title: Extracellular expression and antiviral activity of a bovine interferon-alpha through codon optimization in Pichia pastoris.
abstract: Interferons (IFNs) are the primary line of defense against infectious agents. In particular, IFN-α is an important antiviral cytokine and has a wide range of immune-modulating functions. Porcine and human IFN-α have been successfully prepared and play important roles in the prevention and therapy of viral diseases. To date, there has been limited applied research on bovine IFN-α. To achieve high-level expression of recombinant bovine IFN-α (bIFN-α) in Pichia pastoris for large-scale application, the bIFN-α gene was optimized and synthesized on the basis of codon bias of P. pastoris. Optimized bIFN-α (opti-bIFN-α) was successfully expressed in P. pastoris and directly secreted into the culture supernatant. The amount of extracellular soluble opti-bIFN-α was observed to be 200μg/mL in a shake flask. Expression efficiency of opti-bIFN-α was found to be about three times that of wild-type bIFN-α when the expression yield was compared at the same copies of the targeted gene. In addition, both the original cultural supernatant and purified opti-bIFN-α showed strong antiviral activity in MDBK cells (2×10(6)AU/mL and 1×10(7)AU/mg, respectively) and IBRS-2 cells (3×10(5)AU/mL and 1.5×10(6)AU/mg, respectively) against a recombinant vesicular stomatitis virus expressing the green fluorescence protein. In this study, we demonstrated high-level extracellular expression of opti-bIFN-α by P. pastoris. To the best of our knowledge, the opti-bIFN-α yield observed in this study is the highest to be reported to date. Our results demonstrated that the extracellular opti-bIFN-α with strong antiviral activity could be easily prepared and purified at a low cost and that it may be a potential biological therapeutic drug against bovine viral infections.
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China. Electronic address: ybtu1976@163.com.
authors.forename: Yabin
authors.initials: Y
authors.lastname: Tu
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Yanqun
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Weiye
authors.initials: W
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Chenggang
authors.initials: C
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Shujie
authors.initials: S
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
authors.forename: Zhigao
authors.initials: Z
authors.lastname: Bu
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China. Electronic address: cai139@hvri.ac.cn.
authors.forename: Xuehui
authors.initials: X
authors.lastname: Cai
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003062:Codon; D003470:Culture Media / Q000737:chemistry; D015870:Gene Expression; D007155:Immunologic Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D016898:Interferon-alpha / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D018116:Vesiculovirus / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D003062:Codon; D003470:Culture Media; D007155:Immunologic Factors; D016898:Interferon-alpha; D011994:Recombinant Proteins
keywords: Antiviral activity; Bovine; Codon optimization; Extracellular expression; Interferon-σ; Pichia pastoris