In-bacteria arginylation assay.

ATE1 is an enzyme that catalyzes the post-translational arginylation of proteins by transferring arginine to acidic residues, such as aspartate and glutamate, located at the N-terminus or on side chains. This modification plays important roles in regulating protein stability and function. The mechanisms underlying substrate and site selection by ATE1 remain unclear. An experimental strategy that enables targeted validation of arginylation and site-specific detection on individual substrates is essential for advancing the understanding of ATE1 function. This protocol describes an in-bacteria arginylation assay that enables co-expression of ATE1 with target substrates in E. coli, allowing direct purification of arginylated substrates for downstream analysis. This approach ensures high substrate purity and efficient arginylation, facilitating reliable assessment of arginylation levels and site specificity.