Name: a simple fluorescence-based reporter assay to identify cellular components required for ricin toxin a chain (rta) trafficking in yeast
Uploaded: 2017-12-15T15:00:00
Description: Watch this Scientific Journal Video about A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast at JoVE.com

这项研究的部分得到了德意志 Forschungsgemeinschaft (SFB 1027, A6) 的资助。

注意: 一般实验工作流的概述如图 1所示。
警告: 对人类来说, 贸易协定是剧毒的。安全实验室许可 S2 (生物安全等级2当量) 是必要的。在整个实验中请戴上手套。
1. 在大肠杆菌中他标记的异种的异源表达
<ol>
	<li>用表达质粒 pET24-RTA(其) 6或空向量 pET24a(+)使用标准的电穿孔协议9,<sup cla...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+Reporter+Assay+to+Identify+Cellular+Components+of+Ricin+Toxin+A+Chain+Trafficking.">Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking.</a> Toxins (Basel). 8 (12), (2016).</li><li>Li, X. P., Baricevic, M., Saidasan, H., Tumer, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+depurination+is+not+sufficient+for+ricin-mediated+cell+death+in+Saccharomyces+cerevisiae.">Ribosome depurination is not sufficient for ricin-mediated cell death in Saccharomyces cerevisiae.</a> Infect Immun. 75 (1), 417-428 (2007).</li><li>Lord, J. M., Roberts, L. M., Robertus, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ricin:+structure,+mode+of+action,+and+some+current+applications.">Ricin: structure, mode of action, and some current applications.</a> Faseb J. 8 (2), 201-208 (1994).</li><li>Becker, B., Schmitt, 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=Adapting+yeast+as+model+to+study+ricin+toxin+a+uptake+and+trafficking.">Adapting yeast as model to study ricin toxin a uptake and trafficking.</a> Toxins (Basel). 3 (7), 834-847 (2011).</li><li>Seidman, C. E., Struhl, K., Sheen, J., Jessen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+plasmid+DNA+into+cells.">Introduction of plasmid DNA into cells.</a> Curr Protoc Mol Biol. , (2001).</li><li>Brunelle, J. L., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coomassie+blue+staining.">Coomassie blue staining.</a> Methods Enzymol. 541, 161-167 (2014).</li><li>Shapiro, A. L., Vinuela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem Biophys Res Commun. 28 (5), 815-820 (1967).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.+1979.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. 1979.</a> Biotechnology. 24, 145-149 (1992).</li><li>Ito, H., Fukuda, Y., Murata, K., Kimura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+intact+yeast+cells+treated+with+alkali+cations.">Transformation of intact yeast cells treated with alkali cations.</a> J Bacteriol. 153 (1), 163-168 (1983).</li><li>Vitetta, E. S., Yen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+functional+properties+of+genetically+engineered+ricin+B+chain+lacking+galactose-binding+activity.">Expression and functional properties of genetically engineered ricin B chain lacking galactose-binding activity.</a> Biochim Biophys Acta. 1049 (2), 151-157 (1990).</li><li>Wales, R., Roberts, L. M., Lord, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+an+endoplasmic+reticulum+retrieval+sequence+to+ricin+A+chain+significantly+increases+its+cytotoxicity+to+mammalian+cells.">Addition of an endoplasmic reticulum retrieval sequence to ricin A chain significantly increases its cytotoxicity to mammalian cells.</a> J Biol Chem. 268 (32), 23986-23990 (1993).</li><li>Breslow, D. K., 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=A+comprehensive+strategy+enabling+high-resolution+functional+analysis+of+the+yeast+genome.">A comprehensive strategy enabling high-resolution functional analysis of the yeast genome.</a> Nat Methods. 5 (8), 711-718 (2008).</li><li>Jablonowski, D., Schaffrath, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymocin,+a+composite+chitinase+and+tRNase+killer+toxin+from+yeast.">Zymocin, a composite chitinase and tRNase killer toxin from yeast.</a> Biochem Soc Trans. 35 (Pt 6), 1533-1537 (2007).</li><li>Jablonowski, D., Schaffrath, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+RNA+polymerase+II+is+affected+by+Kluyveromyces+lactis+zymocin.">Saccharomyces cerevisiae RNA polymerase II is affected by Kluyveromyces lactis zymocin.</a> J Biol Chem. 277 (29), 26276-26280 (2002).</li><li>Jablonowski, D., Frohloff, F., Fichtner, L., Stark, M. J., Schaffrath, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kluyveromyces+lactis+zymocin+mode+of+action+is+linked+to+RNA+polymerase+II+function+via+Elongator.">Kluyveromyces lactis zymocin mode of action is linked to RNA polymerase II function via Elongator.</a> Mol Microbiol. 42 (4), 1095-1105 (2001).</li></ol>

在执行上述协议时, 我们建议以下建议, 以取得成功的实验结果。
对于异源蛋白的表达, 重要的是不超过 IPTG 浓度的1毫米。IPTG 的浓度和 #62; 1 mM 抑制启动子诱导的贸易量表达, 导致毒素产量降低。此外, 细胞不应在高于28° c 的温度下培养, 以防止包涵体形成、低效折叠和毒素失活。在较低的温度 (例如, 20-28 ° c) 是可能的, 也导致生物活性毒素的生产。然而, 额外的温度降...

由毒素产生菌感染的病人是每个社会保健系统的严重的医疗和经济负担, 特别是因为有效的治疗治疗仍然很大程度上缺失。为了发展新的治疗策略, 在医学上相关的 a/b 毒素, 如霍乱毒素, 志贺毒素, 或蓖麻毒的复杂中毒机制, 需要在分子水平上充分了解, 基于新的强有力的化验, 必须实施。
近年来, 一些研究试图通过使用耗时和成本密集型的方法, 如放射性毒素标记1,2以及 siRNA-based 筛选来分析酵母和哺乳动物细胞中的 a/b 毒素转运接近3。在某些情况下, 毒素贩运已被可视化的在体内荧光显微镜后, 与荧光, 量子点, 或荧光蛋白的单个毒素亚基的化学和/或遗传耦合4,5。不幸的是, 这种修改往往导致不活泼和/或改变的自然性质的毒素。另一种可以间接回答各种科学问题的优雅方法是使用基于酶的记者系统, 如lacZ、荧光或荧光蛋白 (如GFP 或Discosoma sp. 红色荧光蛋白 (dsRed))。
在这篇手稿中, 一个简单的协议被描述为 extracellularly 在S. 酿酒酵母中的细胞内传输所需的细胞成分进行识别。因此, 荧光-报告质粒含有一个 N 终端 ER 输入信号, 其次是 gfp 作为蛋白质生物合成传感器, 这间接地测量了在体内的 gfp 荧光发射后的蛋白质的翻译抑制. 翻译6。如果与野生型相比, 在特定的酵母缺失突变体中, 吞和/或胞内贩运是消极的 (或积极的), 这可以通过 GFP 荧光发射的增加 (或减少) 检测6。
目前, 所有分析酵母细胞中的区域内转运的方法都被限制在 ER-to-cytosol 的转运过程中。在这样的人工系统中, 含有 ER 输入信号的含氟贸易协定是由一个可诱导的启动子表达的, 导致自杀型表现为1,7。虽然在本手稿中所描述的实验装置中, 蓖麻蛋白的细胞结合 B 亚基同样缺失, 因此, 不完全代表蓖麻毒素 holotoxin 中毒的自然情况8, 从等离子体中运毒膜通过高尔基体对 ER 的仪器可以与这种新的检测严密模仿。有趣的是, 在试点研究中取得的初步结果表明, 贸易协定所使用的贩运途径显示出与蓖麻毒 holotoxin 的中毒路线有惊人的相似之处。
总之, 所描述的方法可以用来确定特定的作用, 选择细胞蛋白在吞和贩运酵母。此外, 这种实验装置可以很容易地适应其他的核糖体灭活毒素产生和分泌的各种酵母和细菌物种, 如 zymocin 或志贺毒素。

<table>
	<tbody>
		<tr>
			<td>Bacterial and yeast strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E coli BL21 DE3(Gold)</td>
			<td>Aligent Technologies</td>
			<td>230130</td>
			<td></td>
		</tr>
		<tr>
			<td>S. cerevisiae BY4742</td>
			<td>Euroscarf</td>
			<td>Y10000</td>
			<td></td>
		</tr>
		<tr>
			<td>S. cerevisiae BY4742 deletion mutants</td>
			<td>Dharmacon</td>
			<td>YSC1054</td>
			<td>whole collection</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids used in this protocol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pET24a(+) (Novagen)</td>
			<td>Millipore</td>
			<td>69772-3</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-RTA(His6)</td>
			<td>Becker et al. (2016)3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase 20T</td>
			<td>USBio</td>
			<td>Z1000.250</td>
			<td>lytic enzyme for cell wall removal</td>
		</tr>
		<tr>
			<td>LB broth medium</td>
			<td>Thermo Scientific</td>
			<td>10855021</td>
			<td>15 g agar was added for plate production</td>
		</tr>
		<tr>
			<td>YNB</td>
			<td>Thermo Scientific</td>
			<td>DF0335-15-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A4418-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast drop-out mix supplemts without leucine</td>
			<td>Sigma-Aldrich</td>
			<td>Y1376-20G</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>05040-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>10197777001</td>
			<td></td>
		</tr>
		<tr>
			<td>D-raffinose</td>
			<td>Sigma-Aldrich</td>
			<td>83400-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S1876-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0750-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Thermo Scientific</td>
			<td>11811031</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Sigma-Aldrich</td>
			<td>I6758-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Roth</td>
			<td>3899.1</td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE ruler prestained</td>
			<td>Fermentas</td>
			<td>26616</td>
			<td>protein ladder used for Western analysis</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material for RTA purification, desalting and reader measurements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer Ultrospec 2100 pro</td>
			<td>Amersham</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Soniprep 150</td>
			<td>MSE</td>
			<td></td>
			<td>old model, other models available</td>
		</tr>
		<tr>
			<td>Fluoroskan Ascent</td>
			<td>Thermo Scientific</td>
			<td>5210470</td>
			<td>old model, not available anymore</td>
		</tr>
		<tr>
			<td>&#196;KTAPurifier</td>
			<td>Thermo Scientific</td>
			<td>28406266</td>
			<td>Product is discontinued and replaced</td>
		</tr>
		<tr>
			<td>HisTRAP HP column</td>
			<td>GE Healthcare</td>
			<td>17-5248-02</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTRAP desalting column</td>
			<td>GE Healthcare</td>
			<td>11-0003-29</td>
			<td></td>
		</tr>
		<tr>
			<td>Midisart sterile filter</td>
			<td>Sartorius</td>
			<td>16534K</td>
			<td>0.2 &#181;m pore size</td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>660 nm assay kit</td>
			<td>Thermo Scientific</td>
			<td>22660</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Thermo Scientific</td>
			<td>260860</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies (optional)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Tetra-His</td>
			<td>Qiagen</td>
			<td>34670</td>
			<td>primary antibody; 1:1,000 dilution</td>
		</tr>
		<tr>
			<td>Anti-mouse-HRP</td>
			<td>Sigma-Aldrich</td>
			<td>A9044-2ML</td>
			<td>secondary antibody, 1:10,000 dilution</td>
		</tr>
	</tbody>
</table>

a simple fluorescence-based reporter assay to identify cellular components required for ricin toxin a chain (rta) trafficking in yeast

细菌和植物 A/B 毒素利用真核细胞的自然贩运途径达到其在胞的细胞内目标, 并最终杀死。这种/B 类毒素一般由酶活性 Asubunit (如、蓖麻毒素 A ()) 和一个或多个细胞结合 Bsubunit (s) 组成, 后者负责对特定细胞表面受体的毒素结合。我们目前对甲乙毒素如何能有效地令人陶醉的细胞的知识帮助科学家了解基本的细胞机制, 如吞和细胞内蛋白在更高的真核细胞中排序。从医学的角度来看, 同样重要的是要找出主要的毒素贩运路线, 为病人找到适当的治疗方案, 或最终发展治疗毒素的癌症治疗应用。
由于对哺乳动物细胞的 a/b 毒素贩运的全基因组分析是复杂、耗时和昂贵的, 因此在酵母模型生物体酿酒酵母中进行了关于 a/b 毒素转运的几项研究。尽管不那么复杂, 酵母和高真核细胞中的基本细胞过程是相似的, 而且在酵母中获得的结果往往可以转移到哺乳动物的情况。
在这里, 我们描述一个快速和易于使用的记者化验分析的细胞内贩运的酵母。这种新的检测方法的一个重要优点是, 不仅有机会从内质网 (er) 中研究胞, 而且从细胞膜到 ER 的吞和逆行毒素的转运。该分析利用了一个记者质粒, 允许间接测量在体内的绿色荧光蛋白 (GFP) 的荧光发射后,在活体翻译。由于贸易协定有效地防止了蛋白质生物合成的 28S rRNA depurination, 这一分析可以通过检测荧光发射的变化来识别细胞内的细胞间贸易中所涉及的宿主细胞膜蛋白。

本手稿中所描述的协议的一般工作流程在图 1中作了说明, 大致总结了成功的贸易协定纯化和随后的 GFP 报告实验的单一步骤。可以在协议中找到每个单独步骤的更详细的描述。图 2说明了通过亲和层析 (图 2A) 和空矢量控件 (图 2B) 成功进行的贸易协定净化的预期结果。...

Watch this Scientific Journal Video about A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast at JoVE.com

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

一个简单的荧光记者的检测方法, 用于识别蓖麻毒素所需的细胞成分 a 链 (贸易协定) 贩运酵母

在手稿中, 我们描述了使用模型荧光记者分析, 以确定涉及的细胞成分的贩运和杀伤过程中的毒素 a 亚基的植物毒蓖麻。

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

Research

JoVE Journal

Biology

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Pyrosequencing: A Simple Method for Accurate Genotyping

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

科研

生物学

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

定量检测，以研究蛋白质：DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

G蛋白偶联受体的基于荧光的钙动员含量表征

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

核酸的分离过滤：一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...