Click Display: A Rapid and Efficient In Vitro Protein Display Method for Directed Evolution

Authors

Yu Zeng
Michael Woolley
Karuppiah Chockalingam
Benjamin Thomas
Srishtee Arora
Magnus Hook
Zhilei Chen

Publication Date

9-8-2023

Journal

Nucleic Acids Research

Abstract

We describe a novel method for in vitro protein display-click display-that does not depend on maintaining RNA integrity during biopanning and yields covalently linked protein-cDNA complexes from double-stranded input DNA within 2 h. The display is achieved in a one-pot format encompassing transcription, translation and reverse transcription reactions in series. Stable linkage between proteins and the encoding cDNA is mediated by a modified DNA linker-ML-generated via a click chemistry reaction between a puromycin-containing oligo and a cDNA synthesis primer. Biopanning of a click-displayed mock library coupled with next-generation sequencing analysis revealed >600-fold enrichment of target binders within a single round of panning. A synthetic library of Designed Ankyrin Repeat Proteins (DARPins) with ∼1012 individual members was generated using click display in a 25-μl reaction and six rounds of library panning against a model protein yielded a panel of nanomolar binders. This study establishes click display as a powerful tool for protein binder discovery/engineering and provides a convenient platform for in vitro biopanning selection even in RNase-rich environments such as on whole cells.

Keywords

DNA, DNA, Complementary, Peptide Library, Protein Engineering, Proteins, Directed Molecular Evolution

Comments

Supplementary Materials

PMID: 37548398