Title:Expression, Purification and Immunological Characterization of Recombinant Shiga Toxin A Subunit
Volume: 22
Issue: 9
Author(s): Vinita Chauhan, Dilip Chaudhary, Mula K. Rao and Ram K. Dhaked
Affiliation:
Keywords:
Cytotoxicity, hemolytic uremic syndrome, IgG isotyping, ribosome inactivating protein, shiga toxin.
Abstract: Shiga toxin family comprises toxins belonging to two major groups, Stx1 and Stx2, produced
by the bacteria Shigella dysenteriae and some strains of Escherichia coli. Shiga toxins are the
leading cause of diarrhea associated with life threatening hemolytic uremic syndrome (HUS). StxA is
a ribosome inactivating protein (RIP) which inhibits the protein synthesis in most species of prokaryotes
and eukaryotes. An in vitro expression system has not been reported to produce full-length biological
active StxA subunit; hence substantial progress has been hampered.
In the present study, a DNA fragment (955 bp Gene Bank Accn No HM017965) encoding for subunit A of Stx was amplified
from Shigella dysenteriae type 1 and subsequently cloned in pGEX-5X-2 vector. We successfully produced recombinant
StxA as GST fusion protein in Escherichia coli using pGEX-5X-2-STXA construct under IPTG induction. For the
purpose of immunization the GST-tag was removed by factor Xa mediated endoproteolytic cleavage from GST-StxA.
Antisera raised against rStxA in mice reacted with recombinant purified protein of rStxA and lysate of Shiga toxin. It was
shown that antisera produced against rStxA significantly recognized Stx producing strains of S. dysenteriae and E. coli.
The antiserum produced effectively neutralized the Shiga toxin’s cytotoxicity towards Vero cells.
