State Key Laboratory of Fine Chemicals, Frontiers Science Centre for Smart Materials Oriented Chemical Engineering, School of Bioengineering, Dalian University of Technology, Dalian 116024, China.
Ningbo Institute of Dalian University of Technology, Ningbo 315016, China.
Nano Lett. 2024 Jul 31;24(30):9237-9244. doi: 10.1021/acs.nanolett.4c01693. Epub 2024 Jul 17.
Self-assembled protein cages are attractive scaffolds for organizing various proteins of interest (POIs) toward applications in synthetic biology and medical science. However, specifically attaching multiple POIs to a single protein cage remains challenging, resulting in diversity among the functionalized particles. Here, we present the engineering of a self-assembled protein cage, Mi3, capable of independently recruiting two different POIs using SpyCatcher (SC)/SpyTag (ST) and DogCatcher (DC)/DogTag (DT) chemistries, thereby reducing variability between assemblies. Using fluorescent proteins as models, we demonstrate controlled targeting of two different POIs onto Mi3 protein cages both in vitro and inside living cells. Furthermore, dual functionalization of the Mi3 protein cage with a membrane-targeting peptide and β-galactosidase resulted in the construction of membrane-bound enzyme assemblies in , leading to a 69.6% enhancement in substrate utilization across the membrane. This versatile protein cage platform provides dual functional nanotools for biological and biomedical applications.
自组装蛋白笼是一种很有吸引力的支架，可以将各种感兴趣的蛋白质（POI）组织起来，应用于合成生物学和医学领域。然而，将多个 POI 特异性地连接到单个蛋白笼上仍然具有挑战性，导致功能化颗粒之间存在多样性。在这里，我们构建了一种自组装蛋白笼 Mi3，该蛋白笼可以使用 SpyCatcher（SC）/SpyTag（ST）和 DogCatcher（DC）/DogTag（DT）化学独立招募两种不同的 POI，从而减少组装之间的变异性。使用荧光蛋白作为模型，我们证明了两种不同的 POI 可以在体外和活细胞内被靶向到 Mi3 蛋白笼上。此外，通过将膜靶向肽和β-半乳糖苷酶双重功能化 Mi3 蛋白笼，构建了在 中具有膜结合酶组装的系统，导致跨膜底物利用率提高了 69.6%。这个多功能的蛋白笼平台为生物和生物医学应用提供了双重功能的纳米工具。
