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


CONCLUSIONS
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.
Yang
You-Feng
YF
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China.
Yuan
Han-Ying
HY
Liu
Nan-Song
NS
Chen
Xiang-Ling
XL
Gao
Bu-Yu
BY
Lu
Hong
H
Li
Yu-Yang
YY
D015195:Drug Design; D006509:Hepatitis B / Q000628:therapy; D006526:Hepatitis C / Q000628:therapy; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics*; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D011994:Recombinant Proteins; D000077596:Thymalfasin; D013947:Thymosin / Q000031:analogs & derivatives* / Q000235:genetics*
D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013947:Thymosin; D000077596:Thymalfasin