Characterization and bioassay of post-translationally modified interferon α-2b expressed in Escherichia coli

Examples of N-terminal acetylation are rare in prokaryotic systems, but in this study, we report one such example in which N-terminal Cys residue of recombinant human interferon α-2b produced in Escherichia coli is a favourite site for N(α)-acetylation. The recombinant protein following Q-sepharose chromatography gave a single band on PAGE analysis. However, on reverse phase HPLC the material separated into three peaks. These were characterized by mass spectrometric techniques as: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue had been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contained an acetyl group. Tryptic digestion of interferon α-2b gave fragments linking Cys(1) to Cys(98) and Cys(29) to Cys(138), while that of N(α)-acetyl-interferon α-2b gave the Cys(1)-Cys(98) fragment with an additional mass of 42 attributed to an acetylated N-terminal. Bioassay of the derivatives showed that N(α)-acetyl-interferon α-2b had 10% of the activity of interferon α-2b. The results suggest that the lower activity derivative seen here in E. coli may also be produced when the protein is produced in yeast.