Engineering human seleno-glutaredoxin containing consecutive rare codons as an artificial glutathione peroxidase

Abstract

The active center of human glutaredoxin (hGrx1) shares a common thioredoxin fold and specific affinity for substrate glutathione (GSH) with natural glutathione peroxidase (GPx). hGrx1 was redesigned to introduce the catalytic selenocysteine residue to imitate the function of antioxidant selenoenzyme GPx in vivo. The human hGrx1 scaffold is a good candidate for potential medical application compared with other animal-originated protein scaffolds. Two consecutive rare codons (AGG-AGG) in the open reading frame of hGrx1 mRNA encoding Arg26-Arg27 residues can reduce seleno-hGrx1 expression level significantly in the Cys auxotrophic Escherichia coli strain BL21cysE51. Therefore, we optimized the rare codons, which resulted in a remarkable increase of the expression level in the Cys auxotrophic cells, which may be sufficient for future medical production. The engineered artificial selenoenzyme displays high GPx catalytic activity, rivaling that of some natural GPx proteins. Kinetic analysis of the engineered seleno-hGrx1 showed a typical ping-pong kinetic mechanism; its catalytic properties are similar to those of some naturally occurring GPx proteins. selenium, selenocysteine, rare codon, artificial enzyme, glutathione peroxidase, glutaredoxin Citation:Zhang W, Luo Q, Wang X P, et al. Engineering human seleno-glutaredoxin containing consecutive rare codons as an artificial glutathione peroxidase.

Heterologous expression and characterization of human cellular glutathione peroxidase mutants

“…Due to the application of Ebselen, several methods for its derivates synthesis have been developed . Glutathione S ‐transferase (GST) and glutaredoxin (Grx) are natural protein scaffolds with intrinsic GSH‐binding sites and widely used to redesign GPx mimics . Furthermore, a number of GPx mimics have been built by a monoclonal antibody and bioimprinting techniques, which introduce the GSH binding site into the models .…”