Biotechnology
Leyla Davarvash; Maghsoud Pazhouhandeh; Mohammad Ahmadabadi
Abstract
Introduction: T7 RNA polymerase from T7 bacteriophage has suitable features for using in recombinant protein expression or transcription systems. This enzyme does not require additional factors to identify its promoter, operates very specific and only express the genes under T7 promoter. The features ...
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Introduction: T7 RNA polymerase from T7 bacteriophage has suitable features for using in recombinant protein expression or transcription systems. This enzyme does not require additional factors to identify its promoter, operates very specific and only express the genes under T7 promoter. The features of T7 expression system make it important in biotechnology. Therefore, due to the increasing importance of this enzyme, its mass production at low cost is greatly important. Materials and Methods: In this research, T7 phage was applied and PCR reaction was performed using T7 RNAPol-specific primers containing the cutting sites for restriction enzymes. After the electrophoresis of PCR product, T7 RNAPol gene and pGEX2TK plasmid were digested by BamHI and EcoRI enzymes and after being purified, the ligation was performed between them. The recombinant pGEX-T7RNAPol plasmid was transformed into the E. coli, by electroporation. Bacterial positive colonies containing the recombinant plasmid were selected using PCR. Results: After confirming the obtained plasmids by PCR and digestion, they were sequenced and the sequence analysis confirmed the exact T7 RNA polymerase gene according to NCBI accession sequence. This recombinant plasmid was transformed into Rosetta strain of E. coli in order to express. Its positive colonies were selected and liquid culture was carried out. T7 RNAPol protein expression in bacteria was done by inducing with IPTG and after extraction, it was confirmed by SDS-PAGE. This research is a prerequisite for T7 RNA Polymerase commercial and mass production that will be start after the optimization of its expression and purification methods.
