Soft Hydrogel Inspired by Elastomeric Proteins.

Elastin polypeptides based on -VPGVG- repeated motifs are widely used in the production of biomaterials because they are stimuli-responsive systems. On the other hand, glycine-rich sequences, mainly present in tropoelastin terminal domains, are responsible for the elastin self-assembly. In a previous study, we have recombinantly expressed a chimeric polypeptide, named resilin, elastin, and collagen (REC), inspired by glycine-rich motifs of elastin and containing resilin and collagen sequences as well. Herein, a three-block polypeptide, named (REC)3, was expressed starting from the previous monomer gene by introducing key modifications in the sequence. The choice was mandatory because the uneven distribution of the cross-linking sites in the monomer precluded the hydrogel production. In this work, the cross-linked polypeptide appeared as a soft hydrogel, as assessed by rheology, and the linear un-cross-linked trimer self-aggregated more rapidly than the REC monomer. The absence of cell-adhesive sequences did not affect cell viability, while it was functional to the production of a material presenting antiadhesive properties useful in the integration of synthetic devices in the body and preventing the invasion of cells.

Fibrillar Self-Assembly of a Chimeric Elastin-Resilin Inspired Engineered Polypeptide.

In the field of tissue engineering, recombinant protein-based biomaterials made up of block polypeptides with tunable properties arising from the functionalities of the individual domains are appealing candidates for the construction of medical devices. In this work, we focused our attention on the preparation and structural characterization of nanofibers from a chimeric-polypeptide-containing resilin and elastin domain, designed on purpose to enhance its cell-binding ability by introducing a specific fibronectin-derived Arg-Gly-Asp (RGD) sequence. The polypeptide ability to self-assemble was investigated. The molecular and supramolecular structure was characterized by Scanning Electronic Microscopy (SEM) and Atomic Force Microscopy (AFM), circular dichroism, state-of-the-art synchrotron radiation-induced techniques X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The attained complementary results allow us to assess as H-bonds influence the morphology of the aggregates obtained after the self-assembling of the chimeric polypeptide. Finally, a preliminary investigation of the potential cytotoxicity of the polypeptide was performed by culturing human fetal foreskin fibroblast (HFFF2) for its use as biomedical device.

Characterization of a Crosslinked Elastomeric-Protein Inspired Polypeptide.

Materials inspired by natural proteins have a great appeal in tissue engineering for their biocompatibility and similarity to extracellular matrix (ECM). Chimeric polypeptides inspired by elastomeric proteins such as silk, elastin, and collagen are of outstanding interest in the field. A recombinant polypeptide constituted of three different blocks, each of them having sequences derived from elastin, resilin, and collagen proteins, was demonstrated to be a good candidate as biomaterial for its self-assembling characteristics and biocompatibility. Herein, taking advantage of the primary amine functionalities present in the linear polypeptide, we crosslinked it with 1,6-hexamethylene-diisocyanate (HMDI). The characterization of the obtained polypeptide was realized by CD spectroscopy, AFM, and SEM microscopies. The obtained results, although not conclusive, demonstrate that the crosslinked polypeptide gave rise to porous networks, thin nanowires, and films not observable for the linear polypeptide. Chirality 28:606-611, 2016. © 2016 Wiley Periodicals, Inc.

Aldehyde modification and alum coadjuvancy enhance anti-TNF-α autovaccination and mitigate arthritis in rat.

Experimental vaccination to induce antibodies (Abs) capable of cytokine antagonism shows promise as a novel immunotherapy for chronic inflammatory disease. We prepared a hybrid antigen consisting of residues 141-235 of rat TNF-α fused to the C-terminus of glutathione-S-transferase (GST), chemically modified to incorporate aldehyde residues, for development of an auto-vaccine eliciting anti-rTNF-α Abs. In rat immunization the soluble aldehyde-modified fusion protein did not generate observable Ab responses. By contrast, vaccination with the aldehyde-modified fusion protein adsorbed on alum induced anti-TNF-α autoAbs with high titer and neutralizing activity. Induction of adjuvant arthritis in rats pre-immunized with unmodified fusion protein or a control protein in alum resulted in severe inflammation and joint damage, whereas the disease induced in rats immunized with the aldehyde-bearing fusion protein in alum was markedly attenuated. Similar results were obtained in a collagen-induced rat arthritis model. Anti-collagen II IgG Ab titers did not deviate significantly in groups pre-immunized with modified fusion protein and control protein, suggesting that anti-TNF vaccination did not skew the immune response related to disease induction. This study demonstrates synergy between particulate alum and protein bound carbonyl residues for enhancement of protein immunogenicity. The antigen-specific co-adjuvant system could prove advantageous for breaking tolerance in emerging auto-vaccination therapies targeting inflammatory cytokines as well as for enhancing a broader category of subunit vaccines. Aldehyde adduction introduces a minimal modification which, together with the established use of alum as a safe adjuvant for human use, could be favorable for further vaccine development.

Design and production of a chimeric resilin-, elastin-, and collagen-like engineered polypeptide.

Protein-inspired biomaterials have gained great interest as an alternative to synthetic polymers, in particular, for their potential use as biomedical devices. The potential inspiring models are mainly proteins able to confer mechanical properties to tissues and organs, such as elasticity (elastin, resilin, spider silk) and strength (collagen, silk). The proper combination of repetitive sequences, each of them derived from different proteins, represents a useful tool for obtaining biomaterials with tailored mechanical properties and biological functions. In this report we describe the design, the production, and the preliminary characterization of a chimeric polypeptide, based on sequences derived from the highly resilient proteins resilin and elastin and from collagen-like sequences. The results show that the obtained chimeric recombinant material exhibits promising self-assembling properties. Young's modulus of the fibers was determined by AFM image analysis and lies in the range of 0.1-3 MPa in agreement with the expectations for elastin-like and resilin-like materials.

Molecular and supramolecular structural studies on significant repetitive sequences of resilin.

Resilin is a member of the family of elastomeric proteins and is found in specialised regions of the cuticle of most insects, and provides low stiffness, high strain and efficient energy storage. It is best known for its role in insect flight and the remarkable jumping ability of fleas and spittle bugs. In common with other elastomeric proteins, the recently identified Drosophila melanogaster proresilin shows glycine-rich repetitive sequences; in particular the N- and C-terminal regions of the protein are dominated by 18 repeats of a 15-residue sequence (SDTYGAPGGGNGGRP) and eleven repeats of a 13-residue sequence (GYSGGRPGGQDLG), respectively. We synthesised and analysed the molecular and supramolecular structure of some polypeptides with sequences belonging to the glycine-rich repeated domain of D. melanogaster resilin. The conformational studies performed by CD, FTIR and NMR spectroscopies pointed to the coexistence of two main conformational features, such as folded beta-turns and (quasi)extended structures (e.g., poly-L-proline II conformation) in common with other elastomeric proteins; this suggests an elasticity mechanism for resilin common to other elastomeric proteins. Our data show that also in the case of resilin, repetitive sequences are characterised by autonomous structures almost independent of the remaining parts of the molecule as already extensively found for elastin. From a supramolecular point of view, a great tendency to aggregate in fibrous structures is observed, particularly for the resilin- inspired polypeptide (PGGGN)(10). This is encouraging for the development of resilin-based biomaterials for the production of biocompatible medical devices, as well as high performing elastic materials.