Study On Refolding Of Recombinant Human Interferon α-2b Inclusion Bodies By Column Chromatography

Interferon alpha-2b (IFNα-2b) is a very important cellular factor. It has manyimportant roles, such as regulating immune response, inhibiting tumor growth andpossessing antivirus. The rhIFNα-2b was expressed in recombinant Escherichia colias inclusion bodies (IBs) which need to be refolded to the activity protein. As a newmethod, the refolding with chromatography can improve the bioactivity yield andsimultaneously accomplish its purification, so it has been paid much attention inrecent years.Here, we reported some on-column chromatographic processes for the refoldingof rhIFNα-2b produced in E.coli and compared them with the traditional way ofdialysis renaturation. The objective of this research is to develop a new method whichassisted protein renaturation and can be used in large scale production.The rhIFNα-2b produced in the E.coli host after induction at 42℃andaccumulated as inclusion bodies. Yield of the rhIFNα-2b IBs was approximately2.4g/L culture. After washed with the detergent buffer the purity of IBs was about60%.The recombinant human interferonα-2b (rhIFNα-2b) was refolded and purifiedsimultaneously by guanidine hydrochloride (Gu-HCl) gradient size-exclusionchromatography. Concerning various factors, such as Gu-HCl gradient length, finalGu-HCl concentration, flow rate and GSH/GSSG ratio, a Gu-HCl gradient gelfiltration method for rhIFNα-2b refolding was established. As a result, under theoptimum condition in the refolding with linearly decreasing Gu-HCl gradient, theprotein mass yield of rhIFNα-2b was 1.5 times of that in the dialysis refolding process.The activity yield was up to 68.7% with specific activity 1.77×10~8IU/mg and thepurity was more than 94%.Refolding processes by Ion exchange chromatography (IEC) with Gu-HClgradient was proposed. The process of refolding and purification could be integratedtogether by IEC. Under the optimum conditions: protein loading 2.24mg, flow rate0.5ml/min and final Gu-HCl concentration 1.5mol/L, the specific activity of rhIFNα-2b was 1.13×10~8IU/mg, the activity recovery was 46.3% and the protein yieldwas 51.2%.The purity of rhIFNα-2b determined by SDS-PAGE was above 90%.Hydrophobic interaction chromatography (HIC) was performed as proteinrefolding system. As a result, the specific activity of rhIFNα-2b was 2.34×10~8IU/mg,the activity recovery was 57.2%, the protein yield was 37.2% and the purity wasabout 92% under such experiment conditions: protein loading 0.644mg, flow rate1ml/min and Gu-HCl concentration in Buffer A and Buffer B 2mol/L.