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

GUAN Zhilong; BAI Shu; SUN Yan; SHI Qinghong

doi:10.11949/j.issn.0438-1157.20170366

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

更新时间：2025-12-10

- Construction and characteristics of alkali-tolerance mutants of Z domain for protein A chromatography
- CIESC Journal Issue 9, Pages: 3459-3465(2017)
- 作者机构：
  
  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
- 作者简介：
- 基金信息：
- DOI：10.11949/j.issn.0438-1157.20170366
  CLC： Q028.8
- Online First：05 September 2017，
  
  Published：05 September 2017
- 稿件说明：
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GUAN Zhilong, BAI Shu, SUN Yan, et al. Construction and characteristics of alkali-tolerance mutants of Z domain for protein A chromatography[J]. CIESC Journal, 2017, (9): 3459-3465.
DOI：

GUAN Zhilong, BAI Shu, SUN Yan, et al. Construction and characteristics of alkali-tolerance mutants of Z domain for protein A chromatography[J]. CIESC Journal, 2017, (9): 3459-3465. DOI： 10.11949/j.issn.0438-1157.20170366.

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（Tm）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SpA色谱配基的分子改造提供了新思路。

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.

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