Genetic fusion of RLP-functionalized coiled-coil constructs that exhibit thermoresponsive behavior

• Arriana Bisram, , University of Delaware

• Kristi Kiick, Department of Biomedical Engineering, University of Delaware; Department of Material Science and Engineering, University of Delaware, University of Delaware

Biomimetic, peptide-based materials have received attention for their usage in biomedical applications due to their biocompatibility, tunability, and cost efficiency compared to synthetic materials. One such peptide-based material which is of interest is resilin-like polypeptides. Resilin is an elastomeric protein derived from insects that demonstrates stimuli responsiveness and elasticity. It has been a point of inspiration for the creation of resilin-like polypeptides (RLPs) based on the CG15920 gene segment of the fruit fly Drosophila melanogaster. RLPs can be conjugated to coiled-coiled bundle forming peptide, which results in the formation of programmable constructs that are responsive to stimuli such as pH, temperature, and chemical environment. This work aims to express, purify, and characterize the propensity for phase separation of RLP-functionalized coiled-coil constructs with various central amino acid substitutions in the resilin units. In addition, we aim to increase the throughput of expressed constructs. To yield the desired protein sequence, recombinant synthesis was utilized and followed by an altered denaturing purification in which the protein-bound resin was separated from the solution before being added to a metal affinity chromatography column. The protein was then analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to characterize the molecular weight and purity of produced constructs. Afterward, Ultraviolet-visible spectroscopy (UV-Vis) was used to investigate the constructs’ phase behavior at various concentrations in response to temperature. The optical density at 350 nanometers was monitored as a function of temperature, allowing us to find the point of phase transition. Results of this investigation exhibit that the altered method of purification increased the throughput of production of these constructs. In addition, the amino acid substitutions exhibit an impact on the UPLC transition temperatures of the constructs at various concentrations.