Farzaneh Farhad, Mirzaie Sako, Dehnavi Ehsan, Aghaeepoor Mojtaba, Farzaneh Shirin, Pourzardosht Navid, Khalili Saeed
Department of Biochemistry, Faculty of Science, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran.
Gene Transfer Pioneers (GTP) Research Group, Shahid Beheshti University of Medical Sciences. Tehran, Iran.
Iran J Biotechnol. 2023 Apr 1;21(2):e3288. doi: 10.30498/ijb.2023.330285.3288. eCollection 2023 Apr.
Over expression of Reteplase enzyme has already been studies in the periplasmic space of (). However, the role different factors in its expresssin rate remained to be elucidated.
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).
The pET21b plasmid was used to sub-clone the designed reteplase gene. Then, the gene was transformed into 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.
Sequence optimization removed all undesirable sequences of the designed gene. Transformation into 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.
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.
已对瑞替普酶在（此处信息缺失）周质空间中的过表达进行了研究。然而，不同因素在其表达率中的作用仍有待阐明。
光密度（OD）、异丙基-β-D-硫代半乳糖苷（IPTG）浓度和表达时间对蛋白质表达率有显著影响。因此，我们旨在使用响应面法（RSM）确定这些因素在瑞替普酶表达中的最佳水平。
使用pET21b质粒亚克隆设计的瑞替普酶基因。然后，将该基因转化到BL21菌株中。用IPTG诱导表达，并通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分析。实验采用响应面法设计，同时使用实时定量聚合酶链反应（Real time-PCR）评估不同条件的影响。
序列优化去除了设计基因中所有不需要的序列。琼脂糖凝胶上1152 bp的条带证实了向BL21的转化。SDS凝胶上39 kDa的表达条带证实了基因表达。进行20次响应面法设计的实验后，确定IPTG浓度和OD的最佳水平分别为0.34 mM和5.6。此外，表达时间的最佳水平为11.91小时。瑞替普酶过表达回归模型的准确性通过F值等于25.31和极小的概率值[（概率>F）<0.0001]得到证实。实时定量聚合酶链反应结果表明，所进行的计算高度准确。
所得结果表明，IPTG浓度、OD和表达时间对重组瑞替普酶表达的增强有显著影响。据我们所知，这是第一项评估这些因素对瑞替普酶表达综合影响的研究。基于响应面法的进一步实验将为瑞替普酶表达的最佳条件带来新的见解。
