Genetic Code Expansion: Inception, Development, Commercialization

Abstract

Virtually all natural proteins are built from only 20 amino acids, and while this makes possible all the functions they perform, the ability to encode other amino acids selected for specific purposes promises to enable the discovery and production of proteins with novel functions, including therapeutic proteins with more optimal drug-like properties. The field of genetic code expansion (GCE) has for years enabled the production of such proteins for academic purposes and is now transitioning to commercialization for the production of more optimal protein therapeutics. Focusing on E. coli, we review the history and current state of the field. We also provide a review of the first generation commercialization efforts, the lessons learned, and how those lessons are guiding new efforts. With continued academic and industrial progress, GCE methodologies promise to make possible the routine optimization of proteins for therapeutic use in a way that has only previously been possible with small-molecule therapeutics.

Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells

“…Site-specific incorporation of unnatural amino acids (Uaas) into proteins in mammalian cells holds much potential to enable both basic science as well as biotechnology applications. [1][2][3][4] Central to this technology is a nonsense-suppressing aminoacyl-tRNA synthetase (aaRS)/tRNA pair which is engineered to charge the Uaa of interest without cross-reacting with any of its host counterparts. Such "orthogonal" aaRS/tRNA pairs are typically imported into the host cell from a different domain of life.…”

Application of Nucleic Acid Frameworks in the Construction of Nanostructures and Cascade Biocatalysts: Recent Progress and Perspective

“…acids (Zhang et al, 2017a;Zhang et al, 2017b;Fischer et al, 2020;Hashimoto et al, 2021;Manandhar et al, 2021), so we can confidently expect the broad application of unnatural components in future construction of nucleic acid scaffolds for building nanostructures and multi-enzyme machineries, both in vitro and in vivo.…”

Peptide Cyclization Methodologies Amenable to in Vitro Display

“…Genetic code reprogramming is a powerful technique which enables incorporation of nonproteinogenic amino acids in translated polypeptides via codon reassignment 32 or expansion. 33,34 The technique has evolved and matured over the years (for recent reviews see these references 35,36 ) in which task of reprogramming is achieved through a combination of an Escherichia coli reconstituted cell-free translation system and pre-aminoacylated tRNA with various nonproteinogenic amino acids facilitated by flexizymes. This system, referred to as FIT (Flexible In-vitro Translation), enables for devising many unique methods for macrocyclization of peptides discussed in the following sections�…”