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.
J Am Chem Soc. 2019 Mar 6;141(9):3875-3884. doi: 10.1021/jacs.8b10734. Epub 2019 Feb 20.
Site-specific protein modification is a widely used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically hindered N termini, such as virus-like particles (VLPs) composed of the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.
定点蛋白质修饰是将药物、成像剂或其他有用的小分子连接到蛋白质载体上的常用策略。N 端修饰是一种特别有用的高产量、定点修饰策略，可与多种蛋白质兼容。然而，这种修饰策略与埋藏或空间位阻 N 端的蛋白质不兼容，例如由研究充分的 MS2 噬菌体衣壳蛋白组成的病毒样颗粒（VLPs）。为了评估与这些技术更兼容的 VLPs，我们基于 N 端脯氨酸残基和三个可变位置的 MS2 衍生蛋白笼生成了一个靶向文库。我们对文库进行了组装、热和化学选择，鉴定出了在不降低热稳定性的情况下高产修饰的变体。紧邻天然 N 端的正电荷对成功延伸出人意料地有益，在表现最好的变体中，超过 50%的变体在该位置含有正电荷。总之，这些研究描述了控制 N 端延伸的非直观设计规则，并确定了具有高修饰潜力的成功延伸。
