重组人成骨蛋白-1复性条件的优化

目的优化重组人成骨蛋白-1(rhOP-1)包涵体蛋白的复性条件。方法将表达rhOP-1的大肠杆菌菌体在冰浴下超声裂解,分离提取包涵体,用8mol/L尿素溶解,纯化后进行梯度透析复性。利用TotalLab软件分析目的蛋白二聚体的含量,用体内法和体外法测定其生物学活性。结果经纯化后,目的蛋白纯度达97%以上。最佳复性条件为4℃,pH9.0,蛋白浓度为0.4～0.8mg/ml,尿素浓度为2mol/L,L-Arg浓度为0.4mol/L;目的蛋白复性率达75%以上,复性后蛋白具有较高的生物学活性。结论已确定了rhOP-1包涵体梯度透析复性的最佳条件。

Optimization of Condition for Refolding of Recombinant Hmnan Osteogenic Protein-1

Objective To optimize the condition for refolding of recombinant human osteogenic protein-1(rhOP-1)in a form of inclusion body.Methods Split the recombinant E.coli for expression of rhOP-1 by ultrasomication in ice bath and extract the inclusion body.Dissolve the inclusion body with 8 mol/L urea,purify by SP-Sepharose chromatography,refold by gradient dialysis.Analyze the content of target protein dimer by TotalLab software and the biological activity by in vitro and in vivo tests.Results The purity of target protein after purification reached more than 97%.The optimal condition for refolding of target protein was as follows:4℃,pH 9.0,protein concentration 0.4-0.8 mg/ml,urea concentration 2 mol/L,L-Arg concentration 0.4 mol/L.The refolding rate of target protein reached more than 75%,and the refolded protein showed high biological activity.Conclusion The condition for refolding of rhOP-1 inclusion body by gradient dialysis was optimized.