The authors would like to acknowledge Natalie Doude and David Westerway (University of Alberta) for providing us Sho cDNA, Christophe Chevalier (INRA) for plasmid construction, Michel Br&#233;mont (INRA) for providing us BL21Sol bacteria; Christine Longin (INRA) for TEM assistance and Edith Pajot-Augy (INRA) for comments and proofreading.

プラスミドとShadoo式の1世代注：遺伝子をコードするネズミShadooタンパク質（Shadoo 25から122）を 、発現ベクターpET-28 11にサブクローニングしました。このクローンは大腸菌に形質転換しました大腸菌 SoluBL21細菌株、11前述したように、N末端 ​​ヘキサヒスチジンタグ、（HHHHHHHHHHSSGHIDDDDKHMKGGRGGARGSARGVRGGARGASRVRVRP APRYGSSLRVAAAGAAAGAAAGVAAGLATGSGWRRTSGPGELGLEDDENGAMGGNGTDRGVYSYWAWTSG）でShadooタンパク質を発現します。プラスミドは、作者から要求することができます。そのアミノ酸配列から算出Shadooの分子量は12,243.2ダあります。 注意：板状流フードの下や近くにブンゼン炎に細菌を持つすべての操作を実行します。 <ol><li>標準の熱ショックプロトコルを使用して、PET-28-Hisの6 -Shadooプラスミドで有能SoluBL21菌を変換します。プラスミドの1〜5μlのこのミックスのために（通常は10 PGに100チューブ中の細菌を50μlにNG）。 45秒間、42℃で熱ショックを実行します。 </li><li>培養物は、40 mg / mlのカナマイシン（ 図1B）を補充したルリア-ベルターニ培地中で0.5〜0.7の600まで37℃で細菌を形質転換しました。 </li><li>培養物に、240 / mlのイソプロピルβ-D-チオガラクトピラノシド、ITPGを加えることによってShadoo発現O / Nを誘導します。 37℃で150rpmで振とうしながら培養菌。注意：一般的に、細菌培養は3 O / Nの600に達しました 。 注：形質転換された細菌は、将来のタンパク質産生のために-80℃で保存することができます。このために、細菌培養物のアリコートに10％を50％（v / v）の滅菌グリセロールを添加し、-80℃で、それらを凍結します。 </li></ol><img alt="図1" src="/files/ftp_upload/53432/53432fig1.jpg" /> Shadoo発現構築物の図1のスキーム、Shadoo発現の細菌形質転換および誘導（ステップ1で説明したように）。 </stroNG&gt;（A）Shadooための発現構築物は、タンパク質、NtとのN末端 ​​に融合したヘキサヒスチジンタグをコードします。スキームはShadooタンパク質がC末端（CT）、続いて基本的なアルギニン/グリシン反復（緑シリンダ）、疎水性ドメイン（HD）、および単一のN-グリコシル化部位（CHO）を含むことを示します。 （B）をpET-28-Hisの6 -Shadooプラスミドを42℃で45秒間熱ショックによってSoluBL21の細菌に形質転換されます。形質転換された細菌は、40 / mlのカナマイシンを含む選択寒天上にプレーティングされています。プレートからの単一コロニーを、1000〜3000 mlの振盪フラスコ培養を接種するために使用されます。組換えタンパク質の発現は、1 mMのIPTGで誘導される。 <a href="https://www.jove.com/files/ftp_upload/53432/53432fig1highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 低圧液体クロマトグラフィーによるShadooタンパク質の2精製（LPLC） <ol><li>細胞溶解とはclusionの体の準備<ol><li>細菌懸濁液を収集し、遠心分離管（ 図2）にそれを転送します。 </li><li> 4℃で20分間、2,500×gでスピン。細胞培養500mlの由来各ペレットのために15ミリリットルバッファー（50 mMトリス、10mMのEDTA、抗プロテアーゼカクテルでpH8）にペレットを再懸濁し、デカンテーションにより上澄み液を除去します。 注：再懸濁したペレットを凍結し、数日間-20℃で保存することができます。凍結は、細菌細胞の溶解を容易にします。 </li><li> SDS-PAGE分析し、クーマシー染色によって、粗製細菌ペレットを用いた組換えタンパク質の過剰発現を確認してください。 <ol><li>このために、細胞をペレット化するために遠心分離機の細菌培養1ミリリットル。 100μlのLaemmliの変性溶液を加える（277.8 mMのトリス-HCl、pH6.8の、4.4％SDSを、44.4％W / Vのグリセロール、0.02％ブロモフェノールブルー）で5分間100℃でペレットを加熱します。 </li><li> 12％アクリルアミドゲルに負荷10μlのサンプルを、実行し、SDS-PAGEおよびVISUアリゼは、クマシー染色によりタンパク質を分離しました。 </li></ol></li><li> ）2.1.2から完全に再懸濁したペレットに0.5％の最終濃度になるように、トリトンX-100を追加します。 </li><li> 30分間37℃の水浴中で懸濁液をインキュベートします。 </li><li>細菌細胞を溶解する氷冷水で6-12ミクロンのピーク・ツー・ピーク振幅を5分間懸濁液を超音波処理します。 </li><li>転送は、クリーン50ミリリットル遠心チューブにサスペンションを超音波処理しました。 </li><li> 4℃で15分間、15,000×gで遠心分離管。 </li><li>デカンテーションにより上清を除去し、チューブ当たり結合バッファー（20 mMトリス - 塩酸緩衝液、pH 7.4、0.5 MのNaCl、8M尿素、5mMのイミダゾール）の5ミリリットルを追加します。 </li><li>回転ホイールのO / Nの上に置いてチューブが完全に封入体からタンパク質を再懸濁します。 </li></ol><img alt="図2" src="/files/ftp_upload/53432/53432fig2.jpg" /> インクルージョンの精製図2のスキームShadoo体を発現する（ステップ2.1に記載のように）形質転換された細菌を遠心分離により回収し、溶解緩衝液中に再懸濁します。懸濁液を37℃で30分間インキュベートし、次いで機械的に、細菌細胞を破壊するために超音波処理されます。超音波処理は、サンプルの加熱を防止するために氷の上で行われます。壊れた細胞を遠心分離により回収し、封入体からタンパク質を可溶化する8 M尿素を含む緩衝液中に再懸濁されている。 <a href="https://www.jove.com/files/ftp_upload/53432/53432fig2highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> </li><li>ニッケル2+ -アフィニティークロマトグラフィー 注：クロマトグラフィーは、5 mlのキレートセファロースカラム（ 図3A）はNi 2+ -charged用いてFPLCシステム上で実行されます。全ての溶液は、1ml /分の流速でカラム上にロードされます。 <ol><li>それを充電するためにセファロースカラムに0.2 MのNiSO 4水溶液を10mlを注入Ni 2+ -イオンを持ちます。 </li><li>イミダゾールの低濃度（20 mMトリス-塩酸緩衝液、pH 7.4、0.5 MのNaCl、8M尿素、5mMのイミダゾール）を含む結合緩衝液の30-50 mlのを注入したNi ​​2+ -イオンを充填したカラムを平衡化します。 </li><li> 50ミリリットル注入ループと尿素可溶化したタンパク質をロードします。 </li><li>完全に非結合タンパク質の最大値を除去するために10 mlの低イミダゾール結合緩衝液（20mMのTris-HCl緩衝液、pH 7.4、0.5 MのNaCl、8M尿素、5mMのイミダゾール）でカラムを洗浄することにより、フロースルー画分を回収カラムから。 </li><li>非特異的にカラムに結合したタンパク質を溶出するために、洗浄緩衝液（20mMのTris-HCl、pH7.4の、0.5MのNaCl、8M尿素、80 mMイミダゾール）50〜mlのカラムを洗浄。 </li><li> 20 mMトリス - 塩酸、pH7.4の、0.5MのNaCl、8M尿素を含有する緩衝液中で80から800 mMのイミダゾールの15分の直線勾配を適用します。勾配適用中に1mlの画分を収集します。注意：Hisタグ - タンパク質は、ウィットに溶出されます（競争相手として使用される）、イミダゾール含有緩衝液の勾配ヘクタール。 </li><li> （2.1.3.1を参照）各画分の8μlのアリコートを取り、4μlの4倍レムリ変性溶液を加えます。 5分間、100℃での熱溶液。 </li><li> 12％アクリルアミドゲルに負荷タンパク質サイズマーカー10μlの各サンプルを10μl、実行SDS-PAGEおよびクーマシー染色によって分離したタンパク質を可視化します。注意：計算されたShadooの分子量は、そのアミノ酸配列は12,243.2ダで形成します。 </li><li> Shadooを含む全ての画分をプール。 </li></ol><img alt="図3" src="/files/ftp_upload/53432/53432fig3.jpg" /> 図3 の精製手順（工程2.2から2.4に記載のように）。 （A）のNi 2+ -イオンはポリヒスチジンタグ融合タンパク質の選択的な精製を可能にするヒスチジン残基によって調整することができます。イミダゾールを介して、タンパク質を溶出するために使用されますニッケル2+ -イオンとの調整と結合したタンパク質を置換する能力。（B）サイズ排除カラム上に分離されている-カラムのNi 2+に保持された様々な流体力学的半径のタンパク質。タンパク質は、最も小さいものに最大から溶出される。（C）使用前に、Shadooを含有する画分をプールし、尿素、イミダゾールおよび塩を除去するために脱塩されます。品質管理アッセイは、SDS-PAGE /クマシー染色、ウエスタンブロッティングを含みます。精製Shadooは、凍結乾燥することができます。 -20℃で保存した場合凍結乾燥Shadooはヶ月間安定である。 <a href="https://www.jove.com/files/ftp_upload/53432/53432fig3highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> </li><li>サイズ排除クロマトグラフィー 注：第二の精製工程は、第1の精製工程の間に他のタンパク質を同時溶出Shadooからタンパク質を分離するサイズ排除クロマトグラフィーからなります（ 図3B）。 <ol><li> 1.5ml /分の流速で、20mMのトリス塩酸緩衝液、pH 7.4、0.5 MのNaCl、8M尿素を250mlを注入することによって、総床体積120 mlとスーパーデックスカラムを平衡化。 </li><li>ロード平衡化したカラムに2.2.9からShadooを含む画分をプールしました。上記緩衝液の一定流量に応じて40ミリリットルのカラム空隙容量後に1mlの画分を収集します。 </li><li>各画分の10μlアリコートを取り、上記のようにShadooを含有する画分を見つけるために、SDS-PAGE分析およびクマシー染色を行います。 </li><li> Shadooを含むプール画分。 </li></ol></li><li>脱塩 注意：脱塩手順は、さらに生物物理学的および生化学的研究（ 図3C）のために適切な緩衝液中の組換えShadooタンパク質を得るために、塩、イミダゾールおよび尿素を除去します。同様の結果は、緩衝液に対して試料を透析することによって得ることができます。 <ol><li> 150ミリリットルと53ミリリットルの脱塩カラムを平衡化/ minで5 mlの流速で10mMの酢酸アンモニウム緩衝液、pH 5の。 </li><li>ロードは、列にShadoo画分をプールしました。酢酸アンモニウム緩衝液の注入時に手動でUV検出器の出力で280nmでの信号の増加を示す画分を収集します。 </li><li>凍結乾燥脱塩Shadooを、-20℃で保管してください。 </li><li>そのような使用前に10mMの酢酸ナトリウム緩衝液、pH 5、などの適切な緩衝液中で凍結乾燥Shadooを溶解します。 注：ポリヒスチジンタグ、エンテロキナーゼを用いて除去することができます。 </li></ol></li></ol>生理的pHでのアミロイド線維にShadoo のインビトロ組立3. 注意：自発的にShadooのタンパク質凝集体とは、pH≥7 11で原線維を形成しています。 <img alt="図4" src="/files/ftp_upload/53432/53432fig4.jpg" /> descriとしてアミロイド線維にリフォールディングShadooの 図4. スキーム（ステップ3のベッド）酸 ​​性溶液では、pH≥7の緩衝液に溶解したとき。精製Shadooが自発的にアミロイド線維に変換し、振盪しながら、Shadooは1 M GDN-HClでのインキュベーション時にアミロイド線維に変換します。品質管理アッセイは、電子顕微鏡とのThT蛍光染色が含まれています。 <a href="https://www.jove.com/files/ftp_upload/53432/53432fig4highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <ol><li> 20mMのTris-HCl緩衝液、pH7.4中2μMの最終濃度までShadooを希釈。 280 nmでの溶液の光学密度を測定すると20970のShadooアミノ酸組成から推定される吸光係数を用いて濃度を測定します。 </li><li>アミロイド構造（ 図4）への自発的なタンパク質の変換を可能にするために、数週間のカップルのために4℃で円錐形のプラスチックチューブ内の溶液500μlをインキュベートします。 </li><li>最終conceにT（ThTの）チオフラビン新たに調製追加Shadoo溶液100μlのアリコートに、10μMのntration。 RTで5分間溶液をインキュベートします。アミロイド構造体17の存在を確認するために、435 nmでの励起波長を使用して、460から520 nmの蛍光発光を測定します。 </li></ol>酸性pHでのアミロイド線維にShadoo のインビトロ組立4. <ol><li> 2μMの最終濃度になるように1 M GdnHCl、20mMの酢酸ナトリウム緩衝液、pH 5でShadooを希釈します。 </li><li> 3-7日のために、37℃で連続的に振盪しながら円錐管で、この溶液500μlをインキュベートします。 </li><li> 3.3のように溶液の一部にのThTを追加することにより、アミロイド線維の存在を確認してください。 </li><li> Shadooアミロイド原線維を精製し、10mMの酢酸ナトリウム緩衝液、pH 5.0に対して得られた懸濁液を透析します。 </li><li> 4℃で精製した原線維を保管してください。 </li></ol><img alt="図5" src="/files/ftp_upload/53432/53432fig5.jpg"/&gt; SDS-PAGEおよびクーマシー染色を用いて分析した図5 の代表的な結果。のNi 2+から溶出したタンパク質画分は、キレートカラム（A）及びサイズ排除カラム（B）を -charged。精製Shadooの同定はSPRN抗Shadoo抗体（C）を用いて評価した。 <a href="https://www.jove.com/files/ftp_upload/53432/53432fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-resistant+prions+selectively+decrease+Shadoo+protein.">Protease-resistant prions selectively decrease Shadoo protein.</a> PLoS Pathogens. 7, e1002382 (2011).</li><li>Westaway, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down-regulation+of+Shadoo+in+prion+infections+traces+a+pre-clinical+event+inversely+related+to+PrP(Sc)+accumulation.">Down-regulation of Shadoo in prion infections traces a pre-clinical event inversely related to PrP(Sc) accumulation.</a> PLoS Pathogens. 7, e1002391 (2011).</li><li>Chevalier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PB1-F2+influenza+A+virus+protein+adopts+a+beta-sheet+conformation+and+forms+amyloid+fibers+in+membrane+environments.">PB1-F2 influenza A virus protein adopts a beta-sheet conformation and forms amyloid fibers in membrane environments.</a> The Journal of Biological Chemistry. 285, 13233-13243 (2010).</li><li>Tarus, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+paths+of+the+nucleoprotein+of+influenza+A+virus.">Oligomerization paths of the nucleoprotein of influenza A virus.</a> Biochimie. 94, 776-785 (2012).</li><li>Biancalana, M., Koide, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+of+Thioflavin-T+binding+to+amyloid+fibrils.">Molecular mechanism of Thioflavin-T binding to amyloid fibrils.</a> Biochimica et Biophysica Acta. 1804, 1405-1412 (2010).</li><li>Minic, J., Sautel, M., Salesse, R., Pajot-Augy, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+system+as+a+screening+tool+for+pharmacological+assessment+of+g+protein+coupled+receptors.">Yeast system as a screening tool for pharmacological assessment of g protein coupled receptors.</a> Current Medicinal Chemistry. 12, 961-969 (2005).</li><li>Schein, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cool+way+to+make+proteins.">A cool way to make proteins.</a> Nature Biotechnology. 22, 826-827 (2004).</li><li>Sorensen, H. P., Mortensen, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+genetic+strategies+for+recombinant+protein+expression+in+Escherichia+coli.">Advanced genetic strategies for recombinant protein expression in Escherichia coli.</a> Journal of Biotechnology. 115, 113-128 (2005).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+glycosylation+enhances+ubiquitin-mediated+proteasomal+degradation+of+Shadoo+in+Scrapie-infected+rodents+and+cultured+cells.">Disruption of glycosylation enhances ubiquitin-mediated proteasomal degradation of Shadoo in Scrapie-infected rodents and cultured cells.</a> Molecular Neurobiology. 49, 1373-1384 (2014).</li><li>Englund, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+and+biosynthesis+of+glycosyl+phosphatidylinositol+protein+anchors.">The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors.</a> Annual Review of Biochemistry. 62, 121-138 (1993).</li><li>Puig, B., Altmeppen, H., Glatzel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GPI-anchoring+of+PrP:+implications+in+sorting+and+pathogenesis.">The GPI-anchoring of PrP: implications in sorting and pathogenesis.</a> Prion. 8, 11-18 (2014).</li></ol>

The authors have nothing to disclose.

組換えマウスShadooタンパク質の大量の発現および精製の双方のための簡単​​で効率的なプロトコルが提供されます。記載されている方法は、成功した可溶化および（彼6）タグ付き組換えShadooタンパク質の精製を可能にします。 、タイトルに指摘したようにそれは、重要なことは、原核生物細菌大腸菌で発現された場合大腸菌 Shadooは、封入体に蓄積します。 Shadoo精製?...

牛で牛海綿状脳症を含むプリオン神経変性疾患、ヒトでの羊とクロイツフェルト・ヤコブ病におけるスクレイピーは、致命的と不治です。プリオン病は、クロスβシートに、主にαヘリックスから成る細胞性プリオンタンパク質の立体構造変化によって特徴付けられる1,2の配座異性アミロイドを豊かに。クロスβ-構造が密に充填し、非常に規則正しい結晶のようなβシートは、水素結合3,4で安定化されています。プリオンアミロイドが原因募集し、単量体タンパク質ユニットを変換するためのテンプレートを提供し、成長エッジにおけるβ鎖に自己複製することができます。 「タンパク質のみ」の仮説によると、プリオンタンパク質のアミロイドコンフォーマーは唯一の感染性​​病原体です。しかしながら、いくつかの他の生体分子は、プリオン疾患に必須であることが提案されています。例えば、細胞膜は場所conversiであると考えられています上で行われ、いくつかの負に荷電した脂質は、変換プロセス5,6を増強することが示されています。加えて、いくつかのタンパク質はまた、プリオン病態に関与し得ます。ドッペルとShadoo 7,8：具体的には、プリオンタンパク質ファミリーの他の二つのメンバーがあります。ドッペルはジスルフィド橋で安定化し、比較的剛直な構造を有しており、アミロイド線維9に自己重合し、集計に失敗します。対照的に、プリオンタンパク質と同様に、Shadooは、アミロイド原線維構造にβシートに富む構造と自己会合を採用することができます。それはShadooがネイティブの条件下または負に帯電した膜10,11を結合する際にフィブリル化できることが示されました。アミロイド様繊維へShadooの変換は、病理に関連してもよいです。実際、アミロイド様構造は、疾患を引き起こすメカニズムはよく理解されていません。 Shadooが疎水性ドメイン（HD）とタンデムのArg /グリシンのシリーズを負い同様のトンを繰り返し、プリオンタンパク質（ 図1A）のN末端 ​​部分、O。プリオンタンパク質のN末端 ​​部分として、Shadooは高度に正に荷電し、ネイティブ非構造タンパク質であると思われる11,12れます。 Shadooは、直接プリオンタンパク質に結合することができるので、プリオン疾患に関連した機能的であると思われます。また、その発現がダウンプリオン病理13,14の間に調節されます。しかし、プリオン病におけるShadooの役割は未だ確立されていません。 我々は、マウスShadoo遺伝子のコード配列を有するプラスミドを開発しました。プラスミドを大腸菌を形質転換しました N末端​​His 6融合Shadooタンパク質の産生のための大腸菌 。この発現系はよく我々の研究室で確立され、一般的に我々の進行中のプロジェクトで使用されている6,15,16。 Shadooの発現のための重要な問題は、Eの選択であります大腸菌菌株。コンピテントBL21細菌株は、一般的に、組換えプロの発現のために使用されますteins Shadooが正常にのみ変換さSoluBL21細菌株から発現および精製しました。 SoluBL21 コンピテントE. 大腸菌は、その発現が、 ​​親BL21中に検出可溶性生成物は得られなかったタンパク質の産生のために開発さBL21宿主株の改良された変異体です。 SoluBL21 大腸菌における Shadooの高レベルの発現大腸菌は、封入体中のタンパク質の蓄積をもたらします。非天然タンパク質を高大腸菌で発現される一般的な特徴として、 大腸菌は 、このタンパク質は、不溶性の封入体中に蓄積する傾向があります。 Shadooはおそらく様々な程度に折り畳まれたタンパク質によって形成された高濃縮動的構造体に非共有結合、疎水性またはイオン性相互作用（またはその両方の組み合わせ）を介して集約します。 （I）変性媒体の他の生体分子からのタンパク質の選択的分離、および（ii）精製されたタンパク質の再生は 、in vitroフォールディングに用いた：結果として、精製は、少なくとも二つの工程を含みます技術。 Shadooの選択的分離は、8M尿素（または6Mグアニジン-HCl）を含む緩衝液中で達成されました。 Shadoo重合を得るために、pHを≥7の溶液中で構造化されていない単量体Shadooタンパク質、または（ii）の再生を得るために酸性pHの溶液中で（I）再生：尿素の除去及びタンパク質の再生は、様々なプロトコルを適用することによって行うことができます特徴的なクロスβシートモチーフを有する安定したアミロイド繊維へ。

<table>
	<tbody>
		<tr>
			<td>AKTA FPLC&#160;</td>
			<td>GE, Amersham</td>
			<td># 1363</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 16/60 Superdex</td>
			<td>GE Healthcare</td>
			<td>17-1069-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTrap IMAC</td>
			<td>GE Healthcare</td>
			<td>GE17-0920-05</td>
			<td></td>
		</tr>
		<tr>
			<td>HiPrep26/10 Desalting column</td>
			<td>GE Healthcare</td>
			<td>GE17-5087-01</td>
			<td></td>
		</tr>
		<tr>
			<td>SoluBL21 Competent E. coli</td>
			<td>Genlantis</td>
			<td>C700200</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-28b+</td>
			<td>Novogen</td>
			<td>69865-3</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Sigma-Aldrich</td>
			<td>I6758</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-Broth medium</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Biophotometar</td>
			<td>Eppendorf</td>
			<td>550507804</td>
			<td></td>
		</tr>
		<tr>
			<td>Trito X-100</td>
			<td>Life Technologies</td>
			<td>85111</td>
			<td></td>
		</tr>
		<tr>
			<td>NiSO4</td>
			<td>Sigma-Aldrich</td>
			<td>656895</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>RES3098T</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>4693116001</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>746398</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazol</td>
			<td>Sigma-Aldrich</td>
			<td>I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>Gdn-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>G4505</td>
			<td></td>
		</tr>
		<tr>
			<td>protein size marker&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>26620</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification and refolding to amyloid fibrils of (his)6-tagged recombinant shadoo protein expressed as inclusion bodies in e. coli

大腸菌発現系は、組換え真核生物のタンパク質の産生のための強力なツールです。我々はそれがShadoo、プリオンファミリーに属するタンパク質を産生するために使用します。 （His）6の精製の ​​ためのクロマトグラフィー法は、封入体が記載されているように、組換えShadooが発現-タグ。封入体は、8M尿素で可溶化し、イオンアフィニティークロマトグラフィーを実行するようにNi 2+ -chargedカラムに結合されています。結合したタンパク質を、イミダゾールの勾配により溶出されます。 Shadooタンパク質を含有する画分を高度に精製されたタンパク質を得るために、サイズ排除クロマトグラフィーに供されます。最後のステップで精製Shadooで塩、尿素およびイミダゾールを除去するために、脱塩します。組換えShadooタンパク質はプリオン病に存在するタンパク質の立体構造の障害の生物物理学的および生化学的研究のための重要な試薬です。多くのレポートは、プリオンの神経変性疾患を刺すの沈着に由来することを実証しましたル、アミロイド線維を命じました。酸性および中性/基本的なpHでアミロイド線維にShadooをフィブリル化する方法を説明するサンプル・プロトコルが提示されています。それは、迅速かつ低コストな方法でタンパク質の大量生産を可能にするためShadooを生成し、フィブリル化する方法についての方法は、プリオン病の作業を実験室での研究を容易にすることができます。

精製Shadooタンパク質の約5〜10 mgの細菌培養1リットル当たり得られます。二段階の精製は、高純度の組換えShadooを得るために必要とされます。最初のステップは、Hisタグタンパク質を保持したNi ​​2+ -chargedカラムを用いたアフィニティークロマトグラフィーによって行われます。溶出した画分をSDS-PAGEに付し、クーマシーブリリアントブルー（ 図5A）で染色します。 Shadoo...

Watch this Scientific Journal Video about Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli at JoVE.com

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

精製とのアミロイド線維にリフォールディング（彼）<sub&gt; 6</sub&gt;内封入体として発現された組換えShadooタンパク質をタグ付き<em&gt;大腸菌</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

purification-refolding-to-amyloid-fibrils-his6-tagged-recombinant

Research

JoVE Journal

Biology

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

14.1K ビュー.  INRA. A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures. 

ビデオ: Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

生物学

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

内タンパク質の定方向進化のための変異導入と機能選択プロトコル E.大腸菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104の精製、タンパク質Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

のタンパク質複合体の同定<em&gt;大腸菌</em&gt;タンデム質量分析との組み合わせでシーケンシャルペプチドアフィニティ精製を用いた

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

のリフォールディング監視タンパク質を結合アッセイ<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

で表現する嚢胞性線維症膜コンダクタンス制御因子タンパク質の精製<em&gt;サッカロマイセス·セレビシエ</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

で産生された組換えジスルフィド豊富な毒タンパク質に印加される高スループット定量的発現スクリーニングおよび精製<em&gt; E.大腸菌の</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

からのペプチドおよびタンパク質との組換えエラスチン様ポリペプチドおよびその融合の非クロマトグラフィー精製<em&gt;エシェリヒア·コリ</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.