# Peptide ligase–mediated display: A cell-free platform for tunable selection of affinity peptides

## TL;DR

A new cell-free method called PL display allows for efficient selection of peptides that bind to specific targets, using covalent linkage and minimal linkers.

## Contribution

The novel contribution is a cell-free platform using peptidyl transferase for covalent genotype-phenotype linkage with a minimal linker.

## Key findings

- HA-tag sequences at 0.01% frequency were fully isolated in one round of FACS selection.
- Consensus binding sequences were enriched from a random library with 1.7 × 10⁶ diversity in two FACS rounds.
- The platform is not limited by cellular physiology or linker protein constraints.

## Abstract

Herein, we report a bead-surface protein display method based on a peptidyl transferase reaction, termed peptide ligase–mediated display (PL display). This technique enables the covalent linkage of genotypic DNA and phenotypic protein variants on beads via a minimal nine–amino acid linker in a fully cell-free system. Using this method, hemagglutinin (HA)-tag sequences introduced at a 0.01% frequency were completely isolated in a single round of selection via fluorescence-activated cell sorting (FACS) against an anti-HA-tag antibody. Furthermore, consensus sequences that bind to the anti-HA-tag antibody were enriched from a random peptide library with a sequence diversity of 1.7 × 106 in two rounds of selection using FACS. This quantitative affinity selection platform using PL display is applicable under diverse conditions, as it is not constrained by cellular physiological properties, fluctuations in gene expression, or the structural and functional limitations of linker proteins involved in genotype–phenotype linkage. These advantages arise from the use of a fully cell-free system and covalent linkage with a minimal linker.