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<p>Avidin is a tetrameric protein known for its strong, specific interaction with biotin. Its multivalent binding properties make it an essential molecular linker in the field of nanobiotechnology. Although avidin can be purified from egg whites, industrial purification is challenging due to its low concentration in the raw material. To address this issue, we have prepared and characterized an affinity chromatography support based on an azo dye. The final structure contains the dye 4′-hydroxy azobenzene-2-carboxylic acid (HABA), which is linked to the Eupergit C matrix via a C-C bond. HABA is prepared by in situ synthesis from anthranilic acid and salicylic acid, and its structure is characterized by spectroscopic techniques. Using the dye affinity matrix, we successfully purified avidin from an egg white protein mixture. The density of the ligand on the solid support impacts the recovery and purity of avidin. Using citrate buffer at pH 5 + 0.25&#xa0;M NaCl as the adsorptive buffer and carbonate buffer at pH 11 + 0.5&#xa0;M NaCl as the elution buffer, we achieved a one-step recovery of avidin with 74% purity. In silico studies revealed that the ligand spacer, cysteamine plus dihydroxy propane, facilitated the proper interaction between avidin and HABA and introduced three new hydrogen bond interactions, restoring the network observed in the avidin/biotin complex.</p>

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Synthesis of an HABA-based affinity matrix for Avidin purification from egg white proteins

  • 期刊论文

  • 发表日期：

    2025年11月19日

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  • P. A. Kikot,
  • D. S. Vazquez,
  • J. G. Carreras,
  • S. L. Laurella,
  • Mariano Grasselli

摘要

Avidin is a tetrameric protein known for its strong, specific interaction with biotin. Its multivalent binding properties make it an essential molecular linker in the field of nanobiotechnology. Although avidin can be purified from egg whites, industrial purification is challenging due to its low concentration in the raw material. To address this issue, we have prepared and characterized an affinity chromatography support based on an azo dye. The final structure contains the dye 4′-hydroxy azobenzene-2-carboxylic acid (HABA), which is linked to the Eupergit C matrix via a C-C bond. HABA is prepared by in situ synthesis from anthranilic acid and salicylic acid, and its structure is characterized by spectroscopic techniques. Using the dye affinity matrix, we successfully purified avidin from an egg white protein mixture. The density of the ligand on the solid support impacts the recovery and purity of avidin. Using citrate buffer at pH 5 + 0.25 M NaCl as the adsorptive buffer and carbonate buffer at pH 11 + 0.5 M NaCl as the elution buffer, we achieved a one-step recovery of avidin with 74% purity. In silico studies revealed that the ligand spacer, cysteamine plus dihydroxy propane, facilitated the proper interaction between avidin and HABA and introduced three new hydrogen bond interactions, restoring the network observed in the avidin/biotin complex.