Production of RXLR effector proteins for structural analysis by X-ray crystallography

Structural analysis of RXLR effector proteins from oomycete plant pathogens is an emerging area of research. These studies are aimed at understanding the molecular basis of how these proteins manipulate plant cells to promote infection and also to help define how they can lead to activation of the plant innate immune system. Here, we describe a medium-throughput procedure for cloning and expression testing oomycete RXLR proteins in Escherichia coli. We also describe methods for purification of soluble protein and crystallization, with the aim of determining three-dimensional structures by X-ray crystallography. The procedures are generally applicable to any research program where the production of soluble recombinant protein in E. coli has proven difficult, or where there is a desire to evaluate E. coli thoroughly as a host before considering alternative hosts for heterologous expression.