内源性 果蝇 瞬时受体电位通道的纯化
Summary
基于INAD蛋白复合物的组装机理，在该方案中，开发了一种修饰的亲和纯化加竞争策略，以纯化内源性 果蝇 TRP通道。
Abstract
果蝇 光转导是已知最快的G蛋白偶联信号通路之一。为了确保该级联反应的特异性和效率，钙（Ca2 +）渗透性阳离子通道瞬时受体电位（TRP）与支架蛋白，失活无后电位D（INAD）紧密结合，并与眼睛特异性蛋白激酶C（ePKC）和磷脂酶Cβ/无受体电位A（PLCβ / NORPA）形成大信号蛋白复合物。然而， 果蝇 TRP通道的生化特性尚不清楚。基于INAD蛋白复合物的组装机理，建立了一种修饰的亲和纯化加竞争策略，以纯化内源性TRP通道。首先，将纯化的组氨酸（His）标记的NORPA 863-1095片段与Ni珠结合并用作诱饵，以从 果蝇 头匀浆中拉下内源性INAD蛋白复合物。然后将过量纯化的谷胱甘肽S-转移酶（GST）标记的TRP 1261-1275片段添加到Ni珠中以与TRP通道竞争。最后，通过尺寸排阻色谱法将上清液中的TRP通道与过量的TRP 1261-1275肽分离。这种方法可以从生化和结构角度研究 果蝇 TRP通道的门控机制。纯化的 果蝇 TRP通道的电生理学特性也可以在未来测量。
Introduction
光转导是吸收的光子被转换成神经元的电码的过程。它专门在脊椎动物和无脊椎动物中中继视蛋白和随后的G蛋白偶联信号级联。在 果蝇中， 通过使用其五个PDZ结构域，支架蛋白灭活无后电位D（INAD）组织了一个超分子信号传导复合物，该复合物由瞬时受体电位（TRP）通道，磷脂酶Cβ/无受体电位A（PLCβ / NORPA）和眼睛特异性蛋白激酶C（ePKC）1组成。这种超分子信号复合物的形成保证了 果蝇 光转导机制的正确亚细胞定位，高效率和特异性。在这种复杂的光敏TRP通道中充当NORPA的下游效应器，并介导钙流入和光感受器的去极化。先前的研究表明， 果蝇 TRP通道的开放是由质子介导的，破坏局部脂质环境，或机械力2，3，4。 果蝇 TRP通道也与钙调蛋白5 相互作用，并通过正反馈和负反馈6，7，8由钙调节。
到目前为止，关于果蝇TRP和TRP样（TRPL）通道的门控机制的电生理学研究是基于切除的膜贴片，来自解离的野生型果蝇光感受器的全细胞记录，以及S2，SF9或HEK细胞2，9，10，11，12，13中的异质表达通道，但不是在纯化通道上。全长果蝇TRP通道的结构信息也尚不清楚。为了研究纯化蛋白质在重构膜环境中的电生理性质并获得全长果蝇TRP通道的结构信息，获得纯化的全长TRP通道是必要的第一步，类似于哺乳动物TRP通道研究中使用的方法14，15，16，17。
近日，基于INAD蛋白复合物18，19，20的组装机理，首次开发了亲和纯化加竞争策略，通过链霉亲和素珠5从果蝇头匀浆中纯化TRP通道。考虑到链霉亲和素微球的低容量和昂贵的成本，这里引入了一种改进的纯化方案，该方案使用His标记的诱饵蛋白和相应的低成本Ni珠，其容量要高得多。该方法有助于从结构角度研究TRP通道的门控机理，并用纯化的蛋白质测量TRP通道的电生理性质。
Protocol
1. 净化带有商品及服务税标签的关税退税和他标记的诺帕片段 纯化 GST 标记的 TRP 1261-1275 片段 使用CaCl 2热冲击转化方法21将pGEX 4T-1 TRP 1261-1275质粒10转化为大肠杆菌（大肠杆菌）BL21（DE3）细胞。在10mL卢里亚贝塔尼（LB）培养基中接种单个菌落，并在37°C下生长过夜。 然后，在37°C的1LLB培养基中扩增10mL接种培养物。 <…
Representative Results
在本文中，演示了一种纯化内源性 果蝇 TRP通道的蛋白质纯化方法（图1）。 首先，应用重组蛋白表达和纯化来获得诱饵和竞争蛋白。然后，将GST标记的TRP 1261-1275片段在LB培养基的大 肠杆菌 BL21（DE3）细胞中表达，并使用谷胱甘肽珠和尺寸排阻柱纯化（图2）。使用考马斯蓝R250染色的SDS-PAGE分析对样品进行了验证。在SDS-…
Discussion
INAD包含五个PDZ域，是 果蝇 光转导机制的核心组织者。先前的研究表明，INAD PDZ3与TRP通道C端尾部结合，具有精细的特异性（KD = 0.3μM）18。INAD PDZ45串联与具有极高结合亲和力（KD = 30 nM）的NORPA 863-1095片段相互作用。这些发现为设计亲和纯化加竞争策略提供了坚实的生化基础，使NORPA CC-PBM片段可用作下拉诱饵，而TRP C末端尾部（片段1261-1275）可用作竞争试剂?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
本研究由国家自然科学基金（no.31870746）、深圳市基础研究基金（JCYJ20200109140414636）和广东省自然科学基金（编号：2021A1515010796）资助。我们感谢LetPub（www.letpub.com）在编写本手稿期间提供的语言帮助。
Materials
| Bacterial strains | |||
| BL21(DE3) Competent Cells | Novagen | 69450 | Protein overexpression |
| Experiment models | |||
| D.melanogaster: W1118 strain | Bloomington Drosophila Stock Center | BDSC:3605 | Drosophila head preparation |
| Material | |||
| 20/30/40 mesh stainless steel sieves | Jiufeng metal mesh company | GB/T6003.1 | Drosophila head preparation |
| 30% Acrylamide-N,N′-Methylenebisacrylamide(29:1) | Lablead | A3291 | SDS-PAGE gel preparation |
| Ammonium Persulfate | Invitrogen | HC2005 | SDS-PAGE gel preparation |
| Cocktail protease inhibitor | Roche | 05892953001 | Protease inhibitor |
| Coomassie brilliant blue R-250 | Sangon Biotech | A100472-0025 | SDS-PAGE gel staining |
| DL-Dithiothreitol (DTT) | Sangon Biotech | A620058-0100 | Size-exclusion column buffer preparation |
| Ethylenediaminetetraacetic acid disodium salt (EDTA) | Sangon Biotech | A500838-0500 | Size-exclusion column buffer preparation |
| Glycine | Sangon Biotech | A610235-0005 | SDS-PAGE buffer preparation |
| Glutathione Sepharose 4 Fast Flow beads | Cytiva | 17513202 | Affinity chromatography |
| Imidazole | Sangon Biotech | A500529-0001 | Elution buffer preparation for Ni-column |
| Isopropyl-beta-D-thiogalactopyranoside (IPTG) | Sangon Biotech | A600168-0025 | Induction of protein overexpression |
| LB Broth Powder | Sangon Biotech | A507002-0250 | E.coli. cell culture |
| L-Glutathione reduced (GSH) | Sigma-aldrich | G4251-100G | Elution buffer preparation for Glutathione beads |
| Ni-Sepharose excel beads | Cytiva | 17371202 | Affinity chromatography |
| N-Dodecyl beta-D-maltoside (DDM) | Sangon Biotech | A610424-001 | Detergent for protein purification |
| N,N,N',N'-Tetramethylethylenediamine (TEMED) | Sigma-aldrich | T9281-100ML | SDS-PAGE gel preparation |
| PBS | Sangon Biotech | E607008-0500 | Homogenization buffer for E.coli. cell |
| PMSF | Lablead | P0754-25G | Protease inhibitor |
| Prestained protein marker | Thermo Scientific | 26619/26616 | Prestained protein ladder |
| Size exclusion column (preparation grade) | Cytiva | 28989336 | HiLoad 26/60 Superdex 200 PG column |
| Size exclusion column (analytical grade) | Cytiva | 29091596 | Superose 6 Increase 10/300 GL column |
| Sodium chloride | Sangon Biotech | A501218-0001 | Protein purification buffer preparation |
| Sodium dodecyl sulfate (SDS) | Sangon Biotech | A500228-0001 | SDS-PAGE gel/buffer preparation |
| Tris base | Sigma-aldrich | T1503-10KG | Protein purification buffer preparation |
| Ultrafiltration spin column | Millipore | UFC901096/801096 | Protein concentration |
| Equipment | |||
| Analytical Balance | DENVER | APX-60 | Metage of Drosophila head |
| Desk-top high-speed refrigerated centrifuge for 15mL and 50mL conical centrifugation tubes | Eppendorf | 5810R | Protein concentration |
| Desk-top high-speed refrigerated centrifuge 1.5mL centrifugation tubes | Eppendorf | 5417R | Centrifugation of Drosophila head lysate after homogenization |
| Empty gravity flow column (Inner Diameter=1.0cm) | Bio-Rad | 738-0015 | TRP protein purification |
| Empty gravity flow column (Inner Diameter=2.5cm) | Bio-Rad | 738-0017 | Bait and competitor protein purification from E.coli. |
| Gel Documentation System | Bio-Rad | Universal Hood II Gel Doc XR System | SDS-PAGE imaging |
| High-speed refrigerated centrifuge | Beckman coulter | Avanti J-26 XP | Centrifugation of E.coli. cells/cell lysate |
| High pressure homogenizer | UNION-BIOTECH | UH-05 | Homogenization of E.coli. cells |
| Liquid nitrogen tank | Taylor-Wharton | CX-100 | Drosophila head preparation |
| Protein purification system | Cytiva | AKTA purifier | Protein purification |
| Refrigerator (-80°C) | Thermo | 900GP | Drosophila head preparation |
| Spectrophotometer | MAPADA | UV-1200 | OD600 measurement of E.coli. cells |
| Spectrophotometer | Thermo Scientific | NanoDrop 2000c | Determination of protein concentration |
| Ultracentrifuge | Beckman coulter | Optima XPN-100 Ultracentrifuge | Ultracentrifugation |
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Diesen Artikel zitieren
Liu, J., Liu, Y., Chen, W., Ding, Y., Lan, X., Liu, W. Purification of Endogenous Drosophila Transient Receptor Potential Channels. J. Vis. Exp. (178), e63260, doi:10.3791/63260 (2021).