Conjugation of proteins to colloidal particles or nanostructures provides enhanced biological activity, stability, and delivery efficacy for applications in biocatalysis, biosensors, and drug delivery. General strategies exploit non-covalent interactions or covalent reactions that require functional groups available on surfaces. In nature, biomineralization forms hybrid structures that specifically interact with proteins, and mimicry of such a method can be used as a versatile tool not only to fabricate novel nanostructures but also to conjugate proteins to the biocompatible inorganics. Here, we demonstrate a facile, general approach to conjugate proteins to nanostructured calcium phosphate via leucine zipper motifs. We first created a triblock polypeptide that consists of two identical basic leucine zipper coiled-coils and a random-coil midblock. The polypeptide was incorporated into nanoflower-shaped hybrids of peptide-calcium phosphate by in situ crystallization, and the nanostructured hybrid particles subsequently assembled into porous supraparticles. In the second step, recombinant model proteins (mCherry and eGFP) fused with an acidic leucine zipper were conjugated to the hybrid particles by simple mixing and incubation. As a result, robust conjugation and high loading of the protein cargos inside the porous particles was achieved. The porous supraparticles are biocompatible and contain hierarchical nanostructures onto which any proteins fused with leucine zippier motifs can be conjugated through highly specific affinity interactions between the coiled-coils. In addition, we were able to create spatial patterns of multiple types of proteins in a single particle by controlling the rate of diffusion and conjugation of proteins inside particles. Besides the fluorescent model proteins, we constructed a therapeutic enzyme fusion protein, which can be conjugated and used for biocatalytic inhibition of a pro-inflammatory cytokine, tumor necrosis factor-a (TNF-a). We discuss effects of conjugation on stability and bioactivity of the enzyme toward efficient modulation of inflammatory responses.

Conjugation of proteins to porous nanostructures using coiled-coil motifs that direct self-assembly of protein-inorganic hybrids

247th National Meeting (2014)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.