(713e) Recombinant Elastin-like Polypeptide Production By Incorporating Non-Standard Amino Acids in Escherichia coli Cell-Free Protein Synthesis

Elastin-like polypeptides (ELPs) are highly biocompatible and stimuli-responsive biopolymers which can be applied for drug delivery and tissue engineering. Typically, ELPs consist of repeats (n) of the pentapeptide sequence (VPGVG)n. This pentapeptide is known to be a key component in elastin and exhibits interesting self-assembly behavior (random coil to helix) above transition temperature. The structure and function of elastin is maintained as long as the glycine and proline residues are present; however, the second valine residue can be replaced with any amino acid. This flexibility in the character of the fourth residue presents an opportunity for modulation of the temperature-responsive properties through introduction of non-standard amino acids (NSAAs). Here, we sought to produce ELPs containing NSAAs using cell-free protein synthesis (CFPS) from a genomically modified Escherichia coli strain, developed for improved NSAA incorporation. We used CFPS, because the open nature of the CFPS platform provides a flexible control of NSAA incorporation with high synthesis yield. We constructed plasmid templates of different repeat sizes of ELPs ranging 32 to 256 repeats via recursive directional ligation method. For example, we produced ELPs containing eight p-acetyl-L-phenylalanine at yields of 280 ± 50 mg/L with more than 95% incorporation efficiency based on quantitative mass spectrometry analysis, and production yield of the modified ELPs was similar to the native ELP production. We will discuss our efforts to both produce and use these materials. We anticipate the NSAA incorporation into peptide-based biomaterials will enable the development of innovative materials with new functions, and that CFPS is a promising platform for the production of novel functional sequence defined polymers.