State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Engineering Research Center for Bio-enzyme Catalysis, Hubei Key Laboratory of Industrial Biotechnology, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, College of Life Sciences, Hubei University, Wuhan, 430062, China.
State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Engineering Research Center for Bio-enzyme Catalysis, Hubei Key Laboratory of Industrial Biotechnology, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, College of Life Sciences, Hubei University, Wuhan, 430062, China.
Enzyme Microb Technol. 2019 Mar;122:55-63. doi: 10.1016/j.enzmictec.2018.12.005. Epub 2018 Dec 10.
Porcine 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 AOX1 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 × 10 IU/mL against the vesicular stomatitis virus (VSV) infected Madin-Darby bovine kidney (MDBK) cells. This strategy provided an efficient way to generate recombinant P. pastoris strains in a non-antibiotics-selection manner, which might also give general guidance for the heterologous expression of other proteins in P. pastoris.
猪干扰素-α（pIFN-α）可用作疫苗佐剂，以增强猪在养猪业中的抗病毒能力。在这里，我们开发了一种组合策略，将密码子优化、多个基因插入、强 AOX1 启动子和有效的分泌信号序列结合起来，以在毕赤酵母 GS115 中获得高水平分泌的 pIFN-α。经密码子优化的 pIFN-α与原始 pIFN-α的序列同一性为 76%，该基因插入到 AOX1 启动子和 MF4I 分泌序列下的毕赤酵母基因组中。我们的结果表明，在重组毕赤酵母菌株中，mRNA 和分泌蛋白水平与基因组整合的 pIFN-α基因的拷贝数之间呈正相关。带有六个 pIFN-α表达盒的重组 opt-pIFN-α-6C 菌株在摇瓶实验中产生了最高的细胞外分泌 pIFN-α，为 3.2±0.1mg/mL，在甲醇诱导 84 小时后的 5L 高密度培养中产生了 17.0±0.8mg/mL。从高密度培养物中分泌的 pIFN-α对感染水疱性口炎病毒（VSV）的 Madin-Darby 牛肾（MDBK）细胞的抗病毒活性约为 2.8±0.9×10 IU/mL。该策略提供了一种在非抗生素选择的情况下产生重组毕赤酵母菌株的有效方法，这也可能为毕赤酵母中其他蛋白质的异源表达提供一般性指导。
