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?...

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. ...

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

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

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

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 plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

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

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 cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

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

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

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

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

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

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by 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

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

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

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

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

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

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

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

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, ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

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

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

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

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

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

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

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

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

人类诺沃克病毒凸，域的生产

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