我们非常感谢 h. Mchaourab 博士提供了获得 EPR 和鹿分光仪和 t 鲍姆博士的机会, 以提供全长半胱氨酸--较少的 TRPV1 质粒。

作者没有什么可透露的。

目前, 哺乳动物膜蛋白的表达和纯化技术使得获得足够数量的蛋白质用于光谱学研究14,15,16,42。在这里, 我们已经适应了这些技术来表达, 纯化, 重建, 并执行光谱分析在 TRPV1。
在《议定书》的关键步骤中, 下面是我们为 TRPV1 诊断的, 可能会对其他蛋白质进行调整。修改 DNA 序列生成?...

近年来, 在单粒子低温电子显微镜 (冷冻 EM) 的研究进展中, 哺乳动物离子通道结构得到了非常的速度。特别是, polymodal 离子通道的结构研究, 如瞬态受体电位辣椒 1 (TRPV1), 已进一步了解其活化机制1,2,3,4,5. 然而, 在膜环境中嵌入离子通道的动态信息需要了解它们的 polymodal 门和药物结合机制。
电子顺磁共振 (EPR) 和双电子-电子共振 (鹿) spectroscopies 提供了一些最明确的机械模型的离子通道6,7,8,9,10,11,12,13. 这些方法主要限于检查原核和 archeal 离子通道, 在细菌抗原时产生大量的洗涤剂纯化蛋白。随着在昆虫和哺乳动物细胞中的真核膜蛋白的发展, 用于功能和结构表征14,15,16, 现在可以获得生物化学用于光谱研究的洗涤剂纯化蛋白质的数量。
EPR 和鹿信号产生于一个 paramagneticspin 标签 (SL) (即methanethiosulfonate) 附着在一个单一的半胱氨酸残留的蛋白质。自旋标签报告三种结构信息: 运动、可访问性和距离。这一信息允许确定残留物是埋藏在蛋白质中, 还是暴露于膜或水环境中的蛋白和配体约束状态13,17,18,19。在高分辨率结构 (如果可用) 的背景下, EPR 和鹿数据提供了在其本机环境中派生动态模型的约束集合, 同时监视可逆的浇口转换 (即闭合、打开、敏化, 和麻木)。此外, 可以通过使用这些环境数据集分配二级结构以及蛋白质20中的位置来获得可能难以由 X 射线晶体学或冷冻 EM 确定的灵活区域。在脂质 nanodiscs 中获得的低温 EM 结构提供了关于离子通道3、21、22、23、24 的浇口的宝贵信息,25;然而, 光谱方法可以提供动态信息从构象状态 (例如, 热变化), 可能是难以确定使用低温 EM。
在去除所有半胱氨酸残留物 (特别是哺乳动物通道中的丰富)、蛋白质产量低、纯化过程中蛋白质不稳定、自旋标记后, 必须克服许多困难, 以实现 EPR 和鹿类, 包括缺乏蛋白质功能。, 以及在洗涤剂或脂质体中的蛋白质聚集。在这里, 我们设计了克服这些关键屏障的协议, 并获得了哺乳动物感官受体的鹿和 EPR 光谱信息。这里的目的是描述的方法, 以表达, 纯化, 标记和重组功能极小的半胱氨酸小鼠 TRPV1 (eTRPV1) 结构的光谱分析。这种方法适用于那些保留其功能的膜蛋白, 尽管除去半胱氨酸残留物或含有半胱氨酸形成二硫键。这些协议的收集可以适应其他哺乳动物离子通道的光谱分析。

<table border="0" cellpadding="0" cellspacing="0" style="width:1048px;" width="1047">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:339px;">QuikChange Lightning Site-Directed Mutagenesis Kit</td>
			<td style="width:292px;">Agilent Technologies</td>
			<td style="width:251px;">210519-5</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2-Propanol (Isopropanol)</td>
			<td>Fisher Scientific</td>
			<td>A416</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Albumin Bovine Serum (BSA)</td>
			<td>GoldBio.com</td>
			<td>A-420-10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Amylose resin</td>
			<td>NEB</td>
			<td>E8021L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Aprotinin</td>
			<td>GoldBio.com</td>
			<td>A-655-25</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Asolectin from Soybean</td>
			<td>Sigma</td>
			<td>11145</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bac-to-Bac Baculovirus Expression System</td>
			<td>Invitrogen Life Technologies</td>
			<td>10359016</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Biobeads SM-2 Adsorbents&#160;</td>
			<td>Bio-Rad</td>
			<td>152-3920</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Borosilicate glass pipettes (3.5&#39;&#39;) (oocyte inyection)</td>
			<td>Drummond Scientific</td>
			<td>3-000-203 G/X</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Borosilicate glass pipettes (oocyte recordings)</td>
			<td>Sutter Instrument</td>
			<td>B150-110-10HP</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CaCl2 2H2O</td>
			<td>Fisher Scientific</td>
			<td>C79</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Carbenicillin (Disodium)</td>
			<td>GoldBio.com</td>
			<td>C-103-5</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cellfectin Reagent</td>
			<td>Invitrogen Life Technologies</td>
			<td>10362-010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">cellSens</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Chloroform</td>
			<td>Fisher Scientific</td>
			<td>C606SK</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Collagenase Type 1</td>
			<td>Worthington-Biochem</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Critiseal</td>
			<td>VWR</td>
			<td>18000-299</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">D-(+)-Glucose</td>
			<td>Sigma&#160;</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">D-(+)-Maltose Monohydrate</td>
			<td>Fisher Scientific</td>
			<td>BP684</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DDM (n-Docecyl-B-D-Maltopyranoside)</td>
			<td>Anatrace</td>
			<td>D310S</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">High glucose medium (Dulbecco&#8217;s Modified Eagle&#8217;s Medium)</td>
			<td>Sigma</td>
			<td>D0572&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Disposable PD-10 Desalting Columns</td>
			<td>GE Healthcare</td>
			<td>45-000-148</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">EGTA</td>
			<td>Fisher Scientific</td>
			<td>O2783</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal Bovine Serum</td>
			<td>Invitrogen Life Technologies</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fluo-4 AM</td>
			<td>Life Technologies</td>
			<td>F-14201</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GenCatch Plus Plasmid DNA Mini-Prep Kit</td>
			<td>Epoch Life Science, Inc</td>
			<td>2160250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GenCatch PCR Cleanup Kit</td>
			<td>Epoch Life Science, Inc</td>
			<td>2360050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gentamicin Sulfate</td>
			<td>Lonza</td>
			<td>17-518Z</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass capillary (25 &#181;l)</td>
			<td>VWR</td>
			<td>53432-761</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass Flask 2800 mL</td>
			<td>Pyrex USA</td>
			<td>4423-2XL</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>BP229</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEK293S GnTl-</td>
			<td>ATCC</td>
			<td>CRL-3022</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td>Sigma&#160;</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IPTG (isopropyl-thio-B-galactoside)</td>
			<td>GoldBio.com</td>
			<td>I2481C25</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kanamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">KCl</td>
			<td>Fisher Chemical</td>
			<td>P217</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LB Broth, Miller</td>
			<td style="width:292px;">Fisher bioReagents</td>
			<td>BP1426</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Leupeptin Hemisulfate</td>
			<td>GoldBio.com</td>
			<td>L-010-5</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamine 2000</td>
			<td>Invitrogen Life Technologies</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MgCl2 6H2O</td>
			<td>Fisher Scientific</td>
			<td>BP214</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MgSO4 7H2O</td>
			<td>Fisher Scientific</td>
			<td>BP213</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">mMESSAGE mMACHINE T7 Kit</td>
			<td>Ambion</td>
			<td>AM1344</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MOPS</td>
			<td>Fisher bioReagents</td>
			<td>BP2936</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MTSL (1-Oxyl-2,2,5,5-tetramethylpyrrolidin-3-yl) Methyl Methanethiosulfonate</td>
			<td>Toronto Research Chemicals, Inc</td>
			<td>O873900</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NaCl</td>
			<td>Fisher Chemical</td>
			<td>S271</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-062</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pepstatin A</td>
			<td>GoldBio.com</td>
			<td>P-020-5</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pluronic Acid F-127 (20%)</td>
			<td>PromoKine&#160;&#160;</td>
			<td>CA707-59004</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PMSF</td>
			<td>GoldBio.com</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Poly-L-lysine Solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Rneasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sealed capillary</td>
			<td>VitroCom</td>
			<td>special order</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SF-900 II SFM (insect cell medium)</td>
			<td>Gibco, Life Technologies</td>
			<td>10902-088</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sf9 Cells (SFM Adapted)</td>
			<td>Invitrogen Life Technologies</td>
			<td>11496-015</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Soybean Polar Lipid Extract</td>
			<td>Avanti Polar Lipids, Inc</td>
			<td>541602C</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S25590</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Superose 6 Increase 10/300 GL</td>
			<td>GE Healthcare</td>
			<td>29091596</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">TCEP HCl</td>
			<td>GoldBio.com</td>
			<td>TCEP1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tetracyclin Hydrochloride</td>
			<td>Fisher Scientific</td>
			<td>BP912-100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tris Base</td>
			<td>Fisher BioReagents</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tryptone</td>
			<td>Difco</td>
			<td>0123-01</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">X-gal</td>
			<td>GoldBio.com</td>
			<td>X4281C</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Xenopus oocytes</td>
			<td>Nasco</td>
			<td>LM00935M</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">XL1 - Blue Competent Cells</td>
			<td>Agilent Technologies, Inc</td>
			<td>200249</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Yeast Extract</td>
			<td>Difco</td>
			<td>0127-01-7</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:339px;">Econo-Pack chromatography column</td>
			<td style="width:292px;">Bio-Rad</td>
			<td>7321010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-PROTEAN TGX Stain-Free Precast Gels</td>
			<td style="width:292px;">Bio-Rad</td>
			<td>17000436</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">pFastBac1 Expression Vector</td>
			<td style="width:292px;">Invitrogen Life Technologies</td>
			<td style="width:251px;">10360-014</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DH10Bac Competent Cells</td>
			<td style="width:292px;">Invitrogen Life Technologies</td>
			<td style="width:251px;">10361-012</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Critiseal capillary tube sealant</td>
			<td>Leica Microsystems</td>
			<td>02-676-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ABI Model 3130XL Genetic Analyzers</td>
			<td>Applied Biosystems</td>
			<td>4359571</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipete</td>
			<td>Fishebrand</td>
			<td>13-711-9AM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanoject II</td>
			<td>Drummond Scientific</td>
			<td>3-000-204</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification and reconstitution of trpv1 for spectroscopic analysis

Polymodal 离子通道传感器不同性质的多种刺激构变化;这些动态构象是挑战, 以确定和仍然很大的未知。近年来, 单粒子低温电子显微镜 (低温 EM) 脱落光对激动剂结合部位的结构特征和几种离子通道的活化机理进行了研究, 确定了其浇口深度动态分析的阶段。使用光谱学方法的机制。电子顺磁共振 (EPR) 和双电子-电子共振 (鹿) 等光谱技术主要局限于可大量纯化的原核离子通道的研究。对大量功能性和稳定的膜蛋白的要求, 阻碍了使用这些方法研究哺乳动物离子通道。EPR 和鹿提供了许多好处, 包括确定移动蛋白区域的结构和动态变化, 虽然在低分辨率, 可能难以获得 X 射线结晶学或低温 EM, 并监测可逆门过渡 (即闭合、开放、敏感和麻木)。在这里, 我们提供的协议, 获取毫克的功能洗涤剂-可溶性瞬态受体电位阳离子通道亚科 V 成员 1 (TRPV1), 可以标记为 EPR 和鹿光谱学。

最小半胱氨酸-少 TRPV1 构造 (eTRPV1) 和单半胱氨酸突变体的功能表征
向光谱学研究迈出的第一步是对功能化和产生生化量蛋白质的半胱氨酸蛋白质结构 (图 2A) 进行设计和表征。eTRPV1 的功能, 由 Ca2 +成像和 TEVC (图 2B-C) 确定。此外, eTRPV1 为 EPR 和鹿实验提供了足够数?...

Watch this Scientific Journal Video about Purification and Reconstitution of TRPV1 for Spectroscopic Analysis at JoVE.com

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

光谱分析 TRPV1 的纯化与重构

本文介绍了获得洗涤剂-可溶性 TRPV1 的生化量的具体方法, 用于光谱分析。这些联合协议提供了生物化学和生物物理工具, 可用于促进膜控制环境中哺乳动物离子通道的结构和功能研究。

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

purification-and-reconstitution-of-trpv1-for-spectroscopic-analysis

Research

JoVE Journal

Biochemistry

7.9K 次观看.  University of Tennessee Health Science Center. 本文介绍了获得洗涤剂-可溶性 TRPV1 的生化量的具体方法, 用于光谱分析。这些联合协议提供了生物化学和生物物理工具, 可用于促进膜控制环境中哺乳动物离子通道的结构和功能研究。

视频: Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

S100A12的表达，纯化和抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

科研

生物化学

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

体外支持性脂质双层自组织蛋白模式的重组

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

神经节苷脂提取、纯化和分析

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode&#39;s thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

活性 秀丽隐杆线 虫核提取物的分离及重建用于 体外 转录

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

用完全纯化的组分重构Msp1提取活性

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

内源性 果蝇 瞬时受体电位通道的纯化

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...