从脂质绑定蛋白和过敏原中去除和替换内源性利根
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 布拉 g 1 克隆 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。 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 。</…
Representative Results
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…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Bla g 1 Gene | Genescript | N/a | Custom gene synthesis service. GenBank Accession no AF072219 Residues 34-216 |
| Affinity purified natural Bla g 1 (nBla g 1) | Indoor biotechnologies | N/a | Custom order |
| Agilent 1100 Series HPLC System | Agilent | G1315B, G1311A, G1322A | UV Detector, Pump, and Degasser |
| Agilent DD2 600 MHz spectrometer | Agilent | N/a | |
| Amicon Ultra-15 Centrifugal Filter Unit | Amicon | UFC-1008 | |
| Ampicillin | Fisher Scientific | BP1760-5 | |
| Benzonase | Sigma-Aldrich | E1014-5KU | |
| Broad- band 5 mm Z-gradient probe | Varian | N/a | |
| ChemStation for LC (Software) | Agilent | N/a | |
| cOmplete Mini Protease Inhibitor Cocktail | Roche | 11836153001 | |
| Distearoylphosphatidylcholine (18:0 PC) | Avanti Polar Lipids | 850365C | |
| E. Coli BL21 DE3 Cells | New England Biolabs | C2530H | |
| Freezone 4.5 Freeze Dry System | Labconco | 7750000 | |
| Glutathione Resin | Genescript | L00206 | |
| Glutathione, Reduced | Fisher Scientific | BP25211 | |
| Isopropyl-β-D-thiogalactopyranoside (IPTG) | Fisher Scientific | 34060 | |
| Jasco CD spectropolarimeter | Jasco | J-815 | |
| Millex Syringe Filter Unit | EMD Millipore | SLGS033SS | |
| NMRPipe (Software) | Delaglio et al. | N/a | Delaglio, F. et al. Nmrpipe – a Multidimensional Spectral Processing System Based On Unix Pipes. J. Biomol. NMR 6, 277–293 (1995). |
| NMRViewJ (Software) | Johnson et al. | N/a | Johnson, B. A. & Blevins, R. A. NMR View: A computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994). |
| Oleic acid | Sigma-Aldrich | O1008 | |
| Pierce BCA Protein Assay | Sigma-Aldrich | BCA1-1KT | |
| Polaris 5 C18-A 250×10.0 mm HPLC Column | Agilent | SKU: A2000250X100 | |
| SD-200 Vacuum Pump | Varian | VP-195 | |
| Sodium Cholate Hydrate | Sigma-Aldrich | C6445 | |
| Sodium Palmitate | Sigma-Aldrich | P9767 | |
| Sodium Stearate | Sigma-Aldrich | S3381 | |
| VnmrJ (Software) | Varian | N/a |
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Cite This Article
Foo, A. C. Y., Thompson, P. M., Mueller, G. A. Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens. J. Vis. Exp. (168), e61780, doi:10.3791/61780 (2021).