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>
	<li>Wang, L., 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=Expanding+the+genetic+code.">Expanding the genetic code.</a> Angew. Chem. Int. Ed. 44 (1), 34-66 (2004).</li><li>Wu, X., 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=Synthesis+at+the+interface+of+chemistry+and+biology.">Synthesis at the interface of chemistry and biology.</a> J. Am. Chem. Soc. 131 (35), 12497-12515 (2009).</li><li>Liu, C. C., 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=Adding+new+chemistries+to+the+genetic+code.">Adding new chemistries to the genetic code.</a> Annu. Rev. Biochem. 79, 413-444 (2010).</li><li>Wang, L., Zhang, Z., Brock, 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=Addition+of+the+keto+functional+groupto+the+genetic+code+of+Escherichia+coli.">Addition of the keto functional groupto the genetic code of Escherichia coli.</a> proc. Natl. Acad. Sci. U.S.A. 100 (1), 56-61 (2003).</li><li>Chin, J. 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=Addition+of+p-azido-L-phenylalanine+to+the+genetic+code+of+Escherichia+coli.">Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli.</a> J. Am. Chem. Soc. 124 (31), 9026-9027 (2002).</li><li>Deiters, 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=In+vivo+incorporation+of+an+alkyne+into+proteins+in+Esshcerichia+coli.">In vivo incorporation of an alkyne into proteins in Esshcerichia coli.</a> Bioorg. Med. Chem. Lett. 15 (5), 1521-1524 (2005).</li><li>Plass, T., Milles, S., Koehler, C., Schultz, C., Lemke, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+copper-free+click+chemistry.">Genetically encoded copper-free click chemistry.</a> Angew. Chem. Int. Ed. 50 (17), 3878-3881 (2011).</li><li>Seitchik, J. L., 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=Genetically+encoded+tetrazine+amino+acid+directs+rapid+site-specific+in+vivo+bioorthogonal+ligation+with+transcyclooctenes.">Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with transcyclooctenes.</a> J. Am. Chem. Soc. 134 (6), 2898-2901 (2014).</li><li>Lee, Y. -. 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=A+genetically+encoded+acrylamide+fuctionality.">A genetically encoded acrylamide fuctionality.</a> ACS Chem. Biol. 8 (8), 1664-1670 (2013).</li><li>Lang, K., 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=Genetically+encoded+norbornene+directs+site-specific+cellular+protein+labelling+via+a+rapid+bioorthogonal+raction.">Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal raction.</a> Nat Chem. 4 (4), 298-304 (2012).</li><li>Lang, K., 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=Genetic+encoding+of+bicyclononynes+and+trans-cyclooctenes+for+site-specific+protein+labeling+in+vitro+and+in+live+mammalian+cells+via+rapid+fluorogenic+Diels-Alder+reactions.">Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions.</a> J. Am. Chem. Soc. 134 (25), 10317-10320 (2012).</li><li>Jewett, J. C., Sletten, E. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Cu-free+click+chemistry+with+readily+synthesized+biarylazacyclooctynones.">Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones.</a> J. Am. Chem. Soc. 132 (11), 3688-3690 (2010).</li><li>Debets, M. F., 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=Azadibenzocyclooctynes+for+fast+and+efficient+enzyme+PEGylation+via+copper-free+(3+++2)+cycloaddition.">Azadibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3 + 2) cycloaddition.</a> Chem. Commun. 46 (1), 97-99 (2010).</li><li>Sletten, E. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+mechanism+to+mouse:+a+tale+of+two+bioorthogonal+reactions.">From mechanism to mouse: a tale of two bioorthogonal reactions.</a> Acc. Chem. Res. 44 (9), 666-676 (2011).</li><li>Debets, M. F., 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=Bioconjugation+with+strained+alkenes+and+alkynes.">Bioconjugation with strained alkenes and alkynes.</a> Acc. Chem. Res. 44 (9), 805-815 (2011).</li><li>Mbua, N. E., Guo, J., Wolfert, M. A., Steet, R., Boons, G. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-promoted+alkyne&#8722;azide+cycloadditions+(SPAAC)+reveal+new+features+of+glycoconjugate+biosynthesis.">Strain-promoted alkyne&#8722;azide cycloadditions (SPAAC) reveal new features of glycoconjugate biosynthesis.</a> ChemBioChem. 12 (12), 1912-1921 (2011).</li><li>Kuzmin, A., Poloukhtine, A., Wolfert, M. A., Popik, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+functionalization+using+catalyst-free+azide-alkyne+cycloaddition.">Surface functionalization using catalyst-free azide-alkyne cycloaddition.</a> Bioconjug. Chem. 21 (11), 2076-2085 (2011).</li><li>Yao, J. Z., 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=Fluorophore+targeting+to+cellular+proteins+via+enzymemediated+azide+ligation+and+strain-promoted+cycloaddition.">Fluorophore targeting to cellular proteins via enzymemediated azide ligation and strain-promoted cycloaddition.</a> J. Am. Chem. Soc. 134 (8), 3720-3728 (2012).</li><li>Hao, Z., Hong, S., Chen, X., Chen, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+bioorthogonal+functionalities+into+proteins+in+living+cells.">Introducing bioorthogonal functionalities into proteins in living cells.</a> Acc. Chem. Res. 44 (9), 742-751 (2011).</li><li>Reddington, S. C., Tippmann, E. M., Jones, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residue+choice+defines+efficiency+and+influence+of+bioorthogonal+protein+modification+via+genetically+encoded+strain+promoted+Click+chemistry.">Residue choice defines efficiency and influence of bioorthogonal protein modification via genetically encoded strain promoted Click chemistry.</a> Chem. Commun. 48 (9), 8419-8421 (2012).</li><li>Chari, R. V. J., Miller, M. L., Widdison, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-drug+conjugates:+an+emerging+concept+in+cancer+therapy.">Antibody-drug conjugates: an emerging concept in cancer therapy.</a> Angew. Chem. Int. Ed. 53 (15), 3751-4005 (2014).</li><li>Tsao, M., Tian, F., 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=Selective+staudinger+modification+of+proteins+containing+p-Azidophenylalanine.">Selective staudinger modification of proteins containing p-Azidophenylalanine.</a> ChemBioChem. 6 (12), 2147-2149 (2005).</li><li>Brustad, E. M., Lemke, E. A., Schultz, P. G., Deniz, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+and+efficient+method+for+the+site-specific+dual-labeling+of+proteins+for+single+molecule+fluorescence+resonance+energy+transfer.">A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer.</a> J. Am. Chem. Soc. 130 (52), 17664-17665 (2008).</li><li>Milles, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+strategies+for+single-molecule+protein+fluorescence.">Click strategies for single-molecule protein fluorescence.</a> J. Am. Chem. Soc. 134 (11), 5187-5195 (2012).</li><li>Jang, S., Sachin, K., Lee, h. J., Kim, D. W., Lee, h. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple+method+for+protein+conjugation+by+copper-free+click+reaction+and+its+application+to+antibody-free+Western+blot+analysis.">Development of a simple method for protein conjugation by copper-free click reaction and its application to antibody-free Western blot analysis.</a> Bioconjug. Chem. 23 (11), 2256-2261 (2012).</li><li>Kazane, S. 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=Self-assembled+antibody+multimers+through+peptide+nucleic+acid+conjugation.">Self-assembled antibody multimers through peptide nucleic acid conjugation.</a> J. Am. Chem. Soc. 135 (1), 340-346 (2012).</li><li>Ko, 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=Efficient+and+site-specific+antibody+labeling+by+strain-promoted+azide-alkyne+cycloaddition.">Efficient and site-specific antibody labeling by strain-promoted azide-alkyne cycloaddition.</a> Bull. Korean Chem. Soc. 36 (9), 2352-2354 (2015).</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> J. Mol. Biol. 395 (2), 361-374 (2010).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Cho, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+extracellular+region+of+HER2+alone+and+in+complex+with+the+Herceptin+Fab.">Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab.</a> Nature. 421 (6924), 756-760 (2003).</li><li>Kolb, H. C., Finn, M. G., Sharpless, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+chemistry+:+Diverse+chemical+function+from+a+few+good+reactions.">Click chemistry : Diverse chemical function from a few good reactions.</a> Angew. Chem. Int. Ed. 40 (11), 2004-2021 (2001).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1. plasmid Construction</td>
			<td></td>
			<td></td>
			<td></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. 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-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 E. coli. J. Mol. Biol. 395 (2), 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>500g</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Alfa Aesar</td>
			<td>L07482</td>
			<td>1g</td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>BD Difco</td>
			<td>244620</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>2. Culture preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.1) Electroporation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pulser&#160;</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>0.1cm electrode gap, pkg. of 50</td>
		</tr>
		<tr>
			<td>Ampicillin Sodium</td>
			<td>Wako</td>
			<td>018-10372</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Alfa Aesar</td>
			<td>B20841</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>SAMCHUN</td>
			<td>214230</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Sigma</td>
			<td>S1797</td>
			<td>100ML</td>
		</tr>
		<tr>
			<td>3. Expression and purification of HerFab-L177AF</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.1 Expression of Herfab-L177AF</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p-azido-L-phenylalanine (AF)</td>
			<td>Bachem</td>
			<td>F-3075.0001</td>
			<td>1g</td>
		</tr>
		<tr>
			<td>L(+)-Arabinose, 99%</td>
			<td>Acros</td>
			<td>104981000</td>
			<td>100g</td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 35~37%</td>
			<td>SAMCHUN</td>
			<td>H0256</td>
			<td>500ml</td>
		</tr>
		<tr>
			<td>3.2 Cell lysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane, 99%</td>
			<td>SAMCHUN</td>
			<td>T1351</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>EDTA disodium salt dihydrate, 99.5%</td>
			<td>SAMCHUN</td>
			<td>E0064</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S9378</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Siyaku</td>
			<td>126-0671</td>
			<td>1g</td>
		</tr>
		<tr>
			<td>3.3 Ni-NTA Affinity Chromatography</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA resin</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td>25ml</td>
		</tr>
		<tr>
			<td>Polypropylene column</td>
			<td>QIAGEN</td>
			<td>34924</td>
			<td>50/pack, 1ml capacity</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>1kg</td>
		</tr>
		<tr>
			<td>Sodium Chloride, 99%</td>
			<td>SAMCHUN</td>
			<td>S2907</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>4. Conjugation of Purified HerFab-L177AF with Alkyne Probes Using Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5.5-ADIBO&#160;</td>
			<td>FutureChem</td>
			<td>FC-6119</td>
			<td>1mg</td>
		</tr>
		<tr>
			<td>5. Purification of Labeled HerFab</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>6. SDS-PAGE Analysis of Labeled HerFab and Fluorescent Gel Scanning</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>10g</td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer, 4X</td>
			<td>Thermofisher</td>
			<td>NP0007</td>
			<td>10ml</td>
		</tr>
		<tr>
			<td>MES running buffer</td>
			<td>Thermofisher</td>
			<td>NP0002</td>
			<td>500ml</td>
		</tr>
		<tr>
			<td>Nupage Novex 4-12% SDS PAGE gels</td>
			<td>Thermofisher</td>
			<td>NO0321</td>
			<td>12well</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Wako</td>
			<td>031-17922</td>
			<td>25g</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

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

20.5K 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

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

ライブセル光親和性標識によって、ネイティブの細胞環境における小分子結合タンパク質の同定

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

生化学

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

リバーシブルジスルフィド架橋ミセルの生産のための簡便かつ効率的なアプローチ

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

治療用抗体の実験室規模の産生および精製

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer&#39;s disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer's disease mouse model and wild-type controls.

拡張サンプル複合前駆同位体標識と同重体タグ付けを使用して組織の多重化（cPILOT）

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

ペプチドマイクロアレイによるヒストン抗体特異性の解析

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

定圧ラベリング、豊富な液体クロマトグラフィー、質量分析法、およびソフトウェアによる定量化して深いプロテオームをプロファイリング

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

高効率巻き戻しと受容体リガンド結合領域の浄化法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

腎糸球体と腎糸球体細胞表面タンパク質の標識体内の分離

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

プロテオーム的分類の単一分子レベルでの均一性の定量化

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.