Construction, expression and characterization of human interferon alpha2b-(G4S)n-thymosin alpha1 fusion proteins in Pichia pastoris

Abstract

Aim: Interferon alpha2b (IFNalpha2b) and thymosin alpha1 (Talpha1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNalpha2b and Talpha1 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 IFNalpha2b-(G4S)n-Talpha1 (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 IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex 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 IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex 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 IFNalpha2b monoclonal antibody and Talpha1 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 IFNalpha2b-(G4S)n-Talpha1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNalpha2b and immunomodulatory activity of Talpha1 in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo.

Figure 1

Construction of (G4S)n-thymosin α1 (n = 1-3) DNA fragments.

Figure 2

PCR analysis of plasmids pPIC9-IFNα2b-(G4S)n-Tα1 (n = 1-3). The electrophoresis was performed on an 8 g/L agarose gel. Lane M: DNA molecular marker; lane 1: pPIC9-IFNα2b-G4S-Tα1; lane 2: pPIC9-IFNα2b-(G4S)2-Tα1; lane 3: pPIC9-IFNα2b-(G4S)3-Tα1.

Figure 3

Expression of the fusion proteins IFNα2b-(G4S)n-Tα1 (n = 1-3) in P. pastoris. A: SDS-PAGE analysis of the fusion proteins. SDS-PAGE was performed on a 150 g/L polyacrylamide gel and stained with Coomassie blue R-250. Lane M: molecular weight marker; lane 1: IFNα2b-G4S-Tα1; lane 2: IFNα2b-(G4S)2-Tα1; lane3: IFNα2b-(G4S)3-Tα1; lane 4, SMD1168/pPIC9 as control; B: Density quantification analysis of lane 2 in A. Peak 4 represents the IFNα2b-(G4S)2-Tα1 fusion protein. Peak 5 represents the degradation fragment.

Figure 4

Identification of expressed fusion proteins IFNα2b-(G4S) n-Tα1 (n = 1-3) by the Western blot. A: IFNα2b monoclonal antibody as the primary antibody; B: Thymosin α1 polyclonal antibody as the primary antibody. Lane 1: IFNα2b-(G4S)3-Tα1; lane 2: IFNα2b-(G4S)2-Tα1; lane 3: IFNα2b-G4S-Tα1.

Figure 5

SDS-PAGE analysis of eluted fractions from the DEAE column by different elution buffers. After fermentation, the supernatant of SMD1168/pPIC9-IFNα2b-(G4S)2-Tα1 was collected and loaded on the DEAE column. The fractions eluted sequentially by elution buffer A and B were analyzed by SDS-PAGE. Lane1-8: the fractions subsequently eluted by elution buffer A (0.05 mol/L Tris-HCl, 0.10 mol/L NaCl, pH 8.0); lane 9-14: the fractions subsequently eluted by elution buffer B (0.05 mol/L Tris-HCl, 0.10 mol/L NaCl, pH 8.0).

Figure 6

Purified fusion proteins IFNα2b-(G4S) n-Tα1 (n = 1-3) after gel filtration. A: SDS-PAGE analysis of purified fusion proteins. Lane M: molecular weight marker; Lane 1: IFNα2b-(G4S)3-Tα1; Lane 2: IFNα2b-(G4S)2-Tα1; Lane 3: IFNα2b-G4S-Tα1; B: Density quantification analysis of lane 2 in A. Peak 1 represents the IFNα2b-(G4S)2-Tα1 fusion protein.

Figure 7

E-rosette analysis of the purified fusion proteins. Experimental details are given in Materials and Methods.