1.质粒构建<ol><li>构建一个表达质粒（的pBAD-HerFab-L177TAG），将表达靶抗体基因（的pBAD-HerFab-WT）用他的6 -tag，并用琥珀密码子取代为亮氨酸177的密码子（TAG）27，用常规定点诱变技术。 见材料表 。 </li><li>构建包含演进的tRNA 酪氨酸和氨酰tRNA合成酶（的aaRS）对所述基因另一个表达质粒（pEVOL-AFRS）。使用专门设计的质粒载体，pEVOL，为UAAs的有效结合。详细的信息载体和克隆的协议在之前的报告中已经提到。 28 </li><li>通过使用可商购的质粒制备试剂盒获得的高质量的质粒DNA。 260/280比率〜1.8的最适合的纯化的DNA。如果需要的话，则执行1％琼脂糖凝胶电泳来检查DNA的纯度。 </li></ol><ol><li> 电穿孔 <ol><li> pEVOL-AFRS 28的pBAD-HerFab-L177TAG各1μLDNA添加到20μL 大肠杆菌菌株DH10β。轻轻混匀，用移液管。质粒可以一起或独立的反应被转化。 </li><li>对于0.1厘米比色皿，设置电设置25μF和2.5千伏。 </li><li>将试管放入震撼室的幻灯片。推滑入室，直到试管使得与室电极紧密接触。 </li><li>按脉冲按钮，直到电机发出哔哔声脉冲一次。 </li><li>加入1毫升含有分解代谢物阻抑（SOC）培养基2％（重量/体积）胰蛋白胨，0.5％（重量/体积）酵母提取物，10mM的氯化钠，2.5mM的氯化钾，10mM的MgCl 2的超级最佳肉汤，和20mM葡萄糖到试管迅速将混合物转移到一个试管中。孵育1小时将细胞在37℃振荡。</li><li>传播转化的大肠杆菌细胞在LB培养基（LB）琼脂含适当的抗生素（35微克/毫升氯霉素和100μg/ mL氨苄青霉素，用于选择pEVOL-AFRS和分别的pBAD-HerFab-L177TAG，）板。孵育在37℃下将板进行12小时。 </li></ol></li><li> 文化准备 <ol><li>接种在5毫升LB培养基含有抗生素的单一转化殖民地。在37℃振荡孵育12小时。 </li></ol></li></ol> 3.表达HerFab-L177AF纯化<ol><li> HerFab-L177AF的表达 <ol><li>传送主培养（5毫升）到200ml的LB培养基含有氨苄青霉素（100微克/毫升）和AF（1毫米），接着温育，在37℃振荡。 注:100mM的AF原液（10毫升）溶液通过在100mM溶解206毫克的AF盐酸（10毫升，最终体积）制备。 </li><li>加入2毫升的1.6M的阿拉伯糖（16毫终浓度）时培养物达到对数期（OD 550 = 0.8，OD =光学密度）。在30℃振荡孵育12小时。 </li><li>收获细胞，离心在11000×g离心5分钟。弃上清，在-20℃冷冻沉淀。 </li></ol></li><li> 细胞裂解 <ol><li>重悬在含有30毫摩尔Tris（pH值8.0），1mM EDTA中，20％的蔗糖和0.2毫克/毫升溶菌酶20毫升周质溶胞缓冲液的细胞沉淀，并在37℃下孵育混合物1小时。 </li><li>离心细胞裂解物以18,000rpm xg离心在4℃下15分钟。将上清转移到新的管中，并弃沉淀。 </li></ol></li><li> 的Ni-NTA亲和层析 <ol><li>添加的Ni-NTA树脂悬浮于每个离心管中（400微升树脂的200mL培养物），并在4℃下1小时轻轻混匀。 </li><li>倒入悬浮液成聚丙烯柱和三次用5mL洗涤蒲式耳洗涤树脂FFER含有50mM的NaH 2 PO 4（pH 8.0）中，20 mM咪唑，和300mM NaCl的。 </li><li>洗脱用含有50mM的NaH 2 PO 4（pH为8.0），250mM咪唑，和300mM NaCl的300微升的洗脱缓冲液的目标抗体。 </li><li>确定由Bradford蛋白测定29或通过测量280nm处的吸光度的突变蛋白的浓度。通过使用用于自动对焦的消光系数（2471 M -1 -1）蛋白质消光系数计算器计算HerFab-L177AF消光系数（75866 M -1 -1）在280纳米。 注:HerFab的氨基酸序列可以在NCBI网站（GI:783282791和GI:783282792）。 </li></ol></li></ol> 4.纯化HerFab-L177AF与炔探头的共轭使用应变促进叠氮炔环加成（SPAAC） <ol><li> H中2 O添加经Cy5.5 Azadibenzocyclooctyne（经Cy5.5艾迪宝）20μL（20081，M终浓度）至HerFab-L177AF10μL的（10μM终浓度）在含有10mM 的 Na 2 HPO 4（pH7.0）中和100mM NaCl的磷酸盐缓冲液的溶液。 注意:作为替代方案，二氟化cyclooctyne（DIFO）和二环[6.1.0] nonyne（BCN）的衍生物也可用于相同的应用。 </li><li>允许应变促进的环加成反应，以在37℃下继续进行6小时。如果光敏探测器 （ 如经Cy5.5）时，用铝箔覆盖的反应容器中。 </li><li>根据步骤5纯化标记HerFab。 </li></ol> 5.净化标记HerFab的<ol><li> （0807）500μL于离心式过滤器离心柱。 </li><li>在14000 xg离心离心旋转柱在4℃15分钟。 </li><li>废弃流过和转移从旋转柱到1.5mL微量离心管中纯化的样品。 </li><li>作为替代步骤5.1- 5.3，执行purifica通过针对相同缓冲液透析和灰。 </li><li>存储纯化的标记HerFab在4℃。 </li></ol>标记HerFab 6. SDS-PAGE分析<ol><li>添加含有106毫摩尔Tris·HCl的，141毫摩尔Tris碱（pH为8.5），2％的LDS，10％甘油，0.51毫摩尔EDTA，0.22毫SERVA蓝G250，和0.175毫摩尔酚红以13微升5微升LDS蛋白质样品缓冲液在存在（+）或不存在纯化的标记HerFab（7.8微米或0.38毫克/毫升）（ - ）2微升二硫苏糖醇（DTT，100mM的终浓度）。孵育在95℃下该混合物10分钟。 </li><li>附加4-12％的Bis-Tris SDS-PAGE凝胶盒的电泳细胞并添加缓冲液。加载轭蛋白质样品与预染的分子量标志物。在200五35分钟进行凝胶电泳 注意:保持在黑暗中的电泳单元中的整个期间，以尽量减少荧光团的光致漂白。 </li><li>电泳后，转移该凝胶的荧光凝胶扫描仪，并在适当的波长扫描荧光发射。对于经Cy5.5，使用扫描仪软件Cy5的模式。 </li><li>染色用市售的蛋白质染色的凝胶。 </li></ol>

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The authors have nothing to disclose.

的非天然氨基酸遗传掺入蛋白质具有在用于蛋白质修饰的其它方法的几个优点。 1-3其中一个重要的优点是它的通用性的任何种类的蛋白质。原则上，存在选择目标蛋白质和该蛋白质的目标部位没有限制。然而，替换为结构上或功能上重要的残基的一个UAA可能导致改变靶蛋白的结构和功能。通常，暴露于溶剂，并且不与其他残基相互作用的残基被选择用于UAAs的掺入。因此，从一个高分辨率晶体结构的结构信息，以便选择为UAA掺入最佳位置被使用。 30由于UAAs的掺入在技术上是容易的，需要一个简单的诱变，多个站点可以容易地筛选，以选择最佳的残基为靶蛋白的期望的功能。&lt;/ P&gt; 在这个协议中，AF是基因掺入抗体片段，和突变片段是位点特异性用荧光团标记，用SPAAC。该方法的缀合产率是没有任何不希望的反应，这是在上一次报告的重要改进定量的。 25这是通过仔细的结构分析，以便获得最佳站点的选择，并增加了反应时间实现。因此，该站点为UAA掺入的选择是用于快速和定量的偶联是至关重要的。另外，利用高效的UAA掺入系统（ 例如 ，pEvol-AFRS）也是蛋白质产量和掺入效率的关键。 28 掺入蛋白质通过基因掺入法对蛋白质缀合其他UAAs也可用于抗体缀合。 4-11在反应速率方面，铜-催化的环加成反应，以及使用四嗪8和应变烯烃或炔烃的逆电子点播Diels-Alder反应，7将比在本研究中使用的SPAAC更好。但是，铜-催化的环加成反应需要铜（I）离子和配体，31和氨基酸的Diels-Alder反应通常是合成具有挑战性的，而不是为了定量结合不够稳定。酮类羟胺缩合4也可用于同样的目的。但是，它需要适度酸性条件下，其反应率比SPAAC的慢。考虑如非天然氨基酸的商业和合成可达性，反应速度，生物正交性，反应条件的生物相容性，和非天然氨基酸掺入效率，使用SPAAC和基因的方法的因素注册成立的AF将是显影改性有用治疗性蛋白质，例如ADC，以及用于一般蛋白标记。

由于在大肠杆菌 p -methoxyphenylalanine的遗传掺入报道，1 100多的非天然氨基酸（UAAs）已经被成功地结合到各种蛋白质。 1-3在这些UAAs，含生物正交功能基的氨基酸已被广泛研究，并代表的比例最大。在UAAs使用的生物正交功能团包括酮，4-叠氮基，5炔，6 cyclooctyne，7四嗪，8α，β不饱和酰胺，9降冰片烯，10 transcyclooctene，11和二环[6.1.0] -nonyne。 11虽然每个官能团都有其优点和缺点，在含叠氮基氨基酸被最广泛地用于蛋白质缀合。 p -Azidophenylalanine（AF），含叠氮基氨基酸之一，是容易得到的，它的掺入efficiency优异。含该氨基酸的突变蛋白可以通过铜催化环加成或与由SPAAC cyclooctynes炔进行反应。 12-20 近日，生物制药已在制药行业中引起极大关注。抗体-药物缀合物（ADC）是一类是有利的治疗性抗体的，由于其对人类癌症21和治疗靶向治疗能力 等疾病。超过50的ADC是目前在临床试验中，并且数量正在迅速增加。 ADC中的发展，需要考虑到最大限度的效力和减少副作用的许多因素。在这些因素中，一个高效的和位点特异性缀合反应以形成抗体之间形成共价键和一个药物是至关重要的。在缀合反应所需的效率和特异性可以通过缀合有生物正交功能组中的一个来实现是专门结合到抗体的非天然氨基酸。 22-26在这里，我们报告一个协议来位点特异性结合 AF成的抗体片段和缀合的突变体抗体片段与生物化学探针。

<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

有可用的化学探针引入蛋白质以研究它们的结构和功能目前许多化学的工具。一种有用的方法是蛋白质缀合通过基因引入含有生物正交官能团的非天然氨基酸。这份报告介绍了位点特异性抗体偶联了详细的方案。该协议包括用于含叠氮氨基酸的遗传掺入，并通过应变促进的叠氮化物 - 炔环加成（SPAAC）的缀合反应的实验细节。该菌株促进的反应通过在生理pH和温度的反应的分子的简单混合进行，并且不需要另外的试剂如铜（I）离子和铜的螯合配体。因此，这种方法将是一般蛋白质偶联和抗体药物偶联物（ADC）的开发是有用的。

<html>在这项研究中，抗体片段是位点特异性通过将一种含叠氮基氨基酸插入片段和突变体抗体片段与应变cyclooctyne反应用荧光团缀合（ 图1）。 HerFab被选为到其中的AF被合并为含叠氮基氨基酸的靶抗体片段。选择在HerFab残余物为带有AF更换，HerFab的X射线晶体结构进行了分析。对残余物30的重要要求包括来自抗体结合位点足够的距离，以尽量减少有效的缀合反应在其结合亲和力的减少和溶剂可及性。亮氨酸在177位是一个体面候选者，因为残余物以及暴露于所述抗体的外面并位于两个免疫球蛋白结构域的界面，这是来自抗体结合位点较远邻近。 内容"FO:保持-together.within页="1"&gt;最初，HerFab具有C-末端His 6 -tag的表达质粒构建，和琥珀密码子是在位置177引入用于通过现场AF掺入-directed诱变。在由共表达的进化的tRNA 酪氨酸和氨酰基tRNA合成酶对的存在1mM的自动对焦的表达含AF突变HerFab。在存在表达的突变蛋白质进行27 Ni-NTA亲和纯化缺席AF及以下SDS-PAGE分析的结果表明是在自动对焦的情况下只得到的全长抗体的片段（ 图2）。然后，将含有AF突变抗体片段进行了评价用于与经Cy5.5偶联反应 - 连接氮杂dibenzocyclooctyne衍生物（经Cy5.5艾迪宝）的抗体片段和经Cy5.5艾迪宝在磷酸盐缓冲液中反应6小时，并通过SDS-PAGE分析反应混合物中的存在（+）和缺席<html> （ - ）DTT（ 图3a）的。 25与野生型抗体片段反应也进行了分析作为对照。荧光图像清楚地显示出与经Cy5.5艾迪宝突变抗体片段缀合，而在与野生型片段在反应中没有观察到结合。电喷雾电离质谱（ESI-MS）分析显示定量缀合没有检测到未结合的片段（ 图3b）。总的来说，这些结果表明，含有AF突变HerFab可以是具有由SPAAC应变cyclooctynes有效和方便地缀合没有任何额外的试剂。 <img alt="图1" src="/files/ftp_upload/54922/54922fig1.jpg" /> 图1:由SPAAC位点特异性抗体标记的示意图。空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54922/54922fig2.jpg" /> 图2: 含AF在演进的tRNA /的aaRS对的存在位置177（L177）突变HerFab的表达。 HerFab-L177AF在LB培养基中表达含有演变的tRNA / AFRS对和1毫米自动对焦。 （ - ）纯化样品通过SDS-PAGE在存在（+）或不存在分析的DTT，并且将凝胶用商业蛋白染色剂染色。图改编自柯，W。 等。 27。 <a href="http://ecsource.jove.com/files/ftp_upload/54922/54922fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54922/54922fig3.jpg" /> 图3: 偶联反应HerFab-L177AF离子与经Cy5.5艾迪宝。 （a）用经Cy5.5艾迪宝野生型HerFab和HerFab-L177AF的偶联反应的SDS-PAGE分析。 （ - ）的反应混合物中的存在（+）和没有进行分析的DTT，以及蛋白质带由商业蛋白染色（左）和荧光成像（右）可视化。 （ 二 ）HerFab-L177AF（左）和HerFab-L177AF标记经Cy5.5艾迪宝（右）。ESI-MS分析:HerFab- L177AF和共轭抗体= 1,190道尔顿，之间预期质量差观测质量差= 1,190达。图改编自柯，W。 等。 27。 <a href="http://ecsource.jove.com/files/ftp_upload/54922/54922fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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).

高效抗体标记由应变叠氮推广 - 炔环加成特定站点

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

20.6K 次观看.  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).

视频: 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.

微流控芯片上捕获环加成反应可逆地固定小分子或多组分结构的生物传感器应用

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

科研

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.

一个优化的协议通过使用金纳米粒子的放射性标记效率<sup&gt; 125</sup&gt;我标记的叠氮辅基

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.

通过Bioorthogonal点击化学化学选择性病毒表面改性

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

免疫与感染

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.

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

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.

用快速 Bioorthogonal 反应生成抗体-药物共轭物的非规范氨基酸的遗传编码

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.

硫醇反应试剂的合成与生物共轭作用--用于原位选择性修饰免疫共轭的生成

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 ...

生物化学

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.

抗体标记用荧光染料使用磁蛋白A和G蛋白珠

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.

使用荧光抗生素探针对细菌耐药性的可视化

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.

高效抗体标记由应变叠氮推广 - 炔环加成特定站点

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