Construction, expression and characterization of human interferon α2b-(G4S)n-thymosin α1 fusion proteins in Pichia pastoris

AIM: Interferon alpha 2b (IFN alpha 2b) and thymosin alpha 1 (T alpha 1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFN alpha 2b and T alpha 1 linked by different lengths of (G4S)n (n = 1-3) were constructed and expressed in Pichia pastoris. METHODS: Using PCR and molecular clone techniques, the fusion genes of IFN alpha 2b-(G4S)n-T alpha 1 (n = 1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFN alpha 2b-(G4S) n-T alpha 1 (n = 1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex (TM) 75 gel filtration and analyzed by SDS-PAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins. RESULTS: DNA sequencing confirmed that the fusion genes of IFN alpha 2b-(G4S)n-T alpha 1 (n = 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFN alpha 2b-(G4S) n-T alpha 1 (n = 1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex (TM) 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFN alpha 2b monoclonal antibody and T alpha 1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay. CONCLUSION: The recombinant IFN alpha 2b-(G4S)n-T alpha 1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFN alpha 2b and immunomodulatory activity of T alpha 1 in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo. (C) 2005 The WJG Press and Elsevier Inc. All rights reserved.