Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo184-8588, Japan.
Graduate School of Engineering Science, Yokohama National University, Yokohama240-8501, Japan.
ACS Nano. 2023 Feb 28;17(4):3358-3367. doi: 10.1021/acsnano.2c07970. Epub 2023 Feb 2.
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 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.
纳米孔传感作为一种快速、简单、无需标记的单分子检测技术引起了广泛关注。为了将纳米孔传感应用于包括多肽在内的广泛目标，需要具有适合目标的大小和结构的纳米孔。我们最近设计了一种β-桶状多肽纳米孔（SVG28），它构建了一个稳定且单分散尺寸的纳米孔。为了开发肽纳米孔的尺寸和功能，需要进行系统的探索。在这里，我们尝试使用一种无细胞合成系统，该系统可以使用转录和翻译来轻易地表达肽。我们使用 PURE 系统设计并表达了 SVG28 的亲水变体。这些肽形成了单分散尺寸的纳米孔，其直径比 SVG28 小 1.1 或 1.5nm。这种可在无细胞条件下合成的肽纳米孔具有实现纳米孔系统定制设计和纳米孔形成肽的全面序列筛选的潜力。
