重组人血管内皮抑素的阳离子交换色谱复性与纯化条件研究  

Refolding and Purification of Recombined Human Endostatin Using Cation Exchange Chromatography

作　　者：陈涛[1,1] 储平[2] 戴清源[1] 李婉珍[1] 李朝军[3] 

机构地区：[1]安徽工程科技学院生物化学工程系,安徽芜湖241000 [2]南京大学生命科学院,江苏南京210008 [3]南京师范大学生命科学院江苏分子医学重点实验室,江苏南京210097

出　　处：《药物生物技术》2010年第2期117-120,共4页Pharmaceutical Biotechnology

基　　金：安徽省教育厅项目资助(KJ2009B147);江苏省博士后科研基金资助(0602025B)

摘　　要：对rhEndostatin包涵体蛋白的色谱复性条件进行研究,摸索出最佳的复性、纯化条件,解决其在大肠杆菌中表达提取时遇到的蛋白变性问题。按实验条件对表达的rhEndostatin包涵体蛋白进行提取、洗涤、变性;利用正交法设计重组蛋白的色谱复性、纯化实验;利用SP-Sepharose Fast Flow阳离子交换柱对rhEndostatin包涵体蛋白进行了色谱复性、纯化。最佳复性条件为rhEndostatin上样浓度为2.5 mg/mL,洗脱体积流量为1/20床体积/min(3.5 mL/min),NaCl浓度0.1 mol/L,GSH/GSSG的浓度比为3/0.3 mmol/L。在此条件下纯度约97%,蛋白回收率为90%,重组蛋白的活性最高,IC50达到5μg/mL。The best refolding and purification condition of recombined human endostatin expressed in engineered Escherichia coli using the cation exchange chromatography was explored in this paper. The inclusion protein was extracted, washed, denatured and partial purified using different buffer firstly, then the condition of chromatography refolding and purification such as elution velocity of flow, concentration of inclusion body, salt, and GSSG were designed by orthogonal method. The denatured recombinant protein was refolded and purified on the column of SP-Sepharose fast flow, and the effects of different refolding facts were analyzed at same time. The best refolding and purification conditions were obtained as fellows： the best elution velocity of flow was 1/20 volume of column/min（about 3.5 mL/min）, and the best concentration of inclusion body, sodium chloride, and GSH/GSSGGSSG were 2.5 mg/mL, 0. 1mol/L, 3/0. 3mmol/L respectively. Under this condition, the IC50 of rhEndostatin was 5 μg/mL, the purity was above 97%, and the active recovery of recombinant protein was 90%.

关 键 词：重组人血管内皮抑素 色谱复性 阳离子交换色谱 

分 类 号：Q51[生物学—生物化学]