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Background: Shiga toxins comprise a family of related proteins produced by bacteria Shigella dysenteriae and some strains of Escherichia coli that cause severe clinical manifestations. Severe Shiga toxin intoxication results in Haemolytic-Uremic Syndrome (HUS), up to 50% of HUS patients manifest some degree of renal failure and ~10% of such cases develop permanent renal failure or death. Objective: In present research work production of biologically active rStx from non-toxic rStxA and rStxB subunits were established that can be used in many biomedical applications. Methods: Purification of Shiga toxin from bacteria is a multistep time consuming process resulting in low yield. To overcome this problem, the rStxA and rStxB protein were separately cloned and expressed in E. coli host and purified through affinity chromatography. GST pull-down assay was performed for interaction study between rStxA and pentameric rStxB. The affinity between A and B subunits of reconstituted recombinant Shiga toxin (AB5) was determined by SPR. The biological activity of the toxin was confirmed in Vero cells and mouse lethality assay. Results: The yield of GST-StxA and His6X-StxB obtained after affinity chromatography was estimated to 2 and 5 mg/l, respectively. Samples analyzed in pull down assay revealed two bands of ~58 kDa (rStxA) and ~7.7 kDa (rStxB) on SDS-PAGE. Affinity was confirmed through SPR with KD of 0.85 pM. This rStx produced from 1:5 molar ratio found to be cytotoxic in Vero cell line and resulted lethality in mouse. Conclusions: Large scale production of rStx using the method can facilitate screening and evaluation of small molecule inhibitors for therapeutics development.