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Volume 22 Issue 3
June 2007
    Citation: Bin FANG, Bu-feng LIANG, Guang-yuan HE. Synonymous Codon Usage Bias and Overexpression of a Synthetic Gene Encoding Interferon α2b in Yeast .VIROLOGICA SINICA, 2007, 22(3) : 226-232.
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    Synonymous Codon Usage Bias and Overexpression of a Synthetic Gene Encoding Interferon α2b in Yeast

    • 1.

      China-UK HUST-RRes Genetic Engineering and Genomics Joint Lab, Huazhong University of Science and Technology (HUST), Wuhan 430074, China

    • 2.

      Joint R & D Lab (Suzhong Pharma Co., Ltd), IHS, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China

    • Corresponding author: Bu-feng LIANG, f288@21cn.com
      Guang-yuan HE, hegy@hust.edu.cn
    • Received Date: 22 November 2006
      Accepted Date: 17 January 2007
      Available online: 01 June 2007

      Fund Project: The National Natural Science Foundation of China 30370807The National '973' Basic Research Program 2002CB111302

    • To achieve higher level expression of Interferon α2b (IFN-α2b) in methylotrophic yeast (Pichia pastoris), a cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P. pastoris and optimized G+C content. The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA, and then integrated into P. pastoris GS115 genome by electroporation. Multi-copy integrants in the Mut+ recombinant P. pastoris strain were screened by high concentrations of Zeocin. 120 hours culturing allowed expression of the IFN-α2b transformant up to 810 mg/L as detected by SDS-PAGE and quantitative methods. In addition, Western blot analysis showed that the recombinant proteins had immunogenicity. The significant antiviral activity of the recombinant IFN-α2b protein was verified by WISH/ VSV system, which was 3.3×105 IU/mL.
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      5. Hao Y Y, Chu J, Wang Y H, et al. 2006. Expression and aggregation of recombinant human consensus interferon-α mutant by Pichia pastoris. Biotechnol Lett, 28: 905-909.
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      12. Sreekrishna K, Brankamp R G, Kropp K E, et al. 1997. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene, 190: 55-62.
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      13. Xiong A S, Yao Q H, Peng R H, et al. 2006. High level expression of a synthetic gene encoding Peniophoralycii phytase in mythylotrophic yeast Pichia pastoris. Appl Microbiol Biotechnol, 72: 1039-1047.
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      14. Yang J O, Wang J W, Deng R Q, et al. 2003. High-level expression, purification, and characterization of porcine somatotropin in Pichia pastoris. Protein Expression and Purification, 32: 28-34.
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      15. Yang Y F, Yuan H Y, Liu N S, et al. 2005. Construction, expression and characterization of human interferonα2b-(G4S) n-thymosin a1 fusion proteins in Pichia pastoris. World J Gastroenterol, 11: 2597-2602.
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      Synonymous Codon Usage Bias and Overexpression of a Synthetic Gene Encoding Interferon α2b in Yeast

        Corresponding author: Bu-feng LIANG, f288@21cn.com
        Corresponding author: Guang-yuan HE, hegy@hust.edu.cn
      • 1. China-UK HUST-RRes Genetic Engineering and Genomics Joint Lab, Huazhong University of Science and Technology (HUST), Wuhan 430074, China
      • 2. Joint R & D Lab (Suzhong Pharma Co., Ltd), IHS, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
      • Received Date: 22 November 2006
      • Accepted Date: 17 January 2007
      Fund Project:  The National Natural Science Foundation of China 30370807The National '973' Basic Research Program 2002CB111302

      Abstract: To achieve higher level expression of Interferon α2b (IFN-α2b) in methylotrophic yeast (Pichia pastoris), a cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P. pastoris and optimized G+C content. The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA, and then integrated into P. pastoris GS115 genome by electroporation. Multi-copy integrants in the Mut+ recombinant P. pastoris strain were screened by high concentrations of Zeocin. 120 hours culturing allowed expression of the IFN-α2b transformant up to 810 mg/L as detected by SDS-PAGE and quantitative methods. In addition, Western blot analysis showed that the recombinant proteins had immunogenicity. The significant antiviral activity of the recombinant IFN-α2b protein was verified by WISH/ VSV system, which was 3.3×105 IU/mL.

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