Engineering Elastin-like Polypeptides to Constitute Sustainable Protein-Based Fibers with Enhanced Mechanics

Engineered protein biopolymers have been of great research interest in recent years. Because of their sequence specificity that enables different structural properties, researchers have successfully applied protein biomaterials in various fields such as tissue engineering and drug delivery. Beyond these biological applications, protein biopolymers have the potential to function as sustainable alternatives to current widely used environmentally hazardous materials. These biopolymers have inherent advantages of being biodegradable and nontoxic novel materials.

Protein-based fibers are one representative example of biopolymers that can enormously mitigate the harmful environmental effects caused by its synthetic counterparts such as nylon and polyester. This research focuses on exploring designed protein fibers constituted by elastin-like polypeptides (ELP) [VPGXG] for use as sustainable materials. ELPs are known for having a lower critical solution temperature (LCST), which enables the production of protein fibers through the creation of viscous solutions of biopolymers at a relatively low temperature. However, materials created from ELPs alone rarely have sufficient mechanical strength to replace current synthetic polymer materials. This research seeks to evaluate assembly and crosslinking methods to improve the strength and ductility of protein fibers. In addition to molecular engineering of the protein sequence to improve mechanics, fiber formation techniques such as electro-spinning will be tested.

Protein sequences that incorporate cationic, anionic, or neutral regions and coiled-coil domains were first designed for rapid self-assembly. The X protein in ELP residues were varied as lysine, glutamate, or glutamine to obtain different charges for self-assembly and reactive sites for enzymatic crosslinking. Secondly, the designed genes were cloned by PCR, expressed in E. coli cells, and purified through Ni-NTA affinity chromatography or inverse transition cycling which takes advantages of ELPs’ LCST behavior. Thirdly, coacervates were formed by mixing cationic and anionic ELP proteins (such as ELP-D/E30 mixed with ELP-K30). Turbidity assay and salt titrations were done in order to study the peak of coacervation and its tolerance of salt. The next step of this research will focus on utilizing enzymes or photo-crosslinking, or electro-spinning techniques to obtain protein fibers from the dense coacervates phase . Green Fluorescent Protein (GFP) will be incorporated into the coacervate phase through partitioning so that bio-functionality of the electro-spun fibers can be measured. Lastly, material properties of the designed protein fibers, such as stress-strain behaviors and thermodynamic properties, will be tested.