1.プラスミド構築<ol><li>彼の6タグを有する標的抗体遺伝子（のpBAD-HerFab-WT）を発現し、アンバーコドン（TAG）とロイシン-177のコドンに代わる発現プラスミド（のpBAD-HerFab-L177TAG）を構築27、使用して従来の部位特異的変異誘発技術。 材料の表を参照してください。 </li><li>進化したtRNA TyrおよびアミノアシルtRNA合成酵素（aaRS）ペアの遺伝子を含む別の発現プラスミド（pEVOL-AFRS）を構築します。 UAAsの効率的な取り込みのために、特別に設計されたプラスミドベクター、pEVOLを使用してください。詳細プラスミド情報およびクローニングプロトコルは、以前の報告書に記載されています。 28 </li><li>市販のプラスミド調製キットを用いて高品質のプラスミドDNAを得ました。 〜1.8の260/280比は、精製されたDNAに最適です。必要であれば、DNAの純度を確認するために1％アガロースゲル電気泳動を行います。 </li></ol><ol><li> エレクトロポレーション <ol><li> 20μL 大腸菌 DH10β株にpEVOL-AFRS 28とのpBAD-HerFab-L177TAGの各1μLのDNAを追加します。ピペットを用いて、穏やかに混合します。プラスミドは、一緒にまたは独立した反応で形質転換することができます。 </li><li> 0.1センチメートルキュベットについては、25μFと2.5 kVでのエレクトロポレーションの設定を行います。 </li><li>衝撃的なチャンバーのスライドにキュベットを挿入します。キュベットは、チャンバ電極としっかり接触するまでチャンバー内にスライドを押してください。 </li><li>エレクトロポレーションマシンのビープ音まで、パルスボタンを押すことで、一度パルス。 </li><li>カタボライト抑制（SOC）培地は、酵母エキス、10mMの塩化ナトリウム、2.5mMの塩化カリウム（w / v）のトリプトン、0.5％（w / v）の、10mMのMgCl 2を2重量％含有する1mLの超最適ブロス、及び20mMのグルコースを追加キュベットへの迅速かつ試験管に混合物を移します。振盪しながら37℃で1時間、細胞をインキュベートします。</li><li> （pEVOL-AFRSそれぞれのpBAD-HerFab-L177TAGを、選択するための35μgの/ mLクロラムフェニコールおよび100μg/ mLのアンピシリン）適切な抗生物質を含む寒天プレートLB培地（LB）のスプレッド形質転換された大腸菌細胞 。 12時間37℃で培養します。 </li></ol></li><li> 文化の準備 <ol><li>抗生物質を含有する5mLのLB培地に単一形質転換コロニーを接種します。振盪しながら37℃で12時間インキュベートします。 </li></ol></li></ol> HerFab-L177AFの3発現および精製<ol><li> HerFab-L177AFの発現 <ol><li>振盪しながら37℃でインキュベートし、アンピシリン（100μg/ ml）およびAF（1ミリモル）を含有する200mLのLB培地の中に初代培養物（5ml）に移します。 注：100mMのAFストック溶液（10mL）を100mM塩酸（10mLの最終体積）に206 mgのAFを溶解することによって調製しました。 </li><li> （16を1.6 Mアラビノースの2 mLを加え培養物が対数期（OD 550 = 0.8、OD =光学密度）に達するmMの最終濃度）。振盪しながら30℃で12時間インキュベートします。 </li><li> 5分間、11,000×gで遠心分離によって細胞を回収します。上清を捨て、-20℃でペレットを凍結します。 </li></ol></li><li> 細胞溶解 <ol><li> 30 mMトリス（pHは8.0）、1mMのEDTA、20％スクロース、および0.2 mg / mlでリゾチームを含む20mLのペリプラズム溶解用緩衝液中で細胞ペレットを再懸濁し、そして37℃で1時間混合物をインキュベートします。 </li><li> 15分間4℃で18,000×gで細胞溶解物を遠心。上清を新しいチューブに移し、ペレットを捨てます。 </li></ol></li><li> Ni-NTAアフィニティークロマトグラフィー <ol><li>各遠心分離管（200 mLの培養のための400μL樹脂）へのNi-NTA樹脂懸濁液を追加し、4℃で1時間穏やかに混合します。 </li><li>ポリプロピレンカラムにサスペンションを注ぎ、5 mLの洗浄府で樹脂を3回洗浄fferの50mMのNaH 2 PO 4（pH8.0）で、20 mMイミダゾール、および300 mMのNaClを含みます。 </li><li> 50mMのNaH 2 PO 4（pHは8.0）、250 mMイミダゾール、および300 mMのNaClを含有する300μLの溶出緩衝液を用いて目的とする抗体を溶出させます。 </li><li> Bradfordタンパク質アッセイ29によって、または280nmでの吸光度を測定することにより変異体タンパク質の濃度を決定します。吸光係数（2471 M -1 cm -1 で ）AF用を使用して、タンパク質の吸光係数の計算により280 nmでHerFab-L177AFの吸光係数（75866 M -1センチ-1）を計算します。 注：HerFabのアミノ酸配列は、NCBIのウェブサイトで見つけることができます（GI：783282791とGI：783282792）。 </li></ol></li></ol>ひずみ促進アジド - アルキン付加環を使用してアルキンプローブを用いて精製したHerFab-L177AFの4コンジュゲート（SPAAC） <ol><li> （H 2 Oで200のCy5.5-Azadibenzocyclooctyne（のCy5.5-ADIBO）の20μLを追加します。81; 10mMののNa 2 HPO 4（pH7.0）に100mMのNaClを含むリン酸緩衝液中HerFab-L177AFの10μL（最終濃度10μM）の溶液にM最終濃度）。 注：代替、ジフルオロシクロオクチン（DIFO）およびビシクロ[6.1.0]ノニン（BCN）誘導体も、同じアプリケーションで使用できます。 </li><li>歪み促進付加環化反応は、37℃で6時間進行させます。感光性プローブ（ 例えば、Cy5.5の）が使用される場合、アルミニウム箔で反応容器を覆います。 </li><li>ステップ5に従って標識HerFabを精製します。 </li></ol>標識HerFab 5.精製<ol><li>遠心分離フィルタースピンカラムにサンプルの500μLを追加します。 </li><li> 15分間、4℃、14,000×gでスピンカラムを遠心します。 </li><li>フロースルーを捨て、1.5 mlのマイクロ遠心チューブにスピンカラムから精製されたサンプルを転送します。 </li><li> 5.3 5.1-ステップに代わるものとして、purificaを行います同緩衝液に対して透析することによりる。 </li><li> 4℃で精製し、標識HerFabを保管してください。 </li></ol>標識HerFabの6 SDS-PAGE分析<ol><li> 106 mMトリス・塩酸、141 mMのトリス塩基（pHは8.5）、2％LDS、10％グリセロール、0.51 mMのEDTA、0.22 mMのSERVAブルーG250、および13μLに赤の0.175 mMのフェノールを含む5μLLDSタンパク質サンプルバッファーを追加します。 2μLのジチオスレイトール（DTT、100mMの最終濃度）の（ - ）の存在（+）または非存在下で標識されたHerFab（7.8μMまたは0.38ミリグラム/ mL）を精製しました。 10分間95℃で混合物をインキュベートします。 </li><li>電気泳動セルに4〜12％のBis-Tris SDS-PAGEゲルカセットを取り付け、バッファを実行して追加します。共役タンパク質試料と前染色分子量マーカーをロードします。 200 Vで35分間電気泳動を行います 注：蛍光体の光退色を最小限にするために全期間の間、暗闇の中での電気泳動セルを保管してください。 </li><li>電気泳動後、転送蛍光ゲルスキャナーにゲル、および適切な波長での蛍光発光をスキャン。 Cy5.5のために、スキャナソフトウェアでCy5のモードを使用します。 </li><li>市販のタンパク質染色でゲルを染色。 </li></ol>

<ol>
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The authors have nothing to disclose.

タンパク質への非天然アミノ酸の遺伝的組込みは、タンパク質修飾のために使用される他の方法に比べていくつかの利点を有しています。 1-3重要な利点の一つは、タンパク質の任意の種類への一般的な適用です。原理的には、標的タンパク質とタンパク質の標的部位を選択するには制限がありません。しかし、UAAと構造的または機能的に重要な残基の置換​​は、標的タンパク質の?...

大腸菌 におけるP -methoxyphenylalanineの遺伝的取り込みが報告されて以来、1、100以上の非天然アミノ酸（UAAs）が正常に様々なタンパク質に組み込まれています。 1-3これらのUAAsの中で、バイオ直交官能基を含むアミノ酸は、広く研究さと最大の割合を表してきました。 UAAsで使用されるバイオ直交官能基は、ケトン、4アジド、5アルキン、6シクロオクチン、7テトラジン、8α、β不飽和アミド、9ノルボルネン、10 transcyclooctene、11およびビシクロ[6.1.0] -nonyneが含まれます。各官能基は、その長所と短所を持っている11が、アジド含有アミノ酸は、最も広範にタンパク質結合に使用されてきました。 P -Azidophenylalanine（AF）、アジド含有アミノ酸の一つは、容易に入手可能であり、その取り込みefficiencyが優れています。このアミノ酸を含有する変異体タンパク質は、銅触媒による付加環化によって、またはSPAACによってcyclooctynesとアルキンと反応させることができます。 12月20日 最近、バイオ医薬品は、製薬業界で大きな注目を集めています。抗体-薬物複合体（ADC）が原因で、ヒトの癌21の治療のための標的治療のためのそれらの能力に有利である治療用抗体のクラスであり、 他の疾患。 50以上のADCは現在臨床試験中であり、その数は急速に増加しています。 ADCの開発では、多くの要因が有効性を最大化し、副作用を最小限にするために考慮される必要があります。これらの要因の中でも、抗体と薬物との間に共有結合を形成するための効率的かつ部位特異的コンジュゲーション反応は重要です。コンジュゲーション反応における所望の効率及び特異性にバイオ直交官能基と結合することによって達成することができます具体的に抗体に組み込まれる非天然アミノ酸。ここで22-26、我々は、部位特異的に組み込むためのプロトコルを報告します 抗体断片にAFとは、生化学的プローブと変異型抗体断片を結合します。

<table><tbody><tr><td colspan="4">&lt;strong&gt;1. Plasmid Construction&lt;/strong&gt;</td></tr><tr><td>plasmid pBAD_HerFab_L177TAG</td><td></td><td></td><td>Optionally contain the amber stop codon (TAG) at a desired position. Ko, W. &lt;em&gt;et al.&lt;/em&gt; Efficient and Site-Specific Antibody Labeling by Strain-promoted Azide-Alkyne Cycloaddition. &lt;em&gt;BKCS.&lt;/em&gt; &lt;strong&gt;36 (9)&lt;/strong&gt;, 2352-2354, doi: 10.1002/bkcs.10423, (2015)</td></tr><tr><td>plasmid pEvol-AFRS</td><td></td><td></td><td>Young, T. S., Ahmad, I., Yin, J. A., and Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in &lt;em&gt;E. coli.&lt;/em&gt; &lt;em&gt;J. Mol. Biol.&lt;/em&gt; &lt;strong&gt;395 (2)&lt;/strong&gt;, 361-374, doi: 10.1016/j.jmb.2009.10.030, (2010)</td></tr><tr><td>DH10B</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&amp;nbsp;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&amp;nbsp;g</td></tr><tr><td colspan="4">&lt;strong&gt;2. Culture Preparation&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;2.1 Electroporation&lt;/strong&gt;</td></tr><tr><td>Micro pulser</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&amp;nbsp;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 colspan="4">&lt;strong&gt;3. Expression and Purification of HerFab-L177AF&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;3.1 Expression of Herfab-L177AF&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-azido-L-phenylalanine (AF)</td><td>Bachem</td><td>F-3075.0001</td><td>1 g</td></tr><tr><td>L(+)-Arabinose, 99%</td><td>Acros</td><td>104981000</td><td>100 g</td></tr><tr><td>Hydrochloric acid, 35~37%</td><td>SAMCHUN</td><td>H0256</td><td>500 mL</td></tr><tr><td colspan="4">&lt;strong&gt;3.2 Cell Lysis&lt;/strong&gt;</td></tr><tr><td>Tris(hydroxymethyl)aminomethane, 99%</td><td>SAMCHUN</td><td>T1351</td><td>500 g</td></tr><tr><td>EDTA disodium salt dihydrate, 99.5%</td><td>SAMCHUN</td><td>E0064</td><td>1 kg</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S9378</td><td>500 g</td></tr><tr><td>Lysozyme</td><td>Siyaku</td><td>126-0671</td><td>1 g</td></tr><tr><td colspan="4">&lt;strong&gt;3.3 Ni-NTA Affinity Chromatography&lt;/strong&gt;</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>Imidazole, 99%</td><td>SAMCHUN</td><td>I0578</td><td>1 kg</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 colspan="4">&lt;strong&gt;4. Conjugation of Purified HerFab-L177AF with Alkyne Probes Using Strain-promoted Azide-alkyne Cycloaddition (SPAAC)&lt;/strong&gt;</td></tr><tr><td>Cy5.5-ADIBO&amp;nbsp;</td><td>FutureChem</td><td>FC-6119</td><td>1 mg</td></tr><tr><td colspan="4">&lt;strong&gt;5. Purification of Labeled HerFab&lt;/strong&gt;</td></tr><tr><td>Amicon Ultra 0.5 mL Centrifugal Filters</td><td>MILLIPORE</td><td>UFC500396</td><td>96/pack, 500 &amp;#956;L capacity</td></tr><tr><td colspan="4">&lt;strong&gt;6. SDS-PAGE Analysis of Labeled HerFab and Fluorescent Gel Scanning&lt;/strong&gt;</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>Typhoon 9210 variable mode imager</td><td>Amersham Biosciences</td><td></td><td></td></tr></tbody></table>

efficient and site-specific antibody labeling by strain-promoted azide-alkyne cycloaddition

その構造と機能を研究するためにタンパク質に化学プローブを導入するために利用可能な多くの化学ツールは現在ありません。有用な方法は、遺伝的にバイオ直交官能基を有する非天然アミノ酸を導入することによって、タンパク質結合です。このレポートには、サイト固有の抗体結合のための詳細なプロトコルを記述しています。プロトコルは、アジド含有アミノ酸の遺伝的取り込み、および歪み促進アジド - アルキン付加環（SPAAC）によって結合反応のための実験の詳細が含まれています。この株促進反応は、生理的pHおよび温度で反応する分子を単純に混合することによって進行し、例えば、銅（I）イオンと銅キレートリガンドなどの追加の試薬を必要としません。したがって、この方法は、一般的なタンパク質の結合および抗体薬物複合体（ADCの）の開発に有用であろう。

<html>本研究では、抗体断片は、部位特異的フラグメントにアジド含有アミノ酸を組み込んでいると歪みシクロオクチン（ 図1）を用いて変異体抗体断片を反応させることにより、フルオロフォアとコンジュゲートしました。 HerFabはAFがアジド含有アミノ酸として組み込んだ標的抗体フラグメントとして選択しました。 AFとの交換のためHerFab中の残留物を選...

Watch this Scientific Journal Video about Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition at JoVE.com

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

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 ...

efficient-site-specific-antibody-labeling-strain-promoted-azide

Research

JoVE Journal

Biochemistry

20.6K ビュー.  Sogang University. 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).

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

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

可逆小分子またはバイオセンサー応用のためのマルチコンポーネント構造を固定化するために、マイクロ流体オンチップキャプチャ - 環化反応

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

化学

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

使用した金ナノ粒子の効率的な放射性標識のために最適化されたプロトコル<sup&gt; 125</sup&gt; I標識アジド補綴グループ

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

Bioorthogonalクリックケミストリーを介したウイルス表面の化学選択的修飾

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

免疫・感染学

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 ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

合成と化反応のサイト選択的に作成するためのチオール反応性の試薬の変更 Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Preparation of Site-Specific Cytotoxic Protein Conjugates via Maleimide-thiol Chemistry and Sortase A-Mediated Ligation

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials.
One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.

Here we provide detailed protocols for a site-specific labeling of proteins with cytotoxic drugs using maleimide-thiol reaction and sortase A-mediated ligation.

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as ...

生化学

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

磁気プロテインA及びプロテインGビーズを用いた蛍光色素で抗体ラベリング

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

蛍光系抗生物質プローブを用いた細菌耐性の可視化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

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.

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

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