Efficient Expression of Glutathione Peroxidase with Chimeric tRNA in Amber-less Escherichia coli

The active center of selenium-containing glutathione peroxidase (GPx) is selenocysteine (Sec), which is is biosynthesized on its tRNA in organisms. The decoding of Sec depends on a specific elongation factor and a Sec Insertion Sequence (SECIS) to suppress the UGA codon. The expression of mammalian GPx is extremely difficult with traditional recombinant DNA technology. Recently, a chimeric tRNA (tRNA^UTu) that is compatible with elongation factor Tu (EF-Tu) has made selenoprotein expression easier. In this study, human glutathione peroxidase (hGPx) was expressed in amber-less Escherichia coli C321.ΔA.exp using tRNA^UTu and seven chimeric tRNAs that were constructed on the basis of tRNA^UTu. We found that chimeric tRNA^UTu2, which substitutes the acceptor stem and T-stem of tRNA^UTu with those from tRNA^Sec, enabled the expression of reactive hGPx with high yields. We also found that chimeric tRNA^UTuT6, which has a single base change (A59C) compared to tRNA^UTu, mediated the highest reactive expression of hGPx1. The hGPx1 expressed exists as a tetramer and reacts with positive cooperativity. The SDS-PAGE analysis of hGPx2 produced by tRNA^UTuT6 with or without sodium selenite supplementation showed that the incorporation of Sec is nearly 90%. Our approach enables efficient selenoprotein expression in amber-less Escherichia coli and should enable further characterization of selenoproteins in vitro.