Protein stability at elevated pressure -- Aggregation, refolding and crystallization.

摘要

The effects of high pressure on protein stability were used to probe the thermodynamic driving forces by which proteins interact to form non-native structure, amorphous precipitate and highly-ordered crystal states. High pressure equipment was designed and implemented to provide novel insight in to these interactions in an attempt to gain a better understanding of both protein behavior at elevated pressures and high pressure applications in the biotechnology industry.;We used wild-type T4 lysozyme and two mutations to elucidate the pressure effects on the conformational and colloidal stability of proteins in solution. From high pressure static light scattering data we found that, at elevated pressures, the reduction of the hydrophobic effect increases the colloidal stability of the protein during chaotrope-induced unfolding. This reduction in the hydrophobic effect reduces the driving force for aggregation allowing (partially) unfolded protein to exist under conditions where aggregation is inhibited due to repulsive monomer-monomer interactions. These results provide insight in to the successful and advantageous use of high hydrostatic pressure to refold protein aggregates and inclusion bodies.;In addition, crystallization of recombinant human growth hormone was studied, as a function of pressure, with PEG as the precipitating agent. We found that iv pressure inhibits crystal formation at lower PEG concentrations whereas, increasing PEG concentration produced solution conditions that favored the formation of crystals at elevated pressures while amorphous precipitate formed in the same solution conditions at atmospheric pressure. Using high pressure analytical techniques, we determined that the decrease in the excluded volume interactions with increasing pressure reduces the thermodynamic driving force for protein crystallization. Increasing the concentration of PEG and protein in solution resulted in an increase in thermodynamic instability resulting in solution conditions that favored crystal formation, over amorphous precipitate, at elevated pressures.;Kinetics of rhGH crystallization at elevated pressures was determined from particle sizing data. An increase in crystallization rate occurred at 250 MPa, relative to crystal formation at 0.1 MPa. Further investigation determined that the increase in crystallization rate is likely due to the increase in the growth rate constant at the higher pressure. Bulk diffusion, adsorption and surface diffusion were discussed as potential reasons for the increase in growth rate constant at elevated pressures. We speculate that the pressure effects on the non-covalent surface interactions (e.g. hydrophobic and electrostatic) increase the surface adsorption and diffusion, ultimately increasing the crystallization rate.