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.
Seed priming has been reported to be a simple technique for enhancing seedling establishment in environment and crop production under stressed condition. In this study, we investigated osmo and hydropriming effect in two salt stressed chickpea cultivars. This research monitors the changes in growth factors, proline content, lipid peroxidation and H 2 O 2 scavenging enzymes activities in osmo (mannitol 4%) and hydro (water) primed plantlets of two salt stressed chickpea cultivars (Arman and Bivanich) under salt treatment (100 mM). Our result shows that osmo and hydropriming under salt stressed, caused improvement in shoots and roots weights in both plantlets cultivars. In both osmo and hydropriming, activities of three major H 2 O 2 scavenging enzymes guaiacol peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT) and proline content were enhanced significantly but lipid peroxidation was reduced. High concentrations of proline accumulation were observed in the Bivanich and had higher H 2 O 2 scavenging enzymes activity improvement than the cv. Arman. Our results show that salt stress causes growth reduction, membrane disorganization, generation of reactive oxygen species and biochemical change.
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