Spontaneous α-N-6-Phosphogluconoylation of a “His Tag” inEscherichia coli:The Cause of Extra Mass of 258 or 178 Da in Fusion Proteins☆

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 α-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 15 N-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 α- 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.