我々 はプロジェクト、生産的な議論と偉大な監督の彼の介入のため教授ローター Willmitzer がのでご了承いただきたいと思います。プロテオーム MS 測定を助けるため博士ダニエル Veyel に感謝しております。夫人 Änne ミハイル LC-MS 測定で非常に貴重な技術サポートご提供くださったお願い申し上げます。さらに、テクニカル サポートの Weronika Jasińska に支援と元の原稿と仕事で関わり博士モニカー Kosmacz と博士 Ewelina Sokołowska に感謝したいと思います。

<ol>
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V., Ong, S. -. E., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin+cleaves+exclusively+C-terminal+to+arginine+and+lysine+residues.">Trypsin cleaves exclusively C-terminal to arginine and lysine residues.</a> Molecular &amp; Cellular Proteomics. 3 (6), 608-614 (2004).</li><li>Cox, J., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MaxQuant+enables+high+peptide+identification+rates,+individualized+p.p.b.-range+mass+accuracies+and+proteome-wide+protein+quantification.">MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.</a> Nature Biotechnology. 26 (12), 1367-1372 (2008).</li><li>Cox, 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=Andromeda:+A+peptide+search+engine+integrated+into+the+MaxQuant+environment.">Andromeda: A peptide search engine integrated into the MaxQuant environment.</a> Journal of Proteome Research. 10 (4), 1794-1805 (2011).</li><li>Tyanova, S., Temu, T., Cox, 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+MaxQuant+computational+platform+for+mass+spectrometry-based+shotgun+proteomics.">The MaxQuant computational platform for mass spectrometry-based shotgun proteomics.</a> Nature Protocols. 11 (12), 2301 (2016).</li><li>Hooper, C. M., 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=SUBAcon:+a+consensus+algorithm+for+unifying+the+subcellular+localization+data+of+the+Arabidopsis+proteome.">SUBAcon: a consensus algorithm for unifying the subcellular localization data of the Arabidopsis proteome.</a> Bioinformatics. 30 (23), 3356-3364 (2014).</li><li>Katajamaa, M., Ore&#353;i&#269;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+processing+for+mass+spectrometry-based+metabolomics.">Data processing for mass spectrometry-based metabolomics.</a> Journal of Chromatography A. 1158 (1-2), 318-328 (2007).</li><li>Szklarczyk, 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=STITCH+5:+augmenting+protein-chemical+interaction+networks+with+tissue+and+affinity+data.">STITCH 5: augmenting protein-chemical interaction networks with tissue and affinity data.</a> Nucleic Acids Research. 1277, (2015).</li><li>Tanaka, K. -. i., 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=Dipeptidyl+compounds+ameliorate+the+serum-deprivation-induced+reduction+in+cell+viability+via+the+neurotrophin-activating+effect+in+SH-SY5Y+cells.">Dipeptidyl compounds ameliorate the serum-deprivation-induced reduction in cell viability via the neurotrophin-activating effect in SH-SY5Y cells.</a> Neurological Research. 34 (6), 619-622 (2012).</li><li>Smith, C. A., 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=METLIN:+A+metabolite+mass+spectral+database.">METLIN: A metabolite mass spectral database.</a> Therapeutic Drug Monitoring. 27, 747-751 (2005).</li><li>Bhattacharyya, S., 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=Transient+protein-protein+interactions+perturb+E.+coli+metabolome+and+cause+gene+dosage+toxicity.">Transient protein-protein interactions perturb E. coli metabolome and cause gene dosage toxicity.</a> Elife. 5, (2016).</li><li>Rigaut, G., 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=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nature Biotechnology. 17 (10), 1030-1032 (1999).</li></ol>

著者が明らかに何もありません。

提案するプロトコルは、ターゲット蛋白質の PP と午後の複合体の並列識別を許します。最終結果へのクローン作成から実験は 8 〜 12 週間ほどで完了ことができます。完全な AP は、我々 のプロトコルを半ばスループット解析に適したレンダリング 12 に 24 サンプルのセットの約 4-6 時間かかります。
にもかかわらず全体的に簡単です、プロトコルには、いくつかの重要な手順があります。(入力蛋白質のアフィニティ ビーズ i) 十分な量は、代謝物の検出のダイナミック レンジに到達する重要です。したがって、効率的なセル換散ですプロシージャの重要なステップです。悪い蛋白質収量は、材料または次善の換散バッファー/材料比率の不十分な粉砕の結果をすることができます。(使用試薬が MS 向けであること、ii) 注意をすべき。彼らは MS 検出に干渉としては、強い洗剤、グリセリン、または塩の過剰を避けるべき。(iii) アガロース ビーズ過剰乾燥の手順を洗う時にする必要がありますいないと真空マニホールドを使用する場合は、ビーズを破壊する複雑な安定性に影響しないように低速流率を適用することが重要です。
提案するプロトコルをいくつかの重要な可能な修正がある: (i) 構成の CaMV35S プロモーターを使用して、餌の蛋白質の量を最大化します。過剰発現、非常に便利ですが、細胞の恒常性28に深刻な影響を与えることができるし、生理学的関連性の低い相互作用の形成に 。ネイティブのプロモーター、機能喪失バック グラウンドで可能な限りが真の生物学的インターアクターを取得するための優れたと見なされますを使用して付けられた蛋白質の表現。植物細胞培養で通常は表される蛋白質のため工場の背景が関連するインターアクターを識別するために必要な証明するかもしれない。(ii) 膜タンパク質を使用する場合、換散バッファーは MS 互換洗剤を補充する必要があります。(iii) 2 番目の親和性の浄化のステップの導入は真陽性率に偽陽性を改善できるし、EV コントロール29のための必要性を排除します。2 つの独立したプロテアーゼ切断サイト新規タンデム タグは、面倒で時間のかかる作業である前田ら201411追加サイズ排除クロマトグラフィー ステップに魅力的な代替手段を提示します。
AP の最も重大な欠点は、偽陽性の率が高いです。理由はたくさんあります。構成の過剰発現は、すでに言及されました。生理学的関連性の低い相互作用の別のソース分離細胞小器官を扱うを除き蛋白質および別の細胞内コンパートメントからの代謝物質の混合物を含む全体セル lysates の準備です。内局は、true インターアクターのフィルターに使用する必要があります。それにもかかわらず、不明確な結合蛋白質とアガロース樹脂の間起因する偽陽性の大半。はじめに 2 番目の精製ステップの前述のように、問題に最適なソリューションを提供しています、しかし時間とスループットを犠牲にして来る。さらに、プロトコルが長くなるにつれて弱い相互作用が失われることがあります。AP のもう一つの注意点は、包括的な情報を提供しています、インタラクトームについてターゲット蛋白質のにもかかわらず餌タンパク質の直接的および間接的なターゲット間の区別は不可能であります。ターゲットを絞った分子アプローチが相互作用を確認するため必要です。
MS ベース メタボロミクスと相まって AP を用いて酵母12.の蛋白質複合体の研究私たち以前観察13同様に脂質を極性、半極性化合物に結合したまま細胞溶解液から分離されたタンパク質複合体とともに、この仕事は、提案するプロトコルの概念的な基礎を提供しました。私達のプロトコルは 3 つのユニークなポイントによって特徴付けられる: (i) 対照的に、酵母に働く12AP は疎水性がまた親水性のタンパク質だけでなく有機配位子を取得するのに適しているを示します。(ii) 3 つの抽出のプロトコルを用いて、単一の AP を使用して餌タンパク質のタンパク質と代謝産物のインターアクターを勉強できます。(iii) は植物細胞にプロトコールを適用しました。
今後の取り組みは、2 つの独立したプロテアーゼ切断サイトで新規タンデム タグの作成について説明します。我々 はまた植物ホルモンなど低豊富小さい分子にプロトコルの適合性を探索したいと思います。

方法はここで説明近く体内細胞ライセート条件で選択の蛋白質の代謝産物やタンパク質のパートナーの同定を目指す。それは、今日特徴付けられるより多くより多くの代謝物が規制の重要な機能の1であることが推測されています。代謝生物学的スイッチ、活動、機能、および/またはその受容体タンパク質2,3,4のローカリゼーションを変更として使用できます。過去 10 年間で識別 PMI体内のまたは近い-生体内での条件でを有効にするいくつかの画期的な方法が開発5をされています。使用可能なアプローチは、2 つのグループに分類できます。最初のグループは構成蛋白質パートナーをトラップするために知られている代謝物餌で始まる技術です。親和性クロマトグラフィー6、薬物親和性応答ターゲット安定性アッセイ7、化学療法プロテオミクス8、9をプロファイリング熱プロテオームなどの方法。2 番目のグループは、小分子リガンド10,11を識別するために知られていた蛋白質で始まる 1 つのメソッドで構成されています。
MS ベース リピドと相まって AP が酵母12.タンパク質脂質複合体の分析に使用されました。開始点として、著者はエルゴステ ロールの生合成に関与する 21 の酵素を表現する酵母を使用、タンデム親和性の浄化 (蛇口) タグに融合した 103 キナーゼ。酵素の 70% と、キナーゼの 20% は、複雑なタンパク質-脂質相互作用ネットワークに光を流して別の疎水性配位子をバインドする発見されました。
以前は、我々 は、同様に脂質を極性、半極性化合物もに結合したまま細胞ライセート13から分離されたタンパク質複合体を示すことができます。我々 は ap 通信を最適化することを決めたこれらの調査結果に基づいて、メソッドは植物細胞と親水性化合物14の10、11以前公開します。この目的のため、バン リーネら2010 年工場 PPI 研究15で正常に使用によって記述されたタップ ベクトルを使用しました。トランスジェニックは行を取得するために必要な時間を短縮、シロイヌナズナ細胞培養しました。我々 採用ワンステップ メチルtert-ブチル エーテル、(MTBE)/メタノール/水抽出法、単一のタンパク質 (ペレット)、脂質 (有機相)、および親水性代謝産物 (水相)16の評価を許可します。親和性の浄化実験。EV の制御線は、偽陽性、単独でのタグに結合する蛋白質などを除外する導入されました。タグ付け (5) の 3 つのコンセプトの証明としてヌクレオシド二リン酸キナーゼ (NDPK1-NDPK3) シロイヌナズナのゲノムに存在します。その他の所見の中で我々 は NDPK1 がグルタチオンSと対話することを示すことができます-トランスフェラーゼとグルタチオン。したがって我々 は NDPK1 は glutathionylation14に服従することを証明できます。
要するに、提案するプロトコルはタンパク質間およびタンパク質-低分子間相互作用ネットワークの特性評価に重要なツールで、既存方法と比べて大きな進歩を構成します。

<table>
	<tbody>
		<tr>
			<td>Murashige and Skoog Basal Salts with minimal organics</td>
			<td>Sigma-Aldrich</td>
			<td>M6899</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Naphthylacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>N1641</td>
			<td></td>
		</tr>
		<tr>
			<td>Kinetin solution</td>
			<td>Sigma-Aldrich</td>
			<td>K3253</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-Aldrich</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Carl Roth</td>
			<td>2189.1</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>3609</td>
			<td></td>
		</tr>
		<tr>
			<td>NaF</td>
			<td>Sigma-Aldrich</td>
			<td>S6776</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
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		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>E-64 protease inhibitor</td>
			<td>Sigma-Aldrich</td>
			<td>E3132</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P9599</td>
			<td></td>
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		<tr>
			<td>Na3VO4</td>
			<td>Sigma-Aldrich</td>
			<td>S6508</td>
			<td></td>
		</tr>
		<tr>
			<td>AcTEV Protease</td>
			<td>Thermo Fischer Scientific</td>
			<td>12575015</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotiphorese&#160;Gel 30 (37,5:1)</td>
			<td>Carl Roth</td>
			<td>3029.2</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Carl Roth</td>
			<td>2367.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Prestained Protein Ladder</td>
			<td>Thermo Fischer Scientific</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>SBP Tag Antibody (SB19-C4)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-101595</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG-HRP</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>B6916</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/Lys-C Mix, Mass Spec Grade</td>
			<td>Promega&#160;</td>
			<td>V5071</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5128</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiourea</td>
			<td>Sigma-Aldrich</td>
			<td>T8656</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>9830</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I1149</td>
			<td></td>
		</tr>
		<tr>
			<td>MTBE&#160;</td>
			<td>Biosolve</td>
			<td>138906</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol&#160;</td>
			<td>Biosolve</td>
			<td>136806</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Biosolve</td>
			<td>232106</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Biosolve</td>
			<td>12006</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid&#160;</td>
			<td>Biosolve</td>
			<td>202341</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid&#160;</td>
			<td>Biosolve</td>
			<td>69141</td>
			<td></td>
		</tr>
		<tr>
			<td>Unimax 2010 Platform Shaker</td>
			<td>Heidolph</td>
			<td>5421002000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Mesh (Wire diameter 34 &#181;M, thickness 55 &#181;M, open area 14%)</td>
			<td>Prosepa</td>
			<td>Custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Funnel, 47 mm, 300 ml</td>
			<td>Restek</td>
			<td>KT953751-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Bottle Top 500 mL 0,2 &#181;M Pes St</td>
			<td>VWR International GmbH</td>
			<td>514-0340</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer Mill MM 400</td>
			<td>Retsch GmbH</td>
			<td>207450001</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG Sepharose 6 Fast Flow</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17-0969-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Mobicol &#34;&#34;Classic&#34;&#34; with 2 different screw caps without filters</td>
			<td>MoBiTec GmbH</td>
			<td>M1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter (small) 35 &#181;M pore size, for Mobicol M 1002, M1003, M1050 &#38; M1053</td>
			<td>MoBiTec GmbH</td>
			<td>M513515</td>
			<td></td>
		</tr>
		<tr>
			<td>Variable Speed Tube Rotator SB 3</td>
			<td>Carl Roth</td>
			<td>Y550.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary dishes for rotators SB 3</td>
			<td>Carl Roth</td>
			<td>Y555.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Resprep 24-Port SPE Manifolds</td>
			<td>Restek&#160;</td>
			<td>26080</td>
			<td></td>
		</tr>
		<tr>
			<td>Finisterre C18/17% SPE Columns 100mg / 1ml</td>
			<td>Teknokroma</td>
			<td>TR-F034000</td>
			<td></td>
		</tr>
		<tr>
			<td>Autosampler&#160;Vials</td>
			<td>Klaus Trott Chromatographie-Zubeh&#246;r</td>
			<td>40 11 01 740</td>
			<td></td>
		</tr>
		<tr>
			<td>Acclaim PepMap 100 C18 LC Column</td>
			<td>Thermo Fischer Scientific</td>
			<td>164534</td>
			<td></td>
		</tr>
		<tr>
			<td>EASY-nLC 1000 Liquid Chromatograph</td>
			<td>Thermo Fischer Scientific</td>
			<td>LC120</td>
			<td></td>
		</tr>
		<tr>
			<td>Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer</td>
			<td>Thermo Fischer Scientific</td>
			<td>IQLAAEGAAPFALGMBDK</td>
			<td></td>
		</tr>
		<tr>
			<td>Acquity UPLC system&#160;</td>
			<td>Waters&#160;</td>
			<td>Custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC HSS C18 Column, 100A, 1.8 &#181;M, 2.1 mM X 100 mM, 1/pkg</td>
			<td>Waters&#160;</td>
			<td>186003533</td>
			<td></td>
		</tr>
		<tr>
			<td>High-power ultrasonic cleaning baths for aqueous cleaning solutions</td>
			<td>Bandelin</td>
			<td>RK 31</td>
			<td></td>
		</tr>
		<tr>
			<td>Genedata Expressionist</td>
			<td>Genedata</td>
			<td>NaN</td>
			<td></td>
		</tr>
		<tr>
			<td>Xcalibur Software</td>
			<td>Thermo Fischer Scientific</td>
			<td>NaN</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQuant</td>
			<td>NaN</td>
			<td>NaN</td>
			<td></td>
		</tr>
	</tbody>
</table>

2 in 1: one-step affinity purification for the parallel analysis of protein-protein and protein-metabolite complexes

細胞はタンパク質、代謝、核酸などの生体分子間の相互作用によって規制されています。タンパク質間相互作用 (PPI) の調査に目新しさはなく、内因性のタンパク質代謝相互作用 (PMI) を特徴付けることを目指して実験的アプローチはむしろ最近開発を構成します。ここで、PPI と選択、餌と呼ばれるタンパクの PMI の同時解析を可能にするプロトコルを提案する.我々 のプロトコルを最適化しましたシロイヌナズナ細胞文化し、親和性の浄化 (AP) を組み合わせた質量分析法 (MS)-基づくタンパク質と代謝産物の検出。一言で言えば、親和性タグに融合餌蛋白を発現するトランスジェニックのシロイヌナズナ ラインの最初ネイティブ細胞抽出物を取得する服従分離します。抗タグ抗体を使用して、餌の蛋白質のタンパク質と代謝産物のパートナーをプルダウンします。ワンステップ メチルtertを用いたアフィニ ティー精製錯体を抽出-ブチル エーテル (MTBE)/メタノール/水法。ながら代謝を極または疎水性相に分離、ペレットの蛋白質を見つけることができます。代謝産物やタンパク質は、超高性能液体クロマトグラフィー質量分析法 (高精度 MS または高精度・ MS/MS) によって分析しています。空のベクトル (EV) 制御線は、偽陽性を除外する使用されます。プロトコルの主な利点は、近く生理的条件 (細胞ライセート) で並行してターゲット蛋白質のタンパク質と代謝産物のパートナーの同定が可能です。提案手法は簡単です高速で、植物細胞培養以外の生物学的システムに簡単に適応することができます。

元の研究では、3 つのシロイヌナズナNDPK 遺伝子が構成 35S プロモーター14 (図 1) の制御の下で PSB L 細胞懸濁培養における過剰発現。タンデム親和性のタグは餌の蛋白質のカルボキシまたはアミノ ターミナル端に融解する.アフィニ ティー精製錯体は、MTBE/メタノール/水抽出16に服従しました。アフィニティ引っ張ったタンパク質と低分子化合物は、MS (テーブル S2 と S3) を使用して識別されました。
偽陽性を修正するには、空白のサンプルは研究室の消耗品と化学物質から低分子汚染物質を除外する使用されました。さらに、代謝産物やタンパク質親和性タグのいずれかにバインドするだけで樹脂はいた EV 制御線を使用して、占めています。真陽性、2 非対スチューデントの t 検定と Benjamini & ホフベルク虚偽の発見率を取得するには、補正を代謝産物 (表 S4) とタンパク質 (テーブル S5) 大幅 NDPKs AP の濃縮を識別するために適用しました。EV の制御線と比較して実験 (NDPKs と N- と C 末端タグ付き) (FDR < 0.1)。前の仕事で、タンパク質と低分子のインターアクターの線引きに不在/プレゼンス基準を使用我々 に注意してください。
代表的な結果は、ジペプチド、我々 のグループで研究小分子レギュレータの新規クラスの代謝産物データ フォーカスしながら NDPK1、ために与えられます。プロテオーム解析では、NDPK1 の 26 と推定される蛋白質パートナーを明らかにしました。さらにフィルター NDPK1 として同じ細胞レベル下コンパートメントの共局在蛋白質 (細胞質)、13 と推定されるタンパク質インターアクターにリストを絞り込みます。識別された蛋白質の間でされたグルタチオンS-トランスフェラーゼ、2 つ伸長因子、チューブリン、aconitate ニトリルヒドラターゼ。4 つのジペプチド ヴァル ・ ルー、イル Glu ルー-イル、イルを NDPK1 とペー具体的共同溶出するメタボローム解析が明らかに (図 2)。すべての 4 つのジペプチドの N 末端、共有結合特異性を示唆している疎水性残基を共有することに注意してください。
我々 既知のタンパク質とタンパク質代謝物の複合体のために、照会された 13 は蛋白質、ステッチ データベース25 (図 3) に対して 4 つのジペプチドを検出しました。いくつかの観測を作ることができる: (i) NDPK1 以前報告されたインターアクターのどれも。(ii) APX1 オーソログがアルデヒド脱水素酵素家族メンバー ALDH7B4、翻訳開始因子 FBR12 遺伝子 AT2G40290 別の翻訳開始因子としながらやり取りする報告されました。(iii) 特定のジペプチドはない蛋白質パートナーを報告しています。共同溶出ジペプチドが取得した植物蛋白質を関連付けられている以前に報告されたないです。しかし、彼らは他の生物に重要な役割を果たして: ルー-イル、例えば、ひと細胞ライン26神経栄養因子活性化の効果があります。実験では、システムの正確なトポロジを識別できないことに注意してください。たとえば、ジペプチドは NDPK1 と直接対話することがありますが、よく共同浄化された蛋白質のいずれかに関連している可能性があります。
一緒に取られて、私達の結果表示設立の手順、質量分析法と AP を用いたタンパク質間およびタンパク質-低分子インターアクターの同定を容易にし、についての広汎な情報の作成を支援します標的タンパク質のインタラクトーム。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57720/57720fig1.jpg"> 図 1。AP MS ワークフローの方式です。(A)植物細胞培養からネイティブ可溶性画分の調製。(B)次の手順の AP。列にサンプルをロードした後 (ポイ) タップ タグ融合タンパク質は、アガロース ビーズに固定化した抗体にバインドします。列の洗浄は、非連結のタンパク質と代謝産物の除去を促進します。AcTEV 胸の谷間を実行した後ポイ タンパク質代謝物の複合体に溶出されます。(C)分離複合体の半定量的な分析に続いてタンパク質と代謝産物の割合。この図の一部は、Luzarowski et al. 201714から再現します。<a href="/files/ftp_upload/57720/57720fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57720/57720fig2.jpg"> 図 2。具体的には共同 NDPK1 と溶出性ジペプチド。ヴァル ・ ルー (A)、イル Glu (B)、ルー-イル(C)、およびイル Phe (D) AP の実験で測定した 4 つのジペプチドの平均強度をプロットしました。すべての 4 つのジペプチド EV コントロールと比較して NDPK1 サンプルの重要な濃縮を表示する (アスタリスクは FDR を表す < 0.1)。誤差範囲を表す六つの測定の標準誤差 (N-3 複製と C 末端の 3 タグ付きタンパク質)。<a href="/files/ftp_upload/57720/57720fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57720/57720fig3.jpg"> 図 3。前のみを考慮したステッチ データベースに対してクエリを実行共同 NDPK1 と溶出性すべての分子の相互作用ネットワーク実験およびデータベース証拠 (自信 > 0.2).高い信頼は相互作用の高い可能性を示し、堆積のデータに基づいて計算されます。<a href="/files/ftp_upload/57720/57720fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
テーブル S1。MaxQuant は出力テーブル"parameters.txt"です。テーブルには識別と定量化、として使用するデータベースについての情報のためのしきい値が含まれています。<a href="/files/ftp_upload/57720/Table_S1.xlsx">このファイルをダウンロードするここをクリックしてください</a>。
テーブル S2。MaxQuant からの情報出力テーブル"proteinGroups.txt"。テーブルには、すべての識別された蛋白質のグループの強度とユニークなペプチドとスコアの数などの追加情報の一覧が含まれています。<a href="/files/ftp_upload/57720/Table_S2.xlsx">このファイルをダウンロードするここをクリックしてください</a>。
テーブル S3。代謝物の分析を含む出力ファイル。テーブルには特定 m/z、RT および強度によって特徴付けられるすべての識別された質量の機能の一覧が含まれます。<a href="/files/ftp_upload/57720/Table_S3.xlsx">このファイルをダウンロードするここをクリックしてください</a>。
表 S4。ジペプチドは餌として使用された NDPK1、NDPK2 または NDPK3 AP サンプルで発見しました。ジペプチド空白のサンプルで現在がリストから除外されました。各 NDPK の 2 つの独立した行 (タグ付きの N 末端または C 領域のいずれかで) は、3 通で実行されました。学生のt-テスト、 pの補正をさらに-Benjamini を使用しての値 & ホフベルク メソッドを用いて NDPKs の大幅充実した入力/終端] パートナーを決定 (FDR < 0.1)。与えられた ΔRT は参照化合物の質量は、Metlin27モノアイソに関連して Δppm に関連して計算します。<a href="/files/ftp_upload/57720/Table_S4.xlsx">このファイルをダウンロードするここをクリックしてください</a>。
テーブル S5。共同 NDPK1 で精製したタンパク質。各 NDPK の 2 つの独立した行 (N 末端または C 領域のいずれかのタグ付き) の 3 通で実行されました。学生のt-テスト、 pの補正をさらに-Benjamini を使用しての値 & ホフベルク メソッドを用いて NDPKs の大幅充実した入力/終端] パートナーを決定 (FDR < 0.1)。<a href="/files/ftp_upload/57720/Table_S5.xlsx">このファイルをダウンロードするここをクリックしてください</a>。

Watch this Scientific Journal Video about 2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes at JoVE.com

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

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

タンパク質とタンパク質代謝の相互作用は携帯電話のすべての機能のために重要です。ここで、選択の蛋白質のこれらの相互作用の平行分析を可能にするプロトコルについて述べる。私達のプロトコルは、植物細胞培養用に最適化されたし、質量分析を用いたタンパク質と代謝物の検出と親和性の浄化を結合します。

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

2-1-one-step-affinity-purification-for-parallel-analysis-protein

Nanyang Technological University

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Biochemistry

11.2K ビュー.  Max Planck Institute of Molecular Plant Physiology. タンパク質とタンパク質代謝の相互作用は携帯電話のすべての機能のために重要です。ここで、選択の蛋白質のこれらの相互作用の平行分析を可能にするプロトコルについて述べる。私達のプロトコルは、植物細胞培養用に最適化されたし、質量分析を用いたタンパク質と代謝物の検出と親和性の浄化を結合します。

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

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other fields. For instance, trehalose analogues bearing detectable tags have been used to detect Mycobacterium tuberculosis and may have applications as tuberculosis diagnostic imaging agents. Hydrolytically stable versions of trehalose are also being pursued due to their potential for use as non-caloric sweeteners and bioprotective agents. Despite the appeal of this class of compounds for various applications, their potential remains unfulfilled due to the lack of a robust route for their production. Here, we report a detailed protocol for the rapid and efficient one-step biocatalytic synthesis of trehalose analogues that bypasses the problems associated with chemical synthesis. By utilizing the thermostable trehalose synthase (TreT) enzyme from Thermoproteus tenax, trehalose analogues can be generated in a single step from glucose analogues and uridine diphosphate glucose in high yield (up to quantitative conversion) in 15-60 min. A simple and rapid non-chromatographic purification protocol, which consists of spin dialysis and ion exchange, can deliver many trehalose analogues of known concentration in aqueous solution in as little as 45 min. In cases where unreacted glucose analogue still remains, chromatographic purification of the trehalose analogue product can be performed. Overall, this method provides a &#34;green&#34; biocatalytic platform for the expedited synthesis and purification of trehalose analogues that is efficient and accessible to non-chemists. To exemplify the applicability of this method, we describe a protocol for the synthesis, all-aqueous purification, and administration of a trehalose-based click chemistry probe to mycobacteria, all of which took less than 1 hour and enabled fluorescence detection of mycobacteria. In the future, we envision that, among other applications, this protocol may be applied to the rapid synthesis of trehalose-based probes for tuberculosis diagnostics. For instance, short-lived radionuclide-modified trehalose analogues (e.g., 18F-modified trehalose) could be used for advanced clinical imaging modalities such as positron emission tomography-computed tomography (PET-CT).

Trehalose analogues are emerging as important molecules for bio(techno)logical and biomedical applications. We describe an optimized protocol for enzymatically synthesizing and purifying trehalose analogues that is simple, efficient, fast, and environmentally friendly. Its application to the rapid production and administration of a probe for the detection of mycobacteria is demonstrated.

迅速なワンステップ酵素合成とトレハロース類似体のすべての水精製

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other ...

生化学

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

HCN1とTRIP8bの間のタンパク質 - タンパク質相互作用の小分子阻害剤を同定するための方法

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

足場リポソームを使用することにより、脂質近位タンパク質 - タンパク質相互作用を再構成します<em&gt;インビトロ</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

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 ...

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

酵素的に活性G4 Resolvase1の精製のためのG-四重鎖DNA親和性のアプローチ

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

精製と TRPV1 の分光学的解析のための再構成

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

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 ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

ビオチンと写真分解リンカーに接続されている RNA を用いた高分子複合体のシングル ステップ浄化

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

生体内細胞からのRNA単離に対するリボソーム親和性精製(TRAP)の翻訳

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

転写におけるタンパク質とタンパク質の相互作用を研究するための生体層干渉計(BLI)の応用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

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

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