Characterization and interaction mechanism of selective protein separation by epsilon-polylysine: The role of hydrophobic attraction

ε-polylysine (EPL) is a nature-occurred cationic polypeptide. Despite its antimicrobial effectiveness being deactivated by phase separation from proteins, this article proposes a novel strategy via selective phase separation (SPS) for protein purification at neutral pH conditions. Since a step-by-step phase separation in the presence of EPL was observed in skim milk, a mixed model comprised of β-casein (BCN), β-lactoglobulin (BLG) and bovine serum albumin (BSA) is expected to elucidate this phenomenon where an SPS in terms of the order: BCN > BLG > BSA was presented. To investigate the phase separation mechanism, we first established phase diagrams for different EPL-protein systems and speculated that the pH and ionic strength dependence of EPL-protein complexation are mainly driven by electrostatic attraction (EA) and hydrophobic attraction (HA). Moreover, although the interaction process is enthalpically driven as the ΔH is highly negative characterized by isothermal titration calorimetry (ITC), we found that the phase separation is a kinetic process following the “nucleation and growth” mechanism. By altering the environmental conditions (1 M NaCl and 8 M urea), Dynamic light scattering (DLS) measurement and molecular dynamics simulation (MDs) indirectly illustrated that the mechanism of phase separation of the EPL-protein system consists of a two-stage process at which the initial complexes (clusters) and subsequent precipitations are driven by EA and HA, respectively. Since the irreversible HA drives the phase separation to fall into the primary energy potential well to minimize the free energy of the system, we proposed that the priority of precipitation arising from HA plays a major role in selectivity. This hypothesis suggests that “hydrophobic patches” from proteins rather than “electrostatic patches” could regulate the SPS via EPL, which provides a novel strategy for protein purification.