Nanopore Group, Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, California, USA.
Nat Biotechnol. 2013 Mar;31(3):247-50. doi: 10.1038/nbt.2503. Epub 2013 Feb 3.
Using nanopores to sequence biopolymers was proposed more than a decade ago. Recent advances in enzyme-based control of DNA translocation and in DNA nucleotide resolution using modified biological pores have satisfied two technical requirements of a functional nanopore DNA sequencing device. Nanopore sequencing of proteins was also envisioned. Although proteins have been shown to move through nanopores, a technique to unfold proteins for processive translocation has yet to be demonstrated. Here we describe controlled unfolding and translocation of proteins through the α-hemolysin (α-HL) pore using the AAA+ unfoldase ClpX. Sequence-dependent features of individual engineered proteins were detected during translocation. These results demonstrate that molecular motors can reproducibly drive proteins through a model nanopore--a feature required for protein sequence analysis using this single-molecule technology.
十多年前就有人提出利用纳米孔对生物聚合物进行测序。最近在基于酶的控制 DNA 易位方面的进展以及使用修饰后的生物孔实现 DNA 核苷酸分辨率方面的进展,满足了功能纳米孔 DNA 测序设备的两个技术要求。人们还设想了对蛋白质进行纳米孔测序。虽然已经证明蛋白质可以穿过纳米孔,但尚未证明一种用于连续易位的蛋白质展开技术。在这里,我们使用 AAA+ 解旋酶 ClpX 描述了通过α-溶血素(α-HL)孔对蛋白质进行的受控展开和易位。在易位过程中检测到了单个工程化蛋白质的序列依赖性特征。这些结果表明,分子马达可以重复地将蛋白质驱动通过模型纳米孔——这是使用这种单分子技术进行蛋白质序列分析所必需的特征。