An efficient method to produce recombinant bacterial effector Tir coupled cytoskeleton protein (TCCP) complexed with host protein insulin receptor tyrosine kinase substrate (IRTKS)

Fang Yao, Jiang Mingming, Wang Haiguang, Cheng Yan, Yu Shu, Li Qian, Lu Dongshui, Mao Xuhu, Gu Jiang
2013 African Journal of Microbiology Research  
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.
more » ... interaction between IRTKS and TCCP is mediated by the binding of the SH3 domain of IRTKS (SH3 IRTKS ) 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 SH3 IRTKS with three PRR TCCP domains. Two recombinant fragments, TCCP3R and TCCP5R, as well as SH3 IRTKS , were successfully expressed in soluble form and purified. In addition, methods were established to prepare two different protein complexes, SH3 IRTKS -TCCP3R and SH3 IRTKS -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.
doi:10.5897/ajmr2013.5463 fatcat:lt2k4xbx5reylpjyeseyjtg5tu