Cell-Free Expression of De Novo Designed Peptides That Form β-Barrel Nanopores

[Image: see text] Nanopore sensing has attracted much attention as a rapid, simple, and label-free single-molecule detection technology. To apply nanopore sensing to extensive targets including polypeptides, nanopores are required to have a size and structure suitable for the target. We recently designed a de novo β-barrel peptide nanopore (SVG28) that constructs a stable and monodispersely sized nanopore. To develop the sizes and functionality of peptide nanopores, systematic exploration is required. Here we attempt to use a cell-free synthesis system that can readily express peptides using transcription and translation. Hydrophilic variants of SVG28 were designed and expressed by the PURE system. The peptides form a monodispersely sized nanopore, with a diameter 1.1 or 1.5 nm smaller than that of SVG28. Such cell-free synthesizable peptide nanopores have the potential to enable the systematic custom design of nanopores and comprehensive sequence screening of nanopore-forming peptides.