耐碱性蛋白A 色谱配基的构建及性能评价Construction and.PDFVIP

研究论文 DOI:10.11949/j.issn.0438-1157 耐碱性蛋白 A 色谱配基的构建及性能评价1 管志龙，白姝，孙彦，史清洪 （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 ） 摘要：本文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Zʹ 和Zʺ。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Zʹ中α-螺旋含量明 显高于突变结构域Zʺ。在pH 6.0 下，突变结构域Zʹ的热转变温度（Tm ）较结构域Z 提高了3.9°C，而突变结构 域Zʺ的Tm 值仅提高了1.6°C。这表明，突变结构域Zʹ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Zʹ具有更好的耐碱性，其耐碱性在突变结构域Zʹ四串联体 融合蛋白中得到了进一步提升。研究工作为SpA 色谱配基的分子改造提供了新思路。 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 中图分类号：TQ028.8 文献标志码：A 文章编号：0438-1157 （2017 ）00-0000-00 Construction and characteristics of alkali-tolerance mutants of Z domainfor protein A chromatography GUAN Zhilong, BAI Shu, SHI Qinghong (Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China) Abstract ：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 Zʹ domain and Zʺ 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 Zʹ domain had higher α-helix content than Zʺ domain. At pH 6.0, heat transition temperature of Zʹ domain increased by 3.9ºC compared with domain Z whilst heat transition temperature of Zʺ domain increased merely by 1.6ºC. It indicated that t