(116n) Lysozyme Crystallization with Neutral Hydrogels Providing Conditions for Counter-Diffusion

Protein crystallization is of much interest due to its importance in describing and understanding a protein as it provides required crystals to perform analytical techniques such as X-ray Diffraction. Improvements in crystallization methods have provided the environment to crystallize some proteins. Counter-diffusion of a precipitating agent has been achieved by suppressing convection and sedimentation by employing common polymers such as agarose. However, such polymers do not provide the means to control the diffusion coefficient of the precipitating agent. This work focuses on the examination of the effects of poly(ethylene glycol) based morphologies on the crystallization of lysozyme. Its main goal is proving that controlling the precipitating agent diffusion coefficient provides a mass transfer profile needed for a better crystallization. Crystallization of lysozyme was achieved utilizing the counter-diffusion method by employing the Granada Crystallization Box (GCB). The precipitating agent was NaCl with acetic acid/sodium acetate buffer at 3.5M and pH of 4.6, respectively, and lysozyme concentration was kept at 40mg/mL. The crystals' growth was studied by varying the morphologies of poly(ethylene glycol) monomethyl ether monomethacrylate (PEGMA) and poly(ethylene glycol) dimethacrylate (PEGDMA). PEGMA (monomer) with MWs of 200, 400, and 1000 g/mol and PEGDMA (cross-linker) of 400 and 1000 g/mol were used. These polymers were synthesized by free radical solution bulk polymerization. The polymer morphologies were modified by varying the monomer and cross-linker ratio. Results indicated that all morphologies were capable of producing crystals. The resulting crystals were examined by X-ray Diffraction. Crystallographic results indicated that crystals obtained in the GCB are of lysozyme and that PEG 1000/1000 crystals had an average mosaicity of 0.227 degrees. These results indicate that cross-linked hydrogel matrices have the potential to provide diffusion controlled environments for crystallization. This study will be repeated using other proteins and, as long term goal, proteins that have never being crystallized.