Recombinant expression, purification, and characterization of human ATE1 arginyltransferase

This chapter presents a straightforward method for expressing and purifying recombinant human Arginyl-tRNA-protein transferase 1 (ATE1) from E. coli. ATE1 is an enzyme that catalyzes the transfer of arginine from arginyl-tRNA to the N-terminal or internal Asp or Glu residues of the substrate proteins, a process that regulates protein turnover or function. The approach enables the efficient purification of milligram-scale quantities of highly soluble, enzymatically active ATE1 with over 98 % purity. Additionally, we describe the procedures for validating human ATE1 activity through an arginylation assay followed by mass spectrometry. We also describe the quantification of ATE1-tRNA binding affinity using Electrophoretic Mobility Shift Assay (EMSA).