Click display: a rapid and efficient in vitro protein display method for directed evolution. Academic Article uri icon


  • 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, translationand 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-l 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.

published proceedings

  • Nucleic Acids Res

altmetric score

  • 6.7

author list (cited authors)

  • Zeng, Y. u., Woolley, M., Chockalingam, K., Thomas, B., Arora, S., Hook, M., & Chen, Z.

citation count

  • 2

complete list of authors

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

publication date

  • September 2023