Efficient and Site-Specific Incorporation of 3-Nitro-Tyrosine Into Recombinant Proteins in Escherichia coli

3-nitro-tyrosine (nitroTyr) is one of numerous oxidative protein modifications implicated in diseases such as cardiovascular disease, cancer, and amyotrophic lateral sclerosis (ALS). Because of this, the ability to site-specifically encode nitroTyr into recombinant proteins is a powerful approach for studying these disease pathways. However, producing proteins with defined nitration sites is technically challenging due to the limitations of traditional chemical nitration via peroxynitrite, which lacks residue and site-specificity. Genetic code expansion (GCE) offers a solution by enabling precise incorporation of nitroTyr at designated TAG codons using engineered aminoacyl-tRNA synthetase/tRNA pairs from Methanocaldococcus jannaschii and Methanomethylophilus alvus. This protocol provides a reliable, optimized workflow for incorporating nitroTyr into proteins in E. coli using GCE. It guides users through key considerations in selecting cell lines, media conditions, and GCE systems to minimize off-target effects such as release factor 1 competition, near-cognate suppression, and chemical reduction of nitroTyr. The method is demonstrated using wild-type and TAG-containing superfolder GFP but is broadly applicable to other proteins of interest. Key features • This protocol offers a practical guide for the recombinant expression of proteins containing site-specific 3-nitro-tyrosine in E. coli. • These methods should be used to characterize the functional and structural consequences of site-specific tyrosine nitration on proteins without having to modify any other residues. • This protocol avoids the use of peroxynitrite as a method to nitrate proteins, which modifies all solvent accessible tyrosine residues to different extents. • Users are guided through the advantages and disadvantages of using different expression strains and genetic code expansion systems depending on specific needs.