RECOMBINANT PROTEIN SEQUENCES CAN TRIGGER METHYLATION OF N-TERMINAL AMINO-ACIDS IN ESCHERICHIA-COLI

Recombinant human hemoglobin rHb1.1 has been genetically engineered wi th the replacement of the wild-type valine residues at all N-termini w ith 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 wa s expressed in Escherichia coli, methylation of the N-Terminal methion ine of the alpha globin was discovered. Another mutant has been engine ered with the alpha globin gene coding for N-terminal methionine follo wed by an insertion of alanine. Characterization of expressed hemoglob in from this variant revealed a methylated N-terminal alanine that occ urred through two posttranslational events: initial excision of the N- terminal methionine, followed by methylation of alanine as the newly g enerated N-terminus. No methylation was observed for variants expresse d with wild-type valine at the N-terminus of the alpha globin. The met hylation of N-terminal amino acids was attributed to a specific protei n sequence that can trigger methylation of proteins expressed in E. co li. Here we demonstrate that proline at position 4 in the protein sequ ence of alpha globin seems an essential part of that signaling. Althou gh N-terminal methylation has been observed previously for native E. c oli proteins with similar N-terminal sequences, methylation of the rec ombinant globins has allowed further delineation of the recognition se quence, and indicates that methylation of heterologous proteins can oc cur in E. coli.