蛋白质是生命的基石,大量的细胞过程由蛋白质-蛋白质相互作用(PPI)处理。通过PPI形成的蛋白质复合物导致纠结的网络,随着他们不断的重塑,建立系统的功能单元。多年来,PPI已经成为许多研究人员感兴趣的领域,导致开发多种体外和体内方法来揭示这些相互作用。酵母双杂交(Y2H)系统是一种以微通量和高通量方式绘制PPI的有效遗传方法。Y2H是一种涉及使用修饰的酵母细胞来鉴定蛋白质-蛋白质相互作用的技术。对于Y2H,只有当特定蛋白质与其相互作用的伴侣之间存在显著相互作用时,酵母细胞才会被工程化以生长。通过响应于恢复的转录因子刺激报告基因在Y2H系统中鉴定PPI。然而,Y2H结果可能受到严格要求的限制,因为通过这种技术进行的数量有限的菌落筛选可能导致消除许多真正的相互作用。因此,可以使用DEEPN(用于评估蛋白质网络的动态富集),提供了在单个Y2H实验中研究多种静态和瞬时蛋白质相互作用的潜力。DEEPN以高通量方式利用下一代DNA测序(NGS)数据,随后应用计算分析和统计建模来识别相互作用的伴侣。该协议描述了定制的试剂和协议,通过它可以有效地和成本有效地利用DEEPN分析。
Proteins are the building blocks of life, and a vast array of cellular processes is handled by protein-protein interactions (PPIs). The protein complexes formed via PPIs lead to tangled networks that, with their continuous remodeling, build up systematic functional units. Over the years, PPIs have become an area of interest for many researchers, leading to the development of multiple in vitro and in vivo methods to reveal these interactions. The yeast-two-hybrid (
Y2H) system is a potent genetic way to map PPIs in both a micro- and high-throughput manner.
Y2H is a technique that involves using modified yeast cells to identify protein-protein interactions. For
Y2H, the yeast cells are engineered only to grow when there is a significant interaction between a specific protein with its interacting partner. PPIs are identified in the
Y2H system by stimulating reporter genes in response to a restored transcription factor. However, Y2H results may be constrained by stringency requirements, as the limited number of colony screenings through this technique could result in the possible elimination of numerous genuine interactions. Therefore, DEEPN (dynamic enrichment for evaluation of protein networks) can be used, offering the potential to study the multiple static and transient protein interactions in a single
Y2H experiment. DEEPN utilizes next-generation DNA sequencing (NGS) data in a high-throughput manner and subsequently applies computational analysis and statistical modeling to identify interacting partners. This protocol describes customized reagents and protocols through which DEEPN analysis can be utilized efficiently and cost-effectively.