JP2004535772A - Soluble reporter gene structure - Google Patents

Abstract

The present invention provides polynucleotides operably linked to a proteolytic response promoter reporter gene and gene reporter systems comprising these polynucleotides along with the expression structure for the target protein. The present invention also provides a cell containing the polynucleotide and the gene reporter system of the present invention. These compositions are useful for monitoring the solubility of a target protein in a cell and identifying variants against a polynucleotide encoding a target protein that alters the solubility of the target protein in cells and the solubility of the target protein. . The invention also provides methods for identifying variations in the protein biosynthesis process that alter the solubility of a target protein, and for screening recombinant library expression libraries for clones that express soluble proteins. Finally, the present invention discloses a novel method for identifying antibiotic reagents.

Description

【表２】

第２表 誤って折畳まれたタンパク質発現の誘導後のコールドショック

【０１０３】
リボゾーム関連遺伝子の誘導
リボゾームに結合する他のヒートショック遺伝子は、翻訳の誤った折りたたみ条件下で誘導される。Ｈｓｐ１５（ｙｒｆＨ）はＲＮＡ（２４）と結合し、そして発生期のポリペプチド鎖を含む自由５０Ｓリボゾームサブユニットに結合する（１６）。ヒートショックは、５０Ｓ及び３０Ｓサブユニットの増加した分解を含むＨｓｐ１５−結合のレベルも増加させる。リボゾームの分解のさらなる提案は、ｆｔｓＪ（ｒｒｍＪ）（ＳＥＱ ＩＤ ＮＯ：１０）に由来する。ｆｔｓＪ遺伝子生成物は、５０Ｓリボゾームサブユニットにおいて含まれる場合のみ、２３ＳｒＲＮＡについて特異的なＲＮＡメチラーゼである（１７，１８）。この酵素は２３Ｓ ｒＲＮＡをリボゾームのペプチジルトランスフェラーゼ中心内に位置する２５５２においてメチル化する（１７）。ｆｔｓＪのメチル化は２３Ｓ ｒＲＮＡを欠き、そして５０Ｓ及び３０Ｓサブユニットの分裂に対応するリボゾーム活性の６５％までの減少を示す（１９）。ｒｐｏＨ変異体において特に顕著なものは、コールドショックタンパク質（ＣＳＰｓ）の転写における大きな増加である（第２表）。これらの遺伝子はヒートショックにより影響を受けないが（９）、翻訳の一時的な停止に関連する。ＣＳＰは、翻訳化されていないメッセージについてのシャペロンとして作用し（２０，２１）、そして抗末端化活性（ａｎｔｉ−ｔｅｒｍｉｎａｔｉｏｎ）を提供するＲＮＡ結合タンパク質である（２２）。シャペロン発現（ｒｐｏＨ６０６）を減少させる条件下でのＣＰＳの増加された発現は、休止した翻訳を表す。まとめて考えると、これらの結果は、或いは脱メチル化の結果として、翻訳の誤った折りたたみの結果として、翻訳の調整応答を提案する。この仮定は、最近の調査における興味深い調整機構である。
【０１０４】
他の誘発された遺伝子
ｙｃｃＶ、ｙｈｄＮ及びｙｒｆＧはヒートショック条件下で発現を増加することを示すが、機能が知られていないものである。これらの公知のヒートショック遺伝子に加え、ｙａｇＵ、ｙｃｉＳ、ｙｂｅＤ、ｙｅｊＧ及びｙｈｇＩは増加した発現を示した。これらのタンパク質のほとんどは比較的小さく、そして一般的に酸性である。ある推測では、これらのタンパク質のうち数種のものは直接的な認識及び誤って折りたたまれたタンパク質の封鎖においてＩｂｐＡＢに類似する役割を果たす。しかし、ＩｂｐＡＢだけが間違って折りたたまれそして凝集したタンパク質と結合する。ｉｂｐＡＢの誘導レベルは非常に高く、そしてこれらの他のタンパク質はより低いレベルで存在する。興味深いことには、ｉｂｐＡＢのノックアウト変異は、細胞成長及び生存力における比較的小さな影響を有し（１４）、そして細胞内の数種の機能的な余剰を意味する。
【０１０５】
タンパク質折りたたみの遺伝子レポーター
プロファイリング結果を確認し及び多数の組換えタンパク質の実験を容易化するために、ｉｂｐＡＢ、ｙｂｅＤ、ｙｈｇＩ及びｙｒｆＧＨＩからのプロモーター領域をベータ−ガラクトシダーゼレポーターベクター内へクローン化した。各場合において、増加されたベータ−ガラクトシダーゼ活性は、折りたたまれたタンパク質ＰＬＡが活性における増加しないにもかかわらず、誤って折りたたまれたタンパク質ＬＣＫの発現が誘導される場合に観察される。これらの結果は、ｉｂｐＡＢ−プロモーターベータ−ガラクトシダーゼ融合物の存在下でともに発現される（ｃｏ−ｅｘｐｒｅｓｓｅｄ）８の誤って折りたたまれたタンパク質及び６の適切に折りたたまれたタンパク質のセットを用いてさらに拡張された。各場合において、増加されたベータ−ガラクトシダーゼ活性は、誤って折りたたまれたタンパク質の発現に相当した。より詳細な説明を以下に示す。そして、観察された応答は、任意の特定のタンパク質に対する特異的な応答よりも、タンパク質の誤った折りたたみの通常の結果を示す。これらのレポーターは、感度の良い酵素アッセイを用いて誤って折りたたまれたタンパク質を同定する単純な方法を提供し、そしてさらなる研究のためのレポーターとしてｉｂｐＡＢプロモーター融合物が選択された。
【０１０６】
溶解性タンパク質についてのＥＬＩＳＡ様アッセイ
組換え環境において改良された折りたたみ特性を有するタンパク質誘導体を同定するために、高処理量スクリーニング機械と融和するＥＬＩＳＡ様アッセイも発展させた。高処理量システムにおける溶解性タンパク質レベルを評価するために、非変性細胞分離物は、ミクロプレートにおける迅速なスクリーニングと融和する条件を使用して製造されなければならない。洗浄剤又は有機リーシスの代わりに、我々は抗生物質カクテルを各ウェルに加え、リーシスを誘導した。溶解性タンパク質融合物を除去し、ミクロプレートに結合し、組換えタンパク質は６Ｘ−ヒスチジンＮ−末端融合物に対するＮｉ−ＨＲＰコンジュゲートの結合を介して検出した。Ｈｉｓ−Ｔａｇは組換えタンパク質間で不均一にアクセス可能ではないことがわかる。それゆえ、ネガティブＮｉ−ＨＲＰ応答は溶解性タンパク質の不存在を表示できないが、タンパク質折りたたみはＨｉｓ−Ｔａｇに対するアクセスを妨げることができる。しかし、我々はこれを観察する必要は無く、一般的な問題である。それからこのアッセイは、ＳＤＳゲルを行うことなく、そしてＨＴスクリーン及びβ−ガラクトシダーゼアッセイに融和する形態で、溶解性組換えタンパク質のレベルの測定を提供する。
【０１０７】
予め測定された発現特性を有するタンパク質の試験
Ｔｈｅｒｍｏｔｏｇａ ｍａｒｉｔｉｍａの全プロテオームをクローニング、発現及び特徴化することにおいて意図する我々の試みの一部として、第３表に示される（６種の溶解性、６種の不溶性及び６種の混合溶解性）１８Ｔ．ｍａｒｉｔｉｍａタンパク質におけるレポーターの効果を試験した。アッセイパラメーターを最適化するために、菌株を９６ウェル内に整列させ、３種の誘導レベル（０．０２％、０．２％及び２％アラビノース）の３種類において及び溶解促進抗生物質添加について４種の後誘発時点（ｐｏｓｔ−ｉｎｄｕｃｔｉｏｎ ｔｉｍｅ）（アラビノース添加後のｔ＝０、３０分、６０分及び１２０分）においてアッセイした。図２は、０．２％アラビノース誘導について、３種のプレート（溶解性、不溶性及び混合）についての平均化された結果を示す。不溶性及び混合プールの両方が、溶解性プールよりも４倍以上β−ガラクトシダーゼ活性を示した（図２Ａ）。逆に、溶解性プールは、不溶性プールに対してＮｉ−ＨＲＰにおける１０倍以上高い応答を示した（図２Ｂ）。溶解性及び不溶性フラクションの両方においておよそ等しく発現されるタンパク質からなる混合プールは、溶解性プールの約半分強度でＮｉ−ＨＲＰ結合を示した。β−ガラクトシダーゼの欠損又はＮｉ−ＨＲＰ活性の存在のいずれかだけが、溶解性タンパク質の測定として使用されるが、良い効果的かつ便利なスクリーンを活性化する２つの比を選択する。
第３表： 予め測定された発現特性を有する Ｔ． ｍａｒｉｔｉｍａタンパク質

【０１０８】
非公知の発現特性を有するタンパク質の試験
我々は、次にこのスクリーンを非公知の折りたたみ特性を有するタンパク質の大きなセットへ適用した。我々は、タンパク質が発現について前もって特徴化されない１８６Ｔ．ｍａｒｉｔｉｍａタンパク質において、上記最適条件下でスクリーンを行った。このスクリーンの結果を、第４表に要約する。ニッケル−キレーティング樹脂からの及び各クローンの溶解された不溶性フラクションからの溶出物のＳＤＳ−ＰＡＧＥは、対応するβ−ガラクトシダーゼ活性、Ｎｉ−ＨＲＰ応答及び１８６クローンについての溶解性スコアに沿って行われる。ゲルの結果に基づき、５７のクローンは可視タンパク質バンドを過剰発現（ｏｖｅｒｅｘｐｒｅｓｓ）せず、６２のクローンは溶解性タンパク質を優勢に発現し、２７のクローンは不溶性凝集物を優勢に発現し、そして４６は溶解性及び不溶性フラクション両方に対しておよそ等しく発現される。Ｎｉ−ＨＲＰアッセイに対するβ−ガラクトシダーゼ活性の比較を、図３に示す。点は溶解性及び不溶性タンパク質フラクションのＳＤＳゲル分析により類別される。スクリーンは、６２の溶解性タンパク質の５４（８７％）をポジティブに同定した。ゲルに従って溶解性である８の残留タンパク質のうち７が、おそらくそれらの融合タンパク質におけるＨｉｓ−ｔａｇのアクセス不能性によって、低いＮｉ−ＨＲＰアッセイを有した。単独では、β−ガラクトシダーゼ活性測定は２７の不溶性タンパク質の２２（８１％）を同定した。部分的な溶解性を示すこれらのタンパク質は、種々の溶解性スコアを表し、部分的な折りたたみがレポーターを通じてβ−ガラクトシダーゼ活性を誘導することを示している。それから、このアッセイは折りたたみ特性を分類する効果的かつ便利な手段を提供する。
第４表： Ｔ． ｍａｒｉｔｉｍａ タンパク質についての平均溶解性スクリーン値

【０１０９】
溶解性タンパク質ドメインの同定
このシステムの有用性は、改良された性質に基づき完全長の遺伝子生成物の変異体、変異又はドメインを同定する能力にある。構造又は生化学的な研究のために、Ｒｅｐ６８（ＧＩ：２０９６１７）、ターゲットＤＮＡ内へのウイルスゲノムの統合に関連する種々の活性を有するアデノアソシエイトウイルス非構造性タンパク質の溶解性フラグメントを同定するためのこのスクリーンの能力を試験した。このタンパク質は、Ｅ．ｃｏｌｉ内の折りたたまれない凝集体として優勢に発現することが、以前に見出された。我々は類似体に関してドメインの選択に基づくランダムなアプローチと合理的なアプローチの両方を行った。Ｒｅｐ６８の３種のドメインを、ＲＰＳ−ＢＬＡＳＴ調査（ｈｔｔｐ／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ｓｔｒｕｃｕｒｅ／ｃｄｄ／ｗｒｐｓｂ．ｃｇｉ）がパルボウイルス非−構造性タンパク質ＮＰ−１に対して相同性を有する内部ドメインを同定した後に選択した。Ｋｙｔｅ−Ｄｏｏｌｉｔｔｌｅ ｈｙｄｒｏｐａｔｈｙプロット（図４）と組み合わせた情報は、各ドメインについての５’ 及び３’ カットオフを整列させることに使用される。残存するＮ末端及びＣ末端残渣は他の２種のドメインを含み、そしてデータベースにおける他の任意のタンパク質に対する重要な相同性を有さない。Ｒｅｐ６８のランダムフラグメントも、ＤＮａｓｅフラグメントによりスクリーンニング用に発生させた。
【０１１０】
３種の予測されるドメイン及びランダムに発生したフラグメントを、Ｒｅｐ６８の溶解性フラグメントの同定に応答してスクリーンした。３種の予測されたドメインは溶解性として同定されたものはなかった（図４）。同時に、ｒｅｐ６８の５６４のランダムに発生したフラグメントもスクリーンした。あるフラグメントは非常に高い溶解性スコアを生じた（第４表）。このクローンは大規模な発現により証明され、そして溶解性及び不溶性フラクションの両方において発現を示した。続けて同定されたクローンのシーケンシングは、それがアミノ酸１−９５に対応するｒｅｐ６８のフラグメントからなることを証明した（図４）。この同定されたフラグメントは完全長タンパク質にわたる溶解性における実質的な改良を示し、結晶化の試みにおいて試験した。
【０１１１】
結論
インビボでのタンパク質折りたたみのほとんどの遺伝子発現の研究は、環境的なストレスの結果としての変性に焦点が当てられてきた。この応答は、常に変化する環境及び非理想的な成長条件を扱うためにインビボで必須である。翻訳の折りたたみの結果は、全てのタンパク質は翻訳されるときに必然的に折りたたまれていない状態であるので、細胞に対して等しく重要である。非天然のタンパク質の発現は、組換え手段又は変異のいずれかを通じ、それ自体においてストレスである。我々は、これらの遺伝子生成物が発生期のポリペプチドの折りたたみにどのように含まれるのかの明確な推理を用いて、ヒートショックのような翻訳の誤った折りたたみに対する細胞応答が公知のシャペロン遺伝子を含むことを示した。さらに、他のヒートショック遺伝子及び非公知の機能の遺伝子が誘導される。これらも折りたたみプロセスに含まれることができる。我々の結果は、転写及び翻訳調整の両方を提案する。ＤｎａＫ−ＲｐｏＨ相互作用は良好に特徴付けられ、転写応答の主要なレギュレーターであることを示す。翻訳ストーリングにおいて複製された遺伝子の変化した発現及びリボゾームの分裂は興味深く、そしてこれらの効果が翻訳で誤って折りたたまれたタンパク質の結果であり得ることを意味する。これらの遺伝子は、５０Ｓリボゾームサブユニットと結合するｙｒｆＨを含む（１６）。ｃｓｐＡＢＧＩは、誘導されるシャペロンの不存在下で、翻訳ストーリング（ｓｔａｌｌｉｎｇ）を提案するｒｐｏＨ６０６において誘導される。ｆｔｓＪも誘導され、それは互いについて２種のサブユニットのより高い親和力を生じる５０Ｓサブユニットの２３ＳｒＲＮＡをメチル化することが公知である（１９）。リボゾーム構造は、ｆｔｓＪのメチル化の位置、２３ＳｒＲＮＡの２５５２部分が興味深くペプチジルトランスフェラーゼ中心に対して閉じており（１８）、誤って折りたたまれたタンパク質用のリボゾームセンサー用の明確な潜在的レギュレーター機構にすることを表す。このようなリボゾームセンサーは、翻訳中にチャージされないｔＲＮＡに対して良好に特徴化された厳重な応答により例証されるように、新規なものではない（２３）。リボゾームストーリングはシャペロン合成及び補充のための時間を許容し、それにより不可逆性の凝集を妨げる機構を提供する。この方法において、細胞は、十分なシャペロンが補充されるまで翻訳リボゾームの比較的防御された環境において新たなタンパク質が折りたたまれるさらなる回収路を維持する。
【０１１２】
異なって調整された同定遺伝子は、細胞タンパク質の折りたたみ状態の新規なレポーターを、特に全体及び過剰に発現された組換えタンパク質として、提供するための価値ある機会を提供する。我々のレポーターアッセイは、ターゲットに対するレポーター遺伝子の直接的なカップリングに依存しないことにより、最近記載されたものとは異なり、それによりレポーターによる干渉を制限する。Ｎｉ−ＨＲＰ及びβ−ガラクトシダーゼアッセイの組合せは、高処理量方法で溶解性組換えタンパク質をアッセイする効果的な方法を提供する。我々は本システムを拡張し、別の誤って折りたたまれた凝集タンパク質の溶解性ドメインを同定するための方法として、単一の遺伝子生成物の変異体及びトランケーションを同定した。このアプローチを使用して、我々はＲｅｐ６８の溶解性フラグメントを同定し、そしてこのアッセイが構造／機能作用のために好適な組換えタンパク質を単離する一般的な手段を提供するものと推測した。
参考文献
１． Ｓａｕｅｒ， Ｒ． Ｔ． ＆ Ｐａｒｓｅｌｌ， Ｄ． Ａ． （１９８９） Ｇｅｎｅｓ Ｄｅｖ． ３，１２２６−１２３２．
２． Ｍｏｇｋ， Ａ．， Ｔｏｍｏｙａｓｕ， Ｔ．， Ｇｏｌｏｕｂｉｎｏｆｆ， Ｐ．， Ｒｕｄｉｇｅｒ， Ｓ．， Ｒｏｄｅｒ， Ｄ． Ｌａｎｇｅｎ， Ｈ． ＆ Ｂｕｋａｕ， Ｂ． （１９９９） ＥＭＢＯ Ｊ． １８，６９３４−６９４９．
３． Ｂｅｃｋｍａｎｎ， Ｒ． Ｐ．， Ｍｉｚｚｅｎ， Ｌ． Ａ． ＆ Ｗｅｌｃｈ， Ｗ． Ｊ． （１９９０） Ｓｃｉｅｎｃｅ ２４８，８５０−８５４．
４． Ｈａｒｔｌ， Ｆ． Ｕ． （１９９６） Ｎａｔｕｒｅ （Ｌｏｎｄｏｎ） ３８１，５７１−５８０．
５． Ｇｅｔｈｉｎｇ， Ｍ．−Ｊ． （１９９７） Ｎａｔｕｒｅ （Ｌｏｎｄｏｎ） ３８８，３２９−３３１．
６． Ｇｏｌｏｕｂｉｎｏｆｆ， Ｐ．， Ｍｏｇｋ， Ａ．， Ｚｕｉ， Ａ． Ｐ． Ｂ．， Ｔｏｍｏｙａｓｕ， Ｔ． ＆ Ｂｕｋａｕ， Ｂ． （１９９９） Ｐｒｏｃ． Ｎａｔｌ．Ａｃａｄ． Ｓｃｉ． ＵＳＡ ９６，１２７３２−１２７３７．
７． Ｌｉｂｅｒｅｋ， Ｋ． ＆ Ｇｅｏｒｇｏｐｏｕｌｏｓ， Ｃ． （１９９３） Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ９０，１１０１９− １１０２３．
８． ＭｃＣａｒｔｙ， Ｊ． Ｓ．， Ｒｕｄｉｇｅｒ， Ｓ．， Ｓｃｈｏｎｆｅｌｄ， Ｈ．−Ｊ．， Ｓｃｈｎｅｉｄｅｒ−Ｍｅｒｇｅｎｅｒ， Ｊ．， Ｎａｋａｈｉｇａｓｈｉ， Ｋ．， Ｙｕｒａ， Ｔ．， ＆ Ｂｕｋａｕ， Ｂ． （１９９６） Ｊ． Ｍｏｌ． Ｂｉｏｌ． ２５６，８２９−８３７．
９． Ｒｉｃｈｍｏｎｄ， Ｃ． Ｓ．， Ｇｌａｎｅｒ， Ｊ． Ｄ．， Ｍａｕ， Ｒ．， Ｈｏｎｇｆａｎ， Ｊ． ＆ Ｂｌａｔｔｎｅｒ， Ｆ． Ｒ． （１９９９） Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ． ２７，３８２１−３８３５．
１０． Ｍａｘｗｅｌｌ， Ｋ． Ｌ．， Ｍｉｔｔｅｒｍａｉｅｒ， Ａ． Ｋ．， Ｆｏｒｍａｎ−Ｋａｙ， Ｊ． Ｄ．， ＆ Ｄａｖｉｄｓｏｎ， Ａ． Ｒ． （１９９９） Ｐｒｏｔｅｉｎ Ｓｃｉ． ８，１９０８−１９１１．
１１． Ｗａｌｄｏ， Ｇ． Ｓ．， Ｓｔａｎｄｉｓｈ， Ｂ． Ｍ．， Ｂｅｒｅｎｄｚｅｎ， Ｊ．， ＆ Ｔｅｒｗｉｌｌｉｇｅｒ， Ｔ． Ｃ． （１９９９） Ｎａｔ．Ｂｉｏｔｅｃｈ． １７，６９１−６９５．
１２． Ｗｉｇｌｅｙ， Ｗ． Ｃ．， Ｓｔｉｄｈａｍ， Ｒ． Ｄ．， Ｓｍｉｔｈ， Ｎ． Ｍ．， Ｈｕｎｔ， Ｊ． Ｆ．， ＆ Ｔｈｏｍａｓ， Ｐ． Ｊ． （２００１） Ｎａｔ． Ｂｉｏｔｅｃｈ． １９，１３１−１３５．
１３． Ａｌｌｅｎ， Ｓ． Ｐ．， Ｐｏｌａｚｚｉ， Ｊ． ＯＬ， Ｇｉｅｒｓｅ， Ｊ． Ｋ． ＆ Ｅａｓｔｏｎ， Ａ． Ｍ． （１９９２） Ｊ． Ｂａｃｔｅｒｉｏｌ． １７４， ６９３８−６９４７．
１４． Ｔｈｏｍａｓ， Ｊ． Ｇ． ＆ Ｂａｎｅｙｘ， Ｆ． （１９９８） Ｊ． Ｂａｃｔｅｒｉｏｌ． １８０，５１６５−５１７２．
１５． Ｖｅｉｎｇｅｒ， Ｌ．， Ｄｉａｍａｎｔ， Ｓ．， Ｂｕｃｈｎｅｒ， Ｊ． ＆ Ｇｏｌｏｕｂｉｎｏｆｆ， Ｐ． （１９９８） Ｊ． ＢＩＯＬ． Ｃｈｅｍ． ２７３， １１０３２−１１０３７．
１６． Ｋｏｒｂｅｒ， Ｐ．， Ｓｔａｈｌ， Ｊ． Ｍ．， Ｎｉｅｒｈａｕｓ， Ｋ． Ｈ． ＆ Ｂａｒｄｗｅｌｌ， Ｊ． Ｃ． Ａ． （２０００） Ｅｍｂｏ Ｊ． １９， ７４１−７４８．
１７． Ｃａｌｄａｓ， Ｔ．， Ｂｉｎｅｔ， Ｅ．， Ｂｏｕｌｏｃ， Ｐ．， Ｃｏｓｔａ， Ａ．， Ｇｅｓｇｒｅｓ， Ｊ． ＆ Ｒｉｃｈａｒｍｅ， Ｇ． （２０００） Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７５，１６４１４−１６４１９．
１８． Ｐｕｇｌｉｓｉ， Ｊ． Ｄ．， Ｂｌａｎｃｈａｒｄ， Ｓ． Ｃ． ＆ Ｇｒｅｅｎ， Ｒ． （２０００） Ｎａｔ． Ｓｔｒｕｃｔ． Ｂｉｏｌ． ７，８５５−８６１．
１９． Ｃａｌｄａｓ， Ｔ．， Ｂｉｎｅｔ， Ｅ．， Ｂｏｕｌｏｃ， Ｐ． ＆ Ｒｉｃｈａｒｍｅ， Ｇ． （２０００） Ｂｉｏｃｈｅｍ． Ｂｉｏｐｈｙｓ． Ｒｅｓ． Ｃｏｍａ． ２７１，７１４−７１８．
２０． Ｗａｎｇ， Ｎ．， Ｙａｍａｎａｋｅ， Ｋ． ＆ Ｉｎｏｕｙｅ， Ｍ． （１９９９） Ｊ． Ｂａｃｔｅｒｉｏｌ． １８１，１６０３−１６０９．
２１． Ｊｉｎｇ， Ｗ．， Ｈｏｕ， Ｙ． ＆ Ｉｎｏｕｙｅ， Ｍ． （１９９７） Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． ２７２，１９６−２０２．
２２． Ｂａｅ， Ｗ．， Ｘｉａ， Ｂ．， Ｉｎｕｏｙｅ， Ｍ． ＆ Ｓｅｖｅｒｉｎｏｖ， Ｋ． （２０００） Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ ９７， ７７８４−７７８９．
２３． Ｃａｓｈｅｌ， Ｍ．， Ｇｅｎｔｒｙ， Ｄ． Ｒ．， Ｈｅｒｎａｎｄｅｚ， Ｖ． Ｊ． ＆ Ｖｉｎｅｌｌａ， Ｄ． （１９９６） Ｔｈｅ ｓｔｒｉｎｇｅｎｔ ｒｅｓｐｏｎｓｅ． ｉｎ Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ａｎｄ Ｓａｌｍｏｎｅｌｌａ Ｃｅｌｌｕｌａｒ ａｎｄ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ， ｅｄ． Ｎｅｉｄｈａｒｄｔ， Ｆ． Ｃ． （ＡＳＭ Ｐｒｅｓｓ， Ｗａｓｈｉｎｇｔｏｎ ＤＣ） ｐｐ． １４５８−１４９６．
２４． ｉｎ Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ （１９８９） ｅｄｓ． Ｓａｍｂｒｏｏｋ， Ｊ．， Ｆｒｉｔｓｃｈ， Ｅ． Ｆ． ＆ Ｍａｎｉａｔｉｓ， Ｔ．， （Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ）， ｐ． １７３５．
２５． Ｌｏｃｋｈａｒｔ， Ｄ． Ｊ．， Ｄｏｎｇ， Ｈ．， Ｂｙｒｎｅ， Ｍ． Ｃ．， Ｆｏｌｌｅｔｔｉｅ， Ｍ． Ｔ．， Ｇａｌｌｏ， Ｍ． Ｖ．， Ｃｈｅｅ， Ｍ． Ｓ．， Ｍｉｔｔｍａｎｎ， Ｍ．， Ｗａｎｇ， Ｃ．， Ｋｏｂａｙａｓｈｉ， Ｍ．， Ｈｏｒｔｏｎ， Ｈ． ＆ Ｂｒｏｗｎ， Ｅ． Ｌ． （１９９６） Ｎａｔ． Ｂｉｏｔｅｃｈｎｏｌ． １４，１６７５−１６８０．
２６． Ｗｏｄｉｃｋａ， Ｌ．， Ｄｏｎｇ， Ｈ．， Ｍｉｔｔｍａｎｎ， Ｍ．， Ｈｏ， Ｍ． Ｈ．， ＆ Ｌｏｃｋｈａｒｔ， Ｄ． Ｊ． （１９９７） Ｎａｔ． Ｂｉｏｔｅｃｈｎｏｌ． １５， １３５９−１３６７．
【０１１３】
本明細書中に記載された例及び具体例は例証のみの目的であり、それらの適切な種々の改変又は変化が当業者に提案され、そしてそれらは本出願の精神及び範囲並びに請求項の範囲内に含まれることが理解される。本明細書中に引用される全ての刊行物、特許及び特許出願は、全ての目的について参考文献として本明細書中に導入される。
【図面の簡単な説明】
【図１】図１は不溶性タンパク質の発現中に誘導される公知のヒートショック遺伝子のプロモーターを示す。
【図２】図２Ａ−Ｃは、予め測定された発現特性を有する１８ Ｔｈｅｒｍａｔｏｇａ ｍａｒｉｔｉｍａタンパク質についてのスクリーニング結果の概要を表す。
【図３】図３は、レポーター株における１８６ Ｔ．ｍａｒｉｔｉｍａタンパク質の発現後に観察される相対Ｎｉ−ＨＲＰ活性に対する相対β−ガラクトシダーゼ活性を表す。
【図４】図４は、第二構造予測のアライメント、並びにＲｅｐ６８の予測された及び同定されたドメインの両方を表す。[0001]
Background of the invention
Field of the invention
The present invention relates to drug discovery and, in particular, to compositions and methods that facilitate the drug discovery process.
[0002]
background
While genetic engineering techniques have provided the ability to modulate the expression of virtually any protein-encoding polynucleotide in selected cells, the key manipulation of protein production in genetically modified cells has been limited. It is observed that it often leads to the formation of properly folded and biologically inactive protein molecules. Often, these mis-folded proteins form insoluble protein aggregates in the cytoplasm of cells. Whether the purpose of manipulation of target protein expression is to alter the phenotype of the cell, to provide a source of biologically active protein or a protein suitable for structural analysis, these insoluble Aggregates are biologically inert, difficult to purify, and difficult to refold into active sequences.
[0003]
Biosynthesis of a functional protein molecule occurs through translation of a polypeptide-encoding messenger RNA molecule. The nascent polypeptide chain folds into a three-dimensional molecule. The ability of a protein to fold into a biologically active sequence is determined by the particular amino acid sequence of the protein and the intracellular conditions during which the protein is produced. In addition, accessory proteins called chaperones have been discovered and can be involved in the process of protein biosynthesis and help to form properly folded protein molecules.
[0004]
Maxwell et al. (1999) Protein Science 8: 1908-1911 describes a fusion protein structure useful for improving the solubility of several insoluble protein targets. However, this approach was limited in its usefulness by relying on fusion of a polynucleotide encoding the chloramphenicol acetyltransferase protein to the gene of interest. Therefore, there is a need for a technique for monitoring or controlling the folding of a target protein into genetically modified cells.
[0005]
Recent advances in understanding heat shock response proteins (Hsp) indicate that there is a link between these certain proteins and protein folding. It is known that cells subjected to elevated temperatures respond by inducing expression of a set of genes known as heat shock genes. The proteins encoded by these genes, heat shock proteins, provide functions to help control the deleterious effects of elevated temperatures and include chaperones and protease molecules. Heat shock responses have been studied in detail in both prokaryotic and eukaryotic systems and are highly maintained throughout development. A detailed analysis of the genes induced by heat shock is described in A set of genes performed in the E. coli genome and induced by this stimulus was identified (Richmond et al. (1999) Nicoleic Acids Res. 27 (19): 3821-3835). The molecular basis of this response is described in E. E. coli (Liberek and Georgopoulos (1993) Proc. Nat'l. Acad. Sci. USA 90: 11019-11023; and McCarty et al. (1996) J. Mol. Biol. 256: 829-837). Altering pH, hypoxia (Lindquist (1986) Ann. Rev. Biochem. 55: 1151-1191) or another stressful such as insoluble proteins (Parcell and Sauer (1989) Genes and Development 3: 1226-1232). It has now been shown that stimulation also induces heat shock protein induction. Studies of alternative conditions for inducing heat shock genes indicate that a common set of genes is induced by various stressful stimuli. Thus, by generating a common set of proteins, including chaperones and protease molecules, cells respond to a wide range of stressful conditions and these proteins function to mitigate the deleterious effects of adverse conditions. (See, for example, Parsell et al. (1994) Nature 372 (6505): 475-478).
[0006]
Various efforts have been made to gain the benefit of heat shock response genes to monitor and protect against toxic conditions. For example, Farr (US Pat. No. 5,589,337) describes a method that utilizes a stress responsive promoter fused to a gene that encodes an assayable product and characterizes and quantifies the toxicity of the compound. In addition, Lindquist (US Pat. No. 5,827,685) describes the use of the yeast Hsp104 promoter and genes that protect cells against potential toxic stress factors such as heat, alcohol and heavy metals. These methods. Are useful for monitoring certain toxic stimuli, but they do not provide a convenient means of measuring or improving the solubility of a range of protein products in cells. There is a need for methods and reagents for measuring and improving the solubility of A. The present invention fulfills these and other needs.
[0007]
Summary of the Invention
The present invention provides methods for determining whether cells, reagents and host cells express a polypeptide of interest, either in soluble or insoluble form. In certain embodiments, the present invention provides: a) a soluble reporter nucleic acid comprising a protein solubility responsive promoter operably linked to a reporter gene; and b) a target polypeptide-expressing nucleic acid comprising a polynucleotide expressing the target polypeptide; A host cell comprising: Expression of the target polypeptide in an insoluble form causes a change in the expression of the reporter gene. In some embodiments of the invention, the solubility responsive promoter is up-regulated when the target polypeptide is expressed in an insoluble form; in other embodiments, the lytic promoter is lysed when the target polypeptide is expressed in an insoluble form. Sex-responsive promoters are down-regulated. Also provided are arrays of two or more sets of such host cells; the host cells of each population differ in the target polypeptide expressed by the host cells.
[0008]
The present invention also provides a method for measuring the solubility of a target polypeptide. These methods comprise a host cell comprising: a) a soluble reporter nucleic acid comprising a protein solubility responsive promoter operably linked to a reporter gene; and b) a target polypeptide-expressing nucleic acid comprising a polynucleotide expressing the target polypeptide. Is cultured under conditions in which the target polypeptide is expressed. The solubility of the expressed target polypeptide is then measured by detecting whether the reporter gene increases or decreases.
[0009]
A further embodiment of the invention provides a method for identifying a variant in a cell that alters the solubility of a target polypeptide. These methods include: a) treating cells with a mutagen; b) a lytic reporter nucleic acid comprising a proteolytic response promoter operably linked to a reporter gene, as well as a polynucleotide encoding a target polypeptide. Introducing the target polypeptide-expressing nucleic acid into the cell; c) culturing the cell under conditions favorable for the expression of the target polypeptide; d) measuring the expression of the reporter gene; e) the expression level of the reporter gene in the cell Comparing the levels observed in non-mutant cells that also contain the soluble reporter nucleic acid and the target polypeptide-expressing nucleic acid to identify cells that contain the mutant that alters the solubility of the target polypeptide.
[0010]
In another embodiment, the invention provides a method of identifying a change to a polynucleotide encoding a target polypeptide that alters the solubility of the target polypeptide. These methods include: a) altering the polynucleotide encoding the target polypeptide to form the altered polynucleotide; b) lysis in the cell comprising a proteolytic response promoter operably linked to a reporter gene. Introducing a target polypeptide-expressing nucleic acid comprising the sex reporter nucleic acid as well as the altered polynucleotide; c) culturing the cells under conditions that favor expression of the target polypeptide; d) measuring the expression of the reporter gene; e) the reporter Compare gene expression levels to levels observed in cells with unaltered polynucleotides encoding the target polypeptide to identify alterations in the polynucleotide that alter the solubility of the encoded target polypeptide Include;
[0011]
The invention also provides a method of identifying a variation in a process for biosynthesis of a target polypeptide that alters the solubility of the target polypeptide. These methods include culturing the host cell under another condition under which the target polypeptide is expressed. The host cell comprises: a) a soluble reporter nucleic acid comprising a protein solubility responsive promoter operably linked to a reporter gene; and b) a target polypeptide-expressing nucleic acid comprising a polynucleotide encoding the target polypeptide. The expression of the reporter gene by the host cells, each grown under different conditions, is then compared to determine the conditions that produce the desired level of target polypeptide solubility.
[0012]
The invention also provides a method of screening an expression library to identify library members that express a soluble target polypeptide. These methods include: a) a plurality of host vectors containing a soluble reporter nucleic acid comprising a protein solubility responsive promoter operably linking a plurality of expression vectors, each comprising a polynucleotide encoding a target polynucleotide, to a reporter gene. B) culturing host cells under conditions in which the target polypeptide is expressed; and c) detecting expression of the reporter gene, thereby dissolving the soluble target polypeptide. Identifying a library member that expresses
[0013]
The present invention also provides a method for identifying an antibiotic. The method comprises: a) contacting a cell containing a soluble reporter nucleic acid comprising a protein solubility responsive promoter operably linked to a reporter gene with a candidate antibiotic reagent; detecting the expression level of the reporter gene. . Changes in reporter gene expression levels in cells that come into contact with the candidate antibiotic reagent, compared to reporter gene expression levels in cells that do not come into contact with the candidate antibiotic reagent, indicate a reagent that suppresses protein folding in the cell I do.
[0014]
The invention also provides a polynucleotide comprising a proteolytic response promoter operably linked to a polynucleotide encoding a detectable or selectable product. The polynucleotide further includes an expression structure for the target protein. The present invention also provides a soluble reporter system having these soluble reporter polynucleotides together with an expression structure for a target protein. The invention also provides a gene delivery vehicle and a genetically modified cell comprising an expression vector and a host or at least a polynucleotide and a gene reporter system of the invention.
[0015]
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the promoter of a known heat shock gene induced during the expression of insoluble proteins. Nucleotide sequences are manually aligned, allowing one gap in the sequence. The sequences are listed at the reduced induction level of the most highly induced member of the operon. The promoter of the non-heat shock gene caused by translation misfolding is represented at the bottom of the figure. Nucleotides retained within the RpoH recognition sequence are represented by gray shades
[0016]
FIGS. 2A-C outline the results of screening for 18 Thermotoga maritima proteins with pre-measured expression characteristics. Average relative β-galactosidase activity (FIG. 2A), Ni-HRP activity (FIG. 2B) and 18T. The resulting solubility score for the maritima protein (FIG. 2C) is represented. Expression characteristics for 18 proteins were previously determined by SDS-PAGE of both soluble and insoluble fractions.
[0017]
FIG. 3 shows 186 T.D. in the reporter strain. Figure 3 shows the relative β-galactosidase activity relative to the relative Ni-HRP activity observed after expression of the maritima protein. Classification of each protein as soluble, insoluble or mixed is based on SDS-PAGE performed on soluble and insoluble isolates after screening.
[0018]
FIG. 4 depicts the alignment of the second structure prediction, as well as both the predicted and identified domains of Rep68. Shown are Chou-Fasman second structure predictions of α-helical and β-sheet structures aligned with a hydrophobic Kyte-Doolittle plot based on the first sequence of Rep68. Aligned below: the full-length Rep68 protein, three predicted domains of Rep68, and a block representing the relative size and location of the Rep68 domain identified by screening for randomly generated fragments of the rep68 gene. It is. The solubility score for the protein is shown.
[0019]
Detailed description
Definition
Throughout this disclosure, various publications, patents and issued patent specifications are referenced by an identifying citation. These publications, patents and published patent specifications are incorporated by reference into this disclosure and fully describe the state of the art to which these inventions pertain.
[0020]
The practice of the present invention will employ, unless otherwise indicated, conventional molecular biology techniques (including recombinant techniques), microbiology, cell biology, biochemistry and immunology which are within the skill of the art. . Such techniques are explained fully in the literature. These methods are described in the following publications: For example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3rd edition (2001); A PRACTICAL APPROACH (M. MacPherson et al., IRL Press at Oxford University Press (1991)); PCR2: A PRACTICAL APPROACH (M. MacPherson, BD D.H.A.I.N.T. , A LABORATORY MANUAL (Harlow and Lane eds. (1988)); and ANIMAL CELL CULTURE (R.I.Freshney ed (1987)), incorporated herein by reference.
[0021]
As used herein, certain terms have the meanings defined below.
[0022]
As used herein and in the claims, the singular forms "a" and "the" include plural referents unless the meaning is explicitly stated otherwise. For example, the term “a cell” includes a plurality of cells and includes a mixture thereof.
[0023]
The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably and refer to a polymeric form of nucleotides of any length. Polynucleotides can include deoxyribonucleotides, ribonucleotides and / or their congeners. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The term "polynucleotide" refers to, for example, single-double stranded and triple helical molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, Vector, any sequence of isolated DNA, any sequence of isolated RNA, nucleic acid probes, and primers. Nucleic acid molecules can also include modified nucleic acid molecules.
[0024]
The term "peptide" is used in a very broad sense and refers to a compound of two or more subunit amino acids, homologs of amino acids, peptidomimetics. Subunits can be linked to peptide bonds. In other embodiments, the subunits can be linked by other bonds, such as, for example, esters, ethers, and the like. As used herein, "amino acid" means a natural and / or unnatural or synthetic amino acid, including both glycine and D or L optical isomers, and amino acid homologs or peptidomimetics. . If the peptide chain is short, a peptide of three or more amino acids is commonly called an oligopeptide. If the peptide chain is long (eg, longer than about 10-20 amino acids), the peptide is commonly called a polypeptide or protein.
[0025]
"Genetically modified" means including and / or expressing an external gene or nucleic acid sequence that in turn modifies the genotype or phenotype or progeny of the cell. In other words, it means any addition, deletion or division of the cell's endogenous polynucleotide. The term "heterologous" also refers to polynucleotides or polypeptides that are not naturally associated with a particular cell or cell component. For example, a promoter that is heterologous to a particular host cell cannot be found in that type of naturally occurring cell. Similarly, a heterologous promoter for a particular protein-encoding polynucleotide is not found attached to the particular polynucleotide in naturally occurring cells. The term "recombination" is often used to refer to nucleic acids containing unrelated polynucleotides in cells that have been unmodified by recombinant methods.
[0026]
As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and translated into a peptide, polypeptide or protein. If the polynucleotide is carried from genomic DNA, expression involves mRNA splicing, if a suitable eukaryotic host is selected. The regulatory elements required for expression include a promoter sequence for binding RNA polymerase and a translation initiation sequence for ribosome binding. For example, bacterial expression vectors include a promoter, such as the lac promoter, and include a Shine-Dalgarno sequence and an initiation codon, ATG, for initiation of transcription and translation (Sambrook et al., Supra (2001)). Similarly, eukaryotic expression vectors include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, an initiation codon AUG, and a termination codon for separation of ribosomes. Such vectors are commercially available and may be collected by the sequences described by methods known in the art, for example, the following methods that make up conventional vectors.
[0027]
A "promoter" is a region in a DNA molecule to which RNA polymerase binds and initiates transcription. The nucleotide sequence of the promoter determines the nature of the attached enzyme and the rate of RINA synthesis. In the present disclosure, the term “promoter” refers to a poly-protein that includes not only an RNA polymerase binding site, but all other flanking sequence elements that interact with factors that modulate transcription initiation, such as inducers or repressors of transcription. Used to mean nucleotide. Thus, a "promoter", as defined herein, is a polynucleotide that contains all of the necessary sequence information to regulate gene expression in the same manner as native elements in the chromosome.
[0028]
The term "proteolytic response promoter" refers to a promoter element that is induced or repressed in cells in response to increasing concentrations of insoluble protein in the cytoplasm.
[0029]
"Under transcription control" is a term well understood in the art, and relies upon the operable linking of transcription of a polynucleotide sequence, usually a DNA sequence, to elements that contribute to the initiation or promotion of transcription. To do so. "Operably linked" means a nearby location in the arrangement for functioning the element.
[0030]
The term "expression structure" refers to a polynucleotide operably linked to a gene. The expression construct can be formatted in various ways, such as in a gene delivery vehicle, and inserted into the chromosome of the cell. The term includes promoter genes produced by any method, including but not limited to recombinant DNA technology, homologous recombination, targeted insertion of genes or promoter elements, or random insertion of genes or promoter elements. It is intended to mean fusion.
[0031]
"Gene delivery vehicle" is defined as any molecule capable of carrying a polynucleotide inserted into a host cell. Examples of gene delivery vehicles include liposomes, biocompatible polymers including natural and synthetic polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial virus envelopes, metal particles, and baculoviruses, adenoviruses and retroviruses. Such bacteria, viruses, bacteriophages, cosmids, plasmids, fungal vectors, and techniques typically used in the techniques described for expression in various eukaryotic and prokaryotic hosts, and genes as well as for simple protein expression Another recombinant vehicle that is also used for therapy.
[0032]
As used herein, "gene delivery", "gene delivery" and the like are terms that refer to the introduction of an exogenous polynucleotide into a host cell, regardless of the method used for the introduction (often referred to as "gene delivery"). Called "transgene"). Such methods are similar to techniques that facilitate the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery and various other techniques used to introduce polynucleotides). Various known techniques such as vector-mediated gene delivery (eg, by viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes). The introduced polynucleotide can be stably or temporarily maintained in the host cell. Stable maintenance means that the introduced polynucleotide contains an origin of replication homologous to the host cell or integrates into a host cell replicon such as an extrachromosomal replicon (eg, a plasmid) or a nuclear or mitochondrial chromosome. Need that. Numerous vectors are known to be capable of mediating the transfer of genes to mammalian cells, as known or described herein.
[0033]
“Viral vector” is defined as a recombinantly produced virus or viral particle containing a polynucleotide that is delivered to a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and the like. In embodiments where gene delivery is mediated by a retroviral vector, the vector structure refers to a polynucleotide comprising the retroviral genome or a portion thereof and a therapeutic gene. As used herein, “retroviral-mediated gene transfer” or “retroviral gene transfer” has the same meaning, and a gene or nucleic acid sequence enters a cell and integrates its genome into the host cell genome. Refers to the process by which the virus stably transfers it into host cells. The virus can enter the host cell via its normal mechanism of infection, and can be modified to bind to a different host cell surface receptor or ligand and enter the cell. As used herein, a retroviral vector refers to a virus particle capable of introducing an exogenous nucleic acid into a cell through a virus or virus-like transfer mechanism.
[0034]
Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse transcribed into a DNA form that is integrated into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.
[0035]
In embodiments where gene delivery is mediated by a DNA viral vector such as adenovirus (Ad) or adenoassociated virus (AAV), the vector structure refers to the polynucleotide comprising the viral genome or portions thereof as well as the transgene. Adenoviruses (Ads) are a relatively well-characterized, homogeneous group of viruses, containing over 50 serotypes. See, for example, WO 95/27071. Ads are easy to grow and do not require integration into the host cell genome. Recombinant Ad-derived vectors, particularly those that reduce the potential for recombination and development of wild-type virus, have also been constructed. See WO 95/00655 and WO 95/11984. Wild-type AAV has a high degree of infectivity and specificity that integrates into the genome of the host cell. Hermonat and Muzyczka (1984) Proc. Nat'l. Acad. Sci. USA 81: 6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8: 3988-3996.
[0036]
Vectors containing both a promoter and a cloning site to which the polynucleotide can be operably linked are known in the art. Such vectors are capable of transcribing RNA in vitro and in vivo, and are commercially available from sources such as, for example, Stratagene (La Jolla, Ca) and Promega Biotech (Madison, WI). To optimize expression and / or in vitro transcription, the 5 'and / or 3' untranslated portions of the clone are removed, added or altered, and extra, potential, either at the level of transcription or translation. Any other inappropriate translation initiation codons or other sequences that may prevent or reduce expression can be removed. Alternatively, a matching ribosome binding site can be inserted immediately 5 'of the start codon to amplify expression.
[0037]
Gene delivery vehicles also include various non-viral vectors, including DNA / liposome complexes, and targeted viral protein DNA complexes. Liposomes that also contain a targeting antibody or fragment thereof can be used in the methods of the invention. To amplify delivery to cells, the nucleic acids or proteins of the invention can be conjugated to antibodies or binding fragments thereof that bind to a cell surface antigen, eg, TCR, CD3 or CD4.
[0038]
As used herein, a "reporter gene" is a polynucleotide that encodes a protein whose expression by a cell is detected and quantified. Therefore, measurement of the reporter expression level indicates the level of activation of the promoter element that directs reporter gene expression. Such detection includes selection for reporter gene expression, for example, by placing cells containing the reporter gene under selective conditions.
[0039]
“Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between bases of nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific manner. The complex can include double strands forming a duplex structure, three or more strands forming a multi-stranded complex, or a combination thereof. Hybridization reactions can continue with steps in a wider range of processes, such as initiating a PCR reaction or enzymatic cleavage of a polynucleotide by a ribozyme.
[0040]
Examples of stringent hybridization conditions include: an incubation temperature of about 25 ° C. to about 37 ° C .; a hybridization buffer concentration of about 6 × SSC to about 10 × SSC; a formaldehyde concentration of about 0% to about 25%; and a wash concentration of about 6 × SSC. Including. Examples of moderate hybridization conditions include: an incubation temperature of about 40 ° C. to about 50 ° C .; a buffer concentration of about 9 × SSC to about 2 × SSC; a formaldehyde concentration of about 30% to about 50%; and a wash concentration of about 5 × SSC to about 2 × SSC; including. Examples of highly stringent hybridization conditions include: an incubation temperature of about 55 ° C. to about 68 ° C .; a formaldehyde concentration of about 55% to about 75%; and a wash concentration of about 1 × SSC to about 0.1 × SSC or deionized water. Including. Generally, hybridization incubation times are from 5 minutes to 24 hours, with one, two or more washing steps, and with a wash incubation time of about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalent SSCs using other buffer systems can be used.
[0041]
A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a predetermined percentage (eg, 80%, 85%, 90% or 95%) of “sequence identity” to another sequence; That means that the bases (or amino acids) when aligned are the same when comparing the two sequences. This alignment and percent homology or sequence identity are described, for example, in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., Eds., 1987) 30th Edition Section 7.7.18, Table 7.7.1. As such, it can be measured using software programs known in the art. A preferred program for sequencing polynucleotides or polynucleotide sequences and measuring percent homology is CLUSRALW, which uses the default parameter. This program, Institute for Biological Computing at Washington University in Saint Louis, MO (www, ibc.wustl.edulmsalclustal.html), Human Genome Sequencing Center of the Baylor College of Medicine in Houston, TX (dot.imgen.bcm.tmc .Edu: 9331 / milti-align / multi-align.html) and the Pasteur Institute in Paris, France (bioweb.pasteur.fr/sequalal/interfaces/classura). Available in a variety of sites on the World Wide Web, such as w-simple.html).
[0042]
A "biologically equivalent" of a reference polynucleotide is at least 75%, or at least 80%, or at least 85%, or at least, as measured using a sequence alignment program under a defect parameter. It is characterized by having 90% or at least 95% sequence identity and corrects for ambiguity in sequence data and changes in nucleotide sequences that do not change function. "Biologically equivalent" polynucleotides can be isolated by hybridization under appropriate or stringent hybridization conditions. In addition to sequence similarity or hybridization with a reference polynucleotide, a biologically equivalent polynucleotide has the same or similar biological function as the reference polynucleotide.
[0043]
Various software programs are available in the art to identify bioequivalent polynucleotides without undue experimentation. Non-limiting examples of these programs include the BLAST family programs including BLASTN, BLASTP, BLASTX, TBLASTN and TBLASTX (BLAST is available from the World Wide Web at http://www.ncbi.nlm.nih.gov/BLASTI). Available), FastA, Compare, DotPlot, BestFit, GAP, FrameAlign, ClustalW and PileUp. These programs are available from GCG Inc. It is commercially available in a comprehensive package of sequence analysis software such as' s Wisconsin. Other similar analysis and alignment programs can be purchased from various providers, such as DNA Star's MegAlign, or the alignment program at GeneJockey. Alternatively, the sequence analysis and alignment program is available at www. sdsc. edu / ResTools / cmshp. at html, accessible through the World Wide Web at sites like CMS Molecular Biology Resources. Any sequence database containing protein sequences that match DNA or genes or segments thereof can be useful for sequence analysis. Commonly used databases include, but are not limited to, GenBank, EMBL, DDBJ, PDB, SWISS-PROT, EST, STS, GSS and HTGS. Sequence similarity can be recognized by aligning the tag sequence against a DNA sequence database. Alternatively, the tag sequence can be translated into six reading frames; the predicted peptide sequence for all possible reading frames is then stored in a protein database such as s done using the BLASTX program. Are compared to the individual sequences performed.
[0044]
Parameters for assuming the degree of similarity exhibited by one or more of the above alignment programs are well established in the art. They include, but are not limited to, p-values, percent sequence identity and percent sequence similarity. The P value is the probability that an alignment will be generated by accident. For a single alignment, the p-value is determined by Karlin et al. (1990) Proc. Nat'l. Acad. Sci. USA 87: 2246. For multiple alignments, it can be calculated using a functional approach such as that programmed in BLAST. Percent sequence identity is defined by the ratio of the number of nucleotides and amino acids that match between the sequence in question and the known sequence when the two are optimally sequenced. When calculating percent similarity, percent sequence similarity is calculated in the same way as percent identity, except that amino acids that are different but similar to the positive are scored.
[0045]
As used herein, "in vivo" gene delivery, gene delivery, gene therapy, etc., involves introducing a vector comprising an exogenous polynucleotide directly into the body of an organism, such as a human or non-human mammal. , Whereby exogenous polynucleotides are introduced into such organisms in vivo.
[0046]
The term “isolated” means separated from components, cells and the like, and usually involves polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof. For example, with respect to a polynucleotide, an isolated polynucleotide is one that is normally separated from the 5 'and 3' sequences that associate on the chromosome. As will be apparent to one of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof need not be "isolated" and distinguished from its naturally occurring counterpart. In addition, "enriched", "isolated" or "diluted" polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof are more concentrated or concentrated per volume than naturally occurring controls. Greater than "isolated" or less than "isolated" than the naturally occurring control. A polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof that differs from a naturally occurring control in the primary sequence or, for example, by a glycosylation pattern, need not exist in an isolated form because it This is because they can be distinguished from generated controls by their primary sequence, or alternatively by other properties, such as glycosylation patterns. Although each invention disclosed in this specification is not explicitly described, it is to be understood that specific examples of each configuration disclosed below and under appropriate conditions are provided by the invention. Would. Thus, a non-naturally occurring polynucleotide is provided as a separate embodiment from an isolated naturally occurring polynucleotide. Proteins produced in bacterial cells are provided as a separate embodiment from naturally occurring proteins that are isolated from naturally occurring eukaryotic cells.
[0047]
A “host cell” or “genetically modified cell” is any individual cell or cell culture that is or has a receptor for a vector or alternatively an exogenous nucleic acid molecule, polynucleotide and / or protein introduction. It is intended to include things. It is intended to include unicellular progeny, and the progeny may, due to natural, accidental or deliberate mutations, be associated with the original parent cell (morphologically or genetically or by total DNA) It need not be exactly the same (in the completion). The cells can be prokaryotic or eukaryotic cells and include, but are not limited to, bacterial cells, yeast cells, animal cells, and mammalian cells such as mice, rats, simians or humans.
[0048]
A “subject” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, simians, humans, farm animals, sports animals and pets.
[0049]
A “control” is an alternative subject or sample used in an experiment for comparison purposes. “Control” can be “positive” or “negative”. For example, if the purpose of the experiment is to determine the correlation of the altered expression level of a gene with a particular type of cancer, it will generally be a positive control (providing such alterations and characterizing the disease). It is preferred to use a subject or a sample from a subject that exhibits a pathological condition) and a negative control (a subject or sample from a subject that lacks altered expression and a significant condition of the disease).
[0050]
The term “culture” refers to the in vitro growth of cells or tissues on or in a wide variety of media. It is understood that the progeny of the cells grown in culture will not be completely identical (morphologically, genetically or in a prototypic manner) to the parent cell. "Expanded" means any growth or division of a cell. "Composition" means an inert (eg, a detectable reagent or label) or combination of active agents, such as active agents and other compounds and compositions, adjuvants.
[0051]
"Pharmaceutical composition" is intended to include a combination of active agents with an inert or active carrier that renders the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
[0052]
As used herein, the term "pharmaceutically acceptable carrier" refers to a phosphate buffered sarin solution, water, and an emulsion such as a water / oil or oil / water emulsion. And any standard pharmaceutical carrier, including various types of wetting agents. The composition can also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin REMINGTON 'S PHARM. SCI. , 15th edition (Mack Publ. Co., Easton (1975)).
[0053]
An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered by one or more administrations, applications or dosages.
[0054]
“Solid growth medium” is a growth medium suitable for culturing organisms, containing agar at a sufficient concentration and providing a solid surface for the purpose of culturing a culture for a clonal population of cells. It is.
[0055]
An "indicator dye" is a chemical that reacts with a reporter gene to produce a compound with changing properties that is easily assayed. An example of a suitable indicator dye is the gene product of the X-gal, lacZ reporter, which reacts with β-galactosidase, producing a blue precipitate.
[0056]
Description of the preferred embodiment
The present invention provides a soluble reporter gene structure that allows one to immediately distinguish whether a protein is produced by a cell in a soluble or insoluble form. A reporter host cell for use in identifying a protein or protein domain produced in a soluble form is also provided, as is a method for measuring the protein solubility state in a cell. In a further embodiment, the invention provides a high-throughput method for determining the solubility of a target protein expressed in a cell.
[0057]
Soluble reporter gene structure
The present invention provides a host cell comprising a soluble reporter structure comprising a promoter which is induced or suppressed depending on whether or not an insoluble protein is present in the cell containing the promoter. These proteolytic responsive promoters are preferably operably linked to a polynucleotide encoding a gene product that is readily detectable when expressed in a cell. When a soluble reporter gene structure is present in a cell that includes a promoter that is up-regulated by the insoluble protein, for example, the presence of the insoluble protein results in an increase in the level of the reporter gene product.
[0058]
To identify a promoter suitable for use in a particular species, gene expression profiles from cells of the species that express the protein known to be expressed in an insoluble form can be compared to cells that do not express the insoluble protein . For example, control cells can express a protein found in a soluble form. Once one or more genes are differentially expressed, depending on whether insoluble or soluble proteins are expressed, the upstream regions of the genes are cloned and used to construct soluble reporter structures. it can. The length of the polynucleotide comprising the upstream region often varies depending on the particular gene and / or species. Once the upstream region has been cloned, its function can be immediately tested by operably linking the upstream region to a reporter structural gene, introducing the structure into host cells and expressing it in an insoluble form To express a protein known to do so. Promoter sequences that respond to misfolded proteins can be identified by, for example, Affymetrix GeneChip®, cDNA arrays, reporter screening, and other approaches known to those of skill in the art.
[0059]
The proteolytic response promoter can be a prokaryotic or eukaryotic promoter. A promoter is utilized that is functional in the particular host cell of interest. For example, for the use of bacterial host cells, proteolytic response promoters can be isolated from Gram negative or Gram positive bacteria. Such Gram-negative bacteria include, for example, members of the familial Enterobacteriaceae family. Examples of members of the family Enterobacteriaceae are Escherichia, Salmonella, Shigella, Klebsiella, or Enterobacteriaceae. Suitable prokaryotes include, but are not limited to, Salmonella typhomurium, Bacillus subtilis and Streptomyces Livedans. One suitable species is the Gram negative bacterium E. coli. It is a promoter element isolated from E. coli. Suitable E. Specific examples of E. coli promoters are, for example, promoters from the following genes: kgtP gene (b2587; SEQ ID NO: 1), gene b3913 (SEQ ID NO: 2), proP (b4111; SEQ ID NO: 3). ExbB (b3006; SEQ ID NO: 4), yegG (b2812; SEQ ID NO: 5), yojH (b2210; SEQ ID NO: 6), ybeD (b0631; SEQ ID NO: 7), yciS (b1279; SEQ) ID NO: 8), yagU (b0287; SEQ ID NO: 9), ftsJ (b3179; SEQ ID NO: 10), grpE (b2614; SEQ ID NO: 11), htpX (b1829; SEQ ID NO: 12), clpB (b2592 SEQ ID NO: 13), fxsA (b4140; SEQ ID NO: 14), hslV (b3932; SEQ ID NO: 15), clpP (b0437; SEQ ID NO: 16), htpG (b0473; SEQ ID NO: 17) , DnaK (b0014; SEQ ID NO: 18), yccV (b0966; SEQ ID NO: 19), yrfG (b3399; SEQ ID NO: 20), ibpA (b3687; SEQ ID NO: 21), and yhdN (b3293; SEQ ID NO: 22).
[0060]
In some embodiments, the proteolytic response promoter comprises an RpoH recognition site. An example of such a promoter is represented in FIG. 1 as SEQ ID NOs 23-43.
[0061]
The present invention relates to Seq. ID. Nos. Also includes the use of polynucleotides that are biologically equivalent to the sequences provided in 1-43, which are identified by sequence homolog search or hybridization under mild or stringent hybridization conditions as described above. Various embodiments of bioequivalent polynucleotides may be at least 75% as measured using, for example, a sequence alignment program under defect parameters that calibrate for ambiguities in the sequence data and changes in nucleotide sequence that do not alter function. % Or at least 80% or at least 85% or at least 90% or at least 95% within the scope of the invention characterized by processing with sequence homology. Bioequivalent means that Seq. ID. Nos. It hybridizes to those that hybridize to 1-43 or their respective complements. Such polynucleotides are tested according to the methods of the invention and identify those that exhibit a desired proteolytic response.
[0062]
For use in eukaryotic cells, proteolytic responsive promoters are usually derived from eukaryotic genes. Many eukaryotic heat shock and other stress-inducing genes are known to those skilled in the art.
[0063]
The present invention provides methods for testing promoters from these or other genes and determining whether they are differentially regulated in response to the presence of insoluble proteins in the cell. These methods include culturing host cells that contain a soluble reporter nucleic acid that includes a putative protein solubility responsive promoter operably linked to a reporter gene. The host cell also contains a target polypeptide-expressing nucleic acid, including a polynucleotide encoding the target polypeptide. The host cells are cultured under conditions in which the target polypeptide is expressed in an insoluble form. The level of reporter gene expression is then detected to determine whether the putative protein solubility responsive promoter is differentially regulated in response to expression of the insoluble polypeptide in the host cell.
[0064]
Suitable eukaryotic cells include, for example, mammalian, insect or plant cells, or microorganisms such as, for example, yeast or fungal cells. Examples of suitable cells include, for example, Azotobacter species (eg, A. vinelandii), Pseudomonas species, Rhizobium species, Erwinia species, Escherichia species (eg, E. coli), and Klebsiella species, among others. Yeast cells can be any of a number of types, including Saccharomyces (eg, S. cerevisiae), Candida (eg, C. utilis, C. parapsilosis, C. krusei, C. versatilis, C. lipolytica, C. zeylanoides, C. guilliermondii, C, albicans and C. humicola), Pichia (eg, P. farinosa and P. ohmeri), Torulopsis (eg, T. candida, T. sphaerica, T. sateria, T. sp. Versatilis), Debaryomyces (eg, D. subglobossus, D. cantarellii, lobousus, D. hansenii and D. japonicus), Zygosaccharomyces (for example, Z. rouxii and Z. bailii), Kluyveromyces (for example, K. marxianus), Hansenulas (for example, H. anomalineb. Anomalus). Further non-limiting examples of suitable eukaryotic cells include Jurkat cells and NIH3T3 cells.
[0065]
The identified protein solubility responsive promoter is operably linked to a reporter gene that functions to identify the presence or absence of the soluble protein in the cytoplasm. Reporter genes include polynucleotides that encode a selectable or detectable polypeptide. Examples of genes useful as "reporter genes" include, but are not limited to, genes encoding metabolic enzymes, antibody resistance factors, photoproteins (eg, luciferase), or fluorescent proteins. Such reporter genes are known in the art, and specific examples are described in Wood (1995) Curr. Opin. Biotechnol. 6 (1): 50-58. In some embodiments, the metabolic enzyme is β-galactosidase. In other embodiments, the metabolic gene complements auxotrophic mutations in the host cell and allows the growth of cells expressing the gene on a selective medium.
[0066]
Methods for detecting and quantifying reporter expression are generally based on measuring the activity of the protein encoded by the reporter. A wide range of detectable markers are known in the art and include fluorescent, radioactive, enzymatic or other ligands such as avidin / biotin, which can provide a detectable signal. In a preferred embodiment, it is desirable to use a fluorescent label or an enzyme tag, such as urease, alkaline phosphate, peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of an enzyme tag, the colorimetric indicator substrate can be used to provide a means that is visible to the human eye or spectrophotometrically, and that a particular nucleic acid-containing sample can be used for specific hybridization. It is known to identify.
[0067]
If the reporter is an enzyme, a substrate for the enzyme that can be metabolized to produce a measurable product can be used. For example, β-galactosidase substrate X-gal, which is cleaved by an enzyme to produce a blue reactive product, is frequently used to assay β-galactosidase reporter expression (Miller J. ed. (1992)). A short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escheichia Coli and Related Bacteria, Cold Spring Harbor Laboratory, Colorado Harbor Laboratory, Canada. Instead, β-galactosidase substrate o-nitrophyl-BD-galactopyranoside (ONPG), which is metabolized by β-galactosidase, produces a compound having a yellow color. The amount of enzyme is measured by spectrophotometrically measuring the optical density of the colored compound or using an ELISA reader. The absorbance at 420 nm is read (Miller J. H. ed. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Other commonly used reporter genes are the antibody resistance factor chloramphenicol acetyltransferase (CAT), firefly luciferase gene, and the jellyfish green fluorescent protein (Valdvia and Falkow (1997) Trends Microbiol. 5). (9): 360-363; Naylor (1999) Biochem. Pharmacol. 58 (5): 749-757; Himes and Shannon (2000) Methods Mol. Biol. 130: 165-174). In addition, various other proteins can also be used as reporters based on their ability to be detected and quantified. Assays for measuring the expression levels of such genes are well-modified and commonly performed by those skilled in the art (Rosenthal (1987) Methods Enzymology 152: 704-720; Davey et al. (1995) Method Mol. Biol. 49: 143-148; and Bronstein et al. (1994) Anal. Biochem. 219 (2): 169-181).
[0068]
Polynucleotides encoding useful reporter genes are available from Life Technologies Inc. (Gaithesburg, MD), Clontech Inc. (Palo Alto, CA), Promega Inc. (Madison, WI), Invitrogen Inc. (Carlsbad, CA) and Strategene Inc. Available from a variety of commercial suppliers of molecular biological reagents, such as (San Diego, CA). In addition, plasmid vectors containing the reporter gene sequence can be obtained from the American Type Culture Collection and E. coli from Yale University. available from gene repositories such as the E. coli strain collection.
[0069]
Soluble reporter nucleic acids of the invention can be used to control additional elements such as ribosome binding sites and polyadenylation sites, such as coding sequences within the same transcription unit, additional transcription units under the control of the same or different promoters, cloning. And sequences that allow expression and transformation of host cells, and any such structures that may be desirable when providing embodiments of the present invention. In some embodiments, the soluble reporter nucleic acid comprises a polynucleotide that encodes a signal peptide that directs a detectable polypeptide encoded by the reporter gene to the surface of a host cell. The detectable polypeptide can then be detected, for example, by a cell sorter. For example, if the reporter gene encodes a fluorescent protein that is displayed on the cell surface in expression, a fluorescence-activated cell sorter is used to separate cells that express the reporter gene from those that do not.
[0070]
The soluble reporter nucleic acid can also include a polynucleotide encoding a molecular tag that facilitates isolation of host cells that do not express the reporter gene. For example, the epitope for an antibody can function as a molecular tag; cells expressing the reporter gene can then be fixed by contacting the cells with a solid support, which is an attached antibody that specifically recognizes the epitope. Other suitable molecular tags are known to those of skill in the art and include, for example, a polyhistidine tag or a FLAG® peptide. If a particular proteolytic response promoter is upregulated in use in response to expression of an insoluble form of the target polypeptide, cells expressing the insoluble target polypeptide are immobilized on the support. Conversely, if a particular protein solubility responsive promoter is down-regulated in use in response to expression of an insoluble form of the target polypeptide, cells expressing the soluble form of the target polypeptide are immobilized on the support. Is done.
[0071]
The present invention also provides a reporter system comprising: a) an isolated polynucleotide comprising at least one proteolytic response promoter operably linked to a reporter gene; and b) an expression structure that directs expression of a target gene. provide. The expression construct can be on either a separate polynucleotide from the promoter and the reporter gene, or the expression construct can be part of a single polynucleotide that also contains the proteolytic response promoter and the reporter gene. Thus, in certain embodiments of the invention, the reporter system comprises a proteolytic response promoter operably linked to the reporter gene, and the isolated polynucleotide further comprises an expression construct.
[0072]
The invention also provides (in vivo, ex vivo or in vitro) a gene delivery vehicle suitable for delivering and / or expressing the polynucleotide of the invention into a cell containing the polynucleotide of the invention. A polynucleotide of the invention can be included in a cloning or expression vector. These vectors (especially expression vectors) are sequentially manipulated and can assume any of a number of forms that can facilitate, for example, delivery and / or introduction into a cell. Examples of suitable expression or delivery vehicles are provided above.
[0073]
The present invention also provides a host or genetically modified cell comprising a proteolytic reporter structure as described above, as well as a target polypeptide-expressing nucleic acid comprising a polynucleotide encoding the identified target polypeptide. An array of cells is also provided, wherein the cells of each population differ in the target polypeptide expressed by the cells. For example, the polypeptides can differ from the reference amino acid sequence by amino acid substitutions, deletions or insertions. Alternatively, the target polypeptide expressed by the population of host cells can be a different fragment of a larger polypeptide.
[0074]
Polynucleotides and sequences embodied in the present invention can be obtained using chemical synthesis, recombinant cloning means, PCR, or any combination thereof. The PCR technique is the subject of U.S. Pat. Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202, and THE POLYMERASE CHAIN REACTION (Mullis Id., Birkhauser Press, Boston (1994)) or MacPherson et al. (1991) and (1995) and in the PCR of references cited therein. Alternatively, one of skill in the art can use the commercial DNA synthesizers provided herein to replicate the sequences and DNA. Thus, the present invention provides a process for obtaining the polynucleotides of the present invention by providing chemicals such as linear sequences of polynucleotides, nucleotides, suitable primer molecules, enzymes, and their replication and chemical replication or appropriate replication. Instructions for linking nucleotides in different directions to obtain a polynucleotide. In a separate embodiment, these polynucleotides are further isolated. In addition, one of skill in the art can insert the polynucleotide into a suitable replicating vector and insert it into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification. The DNA so amplified is isolated from the cells by methods known to those skilled in the art. Methods for obtaining a polynucleotide by this method are provided further herein, as are the polynucleotides thus obtained.
[0075]
RNA is obtained by first inserting a DNA polynucleotide into a suitable host cell. DNA can be introduced by any suitable method, for example, using a suitable gene delivery vehicle (eg, liposomes, plasmids or vectors) or by electroporation. If the cell replicates and the DNA is transcribed into RINA; the RNA is isolated to those skilled in the art by known methods, for example, as described in Sambrook et al. (2001). For example, mRNA can be isolated using various degrading enzymes or chemical solutions described in Sambrook et al. (2001), or extracted with a nucleic acid binding resin, observing the accompanying instructions provided by the manufacturer. You.
[0076]
Compositions comprising the carriers and polynucleotides and sequences of the invention in isolated form or contained in a vector or host or genetically modified cell are further provided herein. When these compositions are used pharmaceutically, they are combined with a pharmaceutically acceptable carrier.
[0077]
Polynucleotides, reporter systems and cells are useful in the methods described below.
[0078]
Industrial use
The structures described herein are useful for quickly and accurately measuring the solubility of a target protein in a cell. To perform this method, cells containing the structures of the present invention are cultured under conditions in which the target protein is expressed and reporter gene expression can be induced. As used herein, the term "inducible" means that reporter gene transcription is initiated in response to a specific stimulus. A particular stimulus that drives transcription of the proteolytic response promoter is an insoluble protein in the cytoplasm of the cell. Gram negative bacteria E. coli Using E. coli cells, for example, cells are grown in liquid media rather than agar plates and become inducible.
[0079]
Reporter gene expression is measured following expression of the target gene. This can be achieved by directly measuring the amount of the protein, such as by measuring the fluorescence of the fluorescent protein or by measuring the reporter gene by an immunoassay such as an ELISA assay. Alternatively, if the reporter gene is an enzyme, the amount of reporter produced will quantify the product produced by enzyme modification, such as the metabolism of X-gal or ONPG by a substrate compound, eg, β-galactosidase. It can be measured using an assay that performs The amount of reporter protein produced is directly proportional to the amount of insoluble target protein in the cytoplasm.
[0080]
The amount of insoluble protein in a particular sample can be measured by first generating a standard curve for target protein insolubility using reporter gene expression levels. This can be achieved by culturing host cells containing the reporter structure along with the target expression structure and generating a series of samples from which various amounts of insoluble target protein are produced. Expression of the protein-insoluble reporter is measured in each of those samples.
[0081]
The amount of soluble and insoluble target protein is determined by lysing the host cells, separating soluble and insoluble material by, for example, centrifugation and filtration, and determining the amount of target protein in each fraction by immunoassay such as, for example, ELISA or Western blot. Is measured quantitatively. Once a standard curve for protein solubility versus reporter expression has been generated, the amount of insoluble protein present in the test sample is determined by measuring the expression of the protein-insoluble reporter in that sample and determining the amount of insoluble protein present from the standard curve. Can be measured by calculating
[0082]
The invention also provides a method of screening for intracellular variants that improve protein solubility. These methods involve treating the mutagen-bearing cell population and identifying those cells that show increased expression of the target protein in a soluble form. “Mutagens” include, but are not limited to, physical reagents such as ionizing radiation, as well as ethyl methanesulfonate, N-methyl-N′-nitroso-guadinine and nitrite. It is intended to include chemical mutagens. In another embodiment, the variant can be introduced into a polynucleotide sequence encoding the target protein. The altered polynucleotide is then tested to determine if the solubility of the target protein is altered. Such mutants are, for example, mutagen-induced mutants; sites directed to mutants that alter particular amino acid residues and remove disulfide bonds, such as mutants of cysteine residues; Deficiencies that remove a specific set of amino acids, such as deficient stretches of hydrophobic amino acids; and fusion of the target protein to a second, especially soluble protein. In each case, the solubility of the target protein is assessed by measuring the expression of the protein soluble reporter nucleic acid as described herein.
[0083]
To identify a mutant that alters the solubility of the target protein, the polynucleotide encoding this protein is expressed under suitable conditions such that the reporter gene is responsive to expression of the insoluble protein. If the mutant induces increased solubility of the target protein, then if the proteolytic response promoter is up-regulated in response to expression of the insoluble protein, then the level of reporter gene expression will be accompanied by the mutation The level of reporter gene expression is reduced compared to the level observed in the host cells prior to treatment of the cells. By selecting a reporter gene whose expression is easily measured in the majority of individual samples, such as β-galactosidase, this method is used to screen the majority of dependent mutants and to lyse target proteins. Changes that improve gender can be identified.
[0084]
This structure is also useful for identifying variations in the method of target protein biosynthesis. The method can vary to modify the solubility of the target protein. Cells containing the proteolytic reporter nucleic acid are cultured under other conditions under which the target protein is expressed and the reporter gene is inducible, and the expression of the reporter gene is measured to improve the solubility of the expressed target protein. Variations in culture conditions to be performed are identified. For example, protein solubility is affected by the temperature, medium composition, or oxygen concentration at which the cells are cultured. A convenient method by which reporter expression is measured allows a variety of alternative conditions to be tested with minimal effort and identifies those conditions that produce the highest percentage of soluble target protein.
[0085]
The construct can be used to compare another cell, perform the method specified herein using at least two other cells, and compare the amount of the expressed reporter gene to increase the amount of the soluble target. Identifying cells that express the protein is useful for identifying cells that synthesize increasing amounts of soluble target protein.
[0086]
The invention also provides a method of screening an expression library of clones and identifying those clones that express soluble proteins. This library consists of modifications in the gene that expresses the target protein of interest. Modification of the gene can be provided by any of a variety of widely used methods. These include making truncations in the gene, random chemical mutagenesis, random mutagenesis through incorrect nucleotide introduction or site-directed mutagenesis. This library of modifications is transformed into cells containing a proteolytic reporter system. Individual clones of the cells to be transformed are then cultured under conditions in which the target gene or its modification is expressed. The level of reporter gene expression in each clone is measured during the expression of the target gene or its modification. Clones expressing increased or decreased levels of the reporter gene are identified by measuring the reporter gene level of each clone and comparing to clones expressing the unmodified target gene. Clones so identified express less insoluble protein and can include more soluble derivatives of the target protein.
[0087]
Selection of the appropriate reporter gene for a protein-soluble reporter system allows for a variety of efficient high-throughput procedures, and rapidly elaborates multiple separate cultures to identify specific samples that produce soluble target proteins. Screening is well known to those skilled in the art. The ease of detection of reporter genes such as β-galactosidase, luciferase and green fluorescent protein is further provided for the development of an automated procedure to screen cells for target protein solubility.
[0088]
In a further aspect, the structures defined herein are useful for identifying antibiotic reagents. Cells containing the proteolytic reporter structure are contacted with the candidate reagent. A potential antibiotic reagent that interferes with the protein folding process results in increased expression of insoluble endogenous cellular proteins, whereby the reporter is under the control of a promoter that is upregulated in response to the presence of the insoluble protein Induces gene expression. Measurement of the reporter gene product is performed after treatment with a potential antibiotic reagent. Cells expressing increased reporter activity with respect to the control agent indicate that the test reagent is a potential antibiotic.
[0089]
A further aspect of the present invention is to use the above process with co-expression of a soluble protein that is a known target of antibiotic therapy. Reagents that interfere with the folding of known target proteins result in increased expression of insoluble proteins and reporter genes. Reagents so identified have potential utility as antibiotics by interfering with the proper folding of these target proteins in the native host.
[0090]
An example
The following is provided by way of illustration, but not limitation.
[0091]
Proper protein folding is the key to generating recombinant proteins for structure determination. In this example, the misfolded recombinant protein, E. coli. The effect on gene expression in E. coli is tested. Comparison of expression patterns shows a unique set of genes that respond to translational misfolding. The response is partly similar to heat shock, and suggests a translational component for regulation. We also used expression information to generate reporters that respond to protein misfolding. These reporters identified properly folded recombinant proteins and generated soluble domains of insoluble proteins for structural studies.
[0092]
Materials and methods
Cloning
Clones expressing the properly folded or misfolded human protein were obtained from GeneStorm collection (Invitrogen). The clones containing the Unigene accession numbers L35545, U18291, M94856, M22146, D87116, M63167, M68520, M60527, M36881, M36981, U35003, S79522, X73460, D14968, M86400 and the M86400 are provided in p.B.A. -Provided inducible expression. T. The martima gene was amplified from genomic DNA and cloned into an expression vector pMH1 encoding a 12 amino acid N-terminal tag containing a 6X-histidine repeat for production and detection. The reporter vector was constructed by inserting a PCR ampli? Er 300 bp upstream of the ibpAB, ybeD, yhgI or yrfGHI gene upstream of beta-galactosidase in the pACYC184 derivative.
[0093]
Rep68 was cloned from a plasmid containing the entire genome of human adeno-associated virus 2 (AAV2). Putative domains containing bases 1-646, 647-1456 and 1457-1611 were amplified from the full-length template and cloned into pMH1. The above template was also used in the amplification of the full length gene for fragmentation. 2 μg of rep68 amplifier was used in each of the five fragmentation reactions containing 1, 0.1, 0.01, 0.001 or 0 units of DNase I (Bperinger Mannheim) as well as Pfu polymerase and dNTP. The reaction was performed on ice with Dnase added immediately before temperature cycling in the following MJ Research thermocycler: 10 minutes @ 25 ° C, 15 minutes @ 95 ° C, and 30 minutes @ 72 ° C. Each reaction was performed on a 1% agarose gel and the fragments corresponding to 1600-1000 bp, 1000-850 bp, 850-600 bp and 600-300 bp were extracted. Each pool was used for blunt cloning and ligation into pMH1 as described above, and was introduced into reporter cell line HK57 for screening.
[0094]
Cell growth and protein expression
E. FIG. E. coli strains MG1655 (Flam rph1) and KY1429 (F-araD139Δ (argF-lac) 169 lam flhD5301 fruA25 relA1 rpsL150 zhh50 :: rpoH606 (ts) deoC1) or LC for expression profiling MK86 (MKA36) with M36A for M36A80 (M). Transformation was performed using the encoding expression plasmid. Cells were cultured at 37 ° C. in Luria Broth with ampicillin. Protein expression was induced by the addition of L-arabinose to a final concentration of 0.1% in 1 hour. KY1429 cells were cultured as described above, except that initial growth continued at 32 ° C. and shifted to 42 ° C. due to non-permissive expression of rpoH606. Top10 cells (FmcrAΔ (mrr-hsdRMS-mcrBC)) containing ibpAB promoter fusion (pHK57) Φ80lacZΔM15 ΔlacX74 deorecrecA1 araD139Δ (ara-leu) . Beta-galactosidase assays were performed essentially as described by Miller (24). Fractionation of soluble and insoluble proteins was performed by centrifugation. Cultures were treated with 50 mM Tris pH 7.9, 50 mM NaCl, 1 mM MgCl₂   Resuspended in 3 mM methionine and sonicated for 2 minutes on ice. Cell debris and insoluble protein aggregates were pelleted by centrifugation at 3000 × g for 15 minutes. The soluble fraction was removed and the pellet was resuspended in an equivalent volume of lysis buffer.
[0095]
Probe production and hybridization and analysis of labeled mRNA
A labeled mRNA is produced and E. coli. E. coli was hybridized to a genome array (Affymetrix) essentially as described (25, 26). Individual gene expression was measured using standard Affymetrix GeneChip analysis software and used to make an aspect comparison of gene expression levels for pre-induced and post-induced samples. Properly folded and misfolded genes were analyzed for individual probe sets.
[0096]
Microplate solubility screening
A 96-well microplate containing 200 μL LB with 100 μg / mL ampicillin and 34 μg / mL chloramphenicol was inoculated into a single colony from above and grown with shaking at 37 ° C. overnight. Inoculate 200 μL of the same medium, average OD of 0.5₆₀₀ Overnight at 37 ° C. until the temperature reached. Cultures were induced with arabinose at a final concentration of 0.2%. Thirty minutes later, a cocktail of cefatriaxone and cefotaxime was added to each well until the respective final concentration was 10 μg / mL, and the plates were incubated for an additional 1.5 hours. Cultures were harvested after a total of 2 hours of induction by centrifugation at maximum speed for 15 minutes and the cell debris at the bottom of the well was pelleted. The soluble isolate was then split into 25 μL into a microplate for β-galactosidase activity screen and 75 μL into a Nunc Maxisorp® ELISA plate for Ni-HRP screening.
[0097]
Isolates were screened for β-galactosidase activity using a variation of the Miller protocol (10). 50 μL of 4 × Z-buffer and 50 μL of 4 × ONPG buffer were added to the microplate containing 25 μL of lysate. After the progression of the yellow color in the positive control wells, the reaction was₂ CO₃ Stopped with pH 8. A₄₂₀ , A₅₅₀ And the reaction time is recorded and the OD₆₀₀ Used along with the data, the β-galactosidase activity was calculated.
[0098]
Ni-HRP screening was performed as in ELISA. 75 μL of the isolate and 25 μL of TBS were bound to the microplate overnight at 4 ° C. and blocked with 1% BSA in TBA at 25 ° C. for 4 hours. Plates were then washed 3 × with TBST, 100 μL of Ni-HRP conjugate (KPL Labs) at a 1: 2500 dilution was added and incubated at 25 ° C. for 1 hour. The plate was then washed with TBST and 100 μL of HRP substrate (KPL Labs) was added and the color was developed until the positive control wells became deep blue. The reaction is stopped with 100 μL of 1N HCl and A₄₂₀ Was measured. The solubility score is determined by Ni-HRPA such that the intensity is one order of magnitude greater than that of the β-galactosidase activity score.₄₂₀ Was calculated and calculated by dividing Ni-HRP absorption by β-galactosidase activity.
[0099]
result
Gene expression analysis
To effect gene expression as a result of a misfolded protein, a representative gene was cloned as a fusion protein into thioredoxin under the control of a tightly regulated arabinose promoter. Human phospholipase A2 (PLA) is almost completely soluble as measured by cell separation and centrifugation. Further evidence of proper folding of this protein is obtained through the dynamic light scattering of the purified protein and the ability to crystallize it from a single similar purification step. Under comparable expression conditions, human cell-specific protein tyrosine kinase (LCK) is almost exclusively expressed as an insoluble protein. Both proteins are expressed at levels sufficient to be excellent translation products. MRNA products from induced and uninduced cultures were prepared and used to probe for gene expression.
[0100]
E. FIG. Recombinant protein expression in E. coli is expected to cause substantial changes in gene expression. Indeed, comparison of gene expression with a pre-induced control showed that in both cases, 6% of all genes showed a> 3-fold difference in expression. For the insoluble recombinant protein, 27 genes showed a> 10-fold change in expression as compared to 10 genes for the soluble recombinant protein. Comparison of the two profiles identified 53 genes listed in Table 1, showing a> 3-fold difference, which are unique in the case of insoluble. Then, these genes are probably responsive to misfolded proteins in the cell, and in dealing with these translational stresses, E. coli. play a role in E. coli.
[0101]
The heat shock transcription factor RpoH is normally suppressed by interaction with the chaperone protein DnaK. In the presence of a misfolded protein, DnaK binds to the protein and causes RpoH to stimulate transcription of the heat shock promoter (7). Many upstream regions of the derived genes in Table 1 indicate the presence of an RpoH-dependent promoter sequence. Further evidence of the important role played by RpoH is provided by expression profiling the results performed from the rpoH606 mutation expressing the misfolded LCK protein (KY1429) compared to non-expression controls. Significantly different expression profiles are seen in the case of the rpoH606 mutant (Tables 1 and 2). The population of genes induced by the misfolded protein in the wild-type strain is imperfectly induced within the rpoH606 mutation, which indicates that it is directly or indirectly under the control of the transcription factor.
[0102]
Heat shock gene induction
Not surprisingly, many of the genes induced by misfolding of transcription are known chaperone activities. These include the well-characterized dnaJ, dnaK and grpE genes. The corresponding proteins interact as a complex with misfolded or denatured proteins in the ATP-dependent repair process. Similarly, the mopAB gene that forms the GroELS folding repair complex is induced under misfolded conditions of translation. IbpAB is a small heat shock polypeptide that binds to the main body aggregate containing recombinant protein (13). Even though they do not show to behave as directly folded chaperones, they bind to misfolded proteins and interact with the DnaJK GrpE protein as a chaperone system (14). The gene product of the Hsp33, yrfI gene, was recently identified as a chaperone protein that responds to oxidative conditions (15). Genes that are affected in the degradation of denatured proteins are also induced by translational misfolding. The lon, clpBP and hslUV protease genes are expressed at increasing levels. Under normal cell growth, these proteases perform important recycling functions. Insoluble aggregates are relatively resistant to proteolysis and the recycling pathway is not effective for recombinant protein expression.
[Table 1]

[Table 2]

Table 2 Cold shock after induction of misfolded protein expression

[0103]
Induction of ribosome-related genes
Other heat shock genes that bind to ribosomes are induced under translational misfolding conditions. Hsp15 (yrfH) binds RNA (24) and binds to the free 50S ribosomal subunit, which contains the nascent polypeptide chain (16). Heat shock also increases the level of Hsp15-binding, including increased degradation of the 50S and 30S subunits. Further proposals for ribosome degradation are from ftsJ (rrmJ) (SEQ ID NO: 10). The ftsJ gene product is an RNA methylase specific for 23S rRNA only when included in the 50S ribosomal subunit (17, 18). This enzyme methylates 23S rRNA at 2552 located within the ribosome peptidyl transferase center (17). Methylation of ftsJ lacks 23S rRNA and shows up to 65% reduction in ribosomal activity corresponding to 50S and 30S subunit cleavage (19). Of particular note in the rpoH variants is a large increase in the transcription of cold shock proteins (CSPs) (Table 2). These genes are not affected by heat shock (9), but are associated with a temporary cessation of translation. CSPs are RNA binding proteins that act as chaperones for untranslated messages (20, 21) and provide anti-termination (22). Increased expression of CPS under conditions that reduce chaperone expression (rpoH606) indicates a quiescent translation. Taken together, these results, or as a result of demethylation, suggest a translational regulatory response as a result of translational misfolding. This assumption is an interesting adjustment mechanism in recent studies.
[0104]
Other induced genes
Although yccV, yhdN and yrfG are shown to increase expression under heat shock conditions, their functions are unknown. In addition to these known heat shock genes, yagU, yciS, ybeD, yejG and yhgI showed increased expression. Most of these proteins are relatively small and are generally acidic. Some speculations suggest that some of these proteins play a role similar to IbpAB in direct recognition and sequestration of misfolded proteins. However, only IbpAB binds misfolded and aggregated proteins. Induced levels of ibpAB are very high, and these other proteins are present at lower levels. Interestingly, knockout mutations of ibpAB have a relatively small effect on cell growth and viability (14) and imply some functional redundancy within the cell.
[0105]
Gene folding reporter for protein folding
To confirm the profiling results and to facilitate experiments with a large number of recombinant proteins, the promoter regions from ibpAB, ybeD, yhgI and yrfGHI were cloned into a beta-galactosidase reporter vector. In each case, increased beta-galactosidase activity is observed when expression of the misfolded protein LCK is induced, despite the fact that the folded protein PLA does not increase in activity. These results were further extended with a set of 8 misfolded proteins and 6 properly folded proteins co-expressed in the presence of the ibpAB-promoter beta-galactosidase fusion. Was done. In each case, increased beta-galactosidase activity corresponded to the expression of misfolded protein. A more detailed description is given below. And the observed response is more indicative of the normal consequences of protein misfolding than the specific response to any particular protein. These reporters provided a simple way to identify misfolded proteins using a sensitive enzyme assay, and the ibpAB promoter fusion was selected as the reporter for further studies.
[0106]
ELISA-like assay for soluble proteins
To identify protein derivatives with improved folding properties in a recombinant environment, an ELISA-like assay compatible with a high-throughput screening machine was also developed. In order to assess soluble protein levels in high-throughput systems, non-denaturing cell isolates must be produced using conditions that integrate with rapid screening in microplates. Instead of detergent or organic lysis we added an antibiotic cocktail to each well to induce lysis. The soluble protein fusion was removed, bound to microplates, and the recombinant protein was detected via binding of the Ni-HRP conjugate to the 6X-histidine N-terminal fusion. It can be seen that His-Tag is not heterogeneously accessible between recombinant proteins. Therefore, a negative Ni-HRP response cannot indicate the absence of soluble protein, but protein folding can prevent access to His-Tag. However, we do not need to observe this, it is a general problem. This assay then provides a measure of the level of soluble recombinant protein without performing an SDS gel and in a form compatible with the HT screen and β-galactosidase assays.
[0107]
Testing for proteins with pre-determined expression characteristics
As part of our attempt at cloning, expressing and characterizing the entire proteome of Thermotoga maritima, it is shown in Table 3 (6 solubilities, 6 insolubles and 6 mixed solubilities). 18T. The effect of the reporter on the maritima protein was tested. To optimize assay parameters, strains were aligned in 96 wells and at three of three induction levels (0.02%, 0.2% and 2% arabinose) and four for lysis enhancing antibiotic addition. Species were assayed at a post-induction time (t = 0, 30, 60 and 120 minutes after addition of arabinose). FIG. 2 shows the averaged results for the three plates (soluble, insoluble and mixed) for 0.2% arabinose induction. Both the insoluble and mixed pools showed more than 4-fold β-galactosidase activity than the soluble pool (FIG. 2A). Conversely, the soluble pool showed a 10-fold higher response in Ni-HRP to the insoluble pool (FIG. 2B). A mixed pool of proteins expressed approximately equally in both the soluble and insoluble fractions showed Ni-HRP binding at about half the intensity of the soluble pool. Only either a lack of β-galactosidase or the presence of Ni-HRP activity is used as a measure of soluble protein, but choose two ratios that activate a good, effective and convenient screen.
Table 3: T. with pre-measured expression characteristics maritima protein

[0108]
Testing for proteins with unknown expression characteristics
We then applied this screen to a large set of proteins with unknown folding properties. We assume that the protein has not been previously characterized for expression. The screen was performed on the maritima protein under the above optimal conditions. The results of this screen are summarized in Table 4. SDS-PAGE of the eluate from the nickel-chelating resin and from the dissolved insoluble fraction of each clone is performed along the corresponding β-galactosidase activity, Ni-HRP response and solubility score for 186 clones. . Based on the gel results, 57 clones did not overexpress the visible protein band, 62 clones predominantly expressed soluble protein, 27 clones predominantly expressed insoluble aggregates, and 46 Is approximately equally expressed for both soluble and insoluble fractions. A comparison of β-galactosidase activity for the Ni-HRP assay is shown in FIG. Points are categorized by SDS gel analysis of soluble and insoluble protein fractions. The screen positively identified 54 (87%) of the 62 soluble proteins. Seven out of eight residual proteins that were soluble according to the gel had a low Ni-HRP assay, presumably due to His-tag inaccessibility in their fusion proteins. Alone, β-galactosidase activity measurements identified 22 (81%) of 27 insoluble proteins. Those proteins that show partial solubility show various solubility scores, indicating that partial folding induces β-galactosidase activity through a reporter. This assay then provides an effective and convenient means of classifying folding properties.
Table 4: T.I. average solubility screen value for maritima protein

[0109]
Identification of soluble protein domains
The utility of this system lies in the ability to identify variants, mutations or domains of the full-length gene product based on the improved properties. For structural or biochemical studies, Rep68 (GI: 209617), to identify soluble fragments of adeno-associated virus nonstructural proteins with various activities associated with the integration of the viral genome into target DNA The ability of this screen was tested. This protein is E. coli. It has previously been found to predominantly express as unfolded aggregates in E. coli. We have taken both random and rational approaches based on domain selection for analogs. The three domains of Rep68 were homologous to the RPS-BLAST survey (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) for the parvovirus non-structural protein NP-1. Were selected after identifying an internal domain with The information combined with the Kyte-Doolittle hydropathy plot (FIG. 4) is used to align the 5 'and 3' cutoffs for each domain. The remaining N- and C-terminal residues contain the other two domains and have no significant homology to any other protein in the database. A random fragment of Rep68 was also generated for screening with the DNase fragment.
[0110]
The three predicted domains and randomly generated fragments were screened in response to the identification of a soluble fragment of Rep68. None of the three predicted domains were identified as soluble (FIG. 4). At the same time, 564 randomly generated fragments of rep68 were also screened. Certain fragments resulted in very high solubility scores (Table 4). This clone was evidenced by large-scale expression and showed expression in both soluble and insoluble fractions. Sequencing of the subsequently identified clone demonstrated that it consisted of a fragment of rep68 corresponding to amino acids 1-95 (Figure 4). This identified fragment showed a substantial improvement in solubility over the full length protein and was tested in a crystallization attempt.
[0111]
Conclusion
Most gene expression studies of protein folding in vivo have focused on degeneration as a result of environmental stress. This response is essential in vivo to handle constantly changing environments and non-ideal growth conditions. The result of translational folding is equally important for cells, as all proteins are necessarily unfolded when translated. Expression of a non-naturally occurring protein is itself a stress, either through recombinant means or mutation. We use clear inferences about how these gene products are involved in the folding of nascent polypeptides to create a known cellular response to translational misfolding such as heat shock. It was shown to include. In addition, other heat shock genes and genes of unknown function are induced. These can also be included in the folding process. Our results suggest both transcription and translation regulation. The DnaK-RpoH interaction is well characterized and indicates that it is a major regulator of the transcriptional response. The altered expression of the replicated gene and the disruption of ribosomes in translational stalling are interesting, and imply that these effects may be the result of misfolded proteins in translation. These genes contain yrfH, which binds to the 50S ribosomal subunit (16). cspABGI is induced in rpoH606, which suggests translation stalling, in the absence of the induced chaperone. ftsJ is also induced, which is known to methylate the 23S rRNA of the 50S subunit resulting in a higher affinity of the two subunits for each other (19). Ribosomal structure is an obvious potential regulator mechanism for ribosomal sensors for misfolded proteins, where the methylation site of ftsJ, the 2552 portion of 23S rRNA is interestingly closed to the peptidyl transferase center (18). It represents that. Such ribosomal sensors are not novel, as exemplified by a well-characterized stringent response to tRNAs that are not charged during translation (23). Ribosome stalling allows time for chaperone synthesis and recruitment, thereby providing a mechanism to prevent irreversible aggregation. In this way, the cells maintain an additional collection pathway for new proteins to fold in the relatively protected environment of the translation ribosome until sufficient chaperones are recruited.
[0112]
Differentially regulated identification genes offer a valuable opportunity to provide novel reporters of the folded state of cellular proteins, especially as whole and overexpressed recombinant proteins. Our reporter assay differs from that recently described by not relying on direct coupling of the reporter gene to the target, thereby limiting reporter interference. The combination of the Ni-HRP and β-galactosidase assays provides an effective way to assay soluble recombinant proteins in a high-throughput manner. We extended this system to identify single gene product variants and truncations as a way to identify the solubility domain of another misfolded aggregate protein. Using this approach, we identified a soluble fragment of Rep68 and speculated that this assay would provide a general means of isolating recombinant proteins suitable for structure / function action.
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[0113]
The examples and embodiments described herein are for illustrative purposes only, and various modifications or alterations thereof will be suggested to those skilled in the art and are not intended to limit the spirit and scope of the present application or the claims. It is understood to be included within. All publications, patents and patent applications cited herein are hereby incorporated by reference for all purposes.
[Brief description of the drawings]
FIG. 1 shows the promoter of a known heat shock gene induced during the expression of insoluble proteins.
FIGS. 2A-C show a summary of the screening results for 18 Thermotoga maritima proteins with pre-measured expression characteristics.
FIG. 3. 186 T.D. in reporter strain. Figure 3 shows the relative β-galactosidase activity relative to the relative Ni-HRP activity observed after expression of the maritima protein.
FIG. 4 depicts the alignment of the second structure prediction, as well as both the predicted and identified domains of Rep68.

Claims (76)

a) a soluble reporter nucleic acid comprising a protein solubility responsive promoter operably linked to a reporter gene; and b) a target polypeptide-expressing nucleic acid comprising a polynucleotide encoding the target polypeptide;
A host cell comprising: a host cell, wherein expression of the target polypeptide in an insoluble form causes a change in reporter gene expression. The host cell of claim 1, wherein the solubility responsive promoter comprises a polynucleotide sequence that is at least 75% identical to a polynucleotide selected from the group consisting of SEQ ID NOS: 1-22. 3. The host cell of claim 2, wherein the solubility responsive promoter comprises a polynucleotide selected from the group consisting of SEQ ID NOS: 1-22. 2. The host cell of claim 1, wherein the solubility responsive promoter comprises a polynucleotide comprising a regulatory region of a gene listed in Table 1. 2. The host cell of claim 1, wherein the solubility responsive promoter comprises a polynucleotide containing an RpoH recognition site. The host cell of claim 5, wherein the solubility responsive promoter comprises a polynucleotide that is at least 75% identical to a polynucleotide selected from the group consisting of SEQ ID NOS: 23-43. 7. The host cell of claim 6, wherein the solubility responsive promoter comprises a polynucleotide selected from the group consisting of SEQ ID NOS: 23-43. 2. The host cell of claim 1, wherein the solubility-responsive promoter is up-regulated when the target polynucleotide is expressed in an insoluble form. 2. The host cell of claim 1, wherein the solubility responsive promoter is down-regulated when the target polynucleotide is expressed in an insoluble form. The host cell of claim 1, wherein the polynucleotide encoding the target polypeptide is heterologous to the host cell. 2. The host cell of claim 1, wherein the target protein expressing nucleic acid comprises a promoter operably linked to a polynucleotide encoding the target polypeptide. 12. The host cell of claim 11, wherein the target protein expressing nucleic acid comprises a promoter that is homologous to the host cell. 12. The host cell of claim 11, wherein the target protein-expressing nucleic acid comprises a promoter that is heterologous to the polynucleotide encoding the target polypeptide. 2. The host cell of claim 1, wherein the proteolytic response promoter is a prokaryotic promoter. 15. The host cell of claim 14, wherein the proteolytic response promoter is a Gram negative bacterial promoter. 16. The host cell of claim 15, wherein the Gram negative bacterium is a familial Enterobacteriaceae. 17. The host cell of claim 16, wherein the member of the familial Enterobacteriaceae is selected from the group consisting of Escherichia, Salmonella, Shigella, Klebsiella, and Enterobacteriaceae. Gram negative bacteria are E. coli. 18. The host cell of claim 17, which is E. coli. 15. The host cell of claim 14, wherein the proteolytic response promoter is a Gram positive bacterial promoter. 2. The host cell of claim 1, wherein the proteolytic response promoter is a eukaryotic promoter. 21. The host cell of claim 20, wherein the promoter is a mammalian, plant, insect, fungal or yeast promoter. 2. The host cell of claim 1, wherein the reporter gene comprises a polynucleotide encoding a selectable or detectable polypeptide. 23. The host cell of claim 22, wherein the selectable or detectable polypeptide is selected from the group consisting of a metabolic enzyme, an antibiotic resistance factor, a chemiluminescent protein, and a fluorescent protein. 24. The host cell of claim 23, wherein the detectable polypeptide is β-galactosidase. 24. The host cell of claim 23, wherein the detectable polypeptide is a luminescent or fluorescent protein. 23. The host cell of claim 22, wherein the reporter gene further comprises a polynucleotide encoding a signal peptide that directs the detectable polypeptide to the surface of the host cell. 27. The host cell of claim 26, wherein the reporter gene further comprises a molecular tag that facilitates separation of a host cell that expresses the reporter gene from a host cell that does not express the reporter gene. The host cell of claim 1, wherein the proteolytic response promoter is from the same species as the host cell. 2. The host cell of claim 1, wherein the target polypeptide comprises a fragment of a larger polypeptide. 30. The host cell of claim 29, wherein the fragment comprises a domain of a larger polypeptide. 31. The host cell of claim 30, wherein the domain is identified by a homolog of another polypeptide, by a hydropathy plot, or by both. 30. The host cell of claim 29, wherein the fragment comprises a polypeptide encoded by a random fragment of a polynucleotide that encodes a larger polypeptide. 2. The host cell of claim 1, wherein the target polypeptide comprises a mutated form of the polypeptide. 2. The two or more arrays of host cells of claim 1, wherein the host cells of each population differ in the target polypeptide expressed by the host cells. 35. The array of claim 34, wherein the polypeptides differ by amino acid substitutions, deletions or insertions as compared to a reference amino acid sequence. 35. The array of claim 34, wherein the target polypeptide expressed by the population of host cells comprises different fragments of a larger polypeptide. a) culturing the host cell of claim 1 under conditions in which the target polypeptide is expressed; and b) determining whether the expression of the reporter gene is increased or decreased, and thereby expressing the expressed target. Measuring the solubility of the polypeptide,
And measuring the solubility of the target polypeptide. 38. The method of claim 37, wherein the host cell is a prokaryotic cell. The host cell is E. coli. 39. The method of claim 38, which is an E. coli cell. 38. The method of claim 37, wherein the solubility responsive promoter comprises a polynucleotide sequence that is at least 75% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 1-43. 41. The method of claim 40, wherein the solubility responsive promoter comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 1-43. 38. The method of claim 37, wherein the host cell is a eukaryotic cell. 38. The method of claim 37, wherein reporter gene expression is measured by performing a quantitative assay, and measuring the amount of a detectable or selectable polypeptide in the cell. 38. The method of claim 37, wherein the host cells undergo cell sorting to separate cells having increased or decreased expression of the reporter gene from cells in which expression of the target polypeptide does not alter expression levels of the reporter gene. 46. The method of claim 44, wherein the reporter gene encodes a fluorescent protein and the cell classification comprises a fluorescence activated cell classification. The soluble reporter nucleic acid further comprises:
a) a polynucleotide encoding a molecular tag; and b) a polynucleotide encoding a signal peptide;
38. The method of claim 37, comprising:
A detectable or selectable polypeptide encoded by a signal polypeptide, a molecular tag, and a reporter gene is expressed as a fusion protein, and the signal polypeptide directs the detectable or selectable polypeptide to the cell surface. A method wherein the host cell is contacted with a solid support to which a molecular tag can be attached, and the cells expressing the reporter gene are fixed on the solid support. 47. The method of claim 46, wherein the solubility responsive promoter is downregulated when the target polypeptide is expressed in an insoluble form, and host cells expressing the target polypeptide in an insoluble form do not bind to a solid support. 47. The method of claim 46, wherein the solubility responsive promoter is up-regulated when the target polypeptide is expressed in an insoluble form, and host cells expressing the target polypeptide in an insoluble form bind to the solid support. 47. The method of claim 46, wherein the molecular tag comprises an epitope for an antibody, a polyhistidine tag or a FLAG® peptide. Furthermore: the host cells are separated under non-denaturing conditions after expression of the target polypeptide, wherein the target polypeptide is in the liquid phase if expressed in a soluble form and in the solid phase if expressed in an insoluble form In; and
Measuring the amount of soluble target polypeptide in the liquid phase;
38. The method of claim 37, comprising: The target polypeptide comprises a molecular tag, and further:
After separation of the cells, a part of the liquid phase is removed;
Contacting the target polypeptide with a detection reagent that binds to the molecular tag and measuring the amount of soluble target polypeptide in the liquid phase;
51. The method of claim 50, comprising: 52. The method of claim 51, wherein the molecular tag comprises an epitope for an antibody, a polyhistidine tag or a FLAG® peptide. 52. The method of claim 51, wherein said moiety is placed on a solid support to which the target polypeptide binds before contacting the polypeptide with the detection reagent. 54. The method of claim 53, wherein the solid support comprises a material selected from the group consisting of glass, plastic, polymer, metal, metalloid, ceramics, and organic. 55. The method of claim 54, wherein the solid support comprises a microtiter plate, a nitrocellulose membrane, a nylon membrane, a derivatized nylon membrane, or agarose particles. A method for identifying an intracellular variant that alters the solubility of a target polypeptide, comprising:
a) treating the cells with a mutagen;
b) introducing into a cell: i) a soluble reporter nucleic acid comprising a protein solubility responsive promoter operably linked to the reporter gene; and ii) a target polypeptide-expressing nucleic acid comprising a polynucleotide encoding the target polypeptide. ;
c) culturing the cells under conditions suitable for expression of the target polypeptide;
d) measuring the expression of the reporter gene; and e) comparing the level of expression of the reporter gene in the cell with the level observed in non-mutant cells containing the soluble reporter nucleic acid and the target polypeptide-expressing nucleic acid. Identify cells containing mutants that alter the solubility of the peptide,
The method comprising: 57. The method of claim 56, wherein the cells are treated with a mutagen after introducing one or both of the soluble reporter nucleic acid and the target polypeptide expressing nucleic acid into the cell. 57. The method of claim 56, wherein the cell is a prokaryotic cell. The cells are E. coli. 59. The method of claim 58 which is an E. coli cell. 57. The method of claim 56, wherein the cell is a eukaryotic cell. 57. The method of claim 56, wherein the solubility is changed to an amplified solubility. 57. The method of claim 56, wherein the solubility is changed to reduced solubility. A method of identifying a change to a polynucleotide encoding a target polypeptide that alters the solubility of the target polypeptide,
a) altering the polynucleotide encoding the target polypeptide to form an altered polynucleotide;
b) introducing into a cell: i) a soluble reporter nucleic acid comprising a protein solubility responsive promoter operably linked to the reporter gene; and ii) a target polypeptide-expressing nucleic acid comprising the altered polynucleotide;
c) culturing the cells under conditions suitable for expression of the target polypeptide;
d) measuring the expression of the reporter gene; and e) comparing the level of expression of the reporter gene with the level observed in a cell having a non-denatured polynucleotide encoding the target polypeptide; Identify changes to the polynucleotide that alter the solubility of the peptide,
The method comprising: A method for identifying a variation in a process for biosynthesis of a target polypeptide that alters the solubility of the target polypeptide,
A host cell comprising: a) a soluble reporter nucleic acid comprising a protein solubility responsive promoter operably linked to a reporter gene; and b) a target polypeptide expression nucleic acid comprising a polynucleotide encoding the target polypeptide; Culturing under selective conditions under which the peptide is expressed;
Comparing reporter gene expression by host cells grown under each selective condition,
The method comprising: 65. The method of claim 64, wherein at least two cells are cultured and the expression of the reporter gene in each cell is compared, thereby identifying cells that express an altered amount of a soluble target polypeptide. A protein solubility responsive promoter is up-regulated when the target polypeptide is expressed in an insoluble form, and expression of the reporter gene at low levels indicates process conditions that result in greater expression of the soluble target polypeptide. 64 methods. A method of screening an expression library to identify library members that express a soluble target polypeptide,
a) introducing a plurality of expression vectors, each containing a polynucleotide encoding a target polypeptide, into a plurality of host cells containing a soluble reporter nucleic acid containing a protein solubility responsive promoter operably linked to a reporter gene; To generate an expression library,
b) culturing the host cells under conditions in which the target polypeptide is expressed; and c) detecting expression of the reporter gene, thereby identifying a library member that expresses the soluble target polypeptide.
The method comprising: The proteolytic response promoter was up-regulated when the target polypeptide was expressed in an insoluble form, and the number of host cells expressing the soluble target polypeptide was reduced as compared to the host cells expressing the insoluble target polypeptide 68. The method of claim 67, wherein the reporter gene is expressed at the level. The proteolytic response promoter was down-regulated when the target polypeptide was expressed in an insoluble form, and the number of host cells expressing the soluble target polypeptide was increased as compared to the host cells expressing the insoluble target polypeptide 68. The method of claim 67, wherein the reporter gene is expressed at the level. 70. The method of claim 69, wherein said reporter gene comprises a selectable marker, and said host cells are grown under selective conditions, thereby selecting for host cells expressing a soluble target polypeptide. A method of identifying an antibiotic reagent, comprising:
Contacting a cell containing a lytic reporter nucleic acid containing a protein lytic response promoter operably linked to the reporter gene with a candidate antibiotic reagent; and detecting the level of expression of the reporter gene, wherein contacting the candidate antibiotic reagent A change in the expression level of the reporter gene in the cell that is expressed is a reagent that inhibits protein folding in the cell, as compared to a reporter gene expression level in the cell that is not contacted with the candidate antibiotic reagent,
The method comprising: 72. The method of claim 71, wherein the proteolytic response promoter comprises a polynucleotide comprising a regulatory region of a gene listed in Table 1. A method for identifying a promoter that is differentially regulated in response to expression of an insoluble polypeptide in a host cell containing the promoter, comprising:
a) a soluble reporter nucleic acid comprising a putative protein solubility responsive promoter operably linked to a reporter gene; and ii) a target polypeptide-expressing nucleic acid comprising a polynucleotide encoding the target polypeptide. Offer to;
b) culturing the host cells under conditions in which the target polypeptide is expressed in an insoluble form;
c) determining whether the expression of the reporter gene is increased or decreased, thereby determining whether the putative proteolytic response promoter is differentially regulated in response to expression of the insoluble polypeptide in the host cell. The method comprising: 74. The method of claim 73, wherein the putative protein solubility responsive promoter is a heat shock promoter. 74. The method of claim 73, wherein the putative protein solubility responsive promoter is a eukaryotic promoter. 74. The method of claim 73, wherein the putative protein solubility responsive promoter is a prokaryotic promoter.

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