The majority of proteins in eukaryote cells are subjected to amino-terminal acetylation. This co-translational modification can affect the stability of a protein and also regulate its biological function. Amino-terminally acetylated recombinant proteins cannot be produced using prokaryote expression systems, such as E. coli, as these cells lack the appropriate N-α-terminal acetyltransferase complexes. Here we describe a simple protocol that allows the recombinant expression and purification of NatB-dependent amino-terminally acetylated proteins from E. coli.