Australian Institute of Bioengineering and Nanotechnology , The University of Queensland , St Lucia , QLD 4072 , Australia.
ACS Nano. 2018 May 22;12(5):4615-4623. doi: 10.1021/acsnano.8b01059. Epub 2018 May 3.
Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages are being developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both in vitro and in vivo cell engineering. However, there is a lack of bionanotechnology platforms that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for in vivo self-sorting of cargo-linked capsomeres of murine polyomavirus (MPyV) that enables controlled encapsidation of guest proteins by in vitro self-assembly. Using Förster resonance energy transfer, we demonstrate the flexibility in this system to support coencapsidation of multiple proteins. Complementing these ensemble measurements with single-particle analysis by super-resolution microscopy shows that the stochastic nature of coencapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable coencapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.
利用工程化的天然蛋白笼进行仿生自组装和自分类系统的开发，用于生物催化和治疗性药物传递。对于体外和体内细胞工程，客体蛋白的包装和细胞内传递都特别重要。然而，目前缺乏既能实现可编程客体蛋白包封，又能有效实现细胞内摄取的生物纳米技术平台。我们报告了一种用于体内自分类的最小肽锚定物，该锚定物用于负载连接的鼠多瘤病毒（MPyV）帽状蛋白的货物，从而能够通过体外自组装实现对客体蛋白的有效封装。通过荧光共振能量转移，我们证明了该系统具有封装多种蛋白质的灵活性。通过超分辨率显微镜的单颗粒分析来补充这些整体测量，表明共封装的随机性是一个主要原则。这对天然和工程化的自分类封装系统的设计和部署以及传染性病毒粒子的组装都有影响。利用普遍存在于哺乳动物细胞表面的唾液酸的编码亲和力，我们证明了自组装的 MPyV 病毒样颗粒能够有效地将客体蛋白递送至原代人细胞的细胞质中。因此，这种可编程共封装和互补生物分子有效胞质内递送的平台在细胞工程中具有巨大的潜力。
