Presenting Author Northern State University Aberdeen, South Dakota
Chitin and carbon nanotubes can both confer extraordinary physical properties upon polymers that contain them. However, these materials are also highly insoluble in most common organic solvents, making them difficult to utilize without first modifying or functionalizing them, which can alter their properties. Natural organisms produce peptides that utilize or demonstrate affinity for these materials, which can be exploited by designing a protein capable of binding to both chitin and carbon nanotubes and of cross-linking to form a biopolymer.
The conserved 65 amino acid motif called the extended Rebers and Riddiford (Ramp;R) Consensus found in arthropod cuticle proteins has been experimentally confirmed to confer chitin-binding ability to recombinant proteins that include it. Similarly, a phage-derived 12 amino acid sequence (P1) has been shown to allow including recombinant proteins to coat and solubilize unfunctionalized carbon nanotubes. This work combines these segments in a single peptide along with flexible linker domains and cross-linking domains derived from insect cuticle proteins to create a novel double-binding cuticle protein (DBCP). Cross-linking can be achieved through [Ru(bpy)3]2+-mediated photo irradiation, a method showing increased promise for the creation of dityrosine cross-links between organic molecules. This will allow ligand-bound proteins to form a stable matrix, capturing chitin and carbon nanotubes to form a resilient biopolymer.
DBCP gene was designed and synthesized for use in heterologous expression in BL21 E. coli. Western blotting will confirm the expression and purification of the target protein. Ligand binding ability will be confirmed by solubilization assays introducing ligands to concentrated protein in common solvents. FTIR will further confirm ligand binding ability and the nature of the bonds or attractions involved. Cross-linking will be induced using [Ru(bpy)3]2+-mediated photo irradiation.
It is anticipated that use of the Ramp;R consensus and P1 phage-derived peptide in conjunction with resilin cross-linking domains will create a recombinant protein capable of binding chitin and carbon nanotubes in addition to forming dityrosine cross-links through [Ru(bpy)3]2+-mediated photo irradiation, resulting in a more rigid biopolymer with than commonly produced protein hydrogels, expanding the range of possible use-cases for laboratory-derived biopolymers.
