Matrix-assisted refolding and purification of proteins by a novel designed anion exchange chromatography

Abstract

Purpose: The refolding of recombinant human interferon α-2b is accompanied by low yield that could be due to high aggregation and multiplicity of steps in downstream processing. It is therefore essential to refold and purify the protein to simultaneously increase the production yield and reduce the required downstream steps. Methods: Inclusion bodies were dissolved in Tris-HCl buffer containing 6 M guanidine and applied to a newly designed poly-arginine anion exchange chromatography system. Different reduced-oxidized glutathione amount were employed for refolding the target protein. The refolded proteins were applied to gel filtration chromatography and subjected to biological activity assessment. Results: By increasing the sample volume, the refolding efficacy, purification factor and potency were significantly decreased. This reduction can be highest when only 2 column volumes of the glutathione redox pair presented in the column. The highest values of these factors were obtained when 4 column volumes of the glutathione redox pair offered to the column. To achieve better results, the proportion of sample volume and the amount of passed glutathione redox pair through column should be optimized. Consistent with the increase of sample volume to 1250 μl at any levels of glutathione redox pair there was a significant drop in all factor values. Conclusion: Poly-arginine anion exchange matrix is a new artificial chaperone that can improve the refolding and purification process simultaneously with high efficacy. Therefore, we present poly-arginine anion exchange chromatography as an alternative method for protein refolding with promising commercial and developmental potential.