Abstract
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.
Highlights
Large scale production of bio-pharmaceuticals has been made possible by recombinant DNA technology
circular dichroism (CD)-spectropolarimetry revealed that with increasing pH of refolding medium, the percent of β-sheet present in the aforementioned protein was decreased whereas relative increment in αhelix can be observed when compared with values at pH 7.0 (Table 3 and Figure 2)
Aggregation and aggregates are the main obstacles in preparation of functional proteins from inclusion bodies which contribute directly to the decrement in production or refolding yield of the target protein [2, 14]
Summary
Large scale production of bio-pharmaceuticals has been made possible by recombinant DNA technology. The aggregated proteins are further processed in order to obtain functional forms of target protein in vitro [2,3,4,5] Such proteins are outcomes of high-level expression of eukaryotic proteins deposited in cytoplasm of E. coli referred to as inclusion bodies (IBs) [3,4,6,7,8]. By addition of 22.5 mL of each refolding buffer at different pH containing 2.5 μL of 80 mM CuSO4 to 2.5 mL of denatured protein (5 mg/mL) in separate 50 mL beakers using a peristaltic pump (Bio-Rad, USA) at the flow rate of 0.06 ml/min for 6 h with gentle stirring using magnetite stirrers with similar size and shape (PLT Scientific, Thailand). After equilibration of the column, the refolded protein at each pH was loaded and washed first with 2 column volumes of PBS and with sodium acetate solution (pH adjusted to 5 with glacial acetic acid). The level of statistical significance was p < 0.05
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