title: Optimization of the Expression of DT386-BR2 Fusion Protein inEscherichia coliusing Response Surface Methodology.
abstract: BACKGROUND
  The aim of this study was to determine the best condition for the production of DT386-BR2 fusion protein, an immunotoxin consisting of catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, for targeted eradication of cancer cells, in terms of the host, cultivation condition, and culture medium.
  
  
  METHODS
  Recombinant pET28a vector containing the codons optimized for the expression of the DT386-BR2 gene was transformed to different strains ofEscherichia coli(E. coliBL21 DE3,E. coliRosetta DE3 andE. coliRosetta-gami 2 DE3), followed by the induction of expression using 1 mM IPTG. Then, the strain with the highest ability to produce recombinant protein was selected and used to determine the best expression condition using response surface methodology (RSM). Finally, the best culture medium was selected.
  
  
  RESULTS
  Densitometry analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expressed fusion protein showed thatE. coliRosetta DE3 produced the highest amounts of the recombinant fusion protein when quantified by 1 mg/ml bovine serum albumin (178.07 μg/ml). Results of RSM also showed the best condition for the production of the recombinant fusion protein was induction with 1 mM IPTG for 2 h at 37°C. Finally, it was established that terrific broth could produce higher amounts of the fusion protein when compared to other culture media.
  
  
  CONCLUSIONS
  In this study, we expressed the recombinant DT386-BR2 fusion protein in large amounts by optimizing the expression host, cultivation condition, and culture medium. This fusion protein will be subjected to purification and evaluation of its cytotoxic effects in future studies.
authors.affiliation: Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Shafiee
authors.affiliation: Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Rabbani
authors.affiliation: Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran; Bioinformatics Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Jahanian-Najafabadi
keywords: DT386-BR2; expression; optimization; response surface methodology