title: Characterization and bioassay of post-translationally modified interferon α-2b expressed in Escherichia coli.
abstract: 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.
authors.affiliation: School of Biological Sciences, University of the Punjab, New Campus, Lahore 54590, Pakistan.
authors.forename: Fatima
authors.initials: F
authors.lastname: Ahsan
authors.affiliation: School of Biological Sciences, University of the Punjab, New Campus, Lahore 54590, Pakistan.
authors.forename: Amina
authors.initials: A
authors.lastname: Arif
authors.affiliation: School of Biological Sciences, University of the Punjab, New Campus, Lahore 54590, Pakistan.
authors.forename: Nasir
authors.initials: N
authors.lastname: Mahmood
authors.affiliation: School of Biological Sciences, University of the Punjab, New Campus, Lahore 54590, Pakistan.
authors.forename: Qurra-Tul-Ann Afza
authors.initials: QT
authors.lastname: Gardner
authors.affiliation: School of Biological Sciences, University of the Punjab, New Campus, Lahore 54590, Pakistan.
authors.forename: Naeem
authors.initials: N
authors.lastname: Rashid
authors.affiliation: School of Biological Sciences, University of the Punjab, New Campus, Lahore 54590, Pakistan; Centre for Biological Sciences, University of Southampton, SO17 1BJ, UK. Electronic address: ma3@soton.ac.uk.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Akhtar
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence / Q000235:genetics; D001681:Biological Assay; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D003545:Cysteine
keywords: Mass spectrometry; Met-interferon α-2b; N(α)-acetyl-interferon α-2b; N-acetyl transferase; Tryptic mapping