Preparesion And Pegylation Of Recombinant Human Interferon-β-1b

Purpose: To prepare human interferon-β-1b(17Ser-IFN-β-1b)by fusion expression in E.coli without denaturation and renaturation process. And then to obtain PEG-β-IFN-1b by modifing target protein with PEG.Methods: Constructing three kinds of TRX fusion expression vectors of pET-32a-β-IFN-1b by PCR to obtain DNA fragments of interferon-β-1b which were inserted into a different proteinase recognition site, including enterokinase (DDDDK), TEV (ENLYFQG) and thrombin (LVPRGS), allowing for the removal of the fusion partner during purification. These vectors were transformed into Origami DE3. The fusion protein (TRX-β-IFN-1b) was expressed in Qrigami DE3, and it was purifyied and digested with different enzyme separately, further screened the engineering plasmid and the engineering bacteria by comparing digestion efficiency and specificity. The rhIFN-β-1b was separated by C18 reverse-phase chromatography from the fusion by cleavage with the protease, and modified with PEG. Meanwhile, its antiviral specific activity was determined with Wish/VSV assay.Result: Recombinant vectors (pET-32a-EK-β-IFN-1b, pET-32a-TEV-β-IFN-1b, pET-32a-thrombin-β-IFN-1b) were successfully constructed with a single protease cut site, which were verified by DNA sequencing. The recombinant plasmids were transformed into Origami DE3. Induced with IPTG, the expression level of fusion protein was over 20% of total bacterial protein. And then pET-32a-EK-β-IFN-1b and pET-32a-EK-β-IFN-1b/Origami DE3 were separately screened as the engineering plasmid and the engineering bacteria by comparing the restriction enzyme digestion efficiency and specificity. The fusion protein was purified by Ni-Chelting column and digested with EK. The digestion efficiency is not less than 90%. The purity of prepared rhIFN-β-1b was more than 98%. Molecular weight of prepared rhIFN-β-1b was consistent with theoretical value. The rhIFN-β-1b was modified with PEG,and its purity was over 90%. The antiviral specific activity results showed that rhβ-IFN-1b reached 4.5×107IU/mg, the antiviral specific activity of PEG-IFN-β-1b was 5.6×106IU/mg.Conclusion: The engineering bacteria was successfully constructed and screened. The rhIFN-β-1b obtained by fusion expression and preparation in this study could be more potential value for the treatment of multiple sclerosis or viral hepatitis.