耐碱性蛋白A色谱配基的构建及性能评价  

Construction and characteristics of alkali-tolerance mutants of Z domain for protein A chromatography

作　　者：管志龙 白姝[1] 孙彦[1] 史清洪[1] 

机构地区：[1]系统生物工程教育部重点实验室天津大学化工学院,天津300072

出　　处：《化工学报》2017年第9期3459-3465,共7页CIESC Journal

基　　金：国家自然科学基金项目(21276189;21476166);天津市应用基础研究计划项目(15JCYBJC48500);生化工程国家重点实验室开放基金(2014KF-03)~~

摘　　要：提出了优化分子内作用力提高蛋白A(SpA)的结构域Z分子稳定性的方法,构建了突变结构域Z'和Z''。圆二色光谱分析结果表明,突变结构域具有与结构域Z一致的特征峰,但突变结构域Z'中α-螺旋含量明显高于突变结构域Z''。在pH 6.0下,突变结构域Z'的热转变温度(T_m)较结构域Z提高了3.9℃,而突变结构域Z''的Tm值仅提高了1.6℃。这表明,突变结构域Z'中第12位引入疏水性更大的Ile残基提高了结构域分子的稳定性。色谱介质耐碱性实验结果表明,突变结构域Z'具有更好的耐碱性,其耐碱性在突变结构域Z'四串联体融合蛋白中得到了进一步提升。研究工作为Sp A色谱配基的分子改造提供了新思路。A novel strategy to optimize intramolecular interaction of Z domain was proposed to improve alkali-tolerance of the domain of Staphylococcal protein A（SpA） and two mutants of Z domain, named as Z＇ domain and Z＂ domain, were prepared to evaluate their alkali tolerance as the ligand for protein A chromatography. The result of circular dichroism showed that both the mutants had the identical characteristic peaks to Z domain whilst Z＇ domain had higher α-helix content than Z＇＇ domain. At pH 6.0, heat transition temperature of Z＇ domain increased by 3.9℃ compared with domain Z whilst heat transition temperature of Z＂ domain increased merely by 1.6℃. It indicated that the introduction of more hydrophobic Ile at position 12 in Z＇ domain improved the stability of the Z＇ domain. Using Z, Z＇ and their tetrameric domains as the ligand for protein A adsorbents, adsorption performance of Sp A adsorbents was evaluated after their exposure in alkaline solutions. The results demonstrated that domain Z＇ had a higher alkali tolerance among three domains and the tolerance was further improved by applying a tetrameric Z＇ domain. The research provided a new clue for the development of alkali-tolerance protein A ligand.

关 键 词：抗体 吸附 热力学 蛋白A色谱 示差扫描量热 圆二色光谱 耐碱性 

分 类 号：TQ028.8[化学工程]