静电耦合亲和层析纯化人血清白蛋白的研究

Study on purification of human serum albumin by novel electrostatic coupled affinity chromatography

人血清白蛋白(Human Serum Albumin,HSA)是人血清中含量最丰富的蛋白,约占血清总蛋白含量的40%~60%。商品化的白蛋白亲和介质(Cibacron Blue F3GA)的配基毒性大、易脱落、载量低,纯化白蛋白回收率较低。本研究制备了一种新型静电耦合亲和介质(DASA-Sepharose,3,5-Diaminobenzoic Acid n-Octyl Succinic Anhydride-Sepharose),并通过一步层析从人血清中高效纯化白蛋白。DASA-Sepharose介质采用3,5-二氨基苯甲酸间隔臂将正辛基琥珀酸酐亲和配体偶联于琼脂糖微球。DASA-Sepharose间隔臂上的羧基通过静电作用吸附白蛋白,进而与正辛基琥珀酸酐亲和配基协同作用实现静电耦合亲和吸附,在保持亲和吸附高特异性的同时大大提高了吸附载量。采用牛血清白蛋白(Bovine Serum Albumin,BSA)为模型蛋白考察了Na Cl浓度和溶液pH对介质静态吸附载量(Qm)的影响,NaCl浓度为0.025~0.06 mol/L时Qm几乎不受影响,而NaCl浓度在0.1 mol/L及以上时Qm显著下降,在pH=5.00的20 mmol/L PBS缓冲体系中,Qm为75.43 mg/mL介质。相比商品化的白蛋白亲和介质Cibacron Blue F3GA的Qm(20 mg/mL介质)提高了约2.7倍。DASA-Sepharose介质能通过一步层析法以较高的收率(94.34%)和纯度(98.20%)从人血清中直接提取白蛋白。圆二色光谱和华法林钠法测定纯化白蛋白的二级结构和小分子药物结合活性,与标准人血清白蛋白基本一致。采用DASA-Sepharose介质可以实现人血清白蛋白的高效分离,本工作为人血清白蛋白的分离纯化提供了新方法。

Human serum albumin(HSA),the most abundant protein in human serum,accounting for about 40%~60%of the total serum protein content.When purified by commercialized albumin affinity medium(Cibacron Blue F3GA),the yield of albumin is low,meanwhile,the ligand of Cibacron Blue F3GA had disadvantages of high toxicity and easily to fall off.In this study,a novel electrostatic coupling affinity medium was prepared and used to purify albumin from human serum by one-step chromatography.New electrostatic coupling affinity medium DASA-Sepharose(3,5-diaminobenzoic acid n-octyl succinic anhydride-Sepharose)was prepared with n-octyl succinic anhydride as affinity ligand coupled to agarose microsphere with 3,5-diaminobenzoic acid as spacer arm.The carboxyl functional group on the DASA-Sepharose spacer arm adsorbed albumin through electrostatic interaction,and then cooperated with the n-octyl succinic anhydride affinity ligand to achieve electrostatic coupling affinity adsorption,which greatly improved the adsorption capacity and maintained the high specificity of affinity adsorption.The effects of different NaCl concentrations and pH values on adsorption equilibrium were investigated with BSA as model protein.When NaCl concentration was 0.025~0.06 mol/L,the saturated adsorption capacity(Qm)was almost unaffected.When NaCl concentration was 0.1 mol/L or above,Qm decreased significantly.The Qm for BSA reached 75.43 mg/mL medium in 20 mmol/L PBS at pH=5.00.Compared with the Qm(20 mg/mL medium)of the commercialized albumin affinity medium Cibacron Blue F3GA,the Qm of DASA-Sepharose increased by about 2.7 times.The HSA could be directly extracted from human serum using the novel electrostatically coupled affinity chromatography medium with high purity(98.20%)and high yield(94.34%).The secondary structure and small molecule drug binding activity of purified albumin were determined by circular dichroism spectrum and warfarin sodium method,which were basically consistent with the standard human serum albumin.The results demonstrated that the electrostatic coupling affinity chromatography can be efficiently used for HSA purification from human serum,which provided a new approach for the HSA separation from plasma.