RESUMEN
Background: Over expression of Reteplase enzyme has already been studies in the periplasmic space of Escherichia coli (E. coli). However, the role different factors in its expresssin rate remained to be elucidated. Objectives: Optical cell density (OD), IPTG concentration, and expression time are highly effective in the protein expression rates. Therefore, we aimed to determine the optimum levels of these factors for reteplase expression using response surface methodology (RSM). Materials and Methods: The pET21b plasmid was used to sub-clone the designed reteplase gene. Then, the gene was transformed into E. coli BL21 strain. Induction of expression was done by IPTG and analyzed by the SDS page. experiments were designed using the RMS, while the effects of different conditions were evaluated using the Real time-PCR. Results: Sequence optimization removed all undesirable sequences of the designed gene. Transformation into E. coli BL21 was confirmed with an 1152 bp band on the agarose gel. A 39 kDa expression band on the SDS gel confirmed the gene expression. Performing 20 RSM-designed experiments, the optimum levels for IPTG concentration and OD were determined as 0.34mM and 5.6, respectively. Moreover, the optimum level of expression time was demonstrated to be 11.91 hours. The accuracy of the regression model for reteplase overexpression was confirmed by an F-value equal to 25.31 and a meager probability value [(Prob > F) < 0.0001]. The real-time-PCR results indicated that the performed calculations were highly accurate. Conclusion: The obtained results indicate that IPTG concentration, OD, and expression time are significantly involved in the augmentation of recombinant reteplase expression. To the best of our knowledge, this is the first study to assess the combined effect of these factors on reteplase expression. Further RSM-based experiments would bring about new insights regarding the best conditions for reteplase expression.
RESUMEN
The influenza H1N1 virus is the causative agent of the flu pandemic in the world. Due to the shortage of effective means of control, it is remained the serious threats to public and avian health. To battle the surge of viral outbreaks, new treatments are crucially needed. The viral RNA polymerase, which is responsible for transcription and replication of the RNA genome, is comprised of subunits PA, PB1 and PB2. PA has endonuclease activity and is a well known target for inhibitor and drug design. In the current study, we employed molecular docking, molecular dynamics (MD), MMPBSA, QMMM and ADME studies to find and propose an inhibitor among 11,873 structures against PA. Our molecular docking, MD, MMPBSA and QMMM studies showed that ZINC15340668 has ideal characteristics as a potent PA inhibitor, and can be used in experimental phase and further development. Also, ADME prediction demonstrated that all physico-chemical parameters are within the acceptable range defined for human use. Molecular mechanism based study revealed that upon inhibitor binding; the flexibility of PA backbone is increased. This observation demonstrates the plasticity of PA active site, and it should be noticed in drug design against PA Influenza A viruses. In the final phase of the study, the efficiency of our proposed hit was tested computationally against mutant drug resistant I38T_PA. Our results exhibited that the hit inhibits the I38T_PA in different manner with high potency.