Preparation of Selenocysteine-Containing Forms of Human SELENOK and SELENOS.

Selenoprotein K (SELENOK) and Selenoprotein S (SELENOS) are the members of the endoplasmic-reticulum-associated degradation (ERAD) complex, which is responsible for translocating misfolded proteins from the endoplasmic reticulum (ER) to the cytosol for degradation. Besides its involvement in the ERAD, SELENOK was shown to bind and stabilize the palmitoyl transferase DHHC6, and thus contributes to palmitoylation. SELENOK and SELENOS reside in the ER membrane by the way of a single transmembrane helix. Both contain an intrinsically disordered region with a selenocysteine (Sec) located one or two residues away from the C-terminus. Here, we describe the preparation of the Sec-containing forms of SELENOS and SELENOK. SELENOK, which contains no native cysteines, was prepared in an E. coli cysteine auxotroph strain by exploiting the codon and the insertion machinery of Cys for the incorporation of Sec. In contrast, the preparation of SELENOS, which contains functionally important cysteine residues, relied on E. coli's native Sec incorporation mechanism.