| Recombinant interferon alpha-2 (IFN-α2) has proven useful for treating a variety of human cancers and viral diseases. IFN-α2 has a short circulating half-life in vivo, which necessitates daily or thrice weekly administration to patients. Increasing the circulating half-life of IFN-α2 was approached initially by modification of the protein with amine-reactive polyethylene glycol (PEG) reagents and by fusion of IFN-α2 to human albumin. However, these products have heterogeneous structures and low specific activities, which increase the cost of the drug and may not maximize the potential therapeutic benefits of the protein. In our research, a free cysteine has been introduced into the structure of recombinant IFN-α2b. We attached a 20kDa PEG on the recombinant IFN-α2b surface without heterogeneous structures by using the reaction between the–SH group of the free cysteine and Maleimide-PEG.Recombinant IFN-α2b was expressed in E. coli as inclusion body. In the process of purification, IEC(Ion Exchange Chromatography), HIC(Hydrophobic Interaction Chromatography) and HAC(Hydroxyapatite Chromatography) have been used to separate the target protein from other impurity. Finally, the resolution of recombinant IFN-α2b increased to 1.5, which means the purity of protein is more than 99%. The recombinant IFN-α2b retained high in vitro bioactivity compared with wild-type IFN-α2. The reaction between recombinant IFN-α2b and the m-PEG has been studied, and more than 75% recombinant IFN-α2b reacted with m-PEG in this reaction.
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