このプロジェクトは、クヌートとアリスバレンベリー財団とスウェーデンの研究評議会によって資金を供給された。

1。ターゲットのクローニングは、融合構築物をgene/ABDz1-tagged <ol><li>発現プラスミドに目的遺伝子（プラスミド含むABDz1は材料移転契約を通じて自由に利用可能である）のN末端ライゲーションのための適当な制限部位に隣接ABDz1遺伝子のPCR断片を準備する。 </li><li>適切な反応バッファーで選択した制限酵素による開裂精製発現ベクターとPCR断片。ライゲーションの前に製品を精製する。 </li><li>目的の遺伝子を含む発現ベクターに制限されたABDz1断片をライゲーションし、E.にライゲーション産物を変換大腸菌 （元のメソッドでRR1ΔM15ひずみが8を使用していた）。選択のための適切な抗生物質を補充した寒天プレート上で形質転換細胞を広げる。 </li><li> PCR画面少数のコロニーとシーケンスは、DNAシーケンシングにより得られた発現カセットを確認してください。 </li><li>シーケンスverifの一晩培養液からプラスミド準備IEDコロニーと好ましい発現株（ヒト遺伝子がコードするタンパク質の産生を向上させるためにプラスミドpRAREをホスティングしているロゼッタ元のメソッド大腸菌 （DE3）、で、使用された）に変換。 </li></ol> 2。タンパク質発現<ol><li> 5g / lの酵母エキスと適切な抗生物質を補充したトリプシン大豆ブロス10mlに単一の細菌コロニーを接種。 37℃で一晩150rpmでインキュベートする。 </li><li> 100ミリリットルの新鮮な培地で一晩培養液1mlを接種し、細胞が対数増殖期に達するとタンパク質発現（lacオペロンを利用したイソプロピル-β- D -チオガラクトシド本来の1mM）を誘導する。 </li><li>遠心分離で25℃一晩と収穫細胞で150rpmでインキュベートする。 </li></ol> 3。直交アフィニティー精製<ol><li> 25ミリリットルランニングバッファー（25mMのトリス- HCl、200 mMのNaCl、1mMのEDTA、0.05％（w / v）のTween 20を、pH8.0）にペレットを再懸濁し、目を混乱させる60％の振幅と3分間1.0/1.0パルスで超音波処理によってEが細胞。サンプルを遠心し、さらに精製する前に、上清を（0.45μM）を含む標的タンパク質をフィルタリング。 </li><li>適切なタンパク質精製システムで、1ml /分でバッファを実行しているの10カラム体積（CV）を使用して、以前に販売業者の勧告によるとHSAとの結合のどちらか1ミリリットルHSAセファロースカラムまたはNHS -活性化カラムを、平衡化させます。 0.5ml /分で細菌のライセートをロードして、流速は1ml /分にリセットされます。 </li><li>洗浄バッファーの5 CV（5 mMのNH 4 AC、pH5.5）に続いて実行されているバッファの5 CVでカラムを洗浄。 </li><li> 1ml /分で溶出バッファー（0.5 M HAC、pHは2.5）でサンプルを溶出し、さらに精製するための溶出ピークからの画分を選択するには280nmで、モニターの吸収を画分を集めた。 </li><li>プール最高タンパク質濃度と分画は、確かにランニングバッファー（20mMリン酸、150mMのNaCl、pH7.2）中で2回希釈し、pHが中性付近であることを確認してください。 pHは必要に応じて増加させる1 Mトリス塩酸pHが8を追加します。 </li><li> 1ml /分で確かにランニングバッファーの10 CVで平衡化した1mlのハイトラップのMabSelect確かカラムに0.5ml /分でサンプルをロードします。ローディング後1 ml / minの流量をリセットします。 </li><li>確かにランニングバッファーの5 CV（ステップ5）でカラムを洗浄し、0.2 M HAC、pHは2.7でタンパク質を溶出させる。敏感な標的タンパク質の場合は溶出液は、トリス- HClの添加によっては採取後、直接中和することができる。 </li></ol>クロマトグラフィー工程はMabSelectの溶出液を逆にしている場合は確かにカラムを緩衝液（ステップ3.1）実行中に希釈し、pHは1 Mトリス- HClを加えて約8に増加することができます。確かに行列は第二段階で採用されている場合、一般的に、狭いピークが観察される。 4。ドデシル硫酸ナトリウムポリアクリルアミドゲル電気泳動（SDS - PAGE）により純度の評価<ol><li>で精製した画分をロードするSDS - PAGEを削減し、必要に応じて、質量分析法により精製物の分子量を分析する。 ABDz1の遊離システインは、非還元条件下での製品の二量体化を引き起こす可能性があるので、それはサンプルを完全に削減することが重要です。 </li></ol> 5。代表的な結果： 原理の証明として、ABDz1タグによって促進された直交アフィニティー精製は、異なる溶解性クラス、分子量および等電点（表1）を表す3つのヒト標的タンパク質を評価した。 ABDz1遺伝子が遺伝的に標的遺伝子に融合され、構造が大腸菌で発現され、図2は、連続してメソッド内のすべてのステップのためのフローチャートを示しています。直交プロトコルによる精製の後、異なる時点で採取した試料は、SDS - PAGE（図3）により分析した。結果は明らかに二重のタグが二高度に特異的な精製段階の有用性を示す。合理的な純度はACであるにもかかわらず、としてゲルから見た初期精製ステップの後にhieved、連続的なステップはよく非常に要求の厳しいアプリケーションに最適な非常に純粋な生成物が得られる。 <img alt="図1" src="/files/ftp_upload/3370/3370fig1.jpg" /> 図1二重特異性ABDz1タグの選択に使用するコンビナトリアルライブラリーのデザイン。 46アミノ酸アルブミン結合ドメインの安定threeヘリックス束に折り目と、それは主に第2ヘリックスに位置するヒト血清アルブミンに対する結合部位が含まれています。遺伝的にファーストとサードヘリックスに位置eleven表面露出アミノ酸をランダム化することにより、新規の結合面が設計されました。 elevenの位置は図に示され、Kraulisらによれば、番号が付けられています。9。ファージ上に発現するコンビナトリアルライブラリとブドウ球菌蛋白質のZ -ドメインの二量体に対するバイオパニングに続いて、二重特異性ABDz1分子が同定された。 <iMG ALT =&quot;図2&quot;SRC =&quot;/ files/ftp_upload/3370/3370fig2.jpg&quot;/&gt; 図2。直交アフィニティー精製プロトコールの簡易フローチャート。 ABDz1配列を含むPCRフラグメントを適当な発現ベクターに目的の遺伝子で（N末端）連結されている。ライゲーション産物を大腸菌に形質転換されるシーケンスの検証とプラスミド調製用の大腸菌 。発現宿主に形質転換後、組換えタンパク質発現のための大量培養は、最初の一晩培養から設定されています。細菌を遠心分離により回収し、標的タンパク質を含む細菌溶解物を生成するために超音波処理によって溶解されています。残留固体粒子を除去する濾過工程の後、ライセートは、連続する二つの精製工程を経ることにより、液体ハンドリングシステム上で直交するアフィニティー精製に供される。両方の精製工程からの溶出ピークをサンプリングし、評価をSDS - PAGEにより純度を評価するとtを比較し彼は、精製前にライセート。 <img alt="図3" src="/files/ftp_upload/3370/3370fig3.jpg" /> ABDz1と融合して発現し、精製した標的タンパク質の図3。SDS - PAGE分析。 異なる特性を表すABDz1、、とABDz1タグ自体に融合で表現三つのタンパク質の細菌溶解物から採取したサンプルは、確実なカラムMabSelect続いてHSA -カラム上で精製から対応するピークからのサンプルと一緒に採取された（） 。レーン1-4 9-12 MabSelect確かに精製（第二段階）から精製、HSA -精製からレーン5-8（第一段階）と車線の前に溶解液からサンプルを表します。 ABDz1 - 141377、ABDz1 - HT875、ABDz1 - HT2375とABDz1：サンプルは次の順序で読み込まれます。さらに、同じ列に逆の順序で精製した同じ溶解物から取得したサンプルを分析した（B）。 1から3レーンは、精製前にライセートからレーン4-6 fをサンプルを表してHSA -精製（第二段階）からROM MabSelect確実な精製（第一段階）とレーン7-9サンプルは、（A）が、タグ自体が含まれていないのと同じ順序でロードされた。これらの結果から、この二段階方式に関係なく手順が適用される順序の高度に精製したタンパク質をもたらすことは明らかである。 <table border="1"><tbody><tr><td> 名前B </td><td> 標的タンパク質 UNIPROT C </td><td> 分子 重量（分子量） </td><td> 溶解度 クラスD </td><td> 分子 融合産物（分子量）の </td><td> 等電点 融合産物の </td></tr><tr><td> 141377 </td><td> B7Z315 </td><td> 17.4 </td><td> 4 </td><td> 23.7 </td><td> 8.5 </td></tr><tr><td> HT875 </td><td> P01040 </td><td> 10.8 </td><td> 3 </td><td> 17.1 </td><td> 4.9 </td></tr><tr><td> HT2375 </td><td> P00740 </td><td> 8.9 </td><td> 5 </td><td> 15.2 </td><td> 6.8 </td></tr></tbody></table><ol class="lalpha"><li>単独でABDz1タグの分子量は6.3 kDaの、等電点6.7です。 </li><li>標的タンパク質は、UniProtのタンパク質の一部を表します。 </li><li> <a href="http://www.uniprot.org">http://www.uniprot.org</a> 。 </li><li> N -末端His 6 ABP 10のタンパク質断片の溶解度クラス。 </li></ol>表1。 ABDz1タグと標的タンパク質との融合製品は、原理の研究の証拠で評価。 異なる分子量、溶解性及び等電点を持つ3つのユニークな人間のタンパク質は、直交する親和性のアプローチによって発現および精製のために選ばれた。

<ol>
	<li>Waugh, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+the+most+of+affinity+tags.">Making the most of affinity tags.</a> Trends Biotechnol. 23, 316-320 (2005).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+engineering+strategies+for+selective+protein+purification.">Protein engineering strategies for selective protein purification.</a> CET. 28, 1315-1325 (2005).</li><li>Porath, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+chelate+affinity+chromatography,+a+new+approach+to+protein+fractionation.">Metal chelate affinity chromatography, a new approach to protein fractionation.</a> Nature. 258, 206-213 (1975).</li><li>Linhult, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+analysis+of+the+interaction+between+albumin-binding+domain+from+streptococcal+protein+G+and+human+serum+albumin.">Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin.</a> Protein Sci. 11, 206-213 (2002).</li><li>Nilsson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+IgG-binding+domain+based+on+staphylococcal+protein.">A synthetic IgG-binding domain based on staphylococcal protein.</a> A. Protein Eng. 1, 107-113 (1987).</li><li>Alm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+bispecific+protein+selected+for+orthogonal+affinity+purification.">A small bispecific protein selected for orthogonal affinity purification.</a> Biotechnol. J. 5, 605-617 (2010).</li><li>Nilsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+affinity+domains+for+the+detection,+purification+and+immobilization+of+recombinant+proteins.">Multiple affinity domains for the detection, purification and immobilization of recombinant proteins.</a> J. Mol. Recognit. 9, 585-594 (1996).</li><li>R&#252;ther, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pUR+250+allows+rapid+chemical+sequencing+of+both+DNA+strands+of+its+inserts.">pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts.</a> Nucleic Acids Res. 10, 5765-5772 (1982).</li><li>Kraulis, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serum+albumin-binding+domain+of+streptococcal+protein+G+is+a+three-helical+bundle:+a+heteronuclear+NMR+study.">The serum albumin-binding domain of streptococcal protein G is a three-helical bundle: a heteronuclear NMR study.</a> FEBS Lett. 378, 190-194 (1996).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+flow+cytometry-based+method+for+analysis+of+expression+levels+in+E+coli+giving+information+about+precipitated+and+soluble+protein.">Novel flow cytometry-based method for analysis of expression levels in E coli giving information about precipitated and soluble protein.</a> J. Biotechnol. 119, 133-146 (2005).</li><li>Carter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+proteolysis+of+fusion+proteins.">Site-specific proteolysis of fusion proteins.</a> ACS Sympos. Ser. 427, 181-193 (1990).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negatively+charged+purification+tags+for+selective+anion-exchange+recovery.">Negatively charged purification tags for selective anion-exchange recovery.</a> PEDS. 17, 779-786 (2004).</li><li>Gr&#228;slund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+engineering+of+a+protein+domain+to+allow+efficient+ion-exchange+recovery.">Charge engineering of a protein domain to allow efficient ion-exchange recovery.</a> Protein Eng. 13, 703-709 (2000).</li></ol>

利害の衝突は宣言されません。

ここで紹介する直交アフィニティー精製プロトコールは、標的タンパク質の広い範囲の効率的な精製が可能になります。新規の結合面とアルブミン結合ドメインの固有の結合部位を組み合わせることで、小さな二重特異性タンパク質のタグが開発されました。それは単に好みの発現ベクターへの関心の任意のタンパク質にクローニングし、融合で表現できるので、タグを利用することは非常に簡単です。ほとんどの研究室で利用できる標準的な装置は、2段階のタンパク質精製に用いることができる。 ABDz1タグの機能を効率的にその二つの結合面を公開するドメインの正しいフォールディングに依存しているので、方法は、可溶性形態で発現されるタンパク質に制限され、変性条件下で精製には適していません。彼らはMabSelect確かマトリックス上にZ -ドメインへABDz1の結合を妨害するので、還元剤も避けるべきである。 直交親和性精製用るには、非常に多くの要求の厳しいアプリケーションでのタンパク質の精製の要件を満たすために、従来の方法に有用な代替手段を提供します。同時に、この方法は、別の精製戦略を連続して使用されているコンビナトリアルタグ付けする必要がなくなります。ネイティブターゲットのタンパク質を必要とするアプリケーションには、プロテアーゼの切断部位は、可能な限りABDz1タグ11の酵素的除去をレンダリングするために導入される可能性があります。さらに、ABDz1タグは、日付に説明した最初の二重特異性小と折り畳まれたタンパク質のドメインです。は、2つのターゲット特異的親和性の相互作用に依存する精製戦略を提供するので、それはユニークです。将来的には、容易に入手可能で安価なクロマトグラフィー樹脂と互換性のある新たな結合面を運ぶ新たな二重特異性タグを開発することにより、この概念を拡張するために興味深いものになるだろう。例えば、塩基性残基とアルブミン結合に関与するアミノ酸を置き換えることにより、私との互換性タグ交換クロマトグラフィーで得られる場合があります。同様の概念はそれぞれ、Z 酸 12と陰イオンと陽イオン交換と互換性のあるZ 基本的な 13として知られているタグを生成するためにZ -ドメインの電荷のエンジニアリングによって効果が証明されています。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> 大腸菌 RR1ΔM15 </td><td>アメリカンタイプカルチャーコレクション</td><td> 35102 </td></tr><tr><td>ロゼッタ大腸菌 （DE3） </td><td> Novagen社</td><td> 70954 </td></tr><tr><td>トリプシン大豆ブイヨン</td><td>ディフコ</td><td> 211822 </td></tr><tr><td>酵母の抽出</td><td>ディフコ</td><td> 212720 </td></tr><tr><td>振動の細胞の音波処理</td><td>ソニックスと材料</td><td> - </td></tr><tr><td> HSAセファロース</td><td>ファルマシアバイオテク</td><td>旧製品</td></tr><tr><td>確かにハイトラップMabSelect </td><td> GEヘルスケア</td><td> 11-0034-93 </td></tr><tr><td>ハイトラップNHS -活性化HPカラム</td><td> GEヘルスケア</td><td> 17-0716-01 </td></tr></tbody></table>

orthogonal protein purification facilitated by a small bispecific affinity tag

プロトコルは、合理化する必要がある組換えタンパク質の精製に伴う高コストのために。ハイスループットな努力のために、標的タンパク質の特定の最適化1を必要としない一般的な方法が求められている。これを達成するために、遺伝的に関心の遺伝子に融合させることができる精製タグは、一般的に2を使用されています。最も広く使用されている親和性のハンドルは、ネイティブと変性条件3の両方での精製に適しているヘキサ-ヒスチジンタグ、です。タグを生成するための代謝負担は低いですが、競合するアフィニティークロマトグラフィーベースの戦略の1,2のような高特異性としては提供されません。 ここでは、46アミノ酸の異なる2つの結合部位を有する二重特異精製タグは、小さなタンパク質ドメインが開発されている。アルブミン結合ドメインは、連鎖球菌プロテインGに由来し、ヒト血清アルブミンへの強力な固有の親和性を有している（HSA）。アルブミン結合4に関与していない十一面に露出したアミノ酸は、コンビナトリアルライブラリーを生成するために遺伝的に無作為に割り付けられた。小説で蛋白質のライブラリをランダムに配置された結合面（図1）は、ファージディスプレイ技術によるバインダーの選択を容易にするために、ファージ粒子上に発現していた。ブドウ球菌性タンパク質5から派生した二量体のZ -ドメインに対するバイオパニングのいくつかのラウンドを通じ、HSAおよび新規ターゲットの両方に対して親和性を有する小、二重特異性分子は、6を同定された。 ABDz1と呼ばれる新規な蛋白質ドメインは、異なる分子量、溶解性及び等電点を持つ標的タンパク質の選択のための精製タグとして評価した。 3つのターゲットタンパク質は、それらのN -末端およびその後アフィニティー精製に融合させた斬新なタグでEscherishia大腸菌で発現させた。固定化HSAまたはZ -ドメインのいずれかで列で初期精製はrelatiをもたらしたvely純粋な製品。二重特異性タグと二段階のアフィニティー精製は、タンパク質の純度の大幅な改善が得られた。固定化Z -ドメインとのクロマトグラフ媒体は、確かに例のMabSelectのために、容易に化学的に第二のマトリックスを提供するためにメディアに結合することができる抗体とHSAの精製のために容易に入手可能である。 回収された標的タンパク質の純度の高需要がある場合は、この方法が特に有利である。発現宿主の代謝負担がタグの小さなサイズのために制限されている間にタグの二機能性は、2つの異なるクロマトグラフィー工程を使用することができます。それは複数のタグを組み合わせて1,7で使用されている、いわゆるコンビナトリアルタギングに競争力のある代替手段を提供します。

Watch this Scientific Journal Video about Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag at JoVE.com

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

小説や高効率二段アフィニティークロマトグラフィーのプロトコルが開発され、詳細に説明されている。方法は2つの固有の親和性を持つ小型の精製タグに基づいており、異なるプロパティを持つ標的タンパク質の広い範囲に適用可能です。

小型二重特異性アフィニティタグによって容易に直交するタンパク質の精製

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

orthogonal-protein-purification-facilitated-small-bispecific-affinity

Research

JoVE Journal

Biology

16.9K ビュー.  Royal Institute of Technology. 小説や高効率二段アフィニティークロマトグラフィーのプロトコルが開発され、詳細に説明されている。方法は2つの固有の親和性を持つ小型の精製タグに基づいており、異なるプロパティを持つ標的タンパク質の広い範囲に適用可能です。

ビデオ: Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

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

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

不安定な多タンパク質複合体の単離で in vivoで制御セルラー架橋及び免疫磁気アフィニティークロマトグラフィー

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

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 Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

タンデムアフィニティー精製を用いたタンパク質相互作用するパートナーの識別

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

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 ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

アフィニティータグ組換えタンパク質のハイスループット精製

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

GST-Hisの精製：全長精製タンパク質を降伏二段階アフィニティー精製プロトコル

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

マイクロスケール熱泳動によって結合親和性の決定のタンパク質精製·フリー法

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

組換えタンパク質ベイトを用いてファージディスプレイおよび親和性選択のためのプロトコル

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

中のタンパク質 - タンパク質相互作用を特定する<em&gt;ショウジョウバエ</emタンデムアフィニティー精製（TAP）による&gt;アダルトヘッド

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...