Expression And PEGylation Of Recombinant Human Interferon

Recombinant interferon-αis used clinically to treat a variety of diseases and cancers because of its antiviral, antiproliferative and immunoregulatory properties. However, the disadvantage of short circulating half-life of IFN-αin vivo makes it not welcomed by most patients. Therefore, the chemical conjugation of polyethylene glycol(PEG) and the protein in this study is used to overcome this problem.The free cysteine residue which is introduced by using site-directed mutagenesis serves as the attachment point for covalent modification of the protein with a cysteine-reactive PEG molecule, resulting in a homogeneous product with a high biological activity we expect.The plasmid containing IFN-αgene was constructed by Bolder Biotechnology, and it was identified by enzyme digestion and DNA sequencing. The plasmid was transformated to Ecoli W3110, and data of SDS-PAGE and Western-blot indicated that a 19KD protein was expressed in the form of inclusion bodies with good immunity. Meanwhile, the expression conditions were also optimizated and the result was that temperature was 37℃,pH of media was 7.0,concentration of IPTG was 0.5mM,induction time was 16h. After denaturation and renaturation of inclusion bodies, the target protein---interferon-αwas obtained utilizing ion exchange chromatography(IEC) and the purity was above 98%. We can get about 22mg protein from 1L media. Finally, IFN-αreacts with maleimide-PEG to produce site-specific, mono-PEGylated proteins.