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>

<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.

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 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

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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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

20.5K 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

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.

小分子结合蛋白的鉴定在天然细胞环境由活细胞光亲和标记

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

科研

生物化学

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.

可逆二硫键交联胶束生产的简便和有效途径

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.

实验室规模的生产和治疗性抗体的纯化

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.

增强组织样品使用组合前体同位素标记和标记等压的复用（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.

用肽微阵列分析组蛋白抗体特异性

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.

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

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.

化学感受器配体结合域的高效复用与纯化方法

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.

肾小球的分离及肾小球细胞表面蛋白的活体标记

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.

单分子水平标记均匀性的全蛋白质定量

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.