减少重组蛋白TP25在Protein L层析中形成多聚体方法的建立
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摘要
目的建立减少重组蛋白TP25在Protein L层析中形成多聚体的方法。方法通过在Protein L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Sephadex G25层析去除洗脱液中Cys。结果去除Protein L层析形成多聚体的最适条件为:用含Cys终浓度为100 mmol/L的20 mmol/L柠檬酸溶液进行直接洗脱,并经0. 5 mmol/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Sephadex G25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Protein L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.
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.
引文
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[2] SU T T,LING W L,LUA W H,et al. The role of antibody Vκframework 3 region towards antigen binding:effects on recombinant production and Protein L binding[J]. Sci Rep,2017,7(1):3766.
[3] ELLERMAN D,MAURER T,SCHEER J M. Assessing activity and conformation of recombinant proteins//Analytical characterization of biotherapeutics[M]. John Wiley&Sons,Inc.,2017:73-109.
[4] GUPTA S K,SHUKLA P. Microbial platform technology for recombinant antibody fragment production:a review[J]. Critical Rev Microbiol,2017,43(1):31-42.
[5] KUMAR N,CHAURASIA S,PATEL R R,et al. Atorvastatin calcium encapsulated eudragit nanoparticles with enhanced oral bioavailability,safety and efficacy profile[J]. Pharm Develop&Technol,2015,22(2):156-167.
[6] WANG M L,XIAO L,ZHOU Q,et al. Preparation of the first Chinese national standard for human albumin[J]. Chin Pharm J,2017,52(6):480-487.(in Chinese)王敏力,肖林,周倩,等.首批人血白蛋白国家参考品的研制[J].中国药学杂志,2017,52(6):480-487.
[7] ZHANG D,CHEN B. Trend and preliminary analysis of the polymer in the human albumin production process[J]. Chin J Blood Transfusion,2016.(in Chinese)张丹,陈蓓.人血白蛋白工艺中多聚体的形成趋势及初步分析[J].中国输血杂志,2016,29(9):909-912.
[8] LV R Y,CHENG L J. Application of mixed mode chromatography to polishing purification of monoclonal antibody[J]. Chin J Biologicals,2011,24(5):609-612.(in Chinese)吕若芸,程立均.新型混合模式层析技术在单克隆抗体精细纯化中的应用[J].中国生物制品学杂志,2011,24(5):609-612.
[9] GAO D,WANG L L,LIN D Q,et al. Evaluating antibody monomer separation from associated aggregates using mixed-mode chromatography[J]. J Chromatogr A,2013,1294(11):70-75.
[10] CHOLLANGI S,PARKER R,SINGH N,et al. Development of robust antibody purification by optimizing protein-A chromatography in combination with precipitation methodologies[J].Biotechnol Bioengi,2015,112(11):2292-2304.
[11] LIU H,FANG G,WU H,et al. L-Cysteine production in Escherichia coli based on rational metabolic engineering and modular strategy[J]. Biotechnol J,2018,13(5):1700695.
[12] MARTINS V D O,GODOI V C,BRUNO F D S,et al. Effects of pH and salt concentration on stability of a protein G variant using coarse-grained models[J]. Biophys J,2018,114(1):65-75.