Name: synthesis of protein bioconjugates via cysteine-maleimide chemistry
Uploaded: 2016-07-20T15:00:00.000+00:00
Duration: 9 min 14 s
Description: Watch this Scientific Journal Video about Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry at JoVE.com

We thank the Australian Research Council (ARC) for ARC Future Fellowship (FT120100101) and ARC Centre of Excellence CE140100036) grants to P.T. and the Mark Wainwright Analytical Centre at UNSW for access to mass spectrometry and NMR facilities.

注意: 如图1下面协议设计用于蛋白质-染料生物共轭的合成，可用于与含有游离表面半胱氨酸蛋白马来酰亚胺的反应的一般协议，与笔记插入在适用与膜蛋白来协助。生物共轭物，蛋白质类聚合物生物缀合物，以及合成的蛋白二聚体（蛋白质 - 蛋白质）生物缀合物。在这种特殊情况下，所述蛋白质异1色素c具有可用下反应一个表面半胱氨酸残基，它允许发生高度特异性的标记。如果感兴趣的蛋白具有多个半胱氨酸残基，相同的协议适用于，尽管有特异性和产品均匀性的损失。化学定位表面赖氨酸残基，用N- -hydroxysuccinimidyl酯或异硫氰酸酯，可以是如果不需要的特异性的简单的方法。 <img alt="图1" src="/files/ftp_upload/54157/54157fig1.jpg" /> <strong&gt;图1. 生物缀反应流程。作为一个例子的情况下，光收集，基于钌天线分子将通过迈克尔加成在基于钌天线分子的侧马来酰亚胺和暴露的半胱氨酸残基附着到细胞色素C（CYS102）上的蛋白。在细胞色素C面的红色区域表示血红素组。 <a href="https://www.jove.com/files/ftp_upload/54157/54157fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 1，细胞色素C的分离纯化注意:此步骤是并不适用于所有的蛋白质。但是，重要的是要知道，从商业供应商获得的蛋白质可含有可能需要通过进一步纯化13去除其他不希望的蛋白同种型。 <ol><li>在1升的超纯水以溶解2.4克磷酸二氢钠（男女 = 120克摩尔-1）公关epare含有20mM的NaH 2 PO 4缓冲溶液。调节pH用1M NaOH至pH 7。 注意:在这个协议中使用的磷酸盐缓冲剂应新鲜每天制备，并通过在使用前用0.2μm的纤维素膜过滤器过滤。 </li><li>溶解在500毫升20毫摩尔的NaH 2 PO 4缓冲29.22克氯化钠（M W =58.44克摩尔-1）做出的20mM的NaH 2 PO 4和1M NaCl的缓冲液中。这是为在步骤1.6）中纯化的洗脱缓冲液。 </li><li>在6ml pH为7的20mM磷酸盐缓冲液中溶解12.0毫克冻干的细胞色素C（色素C）的。 </li><li>分别在95.3微升超纯水溶解14.7毫克二硫苏糖醇（DTT中，M W = 154.25克/摩尔）以制备1M的原液。 注意:DTT原液应新鲜制备，如该试剂易受在水溶液中的氧化失活。 </li><li>果仁ETTE 60微升的1M DTT溶液到该蛋白质溶液中，以减少色素C。溶液的颜色从暗红混合时变为淡红色。 </li><li>注入到任何色谱介质之前过滤通过0.22μm低蛋白结合的PVDF针筒过滤器还原蛋白溶液。 </li><li>附加注射环和一个快速蛋白质液相色谱（FPLC）仪器的UV-Vis检测之间的3.3毫升强阳离子交换柱。 </li><li>用超纯水的3倍柱体积，随后的20mM pH7的磷酸盐缓冲液3倍柱体积平衡色谱柱，使用1毫升/分钟的流速。 </li><li>负载1毫升降低原油色素C的进样环，并开始从328梯度方法- 450毫米氯化钠，超过5柱体积。监视UV-Vis检测的280纳米和410纳米的通道，并收集最大峰。 </li><li>增加盐浓度至1M为2倍柱体积洗脱异2细胞色素权证 ，并且列被刷新后，用20mM磷酸缓冲液2倍柱体积注入下一个粗等分试样之前重新平衡色谱柱。 </li><li>直到所有的粗蛋白已经被净化重复步骤1.9-1.10。 </li><li>池的iso-1含有级分，并用3.5 kDa的分子量截留（MWCO）旋转过滤器，并在3000×g下离心集中起来。 </li><li>加载的浓缩蛋白质进入3.5 kDa的截留分子量透析磁带和透析对超纯水过夜，水2的变化。 </li><li>通过采取10微升，其用超纯水稀释至100μl，并采取的吸收光谱确定的纯度，浓缩蛋白溶液的浓度。使用低容量，100微升石英比色皿，以获得蛋白质的吸收光谱。通常情况下，未稀释的蛋白质浓度是50量级 - 100微米。 <ol><li>使用细胞色素C的特征为410nm的峰值量化concentratioN使用比尔-朗伯定律97.6 -1毫米-1的摩尔吸光值: A =ε×C×ι 其中A是吸光度，ε是摩尔吸光系数，c为以mM的浓度，ι是试管的在厘米13的路径长度。 </li></ol></li><li>此时，划分蛋白质在1ml等份并于-20℃保持冷冻直到需要它们。色素C可以冷冻和没有结构或功能丧失解冻，但重复周期将开始使蛋白质变性。 注:蛋白质忍受冰冻程度不同。例如，绿色荧光蛋白5,9-不应被冻结，而不是保存在冰箱中。 </li></ol> 2，细胞色素C生物共轭物的合成<ol><li>溶解0.9毫克钌（Ⅱ）bisterpyridine maleimIDE（钌（Ⅱ）（TPY）2 -马来酰亚胺，0.975微摩尔，6当量）在600微升的乙腈中。 注意:如果使用的马来​​酰亚胺可溶于水，制备在水代替乙腈原液。为马来酰亚胺可溶于反应缓冲液是重要的。二甲亚砜，N，N-二甲基甲酰胺和乙腈中所有常用的辅助剂，以帮助在低分子量12，常疏水马来酰亚胺反应物的溶解。 </li><li>制备含有100mM磷酸盐缓冲液和100mM乙二胺四乙酸（EDTA中，M W =292.24克/摩尔），其中，当稀释至20mM的pH为7.加入固体氢氧化钠直到EDTA溶解（几克典型地为缓冲溶液）并调节pH至7。 </li><li>溶解2.87毫克三（2-羧乙基）膦盐酸盐（TCEP 15中，M W = 286.65克/摩尔）在1ml超纯水中，以制备10mM储备溶液。这一步是进口蚂蚁，以确保蛋白质上半胱氨酸完全成马来酰亚胺偶联15之前降低。 注意:TCEP原液应新鲜每个反应来制备，因为这种试剂是容易在水溶液中的氧化失活。 </li><li>结合11.4毫升超纯水与50毫升的塑料管3毫升100mM磷酸/ EDTA储液。当与蛋白质和马来酰亚胺储备溶液进一步稀释缓冲液浓度为20毫摩尔。 注意:如果被标记的蛋白是跨膜蛋白，确保一个合适的洗涤剂加入到缓冲以溶解蛋白质。如果不使用洗涤剂的膜蛋白可以慢慢沉淀，这将不利地影响反应产率。使用该洗涤剂中的所有随后的纯化步骤。 </li><li>添加0.15微摩尔（3毫升50微米的溶液，1当量）纯化色素C的对磷酸EDTA缓冲液。 </li><li>加入7.5微升的TCEP股票SOLUT的离子（0.075微摩尔​​，0.5当量）的蛋白质溶液和休假搅拌5分钟，以减少可能由于半胱氨酸氧化二聚化的任何蛋白质。 注意:不要添加TCEP如果感兴趣的蛋白质不容易二聚化。通过非还原条件​​下运行的蛋白质凝胶检查。 </li><li>添加600μl的钌（II）（TPY）2 -马来酰亚胺到细胞色素C的减少，缓冲溶液的乙腈溶液，并留下该反应混合物搅拌，在黑暗中，在室温，24小时。 </li><li>集中使用3.5 kDa的MWCO自旋过滤器将反应混合物，离心以3,000 xg离心，用新鲜的20 mM磷酸盐缓冲液重复2-3次，直到滤液变清。纯化的下一阶段之前，除去从反应混合物中尽可能多的未反应的马来酰亚胺。 注意:在这一点上，将粗生物结合混合物可以储存在冰箱内，在黑暗中。它以消除CENTR未反应的马来酰亚胺染料是重要的ifuge储存前透析作为马来酰亚胺可以在蛋白质的表面上的赖氨酸残基慢慢和非特异性反应。 </li></ol> 3.细胞色素C生物共轭物的分离纯化<ol><li>溶解2.4克磷酸二氢钠和在1升超纯水29.22克氯化钠以制备含有20mM的NaH 2 PO 4和0.5M NaCl的缓冲液。这是用于固定化金属亲和层析（IMAC）纯化的运行缓冲液。 </li><li>溶解在500毫升20毫摩尔的NaH 2 PO 4和0.5M NaCl的缓冲液为17克咪唑（M W =68.077克摩尔-1）。这是对IMAC纯化的洗脱缓冲液。 </li><li>调整两个缓冲液的pH到使用1M的NaOH或HCl pH为7，并在FPLC使用前通过0.22μm的再生纤维素膜过滤。 </li><li>由于购买的IMAC柱出厂时没有加载到山坳任何金属离子UMN，通过用3ml的超纯水，将3ml 100mM的乙酸镍溶液，和6ml的超纯水洗涤柱制备了镍离子为基础的纯化塔。如果该列没有被立即使用冲洗通过3毫升20％乙醇和列存储在冰箱，以防止细菌的生长。 </li><li>附加的Ni 2+ -loaded1毫升IMAC柱的FPLC注入环和UV-Vis检测器之间。 </li><li>用20mM磷酸的3倍柱体积，用0.5毫升/分钟的流速0.5M NaCl的缓冲液平衡该柱。 </li><li>负载100微升对Ni 2+ IMAC柱粗反应混合物（通过0.22μm针筒过滤器过滤）的。用运行缓冲液以洗脱未反应的色素C的3倍柱体积，随后从0咪唑梯度的125mM以上3倍柱体积洗涤柱以洗脱钌bisterpyridine-色素c生物共轭（钌（Ⅱ）-cyt 三 ） 。 </li><li>洗净的Column用5柱体积的250 mM咪唑缓冲液，然后用20 mM磷酸，0.5M NaCl和重复步骤3.7，直到所有的粗生物共轭已被纯化重新平衡色谱柱。 </li><li>凝聚生物共轭级分，并用3.5 kDa的MWCO旋转过滤器，以3000×g的离心分离集中起来。 </li><li>装载集中到生物共轭物3.5 kDa的截留分子量透析磁带和透析对超纯水过夜，水2的变化。 </li><li>确定由UV-VIS纯，浓缩生物共轭溶液的浓度，使用相同的摩尔吸光值作为异1色素c（97.6毫-1厘米-1）在410纳米。通常情况下，生物共轭浓度为50量级 - 100微米。 </li><li>划分成生物共轭物25微升等分，并在-20°C保持冷冻直到需要。 </li></ol> 4.表征细胞色素C的生物共轭<ol><li> 阻止通过MALDI-TOF质谱生物共轭质量mination <ol><li>溶解10毫克的咖啡酸的在1毫升乙腈/水/三氟乙酸溶液（80:20:0.1，V / V / V）。 </li><li>稀释5μl的浓缩的蛋白质溶液与5微升的咖啡酸​​溶液。 </li><li>点0.5微升在MALDI靶盘咖啡酸溶液，并使溶液干燥。 </li><li>现货0.5微升这一咖啡酸点上方的采样/矩阵解决方案，允许当场晾干。发现在此之上，以"三明治"基质的层间的试样另一0.5微升样品基质的，并晾干。 </li><li>使用合适的仪器设置的蛋白质16取得以线性模式质谱。 </li></ol></li><li> 通过凝胶电泳生物缀合物的调查 <ol><li>制备10微升每个蛋白质样品以稀释蛋白质样品与预混合十二烷基硫酸锂（LDS，pH值8.4）缓冲液（4次）T为运行OA的每孔约20微克最终浓度。对于需要还原条件下水井，加入1微升500二硫苏糖醇（DTT）的还原剂（10X）的。 </li><li>热样品在70℃下10分钟。 </li><li>加50预混毫升的1M MES，1M Tris碱，2％SDS，20毫摩尔EDTA（pH值7.7）从商业来源至950毫升的超纯水的运行缓冲液混合物（20倍），以制备凝胶电泳缓冲液。 </li><li>从凝胶中去除塑料梳子，并放置在凝胶运行槽中的凝胶。使孔覆盖有缓冲填充凝胶电泳缓冲液的罐。 </li><li>装载样品仔细到预制12％双Tris，1毫米，使用长枪头10孔凝胶，以协助装载。加载预染10蛋白质分子量标记物（3-188 kDa的）到凝胶的一个中间孔以有助于分析。 </li><li>运行在200伏的恒定电压的凝胶35分钟。 </li><li>染色用2小时商业考马斯蓝溶液的凝胶，并用ultrap洗URE水中48小时。 </li></ol></li><li> 生物共轭纯度通过UV-Vis光谱测定 <ol><li>制备120微升钌（Ⅱ）（TPY）2 -马来酰亚胺，异1细胞色素c和 ​​钌（II）-cyt 的c为5μM的解决方案。 </li><li>测量仅含有超纯水的石英比色皿的基线频谱，从250纳米到650纳米。 </li><li>测量每个元件的光谱，确保了反应杯漂洗和每次测量之间干燥。 </li><li>绘制每个部件的吸光度作为波长的函数，并且与最终产品相比较的原料的线性总和，以确定是否一个1:钌（II）（TPY）2 -马来酰亚胺来色素C的1比已反应。 </li></ol></li></ol>

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(3), 1-21 (2014).</li><li>Peterson, J. R., Thordarson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+the+purification+of+terpyridine-cytochrome+c+bioconjugates.">Optimising the purification of terpyridine-cytochrome c bioconjugates.</a> Chiang Mai J. Sci. 36 (2), 236-246 (2009).</li><li>Hvasanov, D., Mason, A. F., Goldstein, D. C., Bhadbhade, M., Thordarson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+the+synthesis,+polymer+membrane+encapsulation+and+photoreduction+performance+of+Ru(II)-+and+Ir(III)-bis(terpyridine)+cytochrome+c+bioconjugates.">Optimising the synthesis, polymer membrane encapsulation and photoreduction performance of Ru(II)- and Ir(III)-bis(terpyridine) cytochrome c bioconjugates.</a> Org. Biomol. Chem. 11 (28), 4602-4612 (2013).</li><li>Signor, L., Boeri Erba, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+Laser+Desorption/Ionization+Time+of+Flight+(MALDI-TOF)+Mass+Spectrometric+Analysis+of+Intact+Proteins+Larger+than+100+kDa.">Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa.</a> J. Vis. Exp. , e50635 (2013).</li><li>Foucher, M., Verdi&#232;re, J., Lederer, F., Slonimski, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+presence+of+a+non-trimethylated+iso-1+cytochrome+c+in+a+wild-type+strain+of+Saccharomyces+cerevisiae).">On the presence of a non-trimethylated iso-1 cytochrome c in a wild-type strain of Saccharomyces cerevisiae).</a> Eur. J. Biochem. 31, 139-143 (1972).</li><li>M&#252;ller, M., Azzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+labeling+of+beef+heart+cytochrome+oxidase+subunit+III+with+eosin-5-maleimide.">Selective labeling of beef heart cytochrome oxidase subunit III with eosin-5-maleimide.</a> FEBS Lett. 184 (1), 110-114 (1985).</li><li>Shen, B. Q., 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=Conjugation+site+modulates+the+in+vivo+stability+and+therapeutic+activity+of+antibody-drug+conjugates.">Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.</a> Nat. Biotechnol. 30 (2), 184-189 (2012).</li></ol>

The authors have nothing to disclose.

一个生物结合前的原料纯化是非常重要的。从商业的重组来源得到的蛋白质常含有目的蛋白质，其可具有不同的表面化学和反应的其它同种型。例如，在所描述的生物结合，市售细胞色素C包含两个异1和异2色素C 12,14,17的混合物。异2和异1形式的细胞色素c在很大程度上是同源的，与主要的区别是异1细胞色素C的C-末端附近的一个游离半胱氨酸残基的存在?...

生物缀涉及共价连接一种生物分子与另一个或同一个合成的分子，例如染料，药物或聚合物。蛋白质的生物耦合方法现在广泛应用于许多化学，生物和纳米技术研究小组使用，应用范围从荧光染料标记1,2，使蛋白质（抗体）-prodrugs 3（抗体偶联药物- ADC）的蛋白二聚体4,5的合成通过在纳米8和系统化学9中使用的自组装蛋白-聚合物杂种6,7。 用于生物结合化学的特异性，而并非总是关键的，是对大多数功能性蛋白质生物共轭物极为重要，以便不与目标蛋白的活性位点干扰。提供了理想生物缀反应需要满足若干标准，包括:感兴趣的蛋白质上ⅰ）靶向稀有或独特的位点，ⅱ）是有选择性的实现这一目标，ⅲ）非变性条件下进行，以避免蛋白质折叠和iv）是高产作为目标蛋白质通常只适用于子毫摩尔浓度。马来酰亚胺-半胱氨酸迈克尔加成接近满足所有这些标准，并具有该原因长权利生物共轭化学10的字段的特殊状态。这是因为，ⅰ）在其表面上仅含有一个半胱氨酸残基的许多蛋白质​​可以遗传工程那里，ⅱ）在正确的pH值的反应是朝着半胱氨酸高度选择性，ⅲ）它可顺利地进行在水性缓冲液和iv）它是非常快的与马来酰亚胺的二阶速率常数报告超过5000 M -1秒-1在某些情况下11含有半胱氨酸的蛋白质。提供感兴趣的蛋白质可以容忍有机的一个小的（≈5-10％）量的共溶剂12，几乎任何马来酰亚胺官能化的染料，婆lymer，表面或另一种蛋白可以链接到蛋白质。此外，马来酰亚胺是对蛋白质比碘乙酰胺，这是更容易在升高的​​pH值的其他亲核体进行反应的半胱氨酸更具体;比这需要在酸性pH值被保持，以防止二硫键交换13基于二硫键缀合更稳定。 此处我们报告马来酰亚胺官能化的分子的缀合的通用协议包含使用钌（Ⅱ）基的发色团和氧化还原蛋白细胞色素C作为例子之间的反应的单个半胱氨酸残基的蛋白质。这个协议同样适用于含有可接近表面的半胱氨酸残基和相应的马来酰亚胺官能化的靶大多数其他蛋白，无论是另一种蛋白，荧光染料，发色团或合成聚合物。

<table><tbody><tr><td>sodium dihydrogen phosphate</td><td>Sigma-Aldrich</td><td>71496</td><td></td></tr><tr><td>sodium hydroxide</td><td>Sigma-Aldrich</td><td>71691</td><td></td></tr><tr><td>sodium chloride</td><td>Sigma-Aldrich</td><td>73575</td><td></td></tr><tr><td>cytochrome &lt;em&gt;c&lt;/em&gt;, from saccaromyces cerevisiae</td><td>Sigma-Aldrich</td><td>C2436</td><td></td></tr><tr><td>dithiothreitol</td><td>Sigma-Aldrich</td><td>43819</td><td></td></tr><tr><td>TSKgel SP-5PW</td><td>Sigma-Aldrich</td><td>Tosoh SP-5PW, 07161</td><td>3.3 ml strong cation exchange column</td></tr><tr><td>Amicon Ultra-15&amp;nbsp;</td><td>Merck-Millipore</td><td>UFC900308</td><td>3.5 kDa spin filter</td></tr><tr><td>Slide-A-Lyzer mini dialysis units</td><td>Thermo Scientific</td><td>66333</td><td>3.5 kDa dialysis cassetes</td></tr><tr><td>Ru(II) bisterpyridine maleimide</td><td>Lab made</td><td></td><td>see ref (14)</td></tr><tr><td>acetonitrile</td><td>Sigma-Aldrich</td><td>A3396</td><td></td></tr><tr><td>ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>03609</td><td></td></tr><tr><td>tris(2-carboxyethyl)phosphine hydrochloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>93284</td><td></td></tr><tr><td>imidazole</td><td>Sigma-Aldrich</td><td>56749</td><td></td></tr><tr><td>nickel acetate</td><td>Sigma-Aldrich</td><td>244066</td><td></td></tr><tr><td>AcroSep IMAC Hypercell column</td><td>Pall</td><td>via VWR: 569-1008</td><td>1 ml IMAC column</td></tr><tr><td>0.2 micron cellulose membrane filter</td><td>Whatman</td><td>Z697958</td><td>47 mm filter for buffers</td></tr><tr><td>0.2 micron PVDF membrane filter</td><td>Merck-Millipore</td><td>SLGV013SL</td><td>syringe filters for proteins</td></tr><tr><td>hydrochloric acid</td><td>Sigma-Aldrich</td><td>84426</td><td>Extremely corrosive! Use caution.</td></tr><tr><td>caffeic acid</td><td>Sigma-Aldrich</td><td>60018</td><td>MALDI matrix</td></tr><tr><td>trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>91707</td><td>extremely corrosive! Use caution</td></tr><tr><td>SimplyBlue SafeStain</td><td>Thermo Scientific</td><td>LC6060</td><td>Coomassie blue solution</td></tr><tr><td>NuPAGE Novex 12% Bis-Tris Gel</td><td>Thermo Scientific</td><td>NP0342BOX</td><td>precast protein gels</td></tr><tr><td>SeeBlue Plus2 Pre-stained Protein Standard</td><td>Thermo Scientific</td><td>LC5925</td><td>premade protein ladder</td></tr><tr><td>NuPAGE LDS Sample Buffer (4x)</td><td>Thermo Scientific</td><td>NP0008</td><td>premade gel sample buffer</td></tr><tr><td>NuPAGE Sample Reducing Agent (10x)</td><td>Thermo Scientific</td><td>NP0004</td><td>premade gel reducing agent</td></tr><tr><td>NuPAGE MES SDS Running Buffer (20x)</td><td>Thermo Scientific</td><td>NP0002</td><td>premade gel running buffer</td></tr><tr><td>Voyager DE STR MALDI reflectron TOF MS</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>Acta FPLC</td><td>GE</td><td></td><td>Fast Protein Liquid Chromatography</td></tr><tr><td>Cary 50 Bio Spectrophotometer</td><td>Varian-Agilent</td><td></td><td>UV-Vis</td></tr><tr><td>Milli-Q ultrapure water dispenser</td><td>Merck-Millipore</td><td></td><td>ultrapure water</td></tr><tr><td>Low volume UV-Vis Cuvette</td><td>Hellma</td><td>105-201-15-40</td><td>100 microliter cuvette</td></tr></tbody></table>

synthesis of protein bioconjugates via cysteine-maleimide chemistry

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

生物缀合物的合成通过三种主要方法证实:飞行质谱（MALDI-TOF MS），聚丙烯酰胺凝胶电泳的基质辅助激光解吸电离时间，和紫外可见（UV-VIS）光谱， 如图2所示，图3和4。对应于所附小分子的质量的质量增加，并且缺乏未反应蛋白质的演示钌（Ⅱ）的成功的共价键（万吨）2 -马来酰亚胺到细胞色素c和 ​​生物共轭的随后?...

Watch this Scientific Journal Video about Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry at JoVE.com

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

该协议细节对于含有蛋白质马来酰亚胺，包括试剂纯化，反应条件，生物共轭纯化和生物共轭表征半胱氨酸的生物结合所需的重要步骤。

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

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

synthesis-of-protein-bioconjugates-via-cysteine-maleimide-chemistry

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Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Chemical Applications of Statistical Analyses

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38.3K 次观看.  The University of New South Wales. 该协议细节对于含有蛋白质马来酰亚胺，包括试剂纯化，反应条件，生物共轭纯化和生物共轭表征半胱氨酸的生物结合所需的重要步骤。 

视频: Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

合成方法通过固相树脂为基础的方法不对称二茂铁产生的生物共轭系统

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

科研

化学

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

半胱氨酸残基的溶剂可及性分析<em&gt;玉米rayado菲诺病毒</em病毒样颗粒<em&gt;烟草</em植物和交联的肽类病毒颗粒

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

免疫与感染

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

利用光致硫醇-烯/yne Hydrothiolation 构造硫醚类/乙烯基硫化物栓系螺旋肽

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

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 Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

不同二硫 Connectivities µ-芋螺毒素 PIIIA 异构体的合成与结构测定

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

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

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

仿生材料表征细菌宿主相互作用

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

生物工程

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

蛋白质生物缀合物的合成

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