【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。更多还原

【Abstract】 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 3.5mL/min),and the best concentration of inclusion body,sodium chloride,and GSH/GSSGGSSG were 2.5mg/mL,0.1mol/L,3/0.3mmol/L respectively.Under this condition,the IC50 of rhEndostatin was 5μg/mL,the purity was above 97%,and the active recovery of recombinant protein was 90%.更多还原