title: Comparison of two recombinant systems for expression of cholera toxin B subunit from Vibrio cholerae.
abstract: OBJECTIVE
  The aim of this study was to assess the production of recombinant cholera toxin B subunit (rCTB) protein in two different expression systems (pAE_ctxB and pQE_ctxB constructs) in Escherichia coli BL21 (DE3).
  
  
  METHODS
  The ctxB fragment was amplified from Vibrio cholerae O 1 ATCC14035 and cloned in pGETM-T easy vector after which it was transformed to E. coli Top 10F' and grown on LB-ampicillin agar medium. Sequence analysis confirmed the complete ctxB gene sequence in the construct which was further subcloned to pQE-30 vector. The construct was subsequently transformed to E. coli M15 (pREP4). The recombinant pAE_ctxB and pQE_ctxB were transformed to competent E. coli BL21 (DE3) cells to express CTB protein.
  
  
  RESULTS
  Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the maximum expression of rCTB in both systems at 5 h after induction and western blot analysis confirmed the presence of recombinant CTB in blotting membranes.
  
  
  CONCLUSIONS
  Expression of rCTB in pAE_ctxB construct was more efficient (15-fold) than pQE_ctxB, and it seems that Lac UV5 in E. coli BL21 (DE3) is more compatible with the former construct. This expression system can be used to produce recombinant CTB in high yield which may enable us to study the oral tolerance or mucosal adjuvant properties of rCTB using animal models.
authors.affiliation: Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: M
authors.initials: M
authors.lastname: Boustanshenas
authors.forename: B
authors.initials: B
authors.lastname: Bakhshi
authors.forename: M
authors.initials: M
authors.lastname: Ghorbani
authors.forename: D
authors.initials: D
authors.lastname: Norouzian
mesh_terms: D002772:Cholera Toxin / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014734:Vibrio cholerae / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D002772:Cholera Toxin