Department of Chemistry and Biochemistry, University of California, San Diego, CA, USA.
Nat Commun. 2024 Oct 30;15(1):9363. doi: 10.1038/s41467-024-53783-4.
The development of artificial cells has led to fundamental insights into the functional processes of living cells while simultaneously paving the way for transformative applications in biotechnology and medicine. A common method of generating artificial cells is to encapsulate protein expression systems within lipid vesicles. However, to communicate with the external environment, protein translocation across lipid membranes must take place. In living cells, protein transport across membranes is achieved with the aid of complex translocase systems which are difficult to reconstitute into artificial cells. Thus, there is need for simple mechanisms by which proteins can be encoded and expressed inside synthetic compartments yet still be externally displayed. Here we present a genetically encodable membrane functionalization system based on mutants of pore-forming proteins. We modify the membrane translocating loop of α-hemolysin to translocate functional peptides up to 52 amino acids across lipid membranes. Full membrane translocation occurs in the absence of any translocase machinery and the translocated peptides are recognized by specific peptide-binding ligands on the opposing membrane side. Engineered hemolysins can be used for genetically programming artificial cells to display interacting peptide pairs, enabling their assembly into artificial tissue-like structures.
人工细胞的发展为深入了解活细胞的功能过程提供了基础，同时也为生物技术和医学领域的变革性应用铺平了道路。生成人工细胞的一种常见方法是将蛋白质表达系统封装在脂质体中。然而，为了与外部环境进行通信，蛋白质必须穿过脂质膜进行转运。在活细胞中，蛋白质跨膜运输是借助复杂的易位子系统来实现的，而将这些系统重新构建到人工细胞中非常困难。因此，需要有一种简单的机制，使蛋白质能够在合成隔室内部进行编码和表达，但仍能在外部进行展示。在这里，我们提出了一种基于成孔蛋白突变体的遗传可编码的膜功能化系统。我们修饰了α-溶血素的膜转运环，使功能肽能够在脂质膜上转运长达 52 个氨基酸。在没有任何易位子机制的情况下，完全的膜转运会发生，并且转运的肽会被对面膜侧的特定肽结合配体识别。工程化的溶血素可用于遗传编程人工细胞以展示相互作用的肽对，从而将它们组装成类似人工组织的结构。
