Name: directed protein packaging within outer membrane vesicles from escherichia coli: design, production and purification
Uploaded: 2016-11-16T12:30:00.000+00:00
Duration: 10 min 21 s
Description: Watch this Scientific Journal Video about Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification at JoVE.com

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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	<li>Avila-Calderon, E. D., 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=Roles+of+bacterial+membrane+vesicles.">Roles of bacterial membrane vesicles.</a> Arch. Microbiol. 197, 1-10 (2015).</li><li>Kulp, A., Kuehn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+functions+and+biogenesis+of+secreted+bacterial+outer+membrane+vesicles.">Biological functions and biogenesis of secreted bacterial outer membrane vesicles.</a> Annu. Rev. Microbiol. 64, 163-184 (2010).</li><li>Beveridge, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+of+Gram+negative+cell+walls+and+their+derived+membrane+vesicles.">Structures of Gram negative cell walls and their derived membrane vesicles.</a> J. Bacteriol. 181, 4725-4733 (1999).</li><li>Kulkarni, H. M., Jagannadham, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+and+multifaceted+roles+of+outer+membrane+vesicles+from+Gram-negative+bacteria.">Biogenesis and multifaceted roles of outer membrane vesicles from Gram-negative bacteria.</a> Microbiology. 160, 2109-2121 (2014).</li><li>Ellis, T. N., Kuehn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+and+immunomodulatory+roles+of+bacterial+outer+membrane+vesicles.">Virulence and immunomodulatory roles of bacterial outer membrane vesicles.</a> Microbiol. Mol. Biol. Rev. 74, 81-94 (2010).</li><li>Berleman, J., Auer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+bacterial+outer+membrane+vesicles+for+intra-+and+interspecies+delivery.">The role of bacterial outer membrane vesicles for intra- and interspecies delivery.</a> Environ. Microbiol. 15, 347-354 (2013).</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=Bacterial+nanobioreactors-directing+enzyme+packaging+into+bacterial+outer+membrane+vesicles.">Bacterial nanobioreactors-directing enzyme packaging into bacterial outer membrane vesicles.</a> ACS Appl. Mater. Interfaces. 7, 24963-24972 (2015).</li><li>Kim, J. 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=Engineered+bacterial+outer+membrane+vesicles+with+enhanced+functionality.">Engineered bacterial outer membrane vesicles with enhanced functionality.</a> J. Mol. Biol. 380, 51-66 (2008).</li><li>Haurat, 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=Selective+sorting+of+cargo+proteins+into+bacterial+membrane+vesicles.">Selective sorting of cargo proteins into bacterial membrane vesicles.</a> J. Biol. Chem. 286, 1269-1276 (2011).</li><li>Kesty, N. C., Kuehn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+heterologous+outer+membrane+and+periplasmic+proteins+into+Escherichia+coli+outer+membrane+vesicles.">Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles.</a> J. Biol. Chem. 279, 2069-2076 (2003).</li><li>Zakeri, B., 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=Peptide+tag+forming+a+rapid+covalent+bond+to+a+protein,+through+engineering+a+bacterial+adhesion.">Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesion.</a> Proc. Natl. Acad. Sci. U. S. A. 109, e690-e697 (2012).</li><li>Li, L., Fierer, J. O., Rapoport, T. A., Howarth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+and+optimization+of+the+covalent+association+between+SpyCatcher+and+a+peptide+tag.">Structural analysis and optimization of the covalent association between SpyCatcher and a peptide tag.</a> J. Mol. Biol. 426, 309-317 (2014).</li><li>Dumas, D. P., Durst, H. D., Landis, W. G., Raushel, F. M., Wild, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+organophosphorus+nerve+agents+by+the+phophotriesterase+from+Pseudomonas-Diminuta.">Inactivation of organophosphorus nerve agents by the phophotriesterase from Pseudomonas-Diminuta.</a> Arch. Biochem. Biophys. 277, 155-159 (1990).</li><li>Bigley, A. N., Raushel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+mechanisms+for+phosphotriesterases.">Catalytic mechanisms for phosphotriesterases.</a> Biochimica et Biochim. Biophys. Acta, Proteins Proteomics. 1834, 443-453 (2013).</li><li>Minton, N. A., Murray, V. S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+organo-phosphate+poisoning.">A review of organo-phosphate poisoning.</a> Med. Toxicol. Adverse Drug Exper. 3, 350-375 (1988).</li><li>Bigley, A. N., Xu, C., Henderson, T. J., Harvey, S. P., Raushel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+neutralization+of+the+chemical+warfare+agent+VX:+Evolution+of+phosphotriesterase+for+phosphorothiolate+hydrolysis.">Enzymatic neutralization of the chemical warfare agent VX: Evolution of phosphotriesterase for phosphorothiolate hydrolysis.</a> J. Am. Chem. Soc. 135, 10426-10432 (2013).</li><li>Chatterjee, S. N., Chaudhuri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gram-negative+bacteria:+the+cell+membranes.">Gram-negative bacteria: the cell membranes.</a> Outer Membrane Vesicles of Bacteria. 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。接着过滤两次连续离心步骤是典型地足以除去这些污染物。然而，在所有情况下，应注意，当倾析溶液离心后，以尽量减少污染物的转移。同样，最终OMV颗粒Øbtained以下超速离心往往只是松散地附着在离心管的底部。在最后阶段，必须小心除去废培养基时，以确保沉淀在容器的底部仍不受干扰作出。在某些情况下它可能是最好保留最后的培养基，直到使用DLS或纳米粒子跟踪装置，确保颗粒OMV在协议的最后步骤并没有失去样品分析后。下面是可能与这两个特定的协议和其他人都适合研究者的特定需求出现了一些其他问题。 在本系统中使用的PTE的提供了用于有机磷污染区域与PTE是由交替的酶或蛋白质容易替换的环境整治极好模型酶封装。这里描述PTE-SC与外膜蛋白A-ST的共表达导致整体PTE expressi大量增加上以及与更PTE更高囊泡生产水平被包装的小泡内相比PTE和单独表达PTE-SC。通过减少通过OmpA的超​​囊泡的C-末端缺失的外膜和肽聚糖之间的相互作用的数量来实现。这提供了细菌导出重组PTE这减轻了通常在非天然蛋白质的过度表达所观察到的毒性效应的简单的路线。 虽然PTE可以很容易地在这个模型系统来代替需要注意的是并不是所有的酶底物会自由地穿过双层OMV重要的是，它因此，当务之急是每一个独特的酶和底物对由个案基础上进行测试。即使在衬底通过膜不通过该技术仍然可以用于生产和包装的酶进OMV和加法的Triton X-100的或合适的替代表面活性剂，可以在足以勒添加VELS破裂在使用前的囊泡。 在不存在重组蛋白质表达和包装的该协议也可以作为监查生产和纯化来自不同细菌种类的基本方法。替代方法为OMV纯化，如密度梯度分级分离22，膜过滤23，和超过滤24，也存在并且可根据预期的应用更适合的技术。这些替代的纯化技术，也可以通过使用一个双正交合成联动容易地补充到该协议代替OMV的一种蛋白质的定向包装超离心制粒成OMV。 其它修改可以在本协议以及选择的膜束缚蛋白使用的具体的合成联动系统进行。有配对两种不同的蛋白质各种合成策略生物系统中的S中的包括:分裂的蛋白25，连续线圈26，和分割内含肽27，只是列出几个。其它膜结合或跨膜蛋白，如OmpF的和OMPC可能会提供合适的ST修饰位点以及28。在某些情况下，它也可能是必要的交换ST和SC域放置SC上的锚定蛋白和小得多的ST的重组酶/蛋白。也有一些可在这里实现，包括在相同或不同的感应系统中使用的多个质粒的不同克隆策略，与多种蛋白质的单个质粒编码，或者甚至利用同源重组技术表达的，其中包含全部或部分系统到细菌基因组中。 监查的微环境有助于稳定包装酶减少下小于理想ST通常发生酶促失活ORAGE条件，冻融，并冻干29。此外，还预计，该监查将提供大大改善的对蛋白水解切割的抗性作为OMV双层将作为活性酶和存在于外OMV空间蛋白质之间的物理屏障的作用。此协议可进一步扩展到包括一个面向外部的小分子，肽或蛋白质标签以促进OMV包封蛋白和亲和纯化的靶向递送。虽然被选定为这个独特的应用PTE本研究该协议的结果，及内容，可以很容易地被用来设计用于类似的蛋白质包装策略在不同的药物递送， 医疗诊断和环境整治应用30。

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><tbody><tr><td>IPTG</td><td>Any</td><td></td><td>Always prepare fresh or aliquot and freeze.</td></tr><tr><td>L-arabinose</td><td>Any</td><td></td><td>Can be prepared ahead of time and stored at 4 &amp;deg;C.</td></tr><tr><td>Ampicillin</td><td>Any</td><td></td><td>Add immediately prior to use after media cools sufficiently from being autoclaved.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Chloramphenicol&amp;nbsp;</td><td>Any</td><td></td><td>Add immediately prior to use after media cools sufficiently from being autoclaved.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>TB/LB Culture Media</td><td>Any</td><td></td><td>Other growth medias will likely work similarly.</td></tr><tr><td>Triton X-100</td><td>Any</td><td></td><td>One of many potential suitable surfactants.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Baffled culture flasks</td><td>Any</td><td></td><td>The baffles promote higher levels of aeration.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>CHES</td><td>Fisher Bioreagents</td><td>BP318-100</td><td>Optimal buffer used for paraoxon degredation (pH &gt; 8).</td></tr><tr><td>Paraoxon</td><td>Chem Service</td><td>N-12816</td><td>Very toxic substance to be handled carefully and disposed of properly.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Syringe Filter 0.45 &amp;micro;m</td><td>Thermo Scientific</td><td>60183-221 (30 mm)</td><td>Filter diameter will depend on volume of sample. Low protein binding membrane is critical.</td></tr><tr><td>Shaker incubator</td><td>New Brunswick</td><td>Excella E24</td><td>Precise temperature and mixing is essential for reproducable bacterial growth.&amp;nbsp;</td></tr><tr><td>Sorvall Culture Centrifuge</td><td>Thermo Scientific</td><td>RC 5B PLUS</td><td>Large volume (500 ml) culture centrifuge capable of 7,000 x g.</td></tr><tr><td>Sorvall Ultracentrifuge&amp;nbsp;</td><td>Thermo Scientific</td><td>WX Ultra 90</td><td>Capable of centrifugal forces &amp;ge;150,000 x g.</td></tr><tr><td>Ultracentrifuge Rotor</td><td>Thermo Scientific</td><td>AH-629</td><td>Ensure the proper rotor and tubes are used and that everything is properly balanced.</td></tr><tr><td>Ultra-Clear Ultracentrifuge Tubes (25 x 89 mm)</td><td>Beckman Coulter</td><td>344058</td><td>Ensure no stress fractures are present prior to use and that tubes are presicely balanced.</td></tr><tr><td>Spectrophotometer&amp;nbsp;</td><td>Tecan</td><td>Infinite M1000</td><td>Necessary for enzyme kinetic assays.</td></tr><tr><td>DLS/particle tracking</td><td>NanoSight&amp;nbsp;</td><td>LM10</td><td>Necessary for OMV size distribution and concentration determination.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>BL21(DE3)</td><td>NEB</td><td></td><td>Suitable bacterial expression strain.</td></tr><tr><td>pET22</td><td>EMD Millipore</td><td>69744-3</td><td>Other plasmids can be used in place of these.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>pACYC184</td><td>NEB</td><td></td><td>Other plasmids can be used in place of these.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>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。编码外膜锚定蛋白的质粒构建是商业载体pET22，其利用蛋白质表达的控制和规划环境地政局周质定位序列乳糖操纵子。短的ST序列克隆到之前樱雪截短的OmpA蛋白rtion到表达质粒中。在两种质粒中，C-末端六组氨酸序列保持以允许OMV样品中的融合蛋白的同时识别。这些基因结构的构建和特征，包括N-和C-末端ST融合构建到的OmpA，在阿尔维斯等人都充分说明。 7。本文所描述的基因构建体可能不利于所有酶的包装。在某些情况下，可能有必要改变周质定位信号，所述调控元件，或表位标签。此外，某些蛋白质可以是令人望而却步大，无法完全横向内膜。酶活性和监查包装效率不能先验地确定，并需要对每个目标蛋白质凭经验确定。 下面是结果成功地进行Ø后，一个通常会期望UT斯达康此协议。包括的是两个ST / SC分裂的蛋白质结构域的OMV内的示意性的OmpA-ST和PTE-SC融合构建体以及在细菌外膜异肽形成的一个例子，PTE-SC的后续封装（ 图1） 。还包括的是通过扫描电镜纯化OMV形态的代表性结果以及尺寸分布和绝对的OMV的浓度通过粒子追踪软件来确定（ 图2）。监查蛋白质含量和重组蛋白表达的进一步表征可通过SDS-PAGE和Western印迹（ 图3）所示。对于特定于这里提供的协议感兴趣的蛋白质分子量如下:原生外膜蛋白A（37.2 kDa的），外膜蛋白A-ST（23 kDa的），PTE-SC（51 kDa的），外膜蛋白A-ST / PTE-SC（74 kDa的） 。纯化的OMV应该有一个暗带在大约37 kDa的其表示高度丰富天然的OmpA的。根据凝胶的分辨率可能有多个巴NDS存在于胶为外膜蛋白A，OmpF的和OMPC的这个区域都相对丰富，有着相似的分子量。一个附加的频带较大2.2 kDa的比的OmpA-ST还可以观察到由于前导序列作为压倒大肠杆菌表达机械的结果的裂解不当。要注意的是一个成功地纯化OMV样品不会有很多其它高表达的蛋白质是重要的。如果其它频带中存在任一纯化不当进行或应变所利用的细菌可被包装在这个大肠杆菌 BL21（DE3）菌株没有观察到其它蛋白质。 此外，活性测定法和囊泡破裂实验已经提供证明，如果酶底物可以自由地通过跨膜孔蛋白的蛋白质（ 图4）输入的OMV，将被预期代表性结果。对氧磷相对自由地进入OMV通过内源性跨膜孔蛋白的蛋白质与打包PTE，将反应产物， 对硝基苯酚的反应，也可以通过在监查膜相对自由地通过。这种现象不会所有产物，底物和酶集之间无所不在，并且必须通过实验确定。虽然成功OMV断裂和酶的释放已经看到用低浓度的洗涤剂和PTE（这里的Triton X-100），例如增加可能影响其它酶的活性。 质粒图谱也被列入证明利用SC / ST系统（ 图5）双质粒包装策略的设计。 <img alt="图1" src="/files/ftp_upload/54458/54458fig1.jpg" /> 图1:蛋白质的定向包装成OMV。对于在所描述的监查包装STRAT利用的蛋白质晶体结构埃及:外膜蛋白A，PTE，SpyTag（ST）和抓间谍者（SC）; PDB:2GE4，1PTA，4MLI，4MLI分别。形成于细菌的外膜的异肽键的OmpA的-ST和PTE-SC融合构建的示意图被示出。这种膜融合驱动形成OMV内PTE的结合。从阿尔维斯等人图复制（改编）。 ACS申请垫接口 （2015），7（44），第24,963-24,972 7。版权所有2015年美国化学学会。 <a href="https://www.jove.com/files/ftp_upload/54458/54458fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54458/54458fig2.jpg" /> 图2:OMV 表征。 （ 一 ）超离心的SEM从本地大肠杆菌纯化的OMV [BL21（DE3）。 （B）代表颗粒TRAC<html>特大号分布和（C）总囊泡浓度平均超过90秒的采样读取本地OMV，PTE-SC在没有阿拉伯糖激活（PTE-没有A），PTE-SC在阿拉伯糖激活的存在（PTE-A） N-末端的OmpA-ST共转化与PTE-SC在阿拉伯糖和IPTG激活（PTE / OMV-N）的存在下，并超速离心上清液（UC上清液）。该PTE / OMV-N样品中观察到OMV产量显著相比增加本地OMV和PTE-SC单独构建。 OMV在UC上清液进行定量证明OMV在UC颗粒留下很少或几乎没有OMV在上清超速离心后几乎完全恢复。所有数据表示一式三份实验装置（±SD）。从阿尔维斯等人图复制（改编）。 ACS申请垫接口 （2015），7（44），第24,963-24,972 7。版权所有2015年美国化学学会。D / 54458 / 54458fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54458/54458fig3.jpg" /> 图3: 纯化的OMV蛋白质含量测定。 （A）和PTE-SC共表达的C末端的OmpA-ST融合的纯化的监查的UC粒料的SDS-PAGE（PTE / OMV）构建体展示的OmpA-ST，母语的OmpA，PTE-SC和OmpA-的代表丰ST / PTE-SC异肽融合。 （B）利用包括his-标签存在于OmpA的和PTE Western印迹分析构建通过反的6xHis抗体便于可视化。要注意的是PTE已知二聚化，如由较大的分子量his标记物质的存在对印迹证明是重要的。即使有很高的OmpA-ST的表达水平观察到没有的自由完全转化PTE-SC，以膜结合外膜蛋白A-ST / PTE-SC。尽管如此，在整体PTE生产和改进的包装效率的增加表明，虽然共价键的形成不是无所不在的ST和SC域的非共价结合是在监查内定向的包装的一个重要因素。从阿尔维斯等人图复制（改编）。 ACS申请垫接口 （2015），7（44），第24,963-24,972 7。版权所有2015年美国化学学会。 <a href="https://www.jove.com/files/ftp_upload/54458/54458fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54458/54458fig4.jpg" /> 图4:PTE 活动和包装特征。 （ 一 ）总体PTE表达水平和OMV包装效率通过利用对初始速度测量确定氧磷作为显色底物的细胞沉淀，在UC上清液比较PTE本，和纯化的UC OMV粒料。 （ 二 ）有代表性的数据展示了如何使用的Triton X-100（0.5％T100）的破坏OMV双层允许基板的OMV内部通行无阻。这个数据与在不存在T100的进行的测定便于在完好的OMV双层衬底的易位验证如果酶促初始速度保持不变。在对氧磷的情况下，出现了在存在或不存在T100指示对氧磷通过上在监查内源孔自由通过的观察到非常小的活性差异。 （C）PTE-SC动力学数据拟合标准米氏酶动力学方程PTE / OMV-N和PTE-A。 （D）用于确定K M和K 猫 / K M（48一双倒数分析，4.4×10 7; 44微米，4.9×10 7秒-1分别M -1，）证明类似的PTE文献动力学参数作为天然酶（90μM，2.7×10 7秒-1 M -1），在所有情况下，R 2≥0.999。所有数据表示一式三份实验装置（±SD）。从阿尔维斯等人图复制（改编）。 ACS申请垫接口 （2015），7（44），第24,963-24,972 7。版权所有2015年美国化学学会。 <a href="https://www.jove.com/files/ftp_upload/54458/54458fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54458/54458fig5.jpg" /> 图5: 代表质粒构建为这里所描述的示例性系统。抓间谍者修饰酶-SC（PTE-SC）质粒定位为OMV（左）内封装。 SpyTag改性的IED膜锚-ST（OmpA的-ST-N）的质粒（右）。 <a href="https://www.jove.com/files/ftp_upload/54458/54458fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification at JoVE.com

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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Nanyang Technological University

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

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

从构造到晶体 - 迈向β桶外膜蛋白的结构解析

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

科研

化学

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 ...

生物化学

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

监视分泌细菌毒力因子的组装使用站点特定的交联

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

免疫与感染

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

在肽膜前体的维西图洛合成中,用于自主囊泡生长

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

生物工程

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

生物学

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout growth, the outer membrane can bleb to form spherical outer membrane vesicles (OMVs). These OMVs are involved in numerous cellular functions including cargo delivery to host cells and communication with bacterial cells. Recently, the therapeutic potential of OMVs has begun to be explored, including their use as vaccines and drug delivery vehicles. Although OMVs are derived from the OM, it has long been appreciated that the lipid and protein cargo of the OMV differs, often significantly, from that of the OM. More recently, evidence that bacteria can release multiple types of OMVs has been discovered, and evidence exists that size can impact the mechanism of their uptake by host cells. However, studies in this area are limited by difficulties in efficiently separating the heterogeneously sized OMVs. Density gradient centrifugation (DGC) has traditionally been used for this purpose; however, this technique is time-consuming and difficult to scale-up. Size exclusion chromatography (SEC), on the other hand, is less cumbersome and lends itself to the necessary future scale-up for therapeutic use of OMVs. Here, we describe a SEC approach that enables reproducible separation of heterogeneously sized vesicles, using as a test case, OMVs produced by Aggregatibacter actinomycetemcomitans, which range in diameter from less than 150 nm to greater than 350 nm. We demonstrate separation of &#34;large&#34; (350 nm) OMVs and &#34;small&#34; (&#60;150 nm) OMVs, verified by dynamic light scattering (DLS). We recommend SEC-based techniques over DGC-based techniques for separation of heterogeneously sized vesicles due to its ease of use, reproducibility (including user-to-user), and possibility for scale-up.

Bacterial vesicles play important roles in pathogenesis and have promising biotechnological applications. The heterogeneity of vesicles complicates analysis and use; therefore, a simple, reproducible method to separate varying sizes of vesicles is necessary. Here, we demonstrate the use of size exclusion chromatography to separate heterogeneous vesicles produced by Aggregatibacter actinomycetemcomitans.

大小排除色谱分析细菌外膜囊性异质性

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

蛋白质的共表达了多方面的好处<em&gt;大肠杆菌</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

抗菌肽定位的细菌 Spheroplasts 和原生质体的生产与可视化

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

从 大肠杆菌 和其他细菌中可扩展地分离和纯化细胞外囊泡

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

从外膜囊泡内蛋白质导演包装

摘要

探索更多视频

此视频中的章节

从构造到晶体 - 迈向β桶外膜蛋白的结构解析

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

监视分泌细菌毒力因子的组装使用站点特定的交联

将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中

在肽膜前体的维西图洛合成中,用于自主囊泡生长

蛋白复合物的鉴定

大小排除色谱分析细菌外膜囊性异质性

蛋白质的共表达了多方面的好处

抗菌肽定位的细菌 Spheroplasts 和原生质体的生产与可视化

从大肠杆菌和其他细菌中可扩展地分离和纯化细胞外囊泡

从外膜囊泡内蛋白质导演包装

摘要

探索更多视频

此视频中的章节

从构造到晶体 - 迈向β桶外膜蛋白的结构解析

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

监视分泌细菌毒力因子的组装使用站点特定的交联

将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中

在肽膜前体的维西图洛合成中,用于自主囊泡生长

蛋白复合物的鉴定

大小排除色谱分析细菌外膜囊性异质性

蛋白质的共表达了多方面的好处

抗菌肽定位的细菌 Spheroplasts 和原生质体的生产与可视化

从 大肠杆菌 和其他细菌中可扩展地分离和纯化细胞外囊泡

从大肠杆菌和其他细菌中可扩展地分离和纯化细胞外囊泡