摘要
目的建立减少重组蛋白TP25在ProteinL层析中形成多聚体的方法。方法通过在ProteinL层析纯化重组蛋白TP25使用的洗脱缓冲液中添加半胱氨酸(L-cysteine,Cys)等保护剂,并采用碱性溶液中和洗脱液的方法优化多聚体去除条件,经非还原SDS-PAGE检测纯化过程中目的蛋白多聚体的变化情况,葡聚糖凝胶SephadexG25层析去除洗脱液中Cys。结果去除ProteinL层析形成多聚体的最适条件为:用含Cys终浓度为100mmol/L的20mmol/L柠檬酸溶液进行直接洗脱,并经0.5mmol/L的NaOH溶液中和。该条件下纯化的目的蛋白纯度可达95%以上,且多聚体含量大幅减少。葡聚糖凝胶SephadexG25可有效去除半胱氨酸。结论在洗脱液中添加Cys可有效减少重组蛋白TP25在ProteinL层析纯化过程中多聚体的形成。
Objective To develop a method for decreasing the formation of polymer during Protein L chromatography of recombinant protein TP25. Methods Protectants such as L-cysteine(Cys)were added to the elution buffer for Protein L column chromatography,and the eluent was neutralized with alkaline solution to remove the polymers. Non-reduced SDSPAGE was used to detect the changes of polymer of target protein during purification. The L-cysteine in eluent was removed by Sephadex G25 chromatography. Results The optimal condition for removing the formation of polymer during Protein L chromatography was as follows:the samples were eluted directly with 20 mmol/L citrate solution containing Cys at a final concentration of 100 mmol/L,and the eluent was neutralized with 0. 5 mol/L sodium hydroxide solution.The target protein purified under the optimal condition reached a purity of more than 95%,in which the polymer content decreased remarkably. The Cys was effectively removed by Sephadex G25 chromatography. Conclusion The addition of Cys into elution buffer effectively decreased the formation of polymer during Protein L chromatography.
作者
展波
高星星
曹强庚
彭延杰
ZHAN Bo;GAO Xing-xing;CAO Qiang-geng;PENG Yan-jie(Research and Development Center,Shandong Institute of Biological Products,Tai’an 271000,Shandong Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2019年第7期794-797,802,共5页
Chinese Journal of Biologicals
基金
山东省重点研发计划(2016GSF201188)
山东省医药卫生科技发展计划(2017WS275)
