High-level expression in Escherichia coli of selenocysteine-containing ratthioredoxin 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 a nd many other substrates, and is a central enzyme for cell proliferation an d thiol redox control. The enzyme is a selenoprotein and can therefore, lik e all other mammalian selenoproteins, not be directly expressed in Escheric hia coli, since selenocysteine-containing proteins are synthesized by a hig hly species-specific translation machinery. This machinery involves a secon dary structure, SECIS element, in the selenoprotein-encoding mRNA, directin g selenocysteine insertion at the position of an opal (UGA) codon, normally confering termination of translation. It is species-specific structural fe atures and positions in the selenoprotein mRNA of the SECIS elements that h itherto 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 sele noprotein formate dehydrogenase H. A variant of the SECIS element designed to encode the conserved carboxyterminal end of the enzyme (-Sec-Gly-COOH) a nd 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-cont aining TrxR was produced at dramatically higher levels than formate dehydro genase 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. G o-expression with the selA, selB and selC genes (encoding selenocysteine sy nthase, SELB and tRNA(Sec) respectively) further increased the efficiency o f the selenoprotein production and thereby also increased the specific acti vity of the recombinant TrxR to about 25 % of the native enzyme, with as mu ch 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 pr oduction of recombinant selenoproteins. (C) 1999 Academic Press.