Chemical Ligation Synthesis of Isotope‐Labeling Compatible Selenoprotein S

Selenoproteins contain the 21 st amino acid selenocysteine (Sec) and play vital roles in human health, such as antioxidant defense. Although selenoproteins are expressed naturally in the human body, procuring sufficient quantities for biochemical and biophysical characterization by heterologous expression is challenging. Low yield in genetic incorporation is due to the Sec codon being identical to the UGA stop codon in mRNA. An alternative approach for their preparation is chemical ligation, a semi‐synthetic method that relies upon an amide bond formation to generate peptides and proteins from their complementary fragments. Here, we describe a new method for chemical ligation of selenoprotein S (SelS) which is compatible with simple and low cost isotopic labeling and does not rely on complete chemical synthesis of a selenoprotein. SelS is a member of the endoplasmic reticulum associated protein degradation (ERAD) pathway, a protein machinery devoted to relocating misfolded proteins from the ER to a cytoplasmic protein degradation complex. The active site lies close to the C‐terminal where a sole Sec is paired with a Cysteine 13 residues away. Previously we have characterized the activity of SelS prepared by heterologous expression, however, structural characterization with the Sec in place was prohibited by the low yield. In our approach a Sec‐containing fragment is expressed in E. coli and used as a source of the selenopeptide during the chemical ligation. This has the advantage of a high yield, with simple and efficient ligation while being compatible with isotopic‐labeling of selenoproteins. Useful applications of this method include incorporation of Selenium‐77 and Carbon‐13 for structural and biochemical characterization by NMR spectroscopy. Support or Funding Information This work was funded by the National Institute of General Medical Science