title: Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depletedEscherichia coliHost Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation.
abstract: Selenoproteins contain the amino acid selenocysteine (Sec), co-translationally inserted at a predefined UGA opal codon by means of Sec-specific translation machineries. InEscherichia coli, this process is dependent upon binding of the Sec-dedicated elongation factor SelB to aSecinsertionsequence (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-depletedE. colistrain C321.ΔA, we could produce mammalian selenoprotein thioredoxin reductases with unsurpassed purity and yield. We also found that a SECIS element was no longer absolutely required in such a system. Human glutathione peroxidase 1 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.
authors.affiliation: From the Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
authors.forename: Qing
authors.initials: Q
authors.lastname: Cheng
authors.affiliation: From the Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden elias.arner@ki.se.
authors.forename: Elias S J
authors.initials: ES
authors.lastname: Arnér
mesh_terms: D018388:Codon, Terminator; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005979:Glutathione Peroxidase / Q000138:chemical synthesis*; D006801:Humans; D008722:Methods; D016254:Mutagenesis, Insertional; D010454:Peptide Termination Factors / Q000172:deficiency; D012333:RNA, Messenger / Q000235:genetics; D017279:Selenocysteine; D051140:Selenoproteins / Q000138:chemical synthesis; D000094765:Glutathione Peroxidase GPX1
chemical_list: D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D012333:RNA, Messenger; D051140:Selenoproteins; C489005:prfA protein, E coli; D017279:Selenocysteine; D005979:Glutathione Peroxidase; D000094765:Glutathione Peroxidase GPX1
keywords: Escherichia coli (E. coli); anticodon; glutathione peroxidase; recombinant protein expression; selenocysteine; selenocysteine insertion sequence (SECIS); selenoprotein; thioredoxin reductase; transfer RNA (tRNA); translation release factor