Protein crystallization in hydrogel beads.
The use of hydrogel beads for the crystallization of proteins is explored in this contribution. The dynamic behaviour of the internal precipitant, protein concentration and relative supersaturation in a gel bead upon submerging the bead in a precipitant solution is characterized theoretically using a transient diffusion model. Agarose and calcium alginate beads have been used for the crystallization of a low-molecular-weight (14.4 kDa, hen egg-white lysozyme) and a high-molecular-weight (636.0 kDa, alcohol oxidase) protein. Entrapment of the protein in the agarose-gel matrix was accomplished using two methods. In the first method, a protein solution is mixed with the agarose sol solution. Gel beads are produced by immersing drops of the protein-agarose sol mixture in a cold paraffin solution. In the second method (which was used to produce calcium alginate and agarose beads), empty gel beads are first produced and subsequently filled with protein by diffusion from a bulk solution into the bead. This latter method has the advantage that a supplementary purification step is introduced (for protein aggregates and large impurities) owing to the diffusion process in the gel matrix. Increasing the precipitant, gel concentration and protein loading resulted in a larger number of crystals of smaller size. Consequently, agarose as well as alginate gels act as nucleation promoters. The supersaturation in a gel bead can be dynamically controlled by changing the precipitant and/or the protein concentration in the bulk solution. Manipulation of the supersaturation allowed the nucleation rate to be varied and led to the production of large crystals which were homogeneously distributed in the gel bead.
Willaert
Ronnie
R
Department of Ultrastructure, Flanders Interuniversity Institute for Biotechnology, Vrije Universiteit Brussel, B-1050 Brussels, Belgium. ronnie.willaert@vub.ac.be
Zegers
Ingrid
I
Wyns
Lode
L
Sleutel
Mike
M
D000464:Alginates; D003460:Crystallization / Q000379:methods*; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D020100:Hydrogels / Q000737:chemistry*; D008863:Microspheres; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012685:Sepharose
D000464:Alginates; D006603:Hexuronic Acids; D020100:Hydrogels; D011506:Proteins; D020723:Glucuronic Acid; D012685:Sepharose