title: Comparison of the cytoplasmic and periplasmic production of reteplase in Escherichia coli.
abstract: Reteplase is the recombinant type of tissue plasminogen activator variant. In this study, preplasmic and cytoplasmic (as inclusion body: IBs) production and activity of recombinant reteplase in E. coli were investigated and compared using a pET system (pET22b and pET15b). The cDNA of reteplase was cloned by polymerase chain reaction (PCR) amplification, sequenced, inserted into the vector pET 22b and pET15b, and expressed using isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant plasmid was expressed in the form of inclusion body in pET 15b and in periplasmic space in pET22b. The obtained results of inclusion body extraction from recombinant pET22b (rpET22b) and recombinant pET15b (rpET15b) plasmids using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a band of ~39 kD. However, the obtained results of periplasmic space extraction from rpET22b plasmid showed a very weak band, while cytoplasmic expression of reteplase (pET15b) produced a strong protein band confirmed with Western blotting. Consequently, our results demonstrated that the cytoplasmic expression system is efficient for the production of reteplase protein in prokaryote systems and a high amount of reteplase was obtained from the expressed proteins in the form of IBs. The obtained activity of rpET15b plasmid showed a higher enzyme absorbance in comparison to rpET22b plasmid. This suggests rpET15b as an appropriate candidate for reteplase production.
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, IR, Iran.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Khodabakhsh
authors.forename: Mohammad Farid
authors.initials: MF
authors.lastname: Zia
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Moazen
authors.forename: Mohammad
authors.initials: M
authors.lastname: Rabbani
authors.forename: Hamid Mir Mohammad
authors.initials: HM
authors.lastname: Sadeghi
mesh_terms: D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D002479:Inclusion Bodies / Q000201:enzymology; D019897:Periplasm / Q000201:enzymology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; C087896:reteplase; D010959:Tissue Plasminogen Activator