title: Production and characterization of recombinant light chain and carboxyterminal heavy chain fragments of tetanus toxin.
abstract: BACKGROUND
  Light chain (LC) and heavy chain carboxyterminal subdomain (HCC) fragments are the most important parts of tetanus neurotoxin (TeNT) which play key roles in toxicity and binding of TeNT, respectively. In the present study, these two fragments were cloned and expressed in a prokaryotic system and their identity was confirmed using anti-TeNT specific polyclonal and monoclonal antibodies.
  
  
  METHODS
  LC and HCC gene segments were amplified from Clostridium tetani genomic DNA by PCR, cloned into pET28b(+) cloning vector and transformed in Escherichia coli (E. coli) BL21(DE3) expression host. Recombinant proteins were then purified through His-tag using Nickel-based chromatography and characterized by SDS-PAGE, Western blotting and ELISA techniques.
  
  
  RESULTS
  Recombinant light chain and HCC fragments were successfully cloned and expressed in (E. coli) BL21 (DE3). Optimization of the induction protocol resulted in production of high levels of HCC (~35% of total bacterial protein) and to lesser extends of LC (~5%). Reactivity of the His-tag purified proteins with specific polyclonal and monoclonal antibodies confirmed their renatured structure and identity.
  
  
  CONCLUSIONS
  Our results indicate successful cloning and production of recombinant LC and HCC fragments of TeNT. These two recombinant proteins are potentially useful tools for screening and monitoring of anti-TeNT antibody response and vaccine production.
authors.affiliation: Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran ; Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran ; Department of Immunology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran ; These authors equally contributed to this work.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Yousefi
authors.forename: Roya
authors.initials: R
authors.lastname: Khosravi-Eghbal
authors.forename: Azam
authors.initials: A
authors.lastname: Hemmati
authors.forename: Fazel
authors.initials: F
authors.lastname: Shokri
keywords: Fragment C; Light chain; Monoclonal antibody; Tetanus toxin