Cloning, Expression and Purification of the Outer Membrane of Tir Protein from E.coli O157: H7 and Evaluation its Structure

Aim and Background

Enterohemorrhagic E. coli O157: H7 is an important human and animal pathogen that causes hemolytic uremic hemorrhagic diarrhea in humans. The binding to host cells is the first step in stabilizing this bacterium through the secretion system of the type (T3SS) III and through the interactions between secretory proteins. Tir is a protein that is transferred to the host cells after expression in the bacterium and via T3SS and is located in the membrane and plays an important role in the bacterial binding to the host. The purpose of this research is to clone a gene construct that has Tir virulence factors.

Specific primer design for tir gene was performed using oligo softwares and gene amplification was performed by PCR reaction on a DNA template. After the enzyme digestion, the gene was inserted into expression vector pET28a; the construct was transformed to the expression host. After confirmation of gene cloning, the Tir recombinant protein was expressed by induced IPTG induction. The western blot analysis was carried out to confirm Tir protein. The gene of tir was cloned inside pET28a vector.

Cloning of tir gene in pET28a vector was performed appropriately between desired sites and after induction protein, the Tir recombinant protein showed appropriate and significant expression. The antibody used in Western blot was also able to properly detect Tir.

After the induction, the protein of Tir showed a remarkable expression of the recombinant protein. As a result, this structure can be used to produce Tir protein for immunological studies against E.coli O157: H7.