Name: extraction and visualization of protein aggregates after treatment of escherichia coli with a proteotoxic stressor
Uploaded: 2021-06-29T13:30:00
Description: Watch this Scientific Journal Video about Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor at JoVE.com

这项工作得到了伊利诺伊州立大学生物科学学院启动基金、伊利诺伊州立大学新学院倡议赠款和NIAID赠款R15AI164585（J.-U.）的支持。D.).G.M.A.得到了伊利诺伊州立大学本科研究支持计划（对G.M.A.）的支持。K. P. H. 得到了德国学术交流服务局 （DAAD） 提供的 RISE 研究金的支持。作者感谢来自大科技维特里布斯有限公司的乌韦·兰道博士和卡斯滕·迈耶博士提供了AGXX粉末。图1、图2、图3、图4和图5是用生物扳机生成的。

1. 大肠杆菌 株MG1655和CFT073应激处理
<ol><li>接种5mL的利生肉汤（LB）介质，分别与单菌群的共生 大肠杆菌 株MG1655和尿病原性 大肠杆菌 （UPEC）菌株CFT073，并在37°C和300 rpm孵育为14-16小时（过夜）。 注： 大肠杆菌 CFT073是一种人类病原体。CFT073 的处理必须在生物安全 2 级认证实验室中采取适当的生物安全措施。</li>
	<li>将每个菌株稀释成500 mL烧瓶，含有70 mL的3-（N-</e...

<ol>
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本协议描述了一种优化的方法，用于分析蛋白质聚合形成后，处理不同的大肠杆菌菌株与蛋白质毒性抗菌剂。该协议允许同时从经过治疗和未经治疗的大肠杆菌细胞中提取不溶性和可溶性蛋白质分数。与现有的蛋白质聚合分离方案相比，从细胞14，15，16，20，这种方法有几个优点：（?...

细菌不可避免地会受到无数的环境压力，包括低pH值（例如， 在哺乳动物胃）1，2，活性氧和氯物种（ROS/RCS）（例如，在噬菌细胞氧化爆裂期间）3，4，5，温度升高（如在温泉或热休克期间）6，7，和几个有效的抗菌剂（例如，AGXX用于本协议）8。蛋白质特别容易受到这些压力源的影响，暴露会引发蛋白质的无/误折叠，然后播种聚合。所有生物体都采用保护系统，使它们能够处理蛋白质误折叠9。然而，严重的压力会压倒蛋白质质量控制机制，破坏蛋白质的二级和/或三级结构，最终使蛋白质失活。因此，蛋白质聚合物会严重损害细菌生长和生存、抗压和毒性所需的关键细胞功能。因此，关注蛋白质聚合及其在细菌中的后果的研究是一个相关的课题，因为它对传染病控制的潜在影响。
热诱导蛋白的展开和聚合通常是可逆的7。相比之下，其他蛋白质毒性应激，如氧化应激，可以通过氧化特定的氨基酸侧链导致蛋白质脱/误折叠，并最终导致蛋白质聚合4，从而导致不可逆转的蛋白质改变。压力诱导的不溶性蛋白质聚合物的形成，在分子伴奏及其在酵母和细菌11、12、13中的保护作用的背景下得到了广泛的研究。已经发布了几种协议，利用各种生化技术对不溶性蛋白质聚合物14、15、16、17进行分离和分析。现有协议主要用于研究热休克和/或分子伴郎鉴定后的细菌蛋白聚合。虽然这些协议肯定是该领域的一个进步，但在实验程序中存在一些重大不便，因为它们需要（i）高达 10 L14、17、（ii）复杂的物理干扰过程，包括使用细胞干扰器、法国印刷机和/或声波14、15、17或（iii）耗时的重复 洗涤和孵化步骤15，16，17。
本文描述了一个经过修改的协议，旨在解决以前方法的局限性，并允许分析在治疗后与蛋白毒抗菌表面涂层在两种不同的Escherichia大肠杆菌菌株中形成的蛋白质聚合量。涂层由金属银（Ag）和鲁西尼（鲁）组成，以抗坏血酸为条件，其抗菌活性是通过产生活性氧物种8、18来实现的。以下是细菌培养物与抗菌化合物治疗后的准备的详细描述，以及两种大肠杆菌菌株暴露后蛋白质聚集状态的比较，这些菌株具有明显的易感性，可增加抗菌素的浓度。所述方法价格低廉、速度快且可重复，可用于研究其他蛋白质化合物中的蛋白质聚合。此外，可以修改协议，以分析特定基因缺失对各种不同细菌中的蛋白质聚合的影响。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals/Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>67-64-1</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide/Bisacrylamide solution 29:1</td>
			<td>Bio-Rad</td>
			<td>1610156</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Millipore Sigma</td>
			<td>A3678-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase nuclease</td>
			<td>Sigma</td>
			<td>E1014-5KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Bluestain 2 Protein ladder, 5-245 kDa</td>
			<td>GoldBio</td>
			<td>P008-500</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Millipore Sigma</td>
			<td>M6250-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>GoldBio</td>
			<td>B-092-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>MP Biomedicals LLC</td>
			<td>821616</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Millipore Sigma</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sucrose</td>
			<td>Acros Organics</td>
			<td>57-50-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine tetra acetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>SLBT9686</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic acid</td>
			<td>Millipore Sigma</td>
			<td>ARK2183-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>G5516-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>GoldBio</td>
			<td>G-630-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid, ACS reagent</td>
			<td>Sigma-Aldrich</td>
			<td>320331-2.5L</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol (2-Propanol)</td>
			<td>Sigma</td>
			<td>402893-2.5L</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth (Miller)</td>
			<td>Millipore Sigma</td>
			<td>L3522-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth with agar (Miller)</td>
			<td>Millipore Sigma</td>
			<td>L2897-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>GoldBio</td>
			<td>L-040-25</td>
			<td></td>
		</tr>
		<tr>
			<td>10x MOPS Buffer</td>
			<td>Teknova</td>
			<td>M2101</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P-40</td>
			<td>Thomas Scientific</td>
			<td>9036-19-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate, dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>P3786-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate, monobasic</td>
			<td>Acros Organics</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Millipore Sigma</td>
			<td>T9281-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine</td>
			<td>Sigma-Aldrich</td>
			<td>T4625-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Trichloroacetic acid</td>
			<td>Millipore Sigma</td>
			<td>T6399-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>GoldBio</td>
			<td>T-400-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Material/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (15 mL)</td>
			<td>Alkali Scientific</td>
			<td>JABG-1019</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask (125 mL)</td>
			<td>Carolina</td>
			<td>726686</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask (500 mL)</td>
			<td>Carolina</td>
			<td>726694</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer: -80 &#176;C</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads (0.5 mm)</td>
			<td>BioSpec Products</td>
			<td>1107-9105</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Hermle</td>
			<td>Z216MK</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentriguge tubes (1.7 mL)</td>
			<td>VWR International</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentriguge tubes (2.0 mL)</td>
			<td>Axygen Maxiclear Microtubes</td>
			<td>MCT-200-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cuvettes</td>
			<td>Fischer Scientific</td>
			<td>14-377-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>ThermoFisher Scientific</td>
			<td>EC105</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>Alkali Scientific</td>
			<td>RS7235</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking incubator (37 &#176;C)</td>
			<td>Benchmark Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small glass plate</td>
			<td>Bio-Rad</td>
			<td>1653311</td>
			<td></td>
		</tr>
		<tr>
			<td>Spacer plates (1 mm)</td>
			<td>Bio-Rad</td>
			<td>1653308</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermoscientific</td>
			<td>3339053</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop centrifuge for 15 mL centrifuge tubes</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical gel electrophoresis chamber</td>
			<td>Bio-Rad</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Fisher Vortex Genie 2</td>
			<td>12-812</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Benchmark Scientific</td>
			<td>H5000-HC</td>
			<td></td>
		</tr>
		<tr>
			<td>10 well comb</td>
			<td>Bio-Rad</td>
			<td>1653359</td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction and visualization of protein aggregates after treatment of escherichia coli with a proteotoxic stressor

生物体暴露于环境和细胞压力中通常会导致蛋白质平衡的破坏，并可能导致蛋白质聚集。细菌细胞中蛋白质聚合物的积累可导致细胞表型行为发生重大变化，包括生长速率、抗应力和毒性降低。对这些压力源介质表型的检查存在几个实验程序。本文描述了一种优化的检测方法，用于使用含银抗菌素治疗后，从不同的 大肠杆 菌菌株中提取和可溶性蛋白质的提取和可视化。众所周知，这种化合物会产生活性氧物种，并导致广泛的蛋白质聚集。 
该方法结合了从治疗和未经处理的细胞中分离出的蛋白质聚合物和可溶性蛋白质的离心分离，以及随后通过硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和库马西染色进行分离和可视化。这种方法简单、快速，允许对不同 大肠杆菌 株中的蛋白质聚合形成进行定性比较。该方法具有广泛的应用，包括研究其他蛋白质抗菌素对多种细菌 体内 蛋白质聚合的影响的可能性。此外，该协议可用于识别有助于增强对蛋白毒物质抵抗力的基因。凝胶带可用于后续识别特别容易聚集的蛋白质。

<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62628/62628fig06.jpg"> 图6：大肠杆菌株MG1655和UGEC菌株CFT073中抗菌诱导蛋白聚合的代表性结果。大肠杆菌株MG1655和CFT073生长在37°C和300 rpm至OD600= 0.5-0.55在MOPS-g介质之前，他们处理与指示浓度 （-， 0毫克/毫升;+，175毫克/毫升;+，200毫克/毫升）的抗菌剂45分钟。可溶...

Watch this Scientific Journal Video about Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor at JoVE.com

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

本协议描述了用蛋白质毒性抗菌素治疗后从 大肠杆 菌中提取和可溶性蛋白质的提取和可视化。遵循此程序，可以对不同细菌菌株和/或治疗之间的 体内 蛋白质聚合形成进行定性比较。

用蛋白毒性应激器治疗大 肠杆菌 后蛋白质聚合物的提取和可视化

The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein ...

暴露在压力中会导致蛋白质聚集，并导致细菌表型行为发生重大变化。本视频中描述的程序允许在压力处理后提取和可视化蛋白质聚合物。与其他已公布的程序相比，此协议要求较低的细胞数量和避免复杂的物理中断过程，以及耗时的洗涤和孵化步骤。此协议可用于研究各种克阴性和克阳性细菌中的蛋白质聚合。您还可以分析基因缺失的影响或比较蛋白毒化合物的功效。首先接种大肠杆菌株MG1655和尿病原性大肠杆菌株CFT073的单一菌落，每粒进入5毫升的利生肉汤，或LB，介质。在37摄氏度和300转/分下孵育两种肉汤培养物，时间为14至16小时。将每个菌株稀释到 600 纳米（OD600）的光学密度为 0.1，放入包含 70 毫升 MOPS-g 介质的 500 毫升烧瓶中。在 37 度和 300 rpm 下孵化烧瓶，直到达到中日志阶段。孵化后，将每种培养物的20毫升转移到三个预热的125毫升烧瓶中，并在37摄氏度和300转时孵育它们两分钟。在 MOPS-g 介质中准备每毫升两毫克的抗菌化合物溶液。将此解决方案添加到每个培养物中，以达到所需的浓度，并添加未经处理的控制所需的 MOPS-g 介质体积。经过 45 分钟的压力处理后，确定每个培养的 OD600。对于每个样品，4 毫升培养物，其中 OD600 为 1 到 15 毫升离心机管。将细胞颗粒重新用于50微升冰冷裂解缓冲器。在冰上孵化样品30分钟。在样品中加入360微升的冰冷缓冲器A，然后通过管道轻轻混合。将样品转移到两毫升微中心管中，其中约200微升的0.5毫米玻璃珠。在温度混合器中在 8 摄氏度下孵育管子 30 分钟，在 1400 rpm 时进行摇晃。在冰上孵育管子五分钟，不摇晃，以安顿玻璃珠。然后将细胞裂解物的200微升转移到1.7毫升微中心管中。在16，000次克和4摄氏度下离心细胞解解20分钟。收集超纳特，稍后将用作可溶性蛋白质样本。使用移液器在 200 微升冰冷缓冲器 A. Centrifuge 中以 16，000 倍 g 和 4 摄氏度的速度将颗粒重新悬浮在 20 分钟内。然后小心地完全取出超高那。使用移液器小心地将颗粒重新浸入 200 微升冰冷缓冲器 B. Centrifufus 管中，并小心地取出超高分子。然后用缓冲器A.resuspend颗粒在100微升1倍减少SDS样品缓冲器中重复洗涤，并在95摄氏度的热混合器中煮5分钟。将一卷100%TCA与先前收集的四卷可溶性蛋白质样本混合，在4摄氏度下孵育10分钟，用于蛋白质沉淀。将样品在21，000次克和4摄氏度下离心5分钟，以获得沉淀物，并去除超高纳特。使用200微升冰冷丙酮清洗颗粒，去除细胞碎片。再次离心样品以获得沉淀物，并去除超高纳特。要从颗粒中取出剩余的丙酮，请将热混合器中的微中心管保持在 37 摄氏度，并打开盖子。然后加入100微升1倍减少SDS缓冲完全溶解颗粒，并在95摄氏度下煮沸样品5分钟。立即将样品加载到 SDS 聚丙烯酰胺凝胶上进行分离，或将样品存储在零下 20 摄氏度，以便稍后继续。准备文本手稿中描述的两种分离凝胶。使用一毫升移液器将凝胶倒在玻璃板之间，确保上两厘米的凝胶不含混合物。在分离凝胶上添加 70% 乙醇，在两层之间创建均匀的界面。分离凝胶聚合后，使用文本手稿中的说明准备堆叠凝胶。然后从分离的凝胶中取出乙醇，并添加堆叠凝胶溶液。小心地插入一个梳子，用所需的口袋数量，而不引入气泡，并允许凝胶聚合20至30分钟。将每个样品的四微升以及蛋白质梯子装载到单独的井中。然后在室温下以 144 伏特的三甘氨酸运行缓冲器中运行凝胶 45 分钟。使用预加热的费尔班克斯溶液 A 将摇摇器上的凝胶涂上 30 分钟，预加热的费尔班克斯溶液 D 将摇摇器上的凝胶除色，直到达到所需的背景。与未经治疗的细胞相比，当细胞以每毫升175微克和抗菌剂每毫升200微克进行治疗时，蛋白质聚集量有所增加。在更敏感的MG1655菌株中，不溶性蛋白质分数的增加更为明显。与未经治疗的细胞相比，当细胞以每毫升175微克和抗菌剂每毫升200微克进行治疗时，可溶性蛋白质的数量有所减少。在尝试此协议时，请记住，在 600 纳米下对光学密度的正常化对于比较样品中蛋白质聚合的程度非常重要。

Research

JoVE Journal

Biochemistry

Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

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 ...

科研

生物化学

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

人死后脑中洗涤剂不溶性蛋白的富集

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

丰度和收费水平的转移 Rna 在大肠杆菌中的量化

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

利用病毒衍生系统在大肠杆菌中大规模生产圆形脚手架上的重组 rna

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

体外和体内实验的位定向诱变-以大肠杆菌rna相互作用为例

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

通过红外纳米光谱和原子力显微镜对单个蛋白质聚合进行特征化

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在 埃斯切里西亚大肠杆菌

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...

用蛋白毒性应激器治疗大 肠杆菌 后蛋白质聚合物的提取和可视化

用蛋白毒性应激器治疗大肠杆菌后蛋白质聚合物的提取和可视化