JP2005504505A - Protein in porous carrier - Google Patents

Abstract

A protein system is described in which proteins are bound within a matrix material having pores sized to achieve superior properties such as activity, protein density, and stability. In a preferred embodiment, the pore size ranges from 50 to 400 Angstroms. One protein that has shown surprisingly good results in this system is OPH. This protein is well known to degrade organophosphorus compounds found, for example, in chemical weapons and pesticides. A novel method for forming a protein system and a method for producing OPH are also described. Also described is a protein system comprising a porous matrix material and a protein disposed within the porous matrix material, wherein the protein system comprises at least 0.01 mmol protein per gram of matrix material and under the same conditions A protein system that exhibits at least twice as much activity as the protein system formed on the surface of a normal silica matrix material. A method for producing a protein system using non-covalent bonds to incorporate proteins into a porous matrix is also described.

Description

【００５０】
ＢａｍＨＩ制限酵素切断部位を有するＯＰＨ下流プライマー（認識配列は太字の箇所）、２０マー：
【００５１】
【化２】

【００５２】
ＯＰＨ配列を、ＮｄｅＩ、ＢａｍＨＩ制限酵素切断部位を使用してｐＥＴ１１ａ中にサブクローニングした。得られたＯＰＨ配列は、ＯＰＨ酵素の成熟部分をコード化し、すなわち、Ｎ末端の２９個のアミノ酸シグナル配列を有せず、その結果、配列の長さは１０１０bpであり、これは合計３３７個のアミノ酸またはＭＷ３６，４１９Daに相当する。
【００５３】
クローニングの正しい生成物が得られたことの確認を、ＰＣＲ及び制限酵素消化によって行った。
ＯＰＨのプラスミドの図を図２に示す。
発現及び精製：
有機リンヒドロラーゼを、ｏｐｈ−ｐＥＴ１１ａプラスミド及びＢＬ２１（ＤＥ３）ｐＬｙｓｓ^TM、ノバジェンＩｎｃ．、を宿主株として使用して、大腸菌発現系から精製した。（ｐＥＴ−１５ｂプラスミド中にサブクローニングされたＮ末端Ｈｉｓ−Ｔａｇ^TMＯＰＨ）。ＩＰＴＧを用いた誘発は、ＳＤＳゲル表面で明白な移動度を有するタンパク質を生成することを示し、Ｎ末端シグナル配列（約３６kDaまたはＮ末端Ｈｉｓ−Ｔａｇ約３８kDaを有する）を有しない成熟した天然ＯＰＨタンパク質の遺伝子配列に基づく予測と一致した。ＩＰＴＧを用いた誘発の後、組み換え生成物の素性をまた、アミノ酸分析及び未精製の細胞溶解産物中のパラオキソン加水分解活性の出現によって確認した。
【００５４】
バイオ−フロ３’０００発酵槽^TM（Bio-Flo 3'000 fermenter^TM）（ニューブランズウィック、Ｉｎｃ．（New Brunswick Inc.））中での細胞の増殖のために使用する一般的なプロトコールは次の通りだった：
大腸菌細胞を、１２時間、３０℃でフラスコ中で増殖させ、１００μLのグリセロールストック／１LのＬＢ培地、１００μg/mLのアンピシリン、３５μg/mLのクロラムフェニコール、及びこの出発培養を発酵槽の接種材料として使用した。ＯＤ＝０．５を有する５００mLのＯＮ培養を遠心沈殿し、新しいＬＢ培地を用いて洗浄し、再度遠心し、抗生物質を含まない２５０mLのＬＢ中に再溶解させ、発酵槽培地に加えて、総体積＝２．５Lだった。（注意：１mMのＣｏＣｌ₂をＯＮ出発培養用のＬＢ培地に加えないことを確実にする。というのはこれは細胞を殺すからである）。
【００５５】
発酵槽中の細胞は、４時間後に、３７℃で、５g/Lの酵母エキス、１０g/Lのペプトン、５g/LのＮａＣｌ、１mL/Lの泡止め剤、６０mMのＫ₂ＨＰＯ₄、１５mMのＫＨ₂ＰＯ₄、１mMのＣｏＣｌ₂、１．３２μg/mLのチアミン、１００μg/mLのアンピシリン、３５μg/mLのクロラムフェニコール、１０g/Lのグリセロール及び微量金属（１０μMのＮＨ₄Ｍｏ₇Ｏ₂₄、ＣｕＳＯ₄、Ｈ₃ＢＯ₃、ＭｎＣｌ₂、ＺｎＣｌ₂）、５０μMのＦｅＣｌ₃、０．５mMのＣａＣｌ₂、１mMのＭｇＳＯ₄を含む培地中で、中間対数期（mid-log phase）に達した。ＤＯ−撹拌−酸素三重カスケード（DO-agitation-oxygen triple cascade）を使用し、撹拌範囲を最小２００rpm〜最大８００rpmに設定して、酸素レベルを３５％に維持した。培地中の初期グルコース濃度は１０g/Lであり、実験の間中、通常のグルコースストリップ（regular glucose strip）を使用してグルコースレベルをモニターして、２g/L以上になるように維持した。
【００５６】
撹拌が４６７rpm、ＯＤ５５０＝５に達した時に、グルコースレベルは２g/Lだった。３１mLの４０％グルコースを加えて、グルコースレベルを５g/Lにした。撹拌が７００rpm、ＯＤ５５０＝１５に達した時に、グルコースレベルは再度２g/Lに低下した。温度は２８℃に低下し、混合物を、０．２５mMのＩＰＴＧを用いて誘発し、さらに３１mLの４０％グルコースを加えた。２８℃で４時間誘発した後、さらに０．２５mMのＩＰＴＧを加えた（合計０．５mM）。グルコースレベルは再度２g/Lであり、６３mLの４０％グルコースグルコースを加えて、グルコースレベルを１０g/Lにした。さらに２時間誘発した後、細胞は明らかに増殖し続け、温度は２４℃に低下し、細胞をさらに１４時間、発酵槽ＯＮ中に残した。最後に、６，０００rpm、２０分間、４℃の遠心分離によって細胞を集めた。
【００５７】
２．５Lの細胞培養から、約１５０gの湿潤重量の細胞ペーストを単離し、細胞ペーストを−８０℃で貯蔵した。
本発明者らは、６０gの細胞（１Lの培養に対応する）から、１３，２９４．１２酵素単位／mgの活性を有する約９０mgのＯＰＨを精製することができた。この収率を文献と比較できる。Omburo G.A., Kuo J.M., Mullins L.S., and Raushel F.M., in Characterization of the Zinc Binding site of Bacterial Phosphotriesterase. JBC, 1992, v. 267 (5): 13278-13283は、１６０gの細胞から８’０２０酵素単位／mgの活性を有する約２９８mgのコバルトホスホトリエステラーゼを得たことを報告した。
【００５８】
Lai K., Dave K. I., and Wild J.R. (Bimetallic Binding Motifs in Organophosphorous Hydrolase Are Important for Catalysis and Structural Organization. JBC, 1994, 269 (24): 16579-16584)は、比較するのがより困難であるが、５mgのＯＰＨ／１Lの培養（恐らくフラスコ中で増殖させた）を精製したことを報告した。彼らは、Ｔ７のような強いプロモーターに対して、発現用に弱いプロモーター（天然のプラク（native Plac））を使用することを好み、というのは、強いプロモーター構成体を用いて彼らが得たＯＰＨ活性の収率はより低かったからである（データは示さない）。全ての精製工程を、予め冷却した装置及びレブコクロマトグラフィー冷蔵キャビネット（Revco Chromatography Refrigeration cabinet）を使用して４℃で実行した。
【００５９】
細菌細胞（６０g）を、１００mMのＨＥＰＥＳ（pH８．５）、５０μMのＣｏＣｌ₂、１mMのＤＴＴ、抗プロテアーゼカクテル（ペプスタチン、ロイペプチン及びアプロチニン）を含む４２０mLの溶菌緩衝液Ａ中に懸濁させ、フレンチ圧力セル（French pressure cell）を２回使用して細胞を溶菌した。可溶性タンパク質の上澄みを、１時間の１００，０００×gの遠心分離（アバンティ^TM、ベックマン（Avanti^TM, Bechman））によって得、１mMのＤＴＴを５mMのベータ−メルカプトエタノール（２−ｍｅ）の代わりに用いた緩衝液Ａ中で平衡化した５００mLのＨＱ^TM陰イオン交換カラム（パーセプティブ・バイオシステムズ（Perseptive Biosystems））に負荷した。カラム流量は２５mL／分だった。ＯＰＨを含むフロースルーを集め、１MのＭＥＳ（pH５．５）を使用してpHを７．５に調節し、０．１MのＨＥＰＥＳ（pH７．５）、５０μMのＣｏＣｌ₂、５mMの２−ｍｅ中で平衡化した２５０mLのＨＳ陽イオン交換カラム（パースペクティブ・バイオシステムズ（Perspective Biosystems））に適用した。カラム流量は２５mL／分だった。ＨＳカラムのフロースルーを保持し、十分に乾燥した（ＮＨ₄）ＳＯ₄を加え、オリオン １２６、セル ０１２２１０（Orion 126, Cell 012210）導電率計を使用して、試料の最終伝導率を１Mの（ＮＨ₄）ＳＯ₄溶液の伝導率（すなわち、１０５mS/mL）と等しくした。試料を、１Mの（ＮＨ₄）ＳＯ₄、０．１MのＨＥＰＥＳ（pH７．５）、５mMの２−ｍｅ中で平衡化した１８０mLのポリプロピルＡ^TM（ポリエルシー、Ｉｎｃ．）カラムに負荷した。カラム流量は１０mL／分だった。
【００６０】
平衡化緩衝液を用いた３カラム体積の洗浄の後、０．１MのＨＥＰＥＳ（pH７．５）までの１０カラム体積の勾配を利用し、ＯＰＨを勾配の最末端で溶出した。本発明者らは、１００mg近くの純粋なＯＰＨを６０gの細胞（１Lの培養に対応する）から精製することができた。３０ＫＮＭＷＬ（３０kDaカットオフ）膜を用いたミリポア・ウルトラフリー（登録商標）バイオマックス（Millipore Ultrafree（登録商標） Biomax）遠心濃縮器と、２０％グリセロールに対し、０．１MのＨＥＰＥＳ（pH７．５）、５０μMのＣｏＣｌ₂への透析とを使用してタンパク質を３mg/mLまで濃縮した後、タンパク質を分取し、−８０℃で貯蔵した。比活性を測定して１３，２９４．１２酵素単位／mgだった（表Ｉ及びＳＤＳゲルの要約を参照されたい）。
【００６１】
ＨＩＣからピークとして現れたタンパク質の活性と濃縮及び透析後の同じタンパク質の活性との差は恐らく、緩衝液の交換によって説明されよう。１つの場合に、緩衝液は、ＯＰＨ活性の競合阻害剤であるベータ−メルカプトエタノールを含み、ＣｏＣｌ₂を含まず、他の緩衝液は５０μMのＣｏＣｌ₂を含み、２−ｍｅを含まなかった。
【００６２】
【表１】

【００６３】
ＯＰＨ固定化：
使用した媒質：
１．ＳＡＭＭＳ：ＳＨ−、ＣＯＯ−、ＮＨ₂−活性基を用いて誘導体化し、５％及び２０％のコーティングを施した（全ての利用可能なシラン基の５％及び２０％を活性基を用いて修飾したかまたは誘導体化した）。媒質の特性：２５０オングストロームの１２〜１５μmのビーズ、表面積約４５０m²/g
２．ポリエルシーシリカ：購入した未コーティングのものであり、ＮＨ₂−、ＣＯＯ−基を用いてＰＮＮＬ中に誘導体化し、２０％及び１００％のコーティングを施した。媒質の特性：３００オングストロームの１２μmのビーズ、表面積約１００m²/g。
【００６４】
結合した酵素の最高の密度並びに活性の最低の損失及び最低の拡散限度を与えると思われる最良の結合化学に関してふるい分けした後、本発明者らは、ＯＰＨのＮＨ₂−基を介したＯＰＨとＮＨ₂−誘導体化媒質との結合を選択した。
【００６５】
多くの架橋剤を試験し、この中で２つが特に効果的であることが見い出された：すなわち、スルホ−ＢＳＯＣＯＥＳ（ビス［２−（スルホスクシンイミドキシカルボニルオキシ）エチル］スルホン）及びＤＴＳＳＰ（ジチオビス（スルホスクシンイミジルプロピオナート））である。こうした２つの架橋剤の利点は次の通り：
ｉ．両方の架橋剤とも、スペーサーアーム（ＤＴＳＳＰの場合には長さ１２オングストロームであり、ＢＳＯＣＯＥＳの場合には１３オングストローム）を有する。スペーサーアームは、立体障害を避けるために有益である。
【００６６】
ｉｉ．これらの両方は、スルホ官能基（functioning group）が理由となって水溶性である。
ｉｉｉ．ピアース（ロックフォード、ＩＬ）（Pierce (Rockford, IL)）は、ＰＢＳを結合緩衝液（pH７．５）として推奨していた。このpHはＯＰＨにとって好都合であり、というのは、酸性のpHで、すなわち、６．５よりも低いpHで、ＯＰＨは凝集し、活性中心から金属を失う傾向があるからである。より高いpHはさらなる利点を有する：ＮＨＳ−エステルの加水分解はより高いpHでより速く進行する。従って、より高いpHを使用した場合、本発明者らは、架橋剤／タンパク質のより高いモル比を得た。
【００６７】
ｉｖ．両方とも安定な共有結合のアミド結合を生じ、ＤＴＳＳＰの場合には、この結合は、チオール（ＤＴＴ、メルカプトエタノール等）を用いて開裂可能である。この特徴は、特定の用途のために有用である可能性がある。
ＯＰＨとＮＨ ₂ −誘導体化表面とのスルホ−ＢＳＯＣＯＥＳまたはＤＴＳＳＰカップリングのための標準的なプロトコール：
（ピアースのプロトコールをほとんど修正していないもの）
１．スルホ−ＢＳＯＣＯＥＳまたはＤＴＳＳＰ架橋試薬（管入りで入荷）は、−２０℃で、好ましくは乾燥し、窒素下で貯蔵すべきである。実施においては、架橋試薬を用いて作業する（すなわち、最初の管の内容物を分取する）際には、窒素グローブボックスまたは窒素袋（冷蔵室中で窒素タンクを使用して窒素を充填した）を使用するのは良い考えである。管を開ける前に常に試薬を室温にする。
【００６８】
２．ＯＰＨに結合する予定の媒質は、水中で十分に膨潤させるべきである。一般に本発明者らは、５００mgの媒質／５mLのＨ₂Ｏを使用し、ＳＡＭＭＳ用の〜５０％スラリー（v/v）を作製した。このスラリーは、＋４℃で貯蔵した際に非常に安定だった。注意：ポリエルシー媒質の場合、ＳＡＭＭＳと比較して乾燥重量で２倍多い媒質を使用するべきである（すなわち、〜１gのポリエルシー媒質／５mLのＨ₂Ｏ及び〜５０％スラリー（v/v）を作製する）。
【００６９】
３．ＯＰＨ（０９／０２に精製、［３mg/mL］）を２mL分取し、−８０℃で貯蔵し、解凍させ、緩衝液を２５mMのＨＥＰＥＳ（pH８．５）、２０％グリセロール、５０μMのＣｏＣｌ₂から０．１Mのカーブ／重炭酸塩（carb/bicarbonate）（pH９．０）に変更した。本発明者らが使用したＮＡＰ−２５カラムは最大体積１０mLを有した。これを用いて、本発明者らは約２．５〜３．０mLの最大試料体積を利用することができた。
【００７０】
４．新たなミリポア・バイオマックス・ウルトラフリーの４．０mLの３０ＫＣＯ膜ユニットを使用し、１０分間、＋４℃で、ソルバールＣＦ、バッカーローター（Sorvall CF, bucker rotor）を最高速度で使用して２０mg/mLまで濃縮した。
【００７１】
５．切断したイエローチップを用いて、Ｈ₂Ｏ中の５０％スラリー粉末を、２mgに対して９０．１mLの体積の０．１Mのカーブ／重炭酸塩緩衝液（pH９．０）中のＯＰＨに加えた。スラリー粉末のおおよその比は１５０μlの５０％スラリー／２mgのタンパク質である：ポリエルシーシリカ、及びＮＨ₂−ＳＡＭＭＳの２０％被覆の２つのタイプの誘導体化（ＮＨ₂−２０％及び１００％被覆）。
【００７２】
６．１．５mgまたは３mgまたは６mgのスルホ−ＢＳＯＣＯＥＳまたはＤＴＳＳＰを５９０μlの５mMのＭＥＳ（pH５）中に溶解させる。（架橋剤／タンパク質の１０×、２５×、及び５０×のモル比を得る）
７．直ちに架橋剤溶液（１２５μl/２mgのタンパク質）を各エッペンドルフ管（eppendorf tube）に滴下し、混合し、室温で４５分間、管を回転装置に取り付ける。
【００７３】
８．停止液：１mLの１MのＴｒｉｓ（pH８．１）を、１時間室温で加える。
９．ＰＢＳを用いて２回、０．５MのＮａＣｌ−ＰＢＳを用いて１回、及びＰＢＳを用いて１回洗浄する。１００mMのＨＥＰＥＳ（pH８．５）−５０μMのＣｏＣｌ₂中に再懸濁させ、＋４℃で貯蔵する。
【００７４】
１０．ＢＣＡピアースキットを使用して３７℃で３０分間反応させて、樹脂に裏打ちしているタンパク質の量を推定する。
【００７５】
実施例１
スルホ−ＢＳＯＣＯＥＳの２０％コーティングを使用し、５０×モル過剰の架橋剤／タンパク質を使用したＯＰＨとＳＡＭＭＳ−ＮＨ₂誘導体化表面とのカップリング
標準的なプロトコールに次のように従った：
１．スルホ−ＢＳＯＣＯＥＳを含む管から内容物のアリコートを取り出すために、窒素を充填した窒素グローブボックスを使用した。
【００７６】
２．５００mgの媒質を５mLのＨ₂Ｏ中に使用し、〜５０％スラリー（v/v）を作製した。このスラリーは、＋４℃で貯蔵した際に非常に安定だった。
３．ＯＰＨ（０９／０２に精製、［３mg/mL］）を２mL分取し、−８０℃で貯蔵し、解凍させ、緩衝液を２５mMのＨＥＰＥＳ（pH８．５）、２０％グリセロール、５０μMのＣｏＣｌ₂から０．１Mのカーブ／重炭酸塩（pH９．０）に変更し、ファルマシア・セファデックスＧ−２５（Pharmacia Sephadex G-25）カラムを使用した。本発明者らが使用したＮＡＰ−２５カラムは最大体積１０mLを有した。これを用いて、本発明者らは約２．５〜３．０mLの最大試料体積を利用することができた。
【００７７】
４．新たなミリポア・バイオマックス・ウルトラフリーの４．０mLの３０ＫＣＯ膜ユニットを使用し、１０分間、＋４℃で、ソルバールＣＦ、バッカーローターを最高速度で使用して２０mg/mLまで濃縮した。
【００７８】
５．切断したイエローチップを用いて、Ｈ₂Ｏ中の５０％スラリー粉末を、２mgに対して９０．１mLの体積の０．１Mのカーブ／重炭酸塩緩衝液（pH９．０）中のＯＰＨに加えた。スラリー粉末のおおよその比は１５０μlの５０％スラリー／２mgのタンパク質である：ポリエルシーシリカ、及びＮＨ₂−ＳＡＭＭＳの２０％被覆の２つのタイプの誘導体化（ＮＨ₂−２０％及び１００％被覆）。
【００７９】
６．６mgのスルホ−ＢＳＯＣＯＥＳを５９０μlの５mMのＭＥＳ（pH５）中に溶解させる。
７．直ちにスルホ−ＢＳＯＣＯＥＳ溶液（１２５μl/２mgのタンパク質）をエッペンドルフ管に滴下し、混合し、室温で４５分間、管を回転装置に取り付ける。
【００８０】
８．１mLの１MのＴｒｉｓ（pH８．１）を、１時間室温で加える。
９．ＰＢＳを用いて２回、０．５MのＮａＣｌ−ＰＢＳを用いて１回、及びＰＢＳを用いて１回洗浄する。１００mMのＨＥＰＥＳ（pH８．５）−５０μMのＣｏＣｌ₂中に再懸濁させ、＋４℃で貯蔵する。
【００８１】
１０．ＢＣＡピアースキットを使用し、３７℃で反応させて、樹脂と結合したＯＰＨの推定される量＝２５．０mg/mLの媒質及び１２５．０mg/gの媒質だった。
【００８２】
実施例２
スルホ−ＢＳＯＣＯＥＳの２０％コーティングを使用し、２５×モル過剰の架橋剤／タンパク質を使用したＯＰＨとＳＡＭＭＳ−ＮＨ₂誘導体化表面とのカップリング。手順は実施例１と同様だったが、ただし、工程６において３mgのスルホ−ＢＳＯＣＯＥＳを代わりに使用した。樹脂と結合したＯＰＨの推定される量＝１６．０mg/mLの媒質及び８．０mg/gの媒質。
【００８３】
実施例３
スルホ−ＢＳＯＣＯＥＳの２０％コーティングを使用し、１０×モル過剰の架橋剤／タンパク質を使用したＯＰＨとＳＡＭＭＳ−ＮＨ₂誘導体化表面とのカップリング。手順は実施例１と同様だったが、ただし、工程６において１．５mgのスルホ−ＢＳＯＣＯＥＳを代わりに使用した。樹脂と結合したＯＰＨの推定される量＝５．０mg/mLの媒質及び２５．０mg/gの媒質。
【００８４】
実施例４
ＤＴＳＳＰの２０％コーティングを使用し、５０×モル過剰の架橋剤／タンパク質を使用したＯＰＨとＳＡＭＭＳ−ＮＨ₂誘導体化表面とのカップリング。手順は実施例１と同様だったが、ただし、工程６において６mgのＤＴＳＳＰを代わりに使用した。樹脂と結合したＯＰＨの推定される量＝２５．０mg/mLの媒質及び１２５．０mg/gの媒質。
【００８５】
実施例５
ＤＴＳＳＰの２０％コーティングを使用し、２５×モル過剰の架橋剤／タンパク質を使用したＯＰＨとＳＡＭＭＳ−ＮＨ₂誘導体化表面とのカップリング。手順は実施例１と同様だったが、ただし、工程６において３mgのＤＴＳＳＰを代わりに使用した。樹脂と結合したＯＰＨの推定される量＝１６．０mg/mLの媒質及び８０．０mg/gの媒質。
【００８６】
実施例６
ＤＴＳＳＰの２０％コーティングを使用し、１０×モル過剰の架橋剤／タンパク質を使用したＯＰＨとＳＡＭＭＳ−ＮＨ₂誘導体化表面とのカップリング。手順は実施例１と同様だったが、ただし、工程６において１．５mgのＤＴＳＳＰを代わりに使用した。樹脂と結合したＯＰＨの推定される量＝５．０mg/mLの媒質及び２５．０mg/gの媒質。
【００８７】
実施例７
スルホ−ＢＳＯＣＯＥＳ及びＤＴＳＳＰを使用した２０％及び１００％のコーティング表面において、１０×、２５×、及び５０×モル過剰の架橋剤／タンパク質を使用して、ＯＰＨをポリエルシーとカップリングさせた。手順は実施例１〜６と同様だったが、ただし、工程２において５mLのＨ₂Ｏ当り１０００mgのポリエルシーを使用し、〜５０％スラリー（v/v）を作製した。
【００８８】
樹脂と結合したＯＰＨの推定される量を表ＩＩに列記する。
【００８９】
【表２】

【００９０】
本発明の組成物は、従来のシリカ（ポリエルシー）を用いた場合よりも高い密度の負荷が可能であることが分かる。従って、本発明の好適な具体例は、負荷密度を特徴とすることができる。好ましくはタンパク質系は、同じコーティング％を有するポリエルシーよりも２〜約７倍高い（mg/g単位で）、より好ましくは約５〜約７倍高い密度を有する。好適な具体例においては、２０％コーティングで密度を測定する。
【００９１】
実施例８−変性の影響
ＯＰＨ−ＳＡＭＭＳ及び可溶性ＯＰＨの変性条件に対する安定性を、尿素を４M、６M及び８Mの濃度で変性剤として使用して調べた。実施例の節における“可溶性ＯＰＨ”または“ＯＰＨは可溶”という句は、フレンチプレスを用いた細胞破壊の最中に放出され、各精製工程で示される緩衝液中に可溶性である非封入体ＯＰＨを指す。結果を下記の表３に示す。
【００９２】
【表３】

【００９３】
固定化酵素は、遊離タンパク質よりもはるかに安定である。従って、本発明の好適な具体例は、変性剤に対するその安定性を特徴とすることができる。好ましくはタンパク質系は、８Mの尿素中で、遊離タンパク質の少なくとも２倍安定であり、幾つかの具体例においては、約３〜約５倍安定であるような安定性を有する。
【００９４】
実施例９−脱水からの回復
脱水及び回復の実験を、固定化ＯＰＨ−ＳＡＭＭＳ及び可溶性ＯＰＨを使用して行った。可溶性ＯＰＨはその活性の７±１％のみを保持したが、ＯＰＨ−ＳＡＭＭＳはその活性を完全に保持した（１０６±８％）。
【００９５】
実施例１０−動力学的特性
動力学的研究を、ＳＡＭＭＳ表面の固定化ＯＰＨ、ポリエルシー表面の固定化ＯＰＨ、及び可溶性ＯＰＨを用いて実行した。１mMのパラオキソン溶液を２５Ｃで使用して、１００mMのＣＨＥＳ緩衝液（pH８．０）、５０μMのＣｏＣｌ₂中で基質が加水分解されてジエチルホスフェート及びｐ−ニトロフェノラートになる場合に、吸光度の変化を４０５nmでモニターして（吸光係数は１７，０００M^-1cm^-1）、酵素活性を測定した。分析には、耐熱性セルホールダー及びセル撹拌モジュールを備えたヒューレット・パッカードモデル８４５３ＵＶ／Ｖｉｓ分光光度計を動力学モードで（Hewlett-Packard model 8453 UV/Vis spectrophotometer in kinetics mode equipped with the Thermostable Cell Holder and Cell Stirring Module）用いた。基質の新しい希釈物は、測定前３０分以内に作製した。シグマプロット（SigmaPlot）を使用して、データの線形回帰を作図した。ポリエルシー（コロンビア、ＭＤ）シリカは購入した未コーティングのものであり、アミノ基を用いて誘導体化した。１２μmのビーズの孔径は３００オングストロームだった。ＳＡＭＭＳで固定化されたＯＰＨ及びポリエルシーで固定化されたＯＰＨの両方は、可溶性ＯＰＨと同じＫ_mを有するが、より低いＶ_max（ＯＰＨ−ＳＡＭＭＳの場合には２．８３倍低く、ＯＰＨ−ポリエルシーの場合には６．４４倍低い）を証明した。すなわち、ＯＰＨ−ＳＡＭＭＳは、ＯＰＨ−ポリエルシーよりも約２．３倍大きい反応速度を有する。表の質量は、ＯＰＨの質量を指す。
【００９６】
【表４】

【００９７】
実施例１１−アルカリ性pHにおける安定性
この実験において、ＯＰＨ−ＳＡＭＭＳ及び可溶性ＯＰＨを、アルカリ性条件下での安定性に関して試験した。１時間のアルカリ性pH処理（１MのＴｒｉｓ、pH１２．０）の後に、ＯＰＨ−ＳＡＭＭＳは、可溶性ＯＰＨの場合の０．７７％と比較して、その活性の１１．６％を保持することが見い出され、２４時間のこのアルカリ性処理の後に、ＯＰＨ−ＳＡＭＭＳは、可溶性ＯＰＨの場合の０．０３％と比較して、その活性の９．９％を保持することが見い出された。この実施例における条件は、“アルカリ性条件”という用語が本出願において使用される時にこの用語が意味するものを定義する。この実施例ではＯＰＨ系を示すが、本発明のメソ細孔性マトリックスによって提供される安定性の利点は一般的なものであるということは認識できるはずであり、他のタンパク質でも同様の安定性の利点が得られるだろうということは予想される。
【００９８】
実施例１２−熱安定性
熱安定性を研究する実験は、ＯＰＨ−ＳＡＭＭＳは溶液中のＯＰＨよりもかなり安定であることを示した。こうした実験の結果を下記の表に示す。
【００９９】
【表５】

【０１００】
実施例１３−凍結乾燥の影響
ＯＰＨ−ＳＡＭＭＳ及び可溶性ＯＰＨを凍結乾燥条件（１MのＭＥＳ、pH５．０）にさらして、１時間後及び２４時間後にＯＰＨ−ＳＡＭＭＳはその活性の５０％を保持したが、１時間後及び２４時間後に可溶性ＯＰＨはその活性の１５％のみを保持したことが見い出された。
【０１０１】
実施例１４
（１）スルホ−ＢＳＯＣＯＥＳを含む管から内容物のアリコートを取り出すために、窒素を充填した窒素グローブボックスを使用した。
【０１０２】
（２）pH７．５で０．１MのＨＥＰＥＳ／５０μMのＣｏＣｌ₂中のＯＰＨを、０５／０４／０１に精製し、１mL分取し、−８０℃で貯蔵し、冷蔵庫中で解凍させた。酵素ストックは、９２２３．３０酵素単位（０．２９mgのＯＰＨタンパク質の量）／mLを含んだ。
【０１０３】
（３）１．８mLの管中の３００オングストロームの２％ＮＨ₂−ＳＡＭＭＳの２〜４mgのアリコートに、１００〜２００μLの３．２mg/mLのスルホ−ＢＳＯＣＯＥＳ（pH７．５で０．１MのＨＥＰＥＳ／５０μMのＣｏＣｌ₂中）を加え、２分間振とうした。次に、これを１４００rpmで２分間遠心分離し、過剰の架橋剤を含む上澄みを分離し、堆積物を、１００μLの同じ緩衝液を用いて洗浄した。次にこれに１００〜８００μLのＯＰＨストックを加え、１４００分^-1の速度、エッペンドルフサーモミキサー５４３６（Eppendorf Thermomixer 5436）上、２５℃で４５〜９０分間振とうした。
【０１０４】
（４）次に酵素インキュベーション溶液を分離し、得られた堆積物をｎ×４００μLのpH７．５で２０mMのホスフェート／０．１５MのＮａＣｌ（ｎ≧１０）で洗浄した。中間段階で、これを１４Krpmで６分間遠心分離した。最後に、洗浄した堆積物を、１００μLの緩衝液／mgの初期ＳＡＭＭＳで、同じ洗浄用緩衝液中に再度再懸濁させた。徹底的な洗浄の後に懸濁液及び透明な溶液の両方を測定して、しっかり固定化されていないＯＰＨが完全に洗い流されたことを示した。
【０１０５】
（５）共有結合したＯＰＨの推定されるタンパク質の量は、ピアースＢＣＡアッセイキット（ピアース、２３２２７）によって１１．００mg/gのＳＡＭＭＳであり、初期比活性１１３８６．０７酵素単位／mgの結合したＯＰＨを有した。
【０１０６】
実施例１５
（１）１０．１７mgのＧＯＤ（シグマＧ−７０１６）を、５mLのpH７．５で２０mMのホスフェート／０．１５MのＮａＣｌ中にＧＯＤストックとして溶解させた。酵素ストックは、３９５．０５酵素単位（１．０５mgのＯＰＨタンパク質の量）／mLを含んだ。
【０１０７】
（２）実施例１４と同様だが、ＧＯＤストック及び３００オングストロームの２０％ＮＨ₂−ＳＡＭＭＳを代わりに使用した。
（３）ＧＯＤ活性を、シグマ標準法（０８／３０／９６に改訂）を使用して測定した。
【０１０８】
（４）共有結合したＧＯＤの推定されるタンパク質の量は１４．８５mg/gのＳＡＭＭＳであり、初期比活性１１４．３１酵素単位／mgの結合したＧＯＤを有した。
【０１０９】
実施例１６
（１）９．９５mgのＧＯＤ（シグマＧ−７０１６）を、５mLのpH７．０で２０mMのホスフェート／０．１５MのＮａＣｌ中にＧＯＤストックとして溶解させた。酵素ストックは、３７２．２５酵素単位（１．０８mgのＯＰＨタンパク質の量）／mLを含んだ。
【０１１０】
（２）実施例１５と同様だが、共有結合剤（covalent linker）源としてスルホ−ＢＳＯＣＯＥＳ溶液の代わりに２００〜４００μLの５％グルタルジアルデヒド（オールドリッチ、３４０８５−５）（pH７．０で２０mMのホスフェート／０．１５MのＮａＣｌ中）を使用した。
【０１１１】
（３）洗浄溶液は、実施例１５において使用したpH７．５の代わりにpH７．０だった。
（４）共有結合したＧＯＤの推定されるタンパク質の量は８１．６４mg/gのＳＡＭＭＳであり、初期比活性７２．３４酵素単位／mgの結合したＧＯＤを有した。
【０１１２】
実施例１７
（１）ストレプトミセス・ルビギノサス（Streptomyces rubiginosus）から精製したＧＩを、ハンプトン・リサーチ、Ｉｎｃ．（Hampton Research, Inc.）から得た。これを、pH７．５で２０mMのホスフェート／０．１５MのＮａＣｌ／１mMのＭｇＳＯ₄中で透析し、１mL分取し、−８０℃で貯蔵し、使用前に冷蔵庫中で解凍させた。ＧＩストックは、１８０．２２酵素単位（４．１１mgのＧＩタンパク質の量）／mLを含んだ。
【０１１３】
（２）実施例１５と同様だが、ＧＯＤストックの代わりにＧＩストックを使用した。インキュベーションを４０℃で実行した。
（３）洗浄溶液は、pH７．５で２０mMのホスフェート／０．１５MのＮａＣｌ／１〜２０mMのＭｇＳＯ₄だった。
【０１１４】
（４）本条件でのＧＩ活性の定義：１酵素単位は、pH７．５、６０℃、１．９２３MのＤ−フルクトース濃度にて、１分間でＤ−グルコースに転換される１ミクロモルのＤ−フルクトースである。活性を以下のような一般的な手順で測定した：５〜２０μLのＧＩ−ＳＡＭＭＳ懸濁液に、０．５mLの２MのＤ−フルクトース（フルカ４７７３９（Fluka 47739））（pH７．５で２０mMのホスフェート／０．１５MのＮａＣｌ／２０mMのＭｇＳＯ₄中）を加え、次に１４００分^-1の速度、エッペンドルフサーモミキサー５４３６上、６０℃の制御された温度で１５分間振とうした。次に、１０μLの１．８MのＨ₂ＳＯ₄によって反応を停止した。次に、５〜２０μLの反応溶液に、２．０mLのグルコース（ＧＯ）アッセイ試薬（シグマ、Ｇ３６６０）を加え、湯浴中、３７℃で３０分間インキュベートさせた。インキュベーション反応を１．０mLの６MのＨ₂ＳＯ₄によって停止し、吸光度を５４０nmで測定した。グルコース標準曲線から、フルクトースのＧＩ触媒転換によって生じたグルコースの量を得た。
【０１１５】
（５）共有結合したＧＩの推定されるタンパク質の量は４８．４７mg/gのＳＡＭＭＳであり、初期比活性５３．４０酵素単位／mgの結合したＧＯＤを有した。
【０１１６】
実施例１８
（１）実施例１７と同様だが、共有結合剤源としてスルホ−ＢＳＯＣＯＥＳ溶液の代わりに２００〜４００μLの５％グルタルジアルデヒド（オールドリッチ、３４０８５−５）（pH７．５で２０mMのホスフェート／０．１５MのＮａＣｌ中）を使用した。
【０１１７】
（２）共有結合したＧＩの推定されるタンパク質の量は３０．４５mg/gのＳＡＭＭＳであり、初期比活性３９．１９酵素単位／mgの結合したＧＯＤを有した。
【０１１８】
実施例１９
（１）pH７．５で０．１MのＨＥＰＥＳ／５０μMのＣｏＣｌ₂中の精製されたＯＰＨを、１mL分取し、−８０℃で貯蔵し、冷蔵庫中で解凍させた。酵素ストックは、９２２３．３０酵素単位（０．２９mgのＯＰＨタンパク質の量）／mLを含んだ。
【０１１９】
（２）１．８mLの管中の３００オングストロームの２％ＮＨ₂−ＳＡＭＭＳの２〜４mgのアリコートに、１００〜８００μLのＯＰＨストックを加え、１４００分^-1の速度、エッペンドルフサーモミキサー５４３６上、２５℃で２〜３時間振とうした。
【０１２０】
（３）次に酵素インキュベーション溶液を分離し、得られた堆積物をｎ×４００μLのpH７．５で２０mMのホスフェート／０．１５MのＮａＣｌ（ｎ≧１０）で洗浄した。中間段階で、これを１４，０００rpmで６分間遠心分離した。最後に、洗浄した堆積物を、１００μLの緩衝液／mgの初期ＳＡＭＭＳで、同じ洗浄用緩衝液中に再度再懸濁させた。徹底的な洗浄の後に懸濁液及び透明な溶液の両方を測定して、しっかり固定化されていないＯＰＨが完全に洗い流されたことを示した。
【０１２１】
（４）取り込まれたＯＰＨの推定されるタンパク質の量は、ピアースＢＣＡアッセイキットによって９．５２mg/gのＳＡＭＭＳであり、初期比活性１６３７８．７４酵素単位／mgの取り込まれたＯＰＨを有した。
【０１２２】
実施例２０
（１）１０．１７mgのＧＯＤ（シグマＧ−７０１６）を、５mLのpH７．５で２０mMのホスフェート／０．１５MのＮａＣｌ中にＧＯＤストックとして溶解させた。酵素ストックは、３９５．０５酵素単位（１．０５mgのＯＰＨタンパク質の量）／mLを含んだ。
【０１２３】
（２）実施例１９と同様だが、ＧＯＤストック及び３００オングストロームの２０％ＮＨ₂−ＳＡＭＭＳを代わりに使用した。
（３）ＧＯＤ活性を、シグマ標準法（０８／３０／９６に改訂）を使用して測定した。
【０１２４】
（４）取り込まれたＧＯＤの推定されるタンパク質の量は１３．３５mg/gのＳＡＭＭＳであり、初期比活性９９．３３酵素単位／mgの取り込まれたＧＯＤを有した。
【０１２５】
実施例２１
（１）実施例２０と同様だが、９．９５mgのＧＯＤ（シグマＧ−７０１６）を、５mLのpH７．０で２０mMのホスフェート／０．１５MのＮａＣｌ中にＧＯＤストックとして溶解させた。酵素ストックは、３７２．２５酵素単位（１．０８mgのＯＰＨタンパク質の量）／mLを含んだ。
【０１２６】
（２）洗浄溶液は、プロトコール７において使用したpH７．５の代わりにpH７．０だった。
（３）取り込まれたＧＯＤの推定されるタンパク質の量は１４．５９mg/gのＳＡＭＭＳであり、初期比活性１３６．２８酵素単位／mgの取り込まれたＧＯＤを有した。
【０１２７】
実施例２２
（１）ストレプトミセス・ルビギノサスから精製したＧＩを、ハンプトン・リサーチ、Ｉｎｃ．から得た。これを、pH７．５で２０mMのホスフェート／０．１５MのＮａＣｌ／１mMのＭｇＳＯ₄中で透析し、１mL分取し、−８０℃で貯蔵し、使用前に冷蔵庫中で解凍させた。ＧＩストックは、１８０．２２酵素単位（４．１１mgのＧＩタンパク質の量）／mLを含んだ。
【０１２８】
（２）実施例２０と同様だが、ＧＯＤストックの代わりにＧＩストックを使用した。インキュベーションを４０℃で実行した。
（３）洗浄溶液は、pH７．５で２０mMのホスフェート／０．１５MのＮａＣｌ／１〜２０mMのＭｇＳＯ₄だった。
【０１２９】
（４）ＧＩ活性を、プロトコール４において行ったものと同じ方法で測定した。
（５）取り込まれたＧＩの推定されるタンパク質の量は９４．９４mg/gのＳＡＭＭＳであり、初期比活性４３．２６酵素単位／mgの取り込まれたＧＯＤを有した。
【０１３０】
実施例２３
（１）精製されたＯＰＨを、２mL分取し、−８０℃で貯蔵し、冷蔵庫中で解凍させ、pH８．５で２５mMのＨＥＰＥＳ／２０％グリセロール／５０μMのＣｏＣｌ₂からpH７．２で０．１MのＨＥＰＥＳへと透析した。酵素ストックは、４８３０．００酵素単位（０．３９mgのＯＰＨタンパク質の量）／mLを含んだ。
【０１３１】
（２）５０mLの管中の３００オングストロームの２％ＨＯＯＣ−ＳＡＭＭＳの８２．４５mgのアリコートに、１２mLのＯＰＨストックを加え、３５０rpmの速度、イノヴァ４３３０冷蔵インキュベーター振とう機（Innova 4330 refrigerated incubator shaker）上、２５℃で２時間４７分間振とうした。次いで、得られた懸濁液を０．４mL分取した。
【０１３２】
（３）次に酵素インキュベーション溶液を分離し、得られた堆積物をｎ×４００μLのpH７．５で２０mMのＨＥＰＥＳ（ｎ≧１０）で洗浄した。中間段階で、これを１４，０００rpmで６分間遠心分離した。最後に、洗浄した堆積物を、１００μLの緩衝液／mgの初期ＳＡＭＭＳで、同じ洗浄用緩衝液中に再度再懸濁させた。徹底的な洗浄の後に懸濁液及び透明な溶液の両方を測定して、しっかり固定化されていないＯＰＨが完全に洗い流されたことを示した。
【０１３３】
（４）取り込まれたＯＰＨの推定されるタンパク質の量は、４６．７３mg/gのＳＡＭＭＳであり、初期比活性２６７６６．７８酵素単位／mgの取り込まれたＯＰＨを有した。
【０１３４】
実施例２４
（１）３１．０７mgのＧＯＤ（シグマＧ−７０１６）を、１１mLのpH７．０で２０mMのホスフェート中にＧＯＤストックとして溶解させた。酵素ストックは、３９３．０８酵素単位（１．７１mgのＧＯＤタンパク質の量）／mLを含んだ。
【０１３５】
（２）実施例２３と同様だが、ＧＯＤストック及び９８．６７mgの３００オングストロームの２０％ＮＨ₂−ＳＡＭＭＳを代わりに使用した。
（３）洗浄溶液はpH７．０で２０mMのホスフェート緩衝液である。
【０１３６】
（４）ＧＯＤ活性を、シグマ標準法（０８／３０／９６に改訂）を使用して測定した。
（５）取り込まれたＧＯＤの推定されるタンパク質の量は１０７．９４mg/gのＳＡＭＭＳであり、初期比活性７６．２２酵素単位／mgの取り込まれたＧＯＤを有した。
【０１３７】
実施例２５
（１）ストレプトミセス・ルビギノサスから精製したＧＩを、ハンプトン・リサーチ、Ｉｎｃ．から得た。これを、pH７．５で２０mMのホスフェート／０．１５MのＮａＣｌ／１mMのＭｇＳＯ₄中で透析し、１mL分取し、−８０℃で貯蔵した。６つのアリコートを使用前に冷蔵庫中で解凍させ、２２．５mLのpH７．０で２０mMのナトリウム／１mMのＭｇＳＯ₄によって一緒に希釈してＧＩストックとした。ＧＩストックは、４２．１０酵素単位（０．８６mgのＧＩタンパク質の量）／mLを含んだ。
【０１３８】
（２）実施例２４と同様だが、ＧＩストック及び１００．９８mgの３００オングストロームの２０％ＮＨ₂−ＳＡＭＭＳを使用した。インキュベーションを４０℃で実行した。
（３）洗浄溶液は、pH７．５で２０mMのホスフェート／１〜２０mMのＭｇＳＯ₄だった。
【０１３９】
（４）ＧＩ活性を、実施例１７において行ったものと同じ方法で測定した。
（５）取り込まれたＧＩの推定されるタンパク質の量は７７．２６mg/gのＳＡＭＭＳであり、初期比活性４５．０６酵素単位／mgの取り込まれたＧＩを有した。
比較例及び様々な条件を含む試験の結果
ポリエルシーＩｎｃ．から市販されている通常のシリカ（品目番号：ＢＭＳＩ １２０３）をＳＡＭＭＳの場合と同様の方法で官能基化し、比較のために同じ実験条件下で上述のプロトコールにおけるＳＡＭＭＳに代えて使用した。
【０１４０】
孔径、官能性のタイプ及び被覆の影響を調べるために、様々な孔径、官能性単一層のタイプ及び被覆を使用して、同じ実験条件下で上述のプロトコールにおけるＳＡＭＭＳに代えた。
【０１４１】
緩衝液組成、pH、安定性及びイオン強度の影響を調べるために、様々な異なる洗浄溶液を、同じ実験条件下で上述のプロトコールにおける洗浄溶液に代えて使用した。
幾つかの結果を図に示す。以下の表記を使用したことに注意されたい：３００オングストロームの２０％ＮＨ₂−ＳＡＭＭＳは、トリス−（メトキシ）アミノプロピルシランから誘導体化されたＮＨ₂−官能性単一層の２０％被覆を有する孔径３００オングストロームのメソ細孔性シリカを意味し、一方、３００オングストロームの２％ＨＯＯＣ−ＳＡＭＭＳは、トリス−（メトキシ）カルボキシルエチルシランから得たＨＯＯＣ−官能性単一層の２％被覆を有する同じメソ細孔性シリカを意味する。
【０１４２】
図５は、５つの異なるマトリックス表面に固定化されたＯＰＨの比較データを示す。通常のシリカを使用した３つの実施例は４８酵素単位／mgのシリカの活性を有し、一方、ＳＡＭＭＳ表面に固定化されたＯＰＨは１５６及び１２５の活性を証明した。実施例１４及び１９を参照されたい。従って、通常のシリカから製造されたタンパク質系はかなりより低い活性を証明した。
【０１４３】
官能基のタイプ及び官能基化度は、タンパク質系を形成する際に重要なファクターであることが見い出された。２％ではなく２０％官能基化（１００オングストローム、１７０オングストロームまたは３００オングストロームのＳＡＭＭＳの表面で）を用いて実施例１９（活性１５６）を繰り返すと、１未満の活性を得た。同様に、２％ではなく２０％官能基化（１００オングストローム、１７０オングストロームまたは３００オングストロームのＳＡＭＭＳの表面で）を用いて実施例１４（活性１２５）を繰り返すと、２０未満の活性を得た。ＯＰＨを用いて処理した３００オングストロームの２０％ＨＯＯＣ−ＳＡＭＭＳは９７の活性を証明し、一方、ＯＰＨを用いて同様に処理した３００オングストロームの２０％ＮＨ₂−ＳＡＭＭＳは１未満の活性を有した。
【０１４４】
実施例の節におけるデータは、タンパク質の共有結合固定化を利用するかまたは非共有結合取込を利用するかに関係なく（とはいえ、結合のタイプは制御できる別のファクターである）、個々のタンパク質に関し適切な密度で陰イオンまたは陽イオン官能基化を適応させることの重要性を証明した。この適応可能性は、高濃度の活性なタンパク質を多孔質マトリックス中に取り入れることを促進する。得られたタンパク質系は、その活性または他の測定可能な特性によって定義することができる。
【０１４５】
図６及び９は、様々な緩衝液濃度で取り込まれたタンパク質（図６）及び、様々な緩衝液濃度で取り込まれたタンパク質の比活性（pH７．２のＯＰＨの溶液と比較して）（図９）の両方の場合に、活性は緩衝液濃度（イオン強度）に敏感であることを示す。
【０１４６】
図７及び８に示すように、pH７．０で作製した試料は、より高いpHで作製した試料よりも多くのＯＰＨを含んだが、タンパク質系の比活性はほぼpH７．５で最適化されるようである。図８の比活性は、pH７．２のＯＰＨの対照液の比活性で割った固定化ＯＰＨの比活性である。
【０１４７】
非共有結合で保持されようと共有結合で保持されようと、取り込まれたＯＰＨは、溶液中のＯＰＨと比較してかなり良好な安定性を証明した。図１０に示すように、４℃で１１０日間冷蔵した場合に、取り込まれたタンパク質は約３０％未満の活性の損失を示した。室温でさらに４０日経た後に、恐らく再生によって若干の活性を回復した。
【０１４８】
図１１は、６つの異なるマトリックス表面に固定化されたＧＯＤの比較データを示す。通常のシリカを使用した３つの実施例は０．２、０．３及び０．４の活性を有し、一方、ＳＡＭＭＳ表面に固定化されたＧＯＤは２．０及び５．９の活性を証明した。実施例２０及び１５を参照されたい。従って、通常のシリカから製造されたタンパク質系はかなりより低い活性を証明した。同じ条件下で処理した“３００オングストロームのメソシリカ”と名付けられた非官能基化ＳＡＭＭＳ（すなわち、０％ＮＨ₂）は、わずか０．３の活性を示した。１００オングストロームの２０％ＮＨ₂−ＳＡＭＭＳから製造したＧＯＤタンパク質系は、３００オングストロームの２０％ＮＨ₂−ＳＡＭＭＳと同じ活性を示したが、共有結合架橋剤を用いて１００オングストロームの２０％ＮＨ₂−ＳＡＭＭＳから製造したＧＯＤタンパク質系は、３００オングストロームの２０％ＮＨ₂−ＳＡＭＭＳ中に共有結合で架橋された同様に製造したタンパク質の活性５．９と比較して、４．０の活性を示した。図１２に示すように、ＧＯＤの場合、２０％アミノ表面被覆率を有する多孔質マトリックスは、２％表面被覆率よりもかなり良好に機能した。０．１５Mの塩化ナトリウムの存在は、ＮＨ₂−ＳＡＭＭＳ系中のＧＯＤの活性のかなりの低下を引き起こした。図１３に示すように、ＧＯＤ−ＮＨ₂−ＳＡＭＭＳ系の製造の最中にpHを上昇させると、活性の低下をもたらす；しかしながら、ＧＯＤの比活性はより高いpH範囲で低下しなかった（図１４を参照されたい）。再度、図１５に示すように、タンパク質系は、ＧＯＤ溶液と比較して向上した安定性を示し、意外なことに、４℃で冷蔵した後に、非共有結合したタンパク質系は共有結合した系と比較してより良好な安定性を示した。
【０１４９】
従って、官能基化の他にまたは官能基化に加えて、各タンパク質系を共有結合及び非共有結合の点でキャラクタリゼーションするのが望ましいことがある。幾つかの好適な具体例においては、本明細書において説明する手順に従って試験した場合、非共有結合したタンパク質は、共有結合で架橋した系と比較して優れた安定性を有する。
【０１５０】
図１６は、８つの異なるマトリックス表面に固定化されたＧＩの比較データを示す。通常のシリカを使用して同様の条件下で作製した４つの実施例は０．０、０．０、０．３及び０．０の活性を有し、一方、ＳＡＭＭＳ表面に固定化されたＧＩはそれぞれ４．１、１．２及び２．６の活性を証明した。実施例２２、１７及び１８を参照されたい。従って、再度、通常のシリカから製造されたタンパク質系はかなりより低い活性を証明した。同じ条件下で処理した“３００オングストロームのメソシリカ”と名付けられた非官能基化ＳＡＭＭＳ（すなわち、０％ＮＨ₂）は、０．０の活性を示した。ＧＩの場合、孔径に基づいてかなり大きな影響が見られ（図１７）、これは、ＧＩの大きなサイズを考慮すると予想外だったわけではない。図１８に示すように、共有結合した２０％ＮＨ₂−通常のシリカ表面に固定化されたＧＩは、比活性７０を証明し、一方、共有結合した３００オングストロームの２０％ＮＨ₂−ＳＡＭＭＳ表面に固定化されたＧＩは、比活性８９を証明した。ＮＨ₂−ＳＡＭＭＳ中のＧＩは、０．１５Mの塩化ナトリウムの存在によってほとんど影響されないことが見い出された。３００オングストロームの２％ＮＨ₂−ＳＡＭＭＳ及び３００オングストロームの２０％ＨＯＯＣ−ＳＡＭＭＳは、活性がほとんどまたは全く無いことが見い出された。
【０１５１】
ＧＩは頑健な酵素（robust enzyme）であり、ＳＡＭＭＳ中への取込は、試験した期間及び条件に関して比較的にわずかな安定性の向上を示した。しかしながら、ＯＰＨタンパク質系における安定性試験に基づいて、変性剤にさらす等のより困難な条件下での試験を行えば、好ましい本発明のタンパク質系、特にＳＡＭＭＳに基づく系の優れた安定性特性を明らかにすると思われると結論するのは合理的である。本発明を、一般に様々な試験条件下での安定性に関して説明することができる。
【０１５２】
本発明は、本発明の精神及び範囲から逸脱することなく様々な修正及び変更を含むことができる。従って、本発明を特定の説明及び実施例に限定すべきではなく、請求の範囲及び請求の範囲において述べる要素の同等物において述べる限定によって制御されるべきであることは理解できるはずである。
【図面の簡単な説明】
【０１５３】
【図１】多孔質基体中に配置された酵素の概念化された断面図である。
【図２】ＯＰＨプラスミドのリボン図である。
【図３】Ｔ７プロモーターの直ぐ前からＯＰＨ停止コドンの直ぐ向こうまでを包含する、構成体のＢａｍＨ ＩからＢｇｌ ＩＩ部位までの関連ＤＮＡ配列（ＳＥＱ ＩＤ：２）である。
【図４】ＯＰＨのアミノ酸配列（ＳＥＱ ＩＤ：１）である。
【図５】ＯＰＨ固定化に関する通常のシリカとＮＨ₂−ＳＡＭＭＳとの比較を示す。
【図６】ＨＯＯＣ−ＳＡＭＭＳ中に取り込まれたＯＰＨにイオン強度が及ぼす影響を示す。
【図７】ＨＯＯＣ−ＳＡＭＭＳ中へのＯＰＨ取込にpHが及ぼす影響を示す。
【図８】様々なpHでのＨＯＯＣ−ＳＡＭＭＳ中のＯＰＨの固定化効率を示す。
【図９】ＨＯＯＣ−ＳＡＭＭＳ中のＯＰＨ固定化効率にイオン強度が及ぼす影響を示す。
【図１０】ＮＨ₂−ＳＡＭＭＳ中に固定化されたＯＰＨの安定性を示す。
【図１１】グルコースオキシダーゼ（ＧＯＤ）固定化に関する通常のシリカとＮＨ₂−ＳＡＭＭＳとの比較を示す。
【図１２】ＧＯＤ取込にＮＨ₂−ＳＡＭＭＳの官能基の被覆が及ぼす影響を示す。
【図１３】ＮＨ₂−ＳＡＭＭＳ中へのＧＯＤ取込にpHが及ぼす影響を示す。
【図１４】様々なpHでのＮＨ₂−ＳＡＭＭＳ中のＧＯＤの固定化効率を示す。
【図１５】ＮＨ₂−ＳＡＭＭＳ中に固定化されたＧＯＤの安定性を示す。
【図１６】ＧＩ固定化に関する通常のシリカとＮＨ₂−ＳＡＭＭＳとの比較を示す。
【図１７】ＧＩ固定化とＮＨ₂−ＳＡＭＭＳの孔径の影響との比較を示す。
【図１８】ＮＨ₂−ＳＡＭＭＳ中のＧＩの固定化効率を示す。[0001]
Related applications
This application is a continuation-in-part of US patent application Ser. No. 09 / 791,138, filed Feb. 21, 2001, which is hereby incorporated by reference in its entirety.
[0002]
Field of Invention
The present invention relates to a protein in a porous carrier, a method for supporting a protein, and a method using a supported protein. The present invention also provides an improved process for producing organophosphorous hydrolase (“OPH”).
[0003]
Background of the Invention
The usefulness of proteins for promoting chemical reactions in vitro has been well known and has been used very effectively. There is a much greater potential for use of proteins in facilitating more diverse reactions and promoting these reactions on a larger scale. However, there are many problems to overcome before this possibility is fully realized. These problems include the need for highly active protein systems; the need for protein systems that maintain high activity under a range of conditions; and the active protein placed on a porous carrier in high density Ability to do.
[0004]
One example of a protein useful for catalyzing a variety of useful reactions is organophosphorus hydrolase (“OPH”). OPH is an enzyme that can be used to inactivate chemical weapons or organophosphorus pesticides. Chemical weapons (ie nerve gases, especially sarin and VX) and organophosphorus pesticides (eg parathion, paraoxon and acephate) are extremely toxic to higher organisms. Accordingly, there is a need for a method to clean up unwanted releases of chemical weapons and organophosphorus pesticides in the event of accidental spills or contamination of manufacturing plants. OPH enzymes offer the potential to inactivate chemical weapons or organophosphorus pesticides without the need for complex and expensive incineration facilities. Despite that possibility, the lack of appropriate methods for large-scale production of systems with active and stable OPH has limited the use of this enzyme.
[0005]
The present invention provides an improved protein system that can better address the problems described above. Although the present invention is generally applied to immobilized enzyme systems and the like, in some specific examples, the present invention also provides improved methods for producing systems comprising OPH and active OPH.
[0006]
Summary of the Invention
One concept of the invention is the engineering creation of a carrier structure that matches the size of the protein to the pore size of the carrier structure. Surprisingly, it has been found that well-matched sizes can produce protein systems with desirable qualities such as high activity of active proteins, improved stability, and relatively high density. Coupling of proteins in pores that are too small or too large results in poor properties. Other factors such as surface area, pore density, pore uniformity and distribution, protein occupancy within the support, and type and density of cross-linking sites may also be utilized to control the properties of the protein system.
[0007]
In one aspect, the present invention provides a protein system for use in promoting chemical reactions. The system includes a porous matrix material having pores within the solid matrix. In another aspect, the protein system comprises a porous matrix material having a pore volume such that at least 90% of the pore volume is composed of pores having a size in the range of 50-400 Angstroms, and the matrix material And chemically active proteins combined with “Coupled” refers to covalent attachment, ionic attachment, and / or electrostatic attachment to a matrix material. In a preferred embodiment, the protein is covalently bound to the matrix via a coupling group.
[0008]
In another aspect, the protein system comprises a porous matrix material sized such that the protein system contains 0.01-1 mmol protein per gram of matrix material, wherein the protein in the protein system Exhibits an activity of at least 65% of the activity of the protein in the active state.
[0009]
The present invention further provides a protein system comprising a porous matrix material comprising entrapped protein, wherein the protein system is under the same conditions as at least 0.01 mmol protein per gram of matrix material. It is characterized in that it has an activity that is at least twice as large as that of the protein system formed on the surface of a normal silica matrix material. "Regular silica" is an uncoated silica bulk material having a pore size of 300 Angstroms and a bead size of 12 micrometers, and when available, regular silica is available from Poly LLC Inc. of Columbia, Maryland, USA. (PolyLC Inc., Columbia Maryland, USA) should be available under item number BMSI 1203.
[0010]
In another aspect, the present invention provides a protein system comprising a porous matrix material comprising incorporated proteins, wherein the protein system comprises at least 0.01 mmol protein per gram of matrix material. With improved stability as defined by the stability test procedure described in the document.
[0011]
The present invention is also a method of forming a protein system comprising:
Providing a porous matrix material having a pore volume such that at least 90% of the pore volume is composed of pores having a size in the range of 50 to 400 Angstroms; and Reacting so that the protein chemically binds to the porous matrix material. Preferably, the method comprises the steps of reacting a porous matrix material and a cross-linking agent to form a porous matrix material having a cross-linking agent covalently bonded to the surface, and a porous material having a cross-linking agent covalently bonded to the surface. Reacting the matrix material with the protein so that the protein chemically binds to the porous matrix material.
[0012]
In another aspect, the present invention provides a method for producing a protein system by adding protein to a porous matrix material without a crosslinker. It has been found that a stable and active protein system can be obtained in which the protein is incorporated by non-covalent bonds. In one such method, the protein system is a porous matrix material having a pore volume such that at least 90% of the pore volume is composed of pores having a size in the range of 50-400 angstroms. Forming a porous matrix material having a functionalized surface and adding protein so that the protein is incorporated into the porous matrix material by non-covalent bonding.
[0013]
The present invention also provides a method for producing OPH. In this method, host cells are transfected with a vector containing sequences encoding OPH, and the sequences are operably linked to T7 expression control sequences. Transfected host cells are cultured under the control of expression control sequences under conditions that allow expression. OPH is purified from cells or cell culture medium.
[0014]
A protein system is engineered to match the size of individual proteins to the size of individual pores, and in a preferred embodiment, the volume of individual proteins represents 5-40% of the average volume of each pore. Occupy.
[0015]
The present invention also includes methods of using such systems in promoting chemical methods (ie, chemical manufacturing methods) such as hydrolysis, oxidation, hydrogenation, and proteolysis. The present invention also provides porosity within filtration devices that perform detoxification for individual soldiers, persons engaged in the agrochemical field, vehicles, aircraft, ships and buildings such as civil and military defense shelters. Includes the use of active enzymes in the carrier.
[0016]
Various embodiments of the present invention include high protein activity at the surface of a porous carrier; stability under various conditions; high density of active protein; ability to use on an industrial scale; and chemical weapons and organophosphorus Numerous benefits can be provided, including providing an environmentally safe method for destroying pesticides and avoiding the risks associated with burning such materials. Other advantages can be foreseen in view of the following description and examples.
[0017]
Detailed description of the invention
A conceptual diagram of one specific example of the protein system 2 of the present invention is shown in FIG. The matrix material 4 has pores 6 containing proteins 8. Protein 8 is linked to the matrix via a linking portion 10. Many variations of this structure are possible. For example, this figure shows a single protein in each pore, but in many embodiments some pores will contain a large number of proteins while other pores will contain nothing. . The present invention is not limited to the specific example shown in FIG.
[0018]
The porous matrix material is preferably such that at least 90% of the pore volume is composed of pores having a size in the range of 50-400 angstroms, more preferably 100-200, even more preferably 100-120 angstroms. Have a fine pore volume. For the purposes of the present invention, the pore size distribution is determined using a technique well known in the prior art.₂Measure by adsorption. Especially in the case of a material having large pores, in order to obtain an accurate pore size distribution, N₂Adsorption may need to be supplemented with mercury porisimetry or microscopy. As is conventional, “pore diameter” refers to pore diameter. In protein systems, the pore size distribution should be measured without protein in the matrix, and for measurement, the protein can be removed from the matrix by protease or other suitable means. In the case of measurements on protein systems, the coupling agent remains bound to the matrix during the pore size distribution measurement. In order to characterize the method of the invention or to characterize a protein system by a production method, the pore size distribution of the porous matrix material is measured without a coupling agent. The composition of the matrix material can vary, but is preferably an inorganic oxide-containing material. Inorganic oxide-based materials (eg, silica-based materials) offer advantages over many organic supports, which can include mechanical strength and chemical and thermal stability.
[0019]
In preferred embodiments, the protein system includes a coupling agent disposed between the inorganic porous matrix and the protein. Unreacted (ie, before reacting with protein) inorganic oxide supports typically have surface hydroxyl groups. Preferably, such surface hydroxyls are reacted with relatively low molecular weight organic compounds to form a functionalized monolayer. Treatment with a suitable coupling agent can produce selected functionalized moieties on the surface of the porous support. Preferred coupling moieties are mercapto (-SH), amino (-NH₂), Carboxyl (—COOH), hydroxyl (—OH), and azide (—N_Three). A particularly preferred embodiment utilizes a functionalized mesoporous support as described by Feng et al. In “Functionalized Monolayers on Ordered Mesoporous Supports,” Science, vol. 276, 923-926 (1997). As explained in a paper by Feng et al., The surface hydroxyl was converted to mercaptopropyltrimethoxysilane, (MeO)._ThreeSi (CH₂)_ThreeIt can be reacted with SH to form a functionalized surface with terminal mercapto groups. Functionalized surfaces are superior to non-functionalized surfaces because they provide a better and more controllable chemical environment and protein binding.
[0020]
If the surface of the porous matrix material is functionalized, it is measured by the degree of functionalization (surface coverage, as described in the above paper by Feng et al. In addition, surface coverage was measured by transmission electron microscopy) was found to affect the activity level of bound protein. Preferably, the surface coverage is at least 2%, more preferably about 20-70%, more preferably 20-50%. Too much coupling moieties can reduce activity, while too little can reduce protein covalent attachment to the matrix and reduce protein system stability.
[0021]
In particular, in the case of embodiments that do not use a coupling agent, it has been found that a number of factors can affect the ability of the porous matrix to incorporate proteins. A non-exhaustive list of such factors can include degree of functionalization, type of functionalized moiety (eg, amino, carboxyl, etc.), pH, ionic strength, buffer, and pore size. In general, electrostatic interactions can be very important to control protein uptake. For example, carboxyls in the pores of the matrix material can interact with amino on the surface of the protein, and vice versa. Similarly, the hydrophilic / hydrophobic interaction can be controlled to adapt the matrix material to protein uptake; for example, the hydrocarbon chains in the pores of the matrix material can interact with hydrophobic regions on the protein surface. Can interact. The identification of conditions for incorporating the selected protein can be determined by routine experimentation in light of the description set forth herein.
[0022]
Proteins are macromolecular organic compounds that contain more than about 100 amino acid residues and typically have a molecular weight in the range of about 8,000 to about 300,000 daltons. Most interesting in the present invention are chemically active proteins, ie proteins that can promote chemical methods such as hydrolysis, oxidation, reduction, oxygen transport, optical inversion, dehydrogenation, elimination and the like. More preferred are enzymes, ie proteins that catalyze chemical reactions. One particularly preferred protein is organophosphorus hydrolase (OPH), which is well known and reported in the literature. See, for example, Muchandani et al., “Biosensor for direct determination of organophosphate nerve agents. Potentiometric enzyme electrode,” Biosensors & Bioelectronics, 14, 77-85 (1999).
[0023]
Proteins can be composed of amino acids that are all linked through covalent bonds. Proteins can also be composed of subunits that are held together by non-covalent interactions. For example, hemoglobin is a protein composed of four subunits. A protein may also include other components such as metal atoms, porphyrin rings, and other artificial or naturally occurring modifications. The OPH has a diameter of about 45-80 Angstroms and a volume of about 1.95 × 10.^FiveAngstrom^ThreeIs a dimeric enzyme. Thus, if the protein system is designed such that OPH occupies 10% of the average pore volume, the matrix has an average pore volume of about 1.95 × 10⁶Angstrom^ThreeSeems to have.
[0024]
The size of the protein in the present invention is defined in the conventional sense based on the radius of rotation in the native state. In the protein system of the present invention, a preferred type of protein is 0.5 × 10^FiveAngstrom^Three~ 3x10^FiveAngstrom^ThreeEnzymes with a volume in the range are because proteins within this size (volume) range are particularly advantageous in the porous matrix of the protein system of the present invention.
[0025]
The protein in the matrix can be compared to the protein in the “active state”. In the present invention, the definition of immobilized protein activity (or “unit activity”) is the same as the generally accepted definition of non-immobilized protein. The unit of activity is defined by the amount of protein required to produce a product from a known or characterized substrate for a specified time at a specific temperature in a specific buffer condition. There are generally recognized units of activity for many enzymes and classes of enzymes. One source of accepted activity units is available from the Worthington Enzyme Manual (the Worthington Biochemical Corporation, Freehold, NJ). ). This manual contains definitions of the activities of enzymes including glucose oxidase (GOD) and glucose isomerase (GI). In the present invention, the activity of OPH is defined as described in Dumas et al., J. Biol. Chem., V. 264, p19659 (1989); that is, the activity unit is 25 ° C., 100 mM. Of 1 micromole of paraoxon per minute at pH 9 in CHES of CO 2 when the paraoxon substrate is hydrolyzed to diethyl phosphate and p-nitrophenolate anion.₄₀₅= 17,000M^-1cm^-1Assuming that the change in absorbance is typically monitored at 400 nm. In preferred embodiments, the protein of the invention is at least 50%, more preferably at least 75% of the activity in the active state. The present inventors have found that the activity of the OPH-containing protein system has an excellent activity. In some preferred embodiments, the proteins in the systems of the invention have 65-95% activity. In some embodiments, the protein systems of the invention are also produced and tested under the same conditions, including the same functionalization conditions, but with the same mass system using normal silica as the porous matrix material. Preferably the protein system is at least twice (2 ×), more preferably compared to a system that is manufactured identically but with normal silica in the porous matrix material It has at least 5 ×, even more preferably at least 10 ×, even more preferably at least 50 × greater activity. The present invention can also be defined as being able to maintain activity to the same or greater extent as described in the Examples under the same conditions.
[0026]
A non-limiting list of proteins that may be used in various embodiments of the invention is as follows: adenosine aminohydrolase, adenosine deaminase, alcohol oxidase, alcohol dehydrogenase, amino acid oxidase, amino acid deaminase, amino acid decarboxylase, ATP kinase, ATP phosphatase, Bilirubin oxidase, catalase, chymotrypsin, creatine phosphokinase, catecholase, cholesterol oxidase, chitinase, chitodextrinase, chitosanase, cholesterol dehydrogenase, cholesterol esterase, choline oxidase, choline phosphatase, citrase, cocaine esterase, cytochrome C, cytochrome C oxidase, cytochrome C reductase, cytochrome P450, cytochrome P450 reductase, cytochrome B_Five, DNase, DNA gyrase, helicase, DNA joinase, ligase, nucleotidyl exotransferase, DNA nucleotidyl transferase, DNA polymerase, DNA repair enzyme, DNA topoisomerase, endopectin lyase, endopeptidase, endoperoxide isomerase, endoproteinase, endo Ribonuclease, fibrinogen, ferredoxin, ferredoxin reductase, ferredoxin oxidoreductase, ferrooxidase, galactose oxidase, galactose dehydrogenase, GI, glucose dehydrogenase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione Ductase, hemoglobin, heparinase, lactose synthase, lactoperoxidase, lactate dehydrogenase, lactate oxidase, lactase, laccase, myoglobin, maleate dehydrogenase, malic dehydrogenase, malic enzyme, methanol oxidase, Microperoxidase, monophenol monooxygenase, monophenol oxidase, mutarotase, NADH dehydrogenase, oxidase, peroxidase, NADP cytochrome reductase, NADPH oxide reductase, nuclease, nitrate reductase, OPH, oxalate decarboxylase, oxalate oxidase, peroxidase , Thease, proteinase, putidaredoxin, papain, pepsin, plasmin, plasminogen, pectinase, pyruvate carboxylase, decarboxylase, pruvate dehydrogenase, oxidase, kinase, quinone reductase, ribonuclease, salicylate hydroxylase, monooxygenase , Sarcosine dehydroxylase, oxidase, sulfatase, sulfite oxidase, superoxide dismutase, streptolysin O, trypsin, urate oxidase, urease, uricase, xanthine dehydrogenase, oxidase, actin, beta agarase, albumin, bovine serum, aldolase, amylase , Alpha amylase, beta aspartyl Minotransferase, avidin, carbonic anhydrase, carboxypeptidase A, carboxypeptidase B, carboxypeptidase Y, casein, alpha cellulase, cholesterol esterase, cholinesterase, acetylcholinesterase, butyryl, chymotrypsin, clostripain, collagen, collagenase, concanavalin A, Creatine kinase, deoxyribonuclease I, deoxyribonuclease II, T4 DNA ligase, DNA polymerase I, T4 DNA polymerase, dextranase, diaphorase, elastase, elastin, galactosidase, beta glucose-6-phosphate dehydrogenase, beta glucosidase, beta glucuronidase, glutamate de Carboxylase, glyceraldehyde-3-phosphate dehydrogenase, glycerol dehydrogenase, glycerol kinase, hexokinase, hyaluronidase, hydroxysteroid dehydrogenase, leucine aminopeptidase, lipase, luciferase, lysozyme, maleate dehydrogenase, maltase, mucin, NADase, neuraminidase, micrococca Nuclease, S1 nuclease, ovalbumin, oxalate decarboxylase, pectinase, acid phosphatase, alkaline phosphatase, phosphodiesterase I, phosphodiesterase II, phosphoenol pyruvate carboxylase, phosphoglucomutase, phospholipase A2, phospholipase C, plasma amine oxidase , American Yamagobo antiviral toxin, T4 polynucleotide kinase, polyphenol oxidase, protease, S. aureus, proteinase K, pyruvate kinase, reverse transcriptase, ribonuclease, ribonuclease T1, ribonucleic acid, RNA polymerase, RNA polymerase, T7, and xylose isomerase.
[0027]
In various embodiments, the invention can be defined as including (exclusively, substantially, or including) any of the proteins listed above. For example, according to the present invention, the protein may be exclusively (ie consisting of) OPH (or any of the other proteins listed above) or substantially (ie essentially Consisting of OPH (or any of the other proteins listed above) or includes OPH (or any of the other proteins listed above) (ie, Including).
[0028]
There are a great variety of proteins, but there is also a great deal of overlap in the chemical parts that make up the protein structure. The same type of amino acid is common to most proteins. This similarity in the chemical moiety allows the protein to be bound onto the support using the same coupling technique. For example, cysteine, the amino-terminal amino acid and carboxyl-terminal amino acid sulfhydryls, and the amino groups of arginine and lysine react with the coupling agent or matrix surface, generally in the same manner, regardless of the protein in which such moiety is present. can do.
[0029]
In most cases, the protein will not bind directly to the carrier. In some cases, one or more linking moieties are associated with the carrier and the protein. For example, the coupling agent can react with an amine on the protein surface via a hydroxyl moiety on the carrier surface (see, eg, US Pat. No. 5,077,210, which is incorporated herein by reference). Such linking moieties are preferably organic moieties having a chain length of 2 to 20 atoms, more preferably a chain length of 4 to 10 atoms. Preferably, each protein binds to the matrix via at least one coupling moiety, more preferably 2-10 moieties. The number of moieties bound to each protein can be determined by appropriate analytical methods, for example by cleaving the bound protein and analyzing the cleaved molecules by mass spectrometry. There are a number of well-known coupling agents for linking surface hydroxyls to proteins. For example, the coupling agent is a siloxane (—Si (OR)) that forms an oxo bond with the surface._x) End groups, flexible organic chains (eg (CH₂)_n), And thiol (-SH) end groups that bind to proteins.
[0030]
Protein systems that combine a carrier and an incorporated (eg, non-covalently or covalently attached) protein can be difficult to characterize with chemical accuracy. However, the system can be characterized by measurable properties. Measurable properties that can define various embodiments of the present invention include pore size, pore volume, pore size distribution, surface area, activity, stability, protein density in the carrier, system density, and system properties. Strength. It has been discovered that excellent properties can be obtained by engineering a carrier having a pore size (or pore volume) that corresponds to the size of the protein (or protein volume). Preferably, the protein volume is 5-40% of the average pore volume (for this metric, the average pore volume is based only on pores whose size ranges from 50-400 angstroms), More preferably, the protein volume is 10-25% of the average pore volume. Matching the protein size to the pore size in this way can provide surprising improvements in activity and stability. Although the mechanism that produces these improved properties has not been fully elucidated, protein confinement may help direct reactive species into the protein and prevent the protein from irreversibly denatured. It is thought that it may be. Protein volume can be measured by biophysical methods such as analytical ultracentrifugation or X-ray crystallography. Preferably, the activity is at least 60% of the activity of the protein in the active state, measured per protein molecule. Preferably, the system comprises less than 40% protein by volume; more preferably 5-40% protein by volume; even more preferably 10-25% protein by volume. Preferably, when the system is exposed to 8M urea (as described in the Examples), the protein in the protein system is at least twice as stable as the free protein, and in some embodiments about 3 to about 5 It has a stability that is double stable. In other embodiments, the protein in the protein system is at least twice as stable as the free protein (as measured by the rate of denaturation) when placed in solution under any of the conditions described in the examples. More preferably at least 3 times more stable.
[0031]
A further advantage that can be obtained with the present invention is the high surface area (N₂Measured by adsorption). Similar to the pore size, the surface area is measured in terms of functionalized surfaces in the case of protein systems and in the case of the present invention as defined by the method and the system produced by such methods in relation to non-functionalized matrix materials. The surface area is preferably at least 700 m²/ g; more preferably at least 900 m²/ g. The upper surface area limit may be limited by the upper limit of mesoporous matrix materials of the type described by Feng et al. And similar materials. A further advantage of the present invention is that a relatively dense protein system can be produced. Preferably, the protein in the system has a density of at least 0.01 mmol / g; more preferably a density of 0.1-1 mmol / g. The system of the present invention may be characterized by exhibiting some of its properties in any one or various combinations of its properties. For example, in a preferred embodiment, the protein system exhibits an activity of 65-95% of the activity of the active protein and has a density of 0.1-1 mmol / g.
[0032]
In some preferred embodiments, a porous matrix material containing incorporated proteins is placed inside the microchannel. A microchannel is a channel having at least one dimension of 2 mm or less, preferably 1 mm or less. Microchannels can provide benefits such as improved heat transfer and / or mass transfer and design density. Tests for OPH crosslinked in SAMMS placed in a 0.25 mm wide channel show low pressure drop (flow under gravity or under peristaltic pump delivery or less than 1 bar) and greater reaction time. certified. Accordingly, the present invention includes devices and methods in which a porous matrix material containing incorporated proteins is disposed within a microchannel. In this way, for example, the reactants can pass through microchannels to form products.
[0033]
Proteins can be produced by well-known procedures, and in preferred embodiments, no special procedures are required before reacting with one or more coupling agents and binding to a carrier. Preferably, prior to binding within the matrix, the protein should be about 95% pure in an aqueous solution that stabilizes the activity, and the buffer should not interfere with the chemistry of the coupling.
[0034]
In the method of the invention for producing OPH, a host cell is transfected with a vector comprising a sequence encoding OPH, and the sequence is operably linked to a T7 expression control sequence. Transfected host cells are cultured under the control of expression control sequences under conditions that allow expression. OPH is purified from cells or cell culture medium. In a preferred embodiment, the vector is provided with a sequence encoding OPH operably linked to a T7 expression control sequence. Preferably, OPH has an activity of about 13,000 enzyme units / mg (unit / mg). Preferably the vector is a plasmid. The host cell can be a prokaryotic cell, a eukaryotic cell, or a yeast cell. The prokaryotic cell is preferably a bacterium, more preferably the bacterium is E. coli. The yeast cell is preferably Pichia pastoris.
[0035]
The matrix is preferably a mesoporous oxide material made from a soluble precursor. Examples of preferred syntheses are U.S. Patent Nos. 5,645,891 and 5,922,299 and U.S. Patent Application No. 09 / 020,028, all three of which are incorporated below in their entirety, Liu et al., " Molecular Assembly in Ordered Mesoporosity: A New Class of Highly Functional Nanoscale Materials, "J. Phys. Chem., 104, 8328-8339 (Aug. 2000)" and in the above-referenced paper by Feng et al.
[0036]
A typical synthesis of matrix materials was reported by Feng et al., Science, 276, p923 (1997). To make the CTAC / OH solution, the CTAC solution was contacted with a strongly basic ion exchange resin (DOWEX-1; 0.2 g resin / 1 gram 29% CTAC solution). 13 g of colloidal silica, 51 g of tetramethylammonium silicate and 28 g of mesitylene were each added to 100 g of CTAH / OH solution. Teflon mixture^TMAnd sealed in a container lined with, and heated at 105 ° C. for 1 week. The product was collected by suction filtration, dried at ambient temperature and calcined in air at 540 ° C. for 12 hours. The surface of the obtained mesoporous material was functionalized by various methods. For example, the surface can be functionalized with a thiol group by reacting with tris (methoxy) mercaptopropylsilane. The functionalized matrix obtained is called “SAMMS”. % Surface coverage, (i) surface area of support, (ii) weight change after deposition of functionalized monolayer, and (3) ideal loading density that may be realized on a flat surface , Based on the estimation. The% surface coverage can be demonstrated by electron energy-dispersive spectroscopy (EDS).
[0037]
As is well known in the prior art, various approaches can be used to attach proteins to a carrier. In a preferred embodiment, the support is pretreated with a coupling agent such as bis [2- (sulfosuccinimidoxycarbonyloxy) ethyl] sulfone (BSOCOES). Excess coupling agent can be washed away. Subsequently, the protein is reacted with the surface treated with the coupling agent. Alternatively, the protein can be reacted first with a coupling agent and subsequently with the surface of the matrix. Excess protein can be washed away and recovered.
[0038]
In a preferred method, the functionalized porous matrix is first reacted with a crosslinker. For example, amino derivatized SAMMS is reacted with sulfo-BSOCOES or glutardialdehyde. It is desirable to remove any excess crosslinker if present. Next, the protein is added. Preferably, the protein is in a low concentration solution. This procedure reduces or eliminates the possibility of intermolecular cross-linking of proteins by cross-linking agents.
[0039]
In other methods, a porous matrix material (preferably a functionalized porous matrix material) is combined with the protein without the addition of a crosslinking agent. In this way, a protein system in which the protein is non-covalently bound to the porous matrix can be produced. This method is preferably such that the porous matrix creates a complementary environment for the selected protein (eg, the amino groups in the pores of the matrix coincide with the carboxyl groups on the protein surface). And by adapting the electrostatic interaction. Factors that may be considered when adapting electrostatic interactions are discussed above.
[0040]
The protein systems described herein can be produced using any of the methods described herein, and such methods are also part of the present invention.
[0041]
Example
We obtained the OPH gene available from ATCC and subcloned into two vectors purchased from Novagen, pET11a and pET15b. Natural OPH and His-Tag^TMTwo Novagen vectors were used so that both OPH containing can be produced. We have generated a large number of clones of both types. These Novagen vectors contain strong promoters and are designed to maximize the yield of the desired protein. For restriction digests, we definitely subcloned the OPH gene and the resulting construct was the active OPH protein: SEQ ID No. 4 (FIG. 4) was confirmed.
[0042]
Bacterial expression and purification: After binding the recombinant OPH protein to the porous substrate, a purification step was performed. The total expression level achieved was about 4 g / liter of total protein. We purified 10 mg / liter of active protein from the soluble fraction. Thus, the majority of OPH is in inclusion bodies; that is, it exists in an inactive form. The fact that the protein is present in the inclusion bodies simplifies purification. OPH purified directly from washed and centrifuged inclusion bodies appears to be almost as pure as OPH purified by affinity column chromatography as analyzed by SDS polyacrylamide gel electrophoresis. Large scale methods to recover activity from inclusion body proteins can be developed by routine experimentation. This simplified purification procedure is suitable for industrial production.
[0043]
Materials and methods
material:
Diethyl p-nitrophenyl phosphate (paraoxon, 90%), various metal salts, glycerol and all buffers and other salts purchased from Sigma®-Aldrich® did.
[0044]
The components of the fermenter medium (peptone and yeast extract) were obtained from Gibco BRL (Gibco BRL), and the expression vector (pET11a^TM, PET15b^TM) Novagen Inc. , Madison, WI (Novagen Inc., Madison, Wis.). Primers for PCR were purchased from Genosis Inc. (Genosys Inc.)
[0045]
Bulk chromatography media for protein purification was obtained from Perseptive Biosystems (HS^TMAnd HQ^TM).
Polypropyl A^TM(Polypropyl A^TM) Column as well as non-derivatized silica resin for comparison of OPH bonds was obtained from PolyLC Inc. From Columbia, MD.
[0046]
Cross-linking reagents for enzyme immobilization were purchased from Pierce Chemical Company, Rockford, IL.
Abbreviations used:
CTAC, cetyltrimethylammonium chloride
OPH, organophosphorus hydrolase
HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
CHES, 2- (cyclohexylamino) ethanesulfonic acid
IPTG, isopropylthiogalactoside
SAMMS, self-assembled monolayer on mesoporous silica surface
Sulfo-BSOCOES, bis [2- (sulfosuccinimidoxycarbonyloxy) ethyl] sulfone
DTSSP, dithiobis (sulfosuccinimidyl propionate)
β-ME, β-mercaptoethanol
Matrix synthesis:
A typical procedure for producing the mesoporous oxide material used in the examples is as follows. Mesoporous silica with a pore diameter of 300 Angstroms was produced by a liquid crystal template procedure. Pluronic P123, a triblock copolymer (Propylene oxide / ethylene oxide copolymer available from BASF, M_av= 5,600) as the structure directing agent and mesitylene as the pore expending agent. 20 g of Pluronic P123 was dissolved in 150 g of deionized (DI) water and 600 g of 2M HCl solution with stirring at 40 ° C. 31.9 g of mesitylene was then added and stirring was continued at the same temperature. 42.5 g of TEOS was dropped into the cloudy micelle solution and cured with stirring at the above temperature for 20 hours. Teflon the mixture^TMIn an autoclave lined with a, overnight at 100 ° C. without stirring. The white solid was filtered, washed with DI water and air dried. The solid was calcined by gradually raising the temperature at 550 ° C. for 6 hours (1 ° C./min).
[0047]
In a typical preparation of 20% propyl carboxylic acid functionalized mesoporous silica, 2.0 g of mesoporous silica (average pore size = 30 nm, surface area = 533 m)²/ g) was first suspended in toluene (60 mL) and pretreated with approximately two layers of DI water (0.64 mL). The suspension was stirred for 2 hours to distribute water throughout the mesoporous matrix. The hydrated mixture was then treated with 20% (0.288 g) of 1 monolayer's 3-cyanopropyltrimethoxysilane (CPTS) and heated to reflux for 6 hours. The treated mesoporous silica was washed with toluene to remove any unreacted silane. The air dried CPTS-SAMMS material is then 50% H₂SO_FourTreated with solution and refluxed for 3 hours. After washing with DI water for a long time, the white sample was dried under vacuum at 70 ° C. overnight.
[0048]
In the case of 20% aminopropyl (APTS) and mercaptopropyl (MPTS) functionalized silica, the same procedure was applied without the hydrolysis step. To a suspension of 2.0 g mesoporous silica was added separately toluene (60 mL) and 0.64 g water and 0.188 g APTS or 0.206 g MPTS. The mixture was heated to reflux for 6 hours, then filtered off, washed with ethanol and dried under vacuum at 70 ° C. overnight.
OPH subcloning:
OPH sequences were obtained by PCR from the E. coli FM5 (Amgen Inc.) (American Type Culture Collection, Rockville, MD) using the pCMS75 plasmid (ATCC ( (Registered trademark) # 67778)). Primers for PCR reactions, Primer Premier from Premier Biosoft International^TMSelected using software, version 4.04 (Primer Premier software, version 4.04). The primers used are listed as follows:
OPH upstream primer, 26mer:
[0049]
[Chemical 1]

[0050]
OPH downstream primer having a BamHI restriction enzyme cleavage site (recognition sequence is in bold), 20mer:
[0051]
[Chemical 2]

[0052]
The OPH sequence was subcloned into pET11a using NdeI, BamHI restriction enzyme cleavage sites. The resulting OPH sequence encodes the mature part of the OPH enzyme, ie it does not have an N-terminal 29 amino acid signal sequence, so that the length of the sequence is 1010 bp, which is a total of 337 Corresponds to an amino acid or MW 36,419 Da.
[0053]
Confirmation that the correct product of cloning was obtained was performed by PCR and restriction enzyme digestion.
A diagram of the OPH plasmid is shown in FIG.
Expression and purification:
Organophosphorus hydrolase was transformed into the oph-pET11a plasmid and BL21 (DE3) pLyss^TMNovagen Inc. Was purified from the E. coli expression system using. (N-terminal His-Tag subcloned into pET-15b plasmid^TMOPH). Induction with IPTG has been shown to produce a protein with apparent mobility on the SDS gel surface, mature native OPH without an N-terminal signal sequence (having about 36 kDa or N-terminal His-Tag about 38 kDa) Consistent with predictions based on protein gene sequence. Following induction with IPTG, the identity of the recombinant product was also confirmed by amino acid analysis and the appearance of paraoxon hydrolysis activity in the crude cell lysate.
[0054]
Bio-Flo 3'000 fermenter^TM(Bio-Flo 3'000 fermenter^TMThe general protocol used for cell growth in (New Brunswick, Inc.) was as follows:
E. coli cells are grown in flasks at 30 ° C. for 12 hours and 100 μL glycerol stock / 1 L LB medium, 100 μg / mL ampicillin, 35 μg / mL chloramphenicol, and this starting culture are inoculated into the fermentor Used as material. Centrifuge 500 mL ON culture with OD = 0.5, wash with fresh LB medium, centrifuge again, redissolve in 250 mL LB without antibiotics, add to fermentor medium, Total volume = 2.5L. (Note: 1 mM CoCl₂Ensure that is not added to the LB medium for ON starting culture. Because it kills the cells).
[0055]
The cells in the fermenter were 4 hours later at 37 ° C. with 5 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl, 1 mL / L antifoam, 60 mM K.₂HPO_Four, 15 mM KH₂PO_Four1 mM CoCl₂1.32 μg / mL thiamine, 100 μg / mL ampicillin, 35 μg / mL chloramphenicol, 10 g / L glycerol and trace metals (10 μM NH_FourMo₇O_{twenty four}, CuSO_Four, H_ThreeBO_Three, MnCl₂ZnCl₂), 50 μM FeCl_Three0.5 mM CaCl₂1 mM MgSO_FourThe mid-log phase was reached in the medium containing. A DO-agitation-oxygen triple cascade was used and the agitation range was set from a minimum of 200 rpm to a maximum of 800 rpm to maintain the oxygen level at 35%. The initial glucose concentration in the medium was 10 g / L, and glucose levels were monitored and maintained above 2 g / L during the experiment using a regular glucose strip.
[0056]
When the agitation reached 467 rpm and OD550 = 5, the glucose level was 2 g / L. 31 mL of 40% glucose was added to bring the glucose level to 5 g / L. When stirring reached 700 rpm and OD550 = 15, the glucose level dropped again to 2 g / L. The temperature dropped to 28 ° C. and the mixture was induced with 0.25 mM IPTG and an additional 31 mL of 40% glucose was added. After induction for 4 hours at 28 ° C., an additional 0.25 mM IPTG was added (0.5 mM total). The glucose level was again 2 g / L and 63 mL of 40% glucose glucose was added to bring the glucose level to 10 g / L. After another 2 hours of induction, the cells apparently continued to grow, the temperature dropped to 24 ° C. and the cells were left in the fermentor ON for an additional 14 hours. Finally, the cells were collected by centrifugation at 6,000 rpm for 20 minutes at 4 ° C.
[0057]
From a 2.5 L cell culture, about 150 g wet weight of cell paste was isolated and stored at -80 ° C.
We were able to purify about 90 mg of OPH with an activity of 13,294.12 enzyme units / mg from 60 g of cells (corresponding to 1 L culture). This yield can be compared with the literature. Omburo GA, Kuo JM, Mullins LS, and Raushel FM, in Characterization of the Zinc Binding site of Bacterial Phosphotriesterase. JBC, 1992, v. 267 (5): It was reported that about 298 mg of cobalt phosphotriesterase with mg activity was obtained.
[0058]
Lai K., Dave KI, and Wild JR (Bimetallic Binding Motifs in Organophosphorous Hydrolase Are Important for Catalysis and Structural Organization.JBC, 1994, 269 (24): 16579-16584) are more difficult to compare, It was reported that 5 mg of OPH / 1 L culture (probably grown in a flask) was purified. They prefer to use weak promoters for expression (native Plac) versus strong promoters such as T7 because the OPH they obtained using strong promoter constructs This is because the yield of activity was lower (data not shown). All purification steps were performed at 4 ° C. using pre-cooled equipment and a Revco Chromatography Refrigeration cabinet.
[0059]
Bacterial cells (60 g) were washed with 100 mM HEPES (pH 8.5), 50 μM CoCl.₂The cells were lysed by suspending in 420 mL of lysis buffer A containing 1 mM DTT, anti-protease cocktail (pepstatin, leupeptin and aprotinin) and using the French pressure cell twice. The soluble protein supernatant was centrifuged at 100,000 × g for 1 hour (Avanti^TMThe Beckman (Avanti^TMBechman)), 500 mL of HQ equilibrated in buffer A using 1 mM DTT instead of 5 mM beta-mercaptoethanol (2-me).^TMAn anion exchange column (Perseptive Biosystems) was loaded. The column flow rate was 25 mL / min. Collect the flow-through containing OPH, adjust the pH to 7.5 using 1 M MES (pH 5.5), 0.1 M HEPES (pH 7.5), 50 μM CoCl.₂It was applied to a 250 mL HS cation exchange column (Perspective Biosystems) equilibrated in 5 mM 2-me. The column flow rate was 25 mL / min. Hold the HS column flow-through and dry thoroughly (NH_Four) SO_FourAnd using an Orion 126, Cell 012210 conductivity meter to bring the final conductivity of the sample to 1M (NH_Four) SO_FourSolution conductivity (ie, 105 mS/ mL). Samples were washed with 1M (NH_Four) SO_Four180 mL of polypropyl A equilibrated in 0.1 M HEPES (pH 7.5), 5 mM 2-me^TM(PolyLC, Inc.) The column was loaded. The column flow rate was 10 mL / min.
[0060]
After washing 3 column volumes with equilibration buffer, a 10 column volume gradient up to 0.1 M HEPES (pH 7.5) was used to elute OPH at the extreme end of the gradient. We were able to purify nearly 100 mg of pure OPH from 60 g of cells (corresponding to 1 L culture). Millipore Ultrafree® Biomax centrifugal concentrator using a 30 KNMWL (30 kDa cut-off) membrane and 0.1 M HEPES (pH 7.5) for 20% glycerol 50 μM CoCl₂After concentrating the protein to 3 mg / mL using dialysis, the protein was aliquoted and stored at −80 ° C. Specific activity was determined to be 13,294.12 enzyme units / mg (see Table I and SDS gel summary).
[0061]
The difference between the protein activity appearing as a peak from the HIC and the activity of the same protein after concentration and dialysis is probably explained by buffer exchange. In one case, the buffer comprises beta-mercaptoethanol, a competitive inhibitor of OPH activity, and CoCl₂The other buffer is 50 μM CoCl.₂And 2-me was not included.
[0062]
[Table 1]

[0063]
OPH immobilization:
Medium used:
1. SAMMS: SH-, COO-, NH₂-Derivatized with active groups and provided 5% and 20% coatings (5% and 20% of all available silane groups were modified or derivatized with active groups). Medium properties: 250 angstrom 12-15 μm beads, surface area about 450 m²/ g
2. PolyLC silica: purchased uncoated, NH₂-Derivatized in PNNL using COO- groups and applied 20% and 100% coatings. Medium characteristics: 300 angstrom 12 μm beads, surface area about 100 m²/ g.
[0064]
After sieving for the highest density of bound enzyme and the best binding chemistry that would give the lowest loss of activity and the lowest diffusion limit, we found that the NH of OPH₂-OPH and NH via the group₂-The coupling with the derivatization medium was selected.
[0065]
A number of crosslinkers have been tested and two of them have been found to be particularly effective: sulfo-BSOCOES (bis [2- (sulfosuccinimidoxycarbonyloxy) ethyl] sulfone) and DTSSP (dithiobis ( Sulfosuccinimidyl propionate)). The advantages of these two crosslinkers are:
i. Both crosslinkers have spacer arms (12 angstroms long for DTSSP and 13 angstroms for BSOCOES). Spacer arms are beneficial to avoid steric hindrance.
[0066]
ii. Both of these are water soluble because of the sulfo functioning group.
iii. Pierce (Rockford, IL) recommended PBS as a binding buffer (pH 7.5). This pH is favorable for OPH, because at acidic pH, i.e. below 6.5, OPH tends to aggregate and lose metal from the active center. Higher pH has further advantages: NHS-ester hydrolysis proceeds faster at higher pH. Thus, when higher pH was used, we obtained a higher crosslinker / protein molar ratio.
[0067]
iv. Both result in stable covalent amide bonds, which in the case of DTSSP can be cleaved using thiols (DTT, mercaptoethanol, etc.). This feature may be useful for certain applications.
OPH and NH ₂ Standard protocol for sulfo-BSOCOES or DTSSP coupling with derivatized surfaces:
(Pierce's protocol is almost unmodified)
1. Sulfo-BSOCOES or DTSSP cross-linking reagents (stocked in tubes) should be stored at −20 ° C., preferably dry and under nitrogen. In practice, when working with a cross-linking reagent (ie, dispensing the contents of the first tube), a nitrogen glove box or nitrogen bag (filled with nitrogen using a nitrogen tank in a refrigerator) ) Is a good idea. Always bring the reagents to room temperature before opening the tube.
[0068]
2. The medium that will bind to OPH should swell well in water. In general, we have 500 mg medium / 5 mL H₂Using O, a ~ 50% slurry (v / v) for SAMMS was made. This slurry was very stable when stored at + 4 ° C. Note: In the case of poly-LC medium, medium twice as much dry weight as compared to SAMMS should be used (ie ~ 1 g poly-LC medium / 5 mL H₂O and ~ 50% slurry (v / v) are made).
[0069]
3. 2 mL of OPH (purified to 09/02, [3 mg / mL]) is aliquoted, stored at −80 ° C. and thawed, and the buffer is 25 mM HEPES (pH 8.5), 20% glycerol, 50 μM CoCl.₂To 0.1M curve / carb / bicarbonate (pH 9.0). The NAP-25 column used by the inventors had a maximum volume of 10 mL. With this, we were able to utilize a maximum sample volume of about 2.5-3.0 mL.
[0070]
4). Using a new Millipore Biomax Ultra Free 4.0 mL 30 KCO membrane unit, 10 mg at + 4 ° C., using Solval CF, Bucker rotor at maximum speed, 20 mg / mL Until concentrated.
[0071]
5). Using the cut yellow tip,₂50% slurry powder in O was added to OPH in 0.1 M curve / bicarbonate buffer (pH 9.0) in a volume of 90.1 mL for 2 mg. The approximate ratio of slurry powder is 150 μl of 50% slurry / 2 mg protein: PolyLC silica and NH₂-Two types of derivatization (NH₂-20% and 100% coverage).
[0072]
6. Dissolve 1.5 mg or 3 mg or 6 mg sulfo-BSOCOES or DTSSP in 590 μl of 5 mM MES (pH 5). (To obtain crosslinker / protein molar ratios of 10 ×, 25 ×, and 50 ×)
7). Immediately drop the crosslinker solution (125 μl / 2 mg protein) into each eppendorf tube, mix and attach the tube to the rotator for 45 minutes at room temperature.
[0073]
8). Stop solution: 1 mL of 1 M Tris (pH 8.1) is added for 1 hour at room temperature.
9. Wash twice with PBS, once with 0.5M NaCl-PBS, and once with PBS. 100 mM HEPES (pH 8.5) -50 μM CoCl₂Resuspend in and store at + 4 ° C.
[0074]
10. The reaction is performed at 37 ° C. for 30 minutes using a BCA Pierce kit to estimate the amount of protein backing the resin.
[0075]
Example 1
OPH and SAMMS-NH using a 20% coating of sulfo-BSOCOES and a 50 × molar excess of crosslinker / protein₂Coupling with derivatized surfaces
The standard protocol was followed as follows:
1. A nitrogen-filled nitrogen glove box was used to remove an aliquot of the contents from the tube containing sulfo-BSOCOES.
[0076]
2. 500 mg medium with 5 mL H₂Used in O to make ~ 50% slurry (v / v). This slurry was very stable when stored at + 4 ° C.
3. 2 mL of OPH (purified to 09/02, [3 mg / mL]) is aliquoted, stored at −80 ° C. and thawed, and the buffer is 25 mM HEPES (pH 8.5), 20% glycerol, 50 μM CoCl.₂To 0.1 M curve / bicarbonate (pH 9.0) and a Pharmacia Sephadex G-25 column was used. The NAP-25 column used by the inventors had a maximum volume of 10 mL. With this, we were able to utilize a maximum sample volume of about 2.5-3.0 mL.
[0077]
4). A new Millipore Biomax Ultrafree 4.0 mL 30 KCO membrane unit was used and concentrated to 20 mg / mL using Solvar CF, backer rotor at top speed for 10 minutes at + 4 ° C.
[0078]
5). Using the cut yellow tip,₂50% slurry powder in O was added to OPH in a 0.1 M curve / bicarbonate buffer (pH 9.0) in a volume of 90.1 mL for 2 mg. The approximate ratio of slurry powder is 150 μl of 50% slurry / 2 mg protein: PolyLC silica and NH₂-Two types of derivatization (NH₂-20% and 100% coverage).
[0079]
6.6 mg of sulfo-BSOCOES is dissolved in 590 μl of 5 mM MES (pH 5).
7). Immediately drop sulfo-BSOCOES solution (125 μl / 2 mg protein) into Eppendorf tube, mix and attach tube to rotator for 45 minutes at room temperature.
[0080]
8.1 mL of 1M Tris (pH 8.1) is added for 1 hour at room temperature.
9. Wash twice with PBS, once with 0.5M NaCl-PBS, and once with PBS. 100 mM HEPES (pH 8.5) -50 μM CoCl₂Resuspend in and store at + 4 ° C.
[0081]
10. Using a BCA Pierce kit and reacting at 37 ° C., the estimated amount of OPH bound to the resin = 25.0 mg / mL medium and 125.0 mg / g medium.
[0082]
Example 2
OPH and SAMMS-NH using a 20% coating of sulfo-BSOCOES and using a 25x molar excess of crosslinker / protein₂Coupling with derivatized surfaces. The procedure was similar to Example 1, except that 3 mg sulfo-BSOCOES was used instead in step 6. Estimated amount of OPH bound to resin = 16.0 mg / mL medium and 8.0 mg / g medium.
[0083]
Example 3
OPH and SAMMS-NH using a 20% coating of sulfo-BSOCOES and a 10x molar excess of crosslinker / protein₂Coupling with derivatized surfaces. The procedure was similar to Example 1, except that 1.5 mg of sulfo-BSOCOES was used instead in step 6. Estimated amount of OPH bound to resin = 5.0 mg / mL medium and 25.0 mg / g medium.
[0084]
Example 4
OPH and SAMMS-NH using a 20% coating of DTSSP and a 50x molar excess of crosslinker / protein₂Coupling with derivatized surfaces. The procedure was similar to Example 1, except that 6 mg of DTSSP was used in Step 6 instead. Estimated amount of OPH combined with resin = 25.0 mg / mL medium and 125.0 mg / g medium.
[0085]
Example 5
OPH and SAMMS-NH using a 20% coating of DTSSP and a 25x molar excess of crosslinker / protein₂Coupling with derivatized surfaces. The procedure was similar to Example 1, except that 3 mg DTSSP was used instead in step 6. Estimated amount of OPH combined with resin = 16.0 mg / mL medium and 80.0 mg / g medium.
[0086]
Example 6
OPH and SAMMS-NH using a 20% coating of DTSSP and a 10x molar excess of crosslinker / protein₂Coupling with derivatized surfaces. The procedure was similar to Example 1, except that 1.5 mg of DTSSP was used instead in step 6. Estimated amount of OPH bound to resin = 5.0 mg / mL medium and 25.0 mg / g medium.
[0087]
Example 7
On 20% and 100% coated surfaces using sulfo-BSOCOES and DTSSP, OPH was coupled to PolyLC using 10 ×, 25 ×, and 50 × molar excess of crosslinker / protein. The procedure was the same as in Examples 1-6 except that in step 2, 5 mL H₂Using 50 mg of polyLC per O, a ~ 50% slurry (v / v) was made.
[0088]
The estimated amount of OPH bound to the resin is listed in Table II.
[0089]
[Table 2]

[0090]
It can be seen that the composition of the present invention can be loaded with a higher density than when conventional silica (polyLC) is used. Accordingly, preferred embodiments of the present invention can be characterized by load density. Preferably, the protein system has a density that is 2 to about 7 times higher (in mg / g), more preferably about 5 to about 7 times higher than PolyLC with the same% coating. In a preferred embodiment, the density is measured with a 20% coating.
[0091]
Example 8-Effect of denaturation
The stability of OPH-SAMMS and soluble OPH to denaturing conditions was investigated using urea as a denaturant at concentrations of 4M, 6M and 8M. The phrase “soluble OPH” or “OPH is soluble” in the Examples section refers to non-inclusion bodies that are released during cell disruption using a French press and are soluble in the buffer indicated at each purification step. Refers to OPH. The results are shown in Table 3 below.
[0092]
[Table 3]

[0093]
Immobilized enzymes are much more stable than free proteins. Accordingly, preferred embodiments of the present invention can be characterized by their stability to denaturing agents. Preferably, the protein system is stable in 8M urea at least twice as stable as the free protein, and in some embodiments, about 3 to about 5 times more stable.
[0094]
Example 9-Recovery from dehydration
Dehydration and recovery experiments were performed using immobilized OPH-SAMMS and soluble OPH. Soluble OPH retained only 7 ± 1% of its activity, while OPH-SAMMS fully retained its activity (106 ± 8%).
[0095]
Example 10-Kinetic Properties
Kinetic studies were performed using SAMMS surface immobilized OPH, PolyLC surface immobilized OPH, and soluble OPH. Using 1 mM paraoxon solution at 25 C, 100 mM CHES buffer (pH 8.0), 50 μM CoCl₂When the substrate was hydrolyzed to diethyl phosphate and p-nitrophenolate, the change in absorbance was monitored at 405 nm (the extinction coefficient was 17,000 M^-1cm^-1), Enzyme activity was measured. For analysis, a Hewlett-Packard model 8453 UV / Vis spectrophotometer in kinetics mode equipped with the Thermostable Cell Holder and Cell Stirring Module) was used. New dilutions of substrate were made within 30 minutes prior to measurement. A sigma plot (SigmaPlot) was used to plot a linear regression of the data. PolyLC (Colombia, MD) silica was purchased uncoated and derivatized with amino groups. The pore size of the 12 μm beads was 300 Å. Both SAMMS-immobilized OPH and poly-LC immobilized OPH have the same K as soluble OPH._mBut lower V_max(2.83 times lower for OPH-SAMMS and 6.44 times lower for OPH-Polysea). That is, OPH-SAMMS has a reaction rate that is about 2.3 times greater than OPH-PolyLC. The mass in the table refers to the mass of OPH.
[0096]
[Table 4]

[0097]
Example 11-Stability at alkaline pH
In this experiment, OPH-SAMMS and soluble OPH were tested for stability under alkaline conditions. After 1 hour alkaline pH treatment (1 M Tris, pH 12.0), OPH-SAMMS was found to retain 11.6% of its activity compared to 0.77% for soluble OPH. After 24 hours of this alkaline treatment, OPH-SAMMS was found to retain 9.9% of its activity compared to 0.03% for soluble OPH. The conditions in this example define what the term “alkaline conditions” means when used in this application. Although this example shows an OPH system, it should be recognized that the stability benefits provided by the mesoporous matrix of the present invention are general and other proteins have similar stability It is expected that the benefits will be obtained.
[0098]
Example 12-Thermal stability
Experiments investigating thermal stability showed that OPH-SAMMS is much more stable than OPH in solution. The results of these experiments are shown in the table below.
[0099]
[Table 5]

[0100]
Example 13-Effect of lyophilization
OPH-SAMMS and soluble OPH were exposed to lyophilized conditions (1 M MES, pH 5.0), and OPH-SAMMS retained 50% of its activity after 1 hour and 24 hours, while 1 hour and 24 hours. Later it was found that soluble OPH retained only 15% of its activity.
[0101]
Example 14
(1) A nitrogen glove box filled with nitrogen was used to remove an aliquot of the contents from the tube containing sulfo-BSOCOES.
[0102]
(2) 0.1M HEPES / 50 μM CoCl at pH 7.5₂The OPH in was purified to 05/04/01, 1 mL aliquots were stored at −80 ° C. and thawed in the refrigerator. The enzyme stock contained 9223.30 enzyme units (amount of 0.29 mg OPH protein) / mL.
[0103]
(3) 300 Å 2% NH in 1.8 mL tube₂-2-4 mg aliquots of SAMMS, 100-200 μL of 3.2 mg / mL sulfo-BSOCOES (0.1 M HEPES / 50 μM CoCl at pH 7.5)₂Medium) was added and shaken for 2 minutes. This was then centrifuged at 1400 rpm for 2 minutes, the supernatant containing excess crosslinker was separated, and the deposit was washed with 100 μL of the same buffer. Then add 100-800 μL of OPH stock to this for 1400 minutes^-1And shaking on an Eppendorf Thermomixer 5436 at 25 ° C. for 45-90 minutes.
[0104]
(4) The enzyme incubation solution was then separated and the resulting deposit was washed with 20 mM phosphate / 0.15 M NaCl (n ≧ 10) at n × 400 μL pH 7.5. At an intermediate stage, this was centrifuged at 14K rpm for 6 minutes. Finally, the washed sediment was resuspended again in the same washing buffer with 100 μL buffer / mg initial SAMMS. After thorough washing, both the suspension and the clear solution were measured, indicating that OPH that was not firmly immobilized was washed away completely.
[0105]
(5) The estimated protein amount of covalently bound OPH is 11.00 mg / g SAMMS by Pierce BCA assay kit (Pierce, 23227), with an initial specific activity of 11386.07 enzyme units / mg bound OPH. Had.
[0106]
Example 15
(1) 10.17 mg GOD (Sigma G-7016) was dissolved as a GOD stock in 20 mM phosphate / 0.15 M NaCl at 5 mL pH 7.5. The enzyme stock contained 395.05 enzyme units (amount of 1.05 mg OPH protein) / mL.
[0107]
(2) Same as Example 14, but with GOD stock and 300 Å 20% NH₂-SAMMS was used instead.
(3) GOD activity was measured using Sigma standard method (revised to 08/30/96).
[0108]
(4) The estimated protein amount of covalently bound GOD was 14.85 mg / g SAMMS, with an initial specific activity of 114.31 enzyme units / mg bound GOD.
[0109]
Example 16
(1) 9.95 mg of GOD (Sigma G-7016) was dissolved as a GOD stock in 5 mM pH 7.0 in 20 mM phosphate / 0.15 M NaCl. The enzyme stock contained 372.25 enzyme units (amount of 1.08 mg OPH protein) / mL.
[0110]
(2) Same as Example 15, but 200-400 μL of 5% glutardialdehyde (Oldrich, 34085-5) (pH 7.0, 20 mM) instead of sulfo-BSOCOES solution as source of covalent linker Phosphate / 0.15M NaCl) was used.
[0111]
(3) The washing solution had a pH of 7.0 instead of the pH 7.5 used in Example 15.
(4) The estimated protein amount of covalently bound GOD was 81.64 mg / g SAMMS, with an initial specific activity of 72.34 enzyme units / mg bound GOD.
[0112]
Example 17
(1) GI purified from Streptomyces rubiginosus was purchased from Hampton Research, Inc. (Hampton Research, Inc.). This is treated with 20 mM phosphate / 0.15 M NaCl / 1 mM MgSO at pH 7.5._FourDialyzed in, aliquoted 1 mL, stored at −80 ° C., and thawed in refrigerator before use. The GI stock contained 180.22 enzyme units (amount of 4.11 mg GI protein) / mL.
[0113]
(2) Similar to Example 15, but GI stock was used instead of GOD stock. Incubation was performed at 40 ° C.
(3) The wash solution was pH 7.5, 20 mM phosphate / 0.15 M NaCl / 1 to 20 mM MgSO._Fourwas.
[0114]
(4) Definition of GI activity under these conditions: 1 enzyme unit is 1 micromolar D-converted to D-glucose in 1 minute at pH 7.5, 60 ° C., 1.923 M D-fructose concentration Fructose. Activity was measured by the following general procedure: To 5-20 μL of GI-SAMMS suspension, 0.5 mL of 2M D-fructose (Fluka 47739) (20 mM at pH 7.5) was added. Phosphate / 0.15M NaCl / 20mM MgSO_FourMedium) and then 1400 minutes^-1And shaken on an Eppendorf Thermomixer 5436 at a controlled temperature of 60 ° C. for 15 minutes. Next, 10 μL of 1.8 M H₂SO_FourThe reaction was stopped by. Next, 2.0 mL of glucose (GO) assay reagent (Sigma, G3660) was added to 5 to 20 μL of the reaction solution and allowed to incubate at 37 ° C. for 30 minutes in a hot water bath. Incubate the reaction with 1.0 mL of 6M H.₂SO_FourAnd the absorbance was measured at 540 nm. From the glucose standard curve, the amount of glucose produced by GI-catalyzed conversion of fructose was obtained.
[0115]
(5) The estimated protein amount of covalently bound GI was 48.47 mg / g SAMMS, with an initial specific activity of 53.40 enzyme units / mg bound GOD.
[0116]
Example 18
(1) Similar to Example 17, but instead of sulfo-BSOCOES solution as a source of covalent binder, 200-400 μL of 5% glutardialdehyde (Oldrich, 34085-5) (pH 7.5, 20 mM phosphate / 0. 15M NaCl) was used.
[0117]
(2) The estimated amount of covalently bound GI protein was 30.45 mg / g SAMMS with an initial specific activity of 39.19 enzyme units / mg bound GOD.
[0118]
Example 19
(1) 0.1M HEPES / 50 μM CoCl at pH 7.5₂1 mL of the purified OPH was collected, stored at −80 ° C., and thawed in the refrigerator. The enzyme stock contained 9223.30 enzyme units (amount of 0.29 mg OPH protein) / mL.
[0119]
(2) 300 angstroms of 2% NH in a 1.8 mL tube₂Add 100-800 μL of OPH stock to 2-4 mg aliquots of SAMMS, 1400 minutes^-1And shaken on an Eppendorf Thermomixer 5436 at 25 ° C. for 2-3 hours.
[0120]
(3) The enzyme incubation solution was then separated and the resulting deposit was washed with 20 mM phosphate / 0.15 M NaCl (n ≧ 10) at n × 400 μL pH 7.5. At an intermediate stage, this was centrifuged at 14,000 rpm for 6 minutes. Finally, the washed sediment was resuspended again in the same washing buffer with 100 μL buffer / mg initial SAMMS. After thorough washing, both the suspension and the clear solution were measured, indicating that the OPH that was not firmly immobilized was washed away completely.
[0121]
(4) The estimated protein amount of incorporated OPH was 9.52 mg / g SAMMS by Pierce BCA assay kit, with an initial specific activity of 16378.74 enzyme units / mg incorporated OPH.
[0122]
Example 20
(1) 10.17 mg GOD (Sigma G-7016) was dissolved as a GOD stock in 20 mM phosphate / 0.15 M NaCl at 5 mL pH 7.5. The enzyme stock contained 395.05 enzyme units (amount of 1.05 mg OPH protein) / mL.
[0123]
(2) Same as Example 19, but with GOD stock and 300 Å 20% NH₂-SAMMS was used instead.
(3) GOD activity was measured using Sigma standard method (revised to 08/30/96).
[0124]
(4) The estimated protein amount of incorporated GOD was 13.35 mg / g SAMMS, with an initial specific activity of 99.33 enzyme units / mg incorporated GOD.
[0125]
Example 21
(1) Similar to Example 20, but 9.95 mg GOD (Sigma G-7016) was dissolved as a GOD stock in 20 mM phosphate / 0.15 M NaCl at 5 mL pH 7.0. The enzyme stock contained 372.25 enzyme units (amount of 1.08 mg OPH protein) / mL.
[0126]
(2) The wash solution had a pH of 7.0 instead of the pH 7.5 used in protocol 7.
(3) The estimated protein amount of incorporated GOD was 14.59 mg / g SAMMS, with an initial specific activity of 136.28 enzyme units / mg incorporated GOD.
[0127]
Example 22
(1) GI purified from Streptomyces rubiginosus was purchased from Hampton Research, Inc. Obtained from. This is treated with 20 mM phosphate / 0.15 M NaCl / 1 mM MgSO at pH 7.5._FourDialyzed in, aliquoted 1 mL, stored at −80 ° C., and thawed in refrigerator before use. The GI stock contained 180.22 enzyme units (amount of 4.11 mg GI protein) / mL.
[0128]
(2) Same as Example 20, but GI stock was used instead of GOD stock. Incubation was performed at 40 ° C.
(3) The wash solution was pH 7.5, 20 mM phosphate / 0.15 M NaCl / 1 to 20 mM MgSO._Fourwas.
[0129]
(4) GI activity was measured by the same method as performed in protocol 4.
(5) The estimated protein amount of incorporated GI was 94.94 mg / g SAMMS, with an initial specific activity of 43.26 enzyme units / mg incorporated GOD.
[0130]
Example 23
(1) 2 mL aliquots of purified OPH are stored at −80 ° C., thawed in a refrigerator, 25 mM HEPES / 20% glycerol / 50 μM CoCl at pH 8.5₂To 0.1 M HEPES at pH 7.2. The enzyme stock contained 4830.00 enzyme units (amount of 0.39 mg OPH protein) / mL.
[0131]
(2) To an 82.45 mg aliquot of 300 angstrom 2% HOOC-SAMMS in a 50 mL tube, add 12 mL OPH stock, 350 rpm speed, on an Innova 4330 refrigerated incubator shaker (Innova 4330 refrigerated incubator shaker) And shaken at 25 ° C. for 2 hours and 47 minutes. Next, 0.4 mL of the obtained suspension was collected.
[0132]
(3) The enzyme incubation solution was then separated and the resulting deposit was washed with 20 mM HEPES (n ≧ 10) at n × 400 μL pH 7.5. At an intermediate stage, this was centrifuged at 14,000 rpm for 6 minutes. Finally, the washed sediment was resuspended again in the same washing buffer with 100 μL buffer / mg initial SAMMS. After thorough washing, both the suspension and the clear solution were measured, indicating that OPH that was not firmly immobilized was washed away completely.
[0133]
(4) The estimated protein amount of incorporated OPH was 46.73 mg / g SAMMS, with an initial specific activity of 26766.78 enzyme units / mg incorporated OPH.
[0134]
Example 24
(1) 31.07 mg of GOD (Sigma G-7016) was dissolved as a GOD stock in 20 mM phosphate at 11 mL pH 7.0. The enzyme stock contained 393.08 enzyme units (1.71 mg of GOD protein amount) / mL.
[0135]
(2) Same as Example 23 but with GOD stock and 98.67 mg of 300 angstrom 20% NH.₂-SAMMS was used instead.
(3) The wash solution is pH 7.0, 20 mM phosphate buffer.
[0136]
(4) GOD activity was measured using the Sigma standard method (revised to 08/30/96).
(5) The estimated protein amount of incorporated GOD was 107.94 mg / g SAMMS, with an initial specific activity of 76.22 enzyme units / mg incorporated GOD.
[0137]
Example 25
(1) GI purified from Streptomyces rubiginosus was purchased from Hampton Research, Inc. Obtained from. This is pH 7.5, 20 mM phosphate / 0.15 M NaCl / 1 mM MgSO._FourDialyzed in, collected 1 mL and stored at -80 ° C. Six aliquots are thawed in a refrigerator before use, 22.5 mL pH 7.0, 20 mM sodium / 1 mM MgSO_FourTo give a GI stock. The GI stock contained 42.10 enzyme units (amount of 0.86 mg GI protein) / mL.
[0138]
(2) Same as Example 24 but with GI stock and 100.98 mg of 300 Å 20% NH₂-SAMMS was used. Incubation was performed at 40 ° C.
(3) The wash solution was pH 7.5, 20 mM phosphate / 1-20 mM MgSO_Fourwas.
[0139]
(4) GI activity was measured by the same method as performed in Example 17.
(5) The estimated protein amount of incorporated GI was 77.26 mg / g SAMMS with an initial specific activity of 45.06 enzyme units / mg incorporated GI.
Test results including comparative examples and various conditions
PolyLC Inc. Normal silica (Item No .: BMSI 1203) commercially available from is functionalized in the same way as for SAMMS and used for comparison instead of SAMMS in the above protocol under the same experimental conditions.
[0140]
In order to investigate the effects of pore size, functionality type and coating, various pore sizes, functional monolayer types and coatings were used to replace SAMMS in the above protocol under the same experimental conditions.
[0141]
To investigate the effects of buffer composition, pH, stability and ionic strength, a variety of different wash solutions were used in place of the wash solutions in the protocol described above under the same experimental conditions.
Some results are shown in the figure. Note that the following notation was used: 300 Å 20% NH₂-SAMMS is NH derivatized from tris- (methoxy) aminopropylsilane₂-Means 300 angstroms mesoporous silica with 20% coverage of functional monolayer, while 300 angstroms of 2% HOOC-SAMMS is a HOOC-functionalized from tris- (methoxy) carboxylethylsilane Means the same mesoporous silica with a 2% coverage of a functional monolayer.
[0142]
FIG. 5 shows comparative data for OPH immobilized on five different matrix surfaces. Three examples using normal silica had an activity of 48 enzyme units / mg of silica, while OPH immobilized on the SAMMS surface demonstrated 156 and 125 activities. See Examples 14 and 19. Thus, protein systems made from conventional silica have demonstrated much lower activity.
[0143]
The type of functional group and the degree of functionalization have been found to be important factors in forming protein systems. Example 19 (activity 156) was repeated using 20% functionalization (on the surface of a 100, 170 or 300 angstrom SAMMS) instead of 2%, resulting in an activity of less than 1. Similarly, when Example 14 (activity 125) was repeated using 20% functionalization (on the surface of a 100, 170, or 300 angstrom SAMMS) instead of 2%, an activity of less than 20 was obtained. 300 Å 20% HOOC-SAMMS treated with OPH demonstrated 97 activity, while 300 Å 20% NH treated similarly with OPH₂-SAMMS had an activity of less than 1.
[0144]
The data in the Examples section shows the individuality regardless of whether protein immobilization or non-covalent uptake is used (although the type of binding is another factor that can be controlled). The importance of adapting anionic or cationic functionalization at an appropriate density for a number of proteins was demonstrated. This adaptability facilitates the incorporation of high concentrations of active protein into the porous matrix. The resulting protein system can be defined by its activity or other measurable property.
[0145]
FIGS. 6 and 9 show the protein incorporated at various buffer concentrations (FIG. 6) and the specific activity of the protein incorporated at various buffer concentrations (compared to a solution of OPH at pH 7.2) (FIG. 6). In both cases 9) the activity is sensitive to the buffer concentration (ionic strength).
[0146]
As shown in FIGS. 7 and 8, the sample made at pH 7.0 contained more OPH than the sample made at higher pH, but the specific activity of the protein system appears to be optimized at approximately pH 7.5. It is. The specific activity of FIG. 8 is the specific activity of immobilized OPH divided by the specific activity of the control solution of OPH at pH 7.2.
[0147]
Incorporated OPH, whether retained non-covalently or covalently, has demonstrated much better stability compared to OPH in solution. As shown in FIG. 10, incorporated protein showed less than about 30% loss of activity when refrigerated at 4 ° C. for 110 days. After another 40 days at room temperature, some activity was restored, perhaps by regeneration.
[0148]
FIG. 11 shows comparative data for GOD immobilized on 6 different matrix surfaces. Three examples using normal silica have activities of 0.2, 0.3 and 0.4, while GOD immobilized on the SAMMS surface proves activity of 2.0 and 5.9 did. See Examples 20 and 15. Thus, protein systems made from conventional silica have demonstrated much lower activity. Unfunctionalized SAMMS (ie, 0% NH) named “300 Å mesosilica” treated under the same conditions₂) Showed an activity of only 0.3. 100 angstroms 20% NH₂-GOD protein system produced from SAMMS is 300 angstroms of 20% NH₂-Showed the same activity as SAMMS, but with 100 angstroms of 20% NH using a covalent crosslinker₂-GOD protein system produced from SAMMS is 300 angstroms of 20% NH₂-It showed an activity of 4.0 compared to the activity 5.9 of a similarly prepared protein covalently crosslinked in SAMMS. As shown in FIG. 12, in the case of GOD, a porous matrix with 20% amino surface coverage performed much better than 2% surface coverage. The presence of 0.15M sodium chloride is NH₂-Caused a considerable decrease in the activity of GOD in the SAMMS system. As shown in FIG. 13, GOD-NH₂-Increasing the pH during the production of the SAMMS system resulted in a decrease in activity; however, the specific activity of GOD did not decrease in the higher pH range (see Figure 14). Again, as shown in FIG. 15, the protein system exhibits improved stability compared to the GOD solution, and surprisingly, after refrigeration at 4 ° C., the non-covalent protein system is a covalently bonded system. It showed better stability in comparison.
[0149]
Therefore, it may be desirable to characterize each protein system in terms of covalent and non-covalent bonds in addition to or in addition to functionalization. In some preferred embodiments, non-covalently bound proteins have superior stability compared to covalently crosslinked systems when tested according to the procedures described herein.
[0150]
FIG. 16 shows comparative data for GI immobilized on 8 different matrix surfaces. Four examples made under similar conditions using regular silica have activities of 0.0, 0.0, 0.3 and 0.0, while GI immobilized on the SAMMS surface Demonstrated activity of 4.1, 1.2 and 2.6, respectively. See Examples 22, 17, and 18. Thus, again, protein systems made from conventional silica have demonstrated much lower activity. Unfunctionalized SAMMS (ie, 0% NH) named “300 Å mesosilica” treated under the same conditions₂) Showed an activity of 0.0. In the case of GI, a significant effect is seen based on the pore size (FIG. 17), which was not unexpected given the large size of the GI. As shown in FIG. 18, covalently bonded 20% NH₂GI immobilized on normal silica surface proves specific activity 70, while covalently bonded 300 angstrom 20% NH₂-GI immobilized on the SAMMS surface demonstrated a specific activity of 89. NH₂-The GI in SAMMS was found to be hardly affected by the presence of 0.15 M sodium chloride. 300 Angstrom 2% NH₂-SAMMS and 300 Angstroms of 20% HOOC-SAMMS were found to have little or no activity.
[0151]
GI is a robust enzyme, and incorporation into SAMMS showed a relatively slight improvement in stability with respect to the time period and conditions tested. However, if the test is conducted under more difficult conditions such as exposure to a denaturant based on the stability test in the OPH protein system, the excellent stability characteristics of the preferred protein system of the present invention, particularly the system based on SAMMS, can be obtained. It is reasonable to conclude that it seems obvious. The present invention can generally be described in terms of stability under various test conditions.
[0152]
The present invention can include various modifications and changes without departing from the spirit and scope of the invention. Therefore, it should be understood that the invention should not be limited to the specific descriptions and examples, but should be controlled by the limitations set forth in the claims and the equivalents of the elements recited in the claims.
[Brief description of the drawings]
[0153]
FIG. 1 is a conceptualized cross-sectional view of an enzyme disposed in a porous substrate.
FIG. 2 is a ribbon diagram of the OPH plasmid.
FIG. 3 is the relevant DNA sequence (SEQ ID: 2) from the BamH I to Bgl II site of the construct, including just before the T7 promoter and just beyond the OPH stop codon.
FIG. 4 shows the amino acid sequence of OPH (SEQ ID: 1).
FIG. 5: Normal silica and NH for OPH immobilization₂-Comparison with SAMMS is shown.
FIG. 6 shows the effect of ionic strength on OPH incorporated into HOOC-SAMMS.
FIG. 7 shows the effect of pH on OPH uptake into HOOC-SAMMS.
FIG. 8 shows the immobilization efficiency of OPH in HOOC-SAMMS at various pHs.
FIG. 9 shows the effect of ionic strength on OPH immobilization efficiency in HOOC-SAMMS.
FIG. 10: NH₂-Shows the stability of OPH immobilized in SAMMS.
FIG. 11: Normal silica and NH for glucose oxidase (GOD) immobilization₂-Comparison with SAMMS is shown.
Fig. 12 NH for GOD incorporation₂-Shows the effect of SAMMS functional group coating.
FIG. 13 NH₂-Shows the effect of pH on GOD incorporation into SAMMS.
FIG. 14: NH at various pH₂-Indicates the immobilization efficiency of GOD in SAMMS.
FIG. 15 NH₂-Shows the stability of GOD immobilized in SAMMS.
FIG. 16: Normal silica and NH for GI immobilization₂-Comparison with SAMMS is shown.
FIG. 17 GI immobilization and NH₂-Comparison with the effect of SAMMS pore size.
FIG. 18 NH₂-Shows the immobilization efficiency of GI in SAMMS.

Claims (57)

At least 90% of the pore volume comprises a porous matrix material having said pore volume such that it is composed of pores having a size in the range of 50-400 Angstroms;
A protein system further comprising a chemically active protein associated with the matrix material. The protein system comprises 0.01-1 mmol of the protein per gram of matrix material, wherein the protein in the protein system exhibits an activity of at least 65% of the activity of the protein in an active state. The system according to 1. The system of claim 1, wherein the protein occupies 5-40% of the average pore volume. The protein system comprises 0.01-1 mmol of the protein per gram of matrix material, and at least 90% of the pore volume is composed of pores having a size in the range of 100-200 angstroms; The system according to claim 3. The system of claim 3, wherein the protein is an enzyme. 6. The system of claim 5, wherein the enzyme has a volume in the range of 0.5 x 10 < ⁵ > angstrom ^{3 to} 3 x 10 < ⁵ > angstrom ³ . 7. The system of claim 6, wherein the enzyme has an activity that is at least 50% of the activity in the active state. The system according to claim 7, wherein the enzyme is OPH having an activity of 60-95% of the activity in the active state. 7. The system of claim 6, wherein the protein volume is in the range of 10-25% of the average pore volume. The system of claim 6, wherein the surface area of the porous matrix material is at least 700 m ² / g. 6. The system of claim 5, wherein the enzyme is OPH and the system comprises 5-25 mg OPH per cubic centimeter. The enzyme is OPH, having a V _max of 0.15 to 0.66, the system according to claim 5. 6. The system of claim 5, wherein the system retains about 10% of its activity after 24 hours under alkaline conditions. The system of claim 2, wherein the matrix is SAMMS. A chemical process catalyzed by the system of claim 1. In the protein system comprising a porous matrix material sized such that the protein system contains 0.01-1 mmol protein per gram of matrix material, the protein in the protein system is in the active state A protein system that exhibits an activity of at least 65% of the activity of the protein. 17. The protein system of claim 16, wherein the porous matrix material has the pore volume such that at least 90% of the pore volume is composed of pores having a size in the range of 50-400 angstroms. 18. The protein system of claim 17, wherein the protein occupies 5-40% of the average pore volume. 18. A protein system according to claim 17, wherein the enzyme is OPH. 18. A protein system according to claim 17, wherein the matrix is a mesoporous oxide material. A chemical method catalyzed by the system of claim 16. A method of forming a protein system comprising:
Providing a porous matrix material having said pore volume such that at least 90% of the pore volume is composed of pores having a size in the range of 50 to 400 Angstroms;
Reacting the porous matrix material with a protein so that the protein chemically binds to the porous matrix material. 23. The method of claim 22, wherein the porous matrix material comprises surface hydroxyl, and the surface hydroxyl further comprises reacting with a coupling agent to form a functionalized monolayer. 24. The method of claim 23, wherein the functionalized monolayer comprises a reactive moiety selected from the group consisting of mercapto, amino, carboxyl, hydroxyl, and azide. 24. The method of claim 23, wherein the coupling agent comprises mercaptopropyltrimethoxysilane. 24. The method of claim 23, wherein the coupling agent has a chain length having 2 to 20 atoms. 23. The method of claim 22, wherein the porous matrix material has a surface area of at least 900 m < ² > / g prior to the reaction step. 23. A protein system produced by the method of claim 22. An OPH manufacturing method comprising:
Transfecting a host cell with a vector comprising a sequence encoding OPH, said sequence operably linked to a T7 expression control sequence;
Culturing the transfected host cell under the control of the expression control sequence under conditions allowing expression;
Purifying the OPH from the cells or the medium of the cells. 30. The method of claim 29, wherein the vector is provided with the sequence encoding OPH operably linked to the T7 expression control sequence. 30. The method of claim 29, wherein the OPH has an activity of about 13,000 enzyme units / mg. 30. The method of claim 29, wherein the vector is a plasmid. 32. The method of claim 30, wherein the vector is a plasmid. 30. The method of claim 29, wherein the host cell is a prokaryotic cell. 35. The method of claim 34, wherein the prokaryotic cell is a bacterium. 36. The method of claim 35, wherein the bacterium is E. coli. 30. The method of claim 29, wherein the host cell is a eukaryotic cell. 38. The method of claim 37, wherein the eukaryotic cell is a yeast cell. 40. The method of claim 38, wherein the yeast cell is Pichia pastoris. Reacting the porous matrix material with a crosslinking agent to form a porous matrix material having a crosslinking agent covalently bonded to the surface;
23. The method of claim 22, comprising reacting the porous matrix material having a crosslinker covalently bonded to the surface with a protein so that the protein is chemically bonded to the porous matrix material. Method. A method of forming a protein system comprising:
A porous matrix material having said pore volume such that at least 90% of the pore volume is composed of pores having a size in the range of 50 to 400 Angstroms and having a functionalized surface A process of preparing
Adding a protein so that the protein is entrapped into the porous matrix material by non-covalent bonding. 42. The method of claim 41, wherein the porous matrix material comprises a functionalized monolayer comprising a reactive moiety selected from the group consisting of mercapto, amino, carboxyl, hydroxyl, and azide. 42. The method of claim 41, wherein the protein is added under conditions such that the protein in the resulting protein system has an activity that is at least 50% of the protein in an active state. Including a porous matrix material,
In a protein system comprising a protein disposed within the porous matrix material,
The protein system comprises at least 0.01 mmol protein disposed within the matrix material per gram of matrix material, the protein system formed on the surface of a normal silica matrix material under the same conditions A protein system that exhibits at least twice as much activity as 45. The protein system of claim 44, wherein the porous matrix material is disposed in a microchannel. 46. A device comprising the protein system of claim 45. 45. The protein system of claim 44, wherein the protein system exhibits an activity that is at least 10 times greater than that of a protein system formed on a normal silica matrix material surface under the same conditions. The porous matrix material is sized such that the protein system contains 0.01 to 1 mmol protein per gram of matrix material, and the protein in the protein system is the activity of the protein in an active state 45. The protein system of claim 44 that exhibits at least 65% of the activity. 45. The protein system of claim 44, wherein the protein loses less than about 30% activity when refrigerated at 4 ° C for 110 days. 45. The protein system of claim 44, wherein the activity is at least 60% of the activity of the protein in an active state as measured per protein molecule. 45. The protein system of claim 44, wherein the protein is non-covalently bound in a porous matrix. 45. The protein system of claim 44, wherein the protein has a molecular weight in the range of 8,000 to 300,000 daltons. 45. The protein system of claim 44, wherein the protein has a molecular weight of at least 8,000 daltons. 45. The protein system of claim 44, wherein the porous matrix material is functionalized with a surface coverage of 2-80%. A porous matrix material having said pore volume such that at least 90% of the pore volume is composed of pores having a size in the range of 50 to 400 Angstroms and having a functionalized surface 45. The protein system of claim 44, wherein the protein system is prepared by adding a protein, such that the protein is incorporated into the porous matrix material by non-covalent bonding. 45. The protein system of claim 44, wherein the protein is incorporated by covalent bonding with the porous matrix material via a cross-linking agent. 45. The protein system of claim 44, wherein the porous matrix material comprises SAMMS.

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