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Biochemical characterization of in-vitro reconstituted biologically active recombinant Shiga toxin.

Author(s):

Vinita Chauhan, Ritika Chauhan, Priyanka Sonkar and Ram Kumar Dhaked  

Abstract:


The Shiga toxins comprise a family of related protein toxins produced by bacteria Shigella dysenteriae and some strains of Escherichia coli that cause severe clinical manifestations. Severe Shiga toxin intoxication results in hemolytic-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. Purification of Shiga toxin from bacteria is a multistep time consuming process resulting in low yield. E. coli clones expressing recombinant A and B subunits (rStxA and rStxB) were established for in vitro reconstitution of recombinant Shiga toxin (rStx). For this, the rStxA and rStxB protein were expressed and purified in different conditions. An interaction between rStxA and pentameric rStxB was established by GST pull-down and surface plasmon resonance biosensor. In vitro reconstituted recombinant Shiga toxin (AB5) showed cytotoxicity towards Vero cells and lethality in mice.

Keywords:

Shiga toxin, ribosome inactivating protein, cytotoxicity, hemolytic uremic syndrome, GST pulls down.

Affiliation:

Biotechnology Division, Defence Research & Development Establishment, Jhansi Road, Gwalior-474002, MP, Biotechnology Division, Defence Research & Development Establishment, Jhansi Road, Gwalior-474002, MP, Biotechnology Division, Defence Research & Development Establishment, Jhansi Road, Gwalior-474002, MP, Biotechnology Division, Defence Research & Development Establishment, Jhansi Road, Gwalior-474002, MP



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