Name: removal and replacement of endogenous ligands from lipid-bound proteins and allergens
Uploaded: 2021-02-24T13:00:00
Description: Watch this Scientific Journal Video about Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens at JoVE.com

我们要感谢汤姆·柯比博士、斯科特·加贝尔博士和罗伯特·伦敦博士在整个工作中给予的帮助和帮助，以及鲍勃·彼得罗维奇博士和萝莉·爱德华兹博士在本研究中使用的仪器和帮助产生Bla g 1构造。我们感谢安德里亚·亚当斯对质谱学的帮助，感谢尤金·德罗斯博士对NMR仪器的帮助。这项研究得到了国家卫生研究院、国家环境卫生科学研究院（Z01-ES102906）的校内研究计划的支持。内容完全由作者负责，不一定代表国家环境卫生科学研究所的官方观点。

1. 布拉 g 1 克隆
<ol><li>获得蟑螂过敏原Bla g 1.0101（残留物34-216）的基因，表示MA域的一次重复。为了简单起见，Bla g 1 将在整个工作中用于表示此单次重复，而不是整个 Bla g 1.0101 成绩单。</li>
	<li>将Bla g 1基因子将到所需的载体中。在这项研究中，含有 N - 末期谷胱甘肽 S - 转移酶（ GST ）标签的基因与烟草蚀刻病毒（ TEV ）蛋白酶部位入 pGEX 载体以表达如前所述11 。</...

<ol>
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本工作中描述的协议已成功地应用于系统地研究 Bla g 1 的脂质结合特性。这揭示了货物结合、恒温和内分泌处理之间的相关性，后者与已知T细胞表位的产生减少有关，对免疫原性9、18有潜在影响。除了 Bla g 1 之外，其他过敏原（如保诚 p 3 和 Bet v 1）已证明在使用标准亲和力和大小排除色谱方法13、19、20、21、22<sup class...

对过敏原数据库的调查显示，过敏原只存在于所有已知蛋白质家族的2%中，这表明常见的功能和生物物理特性有助于过敏1。在这些特性中，粘合脂质货物的能力在过敏原中似乎被强烈地夸大了，这表明这些货物可能会影响敏感过程1。事实上，已经表明，巴西坚果过敏原Ber e 1需要与它的内源性脂质共同管理，以实现其充分的敏感潜力2。这些脂质可能直接刺激免疫系统，如小虫过敏原Der p 2和Der p 7所示，这两种脂质都与LPS结合蛋白3、4、5具有很强的结构同源性。根据这一观察，建议Derp 2和Der p 7可以结合细菌脂质，并通过TLR4介导信号直接刺激宿主免疫系统，促进5，6的敏感过程。内源性结合脂质也有可能改变过敏性蛋白质本身的生物物理特性。例如，Sin a 2（芥末）和阿拉h 1（花生）与磷脂性粘结体相互作用的能力显著增强了它们对胃和内分泌降解7的抵抗力，而配体与主要的白杨花粉过敏原Bet v 1结合改变了内分泌处理速率和由此产生的肽8的多样性。鉴于稳定性、T细胞表位生成和蛋白质（如Bet v 1和Bla g 1）的过敏性之间的相关性，这与过敏性特别相关：后者将是这项工作的主题9，10。
Bla g 1代表昆虫主要过敏原（MA）蛋白质家族的原型成员，并拥有由12个两栖病态α海片组成的独特结构，包围着异常大的疏水腔9，11。Bla g 1 的可用 X 射线晶体结构显示该腔内的电子密度与绑定磷脂或脂肪酸配体一致：31P-NMR和质谱法证实的猜想。这些货物在性质上是异质的，它们的组成严重依赖过敏原来源，观察到不同的脂质特征，用于大肠杆菌和P.面食表达的重组Bla g 1。奇怪的是，Bla g 1从其天然过敏原源（蟑螂菌）中纯化，在其结合部位内主要含有脂肪酸，棕榈酸盐、油酸盐和凝固物的混合物被确定为其"天然"配体9，11。Bla g 1 在多个净化步骤后保留脂质和脂肪酸的能力阻碍了孤立研究蛋白质的努力。相反，有人提出，Bla g 1（从今称为nMix）的天然棕榈酸盐、石板和油酸盐配体在其过敏性和原生生物功能9中起着关键作用。然而，这些配体在Bla g 1中并不存在，这些配体来自重组来源，因此很难评估这一假设。类似的问题已经观察到其他脂质结合过敏原，如Bet v 112，13。为了促进脂质-过敏原相互作用的系统研究，我们制定了一个协议，通过该协议，过敏原可以定量地剥离其内源性结合脂质，并以Apo形式或装载特定配体进行重组。
过敏原通常使用亲和力色谱和/或大小排除色谱从其自然或重组来源纯化。在这里，我们引入了额外的纯化步骤，以高性能液相色谱 （HPLC） 的形式使用反相 C18 列，从该柱中将过敏原稀释成类似于为脂肪酸结合蛋白14开发的协议的有机溶剂。因此，在脂肪酸和/或磷脂的缺乏或存在的情况下，产生的蛋白质将遭受热退化步骤。除了恢复原生 Bla g 1 折外，升高的温度还增加了脂质货物的溶解性和可访问性，在 Apo 形式中产生 Bla g 1 或均匀装载所需的疏水配体。 31以这种方式净化的Bla g 1的P-NMR光谱确认完全去除内源性结合的配体，并统一更换所需的化合物，而圆形二分法则确认了Bla g 1褶皱的成功恢复。在最近一项工作中，发现货物结合可增强Bla g 1的热敏度和蛋白解电阻，改变T细胞表位生成的动力学，对感化和过敏性9具有潜在影响。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bla g 1 Gene&#160;</td>
			<td>Genescript</td>
			<td>N/a</td>
			<td>Custom gene synthesis service. GenBank Accession no AF072219 Residues 34-216</td>
		</tr>
		<tr>
			<td>Affinity purified natural Bla g 1 (nBla g 1)</td>
			<td>Indoor biotechnologies</td>
			<td>N/a</td>
			<td>Custom order</td>
		</tr>
		<tr>
			<td>Agilent 1100 Series HPLC System</td>
			<td>Agilent</td>
			<td>G1315B, G1311A, G1322A</td>
			<td>UV Detector, Pump, and Degasser</td>
		</tr>
		<tr>
			<td>Agilent DD2 600 MHz spectrometer</td>
			<td>Agilent</td>
			<td>N/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Unit</td>
			<td>Amicon</td>
			<td>UFC-1008</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>Sigma-Aldrich</td>
			<td>E1014-5KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Broad- band 5 mm Z-gradient probe</td>
			<td>Varian</td>
			<td>N/a</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemStation for LC (Software)</td>
			<td>Agilent</td>
			<td>N/a</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Mini Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11836153001</td>
			<td></td>
		</tr>
		<tr>
			<td>Distearoylphosphatidylcholine (18:0 PC)</td>
			<td>Avanti Polar Lipids</td>
			<td>850365C</td>
			<td></td>
		</tr>
		<tr>
			<td>E. Coli BL21 DE3 Cells</td>
			<td>New England Biolabs</td>
			<td>C2530H</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezone 4.5 Freeze Dry System</td>
			<td>Labconco</td>
			<td>7750000</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Resin</td>
			<td>Genescript</td>
			<td>L00206</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione, Reduced</td>
			<td>Fisher Scientific</td>
			<td>BP25211</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl-&#946;-D-thiogalactopyranoside (IPTG)</td>
			<td>Fisher Scientific</td>
			<td>34060</td>
			<td></td>
		</tr>
		<tr>
			<td>Jasco&#160; CD spectropolarimeter</td>
			<td>Jasco</td>
			<td>J-815</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex Syringe Filter Unit</td>
			<td>EMD Millipore</td>
			<td>SLGS033SS</td>
			<td></td>
		</tr>
		<tr>
			<td>NMRPipe (Software)</td>
			<td>Delaglio et al.&#160;</td>
			<td>N/a</td>
			<td>Delaglio, F. et al. Nmrpipe - a Multidimensional Spectral Processing System Based On Unix Pipes. J. Biomol. NMR 6, 277&#8211;293 (1995).</td>
		</tr>
		<tr>
			<td>NMRViewJ (Software)</td>
			<td>Johnson et al.&#160;</td>
			<td>N/a</td>
			<td>Johnson, B. A. &#38; Blevins, R. A. NMR View: A computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603&#8211;614 (1994).</td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Sigma-Aldrich</td>
			<td>O1008</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay</td>
			<td>Sigma-Aldrich</td>
			<td>BCA1-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Polaris 5 C18-A 250x10.0 mm HPLC Column</td>
			<td>Agilent</td>
			<td>SKU: A2000250X100</td>
			<td></td>
		</tr>
		<tr>
			<td>SD-200 Vacuum Pump</td>
			<td>Varian</td>
			<td>VP-195</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cholate Hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C6445</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Palmitate</td>
			<td>Sigma-Aldrich</td>
			<td>P9767</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Stearate</td>
			<td>Sigma-Aldrich</td>
			<td>S3381</td>
			<td></td>
		</tr>
		<tr>
			<td>VnmrJ (Software)</td>
			<td>Varian</td>
			<td>N/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

removal and replacement of endogenous ligands from lipid-bound proteins and allergens

许多主要过敏原与疏水性脂质状分子结合，包括Mus m 1、Bet v 1、Der p 2和Fel d 1。这些配体被强烈保留，并有可能通过直接刺激免疫系统或改变过敏蛋白的生物物理特性来影响敏感过程。为了控制这些变量，需要技术来去除内源性绑定配体，并在必要时用已知成分的脂质代替。蟑螂过敏原Bla g 1封闭了一个大的疏水腔，在使用传统技术纯化时结合了异质内源性脂质的混合物。在这里，我们描述了一种方法，通过这种方法，这些脂质被删除使用反相HPLC，然后热退化产生Bla g 1在其Apo形式或重新加载与用户定义的脂肪酸或磷脂货物的混合物。将该协议与生化检测结果结合，可显著改变Bla g 1的恒温性和蛋白解电阻，对T细胞表位生成速率和过敏性有下游影响。这些结果突出了脂质去除/重新加载协议的重要性，例如在研究来自重组源和自然来源的过敏原时所述的协议。该协议可推广到其他过敏原家族，包括脂蛋白（Mus m 1）、PR-10（投注 v 1）、MD-2（Der p 2）和乌特罗蛋白（Fel d 1），为研究脂质在过敏反应中的作用提供了宝贵的工具。

使用亲和色谱法，重组的 GST-Bla g 1 很容易被隔离到高纯度 （图1A），产生 +2+4 毫克/升的细胞培养产量。在 4 °C 下与 TEV 蛋白酶进行隔夜孵化足以去除 GST 标签，最终产品为 +24 kDa。请注意，在这种情况经和洗涤分数中有大量 GST - Bla g 1 ，这表明谷胱甘肽树脂绑定容量已超出。使用更多的树脂或样品加载和洗脱的多个周期可以为这个问题提供补救措施。
将 Bla g 1 应...

Watch this Scientific Journal Video about Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens at JoVE.com

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

该协议描述了从过敏原中去除内源性脂质，并通过逆相HPLC与热退化用用户指定的配体替换它们。 31P-NMR 和圆形二分法允许快速确认配体去除/装载，并恢复原生过敏原结构。

从脂质绑定蛋白和过敏原中去除和替换内源性利根

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

许多过敏原结合疏水分子。该协议能够完全去除和更换这些配体，使我们能够系统地研究它们对结构和免疫原性的影响。使用逆相HPLC，加上热退化，有两个优点。它去除内源性配体，并有助于溶解配体，以打开否则无法访问的绑定站点。如果我们能确定导致过敏的因素，我们也许能够设计避免这些因素的疗法。许多蛋白质结合脂质配体，这些脂质配体被强烈保留，并可能影响结构和功能。我们的方法允许系统地研究这些相互作用。许多脂质货物的溶解性和可及性有限，可能会阻碍装载过程。在准备这些配衣时一定要小心，并考虑系统特定的适应的需要。由于与疏水和不溶性配体一起工作是一些过敏症患者和生物化学家的调整，因此查看样本在不同阶段的外观是有用的。克隆表达和纯化后，将 12 毫升的裂口 Bla g 1 涂抹到离心滤芯单元中，该过滤器单元的分子重量小于 10 千达尔顿，用于在回转桶转子中多次离心，直到总体积降至 2 毫升以下。将产生的浓缩物加载到 250 乘 10 毫米 HPLC 系统上，该系统配备了 C18 反相色谱列，以每分钟 1.5 至 4 毫秒的流速，以 97% 缓冲 A 和 3% 缓冲 B.Elute Bla g 1 等效，使用 280 纳米的荧光吸收来监测优雅过程。从大约 34 到 40 分钟开始，缓冲 B 浓度高于 74% 收集不超过每个 Bla g 1 分数的 4 毫升。将池中的 Bla g 1 分数引用到玻璃试管中。用石蜡膜盖住管子，用两个孔打孔。然后，将样品在零下80摄氏度下冷冻一小时，然后使用同化剂干燥由此产生的去脂蛋白样本。为了确定预期的 Bla g 1 产量，在 5 毫升的再折叠缓冲中重新暂停裂解脱脂测试。将混合物加热到含有250毫升水的500毫升烧瓶中，加热至95摄氏度的热板上，搅拌和间歇性漩涡。将溶液保持在 95 度，30 至 60 分钟，然后让水浴慢慢平衡到室温。溶液冷却后，通过 0.22 微米注射器过滤器将已脱脂的 Bla g 1 脂质混合物通过去除颗粒物，并使用带有 10 千达吨切口的新型离心滤光片将过滤后的蛋白质三次缓冲到 PBS 中，以去除任何残留的游离脂肪酸和有机溶剂。然后，使用标准蛋白质分析分析评估蛋白质浓度，以确定剩余 Bla g 1 单注的预期产量。要重新组入 Apo-Bla g 1，将 Bla g 1 的别名重新悬挂在每阿里报价的 5 毫升重新折叠缓冲区中，并按演示对样品进行退化。要将 Bla g 1 与磷脂一起装载，将所需的货物加入玻璃试管中 10 毫克，然后溶解在氯仿中，然后蒸发氯仿以产生脂质薄膜。然后，将 PBS 添加到管中，以产生最终的磷脂浓度为 20 毫摩尔。将磷脂加热到脂质货物的相位过渡温度上方，以补充脂质薄膜和涡流，直到溶液变成多云。然后，根据预期产量，加入磷脂货物，产生相对于 Bla g 1 的 20 倍摩尔过多的配体。悬崖可能形成。漩涡在使蛋白质退化前混合， 如所示。要确认通过磷-31 NMR去除或装载货物，请使用离心过滤器将样品浓缩到超过 100 微摩尔（如图所示）。如前所述，准备 PBS 缓冲区中已知浓度的参考磷脂样本，并稀释 Bla g 1 末端参考，将胆碱缓冲器与总容量约 600 微升的 1：1 比率稀释。使用宽带探头获取整理溶解 Bla g 1 样本的一维 31 磷 NMR 光谱和参考磷脂标准， 并将 Bla g 1 31 磷 NMR 光谱与磷脂参考样品获得的光谱进行比较，以确认去除内源性结合的配体和/或根据可见山峰的化学变化对所需配体的结合，然后比较 Bla g 1 光谱的峰值强度和磷脂参考标准的峰值强度，以便确认完全结合的固态测量。使用亲和色色谱，重组的 GST Bla g 1 可以很容易地隔离到高纯度，每升细胞培养产生 2 到 4 毫克的产量。在摄氏四度下用 TEV 蛋白酶进行隔夜孵化足以去除 GST 标签，最终产品产量约为 24 千达吨。将 Bla g 1 应用于反相 C18 列可产生独特的弹性轮廓，其中两个大峰值为 50% 缓冲 B，第二个大峰值为 75% 缓冲 B.磷-31 NMR 光谱 Apo-Bla g 1 显示无可检测的磷脂。使用已知 DSPC 浓度的参考样本，可以从 NMR 生成标准曲线。将DSPC Bla g 1获得的磷31信号强度与此标准曲线进行比较，可以用来产生每蛋白质脂质的结合性血脂。Apo 和脂质加载 Bla g 1 的圆形二分法光谱显示 220 和 210 纳米的微型，表示以阿尔法-赫利科为主的结构。圆形二分法基热变性检测也显示α-赫利科二级结构的合作损失，表明折叠球状域。此外，对由此产生的熔化温度的分析显示，nMix配体结合显著增加，与从其天然过敏原源获得的 Bla g 1 一致。该协议的成功取决于能否克服疏水配体的有限溶解性，以及Bla g 1结合腔的不可接近性。这一程序为免疫学研究（如T细胞增殖检测）奠定了基础，以评估脂质配体对敏感性的影响以及发生这种影响的分子机制。结合此过程与进一步的生物物理检测，我们已经表明，疏水配体的结合增强了Bla g 1的稳定性，对表位生成和过敏的潜在下游影响。

Research

JoVE Journal

Biochemistry

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

从多个小鼠组织分离中间丝状蛋白以研究与老化相关的翻译后修饰

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

科研

生物化学

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

从人体二尖瓣提取蛋白质的优化方案

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

一种简单的分级提取方法，用于从单个样品中综合分析代谢物，脂质和蛋白质

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

用耐热配体测量生物分子 DSC 剖面快速表征折叠和结合的相互作用

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

利用综合沉淀富集系统识别目标蛋白的内源性后向转化剖面

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

肾小球的分离及肾小球细胞表面蛋白的活体标记

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

向外观察:分离氰化物释放的碳水化合物聚合物和蛋白质

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy

使用扩张显微镜对波多细胞蛋白尼腓林、阿克汀和波多辛的成像

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte ...

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

用利甘德分析（DRACALA）的微分径向毛细管作用识别小配体的结合蛋白

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...