Name: production of human norovirus protruding domains in e. coli for x-ray crystallography
Uploaded: 2016-04-19T14:00:00
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?...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Kabsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+Processing+of+Rotation+Diffraction+Data+from+Crystals+of+Initially+Unknown+Symmetry+and+Cell+Constants.">Automatic Processing of Rotation Diffraction Data from Crystals of Initially Unknown Symmetry and Cell Constants.</a> J. Appl. Crystallogr. 26, 795-800 (1993).</li><li>Mccoy, A. 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=Phaser+crystallographic+software.">Phaser crystallographic software.</a> J. Appl. Crystallogr. 40, 658-674 (2007).</li><li>Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+development+of+Coot.">Features and development of Coot.</a> Acta Crystallogr. Sect. D-Biol. Crystallogr. 66, 486-501 (2010).</li><li>Adams, P. 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=PHENIX:+a+comprehensive+Python-based+system+for+macromolecular+structure+solution.">PHENIX: a comprehensive Python-based system for macromolecular structure solution.</a> Acta Crystallogr. Sect. D-Biol. Crystallogr. 66, 213-221 (2010).</li><li>Koromyslova, A. D., Hansman, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanobody+binding+to+a+conserved+epitope+promotes+norovirus+particle+disassembly.">Nanobody binding to a conserved epitope promotes norovirus particle disassembly.</a> J. Virol. 89, 2718-2730 (2015).</li><li>Hansman, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+broad+detection+of+genogroup+II+noroviruses+by+a+monoclonal+antibody+that+binds+to+a+site+occluded+in+the+viral+particle.">Structural basis for broad detection of genogroup II noroviruses by a monoclonal antibody that binds to a site occluded in the viral particle.</a> J. Virol. 86, 3635-3646 (2012).</li><li>Singh, B. K., Leuthold, M. M., Hansman, G. 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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?...

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&#160; 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 cm 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&#945; Competent Cells</td>
			<td>Life Technologies</td>
			<td>18265-017</td>
			<td></td>
		</tr>
		<tr>
			<td>One Shot BL21(DE3) Chemically Competent E. coli</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...

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. ...

Research

JoVE Journal

Biology

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

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

热休克法转化到大肠杆菌质粒DNA

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

科研

生物学

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

非侵入性的三维可视化与亚微米分辨率使用同步加速器X射线断层扫描

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

由紫外线引起的复制中间体的可视化 E。大肠杆菌使用二维的琼脂糖凝胶电泳分析

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

Protein Crystallization for X-ray Crystallography

X射线晶体学的蛋白质结晶

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

基础X射线计算机断层数据的三维打印设置

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

针对性斯尔纳的噬菌体介导的交付构造击倒基因表达<em&gt;电子。大肠杆菌</em

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be ...

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

诱导位点特异性复制遇阻<i&gt;电子。大肠杆菌</i&gt;用荧光阻遏操作员系统

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication ...