(c) Initial velocities (v0) derived from (b) were used to calculate the % of active cutinase remaining after conjugation (5 M cut-GFP, 0C5 M pNP-Q11). fusion SC-514 proteins via a covalent active site-directed capture approach to afford protein-laden nanofibers. These nanofibers could be formulated to present precisely controlled amounts of protein antigen and acted as self-adjuvanting vaccines in mice. Cutinase-pNP reactions were site-selective, allowing antigens to be conjugated without disrupting their tertiary structures, making the approach broadly useful for developing protein-bearing supramolecular materials in a range of applications including immunotherapies. Adjuvants and delivery platforms that present properly folded protein antigens are important in the development of vaccines because they allow for broad immunogenicity in outbred populations compared with peptide vaccines, and because they can include conformational epitopes.[10] Supramolecular assemblies are gaining interest in this regard, because they can be functionalized with a high density of antigens, in some cases without perturbing antigen conformation SC-514 or self-assembly of the material. For example, supramolecular nanoparticle vaccines have been designed to contain both folded protein antigens and peptide antigens that mimic native epitope conformations.[7, 8, 11C13] -sheet-rich nanofibers of peptides and peptide amphiphiles can also act as self-adjuvanting vaccines,[1, 4, 6] and they have an additional advantage of being highly modular, allowing SC-514 the incorporation of multiple different molecular components with negligible compositional drift.[14,15] However, although a few instances of protein-bearing -sheet-rich nanofibers have been reported previously,[16C18] vaccine platforms developed from these materials have employed only peptide antigens to date, which lack any intentionally designed conformation. We developed a general approach to produce supramolecular assemblies containing properly folded proteins using green fluorescent protein (GFP) as a model antigen, and we characterized the materials ability to raise immune responses in mice. Proteins were attached to peptide nanofibers using the chemoselective reaction of cutinase fusion proteins with nanofiber-bound suicide pNP ligands (Figure 1aCb), an approach that has been used previously to conjugate proteins to solid surfaces,[19, 20] but not to construct soft materials. First, we synthesized pNP-Q11, a variant of the -sheet fibrillizing peptide QQKFQFQFEQQ (Q11)[15, 21, 22] having a pNP ligand on its N-terminus, by reacting cysteine-terminated Q11 with maleimido-penta(ethylene glycol)-ethyl-p-nitrophenyl phosphonate, which we also synthesized (Figure 1a, detailed methods in Supplemental Information). In parallel we designed and expressed in a fusion protein containing cutinase and green fluorescent protein domains separated by a flexible linker of glycine and serine residues (cut-GFP). In phosphate-buffered saline, pNP-Q11 self-assembled into individual nanofibers and bundles of nanofibers whose morphologies were similar to previously investigated Q11 materials (Figure 1c).[21, 22] The peptides maintained this fibrillar morphology following reaction with cutinase fusion proteins (Figure 1d), which indicated that the presence of a relatively large appended protein did not perturb Q11 fibrillization. Open in a separate window Figure 1 Protein-bearing self-assembled peptide nanofibersa) pNP-Q11. b) Schematic of the non-covalent assembly of Q11 and pNP-Q11 into nanofibers, and the subsequent covalent capture of cutinase-GFP by pNP-bearing Q11 nanofibers. c-d) TEM of pNP-Q11 nanofibers before (c) and after (d) conjugation with cut-GFP. One SC-514 of the advantages of supramolecular systems is that the relative amounts of different functional components in the final material can often Rabbit polyclonal to ACD be controlled simply by mixing specific combinations of precursor molecules and inducing self-assembly.[23C25] The phosphonate-cutinase system also lent itself to this modularity, as the amount of antigen coupled to the peptide nanofibers could be controlled by specifying the amount of pNP-Q11 co-assembled with non-functionalized Q11 (Figure 2). Protein conjugation was assessed both directly by measuring GFP fluorescence on sedimented nanofibers, and indirectly using a colorimetric assay for residual unreacted cutinase following conjugation. [26] GFP fluorescence additionally served as an indication of proper protein folding. Self-assembled Q11 peptide nanofibers bearing increasing amounts of co-assembled pNP-Q11 bound predictably increasing amounts of cut-GFP, whether measured from the fluorescence of bound GFP (Number 2a) or by residual cutinase activity (Number 2b, c). Q11 fibrils lacking pNP bound negligible amounts of cut-GFP non-specifically, whereas pNP-bearing fibrils incubated having a molar equivalent of cut-GFP bound the protein with about 80% effectiveness (Number 2a). A 3-collapse molar excess of cut-GFP led to nearly complete reaction of the pNP ligand (not shown). In this way, the amount of protein displayed within the fibrils could be controlled with precision in a simple, straightforward manner, by dosing pNP-Q11 into Q11 nanofibers and reacting them with a slight molar excess of cut-GFP. Importantly, the pNP-cutinase conjugation proceeded to the same degree whether cut-GFP was added to freshly dissolved pNP-Q11 or to peptide that had been allowed to assemble into more mature peptide.