Cloning and expression of <b><i>UbiA</i></b> human gene using innovative methodologies for recombinant protein production in <b>PUAST</b> vector

Introduction: A systematic library of Gal4/UAS-regulated transgenes has proven to be a powerful genetic system for identifying genes and defining pathways in development. This system offers valuable insights that highlight the evolutionary conservation between animals and humans. Objectives: The objective of this study was to clone, express, and characterize the UbiA gene. The research presents a highly effective method for cloning genes, using the UbiA-pcDNA3 gene as a model for mammalian cloning. These genes were then integrated into the PUAST vector of Drosophila, an expression vector and eukaryotic cell system commonly used for producing recombinant proteins. Materials and Methods: UbiA was isolated from human cells, and complementary DNA was synthesized. An oligonucleotide primer pair was designed based on the UbiA gene sequence, incorporating XhoI and Xbal restriction sites at the 5´ end of the forward and reverse primers, respectively. The UbiA gene was then amplified by PCR, cloned into the pcDNA3 plasmid, and the resulting recombinant plasmid was sequenced. Subsequently, the gene was sub cloned into the PUAST vector and expressed in S2 cells as a eukaryotic cell system. Protein determination and verification were conducted through western blotting techniques. Results: Confirmation of UbiA gene cloning into the PUAST vector was achieved through colony-PCR and digestion by enzymes. Cloning and sub cloning techniques validated by enzymatic digestion, along with gene sequencing. The identity between cloned UbiA gene and the identical gene exhibited 99%. We revealed a singular band purified protein through western blotting with 60 kDa size. Conclusion: More protein synthesis of the UbiA gene can be achieved by using the eukaryotic expression system provided by the PUAST vector. This technique has been proven to be a suitable platform and can be instrumental in various applications such as therapeutics, pharmacology, and vaccine development.