Abstract
Since the establishment of site-specific mutagenesis of single amino acids to interrogate protein function in the 1970s, biochemists have sought to tailor protein structure in the native cell environment. Fine-tuning the chemical properties of proteins is an indispensable way to address fundamental mechanistic questions. Unnatural amino acids (UAAs) offer the possibility to expand beyond the 20 naturally occurring amino acids in most species and install new and useful chemical functions. Here, we review the literature about advances in UAA incorporation technology from chemoenzymatic aminoacylation of modified tRNAs to in vitro translation systems to genetic encoding of UAAs in the native cell environment and whole organisms. We discuss innovative applications of the UAA technology to challenges in bioengineering and medicine.
Highlights
Since the establishment of site-specific mutagenesis of single amino acids to interrogate protein function in the 1970s, biochemists have sought to tailor protein structure in the native cell environment
This Perspective is not meant to stand in for comprehensive reviews published by researchers in the field.[1−4] we acknowledge that there are several exciting technologies that have been developed over the past several decades for chemical modification of proteins; we speak to only the Unnatural amino acids (UAAs) technology here
The compatibility of the tethered ribosomal complexes with the multisite incorporation of UAAs was evaluated by Jewett and Mankin through the fluorescence analysis of a super folder green fluorescent protein (GFP) variant containing five TAG codons, finding that the tethered translation system is effective in incorporating five pAzF click handles into sfGFP.[112]
Summary
Many different methods have been researched, including using the type 3 polymerase III promoter to more efficiently express orthogonal prokaryotic tRNAs,[51] UAA esterification to increase UAA bioavailability,[63] and optimizing tRNA/synthetase affinity to increase the level of UAA incorporation.[64] Encouraging progress was achieved in this research area through the combined use of these optimized methods.[4] In their letter to the editor of Cell Research, Ye, Wang, Li and co-workers reported the introduction, maintenance, and transmission of the genetic material for code expansion in mice In this work, they integrated the orthogonal pAzFRS/tRNACUA pair into the mouse genome.[59] They demonstrated that, in the presence of pAzF, the suppressor tRNA can decode the UAG amber codon to express a dual fluorescent reporter eGFP-TAG-mCherry in neurons and bone marrow cells of mice.[59]
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