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

Abstract

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

Challenges of site-specific selenocysteine incorporation into proteins byEscherichia coli

“…Unwanted recoding of other translational stop codons to Sec can be avoided by using the engineered E. coli C321.DA strains having all 321 UAG (amber) stop codons in its genome changed to the UAA (ochre) stop codon, and the corresponding release factor 1 (RF1) deleted [54]. Together with changing the anticodon of tRNA Sec or its EF-Tu-compatible variants to pair with UAG, such strains allow unambiguous reassignment of the amber codon to Sec, further improving selenoprotein yields [52,55]. Alternatively, one can redesign E. coli SelB to work efficiently in a SECIS-independent manner.…”

A Facile Method for Producing Selenocysteine‐Containing Proteins

“…[6c,7] Such EF-Tu-compatible variants of E. coli tRNA Sec have enabled the production of bacterial and human selenoproteins in E. coli cells. [6,8] …”

“…[6c,7] Such EF-Tu -compatible variants of E. coli tRNA Sec have enabled the production of bacterial and human selenoproteins in E. coli cells. [6,8] Sec-tRNA Sec is synthesized in two steps in bacteria:seryl-tRNAs ynthetase (SerRS) attaches serine (Ser) to tRNA Sec , and SelA converts the Ser moiety to Sec [9] by using selenophosphate provided by selenophosphate synthase (SelD). [10] One of the essential identity elements for E. coli SelA is the non-canonical 13-base pair (13-bp) branch (acceptor plus T-stems).…”

Eine einfache Methode zur Produktion von Selenoproteinen

“…[6c, 7] Dies ermöglichte anschließend die Produktion bakterieller und humaner Selenoproteine in E. coli . [6a–d, 8] …”

“…[10] Eines der zentralen Erkennungsmerkmale von tRNA Sec für SelA aus E. coli ist das nichtkanonische, aus 13 Basenpaaren (bp) gebildete Motiv aus Akzeptorarm und T-Stamm, [11] das folglich auch in tRNA Sec aus E. coli und dessen EF-Tu-kompatiblen synthetischen Derivaten (UTu, UTuX, UTu6 und SecUx) enthalten ist. [6a,c,d, 8] Während dieses 13-bp-Motiv allerdings für die SelB-vermittelte Umkodierungsreaktion optimiert ist, [12] sind kanonische tRNAs, die ein lediglich 12 bp langes Strukturmotiv aus Akzeptorarm und T-Stamm bilden, aller Wahrscheinlichkeit nach besser für die EF-Tu-vermittelte kanonische Translation geeignet. [13] …”