Improved Refolding Efficacy of Recombinant Human Interferon α-2b via pH Modulation
Purpose: To increase the refolding yield of Recombinant Human Interferon α-2b in order to achieve a highly potent product.
Methods: Interferon α-2b inclusion body was dissolved in tris-HCl buffer containing 6 M guanidine-HCl and CuSO4. Different refolding buffers were employed for refolding the target protein. The refolded proteins were then purified by affinity and gel filtration chromatography. The purified proteins were subjected to circular dichroism (CD) spectropolarimetry and assayed for biological activity in vitro.
Results: Increment of pH to 8.5 improved refolding efficacies from 42.28 % to 71.22 %. However, the relative potency significantly increased up to pH 8.0 (from 19353546 to 28633902, p < 0.05) and then decreased to 21081305.00 at pH 8.5. The CD spectra demonstrated that by increasing pH to 8.5, the secondary structure of the protein was altered, probably due to increase in alpha-helix from 23.7 % at pH 7.0 to 28.1 %.
Conclusion: Employing a low-cost and simple method, such as alteration of refolding buffer pH, results in higher refolding yield in downstream processing of rhIFN α-2b.
Keywords: Recombinant human interferon α-2b, Refolding, Circular dichroism, Spectropolarimetry,Recombinant protein, pH effect