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

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 exces s mass, sometimes accompanied by a smaller fraction with an excess 258 Ha, The preponderance of unmodified material excluded mutation, and the extra m asses were attributed to posttranslational modifications, As both types of modified protein were N-terminally blocked, the alpha-amino group was modif ied 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 Ha in N-15-substituted protein, it was deemed nitrogen-free and possibly carbohydrate-like. Limited period ate 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 synthet ic alpha-N-D-gluconoyl-His tag were identical. Together, these results sugg ested that the +258-Da modification was addition of a 6-phosphogluconoyl gr oup. A plausible mechanism was acylation by 6-phosphoglucono-1,5-lactone, p roduced from glucose g-phosphate by glucose-6-phosphate dehydrogenase (EC 1 .1.1.49). Supporting this, treating a His-tagged protein with excess D-gluc ono-1,5-lactone gave only N-terminal gluconoylation. (C) 1999 Academic Pres s.