Decorating Self-Assembled Peptide Cages with Proteins

Abstract

An ability to organize and encapsulate multiple active proteins into defined objects and spaces at the nanoscale has potential applications in biotechnology, nanotechnology, and synthetic biology. Previously, we have described the design, assembly, and characterization of peptide-based self-assembled cages (SAGEs). These ≈100 nm particles comprise thousands of copies of de novo designed peptide-based hubs that array into a hexagonal network and close to give caged structures. Here, we show that, when fused to the designed peptides, various natural proteins can be co-assembled into SAGE particles. We call these constructs pSAGE for protein-SAGE. These particles tolerate the incorporation of multiple copies of folded proteins fused to either the N or the C termini of the hubs, which modeling indicates form the external and internal surfaces of the particles, respectively. Up to 15% of the hubs can be functionalized without compromising the integrity of the pSAGEs. This corresponds to hundreds of copies giving mM local concentrations of protein in the particles. Moreover, and illustrating the modularity of the SAGE system, we show that multiple different proteins can be assembled simultaneously into the same particle. As the peptide-protein fusions are made via recombinant expression of synthetic genes, we envisage that pSAGE systems could be developed modularly to actively encapsulate or to present a wide variety of functional proteins, allowing them to be developed as nanoreactors through the immobilization of enzyme cascades or as vehicles for presenting whole antigenic proteins as synthetic vaccine platforms.

A Modular Vaccine Platform Combining Self‐Assembled Peptide Cages and Immunogenic Peptides

“…In vitro studies of SAGE uptake by HeLa cells showed them to be non‐toxic,[31b] and so SAGE toxicity with other cell types was assessed using human peripheral blood mononuclear cells (PBMCs) isolated from whole blood. PBMCs were exposed to the polyclonal stimulus phytohemagglutinin (PHA) to induce a response indicative of T cell proliferation, in the presence or absence of SAGE.…”

“…The working hypothesis of SAGE architecture is that each CC‐Tri3 unit is oriented within the peptide lattice such that its N terminus faces the exterior of the particle and its C terminus faces the interior, while CC‐Di‐A and CC‐Di‐B have greater freedom of rotation around the disulfide bond . Subsequently, functionality can be incorporated via CC‐Tri3 to allow for bespoke dose delivery as the antigen dose is controlled via peptide synthesis and conjugation rather than passive encapsulation . The fully synthetic SAGE appears to withstand relatively robust functionalization,[31a] can be predictably modified to alter cellular uptake,[31b] and the modularity of the system allows for fine control of modification …”

“…Subsequently, functionality can be incorporated via CC‐Tri3 to allow for bespoke dose delivery as the antigen dose is controlled via peptide synthesis and conjugation rather than passive encapsulation . The fully synthetic SAGE appears to withstand relatively robust functionalization,[31a] can be predictably modified to alter cellular uptake,[31b] and the modularity of the system allows for fine control of modification …”

Self-Assembly of Constrained Cyclic Peptides Controlled by Ring Size

“…10 These materials have shown great potential for many applications, including energy storage and conversion, 11,12 optical devices, 13,14 and nanomedicine. 15,16 The manipulation of cyclic peptide assembly is emerging as a new direction for hierarchical nanostructure fabrication. 17,18 Cyclic peptides usually possess rigid backbones and have high thermodynamic stability, compared to their linear counterparts; therefore, they are excellent building blocks for constructing advanced selfassembling architectures.…”

Ornamentation of Triskelion Peptide Nanotori to Produce Gold Nanoparticle (AuNP)‐Embedded Peptide Nanobangles

“…Thus any changes in symmetry in the presence of external stimuli can also lead to conformational changes and subsequently change the shape of the obtained nanostructures . Self‐assembly, a common technique to develop symmetrical structures, has already been used for the construction of cagelike structures or more complex microstructures …”