耐碱性蛋白A色谱配基的构建及性能评价

doi:10.11949/j.issn.0438-1157.20170366

化工学报 ›› 2017, Vol. 68 ›› Issue (9): 3459-3465.DOI: 10.11949/j.issn.0438-1157.20170366

耐碱性蛋白A色谱配基的构建及性能评价

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

收稿日期:2017-04-10 修回日期:2017-05-10 出版日期:2017-09-05 发布日期:2017-09-05
通讯作者: 史清洪
基金资助:
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）。

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

GUAN Zhilong, BAI Shu, SUN Yan, SHI Qinghong

Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

Received:2017-04-10 Revised:2017-05-10 Online:2017-09-05 Published:2017-09-05
Contact: 10.11949/j.issn.0438-1157.20170366
Supported by:
supported by the National Natural Science Foundation of China (21276189,21476166),the Tianjin Natural Science Foundation (15JCYBJC48500) and the Open Funding Project of the State Key Laboratory of Biochemical Engineering (2014KF-03).

摘要/Abstract

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T_m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

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 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 SpA 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.

Key words: antibody, adsorption, thermodynamics, protein A chromatography, differential scanning calorimetry, circular dichroism, alkali tolerance

中图分类号:

TQ028.8

管志龙, 白姝, 孙彦, 史清洪. 耐碱性蛋白A色谱配基的构建及性能评价[J]. 化工学报, 2017, 68(9): 3459-3465.

GUAN Zhilong, BAI Shu, SUN Yan, SHI Qinghong. Construction and characteristics of alkali-tolerance mutants of Z domain for protein A chromatography[J]. CIESC Journal, 2017, 68(9): 3459-3465.

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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

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