Protein Crystallization in Agarose Gel with High Strength: Developing an Automated System for Protein Crystallographic Processes

Agarose gel media reduce convection and prevent crystal sedimentation, resulting in the production of high-quality protein crystals. However, crystallographers have only tested agarose gel at concentrations between 0.0 and 0.6% (w/v), where it exhibits low gel strength. The effect of agarose gel on protein structures remains to be elucidated, because only a few structural studies have been performed using gel-grown protein crystals. Here, we crystallize thaumatin and elastase using a variety of crystallization methods in 2.0% (w/v) agarose gels, which are completely gellified and have sufficiently high-strength. This new crystallization approach using semi-solid agarose gels is compatible with several conventional crystallization techniques. A comparison of structures crystallized in non-gelled solution and those crystallized in 2.0% (w/v) agarose gels indicates that the crystal structures were not affected by the high-concentration agarose gels. This technique offers the practical advantages of efficient protection by the semi-solid gel media surrounding the protein crystals, allowing them to be handled and transported without affecting any later crystallographic analysis, and thereby providing an automated system for crystal capturing and mounting.