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 posttranslational 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.
Apostol
I
I
Somatogen, Inc., Boulder, Colorado 80301, USA.
Aitken
J
J
Levine
J
J
Lippincott
J
J
Davidson
J S
JS
Abbott-Brown
D
D
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D008715:Methionine / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry*
D006454:Hemoglobins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008715:Methionine; D000409:Alanine