瞬时表达和重组蛋白的细胞定位在培养的昆虫细胞
Summary
Nonlytic昆虫细胞表达系统没有得到充分利用进行生产，细胞运输/定位，和重组蛋白质功能分析。这里，我们描述的方法来产生在市售鳞翅目细胞系的表达载体和随后的瞬时表达。与亚细胞荧光标记蛋白烟粉虱水通道蛋白的共定位还提出。
Abstract
异源蛋白质表达系统用于生产重组蛋白，细胞运输/定位的解释，和蛋白质在子有机体水平的生化功能的确定。虽然杆状病毒表达系统在众多的生物技术，制药和工业应用中，不涉及病毒感染有明显的好处，但往往被忽视和未充分利用nonlytic系统越来越多地用于生产蛋白质。在这里，我们描述了用于产生nonlytic表达载体和瞬时重组蛋白表达的方法。该协议允许对重组蛋白的高效细胞定位和可用于在细胞内迅速辨别蛋白质运输。我们示出了四个重组蛋白在市售的昆虫细胞系的表达，包括来自昆虫烟粉虱 2水通道蛋白的蛋白质，以及作为特异于细胞质膜亚细胞标志物蛋白和细胞内溶酶体。所有的重组蛋白被生产为具有在它们的羧基末端，其允许直接检测重组蛋白的荧光蛋白标记的嵌合体。细胞与质粒构建携带针对感兴趣和已知的亚细胞标记物的基因的转染双允许活细胞成像，改进的蜂窝蛋白质定位的验证。
Introduction
生产用昆虫细胞表达系统重组蛋白的真核蛋白质的研究提供了许多好处。即，昆虫细胞具有相似的翻译后修饰，处理和排序机制的那些存在于哺乳动物细胞中，这是有利的用于制备正确折叠的蛋白1，2，3。昆虫细胞系统通常还需要更少的资源和较少的时间和精力用于维护比哺乳动物细胞系4,5。杆状病毒表达系统是一种这样的昆虫细胞为基础的系统，它现已被广泛地用于许多学科，包括生产用于蛋白质表征和治疗剂，外源肽和用于疫苗生产的病毒蛋白的免疫原性的介绍，多合成重组蛋白的 – 蛋白质复合物，生产糖基化蛋白等 。 1，2，4，6。有，但是，情况，其中杆状病毒表达可能不适用3,7，以及使用nonlytic和瞬态昆虫表达系统的可能更合适。具体而言，瞬态昆虫细胞表达提供了重组蛋白的快速合成的可能性，需要更少的开发和维护，不涉及病毒强加细胞裂解，并提供了蛋白质合成7，8，9中更好地研究细胞运输的手段， 10。
这个协议描述了使用快速生成的表达载体的两步骤的重叠延伸PCR（OE-PCR） <suP类=“外部参照”> 11和质粒DNA在大肠杆菌中标准的克隆。质粒被用于双重转染市售培养的昆虫细胞，并产生代表的蛋白质。该协议描述了两种不同的荧光标记的亚细胞标志物蛋白的生产和使用，并演示共定位与来自昆虫烟粉虱 2水通道蛋白的蛋白质。以下方案提供了用于OE-PCR，昆虫细胞维持和转染，而对于目标蛋白的细胞定位的荧光显微镜的基本方法。
Protocol
1. OE-PCR用于表达质粒的构建注：请参阅表1在OE-PCR中使用的所有引物。建议对所有扩增使用高保真DNA聚合酶。然而，因为这些酶通常不留下3' A，有必要用Taq DNA聚合酶进行简短的，非放大孵育至‘A-尾’的PCR产物将它们克隆到TA昆虫细胞表达前向量。此协议演示了一个方法，以产生昆虫表达质粒携带嵌合蛋白，与框融合到感兴趣的基因的羧基末端的荧光蛋白（在这…
Representative Results
OE-PCR OE-PCR允许的是，一旦被插入到表达载体中，允许产生对应于目标和荧光标记蛋白的任何测试基因的重组嵌合蛋白的嵌合DNA产物的合成。 图1表示用于生产含有烟粉虱水通道蛋白编码符合读框的序列（BtDrip1和BtDrip2_v1）与荧光蛋白质标记EGFP PIB的表达载体的一般方案。在这种情况下，制备含有EGFP在它们的羧…
Discussion
异源蛋白质表达系统可用于产生在许多下游应用4中使用的重组蛋白的重要工具。从现有的各种表达系统选择取决于感兴趣的蛋白质的最终目标。几个昆虫细胞表达系统是可用的提供灵活的替代原核和真核细胞表达系统5,6。需要杆状病毒感染，以驱动蛋白表达的昆虫系统是目前最流行的和广泛使用的昆虫系统中，用在研究和治疗应用<sup…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢林恩Forlow-Jech和丹尼尔尔·勒罗伊技术援助。植物保护检疫[项目＃2020-22620-022-00D]以商品名称或商品的JAF和JJH提及这篇文章仅仅是为目的 – 这项工作是由基地CRIS资金USDA ARS，国家304计划支持的提供具体资料，也没有通过美国农业部门暗示推荐或认可。美国农业部是一个平等的机会提供者和雇主。
Materials
| KOD DNA Polymerase | EMD Millipore | 71085-3 | High-fidelity DNA polymerase used for PCR amplification of overlap extension PCR products |
| ExTaq DNA Polymerase | TaKaRa-Clontech | RR001B | DNA polymerase used for A-tailing of PCR products |
| EconoTaq PLUS GREEN 2x DNA Polymerase Master Mix | Lucigen | 30033-1 | DNA polymerase used for bacterial colony PCR |
| Biometra TProfessional Gradient Thermocycler | Biometra/LABRepCo | 070-851 | |
| Agarose LE | Benchmark Scientific | A1705 | |
| SYBR Safe DNA Gel Stain | ThermoFisher | S33102 | |
| Montage DNA Gel Extraction Kit | EMD Millipore | LSKGEL050 | |
| pIB/V5-His TOPO TA Expression Kit | ThermoFisher | K89020 | Contains components needed to clone overlap extension PCR products, including linearized and topoisomerase I-activated pIB/V5-His-TOPO vector, One Shot TOP10 chemically competent E. coli, and salt solution. |
| QIAprep Spin MiniPrep Kit | Qiagen | 27104 | |
| QIAcube Robotic Workstation | Qiagen | 9001292 | |
| Purifier Vertical Clean Bench | Labconco | 3970401 | |
| Tni cultured insect cell Line | Allele Biotech | ABP-CEL-10005 | |
| Sf9 cultured insect cell Line | Allele Biotech | ABP-CEL-10002 | |
| Serum-Free Insect Culture Medium | Allele Biotech | ABP-MED-10002 | |
| TNM-FH Insect Culture Medium | Allele Biotech | ABP-MED-10001 | |
| IPL-41 Insect Medium | ThermoFisher | 11405081 | |
| Cellfectin II Transfection Reagent | ThermoFisher | 10362100 | |
| 16 cm Disposable Cell Scrapers | Sarstedt | 83.1832 | Cell scrapers with two-position blade |
| 25 cm2 (T25) Tissue Culture Flasks with Vent Filter Caps | Life Science Products | CT-229331 | |
| Transfer Pipets | Fisher | 1371120 | |
| Sterile, 50 mL Bio-Reaction Tubes | Life Science Products | CT-229475 | |
| PipetteBoy | VWR | 14222-180 | |
| 5 mL Serological Pipettes | Sarstedt | 86.1253.001 | |
| 0.5 mL Flat-Cap PCR Tubes | Fisher | 14230200 | |
| Polypropylene Biohazard Autoclave Bags | Fisher | 01828C | |
| 35 mm #1.5 Glass Bottom Dishes | Matsunami Glass | D35-14-1.5-U | 35 mm dish diameter, 14 mm glass diameter, 1.5 mm glass thickness, uncoated |
| Incubator, Model 1510E | VWR | 35823-961 | |
| Countess II FL Cell Counter | ThermoFisher | AMQAF1000 | |
| Countess Cell Counting Chamber Slides with 0.4% Trypan Blue Reagent | ThermoFisher | C10228 | |
| Fluoview FV10i-LIV Laser Scanning Confocal Microscope | Olympus | FV10i-LIV | |
| HsPLA2/pCS6 plasmid DNA | transOMIC Technologies | TCH1303 | |
| pmCherry Vector | Clontech | 632522 | |
| NucBlue Live ReadyProbes Reagent (Hoechst 33342) | ThermoFisher | R37605 |
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Citar este artigo
Fabrick, J. A., Hull, J. J. Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells. J. Vis. Exp. (122), e55756, doi:10.3791/55756 (2017).