%J Bioorganic Chemistry
%K 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
%L discovery10046551
%I ACADEMIC PRESS INC ELSEVIER SCIENCE
%O This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
%X 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.
%T Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli
%A F Ahsan
%A QA Gardner
%A N Rashid
%A GJ Towers
%A M Akhtar
%V 76
%D 2018
%P 294-302