无细胞生产蛋白脂质体，用于功能分析和靶向膜蛋白的抗体开发
Summary
该协议描述了一种有效的无细胞方法，通过使用小麦无细胞系统和脂质体的双层透析方法生产高质量的蛋白水产体。该方法为膜蛋白的功能分析、药物靶点筛选和抗体开发提供了合适的手段。
Abstract
膜蛋白在各种细胞过程中起着至关重要的作用，并发挥着至关重要的功能。膜蛋白在药物发现中具有医学意义，因为它们是一半以上药物的靶标。对膜蛋白进行生化、生物物理和结构研究以及抗体开发的一个障碍是难以生产大量具有正确构象和活性的高质量膜蛋白。在这里，我们描述了一种“双层透析方法”，使用小麦无生殖细胞系统，脂质体和透析杯在短时间内高效合成膜蛋白并制备纯化的蛋白脂质体，成功率高。膜蛋白可以产生多达几毫克，例如GPCR，离子通道，转运蛋白和四跨蛋白。这种无细胞方法有助于减少制备高质量蛋白水产酶体的时间、成本和精力，并为膜蛋白的功能分析、药物靶标筛选和抗体开发提供了合适的手段。
Introduction
膜蛋白是诊断和治疗中最重要的药物靶点之一。事实上，一半的小复合药物靶标是膜蛋白，例如G蛋白偶联受体（GPCR）和离子通道1。多年来，研究人员一直致力于膜蛋白的生物化学、生物物理和结构研究，以阐明其结构和功能2，3。还积极开发针对膜蛋白的单克隆抗体，以加速功能和结构研究，并开发治疗和诊断应用4，5，6，7，8，9。所有这些研究都需要大量的高质量膜蛋白10。例如，抗体开发需要几毫克具有自然构象的纯化膜蛋白。X射线晶体学需要大量高度纯化的膜蛋白。然而，膜蛋白的大规模生产仍然是膜蛋白研究的瓶颈11。膜蛋白具有具有一个或多个跨膜螺旋的复杂结构，在细胞稳态中起重要作用。膜蛋白的异源过表达导致多种障碍，例如膜蛋白聚集在高局部浓度下积累或干扰细胞信号通路。即使表达成功，后续的样品制备步骤也面临困难。例如，蛋白脂质体的制备需要膜蛋白溶解、纯化和稳定的高水平技能和专业经验，并且需要花费大量的精力和时间12，13。
另一方面，近几十年来出现了一些先进技术，可以在不使用活细胞的情况下生产蛋白质14，15，16，17，18。无细胞蛋白质合成技术在试管中重建翻译反应。由于细胞表达系统没有限制，因此无细胞系统有可能合成各种难以表达或在细胞中显示毒性的蛋白质。纯化的细胞提取物或重组的翻译机制与模板mRNA、氨基酸和能量源混合，并在短时间内合成重组蛋白。关于膜蛋白的合成，某些由脂质或两亲分子组成的支架，例如脂质体、双细胞、纳米盘或共聚物被添加到无细胞反应19，20，21，22，23，24中。合成的膜蛋白与支架相互作用，可以在水中稳定。无细胞合成的膜蛋白广泛用于功能研究和抗体生产25，26，27，28，29，30，31。
在该协议中，我们描述了一种使用小麦无细胞系统和脂质体生产蛋白石体的有效无细胞方法。小麦无细胞蛋白质合成系统是一种强大的体外翻译系统，使用小麦胚芽15，32，33的提取物。小麦胚芽含有大量的翻译机制和很少的翻译抑制剂。作为真核生物成员的小麦中的翻译机制适用于翻译真核蛋白质，其翻译效率几乎不受模板mRNA的密码子使用的影响。使用小麦无细胞系统，我们合成了多种蛋白质，包括蛋白激酶34，35，泛素连接酶36，转录因子37和膜蛋白，成功率很高。对于膜蛋白生产，我们将脂质囊泡脂质体作为支架19，38 添加到翻译混合物中。膜蛋白的疏水结构域与脂质双层相互作用，并与脂质体自发整合。密度梯度离心用于从内源性小麦蛋白中严格分离蛋白石质体，即使翻译反应混合物的普通离心足以简单纯化蛋白石质体20。利用小麦无细胞系统合成了多种完整的膜蛋白，并应用于各种研究和开发25，38，39，40，41，42，43，44。此外，我们还开发了用于大规模生产的“双层透析法”45，46.该方法中，将杯式透析装置浸入底物进料缓冲液中，并在杯中形成两层平移反应混合物和底物进料缓冲液，如图1所示。连续供应底物和去除副产物可以在反应混合物的顶部和底部长时间有效地进行，从而获得出色的转化效果（图2A和图2B）45。
Protocol
1. pEU表达质粒的制备 注意：pEU表达质粒应包括起始密码子，靶膜蛋白的开放阅读框和片段中的终止密码子（见 图1）。需要时，在适当的位置添加检测/纯化标签序列。限制性内切酶切或无缝克隆均适用于亚克隆。在这里，我们描述了一种使用无缝克隆方法的协议。 准备插入片段。使用 cDNA 模板、引物 1 和引物 2 通过 PCR 扩增目的基因。引?…
Representative Results
使用该协议，可以在短时间内获得部分纯化的蛋白脂质体。代表性结果如图 2A所示。采用双层透析法（小规模）成功合成了25个A、B、C类GPCR，并通过离心和缓冲液洗涤进行了部分纯化。虽然合成蛋白质的量因蛋白质类型而异，但当使用大型透析杯时，每次反应通常可以合成50至400μg膜蛋白。由于小麦无细胞系统的高可扩展性，通过增加反应次数可以很容易地产生几毫克的膜?…
Discussion
所提出的协议提供了一种以高成功率生产膜蛋白的方法。该协议简单、可重复且易于扩展。它还有可能减少消耗大量膜蛋白的实验的时间和成本。与双层法或透析法相比，双层透析方法将生产率提高了4-10倍（图2B）45。在极端情况下，双层透析法的离子通道和转运蛋白的产率分别比双层法提高了30倍和20倍（数据未显示）。该方案的高生产率是免疫抗原生产?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究得到了AMED支持药物发现和生命科学研究的平台项目（支持创新药物发现和生命科学研究（BINDS）的基础）的支持，资助号为JP20am0101077。这项工作也得到了JSPS KAKENHI授权号20K05709的部分支持。
Materials
| ×3 SDS-PAGE sample buffer | Containing 10% 2-mercaptoethanol | ||
| 5-20% gradient SDS-PAGE gel | ATTO | E-D520L | |
| 70% ethanol | Diluted ethanol by ultrapure water. | ||
| Agarose | Takara Bio | ||
| Ammonium acetate | Nakalai tesque | 02406-95 | As this reagent is deliquescent, dissolve all of it in water once opened and store it at -30°C. |
| Ampicillin Sodium | Nakalai tesque | 02739-74 | |
| Asolectin Liposome, lyophilized | CellFree Sciences | CFS-PLE-ASL | A vial contains 10 mg of lyophilized liposomes. |
| BSA standard | 1000 ng, 500 ng, 250 ng, 125 ng BSA / 10 µL ×1 SDS-PAGE sample buffer | ||
| CBB gel stain | |||
| cDNA clone of interest | Plasmid harboring cDNA clone or synthetic DNA fragment | ||
| Chloroform | Nakalai tesque | 08402-84 | |
| Cooled incubator | Temperature ranging from 0 to 40 °C or wider. | ||
| Creatine kinase | Roche Diagnostics | 04524977190 | |
| Dialysis cup (0.1 mL) | Thermo Fisher Scientific | 69570 | Slide-A-Lyzer MINI Dialysis Device, 10K MWCO, 0.1 mL |
| Dialysis cup (2 mL) | Thermo Fisher Scientific | 88404 | Slide-A-Lyzer MINI Dialysis Device, 10K MWCO, 2 mL |
| DNA ladder marker | Thermo Fisher Scientific | SM0311 | GeneRuler 1 kb DNA Ladder |
| DpnI | Thermo Fisher Scientific | FD1703 | FastDigest DpnI |
| E. coli strain JM109 | |||
| Electrophoresis chamber | ATTO | ||
| Ethanol (99.5%) | Nakalai tesque | 14713-95 | |
| Ethidium bromide | |||
| Evaporation flask, 100 mL | |||
| Gel imager | |||
| Gel scanner | We use document scanner and LED immuninator as a substitute. | ||
| LB broth | |||
| Lipids of interest | Avanti Polar Lipids | ||
| Micro centrifuge | TOMY | MX-307 | |
| NTP mix | CellFree Sciences | CFS-TSC-NTP | Mixture of ATP, GTP, CTP, UTP, at 25 mM each |
| Nuclease-free 25 mL tube | IWAKI | 362-025-MYP | |
| Nucrease-free plastic tubes | Watson bio labs | Do not autoclave. Use them separately from other experiments. | |
| Nucrease-free tips | Watson bio labs | Do not autoclave. Use them separately from other experiments. | |
| PBS buffer | |||
| PCR purification kit | MACHEREY-NAGEL | 740609 | NucleoSpin Gel and PCR Clean-up |
| pEU-E01-MCS vector | CellFree Sciences | CFS-11 | |
| Phenol/chloroform/isoamyl alcohol (25:24:1) | Nippon Gene | 311-90151 | |
| Plasmid prep Midi kit | MACHEREY-NAGEL | 740410 | NucleoBond Xtra Midi |
| Primer 1 | Thermo Fisher Scientific | Custom oligo synthesis | 5’-CCAAGATATCACTAGnnnnnnnnnnnnnnnnnnnnnnnn-3’ Gene specific primer, forward. Upper case shows overlap sequence to be added for seamless cloning. Lower case nnnn…. (20-30 bp) shows gene specific sequence. |
| Primer 2 | Thermo Fisher Scientific | Custom oligo synthesis | 5'-CCATGGGACGTCGACnnnnnnnnnnnnnnnnnnnnnnnn-3’ Gene specific primer, reverse. Upper case shows overlap sequence to be added for seamless cloning. Lower case nnnn…. (20-30 bp) shows gene specific sequence. |
| Primer 3 | Thermo Fisher Scientific | Custom oligo synthesis | 5'-GTCGACGTCCCATGGTTTTGTATAGAAT-3' Forward primer for vector linearization. Underline works as overlap in seamless cloning. |
| Primer 4 | Thermo Fisher Scientific | Custom oligo synthesis | 5'-CTAGTGATATCTTGGTGATGTAGATAGGTG-3' Reverse primer for vector linearization. Underline works as overlap in seamless cloning. |
| Primer 5 | Thermo Fisher Scientific | Custom oligo synthesis | 5’-CAGTAAGCCAGATGCTACAC-3’ Sequencing primer, forward |
| Primer 6 | Thermo Fisher Scientific | Custom oligo synthesis | 5’- CCTGCGCTGGGAAGATAAAC-3’ Sequencing primer, reverse |
| Protein size marker | Bio-Rad | 1610394 | Precision Plus Protein Standard |
| Rotary evaporator | |||
| seamless cloning enzyme mixture | New England BioLabs | E2611L | Gibson Assembly Master Mix Other seamless cloning reagents are also avairable. |
| SP6 RNA Polymerase & RNase Inhibitor | CellFree Sciences | CFS-TSC-ENZ | |
| Submarine Electrophoresis system | |||
| TAE buffer | |||
| Transcription Buffer LM | CellFree Sciences | CFS-TSC-5TB-LM | |
| Translation buffer | CellFree Sciences | CFS-SUB-SGC | SUB-AMIX SGC (×40) stock solution (S1, S2, S3, S4). Prepare ×1 translation buffer before use by mixing stock S1, S2, S3, S4 stock and ultrapure water. |
| Ultrapure water | We recommend to prepare ultrapure water by using ultrapure water production system every time you do experiment. Do not autoclave. We preparaed ultrapure water by using Milli-Q Reference and Elix10 system. Commercially available nuclease-free water (not DEPC-treated water) can be used as a substitute. Take care of contamination after open the bottle. |
||
| Ultrasonic homogenizer | Branson | SONIFIER model 450D-Advanced | Ultrasonic cleaner can be used as a substitute. |
| UV transilluminator | |||
| Vacuum desiccator | |||
| Wheat germ extract | CellFree Sciences | CFS-WGE-7240 | WEPRO7240 |
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Cite This Article
Zhou, W., Takeda, H. Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins. J. Vis. Exp. (163), e61871, doi:10.3791/61871 (2020).