Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the NIH under award number R01AI114362 and Welch Foundation grant I-1782 to Iv&#225;n D'Orso.

1.めっきセル<ol><li>トランスフェクションの1日前に、10％（v / v）のウシ胎児血清（FBS）および1％（v / v）のペニシリン/ストレプトマイシンを補充したダルベッコ改変イーグル培地（DMEM）10mLに3.5×10 6 HEK293T細胞をプレート100ミリメートル皿当たり（10,000 U / mlのストック）。十分なタンパク質回収を確実にするために、実験/条件当たり5 100mm皿 - プレート3。 注意：プレートの数は、目的のタンパク質複合体の発現レベルおよび溶解度に依存するそれぞれの実験/条件に対して決定されなければなりません。より大規模な精製が必要な場合は別の方法として、細胞株は、スピナーフラスコ2に懸濁液中で増殖するように適合させることができますが、この方法は、一過性トランスフェクションのために動作しません。 </li></ol> 2.細胞をトランスフェクトまたは安定な細胞株を誘導<ol><li>翌日、細胞を顕微鏡下で確認してください。 proce前に、80％コンフルエント - 細胞は70でなければなりませんeding。 </li><li>プラスミドDNAの7μgの100 mmディッシュあたりのトランスフェクション試薬の21μlの総混合物で細胞をトランスフェクトします。トランスフェクション効率のコントロールとしてGFP（ 表1）を発現するベクターで細胞をトランスフェクトします。 注：HEK293-T-REXまたはドキシサイクリン14,15に応答して二つ以上の複合体のサブユニットの発現を誘導する同様の安定した細胞株を使用する場合、誘導物質を細胞に添加し、48時間インキュベートされなければなりません。同様に、誘導物質の添加時にGFPの発現を誘導する安定な細胞株はまた、検証目的のために必要とされるであろう。 2.7に進みます。 </li><li> 100ミリメートル皿当たりトランスフェクション混合物を準備します。 1.5ミリリットルマイクロ遠心チューブでは、（2以上のプラスミドに相当）総プラスミドDNAの7μgのと非補充DMEM500μlのを混ぜます。別々のマイクロ遠心チューブでは、未補充DMEMのwiの500μLを混ぜますトランスフェクション試薬の第21μlの。各トランスフェクション反応のために繰り返します。 </li><li>プラスミドDNA溶液へのトランスフェクション試薬溶液をピペットで（逆の順序でソリューションを混在させないでください）。少なくとも4回ピペッティングして混ぜます。 </li><li>もはや15分より15分、しかし - 10室温でこの混合物をインキュベートしていません。 </li><li>メディアリザーバーを作成して、慎重に細胞を乱すことなく、プレートの内側に滴下トランスフェクション混合物をピペットするためにプレートを傾けます。元の平らな位置に皿を返し、混合物を配布するために穏やかに旋回。 </li><li> （この条件は以下「組織培養インキュベーター」とも呼ばれる）、5％CO 2の加湿細胞培養インキュベーター中、37℃で細胞をインキュベートします。メディア5時間後、トランスフェクション（オプション）を交換してください。 </li></ol> 3.確認トランスフェクションまたは誘導効率<ol><li> 24時間後、トランスフェクションでは、インフルエンザの下で細胞を可視化orescent顕微鏡は、トランスフェクション効率（またはGFPを発現制御HEK293T-REX安定な細胞株の誘導）をチェックします。さらに24時間インキュベートします。 </li></ol> 4. STREPアフィニティー精製（STREP AP） <ol><li> 細胞を収集します <ol><li> 48時間トランスフェクション後に、培地を除去し、冷1×リン酸緩衝生理食塩水（PBS）の8ミリリットルを追加します。プレートから細胞を剥離するために上下にピペット。 </li><li>収集し、15 mLのチューブに細胞懸濁液を移し、4℃で4分間、800×gで細胞をスピンダウン。 </li><li> PBS上清を取り除きます。任意の残留PBSを除去するために（4℃で1分間、800×gで）スピンダウン繰り返します。将来の使用のために-80℃で細胞ペレットを保存したり、タンパク質精製を続行するには、以下の手順に従ってください。 </li></ol></li><li> 細胞を溶解 <ol><li>受動溶解緩衝液の細胞ペレットの体積（プレート当たり〜250μl）を×5を使用して、細胞ペレットを再懸濁し（PLB：20mMトリス塩酸pH7.5で、150mMのNaCl、1.5mMのMgCl 2を、1mMのDTT、プロテアーゼ阻害剤カクテル錠、5％（v / v）のグリセロール、1.0％NP-40）及び細胞懸濁液を転送します1.5ミリリットルマイクロチューブ。 </li><li>簡単に言うと4℃で30分間サスペンションとうなだれるをボルテックス。 </li><li> 4℃で10分間10,000×gで細胞懸濁液をスピンダウン。 </li><li>新しい1.5ミリリットルのマイクロ遠心チューブに可溶性溶解物を移し、細胞ペレットを捨てます。可溶性溶解物の最終体積の10％を保存」STREP AP入力。」 4℃で保存。 </li></ol></li><li> STREPビーズを平衡化 注：最初のAPのステップの使用STREPビーズ！複合体はSTREPビーズでプルダウンFLAGビーズでプルダウンものよりも有意に多くのタンパク質を回収。 <ol><li>アウトテイク（nは40μLを×）+ nは50％STREPビーズスラリーμlの（N =サンプル数）、4℃で2分間、1500×gでスピンダウン。 注：使用ワイドメートルピペットビーズにヒントをouthed。 </li><li>上清を除去し、ビーズにPLBの500μlを添加します。 4℃で5分間ビーズ混合物を章動。 4℃で2分間、1500×gでスピンダウン。 <ol><li>合計3回の洗浄のために繰り返します。 40μlの×n個の最終容量までPLB中のビーズを再懸濁します。 </li></ol></li><li> 4℃で2時間、各細胞溶解物のサンプルとうなだれるに50％STREPビーズスラリー40μlのを追加します。 </li></ol></li><li> STREP AP <ol><li> 4℃で2分間、1500×gでソリューションをスピンダウン。 </li><li> 4°Cで「STREP APフロースルー（FT）」と上清とストアを削除します。 </li><li>ビーズにSTREP AP洗浄緩衝液500μlのI（20mMのトリス塩酸pH7.5で、250mMのNaClを、1.5のMgCl 2、1mMのDTT、5％グリセロールおよび0.2％NP-40）を追加します。 5 4℃で分および4℃で2分間、1500×gで遠心分離するためのビーズ混合物を章動させます。上清を捨てます。 <ol><li> 4回の洗浄の合計のために繰り返します。 <br/&gt;注：第洗浄する前に、タンパク質のバックグラウンドを低減するために、新しい1.5ミリリットルのマイクロチューブにビーズ液を移します。このステップは必須です。 </li></ol></li><li> 500μlの洗浄緩衝液II（100 mMトリス塩酸pH8.0の、150mMのNaCl、および1mM EDTA）を加え、チューブを反転させてビーズを平衡化。 4℃で2分間、1500×gでスピンダウンし、上清を捨てます。 </li><li> STREP溶出緩衝液（100mMのトリス塩酸pH8.0の、150mMのNaCl、1mMのEDTA、および2.5 mMのデスチオビオチン）50μlの目的のタンパク質を溶出させます。 4℃で15分間、800rpmでボルテックス。 </li><li> 4℃で1分間、1500×gでビーズをスピンダウン。上清を収集します。この画分は、「STREP AP溶出」のサンプルに対応します。 </li><li>最初の溶出で得られたタンパク質の量の半分までをもたらし得る2回目の溶出、4℃でビーズ画分を保存します。銀染色および/またはウェスタンブロッティングのためにSTREP AP溶出の一定分量10％16。 </li></ol></li><li> FLAGビーズを平衡化 <ol><li> （NX 16μl）を取り出し+ n個μlのFLAGビーズの溶液（n個のサンプルの=数）と4℃で2分間、1500×gでスピンダウン。 注：ピペットのビーズに広口のヒントを参考にしてください。 </li><li>上清を除去し、FLAG洗浄緩衝液500μlを追加（100mMのトリス塩酸pH8.0の、150mMのNaCl、1mMのEDTA、および0.1％NP-40）ビーズへ。 4℃で2分間、1500×gでスピンダウン。合計3回の洗浄のために繰り返します。 </li><li> NX 16μlの最終体積にFLAG洗浄緩衝液でビーズを再懸濁します。 </li><li> 4℃で残りのSTREP AP溶出とうなだれる一晩にFLAGビーズスラリーの16μLを加えます。 </li></ol></li></ol> 5. FLAG IP <ol><li> 4℃で2分間、1500×gでFLAGビーズ懸濁液をスピンダウン。 4°Cで「FLAG IPフロースルー」（FT）として、上清とストアを保存します。 </li><li> 500を追加します。81;溶液へのFLAG洗浄緩衝液のリットル。 5 4℃で分および4℃で2分間、1500×gで遠心分離するための章動させます。 4回の洗浄の合計のために繰り返して、各洗浄後に上清を除去します。 注：第洗浄する前に、バックグラウンドを低減するために、新しい1.5ミリリットルのマイクロチューブにタンパク質ビーズのソリューションを転送します。このステップは必須です。 </li><li>最後のスピンから上清を除去し、ビーズにFLAGペプチドの200 ngの/μLを含むFLAG洗浄緩衝液の30μLを追加します。 4℃で2時間、800 rpmでボルテックス。 </li><li> 4℃で2分間、1500×gでサスペンションをスピンダウン。 4℃で上清とストアを収穫 &quot;FLAG IP溶出。」 注：溶出サンプルは、標準的なプロトコル16を使用して、銀染色および/またはウェスタンブロットによって確認し、さらにタンパク質アッセイのための準備ができていることができます。 （1）STREP AP入力、（2）STREP AP FT、（3）STREP AP Eluti：ウェスタンブロットを設定する場合は、以下のサンプルのすべてを実行します（4）FLAG IP FT、上、および（5）FLAG IP溶出。ロードされたボリュームは、各実験のために決定する必要があります。すべての収集されたサンプルとビーズは、短期記憶および長期保存のために-80℃で、4℃で保存することができます。 TAPサンプルは、その構成要素の身元を確認するための新規の翻訳後修飾（PTMを）を識別するために、および/または精製された複合体2、17を有する任意の新規なタンパク質相互作用を検出するために質量分析によって分析することができます。この目的のために、クマシーゲルまたは銀染色を実行した後、バンドはきれいなメスを用いてゲルから切り出すことができます。質量分析法を議論するいくつかの優れたレビューは、以前に18、19を発表しました。 </li></ol>

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The authors have nothing to disclose.

真核細胞からのタンパク質複合体の発現および単離のためにここに記載したプロトコルは、このような分子集合体の生化学的特徴付けに限定されるものではなく、それらの機能を調節することができ、新規な相互作用および翻訳後修飾の同定に利用することができます。親和性タグの使用は、このプロトコルに記載されているものに限定されるものではありません。しかし、我々の経験は、?...

<html>タンパク質間相互作用（PPIの）生物学的プロセス1の正確な調節のために重要であり、これらのPPIに関するさらなる研究は、それらの機能2をお知らせすることができます。いくつかのアプローチがのPPIの研究と特徴付けのためだけでなく、新たな規制のタンパク質成分のデノボ識別のために考案されています。 1989年、スタンリーフィールズらは、酵母ツーハイブリッド（Y2H）アッセイ3を報告しました 。このアプローチは、 サッカロマイセス・セレビシエの関心の定義されたタンパク質（ベイト）のための相互作用（獲物）の公正かつ包括的な同定を可能にします。 PPIを発見するために著しい有用性に加えて、Y2Hアッセイは、酵母細胞におけるタンパク質の組を特徴付ける最小相互作用ドメインを定義し、そのような相互作用を廃止突然変異を同定するために使用することができます。 Y2Hアッセイを改変することによって、のPPIはまた、哺乳動物細胞において研究することができますクラス= &quot;外部参照&quot;&gt; 4。 Y2Hアッセイ（ 例えば 、酵母スリーハイブリッドシステム）の変動は、細胞内タンパク質、RNAおよびタンパク質-小有機リガンド相互作用を研究するために適用することができます。 相同システムでのPPIを研究するための別の一般的に使用されるツールは、共免疫沈降（共-IP）アッセイ5です。関心対象のタンパク質を免疫沈降するために抗体を使用することにより、共IPアッセイは、研究者が様々な環境条件および実験の状況のた​​めに細胞中でのPPIを監視することを可能にします。親和性精製（AP）メソッド内のエピトープタグ付きタンパク質の使用（ 例えば 、FLAG、Mycを、STREP、およびとりわけHAは、）、銀染色をウェスタンブロットを含むいくつかの下流のアッセイのための複雑なタンパク質混合物からのタンパク質の単離を容易にしました、および酵素的分析。しかしながら、これらの従来のアプローチのいずれも、in vitroでの Aを含む、さらなる特徴付けのためのタンパク質複合体の大量の単離を可能にしませんssays、質量分析による調節サブユニットの発見、および翻訳後修飾の同定。 AP法の改良版は、2後続のAP 6、7を通して精製された2つのエピトープとの融合タンパク質を作成することでのPPIを研究するための精製技術であるタンデムAP（TAP）と呼ばれています。この記事では、二つのサブユニットは、異なるエピトープでタグ付けした後、2回の連続のAP（STREP AP FLAG IPが続く）を通して精製された精製タンパク質複合体のためのTAP方法のバリエーションを提示します。研究者が興味のあるタンパク質複合体に適用できるように、我々はまず、TAPのミニマル概要（ 図1）と、すべての実験工程（ 図2）の詳細な説明を提供します。 TAP法の適用性を実証するために、我々は、PTと呼ばれる（複雑なよく特徴付けサイクリンCDKを選ん調節サブユニット、サイクリンT1（CycT1）及びキナーゼ（CDK9）から構成され、RNAポリメラーゼII（Pol II）8、9、10による転写の調節に関与するEFBキナーゼ）。 P-TEFbはポルIIプロモーターで転写休止を軽減それに関連する負の伸長因子のC末端ドメインをリン酸化し、それによって転写伸長11、12、13を容易にします 。このことを念頭に置い既知の相互作用により、STREPタグ付きCycT1およびFLAGタグCDK9はHEK293T細胞で過剰発現しました。逆数TAP実験をさらにタンパク質相互作用を利用するエピトープとは無関係であることを検証するためにSTREPタグ付きCDK9およびFLAGタグCycT1を用いて行きました。細胞を48時間トランスフェクション後に回収し、溶解しました。可溶性溶解物をTAPにより精製した（STREP APは、FLAGが続きますIP）。入力および精製されたタンパク質は、ウェスタンブロットおよび銀染色（ 図3）によって分析しました。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>GE Healthcare Life Sciences/Hyclone</td><td>SH3002.2FS</td><td></td></tr><tr><td>Fetal bovine serum</td><td>GE Healthcare Life Sciences/Hyclone</td><td>SH30071</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>MP Biomedicals</td><td>MP091670049</td><td></td></tr><tr><td>PolyJet</td><td>SignaGen Laboratories</td><td>SL100688</td><td></td></tr><tr><td>Protease inhibitor cocktail&amp;nbsp;</td><td>Roche&amp;nbsp;</td><td>11836153001</td><td></td></tr><tr><td>STREP-Tactin Superflow</td><td>IBA Lifesciences</td><td>2-1208-010</td><td></td></tr><tr><td>STREP-tag elution buffer</td><td>IBA Lifesciences</td><td>2-1000-025</td><td></td></tr><tr><td>EZview Red ANTI-FLAG M2 Affinity Gel</td><td>Sigma-Aldrich</td><td>F2426</td><td></td></tr><tr><td>Corning 100 mm &amp;times; 20 mm style dish cell culture treated nonpyrogenic polystyrene 20/sleeve</td><td>Corning&amp;nbsp;</td><td>430167</td><td></td></tr><tr><td>Protein Lo-Bind Eppendorf</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Digital Vortex Mixer</td><td>Fisher Scientific&amp;nbsp;</td><td>02-215-370</td><td></td></tr><tr><td>48 hole micro tube foam rack</td><td>Fisher Scientific</td><td>02-215-386</td><td></td></tr><tr><td>Labquake shaker rotisserie&amp;nbsp;</td><td>Thermo&amp;nbsp;</td><td>415110</td><td></td></tr></tbody></table>

tandem affinity purification of protein complexes from eukaryotic cells

活性なタンパク質-タンパク質およびタンパク質-核酸複合体の精製は、酵素活性および新規サブユニットおよび翻訳後修飾の新規同定の特徴付けのために重要です。細菌系は、単一のポリペプチドおよびタンパク質複合体の広範囲の発現および精製を可能にします。しかし、このシステムは、翻訳後修飾を含有タンパク質サブユニット（ 例えば 、リン酸化およびアセチル化）の精製、および唯一の存在である新規な調節サブユニットの識別を可能に/真核生物系で表現されません。ここでは、真核細胞から一過性または安定的に発現エピトープタグ付きタンパク質とタンパク質複合体の精製を容易に小説、堅牢、かつSTREP-を使用して効率的なタンデムアフィニティー精製（TAP）法およびFLAGタグ融合タンパク質の詳細な説明を提供します。このプロトコルはPROTを特徴付けるために適用することができますEIN複雑な機能、複雑なサブユニット上の翻訳後修飾を発見する、および質量分析によって新たな規制の複雑なコンポーネントを識別するために。特に、このTAP法は、このように下流の実験機会の広い配列を得、真核生物または病原性（ウイルス性および細菌性）成分により形成されるタンパク質複合体を研究するために適用することができます。我々は、タンパク質複合体を扱う研究者は、多くの異なる方法で、この方法を利用することができることを提案します。

この記事では、我々は（また、P-TEFbキナーゼとして知られている）は、複雑なよく特徴付けCycT1-CDK9にTAP法の適用性を実証します。 CDK9-FLAG（CDK9：F）、またはCDK9-STREP（CDK9：S）およびサイクリンT1-FLAG（CycT1：F）：サイクリンT1-STREP（S CycT1）をコードするプラスミド（ 表1）は 、HEK293T細胞にトランスフェクトし...

Watch this Scientific Journal Video about Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells at JoVE.com

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

真核細胞からのタンパク質複合体のタンデムアフィニティー精製

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

tandem-affinity-purification-protein-complexes-from-eukaryotic

Research

JoVE Journal

Biochemistry

14.9K ビュー.  The University of Texas Southwestern Medical Center. We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

ビデオ: Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Cryomilled哺乳動物細胞からのタンパク質複合体の親和性のキャプチャ

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

生化学

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

ブルーネイティブPAGEおよびタンパク質相関プロファイリングによってアフィニティー精製タンパク質複合体の解決

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 の 1: タンパク質・ タンパク質代謝物の複合体の並列解析のための親和性の浄化をワンステップ

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

タップのタグを使用して葉緑体トランスロコン タンパク質複合体の親和性の浄化

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

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 ...

生物学

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 ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBLでMultiBacタンパク質複合体生産プラットフォーム

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

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. ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

タンデムアフィニティータグ法を用いた構造解析のためのネイティブ複合体の精製

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Source: Ma, Z., et al. <a href="https://app.jove.com/v/55236">Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.</a> J. Vis. Exp. (2017).
This video demonstrates tandem affinity purification &#8212; a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

タンパク質間相互作用を研究するためのタンデムアフィニティー精製アッセイ

Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).
This video demonstrates tandem ...

真核細胞からのタンパク質複合体のタンデムアフィニティー精製

要約

さらに動画を探す

この動画の章

Cryomilled哺乳動物細胞からのタンパク質複合体の親和性のキャプチャ

ブルーネイティブPAGEおよびタンパク質相関プロファイリングによってアフィニティー精製タンパク質複合体の解決

2 の 1: タンパク質・タンパク質代謝物の複合体の並列解析のための親和性の浄化をワンステップ

タップのタグを使用して葉緑体トランスロコンタンパク質複合体の親和性の浄化

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

のタンパク質複合体の同定

EMBLでMultiBacタンパク質複合体生産プラットフォーム

中のタンパク質 - タンパク質相互作用を特定する

タンデムアフィニティータグ法を用いた構造解析のためのネイティブ複合体の精製

タンパク質間相互作用を研究するためのタンデムアフィニティー精製アッセイ

真核細胞からのタンパク質複合体のタンデムアフィニティー精製

要約

さらに動画を探す

この動画の章

Cryomilled哺乳動物細胞からのタンパク質複合体の親和性のキャプチャ

ブルーネイティブPAGEおよびタンパク質相関プロファイリングによってアフィニティー精製タンパク質複合体の解決

2 の 1: タンパク質・ タンパク質代謝物の複合体の並列解析のための親和性の浄化をワンステップ

タップのタグを使用して葉緑体トランスロコン タンパク質複合体の親和性の浄化

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

のタンパク質複合体の同定

EMBLでMultiBacタンパク質複合体生産プラットフォーム

中のタンパク質 - タンパク質相互作用を特定する

タンデムアフィニティータグ法を用いた構造解析のためのネイティブ複合体の精製

タンパク質間相互作用を研究するためのタンデムアフィニティー精製アッセイ

2 の 1: タンパク質・タンパク質代謝物の複合体の並列解析のための親和性の浄化をワンステップ

タップのタグを使用して葉緑体トランスロコンタンパク質複合体の親和性の浄化