β-NGF融合蛋白的表达及其三步层析纯化工艺

Expression of β-NGF Fusion Protein and Purification by Three Steps Column Chromatography Technology

采用无血清培养基培养表达HIR-β-NGF融合蛋白的工程细胞株CHO,收集上清后,通过浓缩、过滤预处理,并采用亲和层析、阴离子交换层析和阳离子交换层析三步法工艺对融合蛋白进行纯化,最后用Fc ELISA、CHOP ELISA、Western-blot等方法对目的蛋白进行质量检测.实验结果表明:HIR-β-NGF融合蛋白相对分子质量约为190kDa;蛋白收率达47.0%;经纯化后杂质DNA含量可降低到2.3 ng/mg以下,CHOP蛋白降到2.3 ng/mg以下,Protein A蛋白降低到49.8 ng/mg;产物仅有3.51%发生聚集;免疫印迹证明具有生物活性.该纯化工艺产品收率高,纯度高,质量检验方法稳定可靠,为大规模生产该蛋白提供参考.

CHO cells expressing HIR-β-NGF fusion protein were cultured in serum free medium,then the supernate was collected and purified with a three-step purification process：affinity chromatography,cation exchange chromatography and anion exchange chromatography following concentration and filtering pretreatment;its quality was controlled by Fc ELISA,CHOP ELISA,Western-blot,etc.Experimental results show that the fusion protein relative molecular mass is about 190 kDa;protein recovery is 47.0%;DNA impurities decrease below 2.3 ng/mg;CHOP decreases below 3.2 ng/mg and Protein A decreases to 49.8 ng/mg;only 3.51% fusion protein aggregates;western blot analysis shows HIR-β-NGF kept biological activity.In conclusion,high recovery and purity are acquired and the purification process is stable and reliable,which could provide reference for large-scale production.