Name: effect of fluorescent proteins on fusion partners using polyglutamine toxicity assays in yeast
Uploaded: 2018-11-28T14:45:00
Description: Watch this Scientific Journal Video about Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast at JoVE.com

这项研究得到了加拿大卫生研究所向医学博士和 p. l. 的运营赠款的支持。这里介绍的工作得到了加拿大创新基金会的 john r. evans 领导基金奖和安大略省研究基金向 p. l. y. j. 提供的配套基金的支持, 该基金由舒利奇 m 学院院长颁发的博士转学硕士学位奖学金提供支持在西安大略大学的医学和牙科。s. d. g. 由加拿大 als 博士奖学金提供支持。

1. 在酵母中表达的新荧光标记 httex1 记者的生成
注: 本节已由江泽民等人从议定书中修改。16和 albakri等人19岁
<ol><li>设计引物, 通过 pcr 放大编码荧光蛋白或感兴趣的序列。前引物应包括一个引线序列, 以协助在消化过程中的限制性酶 (gatc), 其次是一个 spei 限制位点 (actag) 和20个基地下游的 atg (不包括 atg) 的荧光蛋白基?...

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本文以各种检测方法进行了各种检测, 以测量 httex1多 q 扩张的聚集及其对酵母生长的影响, 以研究不同荧光蛋白如何在荧光记者的背景下改变其融合伙伴.使用 gfp 变体 (ymsfgfp) 作为阳性对照, 我们表明, 这可以检测到多 q 毒性和不同荧光标记之间聚集的显著变化, 并允许对 polyq-fp 融合性能进行直接和快速的比较。gfp 标记的构造16,19。
<p class="jove...

自从最初的表征绿色荧光蛋白 (gfp) 从aequorea维多利亚1, 一个广泛的调色板的基因编码的 fp 已经开发, 使细胞生物学家同时定位和跟踪多个活细胞中的细胞事件蛋白 2,3。flp 来自多种生物, 从水母到珊瑚, 因此, 显示出特定的生物物理特性, 这些特性广泛地转移到它们各自的荧光光谱之外。这些特性包括亮度、光稳定性和寡聚倾向等 2,4。选择单体 fp 是在设计荧光记者时选择合适标签的一个重要方面, 目的是最大限度地减少融合伙伴功能的不适当相互作用和变化, 最大限度地提高记者的效率。给细胞隔间 4,5,6。虽然随着时间的推移, gfp 的发展是为了最大限度地减少将荧光标记添加到融合伙伴5,7, 8 的影响, 但与 gfp 相比, 新的 fp 变体的性能仍难以评估。
很少有方法来描述 fp 的行为。其中大多数涉及使用生化方法测试 flp 的生物物理特性, 例如超离心和凝胶过滤方案9、10、11、12。这样的方法对在溶液中使用纯化的 fp 有一个警告, 几乎没有提供对它们在完整细胞中的行为的了解。通过测试过度表达的 fp 将内质网小管重组为小管的能力, 对 fp在活细胞 13中的低聚倾向进行了量化评估。oser漩涡 14。该技术可以成功地检测 gfp 和其他 fp 的单体和寡聚变异之间的变化。然而, 它主要依赖于瞬态转染细胞中的过度表达, 除非将该技术作为自动数据收集和分析工作流程, 否则定量和图像分析可能非常耗时。
为了补充这些方法, 我们建立了一种方法, 利用荧光标记对酵母15,16中多 q 扩张的毒性和聚集的影响。多 q 拉伸的扩展与超过36个重复在基因编码亨廷顿蛋白 (htt) 的第一个外显子与亨廷顿病 17,18.酵母中膨胀的 ttex1 的表达导致了错误折叠的 htt 蛋白的强烈聚集, 再加上严重的生长缺陷。有趣的是, 这些表型受到多 q 拉伸两侧序列的强烈影响, 包括 fp15,16。合理地认为, 不同性质的 fp 会对酵母中的多 q 毒性有不同的影响。事实上, 与类似 gfp 的 fp 相比, 红色荧光蛋白及其进化形式的毒性和聚集率降低了 16。这份手稿提供了一个详细的协议, 以评估下一代 fp 对酵母中的多 q 毒性和聚集的影响。该方法可对 fp 变体进行快速且可能含量高的分析, 可与以前表征的技术并行使用, 以优化新的 fp 特性, 并可评估它们与 gfp 相比的性能。

<table>
	<tbody>
		<tr>
			<td>5-alpha Competent E. coli (High efficiency)</td>
			<td>New Englanfd Biolab</td>
			<td>C2987</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeI-HF</td>
			<td>New Englanfd Biolab</td>
			<td>R3133</td>
			<td>High fidelity enzymes are preferred</td>
		</tr>
		<tr>
			<td>SalI-HF</td>
			<td>New Englanfd Biolab</td>
			<td>R0138</td>
			<td>High fidelity enzymes are preferred</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1425</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-Broth</td>
			<td>Fisher Scientific</td>
			<td>BP1426</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicilin</td>
			<td>Fisher Scientific</td>
			<td>BP1760</td>
			<td></td>
		</tr>
		<tr>
			<td>PfuUltra High-fidelity DNA Polymerase</td>
			<td>Agilent Technologies</td>
			<td>600382</td>
			<td></td>
		</tr>
		<tr>
			<td>EPOCH2 microplate spectrophotometer</td>
			<td>BioTek Instruments inc</td>
			<td>EPOCH2TC</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Pin Replicator</td>
			<td>V&#38;P Scientific inc.</td>
			<td>VP407AH</td>
			<td></td>
		</tr>
		<tr>
			<td>SPI imager</td>
			<td>S&#38;P Robotics inc.</td>
			<td>spImager-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM 800 confocal with AryScan</td>
			<td>Carl Zeiss Microscopy</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td>8 well Lab-Tek imaging chambers</td>
			<td>Fisher Scientific</td>
			<td>12565470</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Dot apparatus</td>
			<td>Bio-Rad</td>
			<td>1706545</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemi Doc XRS+</td>
			<td>Bio-Rad</td>
			<td>1708265</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-FLAG M1 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>F3040</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG alexa 555 secondary antibody</td>
			<td>Thermo&#160;</td>
			<td>A32727</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid MiniPrep Kit</td>
			<td>Fisher Scientific</td>
			<td>K0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Gel extraction Kit</td>
			<td>Fisher Scientific</td>
			<td>K0831</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Purification Kit</td>
			<td>Fisher Scientific</td>
			<td>K0702</td>
			<td></td>
		</tr>
		<tr>
			<td>Prizm</td>
			<td>GraphPad</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE (Tris-Acetate-EDTA)</td>
			<td>Fisher Scientific</td>
			<td>BP13354</td>
			<td></td>
		</tr>
	</tbody>
</table>

effect of fluorescent proteins on fusion partners using polyglutamine toxicity assays in yeast

为了利用活细胞成像对蛋白质定位和贩运进行研究, 研究人员通常依靠荧光记者感兴趣的蛋白质融合。不断发展的基因编码荧光蛋白 (fp) 列表为用户提供了几种替代方案, 当涉及到荧光融合设计。每个 fp 都具有特定的光学和生物物理特性, 可以影响由此产生的荧光融合的生化、细胞和功能特性。例如, 若干 fp 往往形成非特异性低聚物, 容易阻碍融合伙伴的功能。遗憾的是, 只有少数方法可以测试 fp 对荧光记者行为的影响。在这里, 我们描述了一个简单的方法, 使快速评估的影响, 使用多谷氨酰胺 (聚 q) 毒性检测的多谷氨酰胺酿酒酵母。聚 q 扩展亨廷顿蛋白与亨廷顿病 (hd) 的发生有关, 亨廷顿蛋白的扩展聚集在有毒的低聚物和包涵体中。酵母中多 q 膨胀的聚集和毒性在很大程度上取决于多 q 区两侧的序列, 包括荧光标记的存在, 从而为研究 fop 对其行为的影响提供了一个理想的实验平台。融合合作伙伴。

fp 具有不同的生物物理特性, 包括其寡聚倾向, 这可能会影响其融合伙伴在荧光记者的背景下的行为。该协议描述了一种简单的方法, 其中多个 fp 可以融合到有毒的聚 q 扩展。由于聚 q 毒性高度依赖于多边形拉伸15两侧的序列, 因此该检测允许对荧光聚 q 融合记者进行快速、直接的比较 (图 1)。非 hd 相关的多 q 长度 (25q) 用作负对照, ?...

Watch this Scientific Journal Video about Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast at JoVE.com

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

本文介绍了评估荧光蛋白对错误折叠的多谷氨酰胺扩张的聚集和毒性的影响的协议, 以便在荧光记者的背景下快速评估一种新的无特征荧光蛋白。

荧光蛋白对酵母中多谷氨酰胺毒性测定的融合伙伴的影响

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

这种方法可以帮助回答荧光蛋白领域的关键问题，例如荧光蛋白如何影响它们的融合伙伴。该技术的主要优点是，它提供了荧光蛋白及其融合伙伴的影响的快速和易于扩展的评估。虽然这种方法可以提供荧光蛋白行为的见解，它也可以应用于其他基因编码标签。同样演示这个程序的还有索尼亚·迪·格雷戈里奥，他是西方大学的研究生。通过这种方法测试的每个荧光蛋白首先被克隆成酵母表达载体，该载体编码了 FLAG 标记 HTT 的半乳糖可读版本，其中一种含有无毒 25Q 重复或亨廷顿病相关的毒性 72Q 重复。克隆通过测序进行选择和验证，随后转化为酵母。为了准备各种测定的细胞培养，在含有酵母选择介质、葡萄糖作为碳源的果糖板上，将携带25Q或72Q标签的酵母克隆带去。同时，带25Q或72Q酵母的条纹酵母优化单体超级折叠器GP作为正对。在30摄氏度下孵育盘子两到三天。从每个板中选择最多三个单菌落，接种五毫升合成完整介质，辅以2%葡萄糖作为碳源。在30摄氏度下孵育一夜之间。第二天，将每个隔夜培养的200微升转移到一个微离心管和离心机，以对细胞进行颗粒。用无菌蒸馏水至少洗三次。重要的是要洗好细胞，以消除所有葡萄糖的痕迹，可能有助于抑制GAL1启动子的诱导。如先前所证明的，准备细胞，并在含有2%半乳糖的合成完整介质中重新悬浮，作为碳源，诱导聚Q融合的表达。作为对照，在含葡萄糖的介质中重新悬浮细胞。在管旋转器中孵育细胞在30摄氏度下过夜。第二天早上，使用分光光度计测量每个培养的600纳米的光学密度。在无菌 96 孔板中，将电池密度均衡到光学密度 600，2/100 微升合成完整介质。通过将样品的20微升从以前的井中移液到下一个井的80微升介质中，准备每个样品的四个五倍稀释。使用酵母固定工具将细胞发现到选择性板上，并在30摄氏度下孵育两天。使用图像文档设备对板进行映像。为此测定准备细胞培养物，然后使用分光光度计测量每个培养物的 OD600。在96井板中将细胞稀释至300微升介质的OD600。以三份三份准备每个样本。在板读卡器培养箱中孵育具有摇动功能的板。将样品数量、温度设定为30摄氏度、吸收度为600纳米、实验长度为24小时、测量间隔为15分钟。选择连续抖动模式。实验完成后，使用科学绘图软件创建生长曲线并量化曲线下的区域。将数据粘贴到具有三个复制值的 XY 表中。增长曲线将显示在左侧的图形文件夹下。要量化曲线下的区域，请选择左上角的分析，然后单击 XY 分析中曲线下的面积。开始这个程序，稀释生长介质中10倍的已准备好的细胞。将每个样品的 200 微升转移到八井成像室。使用标准的宽场荧光显微镜对细胞进行成像。调整图像采集的映像设置。由于 72Q 聚合比漫射的 25Q 信号明亮得多，因此通常需要在不同的质粒之间使用不同的采集设置，以避免荧光信号饱和。使用合适的图像处理软件处理图像。在此协议中，点印用于检查蛋白质表达水平。在解解缓冲液中加入四微摩尔苯基硫化物和蛋白酶抑制剂鸡尾酒，为产生蛋白解酶准备缓冲液。通过离心，使每个过夜培养的五毫升。将细胞重新悬浮在200微升玻璃珠和200微升的解解缓冲液中。漩涡 30 秒 12 轮，在两轮之间结冰。在摄氏4度下以12，000倍G离心10分钟，并收集上光。使用微滤装置在硝基纤维素膜上发现等量的总蛋白质。用 PBS 预湿膜并组装设备。将其连接到真空源，并确保拧紧螺钉。加载样品，打开真空，让样品通过重力过滤膜。在 PBS 05%Tween 5% 无脂肪牛奶中混合膜 30 分钟。在四摄氏度下一夜之间用原发性抗旗抗体孵育膜。第二天，用PBS 05%Tween清洗膜三次，每次10分钟。在室温下用荧光标记的二次抗体在PBS 05%Tween 5%无脂肪牛奶中孵育膜一小时。用 PBS 05%Tween 清洗膜三次，每次 10 分钟。随后，使用氨基印迹记录系统对膜进行成像。酵母表达 25Q 或 72Q HTT exon 一融合到酵母优化单体超级折叠器 GFP 或酵母优化单体标签 BFP2 被培养在葡萄糖或半乳糖介质过夜，或发现在阿加板或进一步孵育在液体介质。而72Q酵母优化单体超级折叠器GP诱导显著的生长缺陷。72Q酵母优化单体标签BFP2显示的生长表型类似于无毒25Q对应物，表明荧光标签的性质可以阻碍细胞中的聚Q膨胀行为。通过荧光显微镜对荧光聚Q融合的聚合评估表明，72Q酵母优化单体超级折叠器GFP表现出显著的聚合，而72Q酵母优化的单体标签BFP2显示与无毒25Q对应体类似的扩散细胞质信号。由于各种聚Q融合的表达水平可能影响毒性，因此对点印进行点印以评估蛋白质水平。显示细胞利素五倍稀释的结果。不要忘记，这种技术允许快速比较非字符和荧光蛋白与GP变种，但它不能直接评估寡聚化。按照这个程序，其他措施，例如，在哺乳动物细胞的溃疡测定可以回答其他问题，如荧光蛋白的单体状态。

Research

JoVE Journal

Biology

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

使用荧光抗体对小鼠表皮石蜡切片的免疫组织化学

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

科研

生物学

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

在甲醇酵母表达的重组蛋白毕赤酵母</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Growth Assays to Assess Polyglutamine Toxicity in Yeast

生长实验，以评估在酵母中的多聚谷氨酰胺毒性

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

SNARE蛋白介导的膜融合酶的细胞融合含量的分析

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物<em&gt;温度。线虫</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

比较GTP酶结合蛋白的使用竞争性试验的亲和

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...