Refolding of proteins from inclusion bodies is favored by a diminished hydrophobic effect at elevated pressures

Abstract

The application of high hydrostatic pressure is an effective tool to promote dissolution and refolding of protein from aggregates and inclusion bodies while minimizing reaggregation. In this study we explored the mechanism of high-pressure protein refolding by quantitatively assessing the magnitude of the protein-protein interactions both at atmospheric and elevated pressures for T4 lysozyme, in solutions containing various amounts of guanidinium hydrochloride. At atmospheric pressure, the protein- protein interactions are most attractive at moderate guanidinium hydrochloride concentrations (approximately 1-2 molar), as indicated by a minimum in B(22) values. In contrast, at a pressure of 1,000 bar no minimum in B(22) values is observed, indicating that high pressures colloidally stabilize protein against aggregation. Finally, experimental values of refractive index increments as a function of pressure indicate that at high pressures, wetting of the hydrophobic surfaces is favored, resulting in a reduction of the hydrophobic effect. This reduction in the hydrophobic effect reduces the driving force for aggregation of (partially) unfolded protein.

Application of High Hydrostatic Pressure to Dissociate Aggregates and Refold Proteins

“…Computer simulations suggest that hydrophobic interactions in an aqueous solvent result in the formation of "vapor layers" and upon pressure treatment of c.a. This hypothesis was supported by static light scattering experiments that resulted in the observation that the dn/dc of T4 lysozymes increased at pressures of approximately 2000 bar, suggesting a change in water structure near hydrophobic contacts [25]. This hypothesis was supported by static light scattering experiments that resulted in the observation that the dn/dc of T4 lysozymes increased at pressures of approximately 2000 bar, suggesting a change in water structure near hydrophobic contacts [25].…”

“…Mechanistically, high pressure treatment is thought to dissociate aggregates by the insertion of water molecules within hydrophobic protein-protein interfaces [23,25]. This hypothesis has been developed by comparison of the adiabatic compressibility of aggregates relative to native proteins [23], computer simulations of water/methane interactions as a function of pressure [26,27], and analysis of changes in refractive index as a function of concentration (dn/dc values) at elevated pressures.…”

“…In contrast, hydrogen bonding and secondary structures are not affected by elevated pressures [14,31,33]. 2000 bar decreases protein-protein interactions of T4 lysozymes relative to chaotrope solutions containing 1-3 M guanidine HCl [25]. The second viral coefficient (B 22 ) provides a measurement of protein-protein interactions determined from the non-ideality of the protein in solution [35].…”

High‐pressure refolding of human vascular endothelial growth factor (VEGF) recombinantly expressed in bacterial inclusion bodies: Refolding optimization, and feasibility assessment

“…Secondly, properly folded rhVEGF is also a covalent homodimer, which requires assembly of the subunits by hydrophobic interaction and disulfide formation. Based on Crisman and Randolph's stipulation that hydrostatic pressure reduces protein-protein interactions through hydration of hydrophobic surfaces and thus a reduction of hydrophobic effect, 19 one might expect refolding of multimeric proteins with high-pressure to require greater optimization due to the balance between solvating aggregates and forming dimer. Of the two published examples of concentration-dependent refolding, IFN-c is a native, noncovalent homodimer.…”

Refolding active human DNA polymerase ν from inclusion bodies

“…Several groups have implemented high throughput screens to identify optimal protein refolding conditions (15, 16). Recently an alternative approach to solubilizing and refolding insoluble proteins using high hydrostatic pressure was reported (17–20). Under the high pressures used in this process a protein will seek a conformation with the smallest specific volume and become a soluble monomer in the absence of chaotropic agents.…”