Study On Separation And Purification Process Of Recombinant Human Interferon α2b

Interferons (IFNs) are a family of soluble glycoproteins produced by different cellsand exhibit antiviral, antitumor and immunomodulation functions. Among type Iinterferons, IFN-α exhibits the strongest antiviral activity and has been used to treatchronic hepatitis B and C infections. The main shortcoming of interferon therapy isthat the half-life of interferon alpha by subcutaneous injuction is very short whichcould impair the efficacy and bioavailability. Increasing the circulating half-life ofinterferon alpha2was approached initially by modification of the protein withamine-reactive polyethylene glycal reagents.The plasmid containing IFN-α gene was constructed by Bolder Biotechnology, nc.and it was identified by enzyme digestion and DNA sequencing. The plasmid wastransformated to Ecoli W3110, and data of non-reducing SDS-PAGE indicated that a19KDa protein was expressed in the form of inclusion bodies with good immunity.Cell suspensions were sonicated to obtain inclusion bodies. Then, the washedinclusion bodies were added the solubilization buffer with6mol/L GnHCl to bedegenerated. Finally, the denatured protein slowly into the refolding solution todiluted20times,48h at4°C for refolding.To separate recombinant human interferon alpha2b and remove the dimer by thehydrophobic interaction chromatography for the further study. Firstly the cationexchange chromatography was employed to purify the refolded human interferonalpha2b by removing of most contaminants. Then a further purification was carriedout to remove the mis-folded recombinants in the renaturation process byhydrophobic interaction chromatography. Furthermore, the effects of the saltconcentration, pH, flow rate and urea concentration on hydrophobic interactionchromatography were investigated. Under the optimal condition that was ammoniumsulfate concentration1.2mol/L, pH6.0, flow rate2.5mL/min and urea2mol/L thepurified recombinant showed a single band on a non-reduced SDS-PAGE. ConclusionThe optimal condition for purifing recombinant human interferon alpha2b by thehydrophobic interaction chromatography was determined, and the recombinant withhigh activity and purity was successfully purified, the purity of interferon is63.07%.