Developing a protocol for maximum non‐specific incorporation of selenium into proteins for Se NMR spectroscopy

Abstract

Selenoproteins contain the rare amino acid selenocysteine, which is predominantly found in enzyme active sites where it provides high chemical reactivity and specificity, most often in redox regulation. In mammals, selenoproteins contribute to anti‐inflammatory and antioxidative defense. Among the twenty‐five human selenoproteins, are the important redox enzymes thioredoxin reductase and glutathione peroxidase. Heterologous expression of selenoproteins is challenging due to the inherent inefficiency of selenium incorporation in vivo. Hence, selenoproteins limited availability complicates their structural and functional characterization. We have developed a general method to enrich proteins with selenium by raising the selenium‐to‐sulfur ratios in the E. coli cells' growth media. As may be expected, selenium toxicity impedes cell growth and protein expression. We have screened the growth conditions and ratio of sulfur/selenium in the media to obtain the desired yield vs. incorporation ratio. The method we have developed, using a ratio of 7:3 selenium to sulfur in the growth media, results in a substitution ratio of 70% selenium to sulfur and the yield is about ½ lower than that in a sulfur media. This method has been tested for several proteins and is both robust and fully compatible with additional isotopic enrichment of recombinant selenoproteins with 77Se, 13C, 15N and 2H.