title: Comparison of Three Escherichia coli Strains in Recombinant Production of Reteplase.
abstract: BACKGROUND
  Escherichia coli (E. coli) is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. Reteplase as a highly disulfide-bonded recombinant protein is an example of difficult to express protein in E. coli.
  
  
  METHODS
  In this study, a codon optimized reteplase gene was synthetically prepared and cloned under the control of an IPTG inducible T7 promoter. The vector was simultaneously transformed and expressed in three different E. coli strains. The ability of strains for expression of this recombinant pharmaceutical was compared. Also, an attempt was made to increase the soluble production of reteplase in SHuffle T7 E. coli with alterations of expression condition like temperature, inducer concentration and oxygen supply.
  
  
  RESULTS
  High amounts of reteplase were expressed as inclusion bodies in all three strains. BL21 (DE3) showed the highest level of expression in inclusion bodies followed by Rosetta-gami (DE3) and Shuffle T7. Changes of expression conditions were insufficient for soluble expression of reteplase in SHuffle T7 as a genetically engineered host for production of disulfide bonded proteins.
  
  
  CONCLUSIONS
  The oxidizing cytoplasm of Rosetta-gami and Shuffle T7 in addition to alterations of cultivation parameters could not result in soluble production of reteplase, although the inclusion bodies produced in these two strains might increase the rate of refolding procedure likely due to formation of folding intermediates.
authors: National Institute of Genetic Engineering and Biotechnology (NIGEB), Shahrak-e Pajoohesh, Tehran, Iran.
authors: Mehrnoosh
authors: M
authors: Fathi-Roudsari
authors: National Institute of Genetic Engineering and Biotechnology (NIGEB), Shahrak-e Pajoohesh, Tehran, Iran.
authors: Asal
authors: A
authors: Akhavian-Tehrani
authors: Neurobiology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors: Nader
authors: N
authors: Maghsoudi
keywords: Escherichia coli; Recombinant proteins; Reteplase; Tissue plasminogen activator