Overexpression of Recombinant Selenoproteins in E. coli

Abstract

Expression of selenoproteins necessitates a process of decoding of a UGA codon from termination of translation to insertion of selenocysteine. The mechanisms of this process pose major challenges with regards to recombinant selenoprotein production in E. coli, which however can be overcome especially if the Sec residue is located close to the C-terminal end, as is the case for several naturally found selenoproteins. This chapter summarizes a method to achieve such a production.

Selective, Modular Probes for Thioredoxins Enabled by Rational Tuning of a Unique Disulfide Structure Motif

“…To design cell-free assays to predict cellular enzyme selectivities, we ensured that the assays were performed (a) with cellular reductant concentrations, (b) using the catalytically powered redox systems rather than only pre-reduced effector proteins, and (c) examining a range of protein isoforms, both isolated from primary tissues as well as recombinantly expressed. The last point is particularly relevant for TrxR since the key selenocysteine (Sec, U) residue in its active site is highly present in isolates of native enzymes or recombinant forms made with novel production methodologies (up to 100% Sec contents), which we employed in this study, rather than the ca. 30% Sec content from standard expression methods .…”

Reprogramming the Biosynthesis of Precursor Peptide to Create a Selenazole-Containing Nosiheptide Analogue

“…Despite the fact that the SelB binding region (SBR, or minimal SECIS) in the SECIS element is sequencespecific and should strictly be positioned 11 nucleotides downstream of UGA, other regions of the nucleotide sequence can be flexible. 35 This flexibility allowed us to have a space for the accommodation of the last two coding codons followed by the stop codon UAG for nosM-Sec1. Other nucleotide sequences, including SBR, remained the same as in the natural SECIS from E. coli.…”

“…In fact, Escherichia coli cells naturally contain selenoprotein synthesis machinery for generating Sec-tRNA Sec and specifically incorporating Sec into formate dehydrogenase H. 34 This system consists of Sec synthase (SelA), Sec-specific elongation factor (SelB), tRNA Sec (SelC), and selenophosphate synthetase (SelD), and the insertion of Sec into the polypeptide chain also requires an opal codon UGA and a unique Sec insertion sequence (SECIS) element in the mRNA to bind with the elongation factor SelB. 35 However, the utilization of Secrelated machinery to generate artificial systems has been limited to the production of selenoproteins to date. 33 Consequently, the functionality of selenoprotein synthesis machinery in either natural or heterologous hosts for producing thiopeptides is unknown.…”

Comprehensive chemical proteomics analyses reveal that the new TRi-1 and TRi-2 compounds are more specific thioredoxin reductase 1 inhibitors than auranofin

“…To measure TXNRD1 activities, the end-point TXN-dependent insulin reduction assay was used, essentially as described previously [ 17 ]. In short, 7.5–9 μg total protein from the cell lysate was incubated with 0.16 mM human insulin (Sigma), 0.33 mM NADPH (Saveen Werner) and 16 μM human TXN (recombinantly produced as previously described [ 81 ]) in TE buffer (50 mM Tris-HCl, 2 mM EDTA, pH 7.5) at a total volume of 50 μL and was then incubated at 37 °C. After 0, 15 and 30 min, 10 μL aliquots were taken and combined with 6 M guanidine-HCl and 2.5 mM DTNB in 96-well plates (PerkinElmer), whereupon the absorbance at 412 nm was measured using microplate spectrophotometer (TECAN).…”