Name: production of human norovirus protruding domains in e. coli for x-ray crystallography
Uploaded: 2016-04-19T14:00:00.000+00:00
Duration: 10 min 46 s
Description: Watch this Scientific Journal Video about Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography at JoVE.com

The funding for this study was provided by the CHS foundation. We acknowledge the protein crystallization platform within the excellence cluster CellNetworks of the University of Heidelberg for crystal screening and the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities.

1等。P域克隆<ol><li>由诺如病毒毒株的序列比对确定为P域编码区（例如，GII.10株，GenBank中:AF504671，PDB-ID:3ONU）6。此外，在为P域（ 图2A）的C-末端除去柔性区域。密码子优化E.脱氧核糖核酸大肠杆菌表达，包括BamHI位，以（N端）和NotI（C端）限制性位点亚克隆在P域编码区进入pMalc2x表达载体6,7。 注意:在P域编码区进行了优化，由商业服务合成。在P域编码区（插入）的长度为约1kb和在标准的转移载体递送。 </li><li>消化转移载体的2微克每1微升BamHⅠ位（20,000单位/毫升）和NotI（10,000单位/ ml）的限制，在37℃酶1小时与制造商提供的缓冲液。 </li><li>分隔在1％琼脂糖凝胶上消化插入在135 V，20分钟和纯化从凝胶使用商业试剂盒的插入DNA。 </li><li>通过消化该载体的2微克每1微升BamHⅠ位（20,000单位/毫升）和NotI（10,000单位/ ml）的限制酶在37℃下1小时，制备pMalc2x表达载体。从琼脂糖凝胶中纯化该载体上述（1.3）中所述。注意:两个样品（1.2和1.4）可以被储存在-20℃。 </li><li>结扎纯化插入成与1微升的T4-DNA连接酶（400000单位/ ml）在室温（RT）（ 图2B和2C）15分钟的BamHI和NotI限制位点的消化的pMalc2x载体。使用至少20纳克pMalc2x矢量和矢量的:插入比为1:3（分子量）。连接混合物通常是20〜微升。 </li><li>变换2微升连接混合物的成50μl的化学感受态大肠杆菌 DH5α使用标准转化方案（在冰上10分钟，热休克在42℃45秒）的细菌细胞和生长在600为在37℃1小时微升SOC培养基。离心转化的细胞在1000 xg离心3分钟，弃去上清液，并且重悬沉淀在SOC培养基30微升。 <ol><li>板在LB琼脂平板上转化混合物，含有100μg/ ml氨苄青霉素选择，并在37℃下生长过夜。选择至少五殖民地。 </li></ol></li><li>对于每一个的五个菌落，接种补充有50μg/ ml的氨苄青霉素（LB-AMP）的LB培养基2-3毫升培养并通过在37℃下以160 rpm振摇过夜生长。 </li><li>提取使用商业试剂盒从过夜培养物中的质粒。验证在P域插入物的存在通过测序用pMalc2x正向引物（5&#39;-TCAGACTGTCGATGAAGC-3&#39;）和反向引物（5&#39;-GATGTGCTGCAAGGCGAT-3&#39;）。 </li></ol> ; 2.P域表达式<ol><li>变换1微升 - 为MBP-他-P域pMalc2x矢量编码（150纳克/微升400毫微克/微升）融合蛋白进入50微升感受态的使用标准的转化方案（10分钟在冰上，热休克在42℃45秒） 的大肠杆菌 BL21细胞，并在37℃下生长在600微升SOC培养基1小时。传代培养到在160的转速和37℃下过夜120毫升LB-放大器。 </li><li>接种9升（每1.5升介质如 6×5升烧瓶）的LB放大器与继代培养（1:100）。生长的细胞在160转和37℃振荡直至OD 600达到0.4 - 0.6。随后，降低温度到22℃〜1小时，然后诱导蛋白表达与0.66毫异丙基β-D-硫代半乳糖苷的（IPTG）8。生长的细胞在22℃（〜18小时）过夜。 注:温度可以是多种多样的，但我们建议使用22℃或更低。 </li><li>通过离心收获细胞（10543 xg离心，15分钟，4℃）。弃去上清液并在-20℃冷冻细胞沉淀。 </li></ol>日纯化步骤和蛋白酶切割<ol><li>准备被在蛋白质纯化步骤使用从储备溶液来保证实验的重复性和稳定性缓冲器。准备用于固定化金属离子亲和层析四个不同的缓冲器（IMAC），每个包含咪唑的不同浓度（10毫米，20毫米，50毫米和250毫米）。对于SEC，具有较高的盐浓度准备凝胶过滤缓冲液（GFB），但没有咪唑。使用去离子水，并用0.45微米的孔尺寸在使用前过滤所有缓冲区。 注意:对于详细的缓冲准备方案，请参考表1。 </li><li>解冻从九个升培养细胞沉淀，并在4℃下在150毫升PBS中溶解。超声处理的细胞悬浮液2分钟（功率130 W，振幅20％，脉冲频率50％）三次，以破碎细胞。保持冰细胞悬浮液在超声。 </li><li> CentrifuGE的超声处理细胞悬浮液（43667 xg离心，30分钟，4℃）以细胞碎片从含有表达蛋白的上清液中分离出来。收集上清液并弃沉淀。 </li><li>洗涤和平衡的镍（Ni）的-NTA琼脂糖珠与层析柱10mM咪唑缓冲液中的10ml（= 1个柱体积[简历]）浆料。将平衡的Ni珠粒从步骤3.3添加到上清液含有表达MB​​P-他-P结构域的融合蛋白，并在4℃下缓慢旋转温育30分钟。 </li><li>孵育后，将整个镍珠蛋白混合物应用于层析柱。用10mM，20mM的，和50mM咪唑缓冲液，开始用10mM，然后为20mM和最后的50mM（ 图3A）的各5个CV慢慢洗柱。 </li><li>洗脱用250mM咪唑缓冲液（ 图3A）的MBP-他-P结构域融合蛋白。期间洗脱，检查OD 280nm的验证融合蛋白的洗脱（在外径28的上升0nm）。继续洗脱直到OD 280nm处下降到0.1〜。用过量的250mM咪唑缓冲液（至少10的CV）中，接着的10mM咪唑缓冲液的至少10的CV洗珠。除第二纯化步骤（第4）珠。 </li><li>使用12％SDS-聚丙烯酰胺凝胶（10×8厘米）9（ 图3A）验证的MBP-他-P结构域融合蛋白与SDS-PAGE的存在。 45 A上进行凝胶电泳和45分钟200伏。 </li><li>集中（ 例如，使用市售的集中器）洗脱MBP-他-P结构域融合蛋白，以〜3毫克/毫升的最终浓度。切割MBP-他-P结构域融合以HRV-3C蛋白酶透析期间对2升的10mM咪唑缓冲液（〜1:100），在4℃（ 图3A）过夜。取决于浓缩的蛋白质的最终体积，在透析盒或透析管进行透析。 注:HRV-3C蛋白酶对蛋白质的量裂解是根据特定蛋白酶活性计算（2U /微升，1单位是足以切割100μg蛋白），并且在表达水平而异，可以从SDS-PAGE的结果来估计洗脱融合蛋白质的量（3.7 ）。 </li></ol> 4. 第二纯化步骤<ol><li>从步骤3.6中的10mM咪唑缓冲液中平衡的Ni珠。 </li><li>孵育来自步骤3.7（含有裂解P域，MBP蛋白，和HRV蛋白酶）与平衡的Ni-珠（4.1）处理30分钟，在4℃下缓慢旋转透析蛋白质。 </li><li>镍珠混合物应用到柱并收集流过（切断P域）（ 图3B）。测量蛋白质的浓度，直到OD 280nm处达到〜0.1脱落的列。 注意:MBP-他应该保持结合到镍珠（ 图3B）。 </li><li>检查切开的P域的使用SDS-PAGE的存在下用12％凝胶上述（ 图3B）中所述。浓缩洗脱的P域至〜3毫克/毫升，并在4℃下针对GFB透析过夜随后SEC纯化。 </li></ol> 5. 第三纯化步骤<ol><li>清洗泵和​​高效液相色谱净化系统管道和预平衡的SEC柱（见材料列表）与GFB。 </li><li>注入在P域到柱以1ml使用超容量（高达12毫升）或环（高达3毫升）/分钟的流速，这取决于浓缩的样品的体积。注射完成后，增加流速2.5毫升/分钟。 </li><li>随着OD增加和P域脱落柱，收集1.5ml的分数。检查使用SDS-PAGE的级分用12％凝胶（ 图4A和4B）。池只有最纯的级分并浓缩至约3mg / ml和〜8毫克/毫升。 注意:在〜110毫升（空隙体积）大多数杂质从与320毫升床体积的SEC柱洗脱。该P域通常洗脱二聚体。在P域二聚体洗脱时间/体积依赖于SEC柱上的制备级（PG）。 </li></ol> 6.在P域的结晶<ol><li>使用在约3毫克/毫升和8毫克/毫升为初始结晶筛选在P域。至少准备100微升每个浓度的P域与384市售的筛选条件初始屏幕。在18℃在96孔板格式，其中所述贮存器包含100微升母液进行筛选和下降是由0.2微升母液和0.2微升的蛋白质。 </li><li>重复和优化成功的结晶条件。因此，使用包含3排15孔板中。设置的第一行以100％母液，第二行用90％母液和10％的水，并在第三排具有80％母溶液和20％的水。使用2微升（微升1蛋白+ 1μL母液）和500的墨滴大小81;母液作为一个水库容积L。 </li><li>使用优化结晶条件，共结晶与配体在P域。在6.2中所述准备板。代替2-微升液滴尺寸，设置含有1μl的母液，1μl的蛋白质，并以1mg / ml的浓度1微升的配体的下降。 </li><li>收集利用同步辐射单晶的数据集。使用公开的P结构域具有高序列相似性作为初始搜索模型6,10-13进行分子置换。 注:配体的存在显示为电子密度的未建模斑点。 </li></ol>

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The authors have nothing to disclose.

在这里，我们描述了诺如病毒P域的高品质和数量表达和纯化的协议。诺如病毒没有很好地研究，不断需要的结构数据。据我们所知，使用其它协议（ 例如 ，GST标签P域）p域的生产一直是个问题，到目前为止，并在诺如病毒宿主相互作用足够的结构数据已经丢失。与这里所描述的方法，我们最近显著贡献的诺如病毒的分子细节的理解糖结合。本协议可以适于多种蛋白质。然而，成功地执行该协议的取决于几个因素的纯化方法的每个部分之内。 表达构建体的设计是重要的第一步。我们执行在P域表达构建的密码子优化，以提高在大肠杆菌中的表达产量大肠杆菌和删除RELE万特的限制性位点，在编码区域中存在。此外，我们在C末端可能结晶过程中表达和蛋白质的包装中是不利的蛋白质折叠移除的柔性区域。表达作为MBP融合蛋白是平均表达过程中保持蛋白质在溶液中。 关于可溶性蛋白表达有要考虑的其他参数。 大肠大肠杆菌 BL21菌株为高产蛋白质表达进行了优化，因此，在该协议中使用。在22℃进行表达过夜，并用于诱导的IPTG的量减少。这是用于蛋白质表达的动力学有利，因此，较少的蛋白质会聚集成包涵体由于错误折叠。因此，用IPTG诱导蛋白质表达之前向培养冷却到22℃是重要的。如果蛋白质收率不令人满意，也能够进一步减少TEMPERATURE和调整的IPTG的量。 某些护理应就纯化柱服用。原则上，镍珠可重复使用数次。然而，结合能力会随时间减少。若Ni的溶液失去了它的标准蓝颜色，珠子可以剥离并使用制造商的手册中的说明充电。此外，为了维持在适当的条件下的SEC柱并定期清洁，以允许高性能是重要的。根据SEC柱上的具有待纯化不同的制备级（PG）的蛋白质的大小应选择。在P域二聚体是〜65 kDa的大小，并且可以从使用​​75 pg柱〜100 kDa的杂质很好地分离，而较大的蛋白质可以更好地利用200 pg柱分离。 优化的序列设计，表达和纯化步骤的组合，可以使用我们的甲基获得非常纯净的，高品质的P域OD。由于纯p域的高品质，另外的研究是合适的，包括免疫抗体产生，核磁共振实验，和基于ELISA的研究。此外，纯p域可用于形成复合物具有的Fab抗体和纳米抗体14,15。据我们所知，这是第一个协议，它允许以高通量的方式P域结晶，使用此协议，我们已经确定在复杂的20多个不同的诺如病毒和lagovirus P域的复杂结构与HBGAs 6,16。 根据我们的经验，该协议可能不限于蛋白质达65 kDa的。然而，不同的杯状病毒17和非结构蛋白，如病毒蛋白的基因组连接的（VPG），蛋白酶，和RNA依赖性RNA聚合酶（RdRp进行）的衣壳蛋白被成功表达并使用此方法（未公开）纯化。当应用这种方法来衣壳其他病毒蛋白质，我吨可能是必要的，以改变和优化多个参数（例如 ，洗脱缓冲液的咪唑浓度），以获得蛋白质的足够量。另外，比GFB（ 例如 ，PBS或TBS）等不同的存储缓冲器，以获得最佳的蛋白质稳定性进行测试。 大多数分析构建体中立方/板状晶体，衍射到高的分辨率得到。因此，本协议提供了一种工具，以获得结晶良好的纯蛋白质。只要现在没有可用健壮细胞培养模型，该方法构成了显著步骤有助于诺罗病毒宿主细胞相互作用的理解。

人类诺如病毒是急性胃肠炎全球1的主要原因。这些病毒属于杯状病毒家族，其中至少有5属，包括诺如病毒 ， 沙波病毒 ，Lagovirus，Vesivirus和Nebovirus。尽管医疗系统，分布范围广对他们的高冲击，诺如病毒的人的研究是由缺乏强有力的细胞培养系统而受到阻碍。迄今为止，没有可批准的疫苗或抗病毒药物策略。 诺罗病毒主要衣壳蛋白，称为VP1，可分为一个壳（S）结构域和一个突出（P）的结构域2。在P域被连接到由柔性铰链（H）的区域中的S结构域。 S区形成围绕病毒RNA的支架，而在P域构成病毒衣壳的最外层的一部分。在细菌中表达时在P域组装成生物学相关二聚体。在P（D）定时器与碳水化合物结构相互作用，称为HISTO-血型抗原（HBGAs）中存在如唾液可溶性抗原和某些宿主细胞3中。在P域HBGA相互作用被认为是感染4重要。事实上，最近的一份报告表明合成HBGAs或HBGA表达在体外 5为人类诺罗病毒感染的细菌中的重要性。 主要与能够在昆虫细胞或大肠杆菌 （ 大肠杆菌 ）中表达的重组P域被表达的病毒样颗粒（VLPs）执行关于诺如病毒的宿主细胞附着目前的研究。为了理解在原子分辨率为P域HBGA相互作用，P域HBGA复杂的结构可以利用X射线晶体学来解决。这里，我们描述的p域的表达和纯化一种协议，它允许使用用于X射线结晶性生产在高数量和质量P域的地理学。此外，可以应用其它杯状病毒P域和非结构蛋白此方法。 为E.在P域是密码子最佳大肠杆菌表达并克隆到一个标准的转移载体。在P域然后重新克隆到编码聚组氨酸（His）的标签和甘露糖结合蛋白（MBP），其后跟一个蛋白酶切割位点的表达载体。的MBP-他-P结构域融合蛋白在大肠杆菌中表达的大肠杆菌 ，后跟三个纯化步骤。的MBP-他-P结构域融合蛋白是使用固定化金属离子亲和层析（IMAC）纯化。接着，该融合蛋白被裂解与人鼻病毒（HRV）-3℃蛋白酶和P域从分离的MBP-他通过额外的IMAC纯化步骤。最后，在P域使用大小排阻层析（SEC）纯化。然后将纯p域可用于X射线晶体。蛋白质结晶条件筛选是PERFORmed指使用不同磷结构域蛋白浓度市售筛查试剂盒。晶体生长观察和最有前途的条件进行了优化。 与这里所描述的方法，有可能从基因去蛋白至小于四周内的结构。因此，我们的P域表达，纯化和结晶的方法是合适的，研究诺罗病毒 - 宿主相互作用在分子水平，并提供重要的数据，以协助在上最新疫苗设计和药物筛选。

<table><tbody><tr><td>P domain DNA</td><td>Life Technologies</td><td></td><td>GeneArt Gene Synthesis</td></tr><tr><td>pMalc2x&amp;nbsp; vector</td><td>On request</td><td></td><td></td></tr><tr><td>BamHI</td><td>New England Biolabs</td><td>R0136L</td><td></td></tr><tr><td>NotI</td><td>New England Biolabs</td><td>R0189L</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>S.O.C. Medium</td><td>Life Technologies</td><td>15544-034</td><td></td></tr><tr><td>Econo-Column Chromatography Column</td><td>Bio-Rad</td><td>7372512</td><td>2.5 x 10 cm, possible to use other size</td></tr><tr><td>Ni-NTA Agarose</td><td>Qiagen</td><td>30210</td><td></td></tr><tr><td>Vivaspin 20</td><td>GE Healthcare</td><td>various</td><td>cutoff of 10 kDa, 30 kDa and 50 kDa used</td></tr><tr><td>Subcloning Efficiency DH5&amp;alpha; Competent Cells</td><td>Life Technologies</td><td>18265-017</td><td></td></tr><tr><td>One Shot BL21(DE3) Chemically Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>Life Technologies</td><td>C6000-03</td><td></td></tr><tr><td>HRV 3C Protease</td><td>Merck Millipore</td><td>71493</td><td></td></tr><tr><td>HiLoad 26/600 Superdex 75 PG</td><td>GE Healthcare</td><td>28-9893-34</td><td>SEC column</td></tr><tr><td>JCSG Core suites</td><td>Qiagen</td><td>various</td><td>4 screens with each 96 wells</td></tr><tr><td>Carbohydrates</td><td>Dextra Laboratories, UK</td><td>various</td><td>Blood group products</td></tr></tbody></table>

production of human norovirus protruding domains in e. coli for x-ray crystallography

诺罗病毒衣壳是由一个单一的主要结构蛋白，称为VP1。 VP1被细分成壳（S）结构域和一个突出（P）的结构域。 S区形成围绕病毒RNA的连续支架，而在P域形式上的S域病毒尖峰和包含抗原和宿主 - 细胞相互作用的决定因素。在P域结合碳水化合物结构， 即 ，HISTO-血型抗原，其被认为是对诺罗病毒的感染很重要的。在这个协议中，我们描述了在高产率生产高品质的诺罗病毒P域的方法。这些蛋白质然后可以为了研究抗原性和宿主 - 细胞相互作用被用于X射线晶体和ELISA。 在P域首先克隆到一种表达载体，然后在细菌中表达。该蛋白质是使用涉及固定的金属离子亲和层析和大小排阻层析三步纯化。在原则上，它可以克隆，表达，纯化，并在不到四个星期，这使得该协议分析新出现的诺如病毒株快速系统结晶的蛋白质。

所描述的协议的示意图在图1中描绘的协议包括6个主要的部件，其中包括所述靶基因的克隆，表达，一个三步纯化和结晶。 图2示出了表达构建体的设计（EC）和在pMalc2x表达载体的特点。所述pMalc2x载体的多克隆位点（MCS）的序列显示限制和蛋白酶切割位点。 图3示出的MBP-他-P结构域融合蛋白的代表性的SDS-PAGE结果和裂解P域与前两个的相应示意图纯化步骤。第三纯化步骤在图4中描绘并涉及纯化方案，纯p域的洗脱色谱和收集的级分的代表性的SDS-PAGE结果。最纯的级分（按SDS-PAGE）汇集，concentrat版，并且用于X射线晶体。 <img alt="图1" src="/files/ftp_upload/53845/53845fig1.jpg" /> 图1原理图诺沃克病毒P域的表达，纯化的。对于诺罗病毒P域的表达和纯化该协议包含六个主要部件，覆盖克隆和表达（1和​​2），纯化（3至5），并结晶（6）。红色三角形代表在P域（基因和蛋白质），而蓝色矩形表示的MBP-His的。被IMAC期间使用（3和 4）被示出为大青色球体的Ni-NTA琼脂糖珠。美国证券交易委员会珠被描述为灰色球体。 <img alt="图2" src="/files/ftp_upload/53845/53845fig2.jpg" /> 图2.设计和表达构建的克隆（EC）。在P域名EC，表达矢量地图和多克隆位点（MCS）所示。A）的诺罗病毒衣壳蛋白（VP1），并说明与C末端缺失的EC（绿色）的设计，在P域EC的安装; b在pMalc2x表达载体）示意图用于大肠杆菌 P域表达式大肠杆菌用氨苄青霉素抗性盒（是AmpR），紫胶操纵子（的lacI），甘露糖结合蛋白（雄性，MBP）和pMalc2x表达载体的MCS的MCS。C）的序列。突出显示的是限制性内切酶切割位点（红色框）和用于HRV 3C蛋白酶（精度，蓝色框）识别序列LEVLFQGP。 <img alt="图3" src="/files/ftp_upload/53845/53845fig3.jpg" /> 对于1 和第 2 次 陈建 图3.原理图和代表结果ification步骤，净化概述和代表性的SDS-PAGE结果显示，A）使用Ni-NTA琼脂糖珠（青色大球）的MBP-他-P域融合蛋白的纯化。在12％SDS-PAGE凝胶显示了MBP-他-P结构域融合蛋白。B）中的MBP-His的（蓝色矩形的从裂解的P域（红色三角形）分离）。裂解P域的洗脱是通过SDS-PAGE分析在12％凝胶上。 <img alt="图4" src="/files/ftp_upload/53845/53845fig4.jpg" /> 对于第三 纯化步骤 图4.原理图和代表性的成果 。使用SEC和相应的SDS-PAGE值结果P域洗脱色谱。黑色数字表明，被收集在SEC柱上的分离。 一 ）示意图馏分（SEC珠被描述成灰色球）。 12％SDS-PAGE显示了本蛋白质在那些SEC洗脱期间收集该级分随后被合并，并用于X射线晶体。B）中的SEC色谱显示了洗脱体积，测定吸光度（黑线）。红线表示当P域在到B的第二个高峰期注入到SEC柱。C）放大。 <a href="https://www.jove.com/files/ftp_upload/53845/53845fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table><tbody><tr><td> 缓冲区名称 </td><td> 的1M Tris pH 7.6中 </td><td> 5 M氯化钠 </td><td> 3.5米咪唑，pH 8 </td></tr><tr><td> 250mM咪唑缓冲</td><td> 20毫升</td><td> 40毫升</td><td> 71毫升</td></tr><tr><td> 50 mM咪唑缓冲</td><td> 20毫升</td><td> 40毫升</td><td> 14毫升</td></tr><tr><td>的20mM咪唑缓冲</td><td> 20毫升</tD&gt; <td> 40毫升</td><td> 5.7毫升</td></tr><tr><td>的10mM咪唑缓冲</td><td> 20毫升</td><td> 40毫升</td><td> 2.8毫升</td></tr><tr><td> GFB（凝胶过滤缓冲） </td><td> 25毫升</td><td> 60毫升</td><td> - </td></tr></tbody></table> 表1移液方案在纯化中常用的缓冲液。Tris-盐酸的储备溶液，如在表中的标题中指示氯化钠（NaCl），和咪唑制备。以制备1升的水所需的缓冲区的需要储备溶液的量来表示。

Watch this Scientific Journal Video about Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography at JoVE.com

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

人类诺沃克病毒凸，域的生产<em&gt;电子。大肠杆菌</em&gt; X射线晶体学

在这里，我们描述了一个方法来表达和纯化高品质的诺如病毒在突出E.（P）域大肠杆菌在X射线晶体学研究中的使用。这种方法可以被应用到其它杯状病毒P域，以及非结构蛋白， 即 ，病毒蛋白基因连接的（VPG），蛋白酶，和RNA依赖性RNA聚合酶（RdRp的）。

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

production-human-norovirus-protruding-domains-e-coli-for-x-ray

Nanyang Technological University

Research

JoVE Journal

Biology

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

8.6K 次观看.  University of Heidelberg and the German Cancer Research Center. 在这里，我们描述了一个方法来表达和纯化高品质的诺如病毒在突出E.（P）域大肠杆菌在X射线晶体学研究中的使用。这种方法可以被应用到其它杯状病毒P域，以及非结构蛋白， 即 ，病毒蛋白基因连接的（VPG），蛋白酶，和RNA依赖性RNA聚合酶（RdRp的）。 

视频: Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

生产，结晶和结构测定<em&gt;℃。难辨</em&gt;通过Microseeding和锌SAD PPEP-1

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

科研

生物化学

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% ...

化学

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

结合 x 射线晶体学与小角度 x 射线散射到模型的非结构化区域的 Nsa1 从s 酿酒酵母

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

NEMO IKK绑定域的生产、结晶和结构确定

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

生物工程

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

免疫与感染

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

替代体外测定病毒衣壳结构完整性的方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

使用流细胞测定量化人类诺如病毒样颗粒与准细菌结合

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

病毒感染的人肠道类器官单层的钙成像

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.

作者聚焦：研究肠道上皮信号传导的病毒破坏 – 研究见解和未来方向 

This protocol describes an approach for performing calcium imaging in virus-infected human intestinal organoids and offers an approach to analysis.

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory ...

人类诺沃克病毒凸，域的生产

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生产，结晶和结构测定

从构造到晶体 - 迈向β桶外膜蛋白的结构解析

结合 x 射线晶体学与小角度 x 射线散射到模型的非结构化区域的 Nsa1 从s 酿酒酵母

NEMO IKK绑定域的生产、结晶和结构确定

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

人类诺沃克病毒的表面上检测拭子采样方法

替代体外测定病毒衣壳结构完整性的方法

使用流细胞测定量化人类诺如病毒样颗粒与准细菌结合

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像