Selenoproteins contain the amino acid selenocysteine (Sec), co-translationally inserted at a predefined UGA opal codon by means of Sec-specific translation machineries. This process is in E. coli dependent upon binding of the Sec-dedicated elongation factor SelB to a SECIS element in the selenoprotein-encoding mRNA and competes with UGA-directed translational termination. Here we found that Sec can also be efficiently incorporated at a predefined UAG amber codon, thereby competing with RF1 rather than RF2. Subsequently utilizing the RF1 depleted E. coli strain C321.∆A we could produce mammalian selenoprotein thioredoxin reductases (TrxR) with unsurpassed purity and yield. We also found that a SECIS element was no longer absolutely required in such system. Human glutathione peroxidase 1 (GPx1) could thereby also be produced, and we could confirm a previously proposed catalytic tetrad in this selenoprotein. We believe that the versatility of this new UAG-directed production methodology should enable many further studies of diverse selenoproteins.
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