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

• DOI: 10.1021/acssynbio.7b00290
• Publication Date: 2017-09-03T19:27:17Z
• Authors: Zhenlin Fan; Jian Song; Tuchen Guan; Xiuxiu Lv; Jingyan Wei
• Keywords: Glutathione Peroxidase; Spectrometry, Mass, Electrospray Ionization; 0303 health sciences; Peptide Elongation Factor Tu; Recombinant Proteins; Selenocysteine; 3. Good health; 03 medical and health sciences; RNA, Transfer; Codon, Terminator; Escherichia coli; Humans; Nucleic Acid Conformation; Electrophoresis, Polyacrylamide Gel

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 (tRNAUTu) 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 tRNAUTu and seven chimeric tRNAs that were constructed on the basis of tRNAUTu. We found that chimeric tRNAUTu2, which substitutes the acceptor stem and T-stem of tRNAUTu with those from tRNASec, enabled the expression of reactive hGPx with high yields. We also found that chimeric tRNAUTuT6, which has a single base change (A59C) compared to tRNAUTu, 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 tRNAUTuT6 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.