改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用
Summary
我们已经修改了传统的酵母双杂交筛选，确定蛋白质相互作用的一种有效的遗传工具。此修改的过程明显缩短，减少了工作量，最重要的是，降低了误报数量。此外，这种方法是重复的，可靠的。
Abstract
Progranulin（PGRN），又称granulin epithelin前体（GEP），是一个593个氨基酸的自分泌生长因子。 PGRN是已知的各种生理和疾病过程中发挥了关键作用，包括早期胚胎发育，伤口愈合，炎症2，3，和主机防御4。 PGRN也可以作为一种神经营养因子，并在PGRN基因导致部分损失的PGRN蛋白引起的额颞叶痴呆6，7突变。我们最近的研究作为重要的调节软骨发育和退化8-11导致PGRN隔离。虽然PGRN，发现近二十年前，起着至关重要的作用，在多种生理和病理条件下，努力开拓PGRN的行动，并了解所涉及的机制已明显阻碍我们无法确定其结合受体（S）。为了解决这个问题，我们开发了莫迪fied酵母双杂交（MY2H）方法的基础上最常使用的GAL4基于2混合动力系统。 MY2H与传统的酵母双杂交屏幕相比，极大地缩短了屏幕的过程，并降低了假阳性克隆数。此外，这种方法是重复的，可靠的，我们已经成功地采用这个系统，在隔离各种诱饵的结合蛋白，包括离子通道，细胞外基质蛋白10，13，和生长因子14。在本文中，我们详细描述了作为一个例子，LED作为第一个已知的PGRN相关受体14日，15 TNFR2识别使用PGRN这一MY2H实验过程。
Protocol
1。背景资料酵母双杂交系统是一个功能强大的基因技术，用于发现蛋白质相互作用16， 17。 2混合动力系统，如lexA为基础的系统，SOS招聘系统，细菌或哺乳动物细胞为基础的2混合动力车，几种商业可用，本文特别侧重于最常用的GAL4基于修改酵母双杂交系统。简而言之，该方法是基于负责可分离DNA结合和转录激活域组成的酵母GAL4蛋白的属性。诱饵蛋白表达作为?…
Discussion
酵母双杂交筛选已被证明是一个有效的工具，在确定蛋白质相互作用16，17。与其他方法确定蛋白结合的合作伙伴，如生化合作，纯化和蛋白质芯片，酵母双杂交系统是一个敏感的遗传筛选非常高的数字编码序列在一个相对简单的的实验方法，可使用;此外，它在体内相互作用检测，也不需要复杂的蛋白质纯化。当然酵母双杂交系统有其局限性，例如，需要翻译后修饰，融合蛋白折叠的不足…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由NIH的研究补助金K01AR053210，R01AR061484和国家牛皮癣基金会的赠款。
Materials
| Name of the reagent | Company | Catalogue number |
| ProQuest two-hybrid system | Invitrogen | 10835 |
| YPD Growth Medium | Clontech | 630409 |
| YPD Agar Medium | Clontech | 630410 |
| Minimal SD agar Base | Clontech | 630412 |
| -Leu DO Supplement | Clontech | 630414 |
| -Leu/-Trp DO Supplement | Clontech | 630417 |
| -His/-Leu/-Trp/-Ura DO Supplement | Clontech | 630425 |
| 3-Amino-1,2,4-Triazole (3AT) | Sigma-Aldrich | A8056 |
| Luria broth (LB) | Sigma-Aldrich | L3022 |
| X-Gal | Invitrogen | 15520-034 |
| Sonicated Salmon Sperm DNA | Stratagene | 201190 |
| Ampicillin | AMRESCO | 0339 |
| Kanamycin Sulfate | Invitrogen | 11815-024 |
| Subcloning Efficiency™ DH5α™ Competent Cells | Invitrogen | 18265-017 |
| Trizma® base | Sigma-Aldrich | T6066 |
| Lithium acetate | Sigma-Aldrich | L4158 |
| Ethylenediaminetetraacetic acid | Sigma-Aldrich | ED |
| Nitrocellulose Membrane | Bio-Rad | 162-0115 |
| 10-cm petri dish | ITI Scientific | CT-903 |
| Incubator (30 °C) | ATR (Ecotron) |
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Tags
Modified Yeast-two-hybrid SystemProteinsInteractingGrowth FactorProgranulinPGRNGranulin Epithelin PrecursorAutocrine Growth FactorPhysiologic ProcessesDisease ProcessesInflammationHost DefenseNeurotrophic FactorPGRN GeneFrontotemporal DementiaCartilage Development And DegradationBinding Receptor(s)Modified Yeast Two-hybrid ApproachGAL4 Based 2-hybrid SystemFalse Positive ClonesReproducible And Reliable System
Citer Cet Article
Tian, Q., Zhao, Y., Liu, C. Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin. J. Vis. Exp. (59), e3562, doi:10.3791/3562 (2012).