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

Abstract

Recombinant human hemoglobin rHbl. I 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 rHbl.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.

Mechanism for the Hydrolysis of a Sulfur-Sulfur Bond Based on the Crystal Structure of the Thiosulfohydrolase SoxB

“…In addition, the recombinant SoxY protein had a mass 14 Da greater than the expected mass value of 14,668 Da. These observations are consistent with the N-terminal methionine of SoxY being methylated, as has been reported previously for proteins that have a positively charged residue as the second amino acid (this residue is arginine in T. thermophilus SoxY) (26).…”

Incorporation of Norvaline at Leucine Positions in Recombinant Human Hemoglobin Expressed in Escherichia coli

“…Amino acid analysis of the di-␣-globin showed low recovery of the methionine (3.4 versus expected 5 residues). This is due to approximately 40% methylation of the aminoterminal methionine in the di-␣ chain (27). In the case of both shouldering fractions, recovery of methionine was discernibly increased relative to the respective main fraction, and in both shouldering fractions the methionine peak was atypically broad, possibly suggesting the presence of an unusual or modified amino acid in these fractions eluting closely to the methionine peak in our amino acid analysis.…”

“…It should be noted that the observed mass of the di-␣ chain (30,329.3 Ϯ 3.5) was significantly higher than expected (30,323.9). We ascribe this discrepancy to methylation of 40% the di-␣-globin amino-terminal methionine (27), which translates to an expected 5.6 Da mass gain over the predicted mass for the di-␣-globin.…”

Carbamylation of Cysteine: A Potential Artifact in Peptide Mapping of Hemoglobins in the Presence of Urea

“…The expression and purification methods used to produce rHb1.1 have been reported (2,3,(13)(14)(15)(16)(17)(18). Apohemoglobin was prepared for digestion as previously described (2,14,19) Alkylation of globins.…”