この議定書と彼の貴重な指導の基礎を築いてくれたウェスリー・ワン博士に感謝します。この研究は、国立衛生研究所(グランツR01GM127575およびR01GM121584)およびウェルチ財団(グラントA-1715)によって部分的に支援されました。

1. プラスミド建設
<ol><li>まず、ヒストンタンパク質がアセチル化され、どのリジン部位でアセチル化されるか決定します。サイト指向変異誘発を用いて、サイトをアンバーストップコドン(TAG)に変異させる。 注:ヒストンタンパク質の発現に利用される4つの以前に設計されたプラスミドがあります。4つのヒストンタンパク質はすべて、N末端ヒスチジンタグを用いてpETDuet-1ベクターにクローン化された。ヒストンH4はまた、SUMOタグ、約40のコピー数を有する複製 colE1 の起源、アンピシリン耐性、およびT7プロモーターを含む。<ol>
<li>4つのヒストンタンパク質プラスミドの1つで、目的の部位にTAG突然変異を含む前方および逆プライマー(既存のリジンコドンをアンバーストップコドンに置き換える)を設計します。</li>
		<li>プラスミドを含むタグを増幅するために、全プラスミドPCRを使用してください。プライマーに適したアニーリング温度を決定します。一般に、プライマーから5°Cを引いた融解温度(Tm)で十分です。PCR産物の入手が困難な場合、T m~5°C付近の温度勾配を使用して、アニーリング温度を最適化して増幅することができます。 注:この実験では、30 sの94°C(変性)、30秒のアニーリング温度、6分間(またはキロベースペアあたり1分)の72°Cの条件で、30サイクルに対して、社内で表現されたPFUポリメラーゼを使用しました。このタイプの PCR 方法の詳細については、劉とネイスミス7を参照してください。</li>
		<li>メーカーのプロトコルに従って PCR クリーンアップ キットを使用して PCR 製品をクリーンアップします。アガロースゲル電気泳動とそれに続く臭化エチジウム染色によりPCR産物を分析します。</li>
		<li>プラスミドを16°Cで一晩T4リガーゼでインキュベートしてリゲートする。</li>
	</ol>
</li>
	<li>まず、プラスミド(AmpR)を含むTAGを化学的に有能な 大腸菌に変換します。変換から最低4コロニーを選び、プラスミドを浄化します。標準的な方法を使用してグリセロールでそれぞれの細胞ストックを作り、-80°Cで保存します。 必要な TAG 変異を確認するために、シーケンス処理のために送信します。</li>
	<li>配列が確認されたら、TAG含有プラスミド(AmpR)をpEVOL-AckRS(ChlR)と共に化学的に有能なCobB( 大腸菌で知られている唯一のヒストンリシンデアセチラーゼ)をヒートショック法を用いてBL21細胞を欠失させる。pEVOL は、コピー番号が低いコピー番号 p15A 原点に中間コピー番号を持つプラスミドです。 注:プラスミドのpEVOLシリーズは、直交aaaRS/tRNATyr ペアの発現増加を示す以前の研究に基づいて構築され、タンパク質の切り捨てを減少させ、変異タンパク質の全体的な収量を増加させる効果があった8。CobB欠失細胞がアクセスできない場合は、化学的に有能なBL21細胞を使用して進みます。CobBは、サーチュイン様ヒストンリシンデアセチラーゼおよびニコチンアミドをヒストンタンパク質発現中に5mMの最終濃度で添加して、代替アプローチ9としてCobBを阻害することができる。</li>
	<li>セルストックを作り、-80°Cで保管してください。</li>
</ol>2. アセチル化ヒストンタンパク質発現
<ol><li>1 Lのオートクレーブ2YT培地(または選択量)を培養フラスコに調製する。</li>
	<li>2YT培地の20mLを、TAG含有プラスミド(AmpR)およびpEVOL-AckRS(ChlR)を有する共形細胞ストックと共形化した細胞ストックを含む20mLを接種し、37°CでOD=0.6(4-6h)に成長する。</li>
	<li>1 Lのオートクレーブ2YT培地に適切な抗生物質を加え、20 mLスターター培養液で接種します。37°CでOD = 0.6-0.8(2-3 h)に成長します。</li>
	<li>誘導剤およびアセチルリシン(AcK)をIPTG 1 mM、0.2%アラビノース、およびAcK 5 mMの最終濃度に加えます。</li>
	<li>37°Cで6〜8時間培養します。</li>
	<li>ペレット細胞は2,700 x g で15分間用いた。</li>
	<li>細胞ペレットを-80 °Cで一晩保管してください。</li>
	<li>このステップ以降は、常に氷の上にサンプルを保ちます。培養液1L当たりヒストン溶解バッファーの50mLに細胞ペレットを溶解する。</li>
	<li>次のサイクルに従ってソニッケート:1 sオン、1 sオフ、時間通りに合計:60%振幅で3分。</li>
	<li>41,600 x g で45分間ペレット包包体。</li>
	<li>上清を捨て、ヒストンリシスバッファーの30 mLで封入体を再中断します。ペレットインクルージョンは41,600 x g で30分間です。</li>
	<li>上清を捨て、30 mLのペレット洗浄バッファーに封入体を再中断します。ペレット包包体は41,600 x g で30分間です。</li>
	<li>上清を捨て、含包体を6Mグアニジン塩酸塩(GuHCl)緩衝液の25 mLに溶解する。37°Cで1時間撹拌してインキュベートする。 注:必要に応じて、封入体は4°Cで一晩攪拌してインキュベーションすることができます。</li>
	<li>ペレットインクルージョンは41,600 x g で45分間です。上清を1 mLのNi-NTA樹脂で2時間6M GuHClバッファーと平衡化してインキュベートします。</li>
	<li>変性条件下でのNi-NTAの精製を進めます。カラム洗浄バッファーの3桁のボリュームでカラムを洗浄します。溶出緩衝液の10 mLでヒストンタンパク質をアセチル化した。 注:ここでタンパク質を濃縮しようとすることはオプションですが、アセチル化ヒストンは非常に予測不可能であり、容易に沈殿することができます。2mLを超えて総量に集中することはお勧めできません。</li>
	<li>アセチル化ヒストンタンパク質を5%の酢酸緩衝液に対して広範囲に透析し、塩を除去する。4°Cで一度に3時間の透析液を、最低6回の新鮮な緩衝液と交換する。 注:タンパク質の純度を向上させるために、純水に対して透析するオプションがあります。しかし、これは、低収量のアセチル化ヒストンタンパク質にとって望ましくない可能性がある有意な沈殿および収量の低下を引き起こす。</li>
	<li>アリコートと凍結乾燥タンパク質を、-80°Cで無期限に保存する。 野生型ヒストンタンパク質は一般に10〜50mg/Lを生成し、アセチル化ヒストンタンパク質は特定のリジン部位に応じて10mg/L未満の収量を示します。例えば、H3K79Acは5mg/Lの平均値を示す。</li>
</ol>3. 野生型ヒストンタンパク質発現
<ol><li>完全なヌクレオソームを組み立てるには、4つのヒストンタンパク質すべてを発現する。野生型ヒストンを表現して浄化する場合、プロトコルは以下を除いてアセチル化されたヒストンと同じです。<ol>
<li>共変換を実行しないでください。野生型ヒストン表現には pEVOL-AcKRS を使用しないでください。それに応じて抗生物質を調整します。</li>
		<li>細胞の発現の誘導時に、CobB欠失細胞株を使用したり、ニコチンアミド、AcK、またはアラビノースを添加したりしないでください。</li>
	</ol>
</li>
</ol>4. 601DNAの作成
注:以前に設計された最適化されたDNA配列は、ヒストンオクターで組み立てられ、高効率で単核組を生成します。この配列は、601 DNAまたはWidom配列と呼ばれる。この配列は、クロマチンリモデリングアッセイから単一分子測定10までのヌクレオソームのインビトロ研究のための標準的なDNA配列となっている。
<ol><li>ヌクレオソームアセンブリ用に601 DNAのかなりの量を調製するには、pGEM-3z/601をトップ10細胞に変換し、プラスミド増幅および抽出のために10 mLの培養を増殖させます。プラスミド抽出キットを使用し、メーカーの推奨に従ってください。</li>
	<li>この増幅プロトコルには、社内で表現されたPFUポリメラーゼ(より効率的)を使用してください。PCR反応を設定する:250 μL反応の場合、207.5 μL オートクレーブMQ H2O、25 μL 10x PFUバッファー、5 μLフォワードプライマー:ctggagaatcccgggcgccg、5 μL リバースプライマー:アサガタタタタトガカック、5 μLNTPミックス、2.5μL PFU酵素を使用します。私たちは通常、組み立てるのに十分なDNAを得るために一度に60の反応を実行します。</li>
	<li>PFUポリメラーゼを使用する場合は、30 sで52°Cのアニーリング温度と延長時間30sを使用してください。それ以外の場合は、メーカーの推奨条件に従ってください。</li>
	<li>PCR が完了したら、PCR 製品を精製するために、選択した PCR クリーンアップ キットを使用します。</li>
</ol>5. ヒストン・オクターマーの組み立て
<ol><li>ヒストンタンパク質ペレットH2A、H2B、H3、およびH4のアリコートを溶解し、総体積100μLのGuHClバッファ内にヒストンタンパク質のそれぞれ別々のストックが存在するようにします。</li>
	<li>A280吸光度を測定して、ヒストンタンパク質の濃度を計算します。</li>
	<li>吸光度がタンパク質に対して1より大きい場合は、より正確な濃度を得るためにGuHClバッファーで希釈する。</li>
	<li>H2AとH2Bのタンパク質を1:1モル比で組み合わせ、合計4μg/μLのタンパク質濃度に希釈します。</li>
	<li>2 M TE バッファーに対して 4 °C で連続して 2 M TE バッファー 2 時間、0.5 M TE バッファー 5 時間。 注:透析の第2および第3のステップは、タンパク質の不安定な立体構造を引き起こし、沈殿します。これらのステップでは大きな降水量が予想されます。</li>
	<li>4°Cで16,800 x g で遠心分離により沈殿物を除去する。</li>
	<li>再度、A280 吸光度を測定するヒストンダイマー(H2A/H2B混合物)と四量体(H3/H4混合物)の濃度を計算する。</li>
	<li>1:1モル比でダイマーと四量体を混合し、固体NaClを加えてNaClを2Mに調整します。</li>
	<li>ヒストン・オクターマーは4°Cで保存でき、ダイマーやテトラマーよりも安定しています。これは分解を引き起こす可能性がありますので、ヒストンオクタマーを凍結しないでください。</li>
	<li>アセチル化ヒストンで組み立てる場合は、野生型タンパク質を手順中のアセチル化タンパク質に置き換えるだけです。</li>
</ol>6. 核組立体
<ol><li>100x TE バッファーと固体 NaCl を使用して、601 DNA を 2M TE バッファーに調整します。2M TE バッファーの 601 DNA をヒストンオクタマーに 0.85:1 ~ 0.90:1 のモル比で加えます。遊離DNAがゲルシフト解析に存在する場合、DNAの比率が低くなる可能性があります。</li>
	<li>DNA-ヒストン混合物を透析袋に移し、2M TEバッファーの約200 mL(または複数のサンプルを組み立てる場合は複数)に入れ、4°Cで非常に穏やかにかき混ぜます。</li>
	<li>塩TEバッファーをゆっくりと滴下するためにパージする蠕動ポンプを設定します。ボリュームがおよそ2倍になったら、半分のボリュームを注ぎます。これを合計で少なくとも4回実行します。ポンプを使用すると、開始体積に応じて4-8時間かかることがあります。ヌクレオソームは、塩濃度が150mM(塩分濃度計で測定)に減少すると形成される。</li>
	<li>塩濃度が150mMに低下した後、透析は一晩で20mMのTEバッファーに対して。</li>
	<li>遠心分離により沈殿物を除去し、プレートリーダーを用いてA260 読み取りによるヌクレオソームの濃度を測定する。</li>
	<li>His-TEVプロテアーゼ(TEV:ヌクレオソーム1:30、w:w)を加え、4°Cで一晩インキュベートしてヒスチジンタグをすべて除去します。ニNTA樹脂プルダウンにより、核組液からヒスチジンタグ不純物を除去します。</li>
	<li>ヌクレオソームを位置決めし、試料を均質化し、60°Cで1時間インキュベートする。 注: 特に不安定なアセチル化されたサイトでは、この手順は推奨されない場合があります。</li>
	<li>4 °Cで、短期間(数週間)のヌクレオソームを保管する。</li>
	<li>長期保存のために、透析用ヌクレオソームは貯蔵バッファーに対して、-80°Cで保存します。</li>
</ol>

<ol>
	<li>Srinivasan, G., James, C. M., Krzycki, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrrolysine+encoded+by+UAG+in+Archaea:+Charging+of+a+UAG-Decoding+specialized+tRNA.">Pyrrolysine encoded by UAG in Archaea: Charging of a UAG-Decoding specialized tRNA.</a> Science. 296 (5572), 1459-1462 (2002).</li><li>Wan, W., Tharp, J. M., Liu, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrrolysyl-tRNA+synthetase:+an+ordinary+enzyme+but+an+outstanding+genetic+code+expansion+tool.">Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool.</a> Biochimica Et Biophysica Acta. 1844 (6), 1059-1070 (2014).</li><li>Umehara, T., 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=N-Acetyl+lysyl-tRNA+synthetases+evolved+by+a+CcdB-based+selection+possess+N-acetyl+lysine+specificity+in+vitro+and+in+vivo.">N-Acetyl lysyl-tRNA synthetases evolved by a CcdB-based selection possess N-acetyl lysine specificity in vitro and in vivo.</a> FEBS Letters. 586 (6), 729-733 (2012).</li><li>Hsu, W. W., Wu, B., Liu, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sirtuins+1+and+2+are+universal+histone+deacetylases.">Sirtuins 1 and 2 are universal histone deacetylases.</a> ACS Chemical Biology. 11 (3), 792-799 (2016).</li><li>Wang, W. W., Zeng, Y., Wu, B., Deiters, A., Liu, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+biology+approach+to+reveal+Sirt6-targeted+histone+H3+sites+in+nucleosomes.">A chemical biology approach to reveal Sirt6-targeted histone H3 sites in nucleosomes.</a> ACS Chemical Biology. 11 (7), 1973-1981 (2016).</li><li>Wang, W. W., 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+click+chemistry+approach+reveals+the+chromatin-dependent+histone+H3K36+deacylase+nature+of+SIRT7.">A click chemistry approach reveals the chromatin-dependent histone H3K36 deacylase nature of SIRT7.</a> Journal of the American Chemical Society. 141 (6), 2462-2473 (2019).</li><li>Liu, H., Naismith, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+one-step+site-directed+deletion,+insertion,+single+and+multiple-site+plasmid+mutagenesis+protocol.">An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.</a> BMC Biotechnology. 8 (1), 91 (2008).</li><li>Young, T. S., Ahmad, I., Yin, J. A., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enhanced+system+for+unnatural+amino+acid+mutagenesis+in+E.+coli.">An enhanced system for unnatural amino acid mutagenesis in E. coli.</a> Journal of Molecular Biology. 395 (2), 361-374 (2010).</li><li>Gallego-Jara, J., &#201;cija Conesa, A., de Diego Puente, T., Lozano Terol, G., C&#225;novas D&#237;az, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+CobB+kinetics+and+inhibition+by+nicotinamide.">Characterization of CobB kinetics and inhibition by nicotinamide.</a> PLoS One. 12 (12), 0189689 (2017).</li><li>Lowary, P. T., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+DNA+sequence+rules+for+high+affinity+binding+to+histone+octamer+and+sequence-directed+nucleosome+positioning.">New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.</a> Journal of Molecular Biology. 276 (1), 19-42 (1998).</li><li>Zhao, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+tight+nuclear+tethering+and+inactivation+of+cGAS.">The molecular basis of tight nuclear tethering and inactivation of cGAS.</a> Nature. 587 (7835), 673-677 (2020).</li><li>Burdette, D. L., Vance, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STING+and+the+innate+immune+response+to+nucleic+acids+in+the+cytosol.">STING and the innate immune response to nucleic acids in the cytosol.</a> Nature Immunology. 14 (1), 19-26 (2013).</li><li>Barber, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+DNA+sensing+pathways:+STING,+AIMII+and+the+regulation+of+interferon+production+and+inflammatory+responses.">Innate immune DNA sensing pathways: STING, AIMII and the regulation of interferon production and inflammatory responses.</a> Current Opinion in Immunology. 23 (1), 10-20 (2011).</li><li>Ablasser, 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=cGAS+produces+a+2'-5'-linked+cyclic+dinucleotide+second+messenger+that+activates+STING.">cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING.</a> Nature. 498 (7454), 380-384 (2013).</li><li>Gao, P., 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=Cyclic+[G(2',5')pA(3',5')p]+is+the+metazoan+second+messenger+produced+by+DNA-activated+cyclic+GMP-AMP+synthase.">Cyclic [G(2',5')pA(3',5')p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase.</a> Cell. 153 (5), 1094-1107 (2013).</li><li>Zhao, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+PLPLRT/SD+motif+of+STING+mediates+the+recruitment+and+activation+of+TBK1.">A conserved PLPLRT/SD motif of STING mediates the recruitment and activation of TBK1.</a> Nature. 569 (7758), 718-722 (2019).</li></ol>

著者の誰かによって宣言する競合する財政的利益はありません。

実験中にこのプロトコルを詳細に従うことは不可欠です。ヌクレオソームは非常に安定しておらず、多くの試行錯誤がこのプロトコルを決定することに入っています。粒子状物は容易に組み立てプロセスを妨げる可能性があるので、すべてのステップ(または観察されるたびに)で沈殿物を除去することが重要です。常にヒストンサンプルを氷の上に置いてください。ヌクレオソームが4°Cで長?...

我々は、PylRSおよびtRNAPylを利用して、部位特異的アセチル化ヒストンを有する再構成単核体を合成および組み立てる。PylRSは、翻訳後修飾(PTM)を用いてタンパク質を産生する遺伝コード拡張ツールとして非常に価値があることが証明されており、約200の異なるncAAを組み込むために遺伝的に進化してきました。PylRSは、アンバーストップコドンで組み込み、翻訳中に他のアミノ酸との競合を除去します。PylRSは、メタノジェニック古風で初めて発見され、その後、新しい反応性化学基をタンパク質1,2に組み込むために化学生物学に利用されている。
MmAcKRS1はメタノサルシナ・メイジ・ピルスから進化し、アセチル化タンパク質3、4、5、6の合成のために私たちの研究室で頻繁に使用されました。MmAcKRS1は、大腸菌での翻訳中に選択したmRNAの中で、黄色の突然変異部位でAcKを提供する。この技術は、以前にK4、K9、K14、K18、K23、K27、K36、K56、K64、およびK79ヒストンH3リシン部位にAcKを組み込み、アセチル化単核体4、5、6にサーチュイン1、2、6、および7の活性を研究するために使用されてきた。ここでは、アセチル化ヒストンを発現し、アセチル化ヌクレオソームを再構成するこの方法を詳述する。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>0.5 M NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8</td>
		</tr>
		<tr>
			<td>1 M TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>1 M NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8</td>
		</tr>
		<tr>
			<td>100x TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>2 M TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>2 M NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8</td>
		</tr>
		<tr>
			<td>20 mM TE Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>20 mM NaCl, 20 mM Tris, 1 mM EDTA, pH 7.8</td>
		</tr>
		<tr>
			<td>6 M GuHCl</td>
			<td></td>
			<td></td>
			<td>6M guanidinium chloride, 20 mM Tris, 500 mM NaCl, pH 8.0</td>
		</tr>
		<tr>
			<td>Acetyllysine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Column Wash Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>6 M urea, 500 mM NaCl, 20 mM Tris, 20 mM imidazole pH 7.8</td>
		</tr>
		<tr>
			<td>Elution Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Variable-Flow Chemical Transfer Pump</td>
			<td>Fischer Scientific</td>
			<td>15-077-67</td>
			<td></td>
		</tr>
		<tr>
			<td>His-TEV protease</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Histone Lysis Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>60 mM Tris, 100 mM NaCl, 0.5% Triton-X100 (v/v), 1 mM PMSF pH 8.0</td>
		</tr>
		<tr>
			<td>Ni-NTA Resin</td>
			<td></td>
			<td></td>
			<td>6 M urea, 500 mM NaCl, 20 mM Tris, 250 mM imidazole, pH 7.8</td>
		</tr>
		<tr>
			<td>PCR Clean-Up Kit</td>
			<td>Epoch Life Sicences</td>
			<td>2360050</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet Wash Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>60 mM Tris, 100 mM NaCl, pH 8.0</td>
		</tr>
		<tr>
			<td>petDUET-His-SUMO-TEV-H4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>petDUET-His-TEV-H2A</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>petDUET-His-TEV-H2B</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>petDUET-His-TEV-H3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pEVOL-AckRS</td>
			<td>Addgene</td>
			<td>137976</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEM-3z/601</td>
			<td>Addgene</td>
			<td>26656</td>
			<td></td>
		</tr>
		<tr>
			<td>Storage Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>20 mM NaCl, 20 mM Tris, 20 mM NaCl, 1 mM EDTA, 20% glycerol, pH 7.8</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

site specific lysine acetylation of histones for nucleosome reconstitution using genetic code expansion in escherichia coli

アセチル化ヒストンタンパク質は、変異体をコードする大腸菌、N ε-アセチルリジン(AcK)特異的メタノサルシナマジピロリシンtRNA合成酵素(MmAcKRS1)およびその同系tRNA(tRNAPyl)を組み立てるために容易に発現することができる。MmAcKRS1 および tRNAPylは、大腸菌での翻訳中に選択した mRNA の中で、黄色の変異部位で AcK を送達します。この技術は、H3リジンサイトでAcKを組み込むために広く使用されています.ピロリシル-tRNA合成酵素(PylRS)は、部位特異的なタンパク質修飾または機能化のために他の非カノニカルアミノ酸(ncAAs)を組み込むために容易に進化させることができる。ここでは、MmAcKRS1システムを用いたAcKをヒストンH3に組み込み、アセチル化H3タンパク質を再構成された単核球体に統合する方法を詳述する。アセチル化再構成単核体は、生化学的および結合アッセイ、構造決定、およびよりに使用することができる。新しい相互作用の発見やエピジェネティクスの理解に関連する実験を設計するには、改変単核体を得ることが重要です。

ダイマー、テトラマー、および八重差は、12% SDS PAGEゲルを実行することによって評価できます (図 1および図 2)。ここでは、アセチル化四量体の一部が他のテトラマーよりも低い収率を持っていることがわかります(図1)。実際、コア領域に近いほど、収率は低く見られます。これは、アセチル化が四量...

Watch this Scientific Journal Video about Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli at JoVE.com

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

ここでは、遺伝子コード拡張を用いてアセチル化ヒストンタンパク質を発現し、再構成されたヌクレオソームをインビトロで組み立てる方法を紹介する。

大腸菌の遺伝子コード拡張を用いた核組換えのためのヒストンの特異的なリジンアセチル化

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...

site-specific-lysine-acetylation-histones-for-nucleosome

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biochemistry

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Unit 1

Structure and Organization of DNA

SCIENTISTS IN ACTION

3.9K ビュー.  Texas A&M University. ここでは、遺伝子コード拡張を用いてアセチル化ヒストンタンパク質を発現し、再構成されたヌクレオソームをインビトロで組み立てる方法を紹介する。

ビデオ: Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

からの外膜小胞内の指向性タンパク質包装<em&gt;エシェリヒア・コリ</em&gt;：デザイン、生産および精製

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

生化学

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

のpH依存性活性の検出<em&gt;エシェリヒア・コリ</em&gt;シャペロンHdeB<em&gt;インビトロ</em&gt;と<em&gt;インビボ</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

ひずみ促進アジド - アルキン環付加することにより、効率的で部位特異的抗体ラベリング

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

示差同位体で標識された姉妹ヒストンとヌクレオソームの再構成

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

豊かさと充電転送エシェリヒア属大腸菌の Rna のレベルの定量化

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

エシェリヒア属大腸菌のウイロイド派生システムを使用して円形の足場に組換え Rna の大量生産

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

サイト指示された突然変異誘発のため生体外でそしてエシェリヒア属大腸菌の RNA 相互作用に代表される生体実験

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

ヌクレオソームヒストンのインビトロユビキチン化とデュービキチン化アッセイ

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein aggregation. The accumulation of protein aggregates in bacterial cells can lead to significant alterations in the cellular phenotypic behavior, including a reduction in growth rates, stress resistance, and virulence. Several experimental procedures exist for the examination of these stressor-mediated phenotypes. This paper describes an optimized assay for the extraction and visualization of aggregated and soluble proteins from different Escherichia coli strains after treatment with a silver-ruthenium-containing antimicrobial. This compound is known to generate reactive oxygen species and causes widespread protein aggregation. 
The method combines a centrifugation-based separation of protein aggregates and soluble proteins from treated and untreated cells with subsequent separation and visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. This approach is simple, fast, and allows a qualitative comparison of protein aggregate formation in different E. coli strains. The methodology has a wide range of applications, including the possibility to investigate the impact of other proteotoxic antimicrobials on in vivo protein aggregation in a wide range of bacteria. Moreover, the protocol can be used to identify genes that contribute to increased resistance to proteotoxic substances. Gel bands can be used for the subsequent identification of proteins that are particularly prone to aggregation.

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

This protocol describes the extraction and visualization of aggregated and soluble proteins from Escherichia coli after treatment with a proteotoxic antimicrobial. Following this procedure allows a qualitative comparison of protein aggregate formation in vivo in different bacterial strains and/or between treatments.

タンパク質毒性ストストレスマによる 大腸菌 治療後のタンパク質凝集体の抽出と可視化

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

細菌細胞での共発現と組み合わせたプルダウンアッセイは、困難なタンパク質間相互作用をテストするための時間効率の良いツールとして

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...