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Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli

Ahsan, F; Gardner, QA; Rashid, N; Towers, GJ; Akhtar, M; (2018) Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli. Bioorganic Chemistry , 76 pp. 294-302. 10.1016/j.bioorg.2017.11.016. Green open access

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Abstract

We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has 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 contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b5 at the N-terminal of interferon (b5-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b5 at the C-terminal of interferon (Met-IFN-b5-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b5-chimera), resolved this problem and gave enhanced biological activity.

Type: Article
Title: Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.bioorg.2017.11.016
Publisher version: https://doi.org/10.1016/j.bioorg.2017.11.016
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: Cytochrome b5, Interferon-cytochrome b5-chimera, Interferon fusion, VSV-G (vesicular stomatitis virus G, glycoprotein) pseudotyped HIV-1 derived vector particles, Interferon bioassay, Protein purification, Mass spectrometry, Protein expression, Protein folding, Protein chemistry
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Infection and Immunity
URI: https://discovery.ucl.ac.uk/id/eprint/10046551
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