Multi‐Site Incorporation of Nonstandard Amino Acids into Protein‐Based Biomaterials

Abstract

The ability of natural biomaterials to shape, support, and orchestrate function inspires our efforts to produce functional materials. Guided by protein‐based biomaterials, template‐directed incorporation of synthetic building blocks, such as nonstandard amino acids (nsAAs), can expand the functions of biomaterials by endowing them with new physical and biophysical properties. In this short review, we describe existing technologies for multi‐site nsAA incorporation into proteins. We then discuss examples of the application of this technology for engineering new functions in artificial biopolymers, for creating bio‐inspired adhesives, and for improving the stability of biomaterials. We conclude by briefly discussing recent advances in synthetic biology that have the potential to expand our ability to design protein‐based biomaterials composed of numerous and increasingly exotic nonstandard monomers.

Tuning the Properties of Protein-Based Polymers Using High-Performance Orthogonal Translation Systems for the Incorporation of Aromatic Non-Canonical Amino Acids

“…Incorporating non-canonical amino acids (ncAAs) into proteins has emerged as a powerful methodology to improve, alter, or introduce new functions into proteins. The incorporation of ncAAs bearing a variety of chemical groups can facilitate the elucidation of protein structure–function relationship ( Debelouchina and Muir, 2017 ; Chen et al, 2018 ) or protein–protein interactions ( Nguyen et al, 2018 ), or the production of protein-based therapeutics ( Huang and Liu, 2018 ) and biomaterials with novel functions ( Connor and Tirrell, 2007 ; Israeli et al, 2019 ), among many other applications ( Johnson et al, 2010 ; Chin, 2017 ; Voller and Budisa, 2017 ; Young and Schultz, 2018 ; Lee et al, 2019 ; Zhou and Deiters, 2021 ). There are two common strategies for expressing recombinant proteins containing ncAAs.…”

“…Multi-site ncAA incorporation is particularly desirable for the design and production of protein-based polymers (PBPs), which consist of tandem repeats of either natural or artificial short peptide motifs, and whose properties are determined by the sequence of the AAs and ncAAs in these motifs ( Connor and Tirrell, 2007 ; Israeli et al, 2019 ; Varanko et al, 2020 ; Chang et al, 2021 ). Although the incorporation of ncAAs in numerous PBPs can be used to expand their range of properties and functions ( Connor and Tirrell, 2007 ; Israeli et al, 2019 ), we focus here on the effect of ncAA incorporation on the properties of two families of bio-inspired, thermo-responsive PBPs: elastin-like polypeptides (ELPs) and resilin-like polypeptides (RLPs). ELPs are arguably the most well-studied family of artificial PBPs that comprise multiple repeats (typically 5–200 repeats) of the VPGXG motif (variations of the tropoelastin-derived VPGVP motif), where X is permissive to any amino acid (AA) except proline ( MacEwan and Chilkoti, 2010 ).…”

Genetic Code Expansion in the Engineered Organism Vmax X2: High Yield and Exceptional Fidelity

“…For others, such as the design of sequence-defined protein materials, multiple copies of one (or more) ncAA may be desired. 82 These applications push the limits of genetic code expansion, as the isolated yields of such materials from standard E. coli strains can be low due to the increased frequency of RF1-mediated termination events. 45 Strains that lack RF1, such as B95 and C321 derivatives, have been reported to support greatly improved yields of model proteins containing multiple copies of a single ncAA such as pAzF.…”

Genetic code expansion in the engineered organism Vmax X2: High yield and exceptional fidelity

“…For others, such as the design of sequence-defined protein materials, multiple copies of one (or more) ncAA may be desired. 69 These applications push the limits of genetic code expansion, as the isolated yields of such materials from standard E. coli strains can be low due to the increased frequency of RF1-mediated termination events. 45 Strains that lack RF1, such as B95 and C321 derivatives, have been reported to support greatly improved yields of model proteins containing multiple copies of a single ncAA such as pAzF.…”