Parallelized identification of on- and off-target protein interactions,Molecular Systems Design & Engineering

当前位置： X-MOL 学术 › Mol. Syst. Des. Eng. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Parallelized identification of on- and off-target protein interactions
Molecular Systems Design & Engineering ( IF 3.2 ) Pub Date : 2019-11-26 , DOI: 10.1039/c9me00118b
Jiayi Dou _{1,

2} , Inna Goreshnik _{1,

2} , Cassie Bryan _{1,

2} , David Baker _{1,

2,

3} , Eva-Maria Strauch _{1,

2,

4,

5}

Affiliation

Genetic selection combined with next-generation sequencing enables the simultaneous interrogation of the functionality and stability of large numbers of naturally occurring, engineered, or computationally designed protein variants in parallel. We display hundreds of engineered proteins on the surface of yeast cells, select for binding to a set of target molecules by flow cytometry, and sequence the starting pool as well as selected pools to obtain enrichment values for each displayed protein with each target. We show that this high-throughput workflow of multiplex genetic selections followed by large-scale sequencing and comparative analysis allows not only the determination of relative affinities, but also the monitoring of specificity profiles for hundreds to thousands of protein–protein and protein–small molecule interactions in parallel. The approach not only identifies new interactions of designed proteins, but also detects unintended and undesirable off-target interactions. This provides a general framework for screening of engineered protein binders, which often have no negative selection or design step as part of their development pipelines. Hence, this method will be generally useful in the development of protein-based therapeutics.

中文翻译：

并行识别靶蛋白和脱靶蛋白相互作用

遗传选择与下一代测序相结合，可以同时检测大量天然存在的、工程化的或计算设计的蛋白质变体的功能和稳定性。我们在酵母细胞表面展示数百种工程蛋白质，通过流式细胞术选择与一组目标分子结合，并对起始池和选定池进行测序，以获得每个展示的蛋白质与每个目标的富集值。我们表明，这种多重遗传选择的高通量工作流程，然后是大规模测序和比较分析，不仅可以确定相对亲和力，还可以监测数百到数千种蛋白质-蛋白质和蛋白质-小分子的特异性谱并行交互。该方法不仅可以识别设计蛋白质的新相互作用，还可以检测意外和不希望的脱靶相互作用。这为筛选工程蛋白结合剂提供了一个通用框架，这些结合剂通常没有负选择或设计步骤作为其开发流程的一部分。因此，该方法通常可用于开发基于蛋白质的疗法。

更新日期：2019-11-26

点击分享查看原文

点击收藏

阅读更多本刊最新论文