Identification of a gluconic acid derivative attached to the N-terminus of histidine-tagged proteins expressed in bacteria

Biochem Biophys Res Commun. 1999 Sep 7;262(3):793-800. doi: 10.1006/bbrc.1999.1304.


Our previous studies have shown that the His tag cleaved from fusion proteins contained two distinct components P1 and P2. P1 has been identified to be a His-tagged peptide of G-H-H-H-H-H-H-H-H-H-H-S-S-G-H-I-E-G-R resulted from initiator methionine deletion, and P2 contains an unknown moiety at the second residue glycine of the tag (x-G-H-H-H-H-H-H-H-H-H-H-S-S-G-H-I-E-G-R, x = 178.0 Da). This study aimed to determine the structure of the modification by using a combination of protein isotope labeling and mass spectrometry. His-tagged FKBP was expressed in (15)N and (13)C labeling growth media respectively. Isotopic labeled His-tagged proteins ((15)N-His-FKBP and (13)C-His-FKBP) were isolated by affinity chromatography and subjected to Xa digestions to release the labeled His tag. Subsequent analyses of the released His tag by MALDI-TOF-MS indicated a mass difference of 178.0 +/- 0.2 Da, between the two (15)N-labeled peptides P1 and P2, suggesting that the modification moiety contained no nitrogen. A mass difference of 184.0 +/- 0.2 Da was observed on MALDI between (13)C-labeled peptide P1 and P2, indicating six carbons in the modification group. Also, comparing the mass shift on MALDI spectra of P1 and P2 after hydrogen/deuterium exchange revealed that the modification moiety had five hydroxyl groups. It was concluded that the modification was a gluconic acid derivative attached to the N-terminus of His-tagged proteins expressed in bacteria. The proposed structure was further confirmed by MALDI analysis of periodate oxidation products of His-tagged peptides.

MeSH terms

  • Amino Acid Sequence
  • Carbon Isotopes
  • Chromatography, Affinity
  • Cloning, Molecular
  • Gluconates*
  • Histidine
  • Immunophilins / biosynthesis*
  • Immunophilins / chemistry
  • Immunophilins / isolation & purification
  • Molecular Sequence Data
  • Nitrogen Isotopes
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Tacrolimus Binding Proteins


  • Carbon Isotopes
  • Gluconates
  • Nitrogen Isotopes
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • Histidine
  • Tacrolimus Binding Proteins
  • Immunophilins
  • gluconic acid