Optimization of affinity chromatography using HABA ligand for avidin purification

Abstract

BACKGROUND: Avidin is a commercially available protein used as a specific linker to immobilise molecules. It is currently used in diagnostic kits. The high strength of the avidin-biotin interaction is essential for assay performance; however, it is a drawback for its purification.RESULTS: This work prepared an affinity matrix based on a low-binding affinity ligand, 4-hydroxyazobenzene-2-carboxylic acid (HABA) bound to agarose. The effect of the space arm on the purity and recovery of avidin from a starting material was studied by preparing HABA-agarose matrices bound with three different spacers. The length of the space arm was optimised experimentally in agreement with in silico analysis. The optimal adsorption conditions of avidin were found using response surface methodology, corresponding to citrate buffer 50 mM pH 5.6 NaCl 425 mM, allowing the best adsorption performance and simultaneously reducing non-specific adsorption. Thermodynamic data from Langmuir isotherm reach a dissociation constant of 25x10-6 M, slightly higher than free HABA-Avidin interaction, as was expected. A maximum binding capacity of 16 mg/mL was determined. The analysis of breakthrough curves suggests that the avidin concentration in the feedstock is a sensitive variable to improve the column performance.CONCLUSION: The affinity matrix based on HABA bound to agarose through a space arm of triethylenetetramine was the best material for avidin recovery from an egg-based feedstock solution. The optimal conditions for affinity chromatography described in this report will be applied in an integrated purification process to obtain avidin.