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Expression and Purification of a Novel Calcium Binding Protein Necessary for Phytopathogenesis in Xanthomonas strain

Recently, we have identified a gene whose sequence is conserved in several species of Xanthomonas that, when expressed, elicits a hypersensitive response (HR) in tomato plants. Normally, HR elicitation is limited to very specific bacterial‐host pairings, but introduction and expression of this gene into Escherichai coli, a bacterial species that does not normally infect tomato plants, serves to elicit HR, indicating that the encoded protein is crucial to the infectious process. Bioinformatic analysis of the protein, which we have named EfhX (EF‐Hand containing protein from Xanthomonas) reveals that the protein is predicted to contain a single transmembrane α‐helix, spanning amino acids 60 to 81, and two calcium binding domains, termed EF‐Hands, in the carboxy‐terminal domain of the protein. In order to better understand the function of this novel protein, we have cloned the efhX gene from Xanthomonas aurantofolia and expressed it in Escherichia coli so as to obtain quantities of pure protein sufficient to grow protein crystals and determine the structure of the protein via x‐ray diffraction. We have successfully purified the protein to homogeneity (>95%) as determined by SDS‐PAGE and anti‐hexahistidine Western Blot. We have initiated crystallization trials to identify solution conditions suitable for growth of crystals for x‐ray diffraction experiments so that the structure of the protein can be determined.