The production of heterologous selenoproteins in Escherichia coli necessitates the design of a secondary structure in the mRNA forming a selenocysteine insertion sequence (SECIS) element compatible with SelB, the elongation factor for selenocysteine insertion at a predefined UGA codon. SelB competes with release factor 2 (RF2) catalyzing translational termination at UGA. Stoichiometry between mRNA, the SelB elongation factor, and RF2 is thereby important, whereas other expression conditions affecting the yield of recombinant selenoproteins have been poorly assessed. Here we expressed the rat selenoprotein thioredoxin reductase, with titrated levels of the selenoprotein mRNA under diverse growth conditions, with or without cotransformation of the accessory bacterial selA, selB, and selC genes. Titration of the selenoprotein mRNA with a pBAD promoter was performed in both TOP10 and BW27783 cells, which unexpectedly could not improve yield or specific activity compared to that achieved in our prior studies. Guided by principal component analysis, we instead discovered that the most efficient bacterial selenoprotein production conditions were obtained with the high-transcription T7lac-driven pET vector system in presence of the selA, selB, and selC genes, with induction of production at late exponential phase. About 40 mg of rat thioredoxin reductase with 50% selenocysteine content could thereby be produced per liter bacterial culture. These findings clearly illustrate the ability of E. coli to upregulate the selenocysteine incorporation machinery on demand and that this is furthermore strongly augmented in late exponential phase. This study also demonstrates that E. coli can indeed be utilized as cell factories for highly efficient production of heterologous selenoproteins such as rat thioredoxin reductase.Many organisms express selenoproteins, carrying a selenocysteine residue, the 21st naturally occurring amino acid (7,16,26,34). Selenocysteine is cotranslationally inserted at the position of an opal (UGA) codon, which normally confers termination of translation. The UGA codon is recoded as selenocysteine by complex translation machineries that differ between gram-negative (7, 16) and gram-positive bacteria (13), archaea (31), and higher eukaryotes (9,12,30). The translation system in Escherichia coli is the most characterized (reviewed in references 7, 16, and 34). Briefly, the mRNA for an E. coli selenoprotein carries a specific sequence after the UGA codon, both encoding the amino acids following the selenocysteine residue and forming a stem-loop secondary structure, a so-called selenocysteine insertion sequence (SECIS) element.The SECIS element binds the SelB elongation factor, the selB gene product. SelB is homologous to elongation factor Tu (EF-Tu) but, in addition, binds the loop of the SECIS element through an additional C-terminal domain. In terms of tRNA substrate, SelB is only functional with the selenocysteine-specific tRNA Sec , the selC gene product, in its selenocysteinylated form. By analogy with ...

The pTRAF provides new opportunities for in-depth studies of transcription factor activities. In this study, we found that upon challenges of cells with several redox-perturbing conditions, Nrf2, HIF, and NF-κB are uniquely responsive to separate stimuli, but can also display marked cross talk to each other within single cells. Antioxid. Redox Signal. 26, 229-246.

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Release factor 2 (RF2), encoded by the prfB gene in Escherichia coli, catalyzes translational termination at UGA and UAA codons. Termination at UGA competes with selenocysteine (Sec) incorporation at Sec-dedicated UGA codons, and RF2 thereby counteracts expression of selenoproteins. prfB is an essential gene in E. coli and can therefore not be removed in order to increase yield of recombinant selenoproteins. We therefore constructed an E. coli strain with the endogenous chromosomal promoter of prfB replaced with the titratable P BAD promoter. Knockdown of prfB expression gave a bacteriostatic effect, while two-to sevenfold overexpression of RF2 resulted in a slightly lowered growth rate in late exponential phase. In a turbidostatic fermentor system the simultaneous impact of prfB knockdown on growth and recombinant selenoprotein expression was subsequently studied, using production of mammalian thioredoxin reductase as model system. This showed that lowering the levels of RF2 correlated directly with increasing Sec incorporation specificity, while also affecting total selenoprotein yield concomitant with a lower growth rate. This study thus demonstrates that expression of prfB can be titrated through targeted exchange of the native promoter with a P BAD -promoter and that knockdown of RF2 can result in almost full efficiency of Sec incorporation at the cost of lower total selenoprotein yield.Studies involving titration of expression of essential Escherichia coli genes must be carefully conducted and naturally cannot be performed using a direct gene knockout procedure. It was shown early that the P BAD promoter is suitable for titration or conditional knockdown of essential genes when used in plasmid-driven complementation of chromosomal gene deletions (13). Recently, additional approaches for chromosomal integration of P BAD aiming at titration and knockdown of essential genes have been described (25,26). One caveat in low-level titration of the transcriptional activity of P BAD (in contrast to knockdown with glucose) is that it generally occurs by an on-or-off effect in individual cells, due to arabinoseinduced induction of the araE transporter, which is needed for uptake of arabinose as an inducer of the P BAD promoter (28). This effect, eliminating the possibility of controlled expressional titration on the cellular level, can be circumvented by use of the BW27783 strain, which has araE constitutively expressed (20). We therefore selected the BW27783 strain as an E. coli host in this work, where we wished to study the impact of the essential (15, 18) prfB gene on selenoprotein expression, utilizing replacement of its native chromosomal promoter with P BAD .Protein translation occurs at the ribosome up to the point of termination of translation, which requires release factor 2 (RF2) as one of two polypeptide chain release factors. Thus, when the protein translation complex reaches an in-frame termination codon in the mRNA and this is exposed at the A site, binding of either RF1, at UAG or UAA codons, or RF2, a...