Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, MD, 21205, USA.
Small. 2023 Mar;19(13):e2202104. doi: 10.1002/smll.202202104. Epub 2022 May 26.
Engineering synthetic interfaces between membranes has potential applications in designing non-native cellular communication pathways and creating synthetic tissues. Here, InterSpy is introduced as a synthetic biology tool consisting of a heterodimeric protein engineered to form and maintain membrane-membrane interfaces between apposing synthetic as well as cell membranes through the SpyTag/SpyCatcher interaction. The inclusion of split fluorescent protein fragments in InterSpy allows tracking of the formation of a membrane-membrane interface and reconstitution of functional fluorescent protein in the space between apposing membranes. First, InterSpy is demonstrated by testing split protein designs using a mammalian cell-free expression (CFE) system. By utilizing co-translational helix insertion, cell-free synthesized InterSpy fragments are incorporated into the membrane of liposomes and supported lipid bilayers with the desired topology. Functional reconstitution of split fluorescent protein between the membranes is strictly dependent on SpyTag/SpyCatcher. Finally, InterSpy is demonstrated in mammalian cells by detecting fluorescence reconstitution of split protein at the membrane-membrane interface between two cells each expressing a component of InterSpy. InterSpy demonstrates the power of CFE systems in the functional reconstitution of synthetic membrane interfaces via proximity-inducing proteins. This technology may also prove useful where cell-cell contacts and communication are recreated in a controlled manner using minimal components.
工程化的膜间界面在设计非天然的细胞通讯途径和构建人工组织方面具有应用潜力。本文介绍了一种名为 InterSpy 的合成生物学工具，它由一个异二聚体蛋白组成，该蛋白经过工程改造后能够通过 SpyTag/SpyCatcher 相互作用在相邻的合成膜和细胞膜之间形成和维持膜-膜界面。在 InterSpy 中包含分裂荧光蛋白片段，允许跟踪膜-膜界面的形成，并在相邻膜之间的空间中重新构建功能性荧光蛋白。首先，通过使用哺乳动物无细胞表达 (CFE) 系统测试分裂蛋白设计来验证 InterSpy。通过利用共翻译螺旋插入，无细胞合成的 InterSpy 片段被掺入脂质体和支持的双层脂膜中，具有所需的拓扑结构。膜之间分裂荧光蛋白的功能性重建严格依赖于 SpyTag/SpyCatcher。最后，通过检测两个表达 InterSpy 组件之一的细胞之间的膜-膜界面处分裂蛋白的荧光重建，在哺乳动物细胞中证明了 InterSpy 的作用。InterSpy 展示了 CFE 系统在通过诱导邻近的蛋白来功能性重建人工膜界面方面的强大功能。这项技术在以最小的组件以受控的方式再现细胞-细胞接触和通讯的情况下可能也很有用。
