کلونینگ و بهینه سازی کدون ژن فنیل آلانین دهیدروژناز در سیستم بیانی E.coli و مقایسه بیان ژن با دو پروموتور T7 و λPR

Abstract Background: This study was performed on the base of importance of phenylalanine dehydrogenase (PheDH) in the outbreak of phenylketonuria (PKU). In order for PKU screening one of the medical applications of enzyme is, identifying and quantifying the exact amount of L-phe in blood serum. The research question was whether the level of protein expression changes after optimization of codon usage in pET-23a and pPR37 expression vectors in E.coli expression system host and the level of expression of the two vectors are different. In this study, experimental approach has been performed. Materials and methods: In this study, pdh gene optimized was used belonging to B. sphaericus that was cloned in the two expression vectors. Codon optimization was performed by online Jcat software. PheDH was purified by Ni-NTA chromatography column. The relative molecular mass of PheDH subunit was about 41KDa by SDS-PAGE 10%.The purity and amount of proteins were assessed by SDS-PAGE. Results: The highest rate of expression was observed at 8h after induction in pETpdh/BL21 (DE3) plysS construction. The level of expression of other construction was very low. Applying this method for purification the specific activity of enzyme was 705 U/mg of protein. Our polyhistidine-tag enzyme can be ideally used for the immobilization on a nickel coated microliter plate surface. Conclusion: On the based of this research, it is recommended for large scale enzyme production. According to the comparison between the two gene construction, presumably pETpdh construction is appropriate for production of PeDH enzyme in large-scale. Keywords: L-phenylalanine dehydrogenase, Expression, Plasmid, Promoter