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Chemie

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

Published: February 25, 2019
doi:
10.3791/58882
, , , ,
1Department of Biological Sciences,California Polytechnic State University, San Luis Obispo, 2Center for Applications in Biotechnology,California Polytechnic State University, San Luis Obispo, 3Department of Chemistry and Biochemistry,California Polytechnic State University, 4Department of Chemical and Biological Engineering,Northwestern University, 5Chemistry of Life Processes Institute,Northwestern University, 6Center for Synthetic Biology,Northwestern University, 7Interdisciplinary Biological Sciences Program,Northwestern University

Summary

该协议详细介绍了在4天或更短的时间内生成大肠杆菌基细胞提取物并实施体外蛋白质合成反应所需的步骤、成本和设备。为了将这一平台的灵活性用于广泛的应用, 我们讨论了可以调整和优化的反应条件。

Abstract

在过去的50年里, 无细胞蛋白合成 (cfps) 已成为一种强大的技术, 可以利用试管内细胞的转录和转化能力。通过避免维持细胞活力的需要, 并通过消除细胞屏障, cfps 已成为新兴应用的基础, 在生物制造传统上具有挑战性的蛋白质, 以及应用于快速原型设计代谢工程和功能基因组学。我们实施基于大肠杆菌的 cfps 平台的方法允许新用户访问其中的许多应用程序。在这里, 我们描述了通过使用浓缩介质、挡板烧瓶和可调谐超声基础细胞裂解的可重复方法制备提取物的方法。该提取物可用于蛋白质表达, 能够在从实验设置到数据分析的短短5小时内产生900μg/ml 或更多的超级文件夹绿色荧光蛋白 (sfgfp), 因为事先已经准备好了适当的试剂储备。获得试剂的启动成本估计为 4, 500 美元, 这将维持数千种反应, 估计每产生的蛋白质成本为0.01 美元, 每微米反应0.1919 美元。此外, 蛋白质表达方法反映了由于优化试剂预混料而在市售系统中看到的反应设置的易用性, 成本只是很小的一小部分。为了使用户能够利用 cfps 平台的灵活性, 实现广泛的应用, 我们确定了该平台的各个方面, 这些方面可以根据可用资源和蛋白质表达结果进行调整和优化期望。

Introduction

无细胞蛋白合成 (cfps) 已成为一项技术, 在过去50年中, 在1、2的时间里, 为蛋白质生产、功能基因组学、代谢工程等带来了许多新的机遇。与标准的体内蛋白质表达平台相比, cfps 提供了三个关键优势: 1) 该平台的无细胞性质使其能够产生对细胞有潜在毒性或与细胞无关的蛋白质3,4 ,5,6;2) 基因组 dna 失活, 并引入编码感兴趣基因的模板 dna, 将所有的系统能量引导到对感兴趣的蛋白质产生的反应中;3) 平台的开放性使用户能够实时修改和监测反应条件和成分7、8.这种直接进入反应的方式支持了生物系统的增强, 增加了化学和氧化还原条件, 用于生产新型蛋白质和调整代谢过程2,9,10. 直接访问还允许用户将 cfps 反应与活动检测结合在单锅系统中, 以便更快速地进行设计-构建-测试周期11。在小批量液滴或纸质设备上执行 cfps 反应的能力进一步支持了高通量发现工作和 12、13、14、15的快速原型设计 ,16。由于这些优势和系统的即插即用性质, cfps 独特地促进了各种生物技术应用, 例如在体内难以溶解表达的蛋白质的生产17 18,19,20, 疾病检测 21,22,23, 按需生物制造18,24 25,26, 27,和教育28,29, 所有这些都显示了无细胞平台的灵活性和实用性。

cfps 系统可以从原核细胞和真核细胞系的各种粗裂解物中生成。这使得选择系统中有不同的选择, 每个选择都有优点和缺点, 这取决于利益的应用。cfps 系统在制备时间、成本和生产率方面也有很大差异。最常用的细胞提取物来自小麦胚芽、兔网状细胞、昆虫细胞和大肠杆菌细胞, 后者是迄今为止最具成本效益的, 同时产生的蛋白质体积产量最高。.虽然其他 cfps 系统可以有利于其固有的翻译后修饰机械, 新兴的应用使用大肠杆菌为基础的机械能够通过产生现场-专门磷酸化和糖基化蛋白按需31,32,33,34,35。

cfps 反应可以以批处理、连续交换无细胞 (cecf) 或连续无流动细胞 (cfcf) 格式运行。批次格式是一个封闭的系统, 其反应寿命是有限的, 由于减少的反应物的数量和反应的抑制副产物的积累。cecf 和 cfcf 方法提高了反应的使用寿命, 从而提高了与批处理反应相比的体积蛋白产量。这是通过允许从反应容器中去除蛋白质合成的副产品来实现的, 而在整个反应过程中, 新的反应物都是在2的过程中提供的。在 cfcf 的情况下, 感兴趣的蛋白质也可以从反应室中去除, 而在 cecf 中, 感兴趣的蛋白质仍然存在于由半透膜36,37组成的反应室中。这些方法对于克服感兴趣的难以表达的蛋白质的体积产量低特别有价值,38、39、40、41、42、 43岁实施 cecf 和 cfcf 方法的挑战是: 1) 虽然它们能更有效地利用负责转录和翻译的生物机械, 但它们需要更多的试剂, 从而增加总体成本, 并且增加 2)与批处理格式44相比, 它们需要更复杂的反应设置和专用设备。为了确保新用户的可访问性, 本文所述的协议侧重于15微米反应量的批处理格式, 并就将反应体积增加到毫升规模提出具体建议。

本文介绍的方法使具有基本实验室技能的非专家 (如本科生) 能够实现大肠杆菌 cfps 系统的细胞生长、提取制备和批量格式反应设置。与商用套件相比, 这种方法具有成本效益, 而不会牺牲基于风筝的反应设置的易用性。此外, 这种方法还可在实验室和现场应用。在决定实施 cfps 时, 新用户应彻底评估传统蛋白表达系统在启动投资方面的有效性, 因为 cfps 可能并非在所有情况下都优于 cfps。这里描述的 cfps 方法使用户能够直接实现各种应用, 包括功能基因组学、高通量测试、可在体内表达的难解蛋白质的生产以及现场应用包括合成生物学的生物传感器和教育套件。其他应用, 如代谢工程, 调整蛋白表达条件, 疾病检测, 并使用 cecf 或 cfc 方法扩大规模仍然是可能的, 但可能需要经验与 cfps 平台进一步修改反应条件。我们的方法结合了丰富的介质和挡板烧瓶的生长, 通过超声相对快速和可重复的细胞裂解方法, 然后是一个简化的 cfps 反应设置, 利用优化的预混料45。虽然细胞生长方法在这一领域已经有些标准化, 但细胞裂解的方法差别很大。除了超声, 常见的裂解方法包括利用法国印刷机, 均质机, 珠虫, 或溶菌酶和其他生化和物理破坏方法46,47, 48,49. 使用我们的方法, 每1升细胞获得大约2毫升的粗细胞提取物。该细胞提取物可支持 4, 100μl cfps 反应, 每个反应从模板质粒 pjl1-sfgfgfp 中产生 ~ 900μgg\ cfml 蛋白。这种方法的成本为0.021 美元/mfgfp (反应为 0.19μl $), 不包括人工和设备成本 (补充图 1)。从零开始, 此方法可以在4天内由一个人实现, 重复 cfps 反应可以在数小时内完成 (图 1)。此外, 该协议可以批量扩展, 以进行更大批次的试剂制备, 以满足用户的需要。重要的是, 这里介绍的协议可以由实验室培训的非专家 (如本科生) 来实施, 因为它只需要基本的实验室技能。下面描述的过程和随附的视频是专门开发的, 以改善大肠杆菌cfps 平台的可访问性, 供广泛使用。

Protocol

1. 培养基制备和细胞生长 第1天 将大肠杆菌BL21*(DE3) 细胞从甘油库存流到 lb 琼脂板上, 在37°c 下孵育至少18小时。 在121°c 的液体循环中准备50毫升 lb 介质和高压灭菌器溶液, 时间为30分钟。在室温下存放。 第2天 如补充信息所述, 准备750毫升的 2x ytp 介质和250毫升 0.4 m d-葡萄糖溶液。 将 2x ytp 介质倒入高压灭?…

Representative Results

我们提出了一个基于声纳的大肠杆菌提取物制备协议, 可以在四天的时间内完成,图 1显示了每天的程序故障。有可利用的步骤, 可以在每天完成与各种暂停点, 但我们发现这个工作流是最有效的执行。此外, 细胞颗粒 (步骤 1.3.18) 和完全准备好的提取物 (步骤 2.10) 在-80°c 下稳定至少一年, 允许用户创建每个的较大库存,</stro…

Discussion

无细胞蛋白合成已成为一种强大的和有利的技术, 可用于从生物制造到生化系统的快速原型设计等各种应用。应用程序的广度由实时监视、操作和扩充蜂窝机械的能力提供支持。尽管这一平台技术的影响越来越大, 但由于在实施这些方法方面存在技术细微差别, 广泛的适应仍然缓慢。通过这一努力, 我们的目标是为在新实验室中建立这一技术提供简单和清晰。为此, 我们的基于大肠杆菌的无细…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

作者希望感谢詹妮弗·范德凯伦博士、安德烈·劳布舍和托尼·图雷托的技术支持, 他们是高卫理、莱恩·威廉姆斯和克里斯托弗·海特的有益讨论。作者还感谢比尔和琳达·弗罗斯特基金、雪佛龙生物技术应用研究捐赠赠款中心、cal poly 研究、学术和创意活动赠款方案 (rsca 2017) 提供的资金支持,和国家科学基金会 (nsf-1708919)。mzl 承认加州州立大学研究生助学金。mcj 感谢陆军研究办公室 w911nf-16-1-072、国家科学基金会提供 mcb-1413563 和 mcb-1716766, 空军研究实验室英才中心赠款 fa8500-15-2-51818, 国防威胁减少机构赠款hdtra1-15-15-10052/p00001, david 和 lucile packard 基金会、camille dreyfus 教师-学者方案、能源部 ber grant de-sc0018249、人类前沿科学方案 (rgp152017)、doe 联合基因组 etop 和芝加哥生物医学联合会, 得到芝加哥社区信托基金的支持。

Materials

Luria Broth ThermoFisher 12795027
Tryptone Fisher Bioreagents 73049-73-7
Yeast Extract Fisher Bioreagents 1/2/8013
NaCl Sigma-Aldrich S3014-1KG
Potassium Phosphate Dibasic Sigma-Aldrich 60353-250G
Potassium Phosphate Monobasic Sigma-Aldrich P9791-500G
D-Glucose Sigma-Aldrich G8270-1KG
KOH Sigma-Aldrich P5958-500G
IPTG Sigma-Aldrich I6758-1G
Mg(OAc)2 Sigma-Aldrich M5661-250G
K(OAc) Sigma-Aldrich P1190-1KG
Tris(OAc) Sigma-Aldrich T6066-500G
DTT ThermoFisher 15508013
tRNA Sigma-Aldrich 10109541001
Folinic Acid Sigma-Aldrich F7878-100MG
NTPs ThermoFisher R0481
Oxalic Acid Sigma-Aldrich P0963-100G
NAD Sigma-Aldrich N8535-15VL
CoA Sigma-Aldrich C3144-25MG
PEP Sigma-Aldrich 860077-250MG
K(Glu) Sigma-Aldrich G1501-500G
NH4(Glu) MP Biomedicals 02180595.1
Mg(Glu)2 Sigma-Aldrich 49605-250G
Spermidine Sigma-Aldrich S0266-5G
Putrescine Sigma-Aldrich D13208-25G
HEPES ThermoFisher 11344041
Molecular Grade Water Sigma-Aldrich 7732-18-5
L-Aspartic Acid Sigma-Aldrich A7219-100G
L-Valine Sigma-Aldrich V0500-25G
L-Tryptophan Sigma-Aldrich T0254-25G
L-Phenylalanine Sigma-Aldrich P2126-100G
L-Isoleucine Sigma-Aldrich I2752-25G
L-Leucine Sigma-Aldrich L8000-25G
L-Cysteine Sigma-Aldrich C7352-25G
L-Methionine Sigma-Aldrich M9625-25G
L-Alanine Sigma-Aldrich A7627-100G
L-Arginine Sigma-Aldrich A8094-25G
L-Asparagine Sigma-Aldrich A0884-25G
Glycine Sigma-Aldrich G7126-100G
L-Glutamine Sigma-Aldrich G3126-250G
L-Histadine Sigma-Aldrich H8000-25G
L-Lysine Sigma-Aldrich L5501-25G
L-Proline Sigma-Aldrich P0380-100G
L-Serine Sigma-Aldrich S4500-100G
L-Threonine Sigma-Aldrich T8625-25G
L-Tyrosine Sigma-Aldrich T3754-100G
Fisherbrand Premium Microcentrifuge Tubes: 2.0 mL Fisher Scientific 05-408-138
Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL Fisher Scientific 05-408-129
Fisherbrand Premium Microcentrifuge Tubes: 0.6 mL Fisher Scientific 05-408-120
PureLink HiPure Plasmid Prep Kit ThermoFisher K210007
Ultrasonic Processor QSonica Q125-230V/50Hz 3.175 mm diameter probe
Avanti J-E Centrifuge Beckman Coulter 369001
JLA-8.1000 Rotor Beckman Coulter 366754
1L Centrifuge Tube Beckman Coulter A99028
Tunair 2.5L Baffeled Shake Flask Sigma-Aldrich Z710822
Microfuge 20 Beckman Coulter B30134
New Brunswick Innova 42/42R Incubator Eppendorf M1335-0000
Cytation 5 BioTek
Strep-Tactin XT Starter Kit IBA 2-4998-000
pJL1-sfGFP Addgene 69496
BL21(DE3) New England BioLabs
DOWNLOAD MATERIALS LIST

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Keywords: Escherichia ColiCell-free Protein SynthesisProtocolPlatform TechnologyFunctional GenomicsBiosensorsMetabolic EngineeringGenetic Code ExpansionCentrifugationS30 BufferDTTResuspensionAliquoting
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Levine, M. Z., Gregorio, N. E., Jewett, M. C., Watts, K. R., Oza, J. P. Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology. J. Vis. Exp. (144), e58882, doi:10.3791/58882 (2019).
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