JP2004507240A - Methods for identifying specific cleavable peptides and uses of such peptide sequences - Google Patents

Abstract

The present invention relates to a method for detecting and identifying a peptide that can be specifically cleaved based on a library of nucleic acid peptide fusion molecules using a predetermined amino acid sequence. According to the method, the various peptide moieties are each encoded by a nucleic acid linked thereto, and a proteolytically active solution is used. The invention relates to the use of said peptides for increased release of chemically active ingredients and for diagnosis.

Description

【００３９】
１．１　ＭＭＰ３鋳型ＤＮＡ及びトロンビン鋳型ＤＮＡのＰＣＲを経た調製
２５０ｎｇのＤＮＡ鋳型、２５ｐｍｏｌの５’と３’プライマー、１０ｍＭ ｄＮＴＰｓ、５μｌの１０×ＰＣＲ緩衝液及び５ Ｕ／μｇ Ｔａｑ ＤＮＡ ポリメラーゼを、５０μｌ反応混合物（Ｐｒｏｍｅｇａ， Ｍａｄｉｓｏｎ， ＵＳＡからの緩衝液と酵素）に組み合わせた。そのＰＣＲを、Ｂｉｏｍｅｔｒａ Ｔ ｇｒａｄｉｅｎｔ ｃｙｃｌｅｒ内で行った：１ ｍｉｎ ９４℃， ２１×（０．５分 ９４℃、０．５分 ５５℃、０．５分 ７２℃）。
【００４０】
【表２】

【００４１】
図１は、２％アガロースエチジウムブロマイドゲル上のＰＣＲ産物を示す。レーン１：５０ｂｐ ＤＮＡマーカー、レーン２：ＭＭＰ３ ＤＮＡ、レーン３：トロンビンＤＮＡ、レーン４：１００ ｂｐ ＤＮＡマーカー
図１が示すように、ＭＭＰ３ ＤＮＡ及びトロンビンＤＮＡは、首尾良く増幅された。そのＰＣＲ産物は、予期されたＭＭＰ３ ＤＮＡに関する１９９ｂｐ及びトロンビンＤＮＡに関する１８９ｂｐの分子量を有する。
【００４２】
１．２　ＭＭＰ３及びトロンビンＤＮＡのｉｎ ｖｉｔｒｏ転写
１００ ｐｍｏｌのＤＮＡをその転写反応に使用した（Ｒｉｂｏｍａｘ Ｔ７／Ｐｒｏｍｅｇａ， Ｍａｄｉｓｏｎ， ＵＳＡ）。そのＤＮＡを、５×Ｔ７緩衝液、ｒＮＴＰｓ及びＴ７ ＲＮＡポリメラーゼと共に５００μｌ混合物にして、３７℃で４時間インキュベートとした。
ＲＮＡは、フェノール／クロロホルム抽出により精製され、そして、６％尿素ゲル（Ｎｏｖｅｘ Ｇｅｌ／ Ｉｎｖｅｔｒｏｇｅｎ， Ｇｒｏｎｉｎｇｅｎ， Ｎｅｔｈｅｒｌａｎｄｓ）上で分析された。
図２は、６％尿素ゲル上の転写後に得られたＲＮＡを示す。レーン１：ＭＭＰ３ ＲＮＡ、レーン２：トロンビンＲＮＡ
図２が示すようにＲＮＡは、予期されたようにその転写反応の後に検出できる。そのＲＮＡは分解していない。
【００４３】
１．３　ＲＮＡのピューロマイシンリンカーでのＵＶ架橋を介したライゲーション
３ｎｍｏｌのＲＮＡ、その配列を有する４．５ｎｍｏｌのピューロマイシン（Ｐｕ）リンカー（ＰＥＧ＝ポリエチレングリコール、Ｐｓｏ＝ソラレン）
【００４４】
【表３】

【００４５】
（Ｉｎｔｒｅｒａｃｔｉｖａ／Ｕｌｍ）、１２μｌの１０×リガーゼ緩衝液（１Ｍ ＮａＣＩ， ２５０ｍＭトリス ｐＨ ７．５）を１２０μｌ混合物に組み合わせて、８５℃で５分間次いで室温で１０分間インキュベートした。ＵＶ架橋は、３６６ｎｍで１５分間行った（ＬＴＦ−Ｌａｂｏｒｔｅｃｈｎｉｋ， １２ Ｗによる手持ちＵＶ ランプ）。
ライゲーション効率は、５％尿素アガロースゲル上で確認された。
図３は、５％尿素ＴＢＥゲル上でのライゲーション反応の分析を示す。レーン１：ＭＭＰ３ ＲＮＡ、レーン２：ピューロマイシンリンカーを持つＭＭＰ３ ＲＮＡ、レーン３：トロンビンＲＮＡ、レーン４：ピューロマイシンリンカーを持つトロンビンＲＮＡ（そのシグナルＬは有色マーカーによるものである）
そのＲＮＡへのピューロマイシンリンカーのライゲーションの後、その分子量の明白な増大が観測され、トロンビンＲＮＡ及びＭＭＰ３ ＲＮＡが首尾良くピューロマイシンに連結していることを示す。
【００４６】
１．４　Ｉｎ ｖｉｔｒｏ翻訳
８μｌのリンカーＲＮＡを２００μｌの赤血球溶解物（Ｐｒｏｍｅｇａ， Ｍａｄｉｓｏｎ， ＵＳＡ Ｌ４１６Ｘ）と混合し、５μｌの^３５Ｓメチオニン（Ｈａｒｔｍａｎｎ， １０．８μＭ， 特異的活性：その特異的活性は７２１８１ ｄｐｍ／ｐｍｏｌ））、６μｌのアミノ酸ミックス（メチオニンなし， １ｍＭ， Ｐｒｏｍｅｇａ）、及び８０μｌのＨ_２Ｏを混合して、次いで、３０℃で３０分間インキュベートする。１３０μｌの２Ｍ ＫＣＩ及び７５μｌのＭｇＣ１_２の添加の後、３０℃で３０分間のインキュベーションを行う。得られた融合分子は、オリゴｄＴセルロースを使用して精製された（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ， Ｆｒｅｉｂｕｒｇ， Ｇｅｒｍａｎｙ）（そのｉｎ ｖｉｔｒｏ翻訳混合物の、ポリＡ配列を含有しないそれら全成分の除去）
【００４７】
１．５　逆転写反応
精製された２００μｌ融合分子を２．５μの１００μＭリバースプライマーと混合し、８０℃で５分間インキュベートした。その反応混合物を氷上で冷やした後、５０μｌの５×ストランド緩衝液、２０μｌのｄＮＴＰｓ（各ケースにおいて１０ｍＭ）、２．５μｌのＲＴＳｕｐｅｒｓｃｒｉｐｔ ｌｌ（すべてＰｒｏｍｅｇａ， Ｍａｄｉｓｏｎ， ＵＳＡからのもの）を加えて、その混合物を４２℃で４０分間インキュベートした。
【００４８】
実施例２
融合分子のトロンビン及びＭＭＰ３による溶液中でのタンパク質分解開裂
調製された融合分子を対応するプロテアーゼと共にインキュベートし、それら反応生成物を分析した（図４）。１０ｎＭ ＭＭＰ３（Ｓｉｇｍａ， Ｄｅｉｓｅｎｈｏｆｅｎ， Ｇｅｒｍａｎｙ）を、そのＭＭＰ３開裂部位を有する５ｐｍｏｌの融合分子に加えて、その混合物を全容量で１５μｌのＭＭＰ３緩衝液（５０ｍＭトリス ｐＨ７， １５０ｍＭ ＮａＣＩ， １０ｍＭ ＣａＣ１_２）中に３７℃で４５分間インキュベートした。１Ｕのトロンビン（１ＮＩＨユニット＝０．３２４μｇ、Ｓｉｇｍａ， Ｄｅｉｓｅｎｈｏｆｅｎ， Ｇｅｒｍａｎｙ）を、そのトロンビン開裂部位を有する５ｐｍｏｌの融合分子に加えて、全容量で１５μｌのトロンビン緩衝液（５０ｍＭトリス ｐＨ８， １５０ｍＭ ＮａＣＩ）中に３７℃で４５分間インキュベートした。それら出発融合分子とタンパク質分解開裂後のそれら融合分子を、ＳＤＳ ＰＡＧＥ後、ホスホイメージャー（ｐｈｏｓｐｈｏｉｍａｇｅｒ）を使用して分析した（図４）。
【００４９】
図４は、溶液中のトロンビン及びＭＭＰ−３での融合分子消化を示す：
４〜２０％トリス−グリシンＳＤＳ ＰＡＧＥ後のホスホイメージャー画像
レーン１：消化前の“ＭＭＰ３融合分子”
レーン２：消化前の“トロンビン融合分子”
レーン３：“トロンビン融合分子”＋トロンビン
レーン４：消化前の“トロンビン融合分子”
レーン５：“ＭＭＰ３融合分子”＋ＭＭＰ３
レーン６：消化前の“ＭＭＰ３融合分子”
（バンドｉは融合分子を示し、それらバンドｉｉはペプチド副産物を示し、そして、バンドｉｉｉはＮ末端融合分子フラグメントに由来する。）
その“ＭＭＰ３及びトロンビン融合分子”は、それぞれＭＭＰ−３プロテアーゼ及びトロンビンプロテアーゼによりタンパク質分解開裂された。
【００５０】
実施例３
タンパク質分解開裂した“トロンビン融合分子”の単離と検出、及びその核酸部分の増幅
特異的にタンパク質分解開裂した融合分子は、３つの連続的工程を行うことにより単離され同定された。図５は、特異的開裂ペプチドを同定するための本実施例による方法の工程を概略図で示す。
【００５１】
工程１．融合分子のストレプトアクチン−セファロース（支持材料）へのＮ末端Ｓｔｒｅｐタグ（ｔａｇ）を介したカップリング
工程２．その支持材料上の“トロンビン融合分子”のトロンビンによるタンパク質分解開裂
工程３．開裂したＣ末端融合分子フラグメントの単離、及びその核酸部分のＰＣＲ増幅後の前記フラグメントの検出
場合により、それら単離したフラグメントは、それらのＨｉｓタグを介して、分析前のニッケル粒子へのアフィニティクロマトグラフィーにより精製し得る。
【００５２】
３．１　“トロンビン融合分子”のストレプトアクチンセファロースへのカップリング
５０μｌの“トロンビン融合分子”（約５ｐｍｏｌに相当する）を、５×ストレプトアクチン緩衝液（１５０ｍＭ ＮａＣＩ， １００ｍＭ トリス ｐＨ８）中で洗浄した５０μｌのストレプトアクチンセファロースと混合し、そして４℃で２時間インキュベートした。未結合の融合分子は、ストレプトアクチン緩衝液で５回洗浄することにより除去された。
【００５３】
３．２　“トロンビン融合分子”のトロンビンによるタンパク質分解消化
ストレプトアクチンセファロースに結合した融合分子を、８０μｌの水中に再懸濁し、そして１０μｌの１０×トロンビン緩衝液（１．５Ｍ ＮａＣＩ， ５００ｍＭ トリス ｐＨ８）及び１０μｌの１００Ｕ／μｌトロンビンと共に３７℃で４５分間インキュベートした。コントロールとして、同じ反応をトロンビン無しで行った。
【００５４】
３．２．１　結合した“トロンビン融合分子”の単離と検出
そのインキュベーション期間後、それら反応混合物を遠心分離させた（３０００ｒｐｍで１分間）。開裂した融合分子のＮ末端フラグメントを含有するペレット化ストレプトアクチンセファロースをストレプトアクチン緩衝液で３回洗浄し、次いで、８０μｌの水中に再懸濁した。ストレプトアクチンセファロースと結合した“トロンビン融合分子”（コントロール混合物）又はトロンビンとのインキュベーション後のストレプトアクチンセファロース結合Ｎ末端フラグメントをＳＤＳ ＰＡＧＥにより画分し、ホスホイメージングにより可視化した（図６）。
【００５５】
図６は、“トロンビン融合分子”及びストレプトアクチンセファロースと結合した融合分子フラグメントを示す。１５μｌの再懸濁化ストレプトセファロースを各ケースで５μｌのＬａｍｍｌｉ ｌｏａｄｉｎｇ緩衝液と混合し、８２℃で５分間加熱し、そして、４〜２０％トリス　グリシンＳＤＳ ＰＡＧＥにより画分した。次いでそのゲルを８０℃で２時間乾燥させ、ホスホイメージングにより可視化した。
【００５６】
レーン１：トロンビンとインキュベーション後の“トロンビン融合分子”
レーン２：トロンビンとのインキュベーションをしていない“トロンビン融合分子”（コントロール）
（バンドｉは無傷の融合分子を示し、バンドｉｉはペプチド副産物からのものであり、そして、バンドｉｉｉはＮ末端融合分子フラグメントに由来する。）
“トロンビン融合分子”をトロンビンとインキュベートした後は、そのＮ末端フラグメント（^３５Ｓ−メチオニンで標識されたもの）だけがストレプトアクチンセファロースと結合したままである。
【００５７】
３．２．２　“トロンビン融合分子”の核酸部分のＰＣＲ分析による検出
ストレプトアクチンセファロースを除去した後、その１５μｌの上澄み又は１５μｌの再懸濁したストレプトアクチンセファロースをＰＣＲに使用した。それら１５μｌ試料のほかに、その５０μｌのＰＣＲ混合物には、５μｌの５’と３’トロンビンプライマー（５ｐｍｏｌ／μｌ）、１０ｍＭ ｄＮＴＰｓ、５μｌの１０×ＰＣＲ緩衝液、及び５Ｕ／μｌ Ｔａｑ ＤＮＡポリメラーゼ（Ｐｒｏｍｅｇａ， Ｍａｄｉｓｏｎ， ＵＳＡからの緩衝液と酵素）も含ませた。その増幅は、下記の条件下：１分 ９４℃、２１×（０．５分 ９４℃、０．５分、５５℃ ０．５分 ７２℃）で行われた。得られたＰＣＲ産物を図７に示す。
【００５８】
図７は、２％アガロースゲル電気泳動後の“トロンビン融合分子”のＰＣＲ産物を示す。レーン１及び２は、それら産物を、明るいバックグラウンド上の黒色シグナルとして示し、レーン３及び４は、それら産物を、異なる画像技術により暗いバックグラウンド上の白色シグナルとして示す。
【００５９】
レーン１：“トロンビン融合分子”＋トロンビン；上澄み
レーン２：“トロンビン融合分子”トロンビンなし（コントロール）；上澄み
レーン３：“トロンビン融合分子”＋トロンビン；ストレプトセファロースと結合したもの
レーン４：“トロンビン融合分子”トロンビンなし；ストレプトセファロースと結合したもの
【００６０】
結果的に、“トロンビン融合分子”はトロンビンとのインキュベーションにより切断されたことが示される。核酸部分を有しないＮ末端フラグメントを除去した後、そのＣ末端部分はその上澄みに残る。従って、強力なＰＣＲ増幅物がその上澄み中に得られるが（例えば、レーン１、図７を見よ）、そのペレット中には非常に弱いＰＣＲ増幅物のみ得られる（例えば、レーン３、図７）。コントロール反応においては、“トロンビン融合分子”はトロンビンとインキュベートされなかった。従って、それら融合分子はストレプトアクチンセファロースに結合したままで、強力なＰＣＲ増幅物がそのペレット中に得られるが（例えば、レーン４、図７）、その一方、その上澄みのＰＣＲ増幅は非常に弱い（例えば、レーン２、図７）。
【図面の簡単な説明】
【図１】図１は、２％アガロースエチジウムブロマイドゲル上のＰＣＲ産物を示す。
【図２】図２は、６％尿素ゲル上の転写後に得られたＲＮＡを示す。
【図３】図３は、５％尿素ＴＢＥゲル上でのライゲーション反応の分析を示す。
【図４】図４は、溶液中のトロンビン及びＭＭＰ−３での融合分子消化を示す。
【図５】図５は、特異的開裂ペプチドを同定するための本実施例による方法の各工程を概略図で示す。
【図６】図６は、“トロンビン融合分子”及びストレプトアクチンセファロースと結合した融合分子フラグメントを示す。
【図７】図７は、２％アガロースゲル電気泳動後の“トロンビン融合分子”のＰＣＲ産物を示す。[0001]
The present invention relates to a method for finding and identifying a peptide having a predetermined amino acid sequence and specifically cleavable by using a proteolytically active solution, and for specific release of a chemically active substance and for diagnosis For the use of said peptides.
[0002]
US 5981200 describes a method for detecting a specific cleavable peptide by using a specific protein structure that fluoresces when it is proteolytically cleaved. The method is based on the fluorescence resonance energy technique (FRET). This method has the advantage that it can also be used for in vivo detection of the occurrence of protein cleavage. The disadvantages, on the other hand, lie in the fact that only certain peptides can be labeled, or that when examining different labeled proteins it would be very complicated to accurately associate the cleavage with the relevant sequence. The method has the further disadvantage that relatively large amounts of labeled substrate must be used to obtain a detectable signal.
Another method that can be used for the selective identification of enzyme substrates is phage display (as shown, for example, in WO 97/47314). In this method, the phage-displayed peptide library, in contrast to other surface-displayed peptide libraries, has a small number of differentiable sequences, and the subsequent acquisition of phage particles and determination of the identification substrate are relatively complicated. Has inconvenience.
[0003]
It is an object of the present invention to develop a method for finding and identifying cleavable peptides that are specifically proteolytic, and to provide substances that allow targeted release of a chemically active substance.
The object is a method for identifying a cleavable peptide that is specifically proteolytic, which is achieved by a method comprising the following steps:
a) incubating a library of fusion molecules comprising a peptide and a nucleic acid encoding said peptide with a proteolytically active sample;
b) isolating the proteolytically detached portion of the fusion molecule;
c) determining the sequence of the nucleic acid portion of the isolated fusion molecule.
[0004]
The method of the invention is based on using a fusion molecule comprising a phenotypic peptide portion and a genotype nucleic acid portion having a sequence encoding said peptide portion. The peptide moiety is linked to the nucleic acid moiety via a suitable linker. The linker used is preferably a protein acceptor, such as puromycin, which is covalently linked to the nucleic acid moiety. The linker may comprise further components, for example a non-coding nucleic acid sequence, preferably a polyA sequence. In a preferred embodiment, the nucleic acid portion comprises additional regions contiguous to the sequence and on one or both sides of the coding region. These contiguous nucleic acid regions can serve, for example, as primer binding regions for performing RCR or as restriction enzyme cleavage sites. For this reason, additional contiguous coding nucleotide sequences can be added. The contiguous nucleic acid sequence may encode a peptide section that allows the peptide portion of the fusion molecule to be easily attached to a solid surface or gives rise to a structural element of interest. Examples of such structural elements are epitopes for antibodies, tags for purifying or detecting those structures, or elements that determine a particular tertiary structure.
[0005]
Such fusion molecules can be prepared according to, for example, WO 98/31700, which describes a system in which a protein acceptor, such as puromycin, is linked to a nucleic acid, preferably RNA, via a suitable linker. This allows the synthetic protein to bind to the coding RNA and characterize it in more detail just before the in vitro translation of the RNA into the corresponding protein has ceased. Examples of equivalent systems that can be used for the present invention are described in DE 196 46 372 C1, WO 98/16636, US Pat. No. 5,843,701 and WO 94/13623.
[0006]
In a preferred embodiment of the method of the invention, the fusion molecules are attached to a surface (support) via their peptide moieties. The term "support" according to the present invention means a material that exists in a solid or gel-like form. Examples of suitable support materials are ceramics, metals, especially semiconductors, noble metals, glass, plastics, crystalline materials, or thin layers of such supports, in particular of said materials, and (bio) molecules such as cellulose and structural proteins. It is a filament. However, suitable supports are not only flat materials but also, for example, materials for column chromatography through which proteins can flow, or particles such as beads made of organic polymers. The support is generally covalently, quasi-covalently, supramolecularly or physically packed.
[0007]
The peptide portion of the fusion molecule can be attached to a support, which can mediate such attachment, such as a metal binding domain (eg, His tag), a terminal cysteine residue, or an epitope recognizable by a specific antibody. Through a domain that is biotin-streptavidin binding but not specific, such as a domain having
In the method of the present invention, it is preferred that the fusion molecule be provided to a library containing possible permutations for the variable portion of the peptide sequence. A practically usable library of such fusion proteins is approximately 10^ThirteenOf the other known peptide libraries.³-10⁴Times (eg phage display) or 10⁶-10⁴It is twice. To cover all recombination, it is preferably provided to a variable peptide portion of less than 11 amino acids, and especially to a variable peptide portion of 7 or 8 amino acids. Of course, it is possible to construct a library in which the fusion molecule has a longer variable peptide portion, but in this case it is no longer possible to completely cover the broad and diverse sequence. Proteases known today that cleave certain peptide bonds require a recognition sequence of at least 4 amino acids and are therefore preferably provided in a library containing a variable peptide portion of at least 4 amino acids.
[0008]
The library of fusion molecules is exposed to a proteolytically active sample in solution or, preferably, attached to a support. Suitable proteolytic activity samples, presamples or comparison samples are, in particular, tissue-specific extracts of animal, human or plant sources, such as cytoplasmic extracts, subcellular extracts, membranes An extract of a constituent, an extract of a particular cell compartment, such as an extracellular extract, or a mixture of such extracts. Also of particular interest are extracts of diseased tissues, such as carcinomas. However, it is also possible to use an extract which is representative of the total proteolytic activity of an organism, in particular of a microorganism such as a virus, bacterium or protist, or a part thereof. The sample, preliminary sample or comparative sample of proteolytic activity that can be used may be a solution containing a known protease.
[0009]
The library is incubated with the proteolytically active sample, preferably under physiological conditions, preferably at a temperature between 0C and 45C. For this purpose, the extracts to be tested for their proteolytic activity may be used directly, or they may be taken up in a suitable solvent such as, for example, saline and used for the incubation. Is also good.
During the incubation, the protease contained in the sample cleaves the variable peptide portion of the fusion molecule. This results in a specific proteolytic cleavage pattern that is characteristic of the sample extract used. The portion of the fusion molecule released by cleavage is encoded by the nucleic acid portion.
After incubating the library with the proteolytically active sample to be examined, the portion of the fusion molecule dissociated by proteolytic cleavage is isolated and the coding nucleic acid portion is sequenced. This identifies a cleavable peptide sequence with the sample.
[0010]
After incubation with the library and the proteolytically active extract in solution, the cleaved fusion protein can be isolated as follows. The nucleic acid regions of the truncated fusion proteins are no longer linked to the remainder of the fusion molecule containing the nucleic acid portion due to, for example, the use of sequential structural features within a particular peptide portion, e.g., proteolytic cleavage. The use of a known epitope for the antibody that is to be lost can be isolated from those of the uncleaved fusion protein. For this purpose, the mixture is subjected to affinity chromatography, for example by using said antibodies. The nucleic acid portion of the uncleaved fusion protein that cannot be bound is separated from the uncleaved protein nucleic acid and is present in the eluate. Also, other structural features may be used, such as, for example, a His tag or Strep tag for its retrieval. The fusion molecule retained on the affinity matrix may be eluted and used further in solution or, advantageously, directly in its attached state.
After incubation of the attached library with extracts, various methods may be used to isolate the truncated fusion protein.
[0011]
In a preferred method of the invention, after incubation with the extract of the attachment library, the nucleic acid portions of the truncated fusion molecules are obtained directly in the eluate.
In the simplest case, the sequences of the cleavage peptides can be directly identified by PCR amplification of the nucleic acid portion of the cleavage fusion protein present in the eluate, and then cloned and sequenced. However, first of all, by purifying the cleaved fusion protein from other components present in the eluate, by first using affinity chromatography and specific structures within the peptide or nucleic acid portion of the cleaved fusion protein Is also possible. For example, after eluting the cleaved fusion proteins from the chromatographic material by incubation with a KOH solution, the cleaved fusion proteins can also be sequenced by PCR, cloning and sequencing.
[0012]
In addition, many additional chromatographic or electrophoretic methods are possible to isolate those portions of the fusion molecule that have dissociated by proteolytic cleavage. For this purpose, the fusion molecules are conveniently labeled or modified for easier identification. Thus, for example, the nucleic acid portion of the fusion molecule may be, for example, fluorescently labeled or radioactively labeled. Magnetic labeling of the nucleic acid portion of the fusion molecule is particularly preferred, which may be used in conjunction with a fluorescent or radioactive label. The portion of the fusion molecule that is dissociated by cleavage is very easily and selectively magnetically isolated. The proteolysis pattern is assessed, for example, via hybridization of the isolated nucleic acid sequences on a biochip such as those available from Affymetrix or Nanogen. Thus, it is possible to directly analyze an isolated mixture of fusion molecule parts containing various nucleic acid sequences dissociated by cleavage.
[0013]
When preparing the extracts to be considered, care must be taken to isolate the proteases in their active form. However, at the same time, the DNA degrading nuclease should be inactivated as much as possible.
For this purpose, a proteolytically active sample can be prepared mechanically as follows. Whole tissue is isolated, where appropriate by washing its outside in cold buffer (50 mM Tris-HQ pH 7.5, 2 mM EDTA, 150 mM NaCI, 0.5 mM DTT; Diagnostic, JD, Preparation of Extracts from Higher Eukaryotes. From: Deutscher, MP (ed.), 19th edition, and a number of gasoline proofing. . The tissue is cut up in cold buffer, where it is suitably removed from components such as, for example, connective tissue, skin and blood vessels. The tissue piece was obtained from Kobayashi et al. (Kobayashi, H., Fujishiro, S., Terao, T., Cancer Res. (1994) 54, 65396548) (0.01 M HEPES buffer, pH 7.2, 0.25 M). Mix with sucrose, 0.5% Triton X100, volume ratio 1: 2 to 1: 5 (w / w) and treat to homogenate in a suitable tissue homogenizer (see below) with ice cooling. This law must be applied to the desired tissue type. Examples of suitable tissue homogenizers are:
-Mixer: the liver can be easily charged, for example, in a mixer and then rubbed through a dense mesh nylon net. Fine tissue homogenates are formed.
-"Potter" homogenizer: The rotating glass rod must be inserted tightly into the fitted container to ensure constant cooling, with tissue breaking between the container wall and the glass rod.
-Disperser: homogenizer with rotating blades on the shaft
[0014]
The homogenized tissue is mixed with additional homogenization buffer and incubated at 4-8 ° C with shaking for 45 minutes. The tissue homogenate is then centrifuged at 4 ° C. and 16,000 g, and the supernatant is used directly or after further purification, for example via column chromatography or dialysis, for analysis according to the invention.
This method should be modified if frozen material is used instead of fresh tissue. Frozen tissue should not be thawed immediately. In this case, the protease released from the lysosome inactivates those enzymes. Preferably, the tissue is ground with constant cooling in a mortar using liquid nitrogen and the powder is lyophilized overnight in a lyophilizer. The dry powder can then be used further.
[0015]
Examples of suitable processes for brain tissue and its separation into nuclear, cytoplasmic, membrane and cytoskeletal fractions are as follows. The extracts obtained may be used as proteolytically active samples, preliminary samples or comparative samples, either directly or with the addition of additional auxiliary substances.
Some preparations (about 50) from brain, eg mice, are introduced into about 25 ml of buffer solution 1 ((SB1) (20 mM Tris-HCl pH 73; 150 mM NaCI, 1 mM EDTA, 1 mM EGTA) It is then homogenized at maximum speed for 1 minute using an Ultraturrax, otherwise using a Potter glass homogenizer for low material.
[0016]
The nuclear fraction can be isolated by centrifuging the homogenate at 1000 × g at 4 ° C. for 10 minutes. The supernatant is collected for further preparation. The centrifuge (nuclear pellet) is resuspended in 12 ml of SB1 and centrifuged as before. The nucleic acid pellet is resuspended in SB2 (20 mM Tris-HCl pH 7.5; 150 mM NaCI, 1 mM EDTA, 1 mM EGTA, 1% NP-40).
The collected supernatant can be used to isolate and purify the cytoplasmic fraction. For this purpose, they are centrifuged at 100,000 × g at 4 ° C. for 1 hour. The supernatants are representative of extracts containing cytoplasmic proteins.
[0017]
The membrane fraction is isolated by washing the pellet from the cytoplasmic fraction removed three times in SB1 and centrifuging at 100,000 × g at 4 ° C. for 1 hour. After the third washing step, the pellet is resuspended in 15 ml of SB2 and lysed overnight before centrifugation at 100,000 × g at 4 ° C. for 1 hour. The supernatant forms the membrane fraction.
The cytoskeletal fraction is obtained by washing the pellet from the membrane fraction removed three times in SB2 and centrifuging at 100,000 × g at 4 ° C. for 1 hour. After the third washing step, the pellet is resuspended in SB3 (12.5 mM Tris-HCl pH 7.5; 0.4% SDS) and dissolved at 95 ° C. This is followed by a final centrifugation at 20,800 xg for 15 minutes at room temperature. The supernatant contains the cytoskeletal fraction.
[0018]
Proteolytically active extracts containing isolated proteases are obtained from cells or tissues, for example, by culturing human cells or cell lines as a monolayer in cell culture. The cells are washed three times with cell culture medium. The medium is then replaced with fresh medium and the secreted protease is secreted into the medium at 37 ° C. A suitable secretion time is 1-3 hours. Tissue pieces or tissue portions can be processed in the same manner. Tissue fluid can be obtained from a tissue mass, for example, by carefully chopping an organ or tissue with scissors or a scalpel. The pieces are washed several times with tissue buffer. The pieces or other organelles are incubated in physiological buffer or medium at 37 ° C for 1-3 hours. After incubation, the cell debris is sedimented into a 15 ml conical container, and the medium can be removed for further study.
[0019]
Alternatively, the tissue may be transferred into a centrifuge tube and centrifuged gently. The supernatant contains interstitial fluid protease from the interstitial space.
Alternatively, the following method: ice-cold RIPA buffer (50 mM Tris-HCI, pH 7.4; 1% NP40, 0.25% sodium cholate, 150 mM NaCI, 1 mM EGTA, 1 mM PMSF, 1 mM Na₃VO₄, 1 mM NaF, and in each case 1 μg / ml aprotonin, leupeptin and pepstatin) by chopping the tissue, freezing the minced tissue and subsequently disrupting it in a mortar, You can also get The tissue is homogenized by adding RIPA buffer in a volume ratio of 1-2, and then the mixture is thawed. After 15 minutes, the lysate is centrifuged at 13,000 × g at 4 ° C. for 10 minutes. The lysate may be aliquoted, shockfrozen and stored in liquid nitrogen.
[0020]
In a further modification, the proteolytic activity of the unexamined sample can be measured by comparison with a comparative sample. The method of differential determination of proteolysis comprises the following steps:
a) incubating a library of fusion molecules comprising a peptide and a nucleic acid encoding said peptide with a proteolytically active sample;
b) making the same library of fusion molecules into at least one parallel mix and incubating with at least one proteolytic activity comparison sample;
c) isolating the proteolytically removed portion of the fusion molecule;
d) 構築 building a bank of differential nucleic acids;
e) determining the sequence of those nucleic acids measured.
[0021]
Differential determination of the proteolytically removed nucleic acid portions of the fusion molecules can be performed, for example, by applying excess single-stranded nucleic acid portions isolated from the comparative sample to a suitable support and freeing the support. After saturating the binding site, it is performed by hybridization with a single-stranded nucleic acid moiety isolated from the sample. The removed non-hybridized nucleic acid portion indicates the only peptide sequence cleaved in the sample.
[0022]
In another method of the invention, the proteolytic activity of a sample is measured in comparison to at least one preliminary sample. The continuous incubation of the fusion molecule with the preliminary sample and the sample makes it possible to determine the differential proteolytic activity between said preliminary sample and the sample. The method comprises the following steps:
a) incubating a library comprising a peptide and a nucleic acid encoding said peptide with at least one preliminary sample of proteolytic activity;
b) 取 り 出 す removing the portion of the fusion molecule that is dissociated by cleavage;
c) {incubating the library thus prepared with a proteolytic sample;
d) isolating the portion of the fusion molecule that dissociates due to proteolytic cleavage;
e) determining the sequence of the nucleic acid molecule portion of the removed fusion molecule.
[0023]
In a preferred embodiment of all three variants of the invention, the nucleic acid part of the fusion molecule part isolated by proteolytic cleavage is doubled by PCR. For this purpose, fusion molecules are used whose coding nucleic acid part has a nucleic acid sequence which is continuous on both sides of the coding nucleic acid sequence as a primer binding site. In order to greatly increase both the amount of fusion molecule excised in the considered extract and the specificity of its selection step (the cleavage in its proteolytically active extract), the isolation of the cleaved fusion protein Preferably, the method of the present invention provides for amplifying the nucleic acid by PCR and translating it in vitro. Starting from the RNA prepared in this way, a new library of fusion proteins is prepared again by the method already described and incubated with the same sample containing the protein activity. This cycle can be repeated several times.
[0024]
In a further variation of the method of the invention, performing one or more of the described cycles of RNA, in vitro transcription, and / or in vitro translation with an unusually high error rate. Is possible. This increases the available peptide sequences to be tested (in vitro protein evaluation).
Similarly, various directed in vitro protein evaluations allow changing the known cleavage sequence in a rapid and simple manner to produce different kinetic properties for the cleaving protease.
[0025]
Additional additives, such as specifically acting protease or nuclease inhibitors, or non-specific RNA and / or DNA that saturates possible nucleases, may be added to the protein activity sample. For example, the addition of an inhibitor that acts specifically on the protease, such as the addition of pepstatin A, can be performed by lysosomal or endosomal proteases (such as aspartate peptidase) whose sample extracts cleave nonspecifically. (Acid peptidase) is particularly recommended. Also, specific inhibition of exopeptidases is advantageous to prevent degradation of proteins, especially other proteases and their peptide cofactors, contained in the sample, comparative sample or whole sample. The addition fusion molecule itself does not need to be protected against exoproteases, since there is no N- or C-terminal peptide terminus. Degradation of the peptide portion of the proteolytically cleaved fusion molecule is not critical to the further progress of the method.
[0026]
Removal of nuclease activity from the tissue extract to protect the nucleic acid pad of the fusion molecule can be achieved by treating the freshly prepared tissue homogenate with an RNAse or DNAse inhibitor between the tissue acquisition and subsequent steps. Can be achieved by doing so. In other embodiments, the nuclease inhibitors can be covalently associated with the particles or suitable column material. Thus, nucleases that bind to the surface-associated nuclease inhibitor are removed from the remaining tissue homogenate. Another approach to nuclease inhibition is protection of the nucleic acid portion of the fusion molecule by auxiliary substances. The auxiliary substances wrap the DNA and RNA to form a complex and protect the DNA and RNA against nucleases (Katayose, S. (1998) J. Pharm. Sci., 87: 160163). Said auxiliary substance is in particular polyethyleneimine or PEGPLL, but may also be a protein or a chaperone. Protection against nucleases is likewise possible by using artificially synthesized nucleic acids, such as PNA in fusion molecules. To increase stability, the nucleic acids may be chemically modified, for example, by methylation.
[0027]
The incubation time of the library with the extract depends on the proteolytic activity of the sample used. In an extended embodiment, the method of the invention is performed repeatedly with varying incubation times. This advantage allows for kinetic presentation of those peptide substances, including the sample and the library of fusion molecules. Peptide materials that are proteolytically cleaved in a short incubation time have a high rate constant for proteolytically active sample components.
Another possibility for making a kinetic presentation of the protein activity of the sample is to vary the concentration of the library fusion molecules that make up the peptide material or, preferably, intended for incubation of the sample. Or varying the concentration.
[0028]
A great advantage of the method of the invention is the fact that the protein activity of the sample under consideration can be demonstrated phenomenologically, ie without knowing all of the proteolytic activity of the sample. When comparing different samples, whether the different proteolytic activities are based on the presence of different proteases or rather on direct mutations of the rare proteases, or whether it is incomplete regulation of the protease gene or It does not matter whether it was caused by a protease inhibitor or a protease activator. The method may also record alterations in proteolytic activity induced via extracellular signals. For example, it is known that binding of a ligand, such as a hormone, to a transmembrane receptor can alter intracellular cytosolic kinase and / or phosphatase activity. The degree of phosphorylation has a decisive effect on protease activity as it plays a crucial part in the control of enzyme activity in cells. The phosphatase activity between deregulated cells and their associated protease activities, for example, play a crucial part in the formation of cancer.
[0029]
Of particular advantage is the determination of peptides that are differential and specifically cleavable with respect to extracts of healthy and diseased tissues and extracts of healthy tissues and those infected with pathogens. Finding specific cleavable peptides when comparing individual organisms is likewise of particular interest.
Furthermore, the method of the invention can be used to determine quickly and easily peptide sequences cleaved by the sample under consideration, especially those that have been specifically cleaved. On the other hand, sequences that have not undergone proteolytic cleavage by that sample examined can be found in a similar manner.
[0030]
In addition to identifying specific protein cleavage peptide sequences, the above methods can also be used to find specifically acting inhibitors or activators, which should be inhibited in each individual case. It is not required that the protease be known. To this end, a potential inhibitor or activator is simply added to the sample solution, resulting in differential proteolytic activity between the sample and the sample containing the potential inhibitor or activator. Is measured. A decrease in the number of cleaved sequences indicates inhibition, and an increase in cleaved sequences indicates activation.
In a preferred embodiment, the inhibitor is identified by using a pre-selected library of fusion molecules obtained after several selection cycles using the proteolytically active sample. Alternatively, the specific cleavage peptide identified by the method of the invention can be used directly for inhibitor screening, for example, in a FRET assay.
[0031]
In this way, screening methods for protease inhibitors are available, which can be performed quickly and easily.
The rapid and simple screening of protease inhibitors also makes it possible to combine many effective inhibitors in a mixture with the activator in order to prevent the emergence of their resistance.
The invention further relates to a proteolytic cleavable substance containing a peptide sequence obtainable using one of the methods of the invention.
Specific proteolytic cleavage peptide sequences can be used for specifically acting inhibitors. For this purpose, the peptide sequence may be chemically modified, for example, to prevent proteolysis, or one or more α-amino acids of the peptide acid may be replaced with β-amino acids. Alternatively, the L-amino acid may be replaced with a D-amino acid (see, eg, Werder M. & Hauser H. (1999) Helvetica Chimica Acta, 82, 1774-1783). Specific proteolytic cleavable peptides that can be identified using this method can be used to construct a drug delivery system to achieve selective release of the active agent at the target site. The target site can be, for example, a particular organ, tissue, a particular cell type, a subcellular component, or a diseased portion compared to a healthy tissue, or can be compared to an uninfected tissue or cell. And may be a part infected with a microorganism.
[0032]
In the case of active agents that can be activated by protease cleavage, the specific proteolytic cleavable agent can be used as a covalent inhibitor. The specific proteolytic cleavable peptide may be used as a linker to link the active agent to a targeting ligand, such as, for example, a specific antibody or a specific receptor ligand. However, it is also possible to construct active substance-loaded systems that close with specific proteolytic cleavable substances (Minko T. et al., (2000) Int. J. Cancer, 86: 108117). A variety of directed in vitro protein evaluations allow one to alter known peptide cleavage sequences to generate different cleavage kinetics. This makes it possible to control the time profile of the active substance release.
[0033]
As a result, it is possible to more effectively control the diseased part with the help of specific proteolytic cleavable peptides and via controlled release of the active substance to the target, where the healthy Proteolytic activity caused by altered or imperfect regulation compared to the state or mutation of a particular protease or their inhibitor is present at the desired site of action. Examples of disorders with disturbed protease activity include cancer such as asthma, osteoporosis, leukemia, breast cancer, intestinal cancer, stroke, neuropathy such as Alzheimer's disease, arthritis, pancreatitis, hypertension, thrombosis, cold, and homeostasis. If you have blood sickness.
The targeted release of the active substance can minimize the side effects of the active substance and reduce the amount of active substance used, thanks to the altered distribution profile within the organism . Of particular interest are pharmaceutically active ingredients, for example against bacteria, fungi, viruses or against affected tissue cells, but may be used in herbicides, antifungals or insecticides.
[0034]
Another use of the method of the invention is as a diagnostic assay. The assay is used, for example, to compare profiles between diseased tissue from cancer cells and a sample to be examined, for example, towards a fusion molecule library with a given composition of extract proteolytic activity. It can be based on However, preference is given to a peptide-based assay that is proteolytically cleavable only by diseased tissue, as compared to a comparative or preliminary sample. In such a selective assay, the intended sequencing step following the removal of the fusion molecule portion dissociated by cleavage can be omitted. In this case, the presence of a particular disease-specific protease activity can be identified using a hybridization probe. Such methods can be highly integrated using many cleavable peptides identified as disease-specific markers in such assays.
[0035]
Alternatively, the specific cleavable peptides identified by the method of the invention can be used directly for inhibitor screening, for example in a FRET assay. Specific cleavable sequences may be used in vivo as agents for FRET assays. Peptides that can be specifically cleaved by a proteolytically active sample may be used to isolate proteases that cleave the peptide sequence (eg, Li YM (2000) Nature, 405: 689694).
For example, US Pat. No. 5,843,701 describes a method applicable for isolating serine proteases. Serine proteases are protein enzymes that catalyze the hydrolysis of peptide bonds in proteins. Frequently, the target protein is selectively cleaved due to specific peptide linkages within the material. These serine proteases include, inter alia, those that activate tissue plasminogen, trypsin, elastase, chymotrypsin, thrombin, and plasmin. Many stages of the disease can be treated, for example, with serine protease inhibitors such as blood clotting disorders. Elastase inhibitors can reduce the clinical progression of emphysema.
[0036]
The protease is coupled to the column material with the specific proteolytic cleavable peptide determined by the method described above, for example, by affinity chromatography of a proteolytically active sample using standard techniques on the column material. By performing the above, it can be isolated. The buffers used during these binding cycles must not be denatured so that the protease is inactivated. Proteases that interact with the peptides can, if necessary, additionally be attached to the attached peptides via common crosslinking methods. For this purpose, it is also possible to use β-amino acid-containing cleavage sequences which are specifically recognizable but not cleavable by proteases.
The present invention is made clear by the following several exemplary embodiments.
[0037]
Example 1
Preparation of a fusion molecule comprising a peptide having a known protease cleavage site and a nucleic acid encoding the peptide
General description
Two types of fusion molecules were prepared. The first fusion molecule is one containing a peptide sequence that is specifically cleaved by the protease thrombin ("thrombin fusion molecule") and the second fusion molecule is specific to matrix metalloprotease-3 (MMM-3). A fusion molecule having a peptide sequence that was cleaved dynamically ("MMM-3 fusion molecule"). The preparation of fusion molecules of this type is described, for example, in WO 98/31700.
Those fusion molecules prepared in this manner have a protease cleavage site for MMM-3 or for thrombin.
MMM-3 protease cleaves between Glu and Leu, and thrombin protease cleaves between Arg and Ser. The peptide sequences and corresponding nucleotide sequences used are as listed below.
[0038]
[Table 1]

[0039]
1.1 Preparation of MMP3 template DNA and thrombin template DNA via PCR
250 ng of DNA template, 25 pmol of 5 ′ and 3 ′ primers, 10 mM dNTPs, 5 μl of 10 × PCR buffer and 5 U / μg Taq DNA polymerase in a 50 μl reaction mixture (buffers and enzymes from Promega, Madison, USA) Combined. The PCR was performed in a Biometra T gradient cycler: 1 min 94 ° C, 21x (0.5 min 94 ° C, 0.5 min 55 ° C, 0.5 min 72 ° C).
[0040]
[Table 2]

[0041]
FIG. 1 shows the PCR products on a 2% agarose ethidium bromide gel. Lane 1: 50 bp DNA marker, Lane 2: MMP3 DNA, Lane 3: Thrombin DNA, Lane 4: 100 bp DNA marker
As FIG. 1 shows, MMP3 DNA and thrombin DNA were successfully amplified. The PCR product has the expected molecular weight of 199 bp for MMP3 DNA and 189 bp for thrombin DNA.
[0042]
1.2 In vitro transcription of MMP3 and thrombin DNA
100 pmol of DNA was used for the transcription reaction (Ribomax T7 / Promega, Madison, USA). The DNA was made up to a 500 μl mixture with 5 × T7 buffer, rNTPs and T7 RNA polymerase and incubated at 37 ° C. for 4 hours.
RNA was purified by phenol / chloroform extraction and analyzed on a 6% urea gel (Novex Gel / Invitrogen, Groningen, Netherlands).
FIG. 2 shows the RNA obtained after transcription on a 6% urea gel. Lane 1: MMP3 RNA, Lane 2: thrombin RNA
As FIG. 2 shows, RNA can be detected after its transcription reaction as expected. The RNA is not degraded.
[0043]
1.3 Ligation of RNA via UV crosslinking with puromycin linker
3 nmol of RNA, 4.5 nmol of puromycin (Pu) linker having the sequence (PEG = polyethylene glycol, Pso = psoralen)
[0044]
[Table 3]

[0045]
12 μl of 10 × ligase buffer (1M NaCl, 250 mM Tris pH 7.5) was combined with 120 μl of the mixture and incubated at 85 ° C. for 5 minutes and then at room temperature for 10 minutes. UV crosslinking was carried out at 366 nm for 15 minutes (LTF-Labortechnik, handheld UV lamp with 12 W).
Ligation efficiency was confirmed on a 5% urea agarose gel.
FIG. 3 shows an analysis of the ligation reaction on a 5% urea TBE gel. Lane 1: MMP3 RNA, Lane 2: MMP3 RNA with puromycin linker, Lane 3: Thrombin RNA, Lane 4: Thrombin RNA with puromycin linker (the signal L is due to the colored marker)
After ligation of the puromycin linker to the RNA, a distinct increase in the molecular weight was observed, indicating that thrombin RNA and MMP3 RNA were successfully linked to puromycin.
[0046]
1.4@In vitro translation
8 μl of linker RNA is mixed with 200 μl of erythrocyte lysate (Promega, Madison, USA L416X) and 5 μl of³⁵S methionine (Hartmann, 10.8 μM, specific activity: its specific activity is 72181 dpm / pmol), 6 μl of the amino acid mix (without methionine, 1 mM, Promega), and 80 μl of H₂Mix O and then incubate at 30 ° C. for 30 minutes. 130 μl of 2M KCI and 75 μl of MgCl₂After addition of, an incubation is performed at 30 ° C. for 30 minutes. The resulting fusion molecule was purified using oligo dT cellulose (Amersham Pharmacia, Freiburg, Germany) (removal of all those components of the in vitro translation mixture that do not contain the poly A sequence).
[0047]
1.5 Reverse transcription reaction
200 μl of the purified fusion molecule was mixed with 2.5 μl of 100 μM reverse primer and incubated at 80 ° C. for 5 minutes. After the reaction mixture was cooled on ice, 50 μl of 5 × strand buffer, 20 μl of dNTPs (10 mM in each case), 2.5 μl of RTSuperscript II (all from Promega, Madison, USA) were added. The mixture was incubated at 42 ° C. for 40 minutes.
[0048]
Example 2
Proteolytic cleavage of fusion molecules in solution by thrombin and MMP3
The prepared fusion molecules were incubated with the corresponding protease, and the reaction products were analyzed (FIG. 4). 10 nM MMP3 (Sigma, Deisenhofen, Germany) was added to 5 pmol of the fusion molecule with its MMP3 cleavage site and the mixture was made up to a total volume of 15 μl MMP3 buffer (50 mM Tris pH 7, 150 mM NaCI, 10 mM CaC1).₂) At 37 ° C. for 45 minutes. 1 U of thrombin (1 NIH unit = 0.324 μg, Sigma, Deisenhofen, Germany) was added to 5 pmol of the fusion molecule with its thrombin cleavage site in a total volume of 15 μl of thrombin buffer (50 mM Tris pH 8, 150 mM NaCI). At 37 ° C. for 45 minutes. The starting fusion molecules and the fusion molecules after proteolytic cleavage were analyzed after SDS PAGE using a phosphoimager (FIG. 4).
[0049]
FIG. 4 shows fusion molecule digestion with thrombin and MMP-3 in solution:
Phosphoimager image after 4-20% Tris-glycine SDS PAGE
Lane 1: “MMP3 fusion molecule” before digestion
Lane 2: “Thrombin fusion molecule” before digestion
Lane 3: “thrombin fusion molecule” + thrombin
Lane 4: “Thrombin fusion molecule” before digestion
Lane 5: “MMP3 fusion molecule” + MMP3
Lane 6: “MMP3 fusion molecule” before digestion
(Band i indicates the fusion molecule, those bands ii indicate the peptide byproduct, and band iii is from the N-terminal fusion molecule fragment.)
The “MMP3 and thrombin fusion molecule” was proteolytically cleaved by MMP-3 protease and thrombin protease, respectively.
[0050]
Example 3
Isolation and detection of proteolytically cleaved "thrombin fusion molecule" and amplification of its nucleic acid portion
Specifically proteolytically cleaved fusion molecules were isolated and identified by performing three sequential steps. FIG. 5 shows schematically the steps of a method according to this example for identifying a specific cleavage peptide.
[0051]
Step 1. Coupling of the fusion molecule to Streptactin-Sepharose (support material) via N-terminal Strep tag (tag)
Step 2. Proteolytic cleavage of "thrombin fusion molecules" on its support material by thrombin
Step 3. Isolation of the cleaved C-terminal fusion molecule fragment and detection of said fragment after PCR amplification of its nucleic acid portion
Optionally, the isolated fragments can be purified via their His tag by affinity chromatography on nickel particles prior to analysis.
[0052]
3.1 Coupling of “thrombin fusion molecule” to streptactin sepharose
50 μl of “thrombin fusion molecule” (corresponding to about 5 pmol) is mixed with 50 μl of streptactin sepharose washed in 5 × streptactin buffer (150 mM NaCI, 100 mM Tris pH 8) and incubated at 4 ° C. for 2 hours did. Unbound fusion molecules were removed by washing 5 times with streptactin buffer.
[0053]
3.2 Proteolytic digestion of “thrombin fusion molecule” with thrombin
The fusion molecule bound to Streptactin Sepharose is resuspended in 80 μl water and incubated for 45 minutes at 37 ° C. with 10 μl 10 × thrombin buffer (1.5 M NaCI, 500 mM Tris pH 8) and 10 μl 100 U / μl thrombin. did. As a control, the same reaction was performed without thrombin.
[0054]
3.2.1 Isolation and detection of bound “thrombin fusion molecule”
After the incubation period, the reaction mixtures were centrifuged (1 min at 3000 rpm). The pelleted streptactin sepharose containing the N-terminal fragment of the cleaved fusion molecule was washed three times with streptactin buffer and then resuspended in 80 μl of water. Streptactin Sepharose-bound "thrombin fusion molecule" (control mixture) or Streptactin Sepharose-bound N-terminal fragment after incubation with thrombin was fractionated by SDS PAGE and visualized by phosphoimaging (Figure 6).
[0055]
FIG. 6 shows a “thrombin fusion molecule” and a fusion molecule fragment bound to streptactin sepharose. 15 μl of resuspended streptosepharose were mixed in each case with 5 μl of Lammli loading buffer, heated at 82 ° C. for 5 minutes and fractionated by 4-20% Tris glycine SDS PAGE. The gel was then dried at 80 ° C. for 2 hours and visualized by phosphoimaging.
[0056]
Lane 1: "Thrombin fusion molecule" after incubation with thrombin
Lane 2: “Thrombin fusion molecule” not incubated with thrombin (control)
(Band i indicates the intact fusion molecule, band ii is from the peptide byproduct, and band iii is from the N-terminal fusion molecule fragment.)
After incubating the “thrombin fusion molecule” with thrombin, its N-terminal fragment (³⁵Only those labeled with S-methionine) remain bound to streptactin sepharose.
[0057]
3.2.2 Detection of nucleic acid portion of “thrombin fusion molecule” by PCR analysis
After removing the streptactin sepharose, 15 μl of the supernatant or 15 μl of the resuspended streptactin sepharose was used for PCR. In addition to the 15 μl samples, the 50 μl PCR mixture contained 5 μl of 5 ′ and 3 ′ thrombin primers (5 pmol / μl), 10 mM dNTPs, 5 μl of 10 × PCR buffer, and 5 U / μl Taq DNA polymerase (Promega). , Madison, USA). The amplification was performed under the following conditions: 1 minute at 94 ° C., 21 × (0.5 minutes at 94 ° C., 0.5 minutes, 55 ° C., 0.5 minutes at 72 ° C.). The resulting PCR product is shown in FIG.
[0058]
FIG. 7 shows the “thrombin fusion molecule” PCR product after 2% agarose gel electrophoresis. Lanes 1 and 2 show their products as black signals on a light background, and lanes 3 and 4 show them as white signals on a dark background with different imaging techniques.
[0059]
Lane 1: “thrombin fusion molecule” + thrombin; supernatant
Lane 2: “thrombin fusion molecule” without thrombin (control); supernatant
Lane 3: “thrombin fusion molecule” + thrombin; bound to streptosepharose
Lane 4: "thrombin fusion molecule" without thrombin; bound to streptosepharose
[0060]
The result indicates that the "thrombin fusion molecule" was cleaved by incubation with thrombin. After removing the N-terminal fragment without the nucleic acid portion, the C-terminal portion remains in the supernatant. Thus, a strong PCR amplification is obtained in the supernatant (eg, lane 1, see FIG. 7), but only a very weak PCR amplification is obtained in the pellet (eg, lane 3, FIG. 7). . In a control reaction, the "thrombin fusion molecule" was not incubated with thrombin. Thus, while the fusion molecules remain bound to the streptactin sepharose, a strong PCR amplification is obtained in the pellet (eg, lane 4, FIG. 7), while the PCR amplification of the supernatant is very weak (Eg, lane 2, FIG. 7).
[Brief description of the drawings]
FIG. 1 shows the PCR products on a 2% agarose ethidium bromide gel.
FIG. 2 shows RNA obtained after transcription on a 6% urea gel.
FIG. 3 shows an analysis of the ligation reaction on a 5% urea TBE gel.
FIG. 4 shows fusion molecule digestion with thrombin and MMP-3 in solution.
FIG. 5 shows schematically each step of the method according to this example for identifying a specific cleavage peptide.
FIG. 6 shows a “thrombin fusion molecule” and a fusion molecule fragment bound to streptactin sepharose.
FIG. 7 shows the “thrombin fusion molecule” PCR product after 2% agarose gel electrophoresis.

Claims (34)

A method for identifying a cleavable peptide that is specifically proteolytic, comprising the steps of:
a) incubating a library of fusion molecules comprising a peptide and a nucleic acid encoding said peptide with a proteolytically active sample;
b) isolating the proteolytically detached portion of the fusion molecule;
c) determining the sequence of the nucleic acid portion of the removed fusion molecule. A method for identifying a cleavable peptide that is specifically proteolytic, comprising the steps of:
a) incubating a library of fusion molecules comprising a peptide and a nucleic acid encoding said peptide with a proteolytically active sample;
b) incubating the same library of fusion molecules in a peer mixture with at least one proteolytic activity control sample;
c) isolating the proteolytically detached portion of the fusion molecule;
d) building a bank of differential nucleic acids;
e) determining the sequence of the nucleic acid measured. A method for identifying a cleavable peptide that is specifically proteolytic, comprising the steps of:
a) incubating a library of fusion molecules comprising a peptide and a nucleic acid encoding said peptide with a preliminary sample of proteolytic activity;
b) removing the portion of the fusion molecule that has been dissociated by cleavage;
c) incubating the library thus prepared with a proteolytically active sample;
d) isolating the portion of the fusion molecule that dissociates due to proteolytic cleavage;
e) determining the sequence of the nucleic acid portion of the detached fusion molecule. 4. The method according to any one of the preceding claims, wherein a library of fusion molecules comprising a peptide and a nucleic acid encoding said peptide and having a known nucleotide sequence flanked on one or both sides is used. A library of fusion molecules comprising a peptide, wherein said peptide is linked via a puromycin molecule to a polyA nucleic acid sequence, which leads to said peptide coding region and one or more known nucleic acid sequences. 5. The method according to claim 1, wherein a rally is used. The method according to any one of claims 1 to 5, wherein a fusion molecule comprising a peptide composed of at least four variable amino acid sequences is used. The method according to any one of claims 1 to 6, wherein a fusion molecule comprising a peptide composed of 7 to 11 variable amino acid sequences is used. The method according to any one of claims 1 to 7, wherein fusion molecules are used, which comprise a peptide having at least one contiguous sequence region in addition to the variable sequence region. 9. The method according to any one of claims 1 to 8, wherein a fusion molecule having a labeled nucleic acid part and / or peptide part is used. 10. A fusion molecule having a magnetic, radioactive, or fluorescent label on the nucleic acid and / or peptide moiety or a label with a specific known nucleic acid sequence and / or a specific amino acid sequence is used. The method described in. The method according to any one of claims 1 to 10, wherein the fusion molecules of the library are attached to a solid surface. 12. The method according to any one of the preceding claims, wherein the fusion molecules of the library are attached to solid particles. The method according to any one of claims 1 to 12, wherein the known nucleic acid sequence is cleaved by a restriction enzyme, and only the cleaved or excised nucleic acid portion is further used. 14. The method according to any one of claims 4 to 13, wherein after isolating the fusion molecule portion cleaved by the proteolytically active sample, PCR is performed to double the nucleic acid portion. 15. The method according to claim 14, wherein the amplified nucleic acid is used to construct a new library of fusion molecules, and the method cycle according to any of claims 1 to 3 is performed using the library. the method of. 15. The method according to claim 14, for in vitro protein evaluation, wherein the PCR, the in vitro transcription, and / or the in vitro translation are performed with an increased error rate. . The proteolytic activity sample, the preliminary sample and the comparative sample used may be a whole cell extract, a cytoplasmic extract, a membrane extract, an extracellular extract, a subcellular extract, a combination of the extracts, or a known protease. The method according to any one of claims 1 to 16, which is a solution of 18. The method according to any one of claims 1 to 17, wherein the samples, preliminary samples and comparative samples used are disease-specific and / or tissue-specific and / or organ-specific and / or biological-specific extracts. The described method. 19. The method according to any one of the preceding claims, wherein the proteolytically active sample and / or preliminary sample used is used in the method under physiological conditions. 20. The method according to claim 1, wherein a further auxiliary substance is added to the sample and / or the preliminary sample. 21. The method according to claim 20, wherein the auxiliary substance added is a specifically acting protease inhibitor and / or nuclease inhibitor. 22. The method of any one of claims 1-21, wherein a potential protease inhibitor or protease activator is added to the sample. 23. The method according to any of the preceding claims, wherein the steps of the method are repeated or with different incubation periods and / or different sample concentrations. Use of the nucleic acid sequence determined by the method according to any one of claims 1 to 23 for preparing a specifically proteolytic cleavable substance. 24. A specifically proteolytic cleavable substance comprising a peptide obtainable by the method according to any one of claims 1 to 23. 26. The specific proteolytic cleavable substance of claim 25, comprising a chemically active substance. 27. The specific proteolytic cleavable substance of claim 26, wherein the chemically active substance is activatable by proteolytic release. 28. The specific proteolytic cleavable substance according to any one of claims 25 to 27, wherein further auxiliary substances and additives are added to the substance. 29. The specific proteolytic cleavable substance according to any one of claims 25 to 28, wherein the substance is asthma, osteoporosis, cancer, stroke, neuropathy, arthritis, pancreatitis, hypertension, thrombosis, cold, or schistosome. Use for the manufacture of a medicament for illness. A diagnostic marker comprising a proteolytic cleavable peptide obtainable by the method according to any one of claims 1 to 13. A method for detecting the specific acting protease according to any one of claims 1 to 23, wherein the method comprises the diagnostic marker according to claim 30. 24. Use of a substance comprising a specific proteolytic cleavable peptide determined by the method of any one of claims 1 to 23 for identifying the protease that cleaves said peptide. Use of a specific proteolytic cleavable peptide determined by the method of any one of claims 1 to 23 for identifying a protease inhibitor. Use of a specific proteolytic cleavable peptide sequence determined by the method according to any one of claims 1 to 23 for preparing an inhibitor.

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