金黄色葡萄球菌蛋白A突变体的表达纯化与亲和色谱填料的制备  

Expression and Purification of Staphylococcus aureus protein A mutant and Preparation of Affinity Chromatography Medium

作　　者：韩依珂 吴勇 赵伟 张喜全 冯军 HAN Yike;WU Yong;ZHAO Wei;ZHANG Xiquan;FENG Jun(China State Institute of Pharmaceutical Industry,Shanghai 201203;Shanghai Duomirui Bio-Technology Co.,Ltd.,Shanghai 201203;Chia-tai Tianqing Pharmaceutical Co.,Ltd.,Nanjing 210038)

机构地区：[1]中国医药工业研究总院,上海201203 [2]上海多米瑞生物技术有限公司,上海201203 [3]正大天晴药业股份有限公司,江苏南京210038

出　　处：《中国医药工业杂志》2019年第7期755-761,共7页Chinese Journal of Pharmaceuticals

摘　　要：对本实验室构建表达金黄色葡萄球菌蛋白A突变体的工程菌E.coli BL21(DE3)/pET28a-SP1SPA进行高密度发酵和重组蛋白的表达。发酵液经2步色谱纯化后获得纯度98.48%的蛋白A突变体。对其与环氧预活化琼脂糖的键合反应条件进行优化,确定最佳参数为:反应体系中每克基质加入50 mg蛋白A突变体至终浓度16 mg/ml,在pH 8.5条件下反应18 h。性能测定结果表明抗体结合时间为6 min时,自制填料的动态结合载量为75 mg/ml填料;抗体纯化回收率为85%;使用0.1 mol/L氢氧化钠溶液在位清洗20轮(每轮5次运行)后,填料动态结合载量保持在初始载量的97%以上。In this work, we use E. coli as the host for secretory expression of ligands,and an analytical HPLC column to deal with the new tetrameric ligand, which is a mutated staphylococcal protein A mutant with a high affinity and alkali resistant capacity. The engineered E.coli BL21(DE3)/pET28a-SP1SPA expressing Staphylococcus aureus protein A mutant(SpAm) was constructed and preserved in our laboratory. The SpAm was produced by the high-density fermentation, and separated by two-step chromatography to obtain 98.48% SpAm. Then the SpAm was bound to epoxyactivated agarose, the optimum immobilization conditions were determined by optimizing the different conditions under the bonding process between the ligand and the matrix: 50 mg of SpAm(final concentration 16 mg/ml)reacted with 1 g of the medium for 18 h at pH 8.5. Based on the above conditions, the self-made SpAm affinity chromatography medium can be obtained, its dynamic binding capacity, recovery rate and alkali resistance are excellent. The results of performance test showed that the dynamic binding capacity of the self-made medium was 75 mg/ml at 6 min antibody binding time. The recovery rate of antibody was 85%. Moreover, the dynamic binding capacity of the medium remained above 97% of the initial loading capacity after 20 rounds of cleaning-in-place with 0.1 mol/L sodium hydroxide solution (5 times per round). Therefore, it has strong industrial application potential.

关 键 词：金黄色葡萄球菌蛋白A突变体 键合反应条件 动态结合载量 

分 类 号：R977.6[医药卫生—药品] O629.73[医药卫生—药学]