Jump to main content
Jump to site search
Access to RSC content Close the message box

Continue to access RSC content when you are not at your institution. Follow our step-by-step guide.


Issue 1, 2020
Previous Article Next Article

Parallelized identification of on- and off-target protein interactions

Author affiliations

Abstract

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.

Graphical abstract: Parallelized identification of on- and off-target protein interactions

Back to tab navigation

Supplementary files

Article information


Submitted
07 Sep 2019
Accepted
26 Nov 2019
First published
26 Nov 2019

Mol. Syst. Des. Eng., 2020,5, 349-357
Article type
Paper

Parallelized identification of on- and off-target protein interactions

J. Dou, I. Goreshnik, C. Bryan, D. Baker and E. Strauch, Mol. Syst. Des. Eng., 2020, 5, 349
DOI: 10.1039/C9ME00118B

Social activity

Search articles by author

Spotlight

Advertisements