Cloning of murine SeGpx cDNA and synthesis of mutated GPx proteins in Escherichia coli.
Glutathione peroxidase (GPx) of mammalian cells and Escherichia coli formate dehydrogenase both contain a selenocysteine (SeCys) in their amino acid (aa) sequence. In these two enzymes, this aa is encoded by a UGA codon, which is usually a stop codon for protein synthesis. We constructed plasmids to test the synthesis of GPx in E. coli. These constructions permitted high-level production of GPx mutants, where the SeCys codon was replaced by cysteine (UGC, UGU) or serine (UCA) codons, but synthesis of selenoprotein could not be detected: our data suggest that signals used for the recognition of the UGA codon as a SeCys codon are not conserved between E. coli and mammalian cells.
Rocher
C
C
Centre de Recherche ROUSSEL-UCLAF, Laboratoire Génie Génétique, Romainville, France.
Faucheu
C
C
Hervé
F
F
Bénicourt
C
C
Lalanne
J L
JL
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon / Q000235:genetics; D003545:Cysteine / Q000031:analogs & derivatives; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D005822:Genetic Vectors; D005979:Glutathione Peroxidase / Q000096:biosynthesis / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D012643:Selenium; D017279:Selenocysteine
D003062:Codon; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D017279:Selenocysteine; D011061:Poly A; D004247:DNA; D005979:Glutathione Peroxidase; D012643:Selenium; D003545:Cysteine