(642c) Protein Refolding in Aqueous Two-Phase Systems: Influence of System Design Parameters Using Invertase and Laccase As Model Proteins

Protein refolding represents the limiting step in the recovery of these biomolecules from inclusion bodies and from denaturing operations. Aqueous two-phase systems (ATPS) besides being a primary recovery operation provide good and affordable physicochemical conditions to refold these molecules. On their part, ATPS are formed when mixing two water-soluble substances (a salt and a polymer or two polymers) above a critical concentration giving as result two immiscible phases. When a biological sample is introduced in these systems, hydrophobic interactions, hydrogen bonding, Van der Waals and electrostatic forces between water, the phase forming chemicals and the sample molecules drive the latter ones to fractionate to either the top or bottom phase of the system once thermodynamic equilibrium is achieved. Traditionally, polyethylene glycol (PEG) is one of the most used polymers in developing ATPS strategies. This polymer additionally confers the system with protein refolding characteristics of importance when dealing with denaturized samples since it binds as an intermediate promoting appropriate folding with an analogous function to that of chaperones in in vivo folding. It therefore becomes important to characterize how the different system design parameters such as polymer molecular weight, tie-line length (TLL), volume ratio (V_R), and system pH influence protein partition and refolding from a denatured state assessed by measuring the partition coefficient (K_P, defined as the concentration ratio of the molecule of interest between the top and bottom phases) and the specific activity of the target biomolecules. In this work, 48 different PEG – potassium phosphate ATPS were tested for the partition and refolding of native and denatured invertase and laccase. The model enzymes in both forms have a partition preference for the bottom salt-rich phase where the polymer molecular weight and the tie-line length (TLL) selected for the system play a preponderant role in partition and refolding. Refolding percentages for invertase of up to 100% can be obtained in the polymer-rich phase and between 50% and 75% in the bottom phase. The achieved refolding percentages for laccase were significantly lower since only up to 35% of the loaded samples were correctly refolded in one of the tested systems. This work seeks to evaluate the partition behavior of native and denatured invertase and laccase in PEG – potassium phosphate ATPS and to characterize the influence of system design parameters upon refolding and enzymatic reactivation en route to establishing a potential strategy to define a simplified protocol for the in situ recovery and refolding of these enzyme from different sources.