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蛋白质在离子交换吸附剂上的多点吸附调控及其在大分子纯化中的应用。

Regulation of protein multipoint adsorption on ion-exchange adsorbent and its application to the purification of macromolecules.

作者信息

Huang Yongdong, Bi Jingxiu, Zhao Lan, Ma Guanghui, Su Zhiguo

机构信息

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, PR China.

出版信息

Protein Expr Purif. 2010 Dec;74(2):257-63. doi: 10.1016/j.pep.2010.07.002. Epub 2010 Jul 15.

DOI:10.1016/j.pep.2010.07.002
PMID:20637872
Abstract

Ion-exchange chromatography (IEC) using commercial ionic absorbents is a widely used technique for protein purification. Protein adsorption onto ion-exchange adsorbents often involves a multipoint adsorption. In IEC of multimeric proteins or "soft" proteins, the intense multipoint binding would make the further desorption difficult, even lead to the destruction of protein structure and the loss of its biological activity. In this paper, DEAE Sepharose FF adsorbents with controllable ligand densities from 0.020 to 0.183 mmol/ml were synthesized, and then the effect of ligand density on the static ion-exchange adsorption of bovine serum albumin (BSA) onto DEAE Sepharose FF was studied by batch adsorption technique. Steric mass-action (SMA) model was employed to analyze the static adsorption behavior. The results showed that the SMA model parameters, equilibrium constant (K(a)), characteristic number of binding sites (υ) and steric factor (σ), increased gradually with ligand density. Thus, it was feasible to regulate BSA multipoint adsorption by modulating the ligand density of ion-exchange adsorbent. Furthermore, IEC of hepatitis B surface antigen (HBsAg) using DEAE Sepharose FF adsorbents with different ligand densities was carried out, and the activity recovery of HBsAg was improved from 42% to 67% when the ligand density was decreased from 0.183 to 0.020 mmol/ml. Taking the activity recovery of HBsAg, the purification factor and the binding capacity into account, DEAE Sepharose FF with a ligand density of 0.041 mmol/ml was most effective for the purification of HBsAg. Such a strategy may also be beneficial for the purification of macromolecules and multimeric proteins.

摘要

使用商业离子吸附剂的离子交换色谱法(IEC)是一种广泛用于蛋白质纯化的技术。蛋白质吸附到离子交换吸附剂上通常涉及多点吸附。在多聚体蛋白质或“软”蛋白质的IEC中,强烈的多点结合会使进一步解吸变得困难,甚至导致蛋白质结构破坏和生物活性丧失。本文合成了配体密度在0.020至0.183 mmol/ml之间可控的DEAE Sepharose FF吸附剂,然后通过分批吸附技术研究了配体密度对牛血清白蛋白(BSA)在DEAE Sepharose FF上的静态离子交换吸附的影响。采用空间质量作用(SMA)模型分析静态吸附行为。结果表明,SMA模型参数,平衡常数(K(a))、结合位点特征数(υ)和空间因子(σ)随配体密度逐渐增加。因此,通过调节离子交换吸附剂的配体密度来调节BSA多点吸附是可行的。此外,使用不同配体密度的DEAE Sepharose FF吸附剂对乙型肝炎表面抗原(HBsAg)进行IEC,当配体密度从0.183 mmol/ml降至0.020 mmol/ml时,HBsAg的活性回收率从42%提高到67%。综合考虑HBsAg的活性回收率、纯化因子和结合容量,配体密度为0.041 mmol/ml的DEAE Sepharose FF对HBsAg的纯化最有效。这种策略可能也有利于大分子和多聚体蛋白质的纯化。

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