Significance: This work describes a facile system for incorporating noncanonical amino acids containing long side-chain thiols using an expanded genetic code. These amino acids begin to overcome the distance and geometric constraints of the cysteine disulfide and can pair with cysteines to cross-link more remote sites in proteins. To demonstrate this notion, we constructed a library of random β-lactamase mutants containing these noncanonical amino acids and grew them at nonpermissive temperatures. We identified a mutant enzyme that is cross-linked by one such extended disulfide bond that has significantly enhanced thermal stability. This study suggests that an expanded set of amino acid building blocks can provide novel solutions to evolutionary challenges.

A propeptide strategy increases uptake of phosphotyrosine and a nonhydrolyzable analog to facilitate their incorporation into proteins by recombinant methods, aided by a specific aminoacyl-tRNA synthetase with a reconfigured active site. Tyrosine phosphorylation is a common protein post-translational modification that plays a critical role in signal transduction and the regulation of many cellular processes. Using a propeptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase–tRNA pair that allows site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray structure of the synthetase reveals a reconfigured substrate-binding site, formed by nonconservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site and determining the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.

A chemical protein-DNA conjugation approach enhances precise genome editing in human cells and mouse zygotes.

The concept of genetic code expansion (GCE) has revolutionized the field of chemical and synthetic biology, enabling the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins, thus opening new avenues in research and applications across biology and medicine. In this review, we cover the principles of GCE, including the optimization of the aminoacyl-tRNA synthetase (aaRS)/tRNA system and the advancements in translation system engineering. Notable developments include the refinement of aaRS/tRNA pairs, enhancements in screening methods, and the biosynthesis of noncanonical amino acids. The applications of GCE technology span from synthetic biology, where it facilitates gene expression regulation and protein engineering, to medicine, with promising approaches in drug development, vaccine production, and gene editing. The review concludes with a perspective on the future of GCE, underscoring its potential to further expand the toolkit of biology and medicine. Through this comprehensive review, we aim to provide a detailed overview of the current state of GCE technology, its challenges, opportunities, and the frontier it represents in the expansion of the genetic code for novel biological research and therapeutic applications.