A new project…

At this time, all the Peg hydrogels have been polymerized and lysozyme crystallization is in progress. Because of this, I will grade me with a 3, in other words, with an intermediate progress for my experimentation. We are now waiting for the new crystals to form to obtain new XRD data and establish if the previous results are reliable.

In addition, during March, instead of working with different proteins -as I mentioned in the previous blog- I will be working in a ‘new project’. This project is related to what I have been doing during the last year, but I will implement a different technique with the Granada Crystallization Box.

The GCB was design to do four major experimentations: batch crystallization, crystallization inside a gel, crystallization inside a capillary while providing counter-diffusion with a precipitant agent inside the gel, and counter-diffusion adding the precipitant agent on top of the gel. The crystals that we will get for the new XRD were obtained with the last technique. In the other hand, during March I will be using agarose with the precipitant agent inside the gel as it was done by García-Ruiz, et al. The difference of my new project to the lattest, is that I will include the hydrogels inside the capillaries with the protein to enhance the crystallization process while simulating microgravity.

In general, the progress of my project has been constant and I have not had difficulties achieving my goals. Finally, below is the abstract of the poster that I will be presenting at the Annual Poster Day.

ABTRACT: 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 the 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 and cannot be exposed to organic agents since they are not chemically bonded. This work focuses on the examination of the effects of poly(ethylene glycol) based morphologies on the crystallization of lysozyme. Lysozyme crystallization 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 MW 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 the crystals obtained with agarose, PEG 400/400, PEG 1000/1000, PEG 200/400, PEG 400/1000 had a mosaicity of 0.213, 0.216, 0.243, 0.279, and 1.268 degrees, respectively. These results indicate that cross-linked hydrogel matrices have the potential to provide diffusion controlled environments for crystallization and that this control depends on both, the monomer and the cross-linker. In addition, the crystals obtained with PEG 200/1000 were unstable and of low quality what implies that short monomers polymerized with long cross-linkers do not provide a well-controlled crystallization and vice-versa. This study will be repeated using other proteins and, as long term goal, proteins that have never being crystallized.