High-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterial-type SECIS elements and co-expression with the selA, selB and selC genes.
Mammalian thioredoxin reductase (TrxR) catalyzes reduction of thioredoxin and many other substrates, and is a central enzyme for cell proliferation and thiol redox control. The enzyme is a selenoprotein and can therefore, like all other mammalian selenoproteins, not be directly expressed in Escherichia coli, since selenocysteine-containing proteins are synthesized by a highly species-specific translation machinery. This machinery involves a secondary structure, SECIS element, in the selenoprotein-encoding mRNA, directing selenocysteine insertion at the position of an opal (UGA) codon, normally conferring termination of translation. It is species-specific structural features and positions in the selenoprotein mRNA of the SECIS elements that hitherto have hampered heterologous production of recombinant selenoproteins. We have discovered, however, that rat TrxR can be expressed in E. coli by fusing its open reading frame with the SECIS element of the bacterial selenoprotein formate dehydrogenase H. A variant of the SECIS element designed to encode the conserved carboxyterminal end of the enzyme (-Sec-Gly-COOH) and positioning parts of the SECIS element in the 3'-untranslated region was also functional. This finding revealed that the SECIS element in bacteria does not need to be translated for full function and it enabled expression of enzymatically active mammalian TrxR. The recombinant selenocysteine-containing TrxR was produced at dramatically higher levels than formate dehydrogenase O, the only endogenous selenoprotein expressed in E. coli under the conditions utilized, demonstrating a surprisingly high reserve capacity of the bacterial selenoprotein synthesis machinery under aerobic conditions. Co-expression with the selA, selB and selC genes (encoding selenocysteine synthase, SELB and tRNA(Sec), respectively) further increased the efficiency of the selenoprotein production and thereby also increased the specific activity of the recombinant TrxR to about 25 % of the native enzyme, with as much as 20 mg produced per liter of culture. These results show that with the strategy utilized here, the capacity of selenoprotein synthesis in E. coli is more than sufficient for making possible the use of the bacteria for production of recombinant selenoproteins.
Arnér
E S
ES
Karolinska Institutet, Stockholm, S-171 77, Sweden. Elias.Arner@mbb.ki.se
Sarioglu
H
H
Lottspeich
F
F
Holmgren
A
A
Böck
A
A
D020413:3' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018388:Codon, Terminator / Q000235:genetics; D004274:DNA, Recombinant / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics* / Q000502:physiology; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis / Q000235:genetics; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012643:Selenium / Q000378:metabolism; D017279:Selenocysteine / Q000378:metabolism*; D051140:Selenoproteins; D013880:Thioredoxin-Disulfide Reductase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D020413:3' Untranslated Regions; D018388:Codon, Terminator; D004274:DNA, Recombinant; D011506:Proteins; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D051140:Selenoproteins; D017279:Selenocysteine; D005560:Formate Dehydrogenases; D013880:Thioredoxin-Disulfide Reductase; D012643:Selenium