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?…
Declarações
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 |
Referências
- Tsunoda, S., et al. A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade. Nature. 388 (6639), 243-249 (1997).
- Huang, J., et al. Activation of TRP channels by protons and phosphoinositide depletion in Drosophila photoreceptors. Current Biology: CB. 20 (3), 189-197 (2010).
- Chyb, S., Raghu, P., Hardie, R. C. Polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. Nature. 397 (6716), 255-259 (1999).
- Hardie, R. C., Franze, K. Photomechanical responses in Drosophila photoreceptors. Science. 338 (6104), 260-263 (2012).
- Chen, W., et al. Calmodulin binds to Drosophila TRP with an unexpected mode. Structure. 29 (4), 330-344 (2021).
- Hardie, R. C. Effects of intracellular Ca2+ chelation on the light response in Drosophila photoreceptors. Journal of Comparative Physiology. A, Sensory, Neural, and Behavioral Physiology. 177 (6), 707-721 (1995).
- Scott, K., Sun, Y., Beckingham, K., Zuker, C. S. Calmodulin regulation of Drosophila light-activated channels and receptor function mediates termination of the light response in vivo. Cell. 91 (3), 375-383 (1997).
- Hardie, R. C., Minke, B. Phosphoinositide-mediated phototransduction in Drosophila photoreceptors: the role of Ca2+ and trp. Cell Calcium. 18 (4), 256-274 (1995).
- Delgado, R., et al. Light-induced opening of the TRP channel in isolated membrane patches excised from photosensitive microvilli from Drosophila photoreceptors. Neurociência. 396, 66-72 (2019).
- Lev, S., Katz, B., Minke, B. The activity of the TRP-like channel depends on its expression system. Channels (Austin). 6 (2), 86-93 (2012).
- Hardie, R. C., Raghu, P. Activation of heterologously expressed Drosophila TRPL channels: Ca2+ is not required and InsP3 is not sufficient. Cell Calcium. 24 (3), 153-163 (1998).
- Gutorov, R., et al. Modulation of transient receptor potential C channel activity by cholesterol. Frontiers in Pharmacology. 10, 1487 (2019).
- Yagodin, S., et al. Thapsigargin and receptor-mediated activation of Drosophila TRPL channels stably expressed in a Drosophila S2 cell line. Cell Calcium. 23 (4), 219-228 (1998).
- Guo, W., Chen, L. Recent progress in structural studies on canonical TRP ion channels. Cell Calcium. 83, 102075 (2019).
- Li, X., Fine, M. TRP channel: The structural era. Cell Calcium. 87, 102191 (2020).
- Liao, M., Cao, E., Julius, D., Cheng, Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature. 504 (7478), 107-112 (2013).
- Cao, E., Liao, M., Cheng, Y., Julius, D. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature. 504 (7478), 113-118 (2013).
- Liu, W., et al. The INAD scaffold is a dynamic, redox-regulated modulator of signaling in the Drosophila eye. Cell. 145 (7), 1088-1101 (2011).
- Ye, F., Liu, W., Shang, Y., Zhang, M. An exquisitely specific PDZ/target recognition revealed by the structure of INAD PDZ3 in complex with TRP channel. Structure. 24 (3), 383-391 (2016).
- Ye, F., et al. An unexpected INAD PDZ tandem-mediated plcβ binding in Drosophila photo receptors. eLife. 7, (2018).
- Rahimzadeh, M., Sadeghizadeh, M., Najafi, F., Arab, S., Mobasheri, H. Impact of heat shock step on bacterial transformation efficiency. Molecular Biology Research Communications. 5 (4), 257-261 (2016).
- Ashburner, M., Golic, K. G., Hawley, S. . Drosophila: A laboratory handbook. Second Edition. 80 (2), (2005).
- Nicolas, G., Sillans, D. Immediate and latent effects of carbon dioxide on insects. Annual Review of Entomology. 34 (1), 97-116 (1989).
- Arachea, B. T., et al. Detergent selection for enhanced extraction of membrane proteins. Protein Expression and Purification. 86 (1), 12-20 (2012).
- Hellmich, U. A., Gaudet, R. Structural biology of TRP channels. Handbook of Experimental Pharmacology. 223, 963-990 (2014).
Citar este artigo
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).