蛋白互作组学研究技术获得突破

2023-05-10 14:56:27

蛋白质互作检测是研究蛋白功能、探索生物分子通路的重要途径。酵母双杂交（Y2H）、免疫沉淀（Co-IP）、Pull-down是验证蛋白互作、寻找靶标蛋白互作蛋白的常用方法。然而，这些方法只能局限于对特定目标蛋白的研究，如果要了解细胞内成千上万种蛋白之间的相互作用，现有的实验方法还很难实现的。

最近，著名生物学家、Salk研究所的Joseph R Ecker团队研究人员开发出一种新型高通量蛋白质互作检测技术，可以在一个实验中实现成千上万种蛋白质之间上百万种互作关系的检测。

该方法将酵母双杂交和DNA高通量测序技术完美结合，通过将编码蛋白质的各种随机组合的基因分别连在两个质粒上再导入细胞。如果两种蛋白在细胞内发生互作，质粒上的 Cre报告基因就会被激活，在它的牵引下两个独立质粒上的两种基因就会连在一起。经过选择培养基筛选发生重组的细胞（细胞内含有相互作用蛋白），再利用高通量DNA测序来识别究竟两种互作蛋白的编码基因。

研究人员利用CrY2H-seq在一个月内对拟南芥中1800多种转录因子蛋白，多达400万个实验排列组合，重复做了10次互作检测，共发现了8000多个蛋白互作。这组互作图谱让我们对拟南芥转录因子互作有了新的认识，可帮助人们回答长期以来某些某些未知功能的转录因子的作用。

该方法将极大提升蛋白互作组学领域的研究进展，增加我们对重要生物学进程的深入理解。

Nature Methods, 26 June 2017

CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping

Author

Shelly A Trigg, Renee M Garza, Andrew MacWilliams……Zhuzhu Z Zhang, Mary Galli & Joseph R Ecker*

*: The Salk Institute for Biological Studies, USA.

Abstract

Broad-scale protein–protein interaction mapping is a major challenge given the cost, time, and sensitivity ctraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interacti en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interacti. By performing ten independent screens testing a total of 36 million binary interaction combinati, and uncovering a network of 8,577 interacti among 1,453 transcription factors, we demtrate CrY2H-seq′s improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interacti derived from diverse methods, expands the number of known plant transcription factor interacti by three-fold, and reveals previously unknown family-specific interaction module associati with plant reproductive development, root architecture, and circadian coordination.

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