Response Surface Methodology to Optimize the Expression Efficiency of Recombinant Reteplase.

Abstract

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.