文章摘要
陈晓超;王友;崔文明;李伟松;刘树滔;饶平凡.重组SOD发酵液去除内毒素的效果研究[J].中国食品学报,2019,19(9):172-178
重组SOD发酵液去除内毒素的效果研究
Removal of Endotoxin in Recombinant SOD Fermentation Broth
  
DOI:
中文关键词: 重组蛋白  毕赤酵母  超氧化物歧化酶  蛋白溶液  内毒素  活性炭  Triton X-114
英文关键词: recombinant protein  Pichia pastoris  superoxide dismutase  protein solution  endotoxin  activated carbon  Triton X-114
基金项目:国家重点研发项目(2016YFD04002000);国家自然科学基金面上项目(31271859,31071497);福建省自然科学基金项目(2015J01134)
作者单位
陈晓超;王友;崔文明;李伟松;刘树滔;饶平凡 福州大学生物科学与工程学院福州350108浙江工商大学食品与生物工程学院杭州310018 
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中文摘要:
      目的:去除重组毕赤酵母表达人Cu,Zn-SOD蛋白溶液中的内毒素,为后续活体动物试验和临床应用提供基础数据。方法:采用Triton X-114萃取与活性炭吸附两种方法单独使用或联合使用的纯化方案,比较不同纯化方案对去除重组蛋白溶液中内毒素以及目标蛋白回收率的效果。结果:通过单因素条件优化,确定重组蛋白溶液最佳操作pH为5.0,活性炭最适添加量为5%以及Triton X-114最适添加量为1%(体积分数)。最后确定活性炭吸附和Triton X-114萃取法联合使用的三步法操作工艺,可高效去除重组蛋白溶液中内毒素。终产品内毒素含量降至26.8EU/10 000 U SOD,去除率达到99.8%,目标蛋白回收率为66%,Triton X-114残余量为5.7 μg/mL。结论:纯化后所得重组SOD蛋白溶液符合中国药典规定的注射用针剂中内毒素限量与Triton残余量的质量要求,可用于潜在的保健食品开发。
英文摘要:
      Objective: To evaluate the removal effect of endotoxin in purification process of Cu, Zn-SOD expressed in recombinant Pichia pastoris. Methods: The active carbon adsorption and Triton X-114 phase separation were separately or jointly adopted to remove the endotoxin from Cu, Zn-SOD protein solution expressed in recombinant Pichia pastoris. Results: Both methods worked well but with different efficiency. Removal rate of endotoxin was 99.8% with the protein recovery rate of 66% after active carbon absorption combined with Trition X-114 phase separations. The endotoxin content of the final product was reduced to 26.8 EU/10 000 U determined by Limulus amebocyte lysate (LAL) assay. The residue of Triton X-114 in protein solution was 5.7 μg/mL measured by HPLC chromatography method. Conclusions: Active carbon adsorption combined with Triton X-114 phase separation is a better and economic method to purify the recombinant proteins and remove endotoxin in large scale. Both endotoxin and Triton X-114 in the purified recombinant Cu, Zn-SOD protein solution were effectively removed and their residues were well below the required limits of Chinese Pharmacopoeia.
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