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Analytical and production seeding techniques

1990; Elsevier BV; Volume: 1; Issue: 1 Linguagem: Inglês

10.1016/s1046-2023(05)80145-8

1095-9130

E.A. Stura, Ian A. Wilson,

Protein Structure and Dynamics

In the crystallization of biomolecules two critical steps, the nucleation of the initial seed and the enlargement of this seed, determine the quality of the final crystal. However, the degree of supersaturation required to nucleate crystals is often higher than the optimal concentration necessary for enlargement. Processes such as precipitation and surface denaturation often compete successfully for protein over the long equilibration times that may precede homogeneous nucleation. Thus, even under conditions suitable for crystal growth, kinetic factors may prevent the onset of nucleation and crystal growth. Alternatively spontaneous nucleation may occur at such high frequency that the resulting microcrystalline precipitates are indistinguishable from their amorphous counterparts by inspection alone. In many situations it is essential to decouple crystal growth from nucleation in order to grow large, regular crystals. One must not only control the number of seeds but also reduce the supersaturation level of the protein, and therefore decrease the incorporation of defects detrimental to crystal quality. Seeding techniques provide a preformed, regular crystal surface onto which further molecules may be aggregated in orderly fashion, generally at a lower degree of supersaturation than is required for nucleation. Such techniques are ideally suited to bypass the nucleation step and hence accomplish the decoupling between nucleation and crystal growth. Three aspects of seeding are discussed: (i) pre-equilibration of the protein solution to be seeded, and the determination of the appropriate supersaturation level for seeding; (ii) the environment and the precautions necessary for any seeding method; (iii) the streak seeding technique and how it can be used in conjunction with microseeding and macroseeding. The use of streak seeding, where an animal whisker is used to rapidly transfer microscopic seeds from a seed source to a pre-equilibrated sitting drop, provides a tool for rapidly optimizing and screening many different conditions. It also provides a means for discriminating between microcrystals and precipitates and is a powerful tool for cross-seeding between related macromolecular species, such as genetic variants.