Login Register Cart 0
Generic placeholder image

Protein & Peptide Letters

Editor-in-Chief

ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Biochemical Characterization of In vitro Reconstituted Biologically Active Recombinant Shiga Toxin

Author(s): Vinita Chauhan, Ritika Chauhan, Priyanka Sonkar and Ram Kumar Dhaked*

Volume 26, Issue 3, 2019

Page: [227 - 234] Pages: 8

DOI: 10.2174/0929866526666181228161834

Price: $65

Abstract

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.

Keywords: Shiga toxin, ribosome inactivating protein, cytotoxicity, hemolytic uremic syndrome, GST pulls down, Shigella dysenteriae.

« Previous Next »
Graphical Abstract

[1]
Strockbine, N.A.; Jackson, M.P.; Sung, L.M.; Holmes, R.K.; O’Brien, A.D. Cloning and sequencing of the genes for Shiga toxin from S. dysenteriae type 1. J. Bacteriol., 1988, 170, 1116-1112.
[2]
Sandvig, K.; Vandeurs, B. Endocytosis, intracellular transport and cytotoxic action of Shiga toxin and ricin. Physiol. Rev., 1996, 76, 949-966.
[3]
Donohue-Rolfe, A.; Keusch, G.T.; Edson, C.D.; Thorley-Lawson, D.; Jacewicz, M. Pathogenesis of Shigella diarrhea. IX. Simplified-high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies. J. Exp. Med., 1984, 160, 1767-1781.
[4]
Austin, P.R.; Jablonski, P.E.; Bohach, G.A.; Dunker, A.K.; Hovde, C.J. Evidence that the A2 fragment of Shiga-like toxin type I is required for holotoxin integrity. Infect. Immun., 1994, 62, 1768-1775.
[5]
Lindberg, A.A.; Brown, J.E.; Stromberg, N.; Westling-Ryd, M.; Schultz, J.E.; Karlsson, K.A. Identification of the carbohydrate receptor for Shiga toxin produced by Shigella dysenteriae type 1. J. Biol. Chem., 1987, 262, 1779-1785.
[6]
Endo, Y.; Tsurugi, K.; Yutsudo, T.; Takeda, Y.; Ogasawara, T.; Igarashi, K. The site of action of a Verotoxin (VT2) from Escherichia coli O157:H7 and of Shiga toxin on eukaryotic ribosomes: RNA N-glycosidase activity of the toxins. Eur. J. Biochem., 1988, 171, 45-50.
[7]
Suh, J.K.; Hovde, C.J.; Robertus, J.D. Shiga toxin attacks bacterial ribosomes as effectively as eucaryotic ribosomes. Biochemistry, 1998, 37, 9394-9398.
[8]
Yoh, M.; Frimpong, E.K.; Honda, T. Effect of antimicrobial agents, especially fosfomycin, on the production and release of Vero toxin by enterohemorrhagic Escherichia coli O157:H7. FEMS Immunol. Med. Microbiol., 1997, 19, 57-64.
[9]
Bielaszewska, M.; Clarke, I.; Karmali, M.A.; Petric, M. Localization of intravenously administered verocytotoxins (Shiga-like toxins) 1 and 2 in rabbits immunized with homologous and heterologous toxoids and toxin subunits. Infect. Immun., 1997, 65, 2509-2516.
[10]
Keusch, G.T.; Donohue-Rolfe, A.; Jacewicz, M.; Kane, A.V. Shiga toxin: Production and purification. Methods Enzymol., 1988, 165, 152-162.
[11]
Miyake, M.; Utsuno, E.; Noda, M. Binding of avian ovomucoid to Shiga-like toxin type 1 and its utilization for receptor analog affinity chromatography. Anal. Biochem., 1988, 281, 202-208.
[12]
Ishikawa, S.; Kawahara, K.; Kagami, Y.; Isshiki, Y.; Kaneko, A.; Matsui, H.; Okada, N.; Danbara, H. Protection against Shiga toxin 1 challenge by immunization of mice with purified mutant Shiga toxin 1. Infect. Immun., 2003, 71, 3235-3239.
[13]
Smith, M.J.; Teel, L.D.; Carvalho, H.M.; Melton-Celsa, A.R.; O’Brien, A.D. Development of a hybrid Shiga holotoxoid vaccine to elicit heterologous protection against Shiga toxins types 1 and 2. Vaccine, 2006, 24, 4122-4129.
[14]
Zhu, C.; Yu, J.; Yang, Z.; Davis, K.; Rios, H.; Wang, B.; Glenn, G.; Boedeker, E.C. Protection against Shiga toxin-producing Escherichia coli infection by transcutaneous immunization with Shiga toxin subunit B. Clin. Vaccine Immunol., 2008, 15, 359-366.
[15]
Zollman, T.M.; Austin, P.R.; Jablonski, P.E.; Hovde, C.J. Purification of recombinant Shiga-like toxin type 1 A1 fragment from Escherichia coli. Protein Expr. Purif., 1994, 5, 291-295.
[16]
Nishikawa, T.; Fujii, J.; Yoshida, S.; Yutsudo, T. Reconstitution of active recombinant Shiga toxin (Stx)1 from recombinant Stx1-A and Stx1-B subunits independently produced by E. coli clones. FEMS Microbiol. Lett., 1999, 178, 13-18.
[17]
Tu, W.; Li, T.; Wang, Q.; Cai, K.; Gao, X.; Wang, H. A simple method for expression and purification of Shiga toxin 1 (Stx1) with biological activities by using a single-promoter vector and native signal peptide. Biotechnol. Appl. Biochem., 2016, 63(4), 539-545.
[18]
Gupta, P.; Singh, M.K.; Singh, P.; Tiwari, M.; Dhaked, R.K. Antibodies against recombinant Shiga toxin subunit B neutralize Shiga toxin toxicity in HeLa cells. Protein Pept. Lett., 2010, 17, 774-781.
[19]
Chauhan, V.; Chaudhary, D.; Rao, M.K.; Dhaked, R.K. Expression, purification and immunological characterization of recombinant Shiga toxin A subunit. Protein Pept. Lett., 2015, 22, 844-852.
[20]
Engedal, N.; Skotland, T.; Torgersen, M.L.; Sandvig, K. Shiga toxin and its use in targeted cancer therapy and imaging. Microb. Biotechnol., 2011, 4(1), 32-46.
[21]
Chauhan, V.; Chaudhary, D.; Pathak, U.; Saxena, N.; Dhaked, R.K. In silico discovery and validation of amide based small molecule targeting the enzymatic site of Shiga toxin. J. Med. Chem., 2016, 59, 10763-10773.
[22]
Oloomi, M.; Bouzari, S.; Ajdary, S. N-terminus leader sequence of Shiga toxin (Stx) 1 is essential for production of active recombinant protein in E. coli. Protein Pept. Lett., 2006, 13, 509-512.
[23]
Smith, P.K.; Krohn, R.I.; Hermanson, G.T.; Mallia, A.K.; Gartner, F.H.; Provenzano, M.D.; Fujimoto, E.K.; Goeke, N.M.; Olson, B.J.; Klenk, D.C. Measurement of protein using bicinchoninic acid. Anal. Biochem., 1985, 150, 76-85.
[24]
Schuck, P. Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules. Annu. Rev. Biophys. Biomol. Struct., 1997, 26, 541-566.

Rights & Permissions Print Cite
Article Metrics
25
1
© 2024 Bentham Science Publishers | Privacy Policy