An important pathogenic process in enterohemorrhagic Escherichia coli (EHEC)associated diseases is the formation of attaching and effacing (A/E) lesions, which are a typical pathological change in host cells. The classical pathway for A/E lesion formation requires the participation of proteins from both bacteria and host cells, namely intimin, translocated intimin receptor (Tir), insulin receptor tyrosine kinase substrate (IRTKS), Tir coupled cytoskeleton protein (TCCP), ARP3/2, and N-WASP. The interaction between IRTKS and TCCP is mediated by the binding of the SH3 domain of IRTKS (SH3IRTKS) to the proline rich repeat (PRR) domain of TCCP, which is important for the induction of A/E lesions. The inability to efficiently produce the purified target complex has hindered the structural determination of these protein complexes. Here, we report an effective method for the generation of a complex consisting of SH3IRTKS with three PRRTCCP domains. Two recombinant fragments, TCCP3R and TCCP5R, as well as SH3IRTKS, were successfully expressed in soluble form and purified. In addition, methods were established to prepare two different protein complexes, SH3IRTKS-TCCP3R and SH3IRTKS-TCCP5R. These methods are a good foundation for future studies on the crystal structure of TCCP-IRTKS. Meanwhile, recombinant TCCP was shown to directly bind to IRTKS in vitro, which provides additional evidence for the interaction between these two molecules.
Key words: Enterohemorrhagic Escherichia coli, Tir coupled cytoskeleton protein, insulin receptor tyrosine kinase substrate, protein complex.
EHEC, Enterohemorrhagic Escherichia coli; EPEC, enteropathogenicEscherichia coli; Tir, translocated intimin receptor; IRTKS, insulin receptor tyrosine kinase substrate; TCCP, Tir coupled cytoskeleton protein.
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