核糖核酸酶A的错流超滤复性

Cross-flow ultrafiltration refolding of ribonuclease A

针对重组蛋白高表达形成的包涵体,基于中空纤维膜的错流超滤复性具有较大的应用潜力。为研究不同条件对基于中空纤维膜的错流超滤复性的影响,以核糖核酸酶A(RNase A)为例,以复性过程的活性收率和质量收率作为考察指标,设计了RNase A复性初始浓度(A)、跨膜压力(B)、循环流速(C)的3因素3水平正交实验。结果表明,错流超滤复性过程中,以上复性条件对RNase A的质量收率基本没有影响,对RNase A的活性收率具有显著影响,其较优组合为A1B1C2,即RNase A复性初始浓度为0.3 mg/mL、跨膜压力34.0 kPa、循环流速935 cm/min。在以上复性条件下,RNase A的活性收率可达92.31%,质量收率为77.56%。

Performance of cross-flow ultrafiltration refolding strategy based on hollow fiber membrane for ribonuclease A was investigated.To improve the refolding efficiency,effects of operating conditions of ultrafiltration refolding on the activity yield and mass recovery were studied.Parameters of RNase A concentration(A),transmembrane pressure(B),circulation velocity(C)were chosen as the three test factors,with three level for each factor an L9(34)orthogonal array was implemented.Results of the orthogonal test showed that these three factors had a significant influence on the activity yield of the ultrafiltration refolding of ribonuclease A,whether based on variance analysis or range analysis.However,results of ANOVA(Analysis of Variance)showed that the three factors had no significant effect on mass recovery rate of ultrafiltration of ribonuclease A.Therefore,the optimal conditions for ultrafiltration of ribonuclease A were A1B1C2,that was,the concentration of refolding was 0.3 mg/mL,the transmembrane pressure was 34.0 kPa and the circulation velocity was 935 cm/min.Verification test was performed under the optimal conditions with the active yield of 92.31%and the mass recovery rate of 77.56%,respectively.Furthermore,the reliability of ultrafiltration refolding was confirmed by characterization of refolding products with circular dichroism,reversed-phase high performance liquid chromatography and high-performance gel filtration chromatography.