the start site can be used to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base pairing. Triple helix structures are useful because they prevent the double helix from spreading sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (eg, Gee, JE et al. (1994) In: Huber, BE and BI Carr,Molecular and ImmunologyPRTSProaches, Futura Publishing Co., Ltd. , Mt. Kisco, NY). Complementary sequences or antisense molecules can also be designed to prevent translation of the mRNA by preventing the transcript from binding to the ribosome.
[0189]
Ribozymes, which are enzymatic RNA molecules, can be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by nucleotide strand breaks. For example, it includes a recombinant hammerhead ribozyme molecule that specifically and effectively catalyzes nucleotide chain cleavage of a sequence encoding PRTS.
[0190]
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites that include the following sequences GUA, GUU, and GUC. Once identified, the short RNA sequence of 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site is evaluated for secondary structural features that render the oligonucleotide dysfunctional Is possible. The suitability of a candidate target can also be assessed by using a ribonuclease protection assay to test the ease of hybridization with a complementary oligonucleotide.
[0191]
The complementary ribonucleic acid molecules and ribozymes of the invention can be made for synthesis of the nucleic acid molecule using methods well known in the art. These methods include methods for chemically synthesizing oligonucleotides such as solid phase phosphoramidite compounds. Alternatively, the RNA moleculein in vitroas well asin VivoSuch DNA sequences, which can be generated by transcription of a DNA sequence encoding PRTS, can be incorporated into a variety of vectors using a suitable RNA polymerase promoter, such as T7 or SP6. Alternatively, those cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into a cell line, cell, or tissue.
[0192]
Modification of an RNA molecule can increase intracellular stability and increase half-life. Possible modifications include the addition of flanking sequences at the 5 'and / or 3' end of the molecule, or modifications using phosphorothioate or 2'O methyl instead of phosphodiester bonds in the backbone of the molecule. Including, but not limited to. This concept inherent in the production of PNAs includes acetyl-, methyl-, thio-, and similar modified forms of adenine, cytidine, guanine, thymine, and uridine that are not readily recognized by endogenous endonucleases, as well as These molecules can be expanded throughout by including non-conventional bases such as inosine, queosine, and wybutosine.
[0193]
A further embodiment of the invention includes a method of screening for a compound that is effective in altering the expression of a polynucleotide encoding PRTS. Compounds that are effective in altering the expression of a particular polynucleotide include, but are not limited to, non-polymeric chemicals, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides that can interact with a particular polynucleotide sequence. Includes nucleotides, transcription factors and other polypeptide transcription regulators. An effective compound can act as an inhibitor or enhancer of polynucleotide expression and alter polynucleotide expression. Therefore, in the treatment of diseases associated with increased PRTS expression or activity, compounds that specifically inhibit the expression of a polynucleotide encoding PRTS are therapeutically useful, and are associated with decreased PRTS expression or activity. In treating disease, compounds that specifically promote the expression of a polynucleotide encoding PRTS may be therapeutically useful.
[0194]
At least one to a plurality of test compounds can be screened to determine the effectiveness of an alteration in the expression of a particular polynucleotide. Test compounds can be obtained by any method known in the art, including chemical modification of an effective compound. Such methods may be based on the chemical and / or structural properties of the target polynucleotide when altering the expression of the polynucleotide, selecting from a library of commercially available or proprietary natural or non-natural compounds, and altering the expression of the polynucleotide. It is effective when designing a compound rationally and further selecting from a library of compounds generated in combination or randomly. A sample comprising a polynucleotide encoding PRTS is obtained by exposing to at least one test compound. Samples include, for example, intact cells, permeabilized cells,in in vitroCell free or reconstituted biochemical systems may be included. Changes in the expression of a polynucleotide encoding PRTS are assayed by any method known in the art. Usually, the expression of a specific nucleotide is detected by hybridization using a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding PRTS. The hybridization yield is quantified, and that value can be a basis for comparing the expression of polynucleotides exposed and unexposed to one or more test compounds. A change in the expression of a polynucleotide that is exposed to the test compound is detected, indicating that the test compound is effective in altering the expression of the polynucleotide. To determine compounds that are effective at altering the expression of a particular polynucleotide, for example,Schizosaccharomyces pombeGene expression system (Atkins, D. et al. (1999) U.S. Pat. No. 5,932,435, Arndt, GM et al. (2000) Nucleic Acids Res. 28: E15) or human cell lines such as HeLa cells (Clarke) , ML, et al. (2000) Biochem. Biophys. Res. Commun. 268: 8-13). A particular embodiment of the present invention involves screening a combinatorial library of each oligonucleotide (deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice). (1997) U.S. Patent No. 5,686,242, Bruice, TW et al. (2000) U.S. Patent No. 6,022,691).
[0195]
Numerous methods for introducing vectors into cells or tissues are available,in Vivo,in in vitro,as well asex VivoEqually suitable for use inex VivoIn the case of treatment with, the vector is introduced into hepatocytes collected from a patient and cloned and propagated to return the same patient by autologous transplantation. Transfection, ribosome injection, or delivery by polycation amino polymer can be performed using methods well known in the art (eg, Goldman, CK et al. (1997) Nature Biotechnology 15: 462-66: See).
[0196]
Any of the treatment methods described above can be applied to all subjects in need of treatment, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
[0197]
Another embodiment of the present invention relates to the administration of a medicament or sterile composition with a pharmaceutically acceptable carrier for all of the above-mentioned therapeutic effects. Such compositions comprise PRTS, antibodies, mimetics, agonists, antagonists of PRTS, or inhibitors of PRTS, and the like. The composition can be administered alone or in combination with one or more other agents, such as stabilizers, to a sterile, biocompatible pharmaceutical carrier. Such pharmaceutical carriers include, but are not limited to, saline, buffered saline, glucose, water, and the like. The composition can be administered alone or with another drug, such as a drug or hormone.
[0198]
The composition used in the present invention can be administered using various routes. This route includes oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal Is not limited to these.
[0199]
Compositions for pulmonary administration can be prepared in liquid or dry powder form. Such compositions typically aerosolize shortly before inhalation by the patient. In the case of small molecules (eg, conventional low molecular weight organic drugs), aerosol delivery of fast acting formulations is well known in the art. In the case of macromolecules (e.g., larger peptides and proteins), the technology for pulmonary transport through the alveolar region of the lung has improved in recent years, making it possible to actually transport drugs such as insulin into the blood ( See Patton, JS, et al., US Patent No. 5,997,848). Pulmonary delivery is advantageous in that it can be administered without needle injection, eliminating the need for potentially toxic penetration enhancers.
[0200]
Suitable compositions for use in the present invention include compositions that contain an effective amount of the active ingredient to achieve its intended purpose. One skilled in the art can determine the effective dose with sufficient ability.
[0201]
Preferably, the composition is prepared in a special form to deliver the macromolecule containing PRTS or a fragment thereof directly into the cell. For example, a liposome formulation containing a cell-impermeable polymer can promote cell fusion and intracellular transport of the polymer. Alternatively, PRTS or a fragment thereof can be linked to the cationic N-terminus of the HIV Tat-1 protein. It has been confirmed that the fusion protein thus produced is transduced into cells of all tissues including the mouse model system brain (Schwarze, SR, et al. (1999) Science 285: 1569-). 1572).
[0202]
For any composition, a therapeutically effective dose can be estimated initially either in tumor cell assays, for example, on tumor cells, or in animal models. Usually, a mouse, rabbit, dog, monkey, pig, or the like is used as an animal model. Animal models can also be used to determine suitable enrichment ranges and routes of administration. Based on such treatment, beneficial dosages and routes for administration in humans can be determined.
[0203]
A medically effective dosage relates to the amount of active ingredient that ameliorates the symptoms or condition, eg, PRTS or a fragment thereof, an antibody to PRTS, an agonist or antagonist of PRTS, an inhibitor, and the like. Medicinal efficacy and toxicity are, for example, ED₅₀(Dose that is pharmaceutically effective in 50% of the population for taking) or LD₅₀(Dose that is lethal to 50% of the population for dosing) can be determined by standard pharmaceutical techniques in cell culture or animal studies, such as calculating statistics. The dose ratio between toxic and therapeutic effects is the therapeutic index and the LD₅₀/ ED₅₀It can be shown. Compositions that exhibit high therapeutic indices are desirable. The data obtained from the cell culture assays and animal studies is used in formulating a range of dosage for human application. Dosages in which such compositions are included may have little or no toxicity, ED₅₀It is desirable that the concentration be in the blood concentration range containing The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
[0204]
The exact dosage will be determined by the practitioner, in light of factors related to the patient requiring treatment. Dosage and administration are adjusted to provide effective levels of the active ingredient or to maintain the desired effect. Factors to consider include dose severity, general health of the patient, age, weight, and gender of the patient, time and frequency of administration, concomitant medications, response sensitivities, and Response is included. Long-acting compositions can be administered once every three or four days, once a week, once every two weeks, depending on the half-life and clearance rate of the particular formulation.
[0205]
Normal dosage amounts will vary depending on the route of administration, but will range up to about 0.1-100,000 μg up to about 1 gram. Guidance on specific dosages and methods of delivery is provided in the literature and is generally available to practitioners in the art. One skilled in the art will utilize formulations of nucleotides that are different from the protein or inhibitor. Similarly, delivery of a polynucleotide or polypeptide will be specific to a particular cell, condition, location, etc.
[0206]
(Diagnosis)
In another embodiment, an assay for diagnosing a disease characterized by the expression of PRTS, or for monitoring a patient being treated with PRTS or an agonist or antagonist or inhibitor of PRTS, wherein the antibody that specifically binds to PRTS is used. Used for Antibodies useful for diagnosis are formulated in the same manner as described for therapy. PRTS diagnostic assays include methods of detecting PRTS from human bodily fluids or from cells or tissues using antibodies and labels. These antibodies can be used with or without modification, and can be labeled by covalent or non-covalent attachment of a reporter molecule. Various reporter molecules known in the art may be used, some of which are described above.
[0207]
Various protocols for measuring PRTS, including ELISA, RIA, and FACS, are well known in the art and provide a basis for diagnosing abnormal or abnormal levels of expression of PRTS. Normal or standard PRTS expression values are determined by combining an antibody against PRTS with body fluids or cells collected from a subject, such as a normal mammal, eg, a human, under conditions suitable for complex formation. I do. The amount of standard complex formation can be quantified by various methods such as photometric. A subject's PRTS expression level, control and disease, samples from biopsy tissue are compared to baseline values. The deviation between the reference value and the subject is a diagnostic indicator.
[0208]
According to another embodiment of the present invention, polynucleotides encoding PRTS may be used for diagnosis. Polynucleotides used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNA. The polynucleotide is used to detect and quantify the expression of the gene in a biopsy tissue that expresses PRTS that can be correlated with the disease. This diagnostic assay is used to check for the presence and overexpression of PRTS and to monitor the modulation of PRTS levels during treatment.
[0209]
In one embodiment, nucleic acid sequences encoding PRTS can be identified by hybridization with a PCR probe capable of detecting a polynucleotide sequence comprising the gene sequence encoding PRTS or a closely related molecule. The specificity of a probe, whether it is made from a highly specific region, for example, the 5 'regulatory region, or a region of relatively low specificity, such as a conserved motif, and the stringency of hybridization or amplification, Will identify only natural sequences that encode PRTS, or only natural sequences that encode alleles and related sequences.
[0210]
Probes may also be used to detect related sequences and have at least 50% sequence identity with any sequence encoding PRTS. The target hybridization probe of the present invention can be DNA or RNA, and can be derived from the sequence of SEQ ID NO: 15-28 or a genomic sequence containing the promoter, enhancer, and intron of the PRTS gene.
[0211]
As a method for preparing a hybridization probe specific to DNA encoding PRTS, there is a method of cloning a polynucleotide sequence encoding PRTS and a PRTS derivative into a vector for producing an mRNA probe. Such vectors are commercially available and are well known to those skilled in the art, and by adding a suitable RNA polymerase and a suitable labeled nucleotide,in in vitroUsed to synthesize RNA probes. Hybridization probes are, for example,³²P or³⁵It can be labeled with a population of various reporters, such as radionuclides such as S, or enzymatic labels such as alkaline phosphatase linked to the probe by an avidin / biotin binding system.
[0212]
The polynucleotide sequence encoding PRTS can be used to diagnose a disease associated with PRTS expression. Such disorders include, but are not limited to, gastrointestinal disorders, cardiovascular disorders, autoimmune / inflammatory disorders, abnormal cell proliferation, developmental disorders, epithelial disorders, neurological disorders, and reproductive disorders, Some gastrointestinal disorders include dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal cancer, indigestion, digestive disorders, gastritis, stomach cancer, anorexia, nausea, vomiting, gastroparesis, sinus or pyloric edema , Abdominal angina, heartburn, gastroenteritis, ileus, intestinal infection, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic cancer, biliary disease, hepatitis, hyperbilirubinemia, cirrhosis, liver Passive congestion, hepatome, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple disease, Mallory-Weiss syndrome, colon cancer, colonic obstruction, irritable bowel syndrome, short small bowel syndrome, diarrhea, constipation , Gastrointestinal bleeding, and later Autoimmune deficiency syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatitis, hepatic steatosis, hemochromatosis, Wilson disease, α-1-antitrypsin deficiency, Reye syndrome, primary sclerosing cholangitis, Hepatic infarction, portal venous occlusion and thrombus, central lobular necrosis, hepatic purpura, hepatic vein thrombosis, hepatic vein obstruction, preeclampsia, eclampsia, gestational acute hepatic fat, gestational intrahepatic cholestasis, and nodularity Regeneration and adenomas, liver cancer including carcinoma, and cardiovascular diseases include arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, venous malformation, arterial dissection, varices, thrombophlebitis and Venous thrombosis, vascular tumor, thrombolysis complications, balloon angioplasty, vascular replacement, aortic coronary artery bypass graft surgery ary artery bypass graft), congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcified aortic stenosis, congenital bicuspid aortic valve, mitral Mitral annular calcification, mitral prolapse, rheumatic fever, rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, systemic lupus erythematosus endocarditis, carcinoid Heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, congenital lung anomalies, pulmonary insufficiency, pulmonary congestion and edema, pulmonary embolism, pulmonary bleeding, Pulmonary infarction, pulmonary hypertension, vascular sclerosis, obstructive pulmonary disease, restrictive pulmonary disease (restrictive pulmona) y disease, chronic obstructive pulmonary disease, emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral pneumonia and mycoplasma pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases ), Pneumoconiosis, sarcoidosis, idiopathic pulmonary fibrosis, exfoliative interstitial pneumonia, hypersensitivity pneumonia, hypereosinophilic obstructive bronchiolitis-organic pneumonia eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage (diffuse pulmonary hemorrhage) age syndromes), Goodpasture's syndrome, idiopathic pulmonary hematosis, pulmonary involvment in collagen-vascular disorders, alveolar proteinosis, lung tumors, inflammatory and non-inflammatory inflammatory effusions effusions, pneumothorax, pleural tumors, drug-induced lung disease, radiation-induced lung disease, and complications of lung transplantation, and autoimmune / inflammatory diseases. Among them are inflammation and actinic keratosis, acquired immunodeficiency syndrome (AIDS) and adrenal insufficiency, adult respiratory distress syndrome, allergyー, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, atherosclerotic plaque rupture, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyglandular endocrine candidal ectoderm dystrophy ( APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, pulmonary emphysema, lymphocotoxin temporary lymphopenia, erythroblastosis, erythema nodosum, atrophic Gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, Articular cartilage degradation, osteoporosis, pancreatitis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus Systemic sclerosis, primary thrombocythemia, thrombocytopenia, ulcerative colitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, viral infections, bacterial infections, fungal infections, parasitic infections, Protozoal infections, helminth infections, trauma, and cell proliferation abnormalities include actinic keratosis and atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), Myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically adrenal gland , Bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, testis, thymus, Includes thyroid and uterine cancers, including developmental disorders. Some include tubular acidosis, anemia, Cushing's syndrome, chondrodysplasia dwarfism, Duchenne-Becker muscular dystrophy, bone resorption, epilepsy, gonad dysplasia, WAGR syndrome (Wilms tumor, aniridia, urogenital abnormalities, Hereditary neuropathies such as mental retardation, Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucosal epithelial dysplasia, hereditary keratosis, Charcot-Marie-Tooth disease and neurofibromatosis , Hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spinal cord fission, anencephaly, cranial spondylolysis, congenital glaucoma, cataract, age-related macular degeneration Includes sensory nerve hearing loss, some epithelial diseases, eczema, eczema, alleles Germinal contact dermatitis, keratosis, melasma, vitiligo, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, seborrheic keratosis, folliculitis, herpes simplex, shingles, Chickenpox, candidiasis, dermatophytosis, scabies, insect sting, senile hemangioma, keloid, dermal fibroma, apical fibrous tenderness, urticaria, transient acantholytic dermatosis, xerosis, eczema , Atopic dermatitis, contact dermatitis, hand eczema, monetary eczema, lichen lichen, asthetic eczema, congestive dermatitis, congestive ulcer, seborrheic dermatitis, psoriasis, lichen planus, rose pity Rash, impetigo, acne, dermatophytosis, nausea, wart, acne vulgaris, red nose, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, bullous pemphigoid, gestational Herpes, herpes dermatitis, linear IgA disease, acquired epidermolysis bullosa, dermatomyositis, lupus erythematosus, scleroderma and Localized scleroderma, erythroderma, alopecia, fixed skin lesions, vasodilatation, hypopigmentation, hyperpigmentation, vesicle / blister, rash, skin drug reaction, papular nodule skin disorder (Papulonodular skin lesions), chronic non-healing wounds, photosensitive diseases, simple epidermolysis bullosa, exfoliative and non-epidermal palmoplantar keratoderma, ichthyosis bulosa of Siemens, exfoliative ichthyosis, palmar Plantar keratosis, palmatoplantar keratoderma (keratosis palmoplantaris or palmoplantar keratoderma), keratosis punctata, Meesmann's cornea dystrophy, congenital nail hyperplasia, white spongiform nevus Includes onset sebaceous cysts, epidermal nevus / epidermal keratinization, moniletbrix, trichothiodystrophy, chronic hepatitis / idiopathic cirrhosis, colorectal hyperplasia, and some neurological disorders include epilepsy, ischemic cerebrovascular disease Disorders, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neuromuscular atrophy, Retinitis pigmentosa, hereditary ataxia, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural pyometra, epidural abscess, purulent intracranial thrombotic vein Inflammation, myelitis and radiculitis, viral central nervous system disease, Kuru and Creutzfeldt-Jakob disease, Gerstmann syndrome, Gerstmann-Strau Prion diseases including sler-Scheinker syndrome, fatal familial insomnia, neurotrophic and metabolic diseases, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastoma, trigeminal neurovascular Syndrome, central nervous system mental retardation including Down syndrome and other developmental disorders, cerebral palsy, nervous skeletal disorders, autonomic nervous system disorders, peripheral neuropathy, dermatomyositis and polymyositis, and hereditary, metabolic, endocrine And toxic myopathy, myasthenia gravis, periodic limb paralysis, psychosis including disorders of mood and anxiety, schizophrenia, seasonal disorders (SAD), restlessness, amnesia, nervousness Disease, diabetic neuropathy, extrapyramidal terminal defect syndrome, dystonia, schizophrenic psychiatric disorder, postherpetic neuralgia, and Poulet's disease includes progressive supranuclear palsy, corticobasal degeneration and familial frontotemporal amnesia, among which reproductive disorders include abnormal production of prolactin, fallopian tube disease and ovulation Abnormalities, endometriosis, abnormal estrus, abnormal menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian cancer, uterine fibroids, autoimmune abnormalities, ectopic pregnancy, infertility including teratogenesis Disease, breast cancer, fibrocystic mastosis, mastolacia, spermatogenesis, physiological sperm abnormalities, testicular cancer, prostate cancer, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, male breast and female And breast cancer. The polynucleotide sequence encoding PRTS can be obtained by Southern, Northern, dot blot, or other membrane-based techniques, PCR, dipstick, pin, ELISA, and the expression of mutant PRTS. Can be used for microarrays utilizing body fluids or tissues collected from patients to detect Such qualitative or quantitative methods are well known in the art.
[0213]
In certain embodiments, the nucleotide sequence encoding PRTS will be useful in assays that detect related diseases, particularly those described above. The nucleotide sequence encoding PRTS could be labeled by standard methods and added to a body fluid or tissue sample taken from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared to a reference value. If the amount of signal in the patient's sample is significantly altered compared to the control sample, the level of mutation in the nucleotide sequence encoding PRTS in the sample will reveal the presence of the associated disease. Such assays can be used to estimate the efficacy of a particular treatment in animal studies, clinical trials, or monitoring the treatment of an individual patient.
[0214]
A normal or standard profile of expression is established to provide a basis for the diagnosis of diseases associated with PRTS expression. This can be achieved by combining a PRTS-encoding sequence or a fragment thereof with a body fluid or cell extracted from a normal animal or human subject under conditions suitable for hybridization or amplification. . Standard hybridization can be quantified by comparing values obtained from normal subjects to values from experiments in which known amounts of substantially purified polynucleotides are used. Standard values obtained from normal samples can be compared to values obtained from subjects exhibiting symptoms of the disease. The deviation between the reference value and the subject's value is used to determine whether the subject is affected.
[0215]
Once the presence of the disease has been determined and the treatment protocol has begun, the hybridization assay can be repeated on a regular basis to estimate if the level of expression in the subject has begun to approach the value shown in a normal patient. is there. The results of repeated assays can be used to see the effect of treatment over a period of days to months.
[0216]
In cancer, abnormal amounts of transcripts in living tissue from an individual are predisposed to the development of the disease and can provide a way to detect the disease before actual clinical symptoms occur. This type of more definitive diagnosis allows medical professionals to use preventative or aggressive treatment early to prevent the development or progression of cancer.
[0217]
Additional diagnostic uses for oligonucleotides designed from PRTS-encoding sequences may include the use of PCR. Such oligomers can be chemically synthesized, enzymatically produced, orin in vitroCan be generated. The oligomer preferably includes a fragment of a polynucleotide encoding PRTS, or a fragment of a polynucleotide complementary to the polynucleotide encoding PRTS, and is used to identify a specific gene or condition under optimal conditions. You. Oligomers can also be used for the detection and / or quantification of closely related DNA or RNA sequences under somewhat mild stringent conditions.
[0218]
In some embodiments, oligonucleotide primers derived from a polynucleotide sequence encoding PRTS can be used to detect single nucleotide polymorphisms (SNPs). SNPs are nucleotide substitutions, insertions, and deletions that often cause congenital or acquired genetic disorders in humans. Non-limiting methods for detecting SNPs include single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, DNA is amplified by the polymerase chain reaction (PCR) using oligonucleotide primers derived from the polynucleotide sequence encoding PRTS. This DNA can be derived, for example, from lesions or normal tissue, biopsy samples, body fluids, and the like. The SNPs in this DNA cause a difference in the secondary and tertiary structure of the single-stranded PCR product. This difference is detectable using gel electrophoresis in a non-denaturing gel. In fSCCP, by labeling oligonucleotide primers with fluorescence, it becomes possible to detect an amplimer with a high-throughput device such as a DNA sequencing device. Further, a sequence database analysis method called in silico SNP (isSNP) can identify polymorphisms by comparing the sequences of individual overlapping DNA fragments used to construct a common consensus sequence. . These computer-based methods eliminate sequence variability due to automated analysis of DNA sequence chromatograms and sequencing errors using statistical models and laboratory DNA preparation. Alternatively, SNPs are detected and characterized by mass spectrometry using, for example, a high-throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
[0219]
Methods that can be used to quantify PRTS expression include radiolabeling or biotin labeling of nucleotides, co-amplification of regulatory nucleic acids, and standard curves with results added (eg, (See Melby, PC et al. (1993) J. Immunol. Methods, 159: 235-44; Duplaa, C. et al. (1993) Anal. Biochem. 229-236). The rate of quantification of large numbers of samples will be faster using high-throughput assays. In this assay, the oligomer or polynucleotide of interest is contained in various diluents and quantitation is rapid by spectrophotometry or non-color response.
[0220]
In yet another example, oligonucleotides or longer fragments from any of the polynucleotide sequences described herein can be used as targets in a microarray. Microarrays can be used in transcription imaging techniques to simultaneously monitor the relative expression levels of a large number of genes as described above. Microarrays can also be used to identify genetic mutations, mutations and polymorphisms. This information is used to determine gene function, elucidate the genetic basis of the disease, diagnose the disease, monitor the progression / regression of the disease associated with gene expression, and develop and develop therapeutic agents in the treatment of the disease. Monitoring. In particular, this information can be used to create a pharmacological genomic profile for the patient in order to select the optimal and effective treatment for the patient. For example, based on a patient's pharmacogenomic profile, one can select a therapeutic that is extremely effective for the patient but has few side effects.
[0221]
In another embodiment, PRTS, fragments of PRTS, antibodies specific to PRTS can be used as elements on the microarray. Microarrays can be used to monitor and measure protein-protein interactions, drug-target interactions and gene expression profiles as described above.
[0222]
Particular embodiments relate to the use of the polynucleotides of the present invention to produce transcribed images of certain tissues or cell types. The transcribed image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number and relative abundance of genes expressed at predetermined times under predetermined conditions (Seilliamer et al., US Patent No. 5,840,484, "Comparative Gene"). Transscript Analysis ", incorporated herein by reference to this patent). Thus, by hybridizing all of the transcripts or reverse transcripts of a particular tissue or cell type with the polynucleotide of the present invention or its complementary sequence, a transcribed image can be generated. In some embodiments, the polynucleotides of the invention or their complements are hybridized on a microarray in a high-throughput format that constitutes a subset of a plurality of elements. The resulting transcribed image can be a profile of gene activity.
[0223]
Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsy samples, or other biological samples. Therefore, the transcribed image isin Vivo, Or in the case of cell linesin in vitroReflects gene expression in
[0224]
Transcript images showing the expression profile of the polynucleotide of the present invention can also be used not only for toxicity testing of synthetic or natural compounds,in in vitroIt can be used in connection with preclinical evaluation of model systems and drugs. All compounds cause characteristic gene expression patterns, often referred to as molecular fingerprints or toxic signatures, suggesting mechanisms of action and toxicity (Nuwaysir, EF et al. (1999) Mol. Carcinog. 24: 153-159, Steiner, S. and N. L. Anderson (2000) Toxicol. Lett. 112-113: 467-471, also incorporated herein by reference. And). If the test compounds have the same signature as the signature of a known toxic compound, it is likely that they share toxic properties. The more useful a fingerprint or signature becomes, the more useful it is and the more accurate it contains expression information from genes and gene families. Ideally, expression should be measured across the genome to provide the highest quality signature. Genes that are not altered by any test compound are equally important. This is because the expression levels of these genes can be used to normalize the remaining expression data. The standardization process is useful for comparing expression data after treatment with different compounds. Assigning gene function to elements of a toxic signature helps to elucidate the mechanism of toxicity, but knowledge of gene function is not required for statistical matching of signatures leading to toxicity prediction (eg, February 29, 2000). See Press Release 00-02, published by the National Institute of Environmental Health Sciences, which is available at http://www.niehs.nih.gov/oc/news/toxchip.htm. Therefore, it is important and desirable to include all expressed gene sequences in a toxicity screen using a toxicity signature.
[0225]
In one example, the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. The nucleic acid expressed in the treated biological sample is hybridized with one or more probes specific for the polynucleotide of the present invention, whereby the level of transcription corresponding to the polynucleotide of the present invention can be quantified. The transcript level in the treated biological sample is compared to the level in an untreated biological sample. Differences in transcript levels between the two samples indicate a toxic reaction caused by the test compound in the treated sample.
[0226]
Another example involves analyzing a tissue or cell type proteome using a polypeptide sequence of the invention. The term "proteome" refers to the global pattern of protein expression in a particular tissue or cell type. Each protein that makes up the proteome can be further analyzed individually. Proteome expression patterns or profiles are analyzed by quantifying the number of proteins expressed at a given time under given conditions and their relative abundance. Thus, a proteomic profile of a cell can be generated by separating and analyzing polypeptides of a particular tissue or cell type. In some embodiments, such separation is performed by two-dimensional gel electrophoresis. In the two-dimensional gel electrophoresis, proteins are first separated from a sample by one-dimensional isoelectric focusing, and then separated according to molecular weight by two-dimensional sodium dodecyl sulfate slab gel electrophoresis (Steiner and above). Anderson). These proteins are usually visualized in the gel as dispersed discretely located spots by staining the gel with a stain such as Coomassie blue or silver or fluorescent stain. The optical density of each protein spot is usually proportional to the protein level in the sample. The optical densities of equipotent protein spots obtained from different samples, eg, biological samples, either treated or untreated with a test compound or therapeutic agent, are compared to determine changes in protein spot density associated with treatment. The proteins in the spots are partially sequenced using standard methods of mass spectrometry, for example, after cleavage chemically or enzymatically. The identity of a protein within a spot can be determined by comparing its partial sequence, which is preferably at least 5 contiguous amino acid residues, to a polypeptide sequence of the invention. In some cases, additional sequences are obtained for definitive protein identification.
[0227]
Proteome profiles can also be generated by quantifying PRTS expression levels using an antibody specific for PRTS. In one embodiment, protein expression levels are quantified using antibodies as elements on the microarray and exposing the microarray to a sample to detect the level of protein binding to each array element (Lueking, A. et al. (1999). ) Anal. Biochern. 270: 103-111, Mendose, LG et al. (1999) Biotechniques 27: 778-788). Detection can be performed in a variety of ways known in the art, for example, reacting proteins in a sample with a thiol-reactive or amino-reactive fluorescent compound to detect the amount of fluorescent binding at each array element .
[0228]
Toxic signatures at the proteome level are also useful for toxicological screening and should be analyzed in parallel with toxic signatures at the transcript level. For certain proteins in certain tissues, the correlation between the abundance of the transcript and the abundance of the protein may be low (Anderson, NL and J. Seilhammer (1997) Electrophoresis 18: 533-537). The proteome toxicity signature may be useful in the analysis of compounds that do not significantly affect the transcribed image but alter the proteome profile. In addition, analysis of transcription in body fluids is difficult due to the rapid degradation of mRNA. Thus, in such cases, proteome profiling is more reliable and informative.
[0229]
In another embodiment, the toxicity of the test compound is assessed by treating a biological sample containing the protein with the test compound. The proteins expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in the untreated biological sample. A difference in the amount of protein in both samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing their amino acid residues and comparing these subsequences to the polypeptides of the invention.
[0230]
In another embodiment, the toxicity of the test compound is assessed by treating a biological sample containing the protein with the test compound. A protein obtained from a biological sample is incubated with an antibody specific for a polypeptide of the invention. The amount of protein recognized by the antibody is quantified. The amount of protein in the treated biological sample is compared to the amount of protein in the untreated biological sample. A difference in the amount of protein between the two samples indicates a toxic response to the test compound in the treated sample.
[0231]
Microarrays are prepared, used and analyzed by methods well known in the art. (See, for example, Brennan, TM et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93: 10614-10619; Baldeschweiler et al. (1995)). PCT Application No. WO95 / 251116; Shalon, D. et al. (1995) PCT Application No. WO95 / 35505; Heller, RA et al. (1997) Proc. Natl. Acad. Sci. 94: 2150-2155; and Heller, M. J. et al. (1997) U.S. Patent No. 5,605,662). Various types of microarrays are well known, and for more informationDNA Microarrays: A Practical Approach, M .; Schena, ed. (1999) Oxford University Press, London. This document is incorporated herein by reference.
[0232]
In another embodiment of the invention, nucleic acid sequences encoding PRTS can also be used to generate hybridization probes useful for mapping native genomic sequences. Although either coding or non-coding sequences can be used, in certain instances, non-coding sequences are preferred over coding sequences. For example, conserved coding sequences between members of a multigene family can result in unwanted cross-hybridization during chromosome mapping. This sequence may be a specific chromosome, a specific region of a chromosome or an artificial chromosome, such as a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a bacterial P1 product, or a single chromosome cDNA library. (Harrington, JJ, et al. (1997) Nat Genet. 15: 345-355, Price, CM (1993) Blood Rev. 7: 127-134, Trask, BJ. (1991) Trends Genet. 7: 149-154 and the like). Once mapped, a nucleic acid sequence of the present invention can be used to create a genetic linkage map that correlates, for example, the inheritance of a disease state with the inheritance of a particular chromosomal region or restriction fragment length polymorphism (RFLP) (Lander , ES and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83: 7353-7357).
[0233]
in sitFluorescence hybridization (FISH) can be correlated with other physical and genetic map data (see, eg, Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or on the World Wide Web site of Online Mendelian Inheritance in Man (OMIM). Since the correlation between the location of the gene encoding PRTS on the physical chromosomal map and a particular disease, or a predisposition to a particular disease, can help determine the DNA region associated with such a disease, additional locations are Determining cloning is performed.
[0234]
Chromosome specimenin sitThe genetic map can also be extended using physical mapping techniques such as hybridization and binding analysis using defined chromosomal markers. Placing a gene on the chromosome of another mammal, such as a mouse, often reveals related markers, even if the exact location of the human chromosome is not known. This information is valuable to researchers working on genetic diseases using positional cloning or other gene discovery techniques. Once the location of one or more genes involved in the disease or syndrome has been roughly determined by gene binding to a particular gene region, such as 11q22-23 in telangiectasia, any sequence mapping for that region (See, eg, Gatti, RA et al. (1988) Nature 336: 577-580) for further investigation. The target nucleotide sequence of the present invention may be used to detect a difference in chromosomal position between a normal person and a carrier, that is, an infected person due to translocation or inversion.
[0235]
In another embodiment of the present invention, PRTS, its catalytic or immunogenic fragments or oligopeptides thereof can be used to screen libraries of compounds in any of a variety of drug screening techniques. The fragments used for such screening are free in solution, fixed to a solid support, retained on the surface of a cell, or exist in a cell. Complex formation due to binding of PRTS to the agent to be tested may be measured.
[0236]
Another method used for drug screening is used to increase the screening capacity of compounds having suitable binding affinity for the protein of interest (eg, Geysen, et al. (1984) PCT Application No. WO84 / 03564). See). In this method, a significant number of different small test compounds are synthesized on plastic pins or other substrates. The test compound is washed after reacting with PRTS or a fragment thereof. Next, the bound PRTS is detected by methods well known in the art. Purified PRTS can also be coated directly on plates used in the drug screening techniques described above. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
[0237]
In another example, a competitive drug screening assay can be used in which neutralizing antibodies capable of binding to PRTS specifically compete with the test compound for binding to PRTS. In this method, the antibody detects the presence of any peptide that shares one or more antigenic determinants with the PRTS.
[0238]
In another embodiment, the nucleotide sequence encoding PRTS is used in developing molecular biology techniques to characterize nucleotide sequences such as, but not limited to, the currently known triplet code and specific base pairing interactions. Can provide new technologies that depend on
[0239]
Those skilled in the art will be able to utilize the present invention to its fullest extent without the need for further explanation. Therefore, the specific preferred embodiments described below are for illustration purposes and do not limit the invention.
[0240]
All patent applications, patents, and publications mentioned above and below, particularly US patent applications Ser. Nos. 60 / 172,055, 60 / 177,334, 60 / 178,844, and No. 60 / 179,903 is incorporated herein by reference.
[0241]
(Example)
1 cDNA Creating a library
Incyte cDNA is derived from the cDNA library contained in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA) and is shown in column 4 of Table 4. Incyte cDNA, shown as SEQ ID NO: 15, was made from small intestine, ovary, lung, skin, breast, prostate epithelium, and mixed uterine muscle tissue such as cord blood and teratocarcinoma cells contained in neural progenitors. From the cDNA library. The in situ cDNA of SEQ ID NO: 17 includes bronchial epithelial first cell line, cutaneous microvascular endothelial cells, pancreas, ileal tissue associated with Crohn's disease, rib tissue associated with Pateau's syndrome, kidney, dorsal root ganglion , And a cDNA library made from corpus cavernosum tissue. The in-site cDNA of SEQ ID NO: 18 is derived from a cDNA library generated from brain tumor tissue. The in-site cDNA of SEQ ID NO: 19 is derived from a cDNA library created from adrenal gland, large intestine, and breast tissue. The in-site cDNA of SEQ ID NO: 20 is derived from a cDNA library prepared from T-lymphocytes, lung, breast, and corpus cavernosum tissues. First, a part of the tissue is homogenized and dissolved in a guanidinium isothiocyanate solution, while another part of the tissue is homogenized and dissolved in phenol, or TRIZOL (Life Technologies) or guanidinium isothiocyanate. And a suitable modifier such as a single phase solution of phenol. The lysate was extracted by centrifugation on cesium chloride or with chloroform. RNA was precipitated from this lysate with either isopropanol or sodium acetate and ethanol, or otherwise.
[0242]
Extraction and precipitation of RNA with phenol were repeated as necessary to increase the purity of the RNA. Optionally, the RNA is treated with a DNase. For most libraries, poly (A +) RNA was isolated using oligo d (T) -linked paramagnetic particles (Promega) or OLIGOTEX latex particles (QIAGEN. Valencia CA) and the OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, isolation was performed directly from tissue lysates using another RNA isolation kit such as the POLY (A) PURE mRNA purification kit (Ambion, Austin TX).
[0243]
In some cases, the RNA was provided to Stratagene and Stratagene generated a corresponding cDNA library. If not, cDNA was synthesized using the UNIZAP vector system (Stratagene) or the SUPERSCRIPT plasmid system (Life Technologies) by recommended or similar methods well known in the art to create a cDNA library. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6). Reverse transcription was started with oligo d (T) or random primer. After binding the synthetic oligonucleotide adapter to the double-stranded cDNA, the cDNA was digested with one or more suitable restriction enzymes. For most libraries, the size of the cDNA (300-1000 bp) was selected by SEPHACRYL S1000 or SEPHAROSE CL2B, SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech), or agarose gel electrophoresis. PBLUESCRIPT plasmids (Stratagene) or pSPORT1 plasmids (Life Technologies), pcDNA2.1 plasmids (Invitrogen Carlsbad CA), PBK-CMV plasmids (Stratagene), plNCY plasmids (Incyte Pharmaceuticals such as Incyte Pharmaceuticals, etc.) The cDNA was ligated to the compatible restriction enzyme sites of the polylinker. This recombinant plasmid was introduced into competent E. coli cells containing XL1-Blue, XL1-BIueMRF, SOLR from Stratagene, or DH5α or DH 10B, ELECTROMAX DH 10B from Life Technologies.
[0244]
2 cDNA Clone isolation
the aboveExample 1Plasmids obtained as described in (1) were obtained using the UNIZAP vector system (Stratagene) or cell lysis.in VivoRecovered from host cells by excision. Magic or WIZARD Minipreps DNA Purification System (Promega), AGTC Miniprep Purification Kit (Edge Biosystems, Gaithersburg MD), QIAGEN Plasmid, QIAPELPlasmid, QIAPELPlasmid, QIAPELPlasmid of QIAGEN The plasmid was purified using at least one of the following. After settling, they were resuspended in 0.1 ml of distilled water and stored at 4 ° C. with or without lyophilization.
[0245]
Alternatively, plasmid DNA was amplified from host cell lysates by a high-throughput direct binding PCR method. (Rao, VB (1994) Anal. Biochem. 216: 1-14). The lysis and thermal cycling process of the host cells was performed in a single reaction mixture. Samples are processed and then transferred to a 384-well plate for storage, and the concentration of amplified plasmid DNA is determined using a PICOGREEN dye (Molecular Probes, Eugene OR) and a Fluoroskan II fluorescence scanner (Labsystems Oy, Helsinki, Finland). Was measured.
[0246]
3 Sequencing and analysis
Example 2The in-site cDNA recovered from the plasmid as described in, was sequenced as shown below. The cDNA sequencing reaction is performed by standard methods, or ABI CATALYST 800 (PE Biosystems) thermal cycler or PTC-Mercer Cycler with a HYDRA microdispenser (Robbins Scientific) or MICROLAB 2200 (Hamilton) liquid transfer device. ) Was performed using a high-throughput apparatus. The cDNA sequencing reaction is carried out using Amersham Pharmacia Biotech's reagent or ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems). Separation of cDNA sequencing reactions by electrophoresis and detection of labeled polynucleotides was performed using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics), the ABI PRISM 373 or 377 sequencing system using standard ABI protocols and base pairing software. (PE Biosystems) or other sequence analysis systems well known in the art. The reading frame within the cDNA sequence was determined using standard methods (Ausubel, 1997, supra, unit 7.7). Select some of the cDNA sequences,Example 8The sequence was extended by the method described in (1).
[0247]
Confirmation of the polynucleotide sequence derived from the insite cDNA can be performed by using programs and algorithms based on BLAST, dynamic programming, and analysis of dinucleotide neighbors, using vectors, linkers, and polyA. This was done by removing the sequence and further masking ambiguous base pairs. Next, the insite cDNA sequences and their translations were compiled from the public databases of the primates, rodents, mammals, vertebrates, and eukaryotes of GenBank, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM. (HMM is a probabilistic method for analyzing the main consensus structure of a gene family. For example, see Eddy, S. et al., Hidden Markov Model (HMM). R. (1996) Curr. Opin. Struct. Biol. 6: 361-365). Such an inquiry was made using a program based on BLAST, FASTA, BLIMPS, and HMMR. The in-site cDNA sequence was assembled to generate a full-length polynucleotide sequence. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (Examples 4 and 5) To extend the insite cDNAs to the full length sequence. Sequence assembly was performed using programs based on Phred, Prap, and Consed, and screened through a set of cDNAs using GeneMark, BLAST, and FASTA-based programs to determine open reading frames. These full-length polynucleotide sequences were translated to obtain the corresponding full-length polypeptide sequences. Next, these polypeptide sequences are queried and analyzed against a GenBank protein database (genpept), a protein family database based on a hidden Markov model (HMM) such as SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and PFAM. did. These full-length polynucleotide sequences were also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Alignments of polynucleotide and polypeptide sequences were made using default parameters specified by the CLUSTAL algorithm incorporated into the MEGALIGN multi-sequence alignment program (DNASTAR), which also calculates the percent identity between the aligned sequences.
[0248]
Table 7 briefly summarizes the tools, programs, and algorithms used to assemble insight cDNAs and analyze full-length sequences, along with their descriptions, references, and threshold parameters. Column 1 in Table 7 shows the tools, programs, and algorithms used, column 2 gives a brief description of them, column 3 shows the cited references which are incorporated herein by reference, and column 4 describes the part. Indicates the score, probability value, and other parameters used to evaluate the degree of match between the two sequences (the higher the score, the lower the probability value, the higher the homology between the sequences) Become).
[0249]
The programs described above for assembling and analyzing full-length polynucleotide and polypeptide sequences can also be used to identify polynucleotide sequence fragments of SEQ ID NO: 15-28. Fragments of about 20 to about 4000 nucleotides that are useful for hybridization and amplification techniques are set forth in Table 4, column 4.
[0250]
4 Genome DNA Identification and editing of coding sequences of origin
Putative proteases were first identified by running the Genscan gene identification program in public genomic sequence databases (eg, gbpri and gbhtg). Genscan is a universal gene identification program for analyzing genomic DNA sequences from various organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268: 78-94, Burge, C. And S. Karlin (1998) Curr. Opin. Struct. Biol. 8: 346-354). This program ligates putative exons to construct an assembled cDNA sequence extending from methionine to a stop codon. The sequences obtained by Genscan become the polynucleotide and polypeptide sequences of the FASTA database. The maximum length of a sequence that can be analyzed at one time by Genscan is set to 30 kb. To determine which of these Genscan deduced cDNA sequences encodes the protease, the encoded polypeptide was interrogated and analyzed for the protease in a PFAM model. Potential proteases were identified based on homology to the in-site cDNA sequence annotated as a protease. These selected Genscan deduced sequences were then compared to sequences in the genept and gbpri public databases using BLAST analysis. If necessary, the Genscan putative cDNA sequence was compared with the sequence that BLAST hit most in genept to correct and edit errors such as extra exons or omitted exons in the Genscan putative sequence. Finding in-site or public cDNA containing the Genscan deduced cDNA sequence using BLAST analysis provides evidence of transcription. If the insite cDNA contains a Genscan deduced cDNA sequence, this information can be used to correct or confirm the Genscan deduced sequence. The full-length polynucleotide sequence isExample 3The Genscan deduced coding sequence and the insite cDNA and / or public cDNA sequence were assembled and prepared using the assembly method described in. Also, the full-length polynucleotide sequence was ultimately derived from the edited and unedited Genscan deduced sequence.
[0251]
5 Genome sequence data and cDNA Assembling with sequence data
Stitch arrangement ( Stitched Sequence )
Partial cDNA sequence,Example 4In the exon estimated by the Genscan gene identification program described in.Example 3The partial cDNA, assembled as described in, was mapped to genomic DNA and placed in multiple clusters containing the relevant cDNA and the relevant putative Genscan exons from one or more genomic sequences. Each cluster is integrated and analyzed with cDNA and genomic information using algorithms based on graph theory and dynamic programming, generating potential splice variants that are later identified, edited or extended to full length. Was prepared. Sequence sections in which the entire length of a section exists in two or more sequences of a cluster are identified, and sections identified by transitivity are considered to be equivalent. For example, if a section exists in each of the cDNA and two genomic sequences, all three sections are considered equivalent. In this way, unrelated but continuous genomic sequences are joined together by cDNA sequences. The sections thus identified are stitched together with a stitching algorithm so that they appear along the parent sequence to create the longest possible sequence and variant sequences. The joining of sections connected along a parent sequence of a certain type (cDNA and cDNA, or a genomic sequence and a genomic sequence) is more preferable than the connection of different parent sequence types (cDNA and genomic sequence). The resulting stitch sequences were translated and compared by BLAST analysis to sequences in the genpept and gbpri public databases. The incorrect exons estimated by Genscan are corrected in genept compared to the BLAST most hit sequences. Such sequences were extended with additional cNDA sequences and examined with genomic DNA as needed.
[0252]
Stretch array ( Stretched Sequence )
The partial DNA sequence was extended to full length by an algorithm based on BLAST analysis. First,Example 3The partial cDNA, assembled as described in, was queried against public databases, such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryotic databases using the BLAST program. Next, the highest homology protein of GenBank isExample 4Were compared with either the insite cDNA or the GenScan exon deduced sequence described above. A chimeric protein was prepared using the obtained plurality of high-scoring segment pairs (HSP), and the translated sequence was mapped on a homologous protein of GenBnak. Insertion or deletion may occur in the chimeric protein with respect to the original GenBnak homologous protein. Both GenBnak homologous and chimeric proteins were used as probes to search for homologous genomic sequences from public human genome databases. In this way, the partial DNA sequence was stretched or extended by the addition of a homologous genomic sequence. The resulting stretch sequences containing the complete gene were examined.
[0253]
6 PRTS Chromosome Mapping of Polynucleotide Encoding
The sequences used to construct SEQ ID NO: 15-28 were compared to sequences in the Insight LIFESEQ database and the public domain database using the BLAST and Smith-Waterman algorithms. Sequences from these databases consistent with SEQ ID NO: 15-28 were incorporated into clusters of contiguous and overlapping sequences using construction algorithms such as Phrap (Table 7). Radiation hybrid and gene mapping data are already clustered using radiation hybrid and gene mapping data available from public sources such as the Stanford Human Genonce Center (SHGC), the Whitehead Institute for Genome Research (WIGR) and Genethon. Find out if. If the cluster contained a sequence that was mapped, all sequences in that cluster (including the specific SEQ ID NO) were assigned to that mapping location.
[0254]
Genetic map locations are represented by ranges, intervals, or human chromosomes. The range of the mapping position indicated by centiMorgan was measured from the end of the short arm (p) of the chromosome. (Centimorgan (cM) is a unit indicating a distance based on the crossover rate between genes on the same chromosome. On average, 1 cM is approximately equal to one megabase of human chromosome, but can vary greatly due to high and low recombination rates). The distance cM is based on Genethon-mapped gene markers that can detect the boundaries of radiation hybrid markers whose sequences are contained in each cluster. Diseases in which the above-mentioned interval has already been identified using publicly available human genetic maps and other information sources such as NCBI “GeneMap99” (http: //www.ncbi.nlm.nih.gpv/genemap). Whether it is located in or near the genetic map can be determined.
[0255]
7. Analysis of polynucleotide expression
Northern analysis is an experimental technique used to detect the presence of a gene transcript, which involves the hybridization of a labeled nucleotide sequence to a membrane to which RNA from a particular cell type or tissue is bound. (See, eg, Sambrook, supra, chapter 7, and Ausubel FM et al., Supra, chapters 4 and 16).
[0256]
Similar computer techniques used for BLAST are used to search for identical or related molecules in a cDNA database such as GenBank or LIFESEQ (Incyte Pharmaceuticals). This analysis is much faster than many membrane-based hybridizations. In addition, the sensitivity of the computer search can be changed to determine whether any particular match is classified as an exact match or a homologous match. Search criteria are:
[0257]
(Equation 1)

Is the product score defined as The product score is a standardized value of 0 to 100, and is obtained as follows. Multiply the BLAST score by the percent identity of the nucleotide sequences and divide the product by 5 times the shorter length of the two sequences. A BLAST score is calculated by assigning a +5 score to each matching base in the high score segment pair (HSP) and -4 to each mismatched base pair. The two sequences may share more than one HSP (separated by a gap). If there is more than one HSP, calculate the product score using the base pair with the highest BLAST score. The product score represents a balance between fragmented overlap and quality of the BLAST alignment. For example, a product score of 100 is only obtained if there is a 100% match over the shorter length of the two compared sequences. The product score 70 is either the case where one end overlaps 70% at 100% match or the other end overlaps 100% at 88% match. The product score 50 is either the case where one end overlaps 50% at 100% match or the other end overlaps 100% at 79% match.
[0258]
Alternatively, the polynucleotide sequence encoding PRTS is analyzed against the tissue from which it was derived. For example, certain full-length sequences are assembled with at least partially overlapping insite cDNA sequences (Example 3See). Each cDNA sequence is derived from a cDNA library made from human tissue. Each human tissue has a cardiovascular system, connective tissue, digestive system, embryo structure, endocrine system, exocrine glands, female genitalia, male genitalia, germ cells, blood and immune system, lung, musculoskeletal system, nervous system, pancreas, respiration Fall into one organism / tissue category such as organs, sensory organs, skin, stomatognathic system, unclassified / mixed, or ureter. Count the number of libraries in each category and divide the total by the number of libraries in all categories. Similarly, each human tissue is categorized into one disease / symptom category, such as cancer, cell line, development, inflammation, neurology, trauma, cardiovascular, pooled, and the number of libraries in each category is counted. , And divide the total number by the number of libraries in all categories. The percentage obtained reflects the disease-specific expression of the cDNA encoding PRTS. cDNA sequence and cDNA library / tissue information can be obtained from the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
[0259]
8 PRTS Of polynucleotides encoding
Full length polynucleotide sequences were generated by extending a suitable fragment of the full length molecule using oligonucleotide primers designed from the suitable fragment of the full length molecule. One primer was synthesized to initiate the 5 'extension of a known fragment and the other primer was synthesized to initiate the 3' extension of a known fragment. The starting primer is about 22 to about 30 nucleotides in length, has a GC content of about 50% or more, using OLIGO 4.06 software (National Biosciences) or other suitable program, and has a GC content of about 68%. It was designed to anneal to the target sequence at a temperature of ７２72 ° C. Nucleotides were extended to avoid hairpin structures and primer-primer dimers.
[0260]
This sequence was extended using a selected human cDNA library. If more than one step extension is required or desired, additional or nested primer sets are designed.
[0261]
Amplification was performed with high fidelity by a PCR method using a method known in the art. PCR was performed in a 96-well block plate using a PTC-200 thermal cycler (MJ Research, Inc.). The reaction mixture was composed of template DNA and 200 nmol of each primer, Mg^{2 +}And (NH₄)₂SO₄And a buffer containing β-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene). Amplification was performed on the primer set, PCI A and PCI B, with the following parameters.
Step 1 @ 94 ° C for 3 minutes
Step 2: 15 seconds at 94 ° C
Step 3-1 minute at 60 ° C
Step 4 @ 68 ° C for 2 minutes
Step 5—Repeat steps 2, 3, and 4 20 times
Step 6 @ 68 ° C for 5 minutes
Step 7 Store at 4 ℃
Alternatively, amplification was performed on the primer set, T7 and SK +, with the following parameters.
Step 1 @ 94 ° C for 3 minutes
Step 2: 15 seconds at 94 ° C
Step 3-1 minute at 57 ° C
Step 4 @ 68 ° C for 2 minutes
Step 5—Repeat steps 2, 3, and 4 20 times
Step 6 @ 68 ° C for 5 minutes
Step 7 Store at 4 ° C.
[0262]
The DNA concentration in each well was determined by adding 100 μl of PICOGREEN quantitative reagent (0.25% (v / v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1 × TE and 0.5 μl of undiluted PCR product to opaque fluorescence The solution is distributed to each well of a photometer plate (Coming Costar, Acton MA) so that the DNA can bind to the reagent and the measurement is performed. The plate is scanned with a Fluoroskan II (Labsystems Oy, Helsinki, Finland) and the fluorescence of the sample is measured to quantify the DNA concentration. An aliquot of 5-10 μl of the reaction mixture is analyzed by electrophoresis on a 1% agarose minigel to determine which reaction has successfully extended the sequence.
[0263]
The extended nucleotides are desalted and concentrated, transferred to a 384-well plate, digested with CviJI choleravirus endonuclease (Molecular Biology Research, Madison WI), and religated to a pUC 18 vector (Amersham Pharmacia Biotech) prior to sonication. Or shear was performed. For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel to cut the fragments and the agar was digested with Agar ACE (Promega). A clone extended using T4 ligase (New England Biolabs, Beverly MA) was religated to a pUC18 vector (Amersham Pharmacia Biotech), and Pfu DNA polymerase (Stratagene) was used to extend the restriction site in E. coli cells by treating with Pfu DNA polymerase (Stratagene). Was transfected. The transfected cells were selected and transferred to a medium containing an antibiotic, and each colony was cut out and cultured in a 384-well plate of LB / 2X carbenicillin culture at 37 ° C. overnight.
[0264]
The cells were lysed, and the DNA was PCR-amplified using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) according to the following procedure.
Step 1 @ 94 ° C for 3 minutes
Step 2: 15 seconds at 94 ° C
Step 3-1 minute at 60 ° C
Step 4-2 minutes at 72 ° C
Step 5—Repeat steps 2, 3, and 4 29 times
Step 6-5 minutes at 72 ° C
Step 7 Store at 4 ° C.
DNA was quantified with the PICOGREEN reagent (Molecular Probes) as described above. Samples with poor DNA recovery were amplified again under the conditions described above. The sample was diluted with 20% dimethylsulfoxide (1: 2, v / v), and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE responsivity quantification cycle kit was used. Thing.
[0265]
Similarly, the full length polynucleotide sequence can be tested using the procedure described above, or the full length polynucleotide sequence can be used to control 5 'regulation using oligonucleotides designed for this extension and a suitable genomic library. The sequence was obtained.
[0266]
9 Labeling and use of individual hybridization probes
A cDNA, mRNA, or genomic DNA is screened using a hybridization probe derived from SEQ ID NO: 15-28. Particular mention is made of the labeling of oligonucleotides of about 20 base pairs, but essentially the same procedure is used for larger cDNA fragments. Oligonucleotides were designed using state-of-the-art software such as OLIGO 4.06 software (National Bioscience), and 50 pmol of each oligomer and 250 μCi of [γ-³²P] Adenosine triphosphate (Amersham, Chicago, IL) and T4 polynucleotide kinase (DuPont NEN, Boston MA) are used in combination for labeling. The labeled oligonucleotide is substantially purified using a SEPHADEX G-25 ultra-fine exclusion dextran bead column (Amersham Pharmacia Biotech). 10 per minute⁷An aliquot containing the labeled probe in counts is typically human genomic DNA cut with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xbal or Pvu II (DuPont NEN). Used in membrane-based hybridization analysis.
[0267]
DNA from each cut is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, blots are sequentially washed at room temperature, for example, under conditions of up to 0.1 × sodium salt citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized and compared by autoradiography or other imaging means.
[0268]
10 microarray
The concatenation or synthesis of array elements on a microarray can be achieved using photolithography, piezo printing (see inkjet printers, see Baldschweiler, supra), mechanical microspotting techniques and derivatives thereof. . In each of the above techniques, the substrate should be a uniform, non-porous solid (Schena (1999), supra). Recommended substrates include silicon, silica, glass slides, glass chips and silicon wafers. Alternatively, arrays similar to dot or slot blots can be used to place and attach elements to the surface of the substrate by thermal, ultraviolet, or chemical or mechanical binding means. Regular arrays can be made using available methods and machines and can include any suitable number of elements (Schena, M. et al. (1995) Science 270: 467-470; Shalon. D. et al. ( 1996) Genome Res. 6: 639-645, Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16: 27-31.).
[0269]
Full-length cDNA, expressed gene sequence fragments (ESTs), or fragments or oligomers thereof, can be elements of a microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array element is hybridized with a polynucleotide in a biological sample. Polynucleotides in a biological sample are conjugated to a fluorescent label or other molecular tag to facilitate detection. After hybridization, unhybridized nucleotides are removed from the biological sample and hybridization at each array element is detected using a fluorescence scanner. Alternatively, hybridization can be detected using laser desorption and mass spectrometry. The degree of complementarity and relative abundance of each polynucleotide that hybridizes to an element on the microarray can be calculated. The preparation and use of the microarray in one embodiment is described in detail below.
[0270]
Preparation of tissue or cell samples
Total RNA was isolated from tissue samples using the guanidinium thiocyanate method and poly (A) was isolated using the oligo (dT) cellulose method.⁺Purify the RNA. Each poly (A)⁺RNA samples were MMLV reverse transcriptase, 0.05 pg / μl oligo (dT) primer (21 mer), 1 × first strand buffer, 0.03 units / μl RNase inhibitor, 500 μM dATP, 500 μM dGTP, Reverse transcription is performed using 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). This reverse transcription reaction was performed using a GEMBRIGHT kit (Incyte) using 200 ng of poly (A).⁺Perform in a 25 ml volume containing RNA. Specific control poly (A)⁺RNA isin in vitroIt is synthesized from non-coding yeast genomic DNA by transcription. After incubation at 370 ° C. for 2 hours, each reaction sample (one Cy3 label and the other Cy5 label) was treated with 2.5 ml of 0.5 M sodium hydroxide and incubated at 850 ° C. for 20 minutes to stop the reaction. To denature the RNA. Samples are purified using two consecutive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA). After binding, the two reaction samples are ethanol precipitated using 1 ml glycogen (1 mg / ml), 60 ml sodium acetate and 300 ml 100% ethanol. The sample is then dried and finished using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 μl 5 × SSC / 0.2% SDS.
[0271]
Preparation of microarray
Array elements are made using the sequences of the present invention. Each array element is amplified from bacterial cells containing the vector containing the cloned cDNA insert. PCR amplification uses primers complementary to the vector sequence adjacent to the cDNA insert. The array elements are amplified from an initial amount of 1-2 ng to a final amount greater than 5 μg by 30 cycles of PCR. The amplified array elements are purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
[0272]
The purified array element is immobilized on a polymer-coated glass slide. Microscope glass slides (Corning) are washed during and after treatment with large amounts of distilled water and ultrasonically in 0.1% SDS and acetone. Slides are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and 0.05% aminopropylsilane in 95% ethanol (Sigma). ). The coated slide glass is cured by heating at 110 ° C.
[0273]
Array elements are added to a coated glass substrate using the method described in US Pat. No. 5,807,522. The specification is incorporated herein by reference. 1 μl of array element DNA having an average concentration of 100 ng / μl is filled into an open capillary printing element by a high-speed mechanical device. The device then dispense approximately 5 nl of array element samples per slide.
[0274]
The microarray is UV cross-linked using the STRATALLINKER UV crosslinker (Stratagene). The microarray is washed once with 0.2% SDS at room temperature and three times with distilled water. Non-specific binding sites were determined by incubating the microarray in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) at 60 ° C. for 30 minutes, followed by 0 minutes as described above. Block by washing with 2% SDS and distilled water.
[0275]
Hybridization
The hybridization reaction solution contained 9 μl of a sample mixture containing 0.2 μg each of Cy3 and Cy5 labeled cDNA synthesis products in a 5 × SSC, 0.2% SDS hybridization buffer. The sample mixture was heated at 65 ° C. for 5 minutes, dispensed aliquots on the microarray surface and then 1.8 cm² Cover with cover glass. The array is transferred to a waterproof chamber with a cavity slightly larger than the microscope slide. 140 μl of 5 × SSC is added to the corner of the chamber to keep the inside of the chamber at 100% humidity. The chamber containing the array is incubated at 60 ° C. for about 6.5 hours. Arrays were washed three times in a first wash buffer (1 × SSC, 0.1% SDS) for 10 minutes at 45 ° C. and in a second wash buffer (0.1 × SSC) for 10 minutes at 45 ° C. And then dried.
[0276]
detection
The reporter-labeled hybridization complex is an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of producing 488 nm spectral lines for exciting Cy3 and 632 nm for exciting Cy3. Detect with a microscope equipped with. Focus the excitation laser light on the array using a 20 × microscope objective lens (Nikon, Inc., Melville NY). The slide containing the array is placed on a computer controlled XY stage of a microscope and raster scanned through an objective. The 1.8 cm × 1.8 cm array used in this example is scanned at a resolution of 20 μm.
[0277]
In two different scans, the mixed gas multi-line laser excites the two phosphors sequentially. The emitted light is split into two photomultiplier tube detectors (PMT R1277, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two phosphors based on wavelength. The signal is filtered using a suitable filter located between the array and the photomultiplier. The maximum emission of the phosphor used is 565 nm for Cy3 and 650 nm for Cy5. The instrument can record spectra from both fluorophores simultaneously, but scans once per fluorophore and filters each array typically twice, using filters suitable for the laser source.
[0278]
The sensitivity of the scan is usually calibrated using the signal intensity generated by the cDNA control species added to the known concentration of the sample mixture. Specific locations on the array include complementary DNA sequences so that the intensity of the signal at that location correlates to a 1: 100,000 weight ratio of hybridizing species. When two samples from different samples (eg, representative of test and control cells) are each labeled with a different fluorophore and hybridized to a single array to identify genes that are differentially expressed The calibration is performed by labeling a cDNA sample to be calibrated having two fluorophores, and adding an equal amount to each of the hybridization mixtures.
[0279]
The output of the photomultiplier is digitized using a 12-bit RTI-835H analog-to-digital (AID) conversion board (Analog Devices, Inc., Norwood MA) installed on an IBM compatible PC computer. The digitized data is displayed as an image in which the signal intensity is mapped to a pseudo-color range from blue (low signal) to red (high signal) using a linear 20-color conversion. The data is also analyzed quantitatively. If two different phosphors are excited and measured simultaneously, the data is first corrected for optical crosstalk between the phosphors (due to overlapping emission spectra) using the emission spectra of each phosphor. .
[0280]
The grid is superimposed on the fluorescent signal image so that the signal from each spot is centered on each element of the grid. The fluorescent signal within each element is integrated to give a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is a GEMTOOOLS gene expression analysis program (Incyte).
[0281]
11 Complementary polynucleotide
A sequence that is complementary to the sequence encoding PRTS or any portion thereof is used to reduce or inhibit the expression of native PRTS. Although the use of oligonucleotides containing from about 15 to about 30 base pairs is noted, essentially the same method can be used for fragments of smaller or larger sequences. The appropriate oligonucleotides are designed using the Oligo 4.06 software (National Biosciences) and the coding sequence of the PRTS. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5 'sequence and used to prevent the promoter from binding to the coding sequence. To inhibit translation, complementary oligonucleotides are designed to prevent ribosomes from binding to transcripts encoding PRTS.
[0282]
12 PRTS Expression of
Expression and purification of PRTS can be performed using a bacterial or viral based expression system. For expression of PRTS in bacteria, the cDNA is subcloned into a suitable vector containing an inducible promoter that increases antibiotic resistance and the level of cDNA transcription. Such promoters include, but are not limited to, the T5 or T7 bacteriophage promoter and the trp-lac (tac) hybrid promoter associated with the lac operator regulatory element. The recombinant vector is transformed into a suitable bacterial host, such as BL21 (DE3). Bacteria with antibiotic resistance express PRTS when induced with isopropyl β-D thiogalactopyranoside (IPTG). Expression of PRTS in eukaryotic cells is commonly known as baculovirus in insect or mammalian cell linesAutographica californicaIt is performed by infecting with a nuclear pleiotropic virus (AcMNPV). The non-essential polyhedrin gene of the baculovirus is replaced with the cDNA encoding PRTS by either homologous recombination or bacterial-mediated gene transfer with transfer plasmid-mediated. The infectivity of the virus is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculoviruses are oftenSpodoptera frugiperda (Sf9) Used for infection of insect cells, but may also be used for infection of human hepatocytes. In the latter case, further genetic modification of the baculovirus is required. (See, for example, Engelhard. EK. Et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3224-2327; Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945.) .
[0283]
In most expression systems, PRTS is a fusion protein synthesized with, for example, glutathione S-transferase (GST) or a peptide epitope tag such as FLAG or 6-His. Can be performed quickly and in one go.Schistosoma japonicumThe 26-kilodalton enzyme, GST, allows purification of fusion proteins with immobilized glutathione while maintaining protein activity and antigenicity (Amersham Pharmacia Biotech). After purification, the GST moiety can be proteolytically cleaved from the PRTS at a specific manipulation site. FLAG, an eight amino acid peptide, allows for immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, allows purification on metal chelating resins (QIAGEN). Methods for protein expression and purification are described in Ausubel (1995, supra, ch 10, 16). The assays of Examples 16, 17, 18, and 19 below can be performed directly using PRTS purified by these methods.
[0284]
13 Function Assay
PRTS function is assessed by expression of PRTS-encoding sequences at physiologically elevated levels in mammalian cell culture systems. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses the cDNA at high levels. Such vectors include pCMV SPORT^TM (Life Technologies.) And pCR 3.1 (Invitrogen, Carlsbad, CA), both containing the cytomegalovirus promoter. 5-10 μg of the recombinant vector is transiently transfected into human cell lines, eg, endothelial or hematopoietic, by liposome preparation or electroporation. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is co-transfected. Expression of the labeled protein allows one to distinguish between transfected and non-transfected cells. In addition, the expression of the cDNA from the recombinant vector can be accurately predicted by the expression of the labeled protein. Such labeled proteins include green fluorescent protein (GFP; Clontech), and CD64 or CD64-GFP fusion protein. Identify transfected cells that express GFP or CD64-GFP using automated flow cytometry (FCM) using laser optics-based technology and evaluate the apoptotic state and other cellular properties of the cells I do. In addition, the FCM detects and measures the incorporation of a fluorescent molecule that diagnoses the phenomenon of preceding or simultaneous cell death. These phenomena include changes in nuclear DNA content as measured by staining of DNA with propidium iodide, down-regulation of DNA synthesis as measured by reduced uptake of bromodeoxyuridine, and specific antibodies. And changes in plasma membrane composition as measured by binding of the fluorescent complex annexin V protein to the cell surface, as measured by the reactivity of E. coli. Flow cytometry is described in Ormerod, M .; G. FIG. By (1994)Flow Cytometry Oxford, New York, NY. It is described in.
[0285]
The effect of PRTS on gene expression can be assessed using a highly purified cell population transfected with the sequence encoding PRTS and either CD64 or CD64-GFP. CD64 or CD64-GFP is expressed on the surface of transformed cells and binds to a conserved region of human immunoglobulin G (IgG). Transformed and non-transformed cells can be separated using magnetic beads coated with either human IgG or an antibody against CD64 (DYNAL. Lake Success. NY). mRNA can be purified from cells by methods well known in the art. Expression of mRNA encoding PRTS and other genes of interest can be analyzed by Northern analysis or microarray technology.
[0286]
14 PRTS Of antibodies specific for
Polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, MG (1990) Methods Enzymol. 1816-3088-495) or other purification techniques using PRTS substantially purified by standard techniques Immunize rabbits with a simple protocol to produce antibodies.
[0287]
Alternatively, the PRTS amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine the region of high immunogenicity, and the corresponding oligopeptide is synthesized and used to generate antibodies using methods well known to those skilled in the art. Produce. The selection of suitable epitopes, such as those near the C-terminus or in adjacent hydrophilic regions, is well known in the art (see, eg, Ausubel, 1995, supra, Chapter 11).
[0288]
Usually, an oligopeptide having a length of about 15 residues is synthesized by an Fmoc method chemistry using an Applied Biosystems ABI 431A peptide synthesizer (PE Biosystems) to give N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). The reaction used binds KLH (Sigma-Aldrich, St. Louis MO) to enhance immunogenicity (see, eg, Ausubel, 1995, supra). Rabbits are immunized with the oligopeptide-KLH complex in Freund's complete adjuvant. In order to examine the anti-peptide activity and anti-PRTS activity of the obtained antiserum, the peptide or PRTS was bound to a substrate, blocked with 1% BSA, reacted with rabbit antiserum, washed, and further radioactively washed. React with iodine-labeled goat anti-rabbit IgG.
[0289]
15 Natural using specific antibodies PRTS Purification
The natural or recombinant PRTS is substantially purified by immunoaffinity chromatography using an antibody specific for PRTS. The immunoaffinity column is formed by covalently binding an anti-PRTS antibody to an activation chromatography resin such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After binding, the resin is blocked and washed according to the manufacturer's instructions.
[0290]
The culture solution containing PRTS is passed through an immunoaffinity column, and the column is washed under conditions that allow preferential adsorption of PRTS (for example, with a buffer having high ionic strength in the presence of a surfactant). The column is eluted under conditions that break the binding between the antibody and PRTS (eg, with a buffer of pH 2-3 or a high concentration of chaotropic ions such as urea or thiocyanate ions) to recover the PRTS.
[0291]
16 PRTS Of molecules that interact with
PRTS or a biologically active fragment thereof,¹²⁵Label with I Bolton Hunter reagent (see, for example, Bolton AE and WM Hunter (1973) Biochem. J. 133: 529). Candidate molecules pre-arranged in a multi-well plate are incubated with the labeled PRTS, washed, and all wells with the labeled PRTS complex are assayed. Using the data obtained at the various PRTS concentrations, values are calculated for the quantity and affinity of PRTS bound to the candidate molecule and for association.
[0292]
Alternatively, molecules that interact with PRTS are identified in Fields, S .; And O. The analysis is performed using a commercially available kit based on a two-hybrid system such as the yeast two-hybrid system described in Song (1989, Nature 340: 245-246) or the MATCHMAKER system (Clontech).
[0293]
PRTS also uses the PATHHCALLING process (CuraGen Corp., New Haven CT) using a high-throughput yeast two-hybrid system to determine all interactions between proteins encoded by two large libraries of genes. (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
[0294]
17 PRTS Demonstration of activity
Protease activity is measured by hydrolysis of an appropriate synthetic peptide substrate conjugated to various dye molecules, where the degree of hydrolysis is determined by the spectrophotometric (fluorescent) absorption of the released chromophore (Beynon, RJ). And JS Bond (1994);Proteolytic Enzymes : A Practical Approach, Oxford University Press, New York NY, pp. 25-55). Peptide substrates include protease activity categories as endopeptidases (serine, cysteine, aspartic protease or metalloprotease), aminopeptidases (leucine aminopeptidase), or carboxypeptidases (carboxypeptidases A and B, procollagen C-proteinase). It is designed according to. Typically, the chromogens used are 2-naphthylamine, 4-nitroaniline, furylacrylic acid. The assay is performed at ambient temperature and includes an aliquot of the enzyme and a suitable substrate in a suitable buffer. The reaction is performed in a fluorescent cuvette and the increase / decrease in absorbance of the chromogen released during hydrolysis of the peptide substrate is measured. The change in absorbance is proportional to the enzymatic activity in the assay.
[0295]
An alternative assay for ubiquitin hydrolase activity measures the hydrolysis of ubiquitin precursor. The assay is run at ambient temperature and contains an aliquot of the appropriate substrate and PRTS in a suitable buffer. Chemically synthesized human ubiquitin-valine is used as a substrate. Cleavage of the C-terminal valine residue from the substrate is monitored by capillary electrophoresis (Franklin, K. et al. (1997) Anal. Biochem. 247: 305-309).
[0296]
Alternatively, assays for protease activity utilize fluorescence resonance energy conversion (FRET). FERT occurs when one donor and one acceptor fluorophore are in close proximity with proper spectral overlap. A flexible peptide linker with a cleavage site specific for PRTS fuses between the red-shifted variant (RSGFP4) and blue mutants of green fluorescent protein.
[0297]
This fused protein has spectral properties that cause energy transfer to occur from BFP5 to RSGFP4. When the fused protein is incubated with the PRTS, the substrate is cleaved and the two fluorescent proteins are separated. This is accompanied by a significant decrease during energy transfer as determined by comparing the emitted spectra before and after the addition of PRTS (Mitra, RD et al. (1996) Gene 173: 13-17). Assays can also be performed in living cells. In such cases, the fluorescent substrate protein is constitutively expressed in the cell and PRTS is provided on an inducible vector, so that FRET can be monitored in the presence and absence of PRTS (Sagot, I et al .: ( 1999) FEBS Lett. 447: 53-57).
[0298]
18 PRTS Substrate identification
A phage display library may be used to identify the optical substrate sequence of PRTS. The random hexamers following the linker and the known antibody epitope are cloned as N-terminal extensions of gene III in a filamentous phage library. The gene III code encodes a coat protein and the epitope may be displayed on the surface of each phage molecule. The library is incubated with PRTS under proteolytic conditions, and the epitope may be removed if the hexameric code for PRTS cleaves the site. An antibody that recognizes the epitope is added with immobilized protein A. Uncleaved phage bearing the epitope are removed by centrifugation. The surface phage is then amplified, generating additional rounds of screening. Individual phage clones are then isolated and sequenced. The kinetics for these peptide substrates may be studied using the assay in Example 17, and optimal cleavage sequences may be obtained (Ke, SH et al. (1997) J. Biol. Chem 272: 16603-16609).
[0299]
in VivoDue to the screening of PRTS substrates, this method uses phage molecules (T7SELECT).^TM10-3 A phage display vector, Novagen, Madison, WI), or expanded to screen a cDNA expression library displayed on the surface of a yeast (pYD1 yeast display vector kit, Invitrogen, Carlsbad, CA). In such a case, the entire cDNA is fused between gene III and the appropriate epitope.
[0300]
19 PRTS Inhibitor identification
Compounds to be tested are arrayed on multi-well plates at varying concentrations along appropriate buffers and substrates as described in the assay of Example 17. PRTS activity is measured in each well and the ability of each compound to inhibit PRTS activity may be determined. Dose-response kinetics are similar. Assays may be used to identify molecules that enhance the activity of PRTS.
[0301]
Alternatively, a phage display library may be used to screen for peptide PRTS inhibitors. Candidates were found in peptides strongly bound to the protease. In this case, each well of the multi-well plate is coated with PRTS and incubated with a random peptide phage display library or a periodic peptide library (Koivunen, E. et al. (1999) Nat. Biotechnol. 17: 768-). 774). Unbound phage are washed away and selected phage are amplified in multiple further rounds and rescreened. Candidates are tested for PRTS inhibitory activity using the assay described in Example 17.
[0302]
Those skilled in the art will be able to make various modifications of the described method and system without departing from the scope and spirit of the invention. Although the invention has been described with reference to certain preferred embodiments, it is to be understood that the scope of the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described embodiments of the invention which are obvious to those skilled in molecular biology or related fields are within the scope of the following claims.
[0303]
(Brief description of the table)
Table 1 shows the systematic names for the polynucleotide and polypeptide sequences of the present invention.
[0304]
Table 2 shows the GenBank identification numbers and annotations of the closest GenBank homologs for each polypeptide of the invention. Also shown is the probability score representing the match between each polypeptide and its GenBank homolog.
[0305]
Table 3 shows the structural features of each polypeptide sequence, including putative motifs and domains, and the methods, algorithms, and searchable databases used to analyze each polypeptide.
[0306]
Table 4 shows a list of cDNA and genomic DNA fragments used to assemble each polynucleotide sequence, and a list of selected fragments of the polynucleotide sequence.
[0307]
Table 5 shows a representative cDNA library of each polynucleotide of the present invention.
[0308]
Table 6 is an appendix showing tissues and vectors used for preparing the cDNA library shown in Table 5.
[0309]
Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the present invention, along with descriptions, references, and threshold parameters.
[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

Claims (28)

An isolated polypeptide,
(A) an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 14 (SEQ ID NO: 1-14);
(B) a natural amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14;
(C) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14;
(D) an isolated polypeptide comprising an amino acid sequence selected from the group consisting of an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14. 2. The isolated polypeptide of claim 1, selected from the group consisting of SEQ ID NOs: 1-14. An isolated polynucleotide encoding the polypeptide of claim 1. An isolated polynucleotide encoding the polypeptide of claim 2. 5. The isolated polynucleotide of claim 4, selected from the group consisting of SEQ ID NOs: 40-78. A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide of claim 3. A cell transformed with the recombinant polynucleotide of claim 6. A transgenic organism comprising the recombinant polynucleotide of claim 6. A method for producing the polypeptide of claim 1,
(A) culturing cells transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1 under conditions suitable for expression of said polypeptide. Steps and
(B) recovering the polypeptide thus expressed. An isolated antibody that specifically binds to the polypeptide of claim 1. An isolated polynucleotide, comprising:
(A) a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 15-28;
(B) a natural polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 15-28;
(C) a polynucleotide sequence complementary to (a),
(D) a polynucleotide sequence complementary to (b);
(E) an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of the RNA equivalents of (a) to (d) above. An isolated polynucleotide comprising at least 60 contiguous nucleotides of the polynucleotide of claim 11. A method for detecting a target polynucleotide having a polynucleotide sequence according to claim 11 in a sample,
(A) hybridizing the sample with a probe, wherein the probe comprises at least 20 contiguous nucleotides comprising a sequence complementary to the target polynucleotide in the sample, wherein the probe and the target The probe specifically hybridizing to the target polynucleotide under conditions in which a hybridization complex is formed with the polynucleotide or a fragment thereof;
(B) detecting whether the hybridization complex is present and, if so, optionally measuring the yield thereof. 14. The method of claim 13, wherein said probe comprises at least 60 contiguous nucleotides. A method for detecting, in a sample, a target polynucleotide having the polynucleotide sequence of claim 11,
(A) amplifying the target polynucleotide or a fragment thereof using polymerase chain reaction amplification;
(B) detecting whether the amplified target polynucleotide or a fragment thereof is present, and, if present, optionally measuring the yield thereof. Method. A composition comprising an effective amount of the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 17. The composition of claim 16, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14. 17. A method for treating a disease or condition associated with reduced expression of a functional PRTS, comprising administering the composition of claim 16 to a patient in need of such treatment. A method for screening a compound effective as an agonist of the polypeptide of claim 1,
(A) exposing a sample comprising the polypeptide of claim 1 to a compound;
(B) detecting agonist activity in the sample. A composition comprising an agonist compound identified by the screening method of claim 19 and a pharmaceutically acceptable excipient. 21. A method for treating a disease or condition associated with reduced expression of a functional PRTS, comprising administering to a patient in need of such treatment the composition of claim 20. A method for screening a compound effective as an antagonist of the polypeptide of claim 1,
(A) exposing a sample comprising the polypeptide of claim 1 to a compound;
(B) detecting antagonist activity in the sample. A composition comprising an antagonist compound identified by the screening method of claim 22 and a pharmaceutically acceptable excipient. 24. A method of treating a disease or condition associated with overexpression of functional PRTS, comprising administering to a patient in need of such treatment the composition of claim 23. A method for screening for a compound that specifically binds to the polypeptide of claim 1,
(A) binding the polypeptide of claim 1 to at least one test compound under suitable conditions;
(B) detecting the binding between the polypeptide of claim 1 and the test compound to identify a compound that specifically binds to the polypeptide of claim 1. A method for screening a compound that changes the activity of the polypeptide according to claim 1,
(A) binding the polypeptide of claim 1 to at least one test compound under conditions permitting its activity;
(B) assessing the activity of the polypeptide of claim 1 in the presence of said test compound;
(C) comparing the activity of the polypeptide of claim 1 in the presence of the test compound to the activity of the polypeptide of claim 1 in the absence of the test compound;
A screening method, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound indicates the presence of a compound that alters the activity of the polypeptide of claim 1. A method for screening a compound effective for altering the expression of a target polynucleotide comprising the sequence of claim 5, comprising:
(A) exposing a sample containing the target polynucleotide to a compound under conditions suitable for expression of the target polynucleotide;
(B) detecting a change in expression of the target polynucleotide;
(C) comparing expression of the target polynucleotide in the presence of various amounts of the compound with expression of the target polynucleotide in the absence of the compound. . A method for evaluating the toxicity of a test compound, comprising:
(A) treating a biological sample containing nucleic acids with the test compound;
(B) hybridizing the treated nucleic acid of the biological sample with a probe comprising at least 20 contiguous nucleotides of the polynucleotide of claim 11, wherein the hybridization comprises the hybridization of the probe and the biological sample. The method is carried out under conditions under which a specific hybridization complex is formed with the target polynucleotide, wherein the target polynucleotide is a polynucleotide comprising the polynucleotide sequence of the polynucleotide of claim 11 or a fragment thereof. Said steps;
(C) quantifying the yield of the hybridization complex;
(D) comparing the yield of the hybridization complex in the treated biological sample with the yield of the hybridization complex in the untreated biological sample.
A method for evaluating toxicity of a test compound, wherein a difference in the yield of the hybridization complex in the treated biological sample indicates the toxicity of the test compound.