Recombinant protein sequences can trigger methylation of N‐terminal amino acids in Escherichia coli

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild‐type valine residues at all N‐termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli , methylation of the N‐terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N‐terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N‐terminal alanine that occurred through two posttrans‐lational events: initial excision of the N‐terminal methionine, followed by methylation of alanine as the newly generated N‐terminus. No methylation was observed for variants expressed with wild‐type valine at the N‐terminus of the alpha globin. The methylation of N‐terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli . Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N‐terminal methylation has been observed previously for native E. coli proteins with similar N‐terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli .