响应面法优化fHBP-A蛋白双水相萃取工艺的探究

Optimization of fHBP-A protein double aqueous phase extraction process by response surface methodology

目的采用双水相萃取技术对重组B群脑膜炎奈瑟菌H因子结合蛋白A(fHBP-A)进行分离纯化,研究不同双水相萃取体系对fHBP-A粗提纯化的影响,应用响应面法优化fHBP-A在PEG/(NH_(4))_(2)SO_(4)双水相体系中的萃取工艺。方法首先建立PEG 2000/(NH_(4))_(2)SO_(4)、PEG 4000/(NH_(4))_(2)SO_(4)、PEG 6000/(NH_(4))_(2)SO_(4)和乙醇/(NH_(4))_(2)SO_(4)共4种双水相萃取体系相图;然后考察PEG相对分子质量、PEG质量分数、(NH_(4))_(2)SO_(4)质量分数、萃取体系pH这4个因素对fHBP-A萃取率、下相中fHBP-A纯度的影响;最后采用Box-Behnken试验设计以及响应面分析对fHBP-A双水相萃取工艺进行优化。结果确定fHBP-A双水相萃取的最佳工艺条件为PEG 4000质量分数为28%、萃取体系pH为6.0、(NH_(4))_(2)SO_(4)质量分数为13%。此条件下fHBP-A纯度达到68.11%。结论采用优化的工艺可以较好地提取及初步纯化fHBP-A,为进一步fHBP-A的纯化研究奠定基础。

Objective To purify recombinant group B Neisseria meningitidis H-factor binding protein A(fHBP-A)by using double-phase extraction technique,to study the effect of different double-phase extraction systems on the crude purification of fHBP-A,and to optimize the extraction process of fHBP-A in PEG/(NH_(4))_(2)SO_(4) double-phase aqueous system by applying response surface methodology.Methods Firstly,phase diagrams of four double aqueous phase extraction systems,PEG 2000/(NH_(4))_(2)SO_(4),PEG 4000/(NH_(4))_(2)SO_(4),PEG 6000/(NH_(4))_(2)SO_(4),and ethanol/(NH_(4))_(2)SO_(4) were established.The effects of PEG relative molecular mass,PEG mass fraction,(NH_(4))_(2)SO_(4) mass fraction and pH of extraction system on fHBP-A extraction rate and fHBP-A protein purity in the lower phase were investigated.Finally,the BoxBehnken experimental design and response surface analysis were used to optimize the fHBP-A dual aqueous phase extraction process.Results The optimal extraction conditions for the double aqueous phase extraction of fHBP-A were determined to be 28%mass fraction of PEG 4000,pH 6.0 of the extraction system and 13%mass fraction of(NH_(4))_(2)SO_(4).The purity of fHBP-A reached 68.11%under these conditions.Conclusion The optimized process can be used for the extraction and preliminary purification of fHBP-A,which can lay the experimental foundation for further research on the purification of fHBP-A.