Department of Chemistry, University of California, Berkeley, California 94720-1460, United States.
Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Technological Institute E136, Evanston, Illinois 60208-3120, United States.
Biomacromolecules. 2020 Oct 12;21(10):4194-4204. doi: 10.1021/acs.biomac.0c00987. Epub 2020 Sep 3.
Peptide insertions in the primary sequence of proteins expand functionality by introducing new binding sequences, chemical handles, or membrane disrupting motifs. With these properties, proteins can be engineered as scaffolds for vaccines or targeted drug delivery vehicles. Virus-like particles (VLPs) are promising platforms for these applications since they are genetically simple, mimic viral structure for cell uptake, and can deliver multiple copies of a therapeutic agent to a given cell. Peptide insertions in the coat protein of VLPs can increase VLP uptake in cells by increasing cell binding, but it is difficult to predict how an insertion affects monomer folding and higher order assembly. To this end, we have engineered the MS2 VLP using a high-throughput technique, called Systematic Mutagenesis and Assembled Particle Selection (SyMAPS). In this work, we applied SyMAPS to investigate a highly mutable loop in the MS2 coat protein to display 9,261 non-native tripeptide insertions. This library generates a discrete map of three amino acid insertions permitted at this location, validates the FG loop as a valuable position for peptide insertion, and illuminates how properties such as charge, flexibility, and hydrogen bonding can interact to preserve or disrupt capsid assembly. Taken together, the results highlight the potential to engineer VLPs in a systematic manner, paving the way to exploring the applications of peptide insertions in biomedically relevant settings.
肽插入物在蛋白质的一级序列中通过引入新的结合序列、化学处理或膜破坏基序来扩展功能。利用这些特性，蛋白质可以作为疫苗或靶向药物递送载体的支架进行工程设计。病毒样颗粒 (VLPs) 是这些应用的有前途的平台，因为它们具有遗传简单性、模拟病毒结构以被细胞摄取的特点，并且可以将多个治疗剂拷贝递送到给定的细胞。VLPs 外壳蛋白中的肽插入物可以通过增加细胞结合来增加 VLP 在细胞中的摄取，但很难预测插入如何影响单体折叠和更高阶组装。为此，我们使用一种称为系统诱变和组装颗粒选择 (SyMAPS) 的高通量技术对 MS2 VLP 进行了工程改造。在这项工作中，我们应用 SyMAPS 来研究 MS2 外壳蛋白中的一个高度可突变环，以展示 9,261 个非天然三肽插入物。该文库生成了此位置允许的三个氨基酸插入的离散图谱，验证了 FG 环是肽插入的有价值位置，并阐明了电荷、灵活性和氢键等特性如何相互作用以维持或破坏衣壳组装。总之，这些结果突出了以系统方式工程改造 VLPs 的潜力，为探索肽插入在生物医学相关环境中的应用铺平了道路。
