African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5233

Full Length Research Paper

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

Yao Fang, Mingming Jiang, Haiguang Wang, Yan Cheng, Shu Yu, Qian Li, Dongshui Lu, Xuhu Mao, and Jiang Gu*
Department of Clinical Microbiology and Immunology, College of Medical Laboratory Science, Third Military Medical University, Chongqing 400038, China.
Email: [email protected]

  •  Accepted: 10 May 2013
  •  Published: 28 May 2013


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 TCCPwhich 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 coliEPEC, enteropathogenicEscherichia coli; Tir, translocated intimin receptor; IRTKS, insulin receptor tyrosine kinase substrate; TCCPTir coupled cytoskeleton protein.