Cera-Manjarres, Andry R.

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    Use of crosslinked hydrogels for the control of macromolecular crystallization
    (2012) Cera-Manjarres, Andry R.; Torres-Lugo, Madeline; College of Engineering; Lopez Garriga, Juan; Ortiz Bermudez, Patricia; Department of Chemical Engineering; Santiago Gabrielini, Wilma
    Crosslinked hydrogels in protein crystallization is an area of continuous growth. The counterdiffusion technique is a novel method for the crystallization of proteins, which entails the minimization of convection effects, resulting in high quality crystals. The three layer implementation of this technique in capillary tubes with different diameters has been accepted for the crystallization of proteins, by using a precipitating solution which diffuses through a protein chamber containing a gel matrix that controls the diffusion process. The diffusional process using such systems can be controlled but cannot be widely manipulated. The use of crosslinked hydrogels in the middle chamber can be used to control the diffusion of the precipitant and the number and size of crystals. This research focuses on the role of the crosslinked hydrogels, which control the nucleation process in the proposed protein crystallization. The effects of polymer concentration and hydrogel mesh size on protein crystal size were studied. Some key experimental details of the proposed method were: (i) to prepare pre-polymer solutions at different concentration using protein solution as the dissolvent. (ii) to employ membranes or plugs of different polymer concentrations using water as the dissolvent to control diffusion of the precipitating agent. The above solutions were exposed to a UV light source to induce polymerization. The supersaturation wave in the protein chamber was followed indirectly by recording the crystallization front advance and the crystal size and morphology along the protein chamber. The polymer and proteins used in this study were Poly-ethyleneglycol, insulin, glucose isomerase and lysozyme. The results showed that when lysozyme protein was employed, the nucleation and crystal growth could be controlled by changing the polymer composition in the plug and the protein chamber respectively. In the case of insulin and glucose isomerase it was found that it is possible to obtain crystals of previously reported morphologies by using PEG hydrogel. Mesh sizes were also measured and they varied from 10-20 Å. This research led to the conclusion that the use of crosslinked hydrogels is a potential option for macromolecular crystallization, given that diffusion control can be readily manipulated and the polymer hydrogel does not affect protein crystal quality.