title: Cloning and Expression of Functional Reteplase in Escherichia coli TOP10.
abstract: BACKGROUND
  Production of tissue Plasminogen Activator protein (t-PA) in prokaryotes systems has many problems such as the lack of active protein production, multiple purification steps, and renaturation process which has been shown to be costly and time-consuming.
  
  
  METHODS
  In this study, reteplase which is the nonglycosylated active domain of t-PA was used to transform TOP10 Escherichia coli (E. coli) bacteria to resolve some of the above mentioned problems. Reteplase cDNA was ligated into pBAD/gIII plasmid which allowed secretion of this protein into the periplasmic space and would allow the correct formation of disulfide bonds in protein structure. The presence of reteplase cDNA in pBAD/gIII plasmid was confirmed by restriction digestion and sequencing. After induction of the expression of this protein by adding 0.0002% L-Arabinose to the medium, the proteins in periplasmic space as well as the inclusion bodies formed inside the cell were extracted. Subsequently, these proteins were purified and detected by Western blot method.
  
  
  RESULTS
  Our results showed that the amount of reteplase extracted from periplasmic space was much lower than the extracted inclusion bodies and large quantities of the recombinant protein were present as inclusion bodies. Therefore, it was more efficient to use inclusion body extraction method for protein isolation and purification.
  
  
  CONCLUSIONS
  We produced active reteplase after its expression in E. coli TOP10 and isolation of inclusion bodies produced the best results for purification and extraction of this protein.
authors.affiliation: Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Sciences Research Centre, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Khodabakhsh
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Dehghani
authors.forename: Mohammad Farid
authors.initials: MF
authors.lastname: Zia
authors.forename: Mohammad
authors.initials: M
authors.lastname: Rabbani
authors.forename: Hamid Mir Mohammad
authors.initials: HM
authors.lastname: Sadeghi
keywords: Arabinose; Escherichia coli; Gene expression; Reteplase
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