Deactivation And Stabilization Of Recombinant Urate Oxidase

This dissertation started with a brief overview of recent advances in protein stabilization and modification techniques as enabling tools for the biopharmaceutical industry. Recombinant urate oxidase (UOX, EC 1.7.3.3), one that is effective for the treatment of gout and hyperuricaemia associated with tumor lysis syndrome but fragile to temperature, proteolysis and acidic environment, was chosen as the model enzyme for the present study of protein modification. A molecular insight into the conformational transition of UOX at above mentioned adverse conditions were pursued via a complementary input of molecular simulation and multidimensional structure characterization. Fabrication of UOX nanogel via aqueous in situ polymerization was attempted while the stability, activity, antigenicity and immunogenicity of UOX nanogel were evaluated, respectively, for the exploration of UOX pharmaceuticals.Thermal deactiviation of recombinant UOX from Aspergillus flavus was studied by means of molecular dynamics (MD) simulations at all-atom level, multidimensional structural characterization, and enzyme activity assay. Intersubunit hydrogen bonding (H bonding) was revealed as the essential interaction underlying UOX conformational transitions and catalytic performance. The unfolding of secondary and tertiary structure was observed by circular dichroism spectrophotometry (CD) and fluorescence spectroscopy (FL) during the thermal deactivation while the integrity of quaternary structure was maintained. It was shown by molecular simulation that the thermal stability of UOX could be enhanced by replacing H2O molecules surrounding UOX surface with solvents of weaker polarity that formed less H bonding with UOX, consequently. The above mentioned deactivation and stabilization mechanism was validated by both molecular simulation and experimental study using methanol and DMSO as the model solvents.A rapid and irreversible deactivation of UOX in acidic environment, accompanied by the dissociation of the subunits, was observed by enzymatic activity assay, FL, isoelectric focusing (IEF) and size exclusion charomatography (SEC). Conformational changes of UOX at acidic pH studied shown by MD simulation indicated that the loss of intersubunit hydrogen bonds led to the dissociation of UOX, followed by the irreversible aggregation of the subunits driven by hydrophobic interactions, as experimentally observed.It was indicated by MD simulation that polyacrylamide network could reinforce the intersubunit H bonding, shield the hydrolytic reaction site and thus give the encapsulated UOX an enhanced stability against high temperature, acidic pH and proteolysis. Encapsulation of UOX into spherical and porous polyacrylamide nanogels ranging from 20 to 40 nm in diameter was accomplished by a two-step in situ polymerization in the presence of oxonic acid potassium salt, an inhibitor of UOX, to protect the active sites. The UOX-polyacrylamide (UOX-PAm) nanogel retained 70% of the initial activity and showed enhanced stability at high temperature and against proteolysis, and the tetrameric structure was maintained at acidic pH.Aimed at improving the biocompatibility of UOX nanogel, PEG was introduced into UOX-PAm by forming a sequential interpenetrating polymer network (IPN) with the polyacrylamide network, as confirmed by SEC, transmission electron microscope (TEM), dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FT-IR). The UOX-PAm/PEG IPN nanogel retained 41% of the initial activity but showed a significantly improved stability against high temperature and proteolysis, as compared to UOX-PAm nanogel. Both UOX-PAm and UOX-PAm/PEG nanogel showed greatly reduced antigenicity and immunogenicity compared to the free UOX.In addition to the molecular insight into the deactivation of UOX at high temperature, acidic pH and proteolysis, which is of fundamental importance for the stabilization and the potential application of UOX, the fabrication of nanogel surrounding a multi-subunit protein, particularly the synthesis of UOX-PAm/PEG IPN nanogel provided effective tools for protein modification. The reduced antigenicity and immunogenicity with increased stability were favorable for the exploration of UOX formulation for clinical application.