Reassigning an amber codon to selenocysteine using a functional cysteine suppression pair in Escherichia coli.

Selenoproteins, characterized by the presence of selenocysteine (Sec) residues, are widely distributed across all domains of life. The unique attributes of selenoproteins are conferred by Sec, which is genetically encoded into nascent polypeptide chains through the recoding of the UGA codon. Human glutathione peroxidase 1 (GPx1) is a selenoprotein, which plays a crucial role in maintaining redox homeostasis. However, its complex recoding mechanism presents a major challenge for efficient selenoprotein expression in Escherichia coli. In this study, we engineered a functional amber suppression system that consists of a cysteinyl-tRNA synthetase variant (M38-18) and its cognate tRNACys variant (M1-1). These components were developed through directed evolution of the native E. coli pair. Using this system in E. coli C321.ΔA.exp, we successfully achieved recombinant expression of GPx1 and a GPx1 mutant, GPx1-C-S-49TAG featuring serine substitutions for all cysteine residues, with enzymatic activities of around 420 U/mg and 290 U/mg, respectively. This work provides an alternative platform for selenoprotein production in bacteria and establishes an innovative strategy for the scalable biosynthesis of GPx and related therapeutics.