Spontaneous alpha-N-6-phosphogluconoylation of a "His tag" in Escherichia coli: the cause of extra mass of 258 or 178 Da in fusion proteins

Anal Biochem. 1999 Feb 1;267(1):169-84. doi: 10.1006/abio.1998.2990.

Abstract

Several proteins expressed in Escherichia coli with the N-terminus Gly-Ser-Ser-[His]6- consisted partly (up to 20%) of material with 178 Da of excess mass, sometimes accompanied by a smaller fraction with an excess 258 Da. The preponderance of unmodified material excluded mutation, and the extra masses were attributed to posttranslational modifications. As both types of modified protein were N-terminally blocked, the alpha-amino group was modified in each case. Phosphatase treatment converted +258-Da protein into +178-Da protein. The modified His tags were isolated, and the mass of the +178-Da modification estimated as 178.06 +/- 0.02 Da by tandem mass spectrometry. As the main modification remained at +178 Da in 15N-substituted protein, it was deemed nitrogen-free and possibly carbohydrate-like. Limited periodate oxidations suggested that the +258-Da modification was acylation with a 6-phosphohexonic acid, and that the +178-Da modification resulted from its dephosphorylation. NMR spectra of cell-derived +178-Da His tag and synthetic alpha-N-d-gluconoyl-His tag were identical. Together, these results suggested that the +258-Da modification was addition of a 6-phosphogluconoyl group. A plausible mechanism was acylation by 6-phosphoglucono-1,5-lactone, produced from glucose 6-phosphate by glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Supporting this, treating a His-tagged protein with excess d-glucono-1,5-lactone gave only N-terminal gluconoylation.

MeSH terms

  • Acylation
  • Amino Acid Sequence
  • Cyclic AMP-Dependent Protein Kinases / chemistry
  • Cyclic AMP-Dependent Protein Kinases / genetics
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Gluconates / metabolism
  • Histidine / chemistry*
  • Humans
  • In Vitro Techniques
  • Magnetic Resonance Spectroscopy
  • Mass Spectrometry
  • Molecular Sequence Data
  • Molecular Weight
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Phosphatidylinositol 3-Kinases / chemistry
  • Phosphatidylinositol 3-Kinases / genetics
  • Phosphatidylinositol 3-Kinases / metabolism
  • Protein Processing, Post-Translational
  • Protein-Tyrosine Kinases / chemistry
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / metabolism
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • ZAP-70 Protein-Tyrosine Kinase
  • beta-Adrenergic Receptor Kinases

Substances

  • Gluconates
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • 6-phosphonoglucono-delta-lactone
  • Histidine
  • Phosphatidylinositol 3-Kinases
  • Protein-Tyrosine Kinases
  • ZAP-70 Protein-Tyrosine Kinase
  • ZAP70 protein, human
  • Cyclic AMP-Dependent Protein Kinases
  • beta-Adrenergic Receptor Kinases