酵母 2-混合筛在批次中比较蛋白质相互作用
Summary
酵母 2-混合筛的分批处理可以直接比较多饵蛋白与高度复杂的猎物融合蛋白的相互作用。在这里, 我们描述了精制方法, 新试剂, 以及如何实现这些屏幕的使用。
Abstract
用酵母 2-杂交法筛选蛋白质-蛋白质相互作用长期以来一直是一个有效的工具, 但它的使用在很大程度上仅限于发现高亲和性的 interactors 在互动的候选库中高度丰富。以传统的形式, 酵母 2-杂交试验可以产生太多的菌落分析时进行低严格的, 在那里可能发现低亲和 interactors。此外, 如果没有对同一图书馆进行全面和彻底的讯问, 对不同的诱饵质粒, 就无法进行比较分析。虽然这些问题中的一些可以使用阵列的牺牲品库来解决, 但操作此类屏幕所需的成本和基础设施却是令人望而却步的。作为替代, 我们已经适应了酵母 2-杂交检测, 同时发现在一个单一的屏幕上的许多瞬态和静态蛋白质相互作用的一个战略称为 DEEPN (动态富集评估蛋白质网络), 这采用高通量 DNA 测序和计算来跟踪编码相互作用伙伴的质粒种群的进化。在这里, 我们描述了定制的试剂和协议, 使 DEEPN 屏幕易于执行和经济高效。
Introduction
对细胞生物学过程的完全理解依赖于寻找其分子机制基础的蛋白质相互作用网络。一种识别蛋白质相互作用的方法是酵母 2-杂交 (Y2H) 法, 它通过组装一个功能的嵌合体转录因子, 一旦两个感兴趣的蛋白质领域绑定到另一个1。一个典型的 Y2H 屏幕是通过创建一个酵母种群, 它既可以将相互作用的蛋白质的质粒库编码成转录激活剂 (e. g, “猎物” 融合蛋白) 和由蛋白质组成的给定的 “诱饵” 质粒。的兴趣融合到 dna 绑定域 (例如, 绑定到 Gal4-upstream 激活序列的 Gal4 DNA 绑定域)。Y2H 方法的一个主要优点是, 在为常规分子生物学工作配备的典型实验室中, 进行2是相对容易和廉价的。但是, 当传统上执行时, 用户会对在选择时出现的单个菌落进行正面 Y2H 交互的采样。这严重限制了可以调查的图书馆 “猎物” 克隆的数量。当一个特定的相互作用的猎物的丰度相对于其他捕食者非常高时, 这一问题就会复杂化, 从而减少了从低丰度猎物质粒中检测相互作用的几率。
在蛋白质组的综合覆盖范围内使用 Y2H 原理的一个解决方案是使用矩阵格式化的方法, 其中包含已知单个猎物质粒的阵列可以进行数字审问。但是, 这种方法需要的基础结构对于那些有意定义少量蛋白质或域3的 interactome 的个体调查人员来说是不容易接近或成本效益高的。此外, 非常复杂的猎物库, 可能编码多个相互作用的蛋白质片段, 将扩大这种矩阵数组的大小不切实际的大小。另一种方法是在批次中执行复杂库的检测, 并使用大规模并行高通量排序4评估交互克隆的存在。这可以用来检测在多个殖民地出现的猎物质粒的存在, 使用典型的 Y2H 格式化的方法, 其中酵母细胞外壳的相互作用对融合蛋白被允许生长在一个板块5,6。这一总体思路可以增强, 以增加对多个诱饵和猎物组件的查询, 同时7,8。
尽管如此, 许多调查还是需要更简单、更专注于少量蛋白质 “诱饵” 的努力, 并且可以通过对单个复杂的猎物库进行详尽而半定量的查询来获得更多的好处。我们已经开发并验证了一种使用 Y2H 原则在批处理格式4中执行广泛的蛋白质交互研究的方法。这使用特定的猎物质粒的膨胀率作为 Y2H 交互作用的相对强度的代理9。在正常生长或选择性生长条件下, 在种群内的所有质粒的深度测序生成完整的克隆, 产生强弱的 Y2H 相互作用。interactors 可以在多个诱饵质粒中得到和直接比较。由此产生的工作流称为 DEEPN (对蛋白质网络进行动态富集评估), 因此可以用来识别同一个猎物库中的差异 interactomes, 以确定蛋白质, 从而使一种蛋白质与另一种蛋白进行比较。
在这里, 我们演示了 DEEPN, 并介绍了有助于其使用的实验室方法的改进, 其中概述了图 1。重大改进包括:
捕食酵母种群的生成.DEEPN 的关键要求之一是产生不同饵料质粒的酵母种群, 它们的粒细胞分布相同。捕食质粒库的等效基线种群对不同饵料的 interactomes 进行精确比较是必不可少的。这是最好的实现, 当一个图书馆的质粒已经安置在单倍体酵母种群和移动一个给定的诱饵质粒进入该人群是通过交配产生一个倍。在这里, 我们提供了一个明确的指南, 如何使这些人口使用的商业图书馆在单倍体酵母。虽然我们发现了产生大量 diploids 的方法, 但这些含有酵母菌的商业文库的整体交配效率却很低。因此, 我们构造了一种新的应变, 可以使猎物库产生更 diploids 的每一个交配反应。
新的诱饵质粒集。许多目前表达 “诱饵” 融合蛋白的质粒是由感兴趣的蛋白质和 DNA 结合的领域组成的, 2µ, 允许它们放大其拷贝数。这个拷贝数在人口中可能是相当可变的并且导致变异在 Y2H 转录反应。这反过来可能会扭曲的能力, 以衡量一个特定的蛋白质相互作用的强度, 根据细胞的生长反应的选择。这可以通过使用低拷贝质粒来部分解决, 其中一些以前曾被描述为商业上可用的 pDEST3210。我们构建了一种新的诱饵质粒 (pTEF GBD), 在TRP1着丝粒基低拷贝质粒中产生 Gal4-DNA-binding 域融合蛋白, 携带 Kanr 抵抗基因, 同时也允许克隆上游和Gal4 DNA 结合领域的下游。
新的高密度 Y2H 片段库.我们构建了一个新的质粒来 Y2H 猎物库, 并利用它建立一个高度复杂的 Y2H 图书馆由随机剪切片段的基因组 DNA 从酿酒酵母。序列分析表明, 该库有100万多个不同的元素, 远比以前描述的酵母基因组 Y2H 质粒库11复杂得多。通过这个新的库, 我们能够证明 DEEPN 工作流足够健壮, 能够以可靠和可重现的方式容纳许多不同质粒的复杂库。
Protocol
Representative Results
Discussion
在这里, 我们提供了如何使用优化方法在批处理中执行 Y2H 化验的指南。在这个过程中有几个关键步骤, 以帮助确保将被放置在选择下的酵母的人口是起始库的代表, 并且足够的开始酵母人口用于接受选择限制变异.重要的是, 这些基准相对容易实现, 同时适应传统的 Y2H 检测方法和材料, 从而使大多数实验室都能使用标准分子生物学的方法。在使用不同的诱饵质粒时, DEEPN 允许从相同的猎物库种群中进…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢人类遗传学研究所内的工作人员对图书馆的准备和测序。我们感谢 Einat Snir 在为这里制作的 Y2H 质粒库编写基因组文库片段方面的专长。这项工作得到国家卫生研究院的支持: NIH R21 EB021870-01A1 和 NSF 研究项目赠款: 1517110。
Materials
| Illumina HiSeq 4000 | Illumina | deep sequencing platform | |
| Monoclonal anti-HA antibodies | Biolegend | 901514 | Primary Antibody to detect expression of HA in pGal4AD constructs |
| Polyclonal anti-myc antibodies | QED Biosciences Inc | 18826 | Primary Antibody to detect expression of MYC in pTEF-GBD constructs |
| NarI | New England BioLabs | R0191S | |
| EcoRI-HF | New England BioLabs | R3101S | |
| BamHI-HF | New England BioLabs | R3236S | |
| XhoI | New England BioLabs | R0146S | |
| Polyethylene Glycol 3350, powder | J.T. Baker | U2211-08 | |
| Salmon Sperm DNA | Trevigen, Inc sold by Fisher Scientific | 50-948-286 | carrier DNA for yeast transformation section 3.2.1. |
| Kanamycin Monosulfate | Research Products International | K22000 | |
| LE Agarose | GeneMate | E-3120-500 | used for making DNA agarose gels |
| Sodium Chloride | Research Products International | S23025 | |
| Tryptone | Research Products International | T60060 | |
| D-Sorbitol | Research Products International | S23080 | |
| Lithium Acetate Dihydrate | MP Biomedicals | 155256 | |
| Calcium Chloride | ThermoFisher | C79 | |
| EDTA Sodium Salt | Research Products International | E57020 | |
| Yeast Extract Powder | Research Products International | Y20020 | |
| Yeast Nitrogen Base (ammonium sulfate) w/o amino acids | Research Products International | Y20040 | |
| CSM-Trp-Leu+40ADE | Formedium | DCS0789 | |
| CSM-Trp-Leu-His+40ADE | Formedium | DCS1169 | |
| CSM-Leu-Met | Formedium | DCS0549 | |
| CSM-Trp-Met | Bio 101, Inc | 4520-922 | |
| L-Methionine | Formedium | DOC0168 | |
| Adenine | Research Products International | A11500 | |
| D-(+)-Glucose | Research Products International | G32045 | |
| Bacto Agar | BD | 214010 | used for making media plates in section 1 |
| Peptone | Research Products International | P20240 | |
| 3-amino-1,2,4 Triazole | Sigma | A8056 | |
| 2-Mercaptoehanol (BME) | Sigma-Aldrich | M6250 | |
| Zymolyase 100T | USBiological | Z1004 | |
| Potassium phosphate dibasic | Sigma | P8281 | |
| Phenol:Chloroform:IAA | Ambion | AM9732 | |
| Ammonium Acetate | Sigma-Aldrich | 238074 | |
| Ethanol | Decon Laboratories, INC | 2716 | |
| RNAse A | ThermoFisher | EN0531 | |
| Urea | Research Products International | U20200 | |
| SDS | Research Products International | L22010 | |
| glycerol | Sigma Aldrich | G5516 | |
| Tris-HCl | Gibco | 15506-017 | |
| bromophenol blue | Amresco | 449 | |
| Gibson Assembly Cloning Kit | New England Biolabs | E5510S | Rapid assembly method for cloning of plasmids in section 2 |
| NEBNext High-Fidelity 2x PCR Master Mix | New England Biolabs | M0541S | Used for amplification of products for Gibson Assembly in Section 2.3 as well assample preparation for DEEPN deep sequencing in section 6.2.1 |
| Ethidium Bromide | Amresco | 0492-5G | |
| QIAquick PCR purification kit | Qiagen | 28104 | Used for purification of pcr products in section 6.2.3 |
| Qiaquick DNA Gel Extraction Kit | Qiagen | 28704 | Used for purification of digested pTEF-GBD in section 2.1 |
| KAPA Hyper Prep kit | KAPA Biosystems | KK8500 | preparation kit for deep sequencing |
| Codon optimization | http://www.jcat.de | ||
| Codon optimization | https://www.idtdna.com/CodonOpt | ||
| gBlocks | Integrated DNA Technologies | DNA fragments used for cloning in Section 2.2 | |
| Strings | Thermofisher | DNA fragments used for cloning in Section 2.2 | |
| GenCatch Plasmid DNA mini-prep Kit | EPOCH Life Sciences | Used to prepare quantities of DNA in Section 2.3 | |
| Covaris E220 | Covaris | high performance ultra-sonicator in section 7 | |
| oligo nucelotide 5’- CGGTCTT CAATTTCTCAAGTTTCAG -3’ |
Integrated DNA Technologies or Thermofisher | used for pcr amplification and sequencing 5' insert pTEF-GBD during plasmid construction | |
| oligo nucelotide 5’-GAGTAACG ACATTCCCAGTTGTTC-3’ |
Integrated DNA Technologies or Thermofisher | used for pcr amplification and sequencing 5' insert pTEF-GBD during plasmid construction | |
| oligo nucelotide 5’-CACCGTAT TTCTGCCACCTCTTCC-3’ |
Integrated DNA Technologies or Thermofisher | used for pcr amplification and sequencing 3' insert pTEF-GBD during plasmid construction | |
| oligo nucelotide 5’-GCAACCGC ACTATTTGGAGCGCTG-3’ |
Integrated DNA Technologies or Thermofisher | used for pcr amplification and sequencing 3' insert pTEF-GBD during plasmid construction | |
| oligonucleotide 5’-GTTCCGATG CCTCTGCGAGTG-3’ |
Integrated DNA Technologies or Thermofisher | 5' Pimer used for insert amplification of pGAL4AD | |
| oligonucelotide 5’-GCACATGCT AGCGTCAAATACC-3’ |
Integrated DNA Technologies or Thermofisher | 3' Pimer used for insert amplification of pGAL4AD | |
| oligonucelotide 5’-ACCCAAGCA GTGGTATCAACG-3’ |
Integrated DNA Technologies or Thermofisher | 5' Pimer used for insert amplification of pGADT7 | |
| oligonucelotide 5’- TATTTAGA AGTGTCAACAACGTA -3’ |
Integrated DNA Technologies or Thermofisher | 3' Pimer used for insert amplification of pGADT7 | |
| PJ69-4A MatA yeast strain | http://depts.washington.edu/yeastrc/ James P, Halladay J, Craig EA: Genomic Libraries and a host strain designed for highly efficient two-hybrid selection in yeast. GENETICS 1996 144:1425-1436 | MATA leu2-3,112 ura3-52 trp1-901 his3-200 gal4D, gal80D, GAL-ADE2 lys2::GAL1-HIS3 met2::GAL7 | |
| pTEF-GBD | Dr. Robert Piper Lab | Gal4-DNA binding doimain expression plasmid | |
| PLY5725 MATalpha yeast strain | Dr. Robert Piper Lab | MATalpha his3∆1 leu2∆0 lys2∆0 ura3∆0 gal4∆ trp1∆ Gal80∆ | |
| pGal4AD (pPL6343) | Dr. Robert Piper Lab | Gal4-activation domain expression plasmid | |
| 100 mm petri dishes | Kord-Vallmark sold by VWR | 2900 | |
| 125 mL PYREX Erlenmeyer flask | Fisher Scientific | S63270 | |
| 250 mL PYREX Erlenmeyer flask | Fisher Scientific | S63271 | |
| 1,000 mL PYREX Erlenmeyer flask | Fisher Scientific | S63274 | |
| 2,000 mL PYREX Erlenmeyer flask | Fisher Scientific | S63275 | |
| 20 X 150 mm Disposable Culture Tube | Thermofisher | 14-961-33 | |
| pipet-aid | Drummond | 4-000-100 | |
| 5 mL Serological Pipette | Denville | P7127 | |
| 10 mL Serological Pipette | Denville | P7128 | |
| 25 mL Serological Pipette | Denville | P7129 | |
| 1,000 mL PYREX Griffin Beaker | Fisher Scientific | 02-540P | |
| 1,000 mL PYREX Reuasable Media Storage Bottle | Fisher Scientific | 06-414-1D | |
| 1,000 mL graduated cylinder | Fisher Scientific | 08-572-6G | |
| SpectraMax 190 | Molecular Devices | used to measure the Optical Density of cells | |
| NanoDrop 2000 | Thermo Scientific | ND-2000 | Spectrophotometer used to quantify amount of DNA |
| Electronic UV transilluminator | Ultra Lum | MEB 20 | used to visualize DNA in an Ethidium Bromide agarose gel |
| P1000 Gilson PIPETMAN | Fisher Scientific | F123602G | |
| P200 Gilson PIPETMAN | Fisher Scientific | F123601G | |
| P20 Gilson PIPETMAN | Fisher Scientific | F123600G | |
| P10 Gilson PIPETMAN | Fisher Scientific | F144802G | |
| 1250 µL Low Retention Pipette Tips | GeneMate | P-1236-1250 | |
| 200 µLLow Retention Pipette Tips | VWR | 10017-044 | |
| 10 µL XL Low Retention Pipette Tips | VWR | 10017-042 | |
| 50 mL conical tube | VWR | 490001-627 | |
| 15 mL conical tube | VWR | 490001-621 | |
| cell scraper | Denville Scientific | TC9310 | |
| 1.5 mL Microcentrifuge tubes | USA Scientific | 1615-5500 | |
| HCl | Fluka Analytical | 318949-1L | |
| NaOH | J.T. Baker | 5674-02 | |
| Wooden applicators | Solon Care | 55900 | |
| Eppendorf microcentrifuge 5424 | Fisher Scientific | 05-400-005 | microcentrifuge |
| Sorvall ST16R | Thermo Fisher Scientific | 75004381 | benchtop centrifuge |
| Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey) | GE Healthcare | NA934-1ML | Secondary Antibody |
| Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep) | GE Healthcare | NA931-1ML | Secondary Antibody |
| SuperSignal West Pico Chemiluminescent Substrate | Thermo Fisher Scientific | 34080 | ECL detection solution |
| Isotemp Incubator | Thermo Fisher Scientific | Incubator | |
| Mutitron 2 | INFORS HT | Shaking incubator | |
| Isotemp Digital-Control Water Bath Model 205 | Fisher Scientific | water bath | |
| Y2H mouse cDNA library in Y187 (pan tissue) | Clontech | 630482 | commercially available cDNA Library |
| Y2H mouse cDNA library in Y187 (mouse brain) | Clontech | 630488 | commercially available cDNA Library |
| pGADT7 AD Vector | Clontech | 630442 | commercially available AD Vector housing many cDNA libraries |
| pGBKT7 DNA-BD Vector | Clontech | 630443 | commercially available DNA-BD Vector |
| Biolase DNA Polymerase | Bioline | BIO-21042 | DNA polymerase used for section 2.4 |
| GeneMate GCL-60 Thermal Cycler | BioExpress | P-6050-60 | pcr machine |
| TempAssure 0.5 mL PCR tubes | USA Scientific | 1405-8100 |
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