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

Journal Article


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 Nα-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 Cys1 to Cys98 and Cys29 to Cys138, while that of Nα-acetyl-interferon α-2b gave the Cys1-Cys98 fragment with an additional mass of 42 attributed to an acetylated N-terminal. Bioassay of the derivatives showed that Nα-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. © 2014 Elsevier B.V.

UOW Authors


  •   Ahsan, Fatima (external author)
  •   Arif, Amina (external author)
  •   Gardner, Qurra Tul Ann Af. (external author)
  •   Rashid, Naeem (external author)
  •   Akhtar, Muhammad (external author)
  •   Mahmood, Nasir (external author)

Publication Date


  • 2014

Citation


  • Ahsan, F., Arif, A., Mahmood, N., Gardner, Q. Tul Ann Af., Rashid, N. & Akhtar, M. (2014). Characterization and bioassay of post-translationally modified interferon α-2b expressed in Escherichia coli. Journal of Biotechnology, 184 11-16.

Scopus Eid


  • 2-s2.0-84901402828

Has Global Citation Frequency


Number Of Pages


  • 5

Start Page


  • 11

End Page


  • 16

Volume


  • 184

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 Nα-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 Cys1 to Cys98 and Cys29 to Cys138, while that of Nα-acetyl-interferon α-2b gave the Cys1-Cys98 fragment with an additional mass of 42 attributed to an acetylated N-terminal. Bioassay of the derivatives showed that Nα-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. © 2014 Elsevier B.V.

UOW Authors


  •   Ahsan, Fatima (external author)
  •   Arif, Amina (external author)
  •   Gardner, Qurra Tul Ann Af. (external author)
  •   Rashid, Naeem (external author)
  •   Akhtar, Muhammad (external author)
  •   Mahmood, Nasir (external author)

Publication Date


  • 2014

Citation


  • Ahsan, F., Arif, A., Mahmood, N., Gardner, Q. Tul Ann Af., Rashid, N. & Akhtar, M. (2014). Characterization and bioassay of post-translationally modified interferon α-2b expressed in Escherichia coli. Journal of Biotechnology, 184 11-16.

Scopus Eid


  • 2-s2.0-84901402828

Has Global Citation Frequency


Number Of Pages


  • 5

Start Page


  • 11

End Page


  • 16

Volume


  • 184