一个从人体细胞分泌蛋白生产的方便，一般的表达平台
Summary
在人类后基因组时代，在本机构的重组蛋白的结构，功能和治疗的研究和发展是至关重要的。在这里，我们描述了测试和大规模的蛋白质在人类胚胎肾细胞293T，可用于生产各种重组蛋白表达系统。
Abstract
重组蛋白在细菌中表达，通常大肠杆菌大肠杆菌 ，一直毫克的蛋白质数量表达的最成功的战略。然而，原核主机通常是不为人类，病毒或真核蛋白质由于外国高分子毒性，在蛋白质折叠机制的差异，或因缺乏特别合作或翻译后的修改，在细菌的适当表达。基于酵母（ 毕赤酵母或酵母 ）1,2，杆状病毒感染的昆虫（S.秋行军虫或 T 妮 ）细胞3，和无细胞体外翻译系统的表达系统2,4已被成功地用于生产哺乳动物蛋白质。直观，最匹配的是，使用哺乳动物的主机，以确保包含正确的翻译后修饰的重组蛋白的生产。一些哺乳动物细胞株人类胚胎小子（滋肾（HEK）293，C V-1细胞在 O rigin携带的 S V40 larget T抗原（COS）中国仓鼠卵巢（CHO），和其他人）已成功地利用过度毫克数量的人体蛋白质数量5-9。然而，使用哺乳动物细胞中的优势往往成本较高，专业实验室设备的需求，降低蛋白质产量，和漫长的时间发展稳定表达细胞株的反击。提高产量和生产蛋白质的速度，同时保持成本低，是许多学术和商业实验室的主要因素。
在这里，我们描述了时间和成本效益，由两部分组成，附着HEK 293T细胞分泌的人类蛋白质表达过程。这个系统是能够生产微克到毫克数量的功能蛋白质结构，生物物理和生物化学研究。第一部分，是产生多个感兴趣的基因结构并行和瞬时转成小规模的附着的HEK 293T细胞。重组蛋白分泌到细胞培养液中的检测和分析，进行印迹分析，使用市售的抗体对矢量编码的蛋白质纯化标签指示。随后，适合大规模蛋白质生产结构瞬时转染使用聚乙烯亚胺（PEI）在10层的细胞工厂。成升体积的条件培养基中分泌蛋白集中到管理大量使用切向流过滤，净化，抗HA的亲和层析。这个平台的效用证明毫克量的细胞因子，细胞因子受体，细胞表面的受体，内在的制约因素，与病毒糖蛋白的表达能力。这种方法也被成功地用于三聚ebolavirus糖5,10结构的决心。
<p c姑娘=的“jove_content”的结论>，此平台提供易于使用，速度和可扩展性，同时最大限度地提高蛋白质的质量和功能。此外，没有额外的设备，比标准培养箱二氧化碳培养箱 ，是必需的。这个过程可能会迅速扩大到更复杂的系统，如蛋白质复合物，抗原和抗体，疫苗生产的病毒样颗粒，或生产困难的细胞株转导的腺病毒或慢病毒表达。Protocol
1。准备工作 – 构造和细胞培养在开始的协议之前，应该感兴趣的基因密码子优化，在哺乳动物细胞中表达，并克隆到一个合适的表达载体，利用标准的分子生物学技术。为了确保最高的成功表达的机会，应产生多个感兴趣的基因变种。许多哺乳动物表达载体可商业化，并有各种净化标签（多聚组氨酸，血凝素，链，晕标签，谷胱甘肽S-转移，等等）。我们更倾向于使用pDISPLAY?…
Discussion
10层的细胞工厂生产毫克的蛋白质数量的有效容器。使用超过其他传统的船只，如滚瓶，摇瓶或微调瓶，电池工厂的一个主要优点是，它们不需要任何额外的实验室设备的采购。一个标准的二氧化碳培养箱（6.0立方英尺）轻松容纳4个10层的细胞工厂（ 图2）。此外，这些船只需要较少的劳动比菜，瓶或滚瓶细胞和蛋白质的产生和空间;一个10层的细胞工厂，相当于使用7.5定期滚?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由安大略省的艾滋病毒治疗网络研究工作津贴（的ROG-G645）和加拿大健康研究的新研究者奖（的MSH-113554）研究院JEL和多伦多大学的奖学金支持HA，FCA和纪委。笔者想感谢在斯克里普斯研究所（拉霍亚加州）马妮福斯科，Dafna先生Abelson和埃里卡Ollmann萨费尔博士提供细胞，ebolavirus; GP表达载体和一般建议。
Materials
| Name of reagent | Company | Catalogue number | Comments |
| Alkaline phosphatase (BCIP/NBT) liquid substrate solution | Sigma | B6404 | |
| Antibiotic/Antimycotic, 100X | Invitrogen | 15240062 | |
| Anti-HA affinity matrix, clone 3F10 | Roche | 1815016 | |
| Anti-HA murine mAb, clone 16B12 | Covance | MMS-101P | |
| Cell culture flask, T75 cm2 tissue culture treated | Corning | 430641 | |
| Cell culture flask, T225 cm2 tissue culture treated | Corning | 431082 | |
| Cell culture plates,6-well tissue culture treated | Corning | 3516 | |
| Cell factory, 10-layer CellSTACK | Corning | 3312 | |
| Centramate Omega 5K Cassette | Pall | OS005C12 | |
| Centramate Omega 30K Cassette | Pall | OS030C12 | |
| Chromatography glass column, 1.0×10 cm | Kontes | 4204001010 | |
| Ciprofloxacin | Sigma | 17850 | |
| CO2 | |||
| Dulbecco’s modified Eagle’s media (DMEM) | Sigma | D5796 | |
| Fetal bovine serum (FBS), heat inactivated | Invitrogen | 12484-028 | |
| FuGENE HD transfection reagent | Promega | 4709691001 | |
| GeneJuice transfection reagent | EMD/Merck | 70967-6 | |
| Glycine | Sigma | G8898 | |
| Goat anti-mouse IgG F(ab’)2 alkaline | Thermo Scientific | 31324 | |
| phosphatase-conjugated antibody | |||
| Hemagglutinin (HA) peptide, 100 mg | Genscript | custom synthesis | |
| (sequence: YPYDVPDYA; 95% purity) | |||
| HEK 293T cells | ATCC | CRL-11268 | |
| Household bleach (4% w/v sodium hypochlorite) | various brands are available | ||
| Immobilon-P PVDF membrane | Millipore | IPVH07850 | |
| MiniPrep plasmid purification kit, PureLink Quick | Invitrogen | K2100-11 | |
| MaxiPrep plasmid purification kit, PureLink HiPure | Invitrogen | K2100-07 | |
| NaN3 | Sigma | S8032 | |
| pDISPLAY expression vector | Invitrogen | V660-20 | |
| Penicillin/streptomycin (pen/strep), 100X | Invitrogen | 15140-122 | |
| Phosphate-buffered saline (PBS), sterile 1X | Sigma | D8537 | |
| Polyethyleneimine (PEI), linear 25 kDa | Polyscience | 23966 | |
| Skim milk dry powder | Carnation | ||
| Stericup-GP PES vacuum filtration unit, | Millipore | SCGPU05RE | |
| 0.22 μm, 500 ml capacity | |||
| Trypan blue | Invitrogen | 15250061 | |
| Trypsin-EDTA, 0.05% (w/v) | Invitrogen | 25300-054 | |
| Tween-20 | Sigma | P7949 | |
| Valproic acid | Sigma | P4543 | |
| Centramate tangential flow system | Pall | ||
| CO2 humidified incubator, standard 6.0 cu. ft. | various brands are available | ||
| Electrophoresis and transfer unit | various brands are available | ||
| Incubator, 37 °C | various brands are available |
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Tags
Convenient Expression PlatformSecreted ProteinsRecombinant Protein ExpressionHuman CellsBacteriaE. ColiProkaryotic HostsHuman ProteinsYeast Expression SystemBaculovirus-infected Insect CellsCell-free In Vitro Translation SystemMammalian HostMammalian Cell LinesHEK 293 CellsCV-1 CellsCHO CellsProtein YieldsCost-effective Production
Cite This Article
Aydin, H., Azimi, F. C., Cook, J. D., Lee, J. E. A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells. J. Vis. Exp. (65), e4041, doi:10.3791/4041 (2012).