Production and purification of homogenous recombinant human selenoproteins reveals a unique codon skipping event in E. coli and GPX4-specific affinity to bromosulfophthalein.

Selenoproteins are translated via animal domain-specific elongation machineries that redefine dedicated UGA opal codons from termination of translation to selenocysteine (Sec) insertion, utilizing specific tRNA species and Sec-specific elongation factors. This has made recombinant production of mammalian selenoproteins in E. coli technically challenging but recently we developed a methodology that enables such production, using recoding of UAG for Sec in an RF1-deficient host strain. Here we used that approach for production of the human glutathione peroxidases 1, 2 and 4 (GPX1, GPX2 and GPX4), with all these three enzymes being important antioxidant selenoproteins. Among these, GPX4 is the sole embryonically essential enzyme, and is also known to be essential for spermatogenesis as well as protection from cell death through ferroptosis. Enzyme kinetics, ICP-MS and mass spectrometry analyses of the purified recombinant proteins were used to characterize selenoprotein characteristics and their Sec contents. This revealed a unique phenomenon of one-codon skipping, resulting in a lack of a single amino acid at the position corresponding to the selenocysteine (Sec) residue, in about 30% of the recombinant GPX isoenzyme products. We furthermore confirmed the previously described UAG suppression with Lys or Gln as well as a minor suppression with Tyr, together resulting in about 20% Sec contents in the full-length proteins. No additional frameshifts or translational errors were detected. We subsequently found that Sec-containing GPX4 could be further purified over a bromosulfophthalein-column, yielding purified recombinant GPX4 with close to complete Sec contents. This production method for homogenously purified GPX4 should help to further advance the studies of this important selenoprotein.