Expression, Purification, Crystallization Of SpiC/SsaM Protein Complex And IpaB/Mad2B Protein Complex

The gene of novel protein SpiC located on Salmonella pathogenicity island 2 was required for the survival of pathogen within macrophages, and it was sufficient to inhibit endosome-endosome fusion in vitro. Althouth any informations of function couldn't be obtained by alignment of protein sequence, the significant interaction with the protein SsaM which was also located in the same operon of SpiC, suggested that a complex involving SsaM and SpiC might function in the formation of translocators and control other effecter protein's order of secretion. For the detailed functional and structural investigation, SpiC and SsaM were purified together. Since the SsaM protein expressed in Escherichia coli formed inclusion bodies, soluble protein couldn't be obtained, the plasmid cloned in the gene of SpiC with N-terminal His X 6 tag and the plasmid cloned in the gene of SsaM without any tag were transformed together to the competent cell. Soluble SsaM could be pulled down by SpiC with N-terminal His X 6 tag. Co-purified SpiC and SsaM complex was more stable than SpiC alone when the digestion assays with gradient concentration of trypsin were performed on SpiC and the complex respectively. Digested complex was purified again by negative ion exchanging column and gel filtration column, and finally the protein of good behavior and large quantity was obtained. The Shigella pathogenicity effector IpaB delivered through the type III secretion system (TTSS) into host cells played multiple roles in Shigella's invasion, colonizing and overcoming the innate host defense system. IpaB secreted into cytoplast of host cell specifically bond to Mad2B, which was an inhibitor of E3 liagase APC (Anaphase-promoting Complex) involving in the regulation of cell cycle. The active form of APC required for substrate-specific activators Cdc20 and Cdhl recruiting targets towards APC. Thus, Mad2B, possessing Cdc20 or Cdhl, achieved the suppression of APC. But, the pathogenicity effector IpaB binding directly to Mad2B, released the two factors Cdc20 and Cdhl from the combination with Mad2B, and disturbed the schedule of APC's activation. Consequently, cell cycle of host was arrested at M phase; the vigorous proliferation of epithelial progenitors was also interrupted. Therefore, Shigella could avoid this intrinsic defense system formed by intestinal epithelium self-renewal mechanism. In order to investigate detailed mechanism of interaction of IpaB and Mad2B from the protein structural aspect, and illustrate the putative transformational change of Mad2B when binding with IpaB, we purified them together and subjected to crystallization.