Name: genetic incorporation of biosynthesized l-dihydroxyphenylalanine (dopa) and its application to protein conjugation
Uploaded: 2018-08-24T15:00:00.000+00:00
Duration: 10 min 24 s
Description: Watch this Scientific Journal Video about Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation at JoVE.com

この研究は、地球フロンティア研究プログラム (NRF 2015M3A6A8065833)、によって支えられた、基本的な科学研究プログラム (2018R1A6A1A03024940) 韓国国立研究権財団 (NRF) を通じて韓国政府によって資金を供給します。

1. プラスミド構築
<ol><li>表現シトロバクター属 freundiiから TPL 遺伝子構成のプロモーターの制御下で緑色蛍光タンパク質 (GFP) 遺伝子発現プラスミド (pBAD-デュアル-TPL-GFP-WT) を構築6-タグの制御の下で、araBAD プロモーター。PBAD-デュアル-TPL-GFP-E90TAG サイト指示された突然変異誘発のプロトコルを使用してオレンジ色のコドン (タグ)、DOPA のサイト (E90) のコドンを交換してください。このプラスミッドの構造の詳細は、当社の以前のレポート18で説明しました。</li>
	<li>ドーパの定款の遺伝して、tRNA/aaRS 表現のプラスミド (pEVOL-DHPRS2)18,の19を構築します。pEVOL は独立したプロモーターの aaRS 遺伝子の 2 つのコピーを表現する特別なプラスミッドのベクトルとプラスミドの詳細な情報は以前報告19に記載されています。</li>
	<li>市販のプラスミド調製キットを使用すると、高純度の保有するプラスミド Dna を取得できます。必要に応じて、agarose のゲルの電気泳動を使用して準備された Dna の純度を確認します。</li>
</ol>2. 文化準備
<ol><li>
エレクトロポレーション
	<ol>
<li>この実験に使用電気有能なエシェリヒア属大腸菌DH10β ひずみ。有能なセルの 20 μ L に 1 μ L のそれぞれのプラスミッド DNA (pEVOL DHPRS2 と pBAD デュアル TPL GFP E90TAG) を追加します。ピペットを使用して、均質の混合物をように優しく混ぜます。</li>
		<li>エレクトロポレーション キュベットに混合物を転送します。キュヴェットをエレクトロポレーション室。キュベットを押し込むようにしっかりキュベット商工会議所商工会議所にお問い合わせください。</li>
		<li>キュベット、25 μ F を使用して衝撃を与えると 2.5 kV 0.1 cm キュヴェットのため。</li>
		<li>2% (w/v) トリプトンを含む prewarmed 超最適スープ (SOC) の 1 mL を追加し、0.5% (w/v) 酵母エキス、10 mM の NaCl、2.5 mM KCl、MgCl2、10 mM と 20 mM グルコース、キュヴェットへ文化管にセルを転送します。セルを救出するには、揺れで 37 ° C で 1 時間インキュベートします。</li>
		<li>35 μ g/mL クロラムフェニ コールと 100 μ g/mL アンピシリンを含むホストゲノム スープ (LB) 寒天培地プレート上救出のセルの 10-100 μ L を広めます。16 h の 37 ° C でセルを孵化させなさい。</li>
	</ol>
</li>
	<li>
スターター文化準備
	<ol>
<li>抗生物質を含む LB 培地 5 ml 変形単一コロニーを接種します。揺れで 37 ° C で 12 h のコロニーを孵化させなさい。</li>
	</ol>
</li>
</ol>3. 発現・精製合成システムによって GFP E90DOPA
<ol><li>
生合成システムによる GFP E90DOPA の発現
	<ol>
<li>ペンシルバニア 5052 中20 (50 mM ナ2HPO4、50 ミリメートル KH2PO4、25 mM [NH4]24、2 mM MgSO40.1% [w/v] 微量金属、0.5% [w/v] グリセロール、100 mL にスターター文化 (2 mL) を転送します。0.05% [w/v] グルコースとアミノ酸 5% [w/v] [pH 7.25]) 続いて 35 μ g/mL クロラムフェニ コール、100 μ g/mL アンピシリン、100 mM ピルビン酸、カテコール 10 mM、25 mM アンモニア、および 300 μ M ジチオトレイトール (DTT) を含む振動 30 ° C で培養。</li>
		<li>0.2% (w/v) L-アラビノースの 2 mL を追加し、550 nm に達すると 0.8 の光学濃度 (OD)。揺れで 30 ° C で 12 h サンプルをインキュベートします。</li>
		<li>スピン ・ ダウン 5 分 11,000 x g で細胞、上澄みを廃棄し、-80 ° C で細胞ペレットを凍結</li>
	</ol>
</li>
	<li>
セル換散
	<ol>
<li>氷の上細胞ペレットを解凍し、50 mM NaH2PO4 (pH 8.0) を含む換散バッファーの 10 mL の細胞を再懸濁します 10 mM のイミダゾールと 300 mM の NaCl。</li>
		<li>25 の 10 分使用 80% 超音波振幅パルスで氷の上再懸濁細胞を超音波 s オフと 35 s。</li>
		<li>細胞ライセート 15 分浄化新鮮なチューブに上清転送のための 4 ° C で 18,000 × g でスピンダウンし、ペレットを破棄します。</li>
	</ol>
</li>
	<li>
Ni NTA アフィニ ティー ・ クロマトグラフィー
	<ol>
<li>上清に Ni 国税庁 (ニトリロ三酢酸) 樹脂 (樹脂懸濁液 100 mL 文化のための 300 μ L) を追加し、1 h の懸濁液を優しく揺することによって蛋白質を 4 ° C で Ni NTA ガレージにバインドします。</li>
		<li>ポリプロピレン列に懸濁液を転送させ、樹脂 3 50 mM NaH2PO4 (pH 8.0) を含む洗浄バッファーの 5 ml x 20 mM のイミダゾールと 300 mM の NaCl。</li>
		<li>3 タンパク質を溶出 300 μ L の溶出バッファーを含む 50 mM NaH2PO4 (pH 8.0) x 250 mM のイミダゾールと 300 mM の NaCl。</li>
		<li>280 で吸光度を測定することによりタンパク質の濃度を決定する nm。280 GFP E90DOPA の消散係数 (24,630 M-1cm-1) nm を求めた蛋白質消光係数電卓 (例えばhttps://web.expasy.org/protparam/) と独立して消光ドーパの coefficiennt (2630 M-1cm-1)。別の方法として、蛋白質の集中の決定のためブラッドフォード蛋白質の試金21を使用します。</li>
	</ol>
</li>
</ol>4. 精製 GFP E90DOPA の重合
<ol><li>H2O (6 mM) の過ヨウ素酸ナトリウムの 1 μ L を追加 (1.0 GFP E90DOPA に当量) GFP E90DOPA の 20 μ L の溶液に (300 μ M) 50 mM Na2HPO4 (pH 8.0) を含むリン酸バッファーで、300 mM の NaCl。</li>
	<li>48 h の 25 ° C で進行する重合反応を許可します。</li>
	<li>反応混合物を分析すると、ナトリウム dodecyl 硫酸塩-ポリアクリルアミドゲル電気泳動によって) (SDS-PAGE) 手順 6 で説明しました。</li>
</ol>5. SPOCQ でアルキン探針を用いた GFP E90DOPA の活用
<ol><li>Cy5.5 リンク azadibenzocyclooctyne (Cy5.5 ADIBO)22 H2O (4.0 mM) 1 μ L を追加 (10 GFP E90DOPA に当量) GFP E90DOPA の 20 μ L の溶液に (20 μ M) 50 mM Na2HPO4 (pH 8.0) を含むリン酸バッファーで、300 mM の NaCl。</li>
	<li>H2O の過ヨウ素酸ナトリウムの 1 μ L を追加 (400 μ M) (1.0 GFP E90DOPA に当量) 反応混合物に。</li>
	<li>SPOCQ 反応を 25 ° C、1 h で実行を許可します。光に敏感なプローブを使用する場合は、アルミ箔で反作用の容器をカバーしてください。</li>
</ol>6. ラベルの GFP の浄化
<ol><li>50 mM Na2HPO4 (pH 8.0) を含むリン酸バッファーを追加して SPOCQ 反応混合物を希薄化と 300 mM NaCl、最大 500 μ L。</li>
	<li>希釈した溶液を遠心フィルター スピン列に転送します。スピン列 4 ° C 15 分で 14,000 × gで遠心分離し、流れを破棄します。任意の余分な Cy5.5 ADIBO を除去するために 3 x この手順を繰り返します。</li>
	<li>1.5 mL 遠心チューブにスピン列から精製サンプルを転送します。</li>
	<li>また、同じバッファーに対して透析による浄化を行います。</li>
	<li>4 ° C で精製された GFP を格納します。</li>
</ol>7. SDS-PAGE 解析およびスキャン蛍光ゲル
<ol><li>SDS-PAGE 分析用蛋白質のサンプルバッファーの 5 μ L を追加することによって蛋白質のサンプルを準備する (材料の表を参照してください)、精製、または原油の 13 μ L に地デジ (1.00 M) 2 μ L ラベル GFP (13.6 μ M または 0.38 mg/mL)。オリゴマーの GFP の解析、GFP (67.8 μ M または 1.9 mg/mL) の高濃度を使用します。95 ° C 10 分にそれらをインキュベートし、蛋白質を変化します。</li>
	<li>電気泳動セルに 4%-12% ビス トリス SDS-PAGE ゲル カセット (カセット購入、あらかじめ作られたゲル) を配置し、実行バッファーを追加 (材料の表を参照してください)。蛋白質のサンプルと分子量マーカーを読み込みます。200 V で 35-40 分間電気泳動を実行します。 注: は、蛍光プローブの光退色を最小限に抑えるため、暗闇の中のすべてのプロセスの遂行によって光に蛋白質のサンプルの任意の露出を避けてください。</li>
	<li>蛍光イメージングのための蛍光スキャナーを使用します。タンパク質ゲル電気泳動からをラップし、蛍光スキャナーの上に置きます。ゲルをスキャンするには、適切な波長の設定を使用します。Cy5.5、スキャナー ソフトウェアで提供されている Cy5 モードを選択します。</li>
	<li>試薬を汚す商業蛋白質のゲルを染色します。</li>
</ol>8. MALDI-TOF MS を用いたトリプシンの消化力
<ol><li>精製の GFP-E90DOPA (2.2 mg/mL または 80 μ M) 50 mM トリス-HCl (pH 8.0) 0.1% を含むバッファー (w/v) SDS のと 60 ° C 1 時間で 25 mM DTT の 100 μ L を孵化させなさい。</li>
	<li>ヨードアセトアミド H2O (IAA、4.0 M) 1 μ L を追加 (500 GFP E90DOPA に当量) 同じバッファーにおける精製 GFP E90DOPA 液 (80 μ M) へ。光からの保護と 30 分のための 25 の ° C で混合物を孵化させなさい。</li>
	<li>トリプシン (1: 100 プロテアーゼの基質タンパク質比/w) を追加することによって GFP E90DOPA サンプルを消化し、4 h の 37 ° C で反応混合物を孵化させなさい。</li>
	<li>C18回転カラムを用いた標準脱塩法による消化ペプチドのサンプルを浄化します。</li>
	<li>マトリックス支援レーザー脱離イオン化飛行時間質量分析 (MALDI-TOF MS) をマトリックス23として α-シアノ-4-ヒドロキシケイ皮酸 (CHCA) を使用して、報告されたプロトコルによって消化ペプチドを分析します。</li>
</ol>

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A protocol for MALDI-MS peptide analysis in proteomics.</a> Analytical Chemistry. 73 (3), 434-438 (2001).</li><li>Mukai, 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=Highly+reproductive+Escherichia+coli+cells+with+no+specific+assignment+to+the+UAG+codon.">Highly reproductive Escherichia coli cells with no specific assignment to the UAG codon.</a> Scientific Reports. 5, 9699 (2015).</li><li>Lajoie, M. 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=Genomically+recoded+organisms+expand+biological+functions.">Genomically recoded organisms expand biological functions.</a> Science. 342 (6156), 357-360 (2013).</li><li>Bryson, D. 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著者が明らかに何もありません。

このプロトコルでは生合成と DOPA の直接の混入を説明します。このメソッドで使用される細菌の細胞は、他のアミノ酸を合成でき、タンパク質合成の不自然なビルディング ブロックとして使用します。遺伝的非天然型アミノ酸定款は、展開された遺伝コードと不自然な有機体の開発のためのキー技術をされています。ただし、このメソッドは技術的に完成されているし、定款の効率を改善し...

ドーパは、動物と植物のカテコールアミンの生合成に関与するアミノ酸です。このアミノ酸はチロシン水酸化酵素と分子酸素 (O2)1により Tyr から合成されます。ドーパはドーパミンの前駆体である、血液脳関門を透過することができますので、2パーキンソン病の治療に使用されています。DOPA はウェットコンディション3,4,5,6,7でムール貝との接着は、ムール貝の接着性蛋白 (地図) にもあります。Tyr は、当初ドーパがマップ内にある tyrosinases8,9によってドーパに変換されます位置でエンコードされます。その興味深い生化学的な特性のためドーパは、さまざまなアプリケーションで使用されています。ドーパの dihydroxyl グループは、化学的に酸化しやすく、アミノ酸、L-dopaquinone、粕の前駆体に簡単に変換します。おかげでその高の有する L dopaquinone およびその誘導体は、架橋とチオール化合物とアミン10、11,12,13と活用のため使用されています。1, 2-ベンゾキノンは環化付加反応のジエンとしても機能することができますおよびひずみ促進酸化制御 cyclooctyne-1, 2-キノン (SPOCQ) 環化付加反応による14化反応に使用されています。さらに、dihydroxyl グループは、Fe3 +と Cu2 +などの金属イオンをキレートすることができます、DOPA を含んでいる蛋白質は薬剤配達および15,16を検出金属イオンのため利用されています。
DOPA を遺伝的直交アミノアシル tRNA (aa tRNA) とアミノアシル tRNA 合成酵素 (aaRS) のペア17を使用して蛋白質に組み込まれて、タンパク質共役および架橋10、11,用12,13。本報告では, 実験結果と安価な開始材料と化反応への応用から DOPA 生合成の遺伝の混入のためのプロトコルを説明します。ドーパは、TPL を使用して大腸菌におけるアンモニア、ピルビン酸、カテコールから生合成されます。生合成されるドーパはドーパの進化した aa tRNA と aaRS のペアを表現することによってタンパク質に直接組み込まれます。また、ドーパを含む生合成タンパク質の蛍光プローブとオリゴマー蛋白質を生成する架橋共役は site-specifically。このプロトコルはドーパを含む動植物の突然変異体蛋白質に蛋白質科学者にとって役に立つ、生化学的プローブまたは医薬・工業用薬剤でタンパク質を共役します。

<table><tbody><tr><td>&lt;strong&gt;1. Plasmid Construction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plasmid pBAD-dual-TPL-GFP-E90TAG</td><td></td><td></td><td>optionally contain the amber stop codon(TAG) at a desired position. Ko, W. et al. Efficient and Site-Specific Antibody Labeling by Strain-promoted Azide-Alkyne Cycloaddition. BKCS. 36 (9), 2352-2354, doi: 10.1002/bkcs.10423, (2015)</td></tr><tr><td>Plasmid pEvol-DHPRS2</td><td></td><td></td><td>1. Young, T. S., Ahmad, I., Yin, J. A., and Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395 (2), 361-374, doi: 10.1016/j.jmb.2009.10.030, (2010) 2. Kim, S., Sung, B. H., Kim, S. C., Lee, H. S. Genetic incorporation of l-dihydroxyphenylalanine (DOPA) biosynthesized by a tyrosine phenol-lyase. Chem. Commun. 54 (24), 3002-3005, doi: 10.1039/c8cc00281a (2018).</td></tr><tr><td>DH10&amp;beta;</td><td>Invitrogen</td><td>C6400-03</td><td>Expression Host</td></tr><tr><td>Plasmid Mini-prep kit</td><td>Nucleogen</td><td>5112</td><td>200/pack</td></tr><tr><td>Agarose</td><td>Intron biotechnology</td><td>32034</td><td>500 g</td></tr><tr><td>Ethidium bromide</td><td>Alfa Aesar</td><td>L07482</td><td>1 g</td></tr><tr><td>LB Broth</td><td>BD Difco</td><td>244620</td><td>500 g</td></tr><tr><td>&lt;strong&gt;2. Culture preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;2.1) Electroporation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micro pulser&amp;nbsp;</td><td>BIO-RAD</td><td>165-2100</td><td></td></tr><tr><td>Micro pulser cuvette</td><td>BIO-RAD</td><td>165-2089</td><td>0.1 cm electrode gap, pkg. of 50</td></tr><tr><td>Ampicillin Sodium</td><td>Wako</td><td>018-10372</td><td>25 g</td></tr><tr><td>Chloramphenicol</td><td>Alfa Aesar</td><td>B20841</td><td>25 g</td></tr><tr><td>Agar</td><td>SAMCHUN</td><td>214230</td><td>500 g</td></tr><tr><td>SOC medium</td><td>Sigma</td><td>S1797</td><td>100 mL</td></tr><tr><td>&lt;strong&gt;3. Expression and Purification of GFP-E90DOPA by biosynthetic system&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;3.1 Expression of GFP-E90DOPA by biosynthetic system&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>L(+)-Arabinose, 99%</td><td>Acros</td><td>104981000</td><td>100 g</td></tr><tr><td>Pyrocatechol, 99%</td><td>SAMCHUN</td><td>P1387</td><td>25 g</td></tr><tr><td>Ammonium sulfate, 99%</td><td>SAMCHUN</td><td>A0943</td><td>500 g</td></tr><tr><td>pyruvic acid, 98%</td><td>Alfa Aesar</td><td>A13875</td><td>100 g</td></tr><tr><td>Sodium phosphate dibasic, anhydrous, 99%</td><td>SAMCHUN</td><td>S0891</td><td>1 kg</td></tr><tr><td>Potassium phophate, monobasic, 99%</td><td>SAMCHUN</td><td>P1127</td><td>1 kg</td></tr><tr><td>Magnesium sulfate, anhydrous, 99%</td><td>SAMCHUN</td><td>M0146</td><td>1 kg</td></tr><tr><td>D(+)-Glucose, anhydrous, 99%</td><td>SAMCHUN</td><td>D0092</td><td>500 g</td></tr><tr><td>Glycerol, 99%</td><td>SAMCHUN</td><td>G0269</td><td>1 kg</td></tr><tr><td>Trace metal mix a5 with co</td><td>Sigma</td><td>92949</td><td>25 mL</td></tr><tr><td>L-Proline, 99%</td><td>SAMCHUN</td><td>P1257</td><td>25 g</td></tr><tr><td>L-Phenylalanine, 98.5%</td><td>SAMCHUN</td><td>P1982</td><td>25 g</td></tr><tr><td>L-Tryptophane</td><td>JUNSEI</td><td>49550-0310</td><td>25 g</td></tr><tr><td>L-Arginine, 98%</td><td>SAMCHUN</td><td>A1149</td><td>25 g</td></tr><tr><td>L-Glutamine, 98%</td><td>JUNSEI</td><td>27340-0310</td><td>25 g</td></tr><tr><td>L-Asparagine monohydrate, 99%</td><td>SAMCHUN</td><td>A1198</td><td>25 g</td></tr><tr><td>L-Methionine</td><td>JUNSEI</td><td>73190-0410</td><td>25 g</td></tr><tr><td>L-Histidine hydrochloride monohydrate, 99%</td><td>SAMCHUN</td><td>H0604</td><td>25 g</td></tr><tr><td>L-Threonine, 99%</td><td>SAMCHUN</td><td>T2938</td><td>25 g</td></tr><tr><td>L-Leucine</td><td>JUNSEI</td><td>87070-0310</td><td>25 g</td></tr><tr><td>Glycine, 99%</td><td>SAMCHUN</td><td>G0286</td><td>25 g</td></tr><tr><td>L-Glutamic acid, 99%</td><td>SAMCHUN</td><td>G0233</td><td>25 g</td></tr><tr><td>L-Alanine, 99%</td><td>SAMCHUN</td><td>A1543</td><td>25 g</td></tr><tr><td>L-Isoleucine, 99%</td><td>SAMCHUN</td><td>I1049</td><td>25 g</td></tr><tr><td>L-Valine, 99%</td><td>SAMCHUN</td><td>V0088</td><td>25 g</td></tr><tr><td>L-Serine</td><td>SAMCHUN</td><td>S2447</td><td>25 g</td></tr><tr><td>L-Aspartic acid</td><td>SAMCHUN</td><td>A1205</td><td>25 g</td></tr><tr><td>L-Lysine monohydrochloride, 99%</td><td>SAMCHUN</td><td>L0592</td><td>25 g</td></tr><tr><td>&lt;strong&gt;3.2 Cell lysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Imidazole, 99%</td><td>SAMCHUN</td><td>I0578</td><td>1kg</td></tr><tr><td>Sodium phosphate monobasic, 98%</td><td>SAMCHUN</td><td>S0919</td><td>1 kg</td></tr><tr><td>Sodium Chloride, 99%</td><td>SAMCHUN</td><td>S2907</td><td>1 kg</td></tr><tr><td>Ultrasonic Processor - 150 microliters to 150 milliliters</td><td>SONIC &amp;amp; MATERIALS</td><td>VCX130</td><td></td></tr><tr><td>&lt;strong&gt;3.3 Ni-NTA Affinity Chromatography&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ni-NTA resin</td><td>QIAGEN</td><td>30210</td><td>25 mL</td></tr><tr><td>Polypropylene column</td><td>QIAGEN</td><td>34924</td><td>50/pack, 1 mL capacity</td></tr><tr><td>&lt;strong&gt;4. Oligomerization of Purified GFP-E90DOPA&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium periodate, 99.8&amp;amp;</td><td>Acros</td><td>419610050</td><td>5 g</td></tr><tr><td>&lt;strong&gt;5. Conjugation of GFP-E90DOPA with an Alkyne Probe by Strain-Promoted Oxidation-Controlled Cyclooctyne&amp;ndash;1,2-Quinone Cycloaddition (SPOCQ)&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cy5.5-ADIBO&amp;nbsp;</td><td>FutureChem</td><td>FC-6119</td><td>1mg</td></tr><tr><td>&lt;strong&gt;6. Purification of Labeled GFP&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amicon Ultra 0.5 mL Centrifugal Filters</td><td>MILLIPORE</td><td>UFC500396</td><td>96/pack, 500ul capacity</td></tr><tr><td>&lt;strong&gt;7. SDS-PAGE Analysis and Fluorescence Gel Scanning&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1,4-Dithio-DL-threitol, DTT, 99.5 %</td><td>Sigma</td><td>10708984001</td><td>10 g</td></tr><tr><td>NuPAGE LDS Sample Buffer, 4X</td><td>Thermofisher</td><td>NP0007</td><td>10 mL</td></tr><tr><td>MES running buffer</td><td>Thermofisher</td><td>NP0002</td><td>500 mL</td></tr><tr><td>Nupage Novex 4-12% SDS PAGE gels</td><td>Thermofisher</td><td>NO0321</td><td>12 well</td></tr><tr><td>Coomassie Brilliant Blue R-250</td><td>Wako</td><td>031-17922</td><td>25 g</td></tr><tr><td>G:BOX Chemi Fluorescent &amp;amp; Chemiluminescent Imaging System</td><td>Syngene</td><td></td><td>G BOX Chemi XT4</td></tr><tr><td>&lt;strong&gt;8. MALDI-TOF MS analysis by Trypsin Digestion&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;8.1 Preparation of the digested peptide sample by trypsin digestion&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane, 99%</td><td>SAMCHUN</td><td>T1351</td><td>500 g</td></tr><tr><td>Hydrochloric acid, 35~37%</td><td>SAMCHUN</td><td>H0256</td><td>500 mL</td></tr><tr><td>Dodecyl sulfate sodium salt, 85%</td><td>SAMCHUN</td><td>D1070</td><td>250 g</td></tr><tr><td>Iodoacetamide</td><td>Sigma</td><td>I6125</td><td>5 g</td></tr><tr><td>Trypsin Protease, MS Grade</td><td>Thermofisher</td><td>90057</td><td>5 x 20 &amp;micro;g/pack</td></tr><tr><td>C-18 spin columns</td><td>Thermofisher</td><td>89870</td><td>25/pack, 200 &amp;micro;L capacity</td></tr><tr><td>&lt;strong&gt;8.2 Analysis of the digested peptide by MALDI-TOF&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetonitirile, 99.5%</td><td>SAMCHUN</td><td>A0125</td><td>500 mL</td></tr><tr><td>&amp;alpha;-Cyano-4-hydroxycinnamic acid</td><td>Sigma</td><td>C2020</td><td>10 g</td></tr><tr><td>Trifluoroacetic acid, 99%</td><td>SAMCHUN</td><td>T1666</td><td>100 g</td></tr><tr><td>MTP 384 target plate ground steel BC targets</td><td>Bruker</td><td>8280784</td><td></td></tr><tr><td>Bruker Autoflex Speed MALDI-TOF mass spectrometer</td><td>Bruker</td><td></td><td></td></tr></tbody></table>

genetic incorporation of biosynthesized l-dihydroxyphenylalanine (dopa) and its application to protein conjugation

L-ジヒドロキシフェニルアラニン (DOPA) は、動物や植物中のカテコールアミンの合成に存在するアミノ酸です。アミノ酸の特定の生化学的な特性のため生化学的アプリケーションで複数用途があります。このレポートでは、生合成されるドーパの定款の遺伝的プロトコルとその共役タンパク質への応用をについて説明します。ドーパは、チロシン フェノール-リアーゼ (TPL) カテコール、ピルビン酸とアンモニア、によって生合成とアミノ酸は直接進化したアミノアシル tRNA とアミノアシル tRNA 合成酵素のペアを使用して遺伝的結合法による蛋白質に組み込まれています。この直接混入システムは少し定款その他天然アミノ酸と DOPA の前の遺伝的結合システムよりもより良い蛋白質収量、DOPA 効率的に組み込まれています。ドーパ含有タンパク質とタンパク質共役なサイトと様々 な用途にその有用性を示しています。このプロトコルは、蛋白質科学者目的サイト、医薬・工業用の活用でドーパを含む突然変異体蛋白質の効率的な生合成のための詳細な手順を提供します。

直接混入防止から DOPA 生合成の発現システムを図 1に示します。進化した aa tRNA と aaRS のペアのための遺伝子はプラスミドに配置され、GFP 蛍光で 90 の位置に黄色のコドンを含む GFP 遺伝子 (GFP E90TAG) DOPA の取り込みを評価するもう一つのプラスミドであります。TPL 遺伝子は GFP 遺伝子を含んでいる同じ発現プラスミドと DOPA の生合成の収率を最大...

Watch this Scientific Journal Video about Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation at JoVE.com

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation

遺伝的定款生合成 L-ジヒドロキシフェニルアラニン (DOPA) とタンパク質接合への応用

ここでは、単純な開始材料から L-ジヒドロキシフェニルアラニン生合成の遺伝的定款及びタンパク質接合への応用のためのプロトコルを提案する.

L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical ...

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Biochemistry

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine (DOPA) and Its Application to Protein Conjugation

7.8K ビュー.  Sogang University. ここでは、単純な開始材料から L-ジヒドロキシフェニルアラニン生合成の遺伝的定款及びタンパク質接合への応用のためのプロトコルを提案する.

ビデオ: Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation

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 ...

生化学

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

タンパク質のバイオコンジュゲートの合成<em&gt;経由で</em&gt;システイン - マレイミドケミストリー

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

化学

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

光橋マッピングおよび生きている哺乳類細胞における Bioorthogonal 化学の Gpcr にプローブを化学物質の遺伝的結合の最適化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different positions. Preclinical studies have shown that the therapeutic index of these traditional ADCs can be improved by the site-specific linkage of toxins. However, current approaches to produce homogeneous ADCs have several limitations, such as low protein expression and slow reaction kinetics. In this protocol we describe how to set up an expression system to incorporate a cyclopropene derivative of lysine (CypK) into antibodies using genetic code expansion. This minimal bioorthogonal handle allows rapid conjugation of tetrazine derivatives through an inverse-demand Diels-Alder cycloaddition. The expression system here reported enables the facile production and purification of trastuzumab bearing CypK in each of the heavy chains. We explain how to link the antibody to the toxin monomethyl auristatin E and characterize the immunoconjugate by hydrophobic interaction chromatography and mass spectrometry. Finally, we describe assays to assess the stability in human serum of the dihydropyridazine linkage resulting from the conjugation and to test the selective cytotoxicity of the ADC for breast cancer cells with high levels of HER2 receptor.

Incorporating a cyclopropene derivative of lysine into antibodies allows the site-specific, rapid and efficient linkage of tetrazine-bearing molecules to generate antibody-drug conjugates.

高速 Bioorthogonal 反応により抱合体の新世代抗体医薬の非標準アミノ酸の遺伝的符号化

Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

OaAEP1-媒介酵素合成と単分子力分光法のための重合タンパク質の固定化

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

非正規のアミノ酸の選択圧による 'ゴールデン' 蛍光と質量分析法と蛍光タンパク質解析工学

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

生物工学

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

ニューロンに遺伝的にコードされた非天然アミノ酸を用いた神経タンパク質の光制御

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

神経科学

Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new protein functions or to have access to protein conjugates. Stop codon suppression, in particular amber codon suppression, has emerged as the most popular method to genetically introduce UAAs at defined positions. This methodology is herein applied to the preparation of a carrier protein containing an UAA harboring a bioorthogonal functional group. This reactive handle can next be used to specifically and efficiently graft a synthetic oligosaccharide hapten to provide a homogeneous glycoconjugate vaccine. The protocol is limited to the synthesis of glycoconjugates in a 1:1 carbohydrate hapten/carrier protein ratio but amenable to numerous pairs of biorthogonal functional groups. Glycococonjugate vaccine homogeneity is an important criterion to ensure complete physico-chemical characterization, thereby, satisfying more and more demanding drug regulatory agency recommendations, a criterion which is unmet by classical conjugation strategies. Moreover, this protocol makes it possible to finely tune the structure of the actual conjugate vaccine, giving rise to tools to address structure-immunogenicity relationships.

Genetic code expansion is applied for the introduction of an unnatural amino acid bearing a biorthogonal functional group on a carrier protein at a defined site. The biorthogonal function is further used for the site-selective coupling of a carbohydrate antigen to provide a homogeneous glycoconjugate vaccine.

ワクチン用非天然アミノ酸組み合わせとクリック化学を組み合わせた均質なグリココンジュゲート

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new ...

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

蛍光タンパク質中のプロリン類似体によるプロリンの残基特異的交換:骨格の「分子手術」が折り畳みと安定性にどのように影響するか

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

遺伝的定款生合成 L-ジヒドロキシフェニルアラニン (DOPA) とタンパク質接合への応用

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