This research aimed to produce an‎d optimize recombinant T7 RNA polymerase (T7RNAP), a key tool in modern biotechnology. T7 RNA polymerase (T7RNAP) is widely used in RNA synthesis, mRNA vaccine development, protein expression systems, an‎d molecular biology studies. Due to limited access to commercial enzymes in the country, high costs, an‎d the presence of impurities such as glycerol that negatively affect cell-free systems, the need for a domestic, cost-effective version of this enzyme has become evident. In this study, the T7RNAP gene was cloned into the vectors pAK-T7RNAP an‎d pET28a-T7RNAP, an‎d the resulting constructs were introduced into various E. coli strains (DH5α for plasmid propagation an‎d BL21 for protein expression). To increase production yield, the enzyme expression was eva‎luated under different conditions, including varying temperatures (37°C an‎d 20°C). Results showed that lowering the temperature to 20°C not only enhanced protein stability an‎d solubility but also enabled the production of higher amounts of soluble recombinant protein. Protein purification through histidine-tag-based systems an‎d chromatography was successful, an‎d the final protein concentration reached around 7 mg per 80 ml of culture medium, indicating potential for industrial-scale production. For functional testing, the recombinant enzyme was eva‎luated in in vitro transcription reactions. The results suggested that the enzyme exhibited activity comparable to that of commercial samples an‎d synthesized RNA of the desired length with high accuracy. A significant finding was the absence of non-specific RNA products, a common issue in commercial enzymes, which is often attributed to the high glycerol content. Additional investigations revealed that glycerol concentrations above 10% led to the formation of unwanted RNA products an‎d a decrease in product quality. Moreover, circular DNA templates yielded higher RNA yields than linear DNA templates, providing an effective strategy for future template design. The findings confirm that domestic production of T7RNAP is feasible, an‎d the recombinant enzyme outperforms commercial samples in terms of purity, activity, an‎d suitability for cell-free protein expression systems. The importance of this outcome lies in cost reduction, scientific an‎d industrial independence, an‎d the establishment of a sustainable infrastructure for the development of novel technologies such as mRNA vaccines, RNA-based therapies, an‎d nanobiotechnology. Overall, this research not only presents a practical route for producing key biotechnological enzymes but also paves the way for innovative methods in protein expression, RNA synthesis, an‎d future therapeutic applications. The results indicate the high potential of this enzyme to meet the countryʹs research an‎d industrial needs, providing a foundation for national production of RNA-based kits an‎d products.

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مهديه غلامي

ابوذر سورني , حسين رضوي زادگان جهرمي