Expression and Characterization of Helicobacter pylori Adhesin Protein Linked to Cholera Toxin A2/B Subunits in Escherichia coli

  • Published : 2000.02.01

Abstract

The hpa gene genetically linked to the ctxa2b gene was cloned into the pTED expression vector, and the constructed pTEDhpa/ctxa2b was transformed into Excherichia coli. The fusion protein, the adhesin fused to the cholera toxin subunit A2B (CTXA2B) subunit, was expressed to high levels as inclusion bodies in E. coli. The expressed protein was partially purified by washing the inclusion bodies with working solution containing 8M Urea and 0.1M DTT. Refolding of denatured fusion protein was carried out in the presence of glutathione redox buffer. The refolded fusion protein was purified by size exclusion chromatography. The expressed fusion protein was verified by SDS-PAGE, western blotting with antibodies to both antigenic components of adhesin and cholera toxin subunit B (CTXB), and its N-terminal amino acid sequence was analyzed. The orderly assembled fusion protein was confirmed by modified Gm1-ganglioside ELISA with Abs to adhesin. The results indicate that the purified fusion protein is an Adhesin/CTXA2B protein containing the H. pylori adhesin and $G_{m1}4-ganglioside binding activity of CTXB and the expressed fusion protein in E. coli could be easily purified by the refolding process, Its molecular weight was 168kDa as estimated by size exclusion chromatography. The Adhesin/CTXA2B protein may be used as a candidate antigen for oral immunization against H. pylori.

Keywords

References

  1. Rev. Infect. Dis. v.12 Epidemiology and pathophysiology of Campylobacter pylori infections. Blaser, M. J.
  2. Vaccine v.16 Systemic immunization with urease protects mice against Helicobacter pylori infection. Bruno, G.;M. Gatherine;F. Sophine;H. Jean;V. L. Elisabeth;R. Bachre
  3. Infect. Immun. v.57 Oral acministration of a streptococcal antigen coupled to cholera toxin B subunit evokes strong antibody responeses in salivary glands and extramucosal tissues. Czerkinsky, C.;M. W. Russell;N. Lycke;M. Lindblad;J. Holmgren.
  4. Vaccine v.11 Protection of germ-free mice from ingection by Helicobacter felis after active oral or passive IgA immunization. Czinn, S. J.;A. Cai.;J. G. Nedrud.
  5. J. Bcteriol. v.175 Cloning, nucleotide sequence, and expression of a gene encoding an adhesin subunit protein of Helicobacter pylori. Evans, D. G.;T. K. Karjalainen;D. G. Evans Jr.;D. Y. Graham;C. H. Lee
  6. Infect. Immun. v.57 Serum antibody responses to the Nacetylneuraminyllactose-binding of Compylobacvter pylori. Evans, D. J.;D. G. Evans;K. E. Smith;D. Y. Graham.
  7. J. Immunology v.154 Mucosal immunization with a bacterial protein antigen genetically coupled to cholera toxin A2/B subunits. Hajishengallis, G.;S. K. Hollingshead;T. Koga;M. W. Russell.
  8. Eur. J. Clin. Microbiol. Infect. Dis. v.11 Effect of antimicrobial therapy on the specific serological response to Helicobacter pylori infection. Glupcznsik, Y.;A. Burette;H. Goossens;C. DePrez;J. P. Butzler
  9. Lancet v.339 Eradicating Helicobacter pylori. Glupcznski, Y.;A. Burette.
  10. Nature v.292 Actions of cholera toxin and the prevention and treatment of cholera. Holmgren, J.
  11. $8^th$ International Workshop on Compylobacter, Helicobacter and Related Organisms. Gene cloning of a flagellar sheath protein of Helicobacter pylori Jones, A.;C. J. Luke;C. Cockayne;D. J. Constantinidou;C. W. Penn.;Newell, D. G.(ed);J. Ketley(ed);R. A. Feldman(ed)
  12. J. Microbgiol. Biotechnol. v.8 Separate expression and in vitro activation of recombinant Helicobacter pylori urease structural subunits. Lee, K. K.;J. S. Son;Y. J. Chang;S. U. Kim;K. H. Kim
  13. Infect. Immun. v.61 Receptors affinity purification of a liqid-binding adhesion from Helicobacter pylori. Lingwood, C. A.;G. Wasfy;H. Han;M. Huesca.
  14. Helicobacter pylori : Biology and Clinical Practice. Helicobacter pylori: Receptors and adhesins Lingwood, C. A.;Goodwin C. S.(ed);B. W. Worsley(ed)
  15. J. Infect. Dis. v.157 Restriction endonuclease analysis of the genome of Comphylobacter pylori with a rapid extraction method: Evidence for considerable genomic variation. Majewski, S. I. H.;C. S. Goodwin
  16. Microbios. Lett. v.25 Original isolation of Campylobacter pylordis from human gastric mucosa. Marshall, B. J.;H. Royce;D. I. Annear;C. S. Goodwin;J.W. Pearman;J.R. Warren;J. A. Armstron.
  17. Biochem. J. v.240 The purification of eukaryotic polypeptides synthesized in Escherichia coli. Marston, F. A. O.
  18. J. Immunol. v.133 Cholera toxin B subunit as a carrier protein to stimulate a mucosal immune response. McKenzie, S. J.;J. F. Halsey.
  19. Nature v.306 Cholera toxin genes: Nucleotide sequence, deletion analysis and vaccine development. Mekalanos, J. J.;D. J. Swartsz;G. D. N. Pearson;N. Harford;F. Groyne;M. de Wilde
  20. J. Clin. Immunol. v.7 The common mucosal immune system and current strategies for induction of immune response in external secretions. Mestecky, J.
  21. Vaccine v.16 Protection against Helicobacter pylori ingection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heatlabile enterotoxin(LT) as adjuvant. Michetti, M.;M. Rossi;V. Giannelli;M. M. Giuliani;M. Pizza;S. Censini;A. Covacci;P. Massari;C. Pagliaccia;R. Manetti;J. L. Telford;G. Douce;G. Dougan;R. Rappuoli;P.Ghiara
  22. Proceedings of International Symposium on R&D in the Pharmaceutical Industry of Korea and Hungary Large scale production of recombinant human epidermal growth factor and its development as a new drug Park, M. H.;S. K. Park.;Y. H. Yu;Y. W. Koh;C. H. Kown;S. H. Kim;J. Y. Jung;S. W. Yoon;T. Y. Koo
  23. Protein Expres. Purif v.5 Recombinant cholera toxin B subunit in Escherichia coli:High-level secretion, purification, and characterization. Philippe, S. D.;S. N. Accart;H. V. J. Kolbe;D. Schubnel;B. Bernadette;R. Bischoff;K. Marie-Paule
  24. Infect. Immun. v.59 Distribution, persistence, and recall of serum and salivary antibody reponses to peroral immunization with protein antigen Ⅰ/Ⅱ of Streptococcus mutans coupled to the cholera toxin B subunit. Russell, M. W.;H. Y. Wu
  25. A Laboratory Manual (2nd ed) Molecular Cloning Sambrook, J.;E. F. Fritsch; T. Maniatis.
  26. Epidemiol. Rev. v.13 The epidemiology of Helicobacter pylori infection. Taylor, D. N.;M. J. Blaser
  27. Trends Biotechnol. v.12 Unravelling the pathogenic role of Helicobacter pylori in peptic ulcer; potential for new therapies and vaccines. Telford, J. L.;A. Covacci;P. Ghiara;C. Montecucco;R. Rappuoli
  28. Infect. Immun. v.61 Induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholera toxin B subunit. Wu, H. Y.;M. W. Russell.