Crystal screening

Supersaturation and protein phases

Supersaturated solution is needed to initiate crystallization of any compound. Typical methods for creating supersaturation in protein solution are: (1) instantaneous mixing of the protein solution with a precipitant solution; and (2) gradual changing of precipitant concentration, pH or temperature.

Supersaturation is a thermodynamically unstable state. When it returns to equilibrium, various phases (amorphous precipitate, liquid droplets, gel or crystals) may develop in the protein solution. These phases can be identified by microscopy.

A crystal is a specific, highly-ordered structure. Careful optimization of conditions is needed for producing crystals.

Techniques

After purifying the protein, the crystallizing conditions are explored with any of the micromethods which are convenient to use. Typically 'hanging drop' or 'sitting drop' vapour diffusion methods are used. The screening procedure gives information of the reagents which have potential to produce crystals from the protein. The procedures themselves are not possible in large scale. The idea of the small scale methods is to approach the supersaturation gradually to avoid the formation of amorphous precipitate or large number of tiny crystals.

Crystal morphology and size

Crystalline proteins have various crystal forms like prisms, plates, rods and needles. Even the same protein may crystallize in different crystal forms depending on the crystallization conditions (see Crystal Gallery).

The chemical and physical environment has an effect on crystal size. Typically large number of crystal nuclei form spontaneously at high supersaturation region and clearly less (if any) crystal nuclei form at lower supersaturation region. Quantity of the crystal nuclei will influence on the ultimate size of forming crystals. Crystallization is often controlled by seeding.

Desired crystal size, form and quality depend on requests of handling properties and the end use of protein product. Small crystals or crystal aggregates are suitable for many industrial purposes.