Deci Michael B, Ferguson Scott W, Liu Maixian, Peterson Damian C, Koduvayur Sujatha P, Nguyen Juliane
Department of Pharmaceutical Sciences, School of Pharmacy, University at Buffalo, The State University of New York, Buffalo, NY, 14214, USA.
Department of Electrical and Computer Engineering, Stevens Institute of Technology, Hoboken, NJ, 07030, USA.
Biomaterials. 2016 Nov;108:120-8. doi: 10.1016/j.biomaterials.2016.08.044. Epub 2016 Aug 28.
The simultaneous and spatially controlled display of different proteins on nanocarriers is a desirable property not often achieved in practice. Here, we report the use of clathrin triskelions as a versatile platform for functional protein display. We hypothesized that site-specific molecular epitope recognition would allow for effective and ordered protein attachment to clathrin triskelions. Clathrin binding peptides (CBPs) were genetically fused to mCherry and green fluorescent protein (GFP), expressed, and loaded onto clathrin triskelions by site-specific binding. Attachment was confirmed by surface plasmon resonance. mCherry fusion proteins modified with various CBPs displayed binding affinities between 470 nM and 287 μM for the clathrin triskelions. Simultaneous attachment of GFP-Wbox and mCherry-Cbox fusion constructs to the clathrin terminal domain was verified by Förster resonance energy transfer. The circulating half-lives, area under the curve, and the terminal half-lives of GFP and mCherry were significantly increased when attached to clathrin triskelions. Clathrin triskelion technology is useful for the development of versatile and multifunctional carriers for spatially controlled protein or peptide display with tremendous potential in nanotechnology, drug delivery, vaccine development, and targeted therapeutic applications.
在纳米载体上同时且在空间上可控地展示不同蛋白质是一种理想的特性，但在实践中并不常能实现。在此，我们报道了使用网格蛋白三脚蛋白复合体作为功能性蛋白质展示的通用平台。我们推测位点特异性分子表位识别将允许有效且有序地将蛋白质附着到网格蛋白三脚蛋白复合体上。将网格蛋白结合肽（CBP）与mCherry和绿色荧光蛋白（GFP）进行基因融合、表达，并通过位点特异性结合加载到网格蛋白三脚蛋白复合体上。通过表面等离子体共振确认了附着情况。用各种CBP修饰的mCherry融合蛋白对网格蛋白三脚蛋白复合体的结合亲和力在470 nM至287 μM之间。通过Förster共振能量转移验证了GFP-Wbox和mCherry-Cbox融合构建体同时附着到网格蛋白末端结构域。当附着到网格蛋白三脚蛋白复合体上时，GFP和mCherry的循环半衰期、曲线下面积和末端半衰期显著增加。网格蛋白三脚蛋白复合体技术对于开发用于空间可控蛋白质或肽展示的通用多功能载体很有用，在纳米技术、药物递送、疫苗开发和靶向治疗应用中具有巨大潜力。
