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.