Protein-Based Nanostructures: Column-free Biosynthesis of Bundlemer Peptides with Programmable, Orthogonally Reactive Handles for Nanomaterial Construction

Nature's protein sequences dictate structure and function across scales, from the nanometer-scale of viral capsids to micrometer- to millimeter-scale architectures of extracellular matrices and tissues. This multiscale precision underlies diverse functions, including molecular recognition, mechanical support, and dynamic responsiveness. Mimicking this complexity synthetically remains challenging. To address this, we engineered biosynthetic coiled-coil 'bundlemer' peptides as modular building blocks that can be conjugated and assembled into complex, higher-ordered materials. Using recombinant expression, we produced peptides and peptide fusion constructs that are otherwise difficult to achieve via solid-phase peptide synthesis. An N-terminal fusion protein with a pH-cleavable intein-inclusion body facilitated simple, scarless purification directly from insoluble fractions. We further incorporated SpyTag (-VPTIVMVDAYKRYK-) and sortase-recognition motifs (GGG-, -LPETGG) into the bundlemers, enabling precise, programmable assembly using genetically encoded and enzymatic ligation strategies. Using sortase-mediated ligation, we polymerized a single bundlemer into fibrillar structures, which were confirmed via TEM, SEC-MALS, SDS-PAGE, and native gels. Additionally, we constructed multicomponent architectures with a layered bundlemer and EGFP on an E2 nanocage using orthogonal linking chemistries, specifically SpyCatcher-SpyTag ligation and sortase-mediated ligation, and the associated increase in particle size and molecular weight was confirmed via TEM, DLS, and SDS-PAGE. This platform establishes a versatile framework for designing complex, protein-based nanostructures with defined architecture and function, offering possibilities in biomaterials engineering, targeted drug delivery, and synthetic biology.