我々はこのプロジェクトで彼らの助けと入力用のアシュトンブリュー、カレンジャック、Sharmishtha Musalga、セルゲイSulima、シヴァーニレディ、そしてマイケルRhodin含むDinmanの研究室のメンバーのすべてを、感謝します。この作品は、米国心臓協会（AHA 0630163N）からAMにと国立衛生研究所（5R01 GM058859 - 12）からしてくれたための補助金によって支えられている。 JALは、親の助成金から2009サプリメントのアメリカの再投資および回復法（3R01GM058859 - 11S1）によってサポートされていました。

1。 Sulfolink樹脂の帯電システインの準備<ol><li>室温でSulfolink樹脂（Pierce）を準備します。 </li><li>各バイアルに分散5mlで、として2 10ミリリットルのプラスチックバイアルの製造元によって提供されるSulfolinkカップリングゲルの50％のスラリーを10mlの合計を置きます。 </li><li>簡単に言えば850 × gでバイアルを遠心し、注意深く上清を取り除く。 </li><li> 、5mlにビーズを再懸濁XGの850℃を軽く遠心し、ピペットで上清を除去することにより（50 mMトリス、pH 8.5、5mMのEDTA）カップリングバッファーでゲル3回洗浄する。 </li><li>各チューブにカップリング緩衝液中のL -システインの50 mM溶液五mlを加え、25℃で1時間スラリーを混合℃に</li><li>上記のステップ1.4で説明したように洗浄することにより残存L -システインを削除します。 </li><li>結合緩衝液10mlでゲル[20mMのHEPES、pHは7.6、5 mMのマグネシウム（OAC）2、60mMのNH 4 Clを 、2mMのDTT]を再懸濁し、そして結合緩衝液10mlで洗浄することによって樹脂を平衡化3回は、ステップ1.4で説明したように。デカント10 mlの遠心カラムへの樹脂、キャップと4℃で保存RNA結合用樹脂の能力は〜20ミリリットル当たり260単位です。 </li></ol> 2。 Sulfolink樹脂の帯電システインを使用して、リボソームのクロマトグラフィー精製注：プロテアーゼ活性が問題であると証明する菌株を扱う場合、プロテアーゼ阻害カクテルは、後続のすべてのバッファで使用することができます。 <ol><li>氷の上ですべてのコンポーネントを維持し、RNaseフリー水、無菌ろ過を使用して、すべてのソリューションを準備する。 </li><li>適切なメディアで-中期対数増殖期（1.2 OD 595 = 0.6）に一夜酵母細胞を成長する。ペレットを遠心分離により細胞、および4℃で5分間3700 × gで結合バッファーと遠心で1グラムの細胞を洗浄℃に</li><li>結合緩衝液1mlで2 mlのおおよその合計量に対して、および4に冷やして0.5 mmのガラスビーズの等量を使用して混乱させる再懸濁し、細胞° C UBiospecミニビーズビーター（バートル、[OK]を）歌う。必要に応じてサンプルは複数のチューブに分割することができます。 </li><li> Beckman - Coulter社オプティママックスEの超遠心機（フラートン、カリフォルニア州）で30分間、30,000 xgで遠心分離によって破砕されなかった細胞、オルガネラ、および細胞の残骸を削除します。 </li><li>使用直前に、ペレットは1,000で1分間樹脂は、ストレージソリューションを削除するには、XGの。樹脂の二mlの細胞のすべての1グラムのために使用されます。 </li><li>直接充電Sulfolinkスラリー中に非常に上部または細胞残渣管の下部にある脂質画分のいずれかからの汚染、および場所を最小限に抑えるように注意しながら、30,000 × gでのスピン（S30）から細胞ライセートの上清を取り除く。脂質層を除去したり、先端が上清中に交差した後、ピペットから押し出すことで回避できます。 </li><li> 15分間混合することなく氷上にS30/Sulfolinkの混合物をインキュベートする。 </li><li> 15 mlコニカルチューブにカラムを配置し、1,000 × gで遠心分離R 1分。 </li><li>バック列を、手で混合し、氷上でさらに15分間インキュベートにフロースルー画分を置きます。 </li><li> 15 mlコニカルチューブにカラムを置き、1分間1,000 × gで遠心。ろ過液を廃棄します。 </li><li>キャップの列と結合バッファー5 mlを加える。再懸濁し、キャップを外して、そして1分間1,000 × gでのカラムの遠心機を遠心分離するまで、手で列を混在させること。この洗浄プロトコルをあと2回繰り返します。 </li><li>各カラムに溶出バッファーを1.5 ml（20 mMのHEPES - KOH、pHは7.6、10mMのマグネシウム（OAC）2、500mMのKClを、2mMのDTT、0.5 mg / mlのヘパリン）を追加します。手でスラリーを混合し、氷上で2分間インキュベート</li><li> 1分間1,000 × gで新しい15 mlコニカルチューブと遠心分離機に入れます列。溶出液を集める。もう一度最終的な混合サンプルのボリュームが〜3ミリリットルであることを溶出ステップを繰り返します。使用される樹脂は、ヘパリンなしで溶出緩衝液で洗浄し、結合buffeで平衡化を続けることができますR、および後で再利用するため4で結合バッファー10 mlのボリューム° Cに保存。 </li></ol> 3。ピューロマイシンの治療注：代替方法汚染のtRNA種を除去するためには、メディアがリボソーム流出を促進するために枯渇グルコースへの豊富なグルコースから切り替えることです。しかし、これはリボソームの機能と濃度に影響を与える可能性の酵母細胞の代謝状態を変更しません。 内因性ペプチジル- tRNAのからリボソームを除去するために、ピューロマイシンによる治療が行われます。 <ol><li>室温で1M KOH滴下してpH7.5の100mMのピューロマイシンのソリューションに中和する。 </li><li>溶出液中の〜1mMの最終濃度になるように中和ピューロマイシン溶液を加える。 </li><li> 1mMの最終濃度を100 mMのGTPを追加。 </li><li> 30℃で30分間インキュベート℃、 </li></ol> 4。グリセロールのクッションを介して沈降によるリボソームの精製<ol><li>場所1ミリリットルクッションの緩衝液（20mM HEPES、pHは7.6、10mMのマグネシウム（OAC）2、500mMの塩化カリウム、2mMのDTT、25％グリセロール）の4 mlの容積ポリカーボネート超遠心チューブに。 </li><li>一晩4 XGの100,000クッションの上に溶出分画、及び遠心分離サンプルを扱う優しく層ピューロ℃に</li><li>遠心ローターからチューブを外し、上清を吸引除去する。 </li><li>クッション緩衝液1mlで2回精製リボソームを含むペレットを洗う。 </li><li>ガラス棒を使用して穏やかな破壊によってクッションバッファー100μlでリボソームを再懸濁します。 </li><li>中断した後、パラフィルムでチューブをカバーし、一時間低温室の渦の中程度のスピードで振る。 </li><li> 、マイクロ遠心チューブに内容を移し、4℃での最大速度で5分間遠心し、そして新鮮なチューブに上清を取り除く。 </li><li> 2クッションの緩衝mlのと、保存緩衝液（50mMのHEPES - KOH pHは7.6、50mMのNH 4 Clを、5mMのMgのその100μlを除いてを使用して、ステップ4.1から4.7を繰り返します。（OAC）2、1mMのDTT、25％グリセロール）が代わりにクッションのバッファのステップ4.4および4.5で使用されています。 </li><li>定量精製リボソーム分光光度法（1 A 260 = 20酵母のリボソームのピコモル）、-80℃で保存します。酵母の1グラムの標準的な結果は、リボソームの300〜400ピコモルです。 </li></ol> 5。代表的な結果： プロトコルの3つの主要ステップのそれぞれから抽出したRNA種の例を図2に示されています。 rRNAsは、これらはまた、他のRNA種の多数を含む全細胞ライセート（T）内に存在する主要な種である一方。 Sulfolink列（SL）から精製されたリボソームはまた、同様にこれらの種のカラムベッドの高い親和性によるtRNAの大量が含まれています。ペプチジル- tRNAのからのペプチドの加水分解におけるピューロマイシンの結果、この画分の治療、およびリボソームからこれらの種の解離を促進する。ピューロマイシン処理した試料（PM）の欠如共同purifyinこのようにグラムのtRNA種、および完全に純粋なリボソームを表す。以前に8を報告したように、これらのリボソームは、それらの詳細な機能と構造解析のための理想的な基板製造、無傷および生化学的にアクティブになります。 <img src="/files/ftp_upload/3214/3214fig1.jpg" alt="図1"/> 図1。方法のフローチャート。sulfolinkの樹脂が描かれているリンクされたシステインを使用して、リボソームのクロマトグラフィー精製。 <img src="/files/ftp_upload/3214/3214fig2.jpg" alt="図2"/> 図2。リボソームの調製の代表的な分析全 RNA種が続いてピューロマイシンによる治療を受けた全細胞ライセート（T）、Sulfolink精製リボソーム（SL）とSulfolink精製がリボソームから抽出され、グリセロールのクッション（PM）を介して沈降させた。レーンMはRNAサイズマーカーを表します。 25Sと18S rRNAs、同様のtRNAおよび他のRNA種を表すバンドが示されている。すべてレーンは、RNAを2μgを充填した。

<ol>
	<li>Palade, G. E., Siekevitz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+microsomes;+an+integrated+morphological+and+biochemical+study.">Liver microsomes; an integrated morphological and biochemical study.</a> J. Biophys. Biochem. Cytol. 2, 171-200 (1956).</li><li>Fabry, M., Kalvoda, L., Rychlik, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interactions+of+Escherichia+coli+ribosomes.">Hydrophobic interactions of Escherichia coli ribosomes.</a> Biochim. Biophys. Acta. 652, 139-150 (1981).</li><li>Jelenc, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+of+highly+active+ribosomes+from+Escherichia+coli.">Rapid purification of highly active ribosomes from Escherichia coli.</a> Anal. Biochem. 105, 369-374 (1980).</li><li>Le goffic, F., Moreau, N., Chevelot, L., Langrene, S., Siegrist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+bacterial+ribosomes+using+chloramphenicol+and+erythromycin+columns.">Purification of bacterial ribosomes using chloramphenicol and erythromycin columns.</a> Biochimie. 62, 69-77 (1980).</li><li>Meskauskas, A., Petrov, A. N., Dinman, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+functionally+important+amino+acids+of+ribosomal+protein+L3+by+saturation+mutagenesis.">Identification of functionally important amino acids of ribosomal protein L3 by saturation mutagenesis.</a> Mol. Cell Biol. 25, 10863-10874 (2005).</li><li>Algire, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+reconstituted+yeast+translation+initiation+system.">Development and characterization of a reconstituted yeast translation initiation system.</a> RNA. 8, 382-397 (2002).</li><li>Maguire, B. A., Wondrack, L. M., Contillo, L. G., Xu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chromatography+system+to+isolate+active+ribosomes+from+pathogenic+bacteria.">A novel chromatography system to isolate active ribosomes from pathogenic bacteria.</a> RNA. 14, 188-195 (2008).</li><li>Leshin, J. A., Rakauskaite, R., Dinman, J. D., Meskauskas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+purity,+activity+and+structural+integrity+of+yeast+ribosomes+purified+using+a+general+chromatographic+method.">Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method.</a> RNA. Biol. 7, 354-360 (2010).</li><li>Triana, F., Nierhaus, K. H., Chakraburtty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+RNA+binding+to+80S+ribosomes+from+yeast:+evidence+for+three+sites.">Transfer RNA binding to 80S ribosomes from yeast: evidence for three sites.</a> Biochem. Mol. Biol. Int. 33, 909-915 (1994).</li><li>Azzam, M. E., Algranati, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+puromycin+action:+fate+of+ribosomes+after+release+of+nascent+protein+chains+from+polysomes.">Mechanism of puromycin action: fate of ribosomes after release of nascent protein chains from polysomes.</a> Proc. Natl. Acad. Sci. U. S. 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A. 45, 1721-1729 (1959).</li><li>Becker-Ursic, D., Davies, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+phosphorylation+of+ribosomal+proteins+by+protein+kinases+from+Saccharomyces+cerevisiae.">In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.</a> Biochemistry. 15, 2289-2296 (1976).</li></ol>

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リボソームの精製プロトコルは基本的に低速のスピンからサ ​​イトゾル画分を収穫し、溶解細胞が関与し、高速遠心分離1によってリボソームをペレット化。いくつかの新規な方法は細菌のリボソームを精製するのに使用されているが、同じことが真核生物2〜4真されていませんでした。追加の手順が長年、例えば塩の洗浄、およびグリセロールのクッション（例えば5?...

<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> Sulfolinkカップリング樹脂</td><td>ピアース</td><td> 20402 </td></tr><tr><td>ミニビーズビーター16 </td><td> Biospec </td><td> 607 </td></tr><tr><td>オプティママックスE超遠心機</td><td>ベックマンコールター</td><td></td></tr><tr><td>レジェンドRT </td><td>ソーバル</td><td></td></tr><tr><td> 0.5 mmのガラスビーズ</td><td> Biospec </td><td> 11079105 </td></tr><tr><td>トリス</td><td>シグマアルドリッチ</td><td> 252859 </td></tr><tr><td> EDTA </td><td>シグマアルドリッチ</td><td> E9884 </td></tr><tr><td> DTT </td><td>シグマアルドリッチ</td><td> 43815 </td></tr><tr><td> HEPES </td><td>シグマアルドリッチ</td><td> 54459 </td></tr><tr><td> NH 4 Clを </td><td>シグマアルドリッチ</td><td> A9434 </td></tr><tr><td>塩化カリウム</td><td>シグマアルドリッチ</td><td> 60128 </td></tr><tr><td> MG（OAC）2 </td><td>シグマアルドリッチ</td><td> M5661 </td></tr><tr><td> KOH </td><td>シグマアルドリッチ</td><td> 221473 </td></tr><tr><td>グリセロール</td><td>シグマアルドリッチ</td><td> G5516 </td></tr><tr><td>ピューロマイシン</td><td>シグマアルドリッチ</td><td> P7255 </td></tr><tr><td> GTP </td><td> Fermentas </td><td> R0461 </td></tr></tbody></table>

chromatographic purification of highly active yeast ribosomes

真核生物のリボソームは、このように研究者に大きな課題を投げかけ、はるかに不安定な彼らの真正細菌とarchaelの対応と比較しています。特に厄介な細胞の溶解は、リボソームを低下させる可能性プロテアーゼおよびヌクレアーゼの多数のリリースという事実です。従って、それは可能な限り迅速にこれらの酵素からリボソームを分離することが重要です。残念ながら、従来の差動遠心分離法は、それらの構造的完全性と機能性に影響を与える、時間の許容できないほど長時間、これらの酵素にさらされるリボソームを残します。この問題に対処するため、我々はSulfolink樹脂充電システインを使用してクロマトグラフィー法を利用しています。このシンプルかつ迅速なアプリケーションが大幅にそのまま、非常に生化学的にアクティブ酵母のリボソームの高収量の生産、CO浄化のタンパク質分解と核酸分解活性を低減します。我々は、このメソッドはまた、哺乳動物リボソームに適用されるべきことを示唆している。のシンプルさクロマトグラフィーで精製したリボソームの方法、および強化された純度と活性は、真核生物のリボソームの研究のための重要な技術的進歩を表しています。

Watch this Scientific Journal Video about Chromatographic Purification of Highly Active Yeast Ribosomes at JoVE.com

Chromatographic Purification of Highly Active Yeast Ribosomes

CO浄化ヌクレアーゼ及びプロテアーゼによって従来の方法で精製した真核生物リボソームの調製品の汚染は否定的に下流の生化学的および構造解析に影響を与えます。迅速かつ簡単なクロマトグラフィー精製法は、モデル系として、酵母のリボソームを使用してこの問題を解決するために使用されます。

高活性酵母のリボソームのクロマトグラフィー精製

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

chromatographic-purification-of-highly-active-yeast-ribosomes

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16.7K ビュー.  University of Maryland. CO浄化ヌクレアーゼ及びプロテアーゼによって従来の方法で精製した真核生物リボソームの調製品の汚染は否定的に下流の生化学的および構造解析に影響を与えます。迅速かつ簡単なクロマトグラフィー精製法は、モデル系として、酵母のリボソームを使用してこの問題を解決するために使用されます。

ビデオ: Chromatographic Purification of Highly Active Yeast Ribosomes

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

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

酵母細胞からミトコンドリアの精製

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

ミトコンドリアは密結合のラットの心臓の準備

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

機能と構造解析のためにペプチジル- tRNAを含む翻訳リボソームの分離

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

酵母のコロニーの埋め込み法

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

アクティブT.の試験管内再構成でcastaneumのテロメラーゼ

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

ハイスループット酵母プラスミドの過剰発現の画面

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

バーコード酵母ライブラリーの競争力のあるゲノム画面

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

機能の研究、相互作用、および翻訳後修飾のため出芽酵母タンパク質の抽出と精製

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

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

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

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

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

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

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

Methods Article

高活性酵母のリボソームのクロマトグラフィー精製