Utilizing clathrin triskelions as carriers for spatially controlled multi-protein display

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.