Basic Research
Copyright ©2005 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. May 7, 2005; 11(17): 2597-2602
Published online May 7, 2005. doi: 10.3748/wjg.v11.i17.2597
Construction, expression and characterization of human interferon α2b-(G4S)n-thymosin α1 fusion proteins in Pichia pastoris
You-Feng Yang, Han-Ying Yuan, Nan-Song Liu, Xiang-Ling Chen, Bu-Yu Gao, Hong Lu, Yu-Yang Li
You-Feng Yang, Han-Ying Yuan, Nan-Song Liu, Xiang-Ling Chen, Bu-Yu Gao, Hong Lu, Yu-Yang Li, State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China
Author contributions: All authors contributed equally to the work.
Correspondence to: Dr. Hong Lu, State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China. honglv@fudan.edu.cn
Telephone: +86-21-65642505 Fax: +86-21-65643436
Received: September 13, 2004
Revised: September 14, 2004
Accepted: December 3, 2004
Published online: May 7, 2005
Abstract

AIM: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and Tα1 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 IFNα2b-(G4S)n-Tα1 (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 IFNα2b-(G4S)n-Tα1 (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 IFNα2b-(G4S)n-Tα1 (n = 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1 (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 IFNα2b monoclonal antibody and Tα1 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 IFNα2b-(G4S)n-Tα1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNα2b and immunomodulatory activity of Tα1 in vitro. These results will be the basis for further evaluation of the fusion proteins’ function in vivo.

Keywords: Fusion protein; Interferon α2b; Thymosin α1; Antiviral assay; E-rosette assay