期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 292, 期 13, 页码 5476-5487出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M117.776310
关键词
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资金
- Swedish Research Council
- Swedish Cancer Society
- Knut Foundation
- Alice Wallenberg Foundation
- Karolinska Institutet
Selenoproteins contain the amino acid selenocysteine (Sec), co-translationally inserted at a predefined UGA opal codon by means of Sec-specific translation machineries. In Escherichia coli, this process is dependent upon binding of the Sec-dedicated elongation factor SelB to a Sec insertion sequence (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. Delta A, 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.
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