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. 2022 Jan 1;26(1):85-90.
doi: 10.52547/ibj.26.1.85.

A Simplified Process for Purification and Refolding of Recombinant Human Interferon-α2b

Mohammad Reza Fazeli  1 , Nima Hezarjaribi  2
Affiliations

A Simplified Process for Purification and Refolding of Recombinant Human Interferon-α2b

Mohammad Reza Fazeli et al. Iran Biomed J. .

Abstract

Background: Interferon α-2b is a vital biotherapeutic produced through the recombinant DNA technology in E. coli. The recombinant IFN-α2b normally appears as intercellular IBs, which requires intensive refolding and purification steps.

Method: Purification of IFN-α2b from solubilized IB was performed using two-phase extraction. To optimize refolding conditions, the effects of pH and different additives, including cysteine, cystine, urea, glycerol, Triton X-100, NaCl, and arginine, were investigated. Optimal refolding buffer (0.64 mM of urea, 5.57 mM of cysteine , and 1.8 mM of cystine) was obtained using RSM. The refolding process was performed by an optimized refolding buffer in the dilution and fed-batch refolding method at different protein concentrations (25-1000 µg/mL).

Result: At a final protein concentration of 500 µg/mL, the fed-batch refolding method yielded in a biological activity of 2.24 × 108 IU/mg, which was nearly twice that of dilution method.

Conclusion: Fed-batch refolding method resulted in the biologically active IFN-α2b with high purity, which can be used for research and industrial purposes.

Keywords: Inclusion bodies; Interferon alpha-2b; Protein refolding.

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Conflict of interest statement

None declared.

Figures

Fig. 1
Fig. 1
SDS-PAGE analysis of IFN-α2b purification by two-phase extraction method. The best IFN-α2b purity was obtained in the organic phase. The Figure shows the effect of different pHs of organic (A) and aqueous (B) phases on IFN-α2b purity, respectively. Lane 1, protein marker; lane 2, pH 3.5; lane 3, pH 4.5; lane 4, pH 5.5; lane 5, pH 6.5; lane 6, pH 7.5; lane 7, pH 8.5. IFN-α2b protein molecular weight is 19 kDa
Fig. 2
Fig. 2
The analysis of different concentrations of cysteine/cystine and urea on IFN-α2b refolding by Minitab 2017 software. Urea concentration: (A) 0.75 M, (B) 1.5 M, and (C) 2.25 M; cysteine concentration: 1, 2, 3, 4, and 5 mM; cystine concentration: 0.5, 1, and 1.5 mM for A, B, and C
Fig. 3
Fig. 3
The far UV CD spectrum of IFN-α2b protein
See this image and copyright information in PMC

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