Functional expression of porcine interferon-α using a combinational strategy in Pichia pastoris GS115

Publication date: Available online 10 December 2018Source: Enzyme and Microbial TechnologyAuthor(s): Huahua He, Chao Zhai, Meng Mei, Yi Rao, Yao Liu, Fei Wang, Lixin Ma, Zhengbing Jiang, Guimin Zhang, Li YiAbstractPorcine interferon-α (pIFN-α) could be used as the vaccine adjuvant to enhance the antiviral ability of porcine in swine industry. In here, a combinational strategy integrating codon optimization, multiple gene insertion, strong AOX1 promoter, and efficient secretion signal sequence was developed to obtain high-level secreted pIFN-α in Pichia pastoris GS115. The codon optimized pIFN-α shared 76% sequence identity with the original pIFN-α, which was inserted into the P. pastoris genome under AOX1d1-2x201 promoter and MF4I secretion sequence. Our results showed positive correlation between the mRNA and secreted protein levels with the copy numbers of genome-integrated pIFN-α gene in the recombinant P. pastoris strains. The recombinant opt-pIFN-α-6C strain bearing six copies of pIFN-α expression cassette produced the highest extracellular secretion of pIFN-α of 3.2 ± 0.1 mg/mL in shake flask experiment, and 17.0 ± 0.8 mg/mL in a 5 L high-cell-density cultivation after methanol induction of 84 h. The antiviral activity of secreted pIFN-α from the high-cell-density cultivation was determined to be approximately 2.8 ± 0.9&t...
Source: Enzyme and Microbial Technology - Category: Biotechnology Source Type: research

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