This research was funded by the Office of Naval Research through Core funds provided to the Naval Research Laboratory.

1质粒的制备<ol><li>制备融合到双正交联动域的质粒（ 例如，pET22）含锚蛋白（OmpA的）（在这个协议称为锚-ST），表位标记（如用于纯化和鉴定的6xHis，myc基因或FLAG标签），周质定位标记，抗生素抗性，和基于所选择的细菌菌株7复制的适当的起源。 <ol><li>使用按照制造商的协议市售的DNA分离试剂盒过夜培养物中提取质粒DNA。 </li></ol></li><li>制备的质粒（ 例如，pACYC184的）含有酶/蛋白质（PTE）稠合到互补生物正交联动域到该锚蛋白的OMV中打包（在该协议作为酶-SC称为），表位标记，周质定位标记，抗生素抗性比该锚蛋白的质粒不同，以及适当的原稿在复制的基础上，选择的细菌菌株7。 <ol><li>使用按照制造商的协议7市售的DNA分离试剂盒过夜培养物中提取质粒DNA。 </li></ol></li></ol> 2.生成一个OMV包装大肠杆菌文化<ol><li>质粒隔离<ol><li>净化编码酶-SC构建和锚从ST使用商业质粒提取试剂盒根据制造商的协议7单独的维护细胞株构建质粒。通过在260nm和280nm测量吸光度确定DNA的浓度和纯度。 </li></ol></li><li>共转化<ol><li>选择的大肠杆菌菌株是市售便于蛋白的表达。 注:BL21（DE3）细胞这里利用。 T7启动溶源是所必需的锚-ST构建体的表达，这依赖于T7启动子和T7 RNA聚合酶prot的EIN表达。其他细菌菌株的使用需要T7启动溶源基因的任一的存在或使用除pET22 7其它的质粒。 </li><li>稀释纯化的质粒DNA，以当量摩尔浓度然后将其转化为按照制造商的对所选择的感受态细胞协议通过或者热休克转化或电穿孔感受态细菌细胞。 </li><li>板转化细胞上的Luria-BERTANI（LB）琼脂既氨苄青霉素（pET22）和氯霉素（pACYC184的）平板含抗生素的;同时存在以25微克/毫升的最终浓度。 </li><li>放置在37℃的培养皿过夜以分离含有两种质粒仅克隆。 注:菌落存活下来抗生素选择将被用于以后所有的OMV制剂。抗生素（氨苄青霉素和氯霉素）将被添加到所有液体培养，以保持选择压力，并确保两种质粒的存在。 </li></ol></li></ol> 3.生产OMV <ol><li>殖民地扩张<ol><li>接种5ml含在上述浓度抗生素的培养基（通常了不起肉汤（TB）或LB）（步骤2.2.3）的与来自2.2.4中描述的选择平板的单一菌落。 </li><li>执行1:隔夜发酵剂培养物100倍稀释到百思不得其解培养瓶中。 注:使用的媒体量250-500毫升之间。 </li><li>保持在37℃以250rpm振荡培养，以确保被达到的培养的足够通风。 </li></ol></li><li>每个质粒的感应<ol><li>允许细菌培养到主生长烧瓶接种后生长到OD的0.6-0.8 600，约3小时的增长。 </li><li>以改善PTE-SC加载囊泡的最终产率，离心整个培养在7000 xg离心15分钟。重悬在预热，新鲜培养基将细胞沉淀。 注:此选择有理步骤有助于除去任何空OMV中存在的前PTE-SC诱导培养基。 </li><li>使L-阿拉伯糖在水和无菌过滤器使用0.22微米过滤器的20％的原液。在4℃下2-4周商店L-阿拉伯糖溶液。 </li><li>的L-阿拉伯糖溶液向培养瓶中添加至最终0.2％的浓度来启动PTE-SC的生产。 注意:这有助于与PTE-SC周质空间预加载通过突变外膜蛋白A-ST诱导促进更多灸前。 </li><li>使用0.22微米过滤器中的水和无菌过滤的1M异丙基β-D-1-硫代半乳糖苷（IPTG）溶液。 注:IPTG必须立即使用一次保湿。 IPTG的等分试样可用于3-6个月被冷冻储存，并且可以立即解冻使用前。 </li><li>添加IPTG额外的3小时温育期以0.5mM的终浓度后启动生产的OmpA-ST的。 </li><li>允许CULTURE一边摇动在37℃下额外8-14小时以生长。 </li></ol></li></ol> 4.净化OMV <ol><li>完整的细菌去除<ol><li>离心15分钟，细菌培养介质在7000 xg离心在4℃以沉淀完整细菌。 </li><li>倒出，并保存，媒体脱细胞沉淀后离心，小心的顶部以不干扰细胞沉淀。 </li><li>重复步骤4.1.1和4.1.2一个附加离心循环，以确保所有的完整细菌从培养介质是确保倒出并保存介质中移除。 </li></ol></li><li>蛋白聚集体去除<ol><li>用0.45μm的膜过滤器，以除去残留的细菌和不需要的大蜂窝材料进一步纯化细菌培养基。 注意:使用的生物相容的膜材料为更高的回收率和较低的蛋白质吸附，如乙酸纤维素（CA）或聚偏二氟乙烯（PVDF）。注射器过滤体积小于100 ml和真空过滤量超过100毫升以上。 <ol><li>注射器过滤器，吸取培养液到无菌10-50毫升注射器。附加0.45微米的过滤器，注射器的露儿结束。按下柱塞和收集新船流通。 </li><li>真空过滤，培养基转移到一个无菌过滤装置。附加单位真空泵抽气下沉产生吸力。过滤介质转移到新船。 </li></ol></li></ol></li><li>通过监查超速离心分离<ol><li>经过滤的培养基加入到离心管小心在离心机来平衡对立管。 </li><li>在使用相应的转子匹配都在仪器和管道超速离心机沉淀在4℃〜15万xg离心3小时得到利用。 </li><li>从OMV颗粒倒出OMV耗尽培养基，小心不要去打扰OMV颗粒，立即在完成Ô˚F离心作为允许OMV粒料保留在媒体将软化颗粒减少OMV恢复。 注:该颗粒将是浅棕色，并根据细菌培养的起始量可以或可以不通过眼是可见的。直接从离心管顶部吸媒体也记住了更多的媒体从OMV去除沉淀的更纯净的最终OMV样本将是一种选择。 </li><li>加入以后动态光散射（DLS）检测的离心上清，以验证OMV颗粒完全从媒体耗尽。 </li><li>添加0.5-1毫升的pH 8.0的无菌过滤磷酸盐缓冲盐水（PBS）的pH为7.4或100mM的ñ -环己基-2-氨基乙磺酸（CHES）缓冲剂，以在监查粒料使其在室温下孵育30分钟的。确保颗粒不涸。 注:在溶液中的离子强度，pH值和极端温度可能会导致降低OMV溶解度和促进AGGREgation。 </li><li>小心吸取从离心管的纯化的OMV溶液，混合轻轻以确保整个粒料已经被溶解。 </li></ol></li></ol> 5. OMV表征<ol><li>使用任何市售DLS或纳米粒子跟踪软件，下面每个乐器制造商提供的协议，以确定OMV粒度分布和颗粒浓度。 注:在这里，决心利用NanoSight LM10纳米粒子跟踪和NTA 2.3分析软件OMV大小分布和浓度。 <ol><li>打开软件。 </li><li>打开显微镜和清洁显微镜和纳米粒子跟踪单元的所有表面。 </li><li>添加〜0.5直接OMV样品通过露儿用1毫升无菌注射器是一定要覆盖整个视窗没有任何注入空气接头粒子跟踪装置的视窗毫升泡到系统中。 </li><li>地方ŧ他纳米颗粒在显微镜舞台上跟踪装置和打开激光。 </li><li>在软件中点击"捕获"。 </li><li>调整显微镜的焦点和舞台可视化上存在的软件窗口的预览屏幕的颗粒。 </li><li>调整"相机快门"和"相机增益"，直到光明颗粒显然在深色背景上显示。 </li><li>捕获调整持续时间60秒。 </li><li>点击"记录"。 </li><li>出现提示时，输入采样/设备的温度，标签样品，并在每完成保存视频数据运行到用户指定的文件夹。 </li><li>保留所有的分析参数（检测阈值，模糊，闵履带长度，闵预期粒径）在自动检测模式，然后单击"进程序列。" 注意:请参阅如何每个参数都可以独立调节以微调的分析结果的手册。 </li><li>记录的粒度分布和颗粒/米作为由软件确定升浓度。 注:典型的OMV流体力学尺寸范围为30-200纳米之间具有约10×10 8颗粒/ ml的浓度。 </li></ol></li><li> OMV确定蛋白质含量<ol><li>使用标准SDS-PAGE和Western blot协议，以评估OMV纯度，产酶和SC-ST交联效率7。 注:交联效率不会100％7。 </li></ol></li></ol> 6.酶包装验证<ol><li> PTE活性测定<ol><li>在合适的缓冲液如100毫CHES缓冲液（pH 8）在25℃进行所有酶测定在任一比色皿或在96/384孔板多路复用。 </li><li>加入5微升1:在CHES缓冲氧磷的1000稀释到CHES 90和5μl纯化的OMV的（〜36倍浓缩相比OMV的浓度存在于培养基中）。 注意:对氧磷是一种有毒的农药必须按照制造商和机构的指导方针进行处理。 </li><li>采取定期吸光度读数（每20秒为〜2小时总反应时间），在405纳米（ε= 18.1毫米-1厘米-1）和348纳米（等色点ε= 5.4毫米-1厘米-1），以监视生色氧磷分解产物， 对硝基苯酚的反应级数。 注:在8以上的pH值P1硝基苯酚的优势去质子化形式存在，允许精确监测在405纳米。在所有其它复杂或较低pH溶液，有必要利用348纳米的等吸光点作为p硝基苯酚的多种形式将存在。 </li><li>通过确定从步骤6.1.3进度曲线的线性部分的斜率来比较每个样品，包装效率，和总的PTE生产PTE的相对量计算初始反应速度。 </li><li>执行标准的酶分析方案确定K M，V 最大和k 猫的OMV封装PTE。 注:双倒数分析可以用于测定这些动力学参数，其中y截距= 1 / V max和斜率= K M / V 最多 20个 。 </li></ol></li><li>跨膜基板/产品扩散。 <ol><li>如按体积步骤利用在CHES缓冲0-3％的Triton X-100的浓度逐渐增加6.1所述的相同的动力学测定分析。 注:加入Triton X-100，以溶解的监查的功能破坏脂质双层允许监查的内容是到反应溶液中自由地进行访问。 </li><li>计算并对比酶活性比较我在每次的Triton X-100级的初始速度，以验证在酶的活性变化最小（初始速度）指定的底物/产物的跨膜通道n中没有的Triton X-100。如果在酶的活性大幅增加的Triton X-100的存在下观察到的，很可能在衬底不能自由​​扩散到OMV。 </li><li>测定游离酶的活性（如以上在第6.1节中描述）中的Triton X-100的存在，以验证酶活性不是由表面活性剂本身抑制。 注意:如果活动很大程度上受到的Triton X-100，可替代的添加剂，如皂甙或各种聚山梨醇冲击/吐温表面活性剂可以更适合于破裂OMV膜。 </li></ol></li></ol> 7.存储OMV <ol><li>冷冻<ol><li>纯化OMV的地方〜100微升等分试样直接入液氮捕捉冷冻等分试样，确保没有完全淹没小瓶或接触任何液态氮与样品本身。 注:纯净OMV可卡入缓冲区直接冻结被选定为purificat中溶解的颗粒OMV的必要的7没有额外的冷冻保护剂离子的过程。 </li><li>储存在-80℃的冷冻单元等分。 </li><li>通过将它们在室温为确保不加热样品解冻从步骤7.1.1冷冻等分试样。 </li><li>在此之前准备的所有样本，储存以这种方式执行步骤6.1前相比初始速度和冻结，以验证是否存在酶活性后冻融无显著丢失后描述的酶活性测定。 注:储存在4℃下进行纯化的OMV如果在酶的活性降低是由于冷冻观察。 </li></ol></li><li>冻干<ol><li>冻干利用市售冷冻干燥设备21捕捉冻结纯化OMV等分。 </li><li>商店在RT冻干等份数周或在-80℃下许多个月。 </li><li>精制水同样体积添加至冻干的OMV作为冷冻干燥前，静置在室温30分钟以再水合吨他品尝。轻轻混匀，在必要的时候，确保没有涡旋或超声处理的OMV。 </li><li>直接加入冻干粉OMV点照护的应用场合之前，稀释/溶解是没有必要的。 </li></ol></li></ol>

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SpringerBriefs in Microbiology. , 15-34 (2012).</li><li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+OmpA+in+Escherichia+coli.">The function of OmpA in Escherichia coli.</a> Biochem. Biophys. Res. Commun. 292, 396-401 (2002).</li><li>Danoff, E. J., Fleming, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+soluble,+periplasmic+domain+of+OmpA+folds+as+an+independent+unit+and+displays+chaperone+activity+by+reducing+the+self-association+propensity+of+the+unfolded+OmpA+transmembrane+beta-barrel.">The soluble, periplasmic domain of OmpA folds as an independent unit and displays chaperone activity by reducing the self-association propensity of the unfolded OmpA transmembrane beta-barrel.</a> Biophys. Chem. 159, 194-204 (2011).</li><li>Alves, N. J., Kline, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+on+the+inhibition+of+plasmin+and+delta-plasmin+via+benzamidine+derivatives.">Comparative study on the inhibition of plasmin and delta-plasmin via benzamidine derivatives.</a> Biochem. Biophys. Res. Commun. 457, 358-362 (2015).</li><li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lyophilization+and+development+of+solid+protein+pharmaceuticals.">Lyophilization and development of solid protein pharmaceuticals.</a> Int. J. Pharm. 203, 1-60 (2000).</li><li>Goormaghtigh, E., Scarborough, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density-based+separation+of+liposomes+by+glycerol+gradient+centrifugation.">Density-based separation of liposomes by glycerol gradient centrifugation.</a> Anal. Biochem. 159, 122-131 (1986).</li><li>Alves, N. 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=Functionalized+liposome+purification+via+Liposome+Extruder+Purification+(LEP).">Functionalized liposome purification via Liposome Extruder Purification (LEP).</a> Analyst. 138, 4746-4751 (2013).</li><li>Mayer, L. D., StOnge, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+free+and+liposome-associated+doxorubicin+and+vincristine+levels+in+plasma+under+equilibrium+conditions+employing+ultrafiltration+techniques.">Determination of free and liposome-associated doxorubicin and vincristine levels in plasma under equilibrium conditions employing ultrafiltration techniques.</a> Anal. Biochem. 232, 149-157 (1995).</li><li>Blakeley, B. D., Chapman, A. M., McNaughton, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-superpositive+GFP+reassembly+is+a+fast,+efficient,+and+robust+method+for+detecting+protein-protein+interactions+in+vivo.">Split-superpositive GFP reassembly is a fast, efficient, and robust method for detecting protein-protein interactions in vivo.</a> Mol. BioSyst. 8 (2036), (2012).</li><li>De Crescenzo, G., Litowski, J. R., Hodges, R. S., O'Connor-McCourt, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+the+interacation+of+two-stranded+de+novo+designed+coiled-coils:+effect+of+chain+length+on+the+kinetic+and+thermodynamic+constants+of+binding.">Real-time monitoring of the interacation of two-stranded de novo designed coiled-coils: effect of chain length on the kinetic and thermodynamic constants of binding.</a> Biochemistry. 42, 1754-1763 (2003).</li><li>Charalambous, A., Antoniades, I., Christodoulou, N., Skourides, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-Intein+for+simultaneous+site-specific+conjugation+of+quantum+dots+to+multiple+protein+targets+in+vivo.">Split-Intein for simultaneous site-specific conjugation of quantum dots to multiple protein targets in vivo.</a> J. Nanobiotechnol. 9, 1-14 (2011).</li><li>Lee, E. Y., 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=Global+proteomic+profiling+of+native+outer+membrane+vesicles+derived+fromEscherichia+coli.">Global proteomic profiling of native outer membrane vesicles derived fromEscherichia coli.</a> Proteomics. 7, 3143-3153 (2007).</li><li>Alves, N. J., Turner, K. B., Medintz, I. L., Walper, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+enzymatic+function+through+directed+packaging+into+bacterial+outer+membrane+vesicles.">Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles.</a> Sci. Rep. 6, 24866 (2016).</li><li>Alves, N. J., Turner, K. B., Medintz, I. L., Walper, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+therapeutic+delivery+capabilities+and+challenges+utilizing+enzyme/protein+packaged+bacterial+vesicles.">Emerging therapeutic delivery capabilities and challenges utilizing enzyme/protein packaged bacterial vesicles.</a> Ther. Delivery. 6, 873-887 (2015).</li></ol>

作者什么都没有透露。

这个协议功能表现出代表涉及在其中感兴趣的酶产生和由大肠杆菌包装到OMV封装技术。与许多复杂的技术有多个区域，其中，协议可以被修改以适应不同的独特的应用，其中一些在下面详述使用。而OMV包装和酶包封的机构可以适于特定需要有该协议内的几个步骤，是成功的关键。最初的去除细菌的完整和细胞碎片的是显著的重要性，并会影响在定量或样品分析的所有下游的尝试。接着过滤?...

这里介绍的是进行设计，生产和酶加载细菌外膜囊泡（OMV）的纯化方法。 OMV是小，主要是单层，脂蛋白，从30-200纳米1,2的尺寸范围。已研究了迄今为止所有革兰氏阴性和革兰氏阳性菌都从它们的表面3,4-表明要么OMV或胞外囊泡（EV）的释放。由OMV产生的确切机理还没有被完全阐明由于分泌它们以及它们所服务的不同功能的不同的细菌种群。 OMV已经显示小分子，肽和蛋白质运输各种各样的货物，以提供各种复杂的信号，基因易位遗传物质，和毒力功能5,6-。 OMV生物合成的确切机制尚不充分表征，并出现细菌种类之间不同。尽管THIs in issue争议，我们已经开发了通过创建目的蛋白质和高度丰富的蛋白内源性细菌外膜和随后的OMV之间的合成联动增强的重组蛋白的包装效率成OMV的方法。在不存在的合成联动，或人工引入的亲和力，所述重组表达蛋白和监查之间所观察到的包装效率很低7。这个结果是可以预料的OMV中的蛋白质通过在在细菌表面或通过定向包装OMV形成的精确时刻随机机会封装或者发生由机制未充分了解的掺入。一些成功，在这依赖于机缘凑巧封装，但有效的包装周质空间内过度表达只需通过包装蛋白被发现是高度比别人届蛋白依赖的一些蛋白质高效率包装在不包装在所有8-10。通过利用共同合成生物学技术我们试图工程师大肠杆菌 （ 大肠杆菌 ），以同时生产，包装和分泌感兴趣成OMV活性酶绕过关于OMV是如何形成的，以及如何货物由细菌对选定的当前知识限制打包。 对于本申请的目的，选择了分裂蛋白生物结合系统作为选择的合成联动，以促进定向包装入OMV。顾名思义分裂蛋白生物结合系统包括与彼此交互的两个互补亚基域。选择用于此协议的目的，分裂蛋白结构域称为抓间谍者（SC）和SpyTag（ST）的结构域，并且从化脓性链球菌纤连蛋白结合蛋白（FbaB）11而得。这种分裂蛋白系统是使W异常母鸡两个亚单位是接近度之内的异肽键的近端天冬氨酸和赖氨酸的氨基酸残基建立共价键之间自发地形成。异肽键的形成不需要另外伴侣蛋白，催化酶或辅因子，并且可以在室温（RT）和在宽范围的生理相关条件下12容易发生。 作为概念验证，选择从Brevundimonas微小假磷酸三酯酶（PTE）（EC 3.1.8.1）被打包成衍生OMV 13 大肠杆菌 。 PTE包含双核锌/锌活性部位，并具有通过水解反应转化aryldialkylphosphates成dialkylphosphates和芳基醇14分解有机磷酸酯的能力。接触有机磷酸酯通过神经肌肉接头处出不来乙酰胆碱酯酶抑制乙酰胆碱水解损害神经递质适当的功能摹有机磷衍生化合物极其危险的15。长期或显著暴露于有机磷通常会导致不可控制的抽搐，通常通过窒息引起死亡。而PTE呈现朝向对氧磷的最高的催化活性，一种非常有效的杀虫剂，也是能够水解各种其它农药和V / G型化学神经剂16的。为了便于OMV的包装，一个细菌质粒被设计编码包含诱导型启动子，周质定位序列，和一个短的多克隆位点的SC基因序列上游的基因构建体。领导者和SC序列允许建立一个基因开关为目标的PTE-SC融合蛋白为OMV包装周质空间之间的PTE基因的插入。而这里描述的努力集中在PTE中，酶的基因是可互换的，并且可以很容易地与其他基因序列置换到FAC备用酶或蛋白质的ilitate包装。 作为合成连杆的第二部分，一种丰富的外膜蛋白（OmpA的）被选择为呈现ST肽序列。而锚蛋白可以改变的选择，这是至关重要的，该蛋白具有在周质空间内呈现出宽松的域，耐受融合构建而不诱导细胞毒作用，是已知的存在于监查，并且不聚集时，它是重组产生的。的OmpA是已知在细菌外膜和随后的OMV 17被高度表达的37.2 kDa的跨膜孔蛋白的蛋白质。它是在小分子的运输牵连，&lt;2纳米的尺寸，穿过细菌膜18。天然的OmpA有两个结构独特结构域，跨膜β桶基序和已知与肽聚糖19相互作用的periplasmically可溶性C末端部分。在突变的OmpA-ST融合designeð这里的OmpA的C-末端部分被删除和ST被融合到periplasmically面对N-或C-末端。删除的OmpA的周质部分减小外膜，并导致膜去稳定化，导致超囊泡7肽聚糖之间相互作用的次数。基因组的OmpA保持除重组表达O​​mpA的-ST的构建，以减轻毛膜不稳定。

<table border="0" cellpadding="0" cellspacing="0" width="617">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">IPTG</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Always prepare fresh or aliquot and freeze.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">L-arabinose</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Can be prepared ahead of time and stored at 4C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Ampicillin</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Add immediately prior to use after media cools sufficiently from being autoclaved.&#160;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Chloramphenicol&#160;</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Add immediately prior to use after media cools sufficiently from being autoclaved.&#160;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">TB/LB Culture Media</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Other growth medias will likely work similarly.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Triton X-100</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">One of many potential suitable surfactants.&#160;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Baffled culture flasks</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">The baffles promote higher levels of aeration.&#160;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">CHES</td>
			<td style="width:93px;">Fisher Bioreagents</td>
			<td style="width:96px;">BP318-100</td>
			<td style="width:273px;">Optimal buffer used for paraoxon degredation (pH &#62; 8).</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Paraoxon</td>
			<td style="width:93px;">Chem Service</td>
			<td style="width:96px;">N-12816</td>
			<td style="width:273px;">Very toxic substance to be handled carefully and disposed of properly.&#160;&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:155px;">Syringe Filter 0.45 &#181;m</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td style="width:96px;">60183-221&#160;&#160;&#160;&#160;&#160;&#160; (30 mm)</td>
			<td style="width:273px;">Filter diameter will depend on volume of sample.&#160; Low protein binding membrane is critical.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:155px;">Shaker incubator</td>
			<td style="width:93px;">New Brunswick</td>
			<td style="width:96px;">Excella E24</td>
			<td style="width:273px;">Precise temperature and mixing is essential for reproducable bacterial growth.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Sorvall Culture Centrifuge</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td style="width:96px;">RC 5B PLUS</td>
			<td style="width:273px;">Large volume (500 mL) culture centrifuge capable of 7,000 x g.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Sorvall Ultracentrifuge&#160;</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td style="width:96px;">WX Ultra 90</td>
			<td style="width:273px;">Capable of centrifugal forces &#8805;150,000 x g.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:155px;">Ultracentrifuge Rotor</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td style="width:96px;">AH-629</td>
			<td style="width:273px;">Ensure the proper rotor and tubes are used and that everything is properly balanced.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:155px;">Ultra-Clear Ultracentrifuge Tubes&#160;&#160;&#160;&#160;&#160;&#160;&#160; (25 x 89 mm)</td>
			<td style="width:93px;">Beckman Coulter</td>
			<td style="width:96px;">344058</td>
			<td style="width:273px;">Ensure no stress fractures are present prior to use and that tubes are presicely balanced.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Spectrophotometer&#160;</td>
			<td style="width:93px;">Tecan</td>
			<td style="width:96px;">Infinite M1000</td>
			<td style="width:273px;">Necessary for enzyme kinetic assays.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">DLS / particle tracking</td>
			<td style="width:93px;">NanoSight&#160;</td>
			<td style="width:96px;">LM10</td>
			<td style="width:273px;">Necessary for OMV size distribution and concentration determination.&#160;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">BL21(DE3)</td>
			<td style="width:93px;">NEB</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Suitable bacterial expression strain.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">pET22</td>
			<td style="width:93px;">EMD Millipore</td>
			<td style="width:96px;">69744-3</td>
			<td style="width:273px;">Other plasmids can be used in place of these.&#160;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">pACYC184</td>
			<td style="width:93px;">NEB</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Other plasmids can be used in place of these.&#160;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Gel Extraction Kit</td>
			<td style="width:93px;">Qiagen</td>
			<td style="width:96px;">28704</td>
			<td style="width:273px;">Example kit.</td>
		</tr>
	</tbody>
</table>

directed protein packaging within outer membrane vesicles from escherichia coli: design, production and purification

在应用合成生物学技术，外膜囊泡（OMV）进行编程的兴趣日益增加正在导致对OMV在传统的纳米颗粒被证明太难合成了一些非常有趣和独特的应用。迄今为止，所有革兰氏阴性细菌已被证明产生的各种货物，包括小分子，肽，蛋白质和遗传物质的OMV示范包装。根据其不同的货物，OMV有牵连的许多生物过程，从细胞 - 细胞通信的基因转移和取决于所细菌生产的OMV毒力因子递送。直到最近，细菌的OMV成为在广泛的应用中使用通过的技术的发展，以控制和重组蛋白的直接包装成OMV访问。这个协议描述了生产，纯化的方法，以及利用酶包装OMV的提供改进overa所有的生产重组酶，增加灸，提高酶的稳定性。这个协议的成功利用将导致创建了同时产生的重组蛋白，并指示它对于OMV封装通过创造重组蛋白和外膜锚蛋白之间的合成键的细菌菌株的。该协议还详细介绍了从细菌培养以及正确的处理技术和东西隔离OMV适应这个协议使用了其他独特的应用程序，如时要考虑的方法:药物给药，医疗诊断和环境整治。

两种重组蛋白的同时表达，因为需要在本协议中详述的OMV包装策略，可以通过许多不同的途径来实现的。在这里，两个向量系统使用与复制和独立诱导的基因盒兼容的起源。用于PTE-SC的表达构建商用质粒骨架（pACY184）溶液工程化以包括阿拉伯糖诱导性基因盒和精氨酸周质定位前导序列的双后面是一系列的独特限制性位点以促进酶基因的克隆作为融合到C-末端的SC蛋白。编?...

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Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

从外膜囊泡内蛋白质导演包装<em&gt;大肠杆菌</em&gt;:设计，生产和纯化

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

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Research

JoVE Journal

Biochemistry

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

13.3K 次观看.  Naval Research Laboratory. A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented. 

视频: Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

从细胞使用寡核苷酸定向洗脱同源RNA-蛋白质复合物的隔离

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

从真核细胞蛋白质复合体串联亲和纯化

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

丰度和收费水平的转移 Rna 在大肠杆菌中的量化

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

利用病毒衍生系统在大肠杆菌中大规模生产圆形脚手架上的重组 rna

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

体外和体内实验的位定向诱变-以大肠杆菌rna相互作用为例

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

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.

通过组合单域抗体库的分步噬菌体选择创建高度特异性化学诱导蛋白分解系统

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

This article reports a simple method to isolate the outer and inner perivitelline sublayers of chicken eggs while minimizing structural alteration and to optimize protein solubilization of each sublayer for proteomic analyses.

来自母鸡蛋的外层和内围线子层的机械分离和蛋白质溶解

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. ...