Sf9纯化超加工驱动蛋白-3系列电机的单分子分析
Summary
本研究详细介绍了使用Sf9杆状病毒表达系统纯化驱动蛋白-3家族成员KIF1A（1-393LZ）。这些纯化的马达的 体外 单分子和多马达滑动分析表现出与哺乳动物细胞裂解物的马达相当的稳健运动特性。因此，Sf9杆状病毒系统适合表达和纯化感兴趣的运动蛋白。
Abstract
复杂的细胞环境给单分子运动分析带来了挑战。然而，成像技术的进步改善了单分子研究，并在检测和理解荧光标记分子的动态行为方面获得了极大的普及。在这里，我们描述了使用全内反射荧光（TIRF）显微镜对驱动蛋白-3家族马达进行体外 单分子研究的详细方法。Kinesin-3是一个大家族，在细胞和生理功能中起着关键作用，从细胞内货物运输到细胞分裂再到发育。我们之前已经证明，使用在哺乳动物细胞中表达马达制备的细胞裂解物，组成型活性二聚体驱动蛋白-3马达在单分子水平上表现出快速和超加工运动，具有高微管亲和力。我们的实验室使用细胞，生化和生物物理方法研究驱动蛋白-3马达及其调节机制，此类研究需要大规模纯化的蛋白质。使用哺乳动物细胞表达和纯化这些马达将是昂贵且耗时的，而在原核表达系统中的表达会导致显着聚集和无活性的蛋白质。为了克服细菌纯化系统和哺乳动物细胞裂解物带来的局限性，我们建立了一个强大的Sf9杆状病毒表达系统来表达和纯化这些马达。驱动蛋白-3 电机用 3 串联荧光蛋白（3xmCitirine 或 3xmCit）进行 C 末端标记，可提供增强的信号并减少光漂白。Sf9纯化蛋白的 体外 单分子和多马达滑动分析表明，驱动蛋白-3马达具有快速和超强的合成性，类似于我们之前使用哺乳动物细胞裂解物的研究。使用这些测定的其他应用包括电机低聚物条件的详细知识、平行生化研究的特定结合伙伴及其动力学状态。
Introduction
极其拥挤的细胞环境给分选注定的蛋白质和分子带来了许多挑战。分子马达和细胞骨架轨迹促进了细胞质内分子组织和时空分布的这种高强度工作量。分子马达是水解ATP等能量货币并在运动和力产生过程中利用该能量的酶1。基于氨基酸序列的相似性，驱动蛋白分为14个家族，尽管有这种相似性，但每个电机都对细胞的功能做出了独特的贡献。Kinesin-3 系列电机是最大的电机之一，包括五个亚家族（KIF1、KIF13、KIF14、KIF16 和 KIF28）2，与多种细胞和生理功能相关，包括囊泡转运、信号传导、有丝分裂、核迁移和发育 3，4，5。驱动蛋白-3转运功能的损害与许多神经退行性疾病，发育缺陷和癌症疾病有关6，7，8，9。
最近的研究表明，与传统的驱动蛋白10，11，12，13相比，驱动蛋白-3电机是单体，但经过货物诱导的二聚化，并导致快速和超加工运动。它们的生化和生物物理表征需要大量纯化的活性蛋白质。然而，它们在原核表达系统中的产生导致无活性或聚集的马达，可能是由于不相容的蛋白质合成，折叠和修饰机制14，15，16，17，18。为了规避这些限制并提高产量，我们在这里建立了一个强大的Sf9杆状病毒表达系统来表达和纯化这些马达。
杆状病毒表达系统使用Sf9昆虫细胞系作为高通量真核重组蛋白表达的宿主系统19，20。杆状病毒具有强大的多面体启动子，有助于异源基因表达和可溶性重组蛋白的产生17。由于其成本效益，操作安全且活性蛋白表达量高，它已成为一种强大的工具21。为了表达感兴趣的蛋白质，关键步骤是生成重组杆粒。由于市售的杆粒生成试剂盒价格昂贵，我们将使用更多样品，因此我们开发了一种内部协议，用于将驱动蛋白-3 电机的大尺寸和小型插入到杆粒中。Sf9纯化的驱动蛋白-3马达用于使用全内反射荧光（TIRF）显微镜表征体外单分子和多马达微管滑动特性。电机用 3 串联荧光分子 （3xmCit） 进行 C 端标记，以提供增强的信号并减少光漂白。由于其更高的信噪比，更少的光毒性以及对靠近盖玻片的非常小区域的选择性成像，TIRF成像已被广泛用于体内和体外单分子水平的蛋白质动力学可视化。
本研究讨论了利用Sf9杆状病毒表达系统以及使用TIRF显微镜对马达进行 体外 单分子成像和多马达滑动分析来纯化驱动蛋白-3马达。总之，该研究表明Sf9纯化马达的运动特性与由哺乳动物细胞裂解物制备的马达相同。因此，我们相信Sf9杆状病毒系统可以适应表达和纯化任何感兴趣的运动蛋白。
Protocol
1. Sf9培养、转染和病毒生成 注意：将Sf9细胞保持在30mL的Sf-900 / SFM培养基中，在100mL无菌一次性锥形烧瓶中，没有任何抗生素/抗真菌剂，温度为28°C。 将悬浮培养物保持在90rpm的轨道振荡器中。无需供应CO 2 和湿度维护。细胞通常每四天传代培养一次，接种0.5 x 10 6个细胞/mL，以在第4天达到2.0 x 106个细胞/mL的密度。 P0 病毒库存?…
Representative Results
为了使用Sf9杆状病毒表达大规模表达和纯化活性和功能性重组运动蛋白，该系统需要产生稳定携带编码序列的病毒颗粒以感染Sf9细胞。为了实现这一目标，Sf9细胞用编码KIF1A（1-393LZ）-3xmCit-FLAG的重组杆粒转染。72小时后，显着的细胞群显示出绿色荧光蛋白（mCitrine）的表达，细胞和细胞核扩大（图1 和 图2），表明病毒颗粒（P0）的成功生成。这些病毒颗…
Discussion
Sf9杆状病毒表达系统是高通量蛋白质生产中最通用和最成功的方法之一19，36，37。Sf9细胞的翻译后修饰能力与哺乳动物系统高度相当15。使用该系统的一个相当大的缺点是它速度慢且对污染敏感。最关键的步骤之一是在Sf9细胞中的有效感染和成功表达，这需要正确处理和维护Sf9细胞而没有任何污染。该系统需?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
V.S. 和 P.S. 感谢 Kristen J. Verhey 教授（密歇根大学，密歇根州安娜堡，美国）和 Roop Mallik 教授（印度理工学院孟买分校 （IITB），印度孟买）在整个研究过程中的无条件支持。附言感谢Sivapriya Kirubakaran博士在整个项目中的支持。V.S.承认通过DBT（赠款号：BT / PR15214 / BRB / 10 / 1449 / 2015和BT / RLF / Re-entry/45 / 2015）和DST-SERB（赠款号：ECR / 2016 / 000913）获得的资金。P.K.N感谢ICMR的资助（拨款号5/13/13/2019/NCD-III）。附言感谢DST的资助（拨款号：SR/WOS-A/LS-73/2017）。D.J.S承认IIT Gandhinagar的奖学金。
Materials
| Sf9 culture and transfection materials | |||
| anti-FLAG M2 affinity | Biolegend | 651502 | For motility purification |
| Aprotinin | Sigma | A6279 | For motility assay and purification |
| Cellfectin | Invitrogen | 10362100 | For Sf9 transfection |
| DTT | Sigma | D5545 | For motility assay mixture |
| FLAG peptide | Sigma | F3290 | For motility purification |
| Glycerol | Sigma | G5516 | For motility purification |
| HEPES | Sigma | H3375 | Preparing lysing Sf9 cells |
| IGEPAL CA 630 | Sigma | I8896 | Preparing lysing buffer for Sf9 cells |
| KCl | Sigma | P9541 | For motility purification |
| Leupeptin | Sigma | L2884 | For motility assay and purification |
| MgCl2 | Sigma | M2670 | For preparing lysis buffer |
| NaCl | Sigma | S7653 | For preparing lysis buffer |
| PMSF | Sigma | P7626 | For motility purification |
| Sf9 cells | Kind gift from Dr. Thomas Pucadyil (Indian Institute of Science Education and Research, Pune, India). | For baculovirs expression and purification | |
| Sf9 culture bottles | Thermo Scientific | 4115-0125 | For suspension culture |
| Sf-900/SFM medium (1X) | Thermo Scientific | 10902-096 -500ml | For culturing Sf9 cells |
| Sucrose | Sigma | S1888 | Preparing lysing buffer for Sf9 cells |
| Unsupplemented Grace’s media | Thermo Scientific | 11595030 -500ml | For Sf9 transfection |
| Mirotubule Polymerization and Single molecule assay materails | |||
| ATP | Sigma | A2647 | For motility and gliding assay |
| BSA | Sigma | A2153 | For blocking motility chamber |
| Catalase | Sigma | C9322 | For motility and gliding assay |
| DMSO | Sigma | D5879 | For dissolving Rhodamine |
| EGTA | Sigma | 3777 | For preparing buffers |
| Glucose | Sigma | G7021 | For motility and gliding assay |
| Glucose oxidase | Sigma | G2133 | For motility and gliding assay |
| GTP | Sigma | G8877 | For microtubule polymerization |
| KOH | Sigma | P1767 | Preparing PIPES buffer pH 6.9 |
| PIPES | Sigma | P6757 | For motility and gliding assay |
| Microtubule gliding assay materials | |||
| 26G needle | Dispovan | For shearing microtubules | |
| Casein | Sigma | C3400 | For microtubule glidning assay |
| GFP nanobodies | Gift from Dr. Sivaraj Sivaramakrishnan (University of Minnesota, USA) | For attaching motors to the coverslip | |
| Rhodamine | Thermo Scientific | 46406 | For preparing labelling tubulin |
| Microscope and other instruments | |||
| 0.5ml, 1.5 and 2-ml microcentrifuge tubes | Eppendorf | For Sf9 culture and purification | |
| 10ml disposable sterile pipettes | Eppendorf | For Sf9 culture and purification | |
| 10ul, 200ul, 1ml micropipette tips | Eppendorf | For Sf9 culture and purification | |
| 15ml concal tubes | Eppendorf | For Sf9 culture and purification | |
| 35mm cell culture dish | Cole Palmer | 15179-39 | For Sf9 culture |
| Balance | Sartorious | 0.01g-300g | |
| Benchtop orbial shaking incubator | REMI | For Sf9 suspenculture at 28oC | |
| Camera | EMCCD Andor iXon Ultra 897 | For TIRF imaging and acquesition | |
| Double sided tape | Scotch | For making motility chamber | |
| Glass coverslip | Fisherfinest | 12-548-5A | size; 22X30 |
| Glass slide | Blue Star | For making motility chamber | |
| Heating block | Neuation | Dissolving paraffin wax | |
| Inverted microscope | Nikon Eclipse Ti- U | To check protein expression | |
| Lasers | 488nm (100mW) | For TIRF imaging | |
| Liquid nitrogen | For sample freezing and storage | ||
| Microcapillary loading tip | Eppendorf | EP022491920 | For shearing microtubules |
| Microscope | Nikon Eclipse Ti2-E with DIC set up | For TIRF imaging | |
| Mini spin | Genetix, BiotechAsia Pvt.Ltd | For quick spin | |
| Objective | 100X TIRF objective with 1.49NA oil immersion | For TIRF imaging | |
| Optima UltraCentrifuge XE | Beckman Coulter | For protein purification | |
| Parafilm | Eppendorf | ||
| pH-meter | Corning | Coring 430 | To adjust pH |
| Pipette-boy | VWR | For Sf9 culture and purification | |
| Sorvall Legend Micro 21 | Thermo Scientific | For protein purification | |
| Sorvall ST8R centrifuge | Thermo Scientific | Protein purification | |
| ThermoMixer | Eppendorf | For microtubule polymerization | |
| Ultracentrifuge rotor | Beckman coulter | SW60Ti rotor | |
| Ultracentrifuge tubes | Beckman | 5 mL, Open-Top Thinwall Ultra-Clear Tube, 13 x 51mm | |
| Vortex mixer | Neuation | Sample mixing | |
| Wax | Sigma | V001228 | To seal motility chamber |
References
- Vale, R. D. The molecular motor toolbox for intracellular transport. Cell. 112, 467-480 (2003).
- Miki, H., Okada, Y., Hirokawa, N. Analysis of the kinesin superfamily: insights into structure and function. Trends in Cell Biology. 15 (9), 467-476 (2005).
- Hirokawa, N., Niwa, S., Tanaka, Y. Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease. Neuron. 68 (4), 610-638 (2010).
- Hirokawa, N., Takemura, R. Molecular motors and mechanisms of directional transport in neurons. Nature Reviews Neuroscience. 6 (3), 201-214 (2005).
- Patel, N. M., et al. KIF13A motors are regulated by Rab22A to function as weak dimers inside the cell. Scientific Advances. 7 (6), (2021).
- Franker, M. A., Hoogenraad, C. C. Microtubule-based transport – basic mechanisms, traffic rules and role in neurological pathogenesis. Journal of Cellular Science. 126, 2319-2329 (2013).
- Gunawardena, S., Anderson, E. N., White, J. Axonal transport and neurodegenerative disease: vesicle-motor complex formation and their regulation. Degernative Neurological and Neuromuscular Disease. 4, 29-47 (2014).
- Rath, O., Kozielski, F. Kinesins and cancer. Nature Reviews Cancer. 12 (8), 527-539 (2012).
- Wang, Z. Z., et al. KIF14 promotes cell proliferation via activation of Akt and is directly targeted by miR-200c in colorectal cancer. International Journal of Oncology. 53 (5), 1939-1952 (2018).
- Guo, S. K., Shi, X. X., Wang, P. Y., Xie, P. Run length distribution of dimerized kinesin-3 molecular motors: comparison with dimeric kinesin-1. Scientific Reports. 9 (1), 16973 (2019).
- Scarabelli, G., et al. Mapping the processivity determinants of the Kinesin-3 motor domain. Biophysical Journal. 109 (8), 1537-1540 (2015).
- Soppina, V., et al. Dimerization of mammalian kinesin-3 motors results in superprocessive motion. Proceedings of the National Academy of Sciences of the United States of America. 111 (15), 5562-5567 (2014).
- Soppina, V., Verhey, K. J. The family-specific K-loop influences the microtubule on-rate but not the superprocessivity of kinesin-3 motors. Molecular Biology Cell. 25 (14), 2161-2170 (2014).
- Soppina, V., et al. Kinesin-3 motors are fine-tuned at the molecular level to endow distinct mechanical outputs. BMC Biology. , (2022).
- Korten, T., Chaudhuri, S., Tavkin, E., Braun, M., Diez, S. Kinesin-1 Expressed in insect cells improves microtubule in vitro gliding performance, long-term stability and guiding efficiency in nanostructures. IEEE Transactions on Nanobioscience. 15 (1), 62-69 (2016).
- Schmidt, F. R. Recombinant expression systems in the pharmaceutical industry. Applied Microbiology and Biotechnology. 65 (4), 363-372 (2004).
- Kurland, C., Gallant, J. Errors of heterologous protein expression. Current Opinions in Biotechnology. 7 (5), 489-493 (1996).
- Tao, L., Scholey, J. M. Purification and assay of mitotic motors. Methods. 51 (2), 233-241 (2010).
- Kost, T. A., Condreay, J. P., Jarvis, D. L. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nature Biotechnology. 23 (5), 567-575 (2005).
- Felberbaum, R. S. The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors. Biotechnology Journal. 10 (5), 702-714 (2015).
- Kumar, N., Pandey, D., Halder, A., Kumar, D., Gong, C. . Trends in Insect Molecular Biology and Biotechnology. , 163-191 (2018).
- Nagano, T. Development of fluorescent probes for bioimaging applications. Proceedings of the Japan Academy. Series B. Physical and Biological Sciences. 86 (8), 837-847 (2010).
- Hassan, M., Klaunberg, B. A. Biomedical applications of fluorescence imaging in vivo. Comparative Medicine. 54 (6), 635-644 (2004).
- Yang, Z., Samanta, S., Yan, W., Yu, B., Qu, J. Super-resolution microscopy for biological imaging. Advances in Experimental Medicine and Biology. 3233, 23-43 (2021).
- Ettinger, A., Wittmann, T. Fluorescence live cell imaging. Methods in Cell Biology. 123, 77-94 (2014).
- Waters, J. C. Live-cell fluorescence imaging. Methods in Cell Biology. 114, 125-150 (2013).
- Zheng, Q., Jockusch, S., Zhou, Z., Blanchard, S. C. The contribution of reactive oxygen species to the photobleaching of organic fluorophores. Photochemistry and Photobiology. 90 (2), 448-454 (2014).
- Wojtovich, A. P., Foster, T. H. Optogenetic control of ROS production. Redox Biology. 2, 368-376 (2014).
- Cai, D., Verhey, K. J., Meyhofer, E. Tracking single Kinesin molecules in the cytoplasm of mammalian cells. Biophysical Journal. 92 (12), 4137-4144 (2007).
- Bohm, K. J., Stracke, R., Baum, M., Zieren, M., Unger, E. Effect of temperature on kinesin-driven microtubule gliding and kinesin ATPase activity. FEBS Letters. 466, 59-62 (2000).
- Porter, M. E., et al. Characterization of the microtubule movement produced by sea urchin egg kinesin. The Journal of Biological Chemistry. 262 (6), 2794-2802 (1987).
- Muyldermans, S. Nanobodies: natural single-domain antibodies. Annual Review of Biochemistry. 82, 775-797 (2013).
- Verma, S., Kumar, N., Verma, V. Role of paclitaxel on critical nucleation concentration of tubulin and its effects thereof. Biochemical and Biophysical Research Communications. 478 (3), 1350-1354 (2016).
- Parness, J., Horwitz, S. B. Taxol binds to polymerized tubulin in vitro. Journal of Cell Biology. 91 (2), 479-487 (1981).
- Schiff, P. B., Fant, J., Horwitz, S. B. Promotion of microtubule assembly in vitro by taxol. Nature. 277 (5698), 665-667 (1979).
- Kato, T., Kageshima, A., Suzuki, F., Park, E. Y. Expression and purification of human (pro)renin receptor in insect cells using baculovirus expression system. Protein Expression and Purification. 58 (2), 242-248 (2008).
- Liu, F., Wu, X., Li, L., Liu, Z., Wang, Z. Use of baculovirus expression system for generation of virus-like particles: successes and challenges. Protein Expression and Purification. 90 (2), 104-116 (2013).
- Terpe, K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology. 60 (5), 523-533 (2003).
- Huang, S. T., et al. Liposomal paclitaxel induces fewer hematopoietic and cardiovascular complications than bioequivalent doses of Taxol. International Journal of Oncology. 53 (3), 1105-1117 (2018).
Cite This Article
Soppina, P., Shewale, D. J., Naik, P. K., Soppina, V. Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors. J. Vis. Exp. (185), e63837, doi:10.3791/63837 (2022).