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

Ahsan, Fatima; Arif, Amina; Mahmood, Nasir; Gardner, Qurratulann Afza; Rashid, Naeem; Akhtar, Muhammad

doi:10.1016/j.jbiotec.2014.05.001

Cited by 15 publications

(8 citation statements)

References 24 publications

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“…However, the N-terminal Met can never be completely removed. Consequently, this modification can still be observed in substantial amounts in mature IFNα2b obtained from E. coli expression systems [17], which was further confirmed by this study. N-terminal Met residue was detected in samples 1E~5E expressed in E. coli and 6P expressed in Pseudomonas at peptide level, although not detected in sample 1E, 3E, and 5E at intact protein level.…”

Section: Discussionsupporting

confidence: 83%

Analysis of Molecular Heterogeneity in Therapeutic IFNα2b from Different Manufacturers by LC/Q-TOF

Tao

Zhao

et al. 2020

Molecules

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Recombinant human IFNα2b (rhIFNα2b), as an important immune-related protein, has been widely used in clinic for decades. It is also at the forefront of the recent emergence of biosimilar medicines, with numerous products now available worldwide. Although with the same amino acid sequence, recombinant proteins are generally heterogeneous due to post-translational modification and chemical reactions during expression, purification, and long-term storage, which could have significant impact on the final product quality. So therapeutic rhIFNα2b must be closely monitored to ensure consistency, safety, and efficacy. In this study, we compared seven rhIFNα2b preparations from six manufacturers in China and one in America, as well as four batches of rhIFNα2b preparations from the same manufacturer, measuring IFNα2b variants and site-specific modifications using a developed LC/Q-TOF approach. Three main forms of N-terminus, cysteine, methionine, and acetylated cysteine were detected in five rhIFNα2b preparations produced in E. coli (1E~5E) and one in Pseudomonas (6P), but only the native form with N-terminal cysteine was found in rhIFNα2b preparation produced in Saccharomyces cerevisiae (7Y). Two samples with the lowest purity (4E and 6P), showed the highest level of acetylation at N-terminal cysteine and oxidation at methionine. The level of oxidation and deamidation varied not only between samples from different manufacturers but also between different batches of the same manufacturer. Although variable between samples from different manufacturers, the constitution of N-terminus and disulfide bonds was relatively stable between different batches, which may be a potential indicator for batch consistency. These findings provide a valid reference for the stability evaluation of the production process and final products.

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Section: Discussionsupporting

confidence: 83%

Analysis of Molecular Heterogeneity in Therapeutic IFNα2b from Different Manufacturers by LC/Q-TOF

Tao

Zhao

et al. 2020

Molecules

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“…In the light of these observations, interferon derivative containing Met-Phe was engineered, expressed in E. coli and the inclusion bodies solubilized at pH 11.00. We serendipitously discovered that use of pH 11 greatly improved the yield of the correctly folded protein compared to pH 8.0, used in our previous study [1]. Unlike the HPLC profile of the refolded Met-IFN Figure 2B).…”

Section: Expression Of the Gene For Met-phe-ifn (3): Analysis Of The mentioning

confidence: 80%

“…Interferon derivatives were engineered using two plasmids: pET-met-ifn, which contained the human interferon α-2b gene, cloned in the pET-21a expression vector [1] and a pT7-7 derivative, harbouring the complete gene for the membrane-bound form of human cytochrome b5 (a gift from Mrs Monika Akhtar, Biological Sciences, University of Southampton, UK). T7-promoter and ChimR2 primers (Table S1, supplementary) were used for PCR amplification of the soluble domain of cytochrome b5.…”

Section: Construction Of Interferon Derivativesmentioning

confidence: 99%

“…E. coli BL21 codon plus® harboring the appropriate plasmids were grown and inclusion bodies isolated as described previously [1]. It was found that solubilization of inclusion bodies at pH 11 had a beneficial effect on the proper folding and yield of native IFN as well as Met-Phe-IFN (1a and 3 Figure 1 respectively).…”

Section: Expression and Purification Of Interferon Derivativesmentioning

confidence: 99%

“…25 ml of the solubilized protein above (5 mg of protein /ml) was transferred to the refolding sink to 60 percent of the starting inclusion bodies. For the identification of properly refolded proteins, tryptic digestion of various interferon constructs was performed as described in [1] and MALDI mass spectrometric analysis of fragment containing correct disulphide bridges was performed in linear positive mode as described previously [19].…”

Section: Expression and Purification Of Interferon Derivativesmentioning

confidence: 99%

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

Ahsan

Gardner

Rashid

et al. 2018

Bioorganic Chemistry

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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 b at the N-terminal of interferon (b-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b at the C-terminal of interferon (Met-IFN-b-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b-chimera), resolved this problem and gave enhanced biological activity.

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Removal of N-terminal methionine of human interferon α-2b by co‐producing with Pyrococcus furiosus methionine aminopeptidase in Escherichia coli

2021

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

Cited by 15 publications

References 24 publications

Analysis of Molecular Heterogeneity in Therapeutic IFNα2b from Different Manufacturers by LC/Q-TOF

Analysis of Molecular Heterogeneity in Therapeutic IFNα2b from Different Manufacturers by LC/Q-TOF

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

Removal of N-terminal methionine of human interferon α-2b by co‐producing with Pyrococcus furiosus methionine aminopeptidase in Escherichia coli

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