Selective Protein Separation Based on Charge Anisotropy by Spherical Polyelectrolyte Brushes

Protein purification is of vital importance in the food industry, drug discovery, and other related fields. Among many separation methods, polyelectrolyte (PE)-based phase separation was developed and recognized as a low-cost purification technique. In this work, spherical polyelectrolyte brushes (SPBs) with a high specific surface area were utilized to study the protein accessibility and selective protein binding on highly charged nanoparticles (NPs) as well as the selective phase separation of proteins. The correlation between charge anisotropy, protein binding, and phase separation was investigated on various protein systems including those proteins with similar isoelectric points (pI) such as bovine serum albumin (BSA) and β-lactoglobulin (BLG), proteins with similar molecular weights such as BSA and hemoglobin (HB), and even protein variants (BLG-A and -B) with a tiny difference of amino acids. The nonspecific electrostatic interaction studied by turbidimetric titrations and isothermal calorimetry titration (ITC) indicates a specific binding between proteins and SPBs arising from the charge anisotropy of proteins. An optimized output based on selective protein binding on SPBs could be correlated for efficient protein separation through tuning external conditions including pH and ionic strength. These findings, therefore, proved that phase separation based on selective protein adsorption by SPBs was an efficient alternative for protein separation compared with the traditional practice.