Dual Site-Selective Presentation of Functional Handles on Protein-Engineered Cowpea Chlorotic Mottle Virus-Like
Daan F M Vervoort1, Robin Heiringhoff1, Suzanne B P E Timmermans1
1Eindhoven University of Technology, Institute for Complex Molecular Systems, PO Box 513 (STO 3.41), 5600 MB Eindhoven, The Netherlands.
Abstract:
Protein cages hold much promise as carrier systems in nanomedicine, due to their well-defined size, cargo-loading capacity, and inherent biodegradability. In order to make them suitable for drug delivery, they have to be stable under physiological conditions. In addition, often surface modifications are required, for example, to improve cell targeting or reduce the particle immunogenicity by PEGylation. For this purpose, we investigated the functionalization capacity of the capsid of cowpea chlorotic mottle virus (CCMV), modified at the interior with a stabilizing elastin-like polypeptide (ELP) tag, by employing a combination of protein engineering and bio-orthogonal chemistry. We first demonstrated the accessibility of the native cysteine residue in ELP-CCMV as a site-selective surface-exposed functional handle, which was not available in the native CCMV capsid. An additional bio-orthogonal functional handle was introduced by incorporation of the noncanonical amino acid, azido-phenylalanine (AzF), using the amber suppression mechanism. Dual site-selective presentation of both a cell-penetrating TAT peptide and a fluorophore to track the particles was demonstrated successfully in HeLa cell uptake studies.
