Purification of Proteins for Crystallographic Applications

One of the most important parameters correlated with success in protein crystallization experiments is sample purity and monodispersity. Heterologous expression systems have allowed investigators to produce engineered proteins in sufficient quantities which simplify the purification process compared with the days when macromolecules had to be extracted from source tissue. Improvements in the areas of chromatographic media and instrumentation have also dramatically improved throughput and protein yields while maintaining analytical resolution. In a drug discovery setting, efforts can be focused on either a single protein or family of proteins. This requires the development and refinement of general purification methods that can be applied to multiple proteins or construct variants until readily crystallizable forms of the target protein are discovered. It is the aim of this chapter to provide a practical introduction to the techniques and methods used to purify proteins for crystallographic applications. Additionally, a protocol describing the expression, purification, and crystallization of the ATP-binding domain of the important cancer target Hsp90 provides the reader with an example of methods that can be adapted to a wider set of crystallographic target proteins.