TABLE 2.

Comparison of the method developed here with other approaches for expression of heterologous selenoproteins in E. coli

FDH is formate dehydrogenase; EF-Tu is elongation factor Tu.

Approach	Selenoproteins expressed	Proof of Sec incorporation	Specificity in Sec incorporation	Total yield of selenoprotein	Competing events reported	Refs.
16S rRNA mutations for improved Sec insertion efficiency at the ribosome	Luciferase reporter downstream of SECIS element, endogenous FDH	Luciferase activity of reporter, benzyl viologen assay, for endogenous FDH activities	Best mutants, ≈30%	Unclear, not quantified	UGA truncation, Trp-mediated UGA suppression	Thyer et al. (31)
Synthetic tRNAUtu species for EF-Tu-mediated Sec insertion at either UGA or UAG codons	Endogenous FDH, GPx1 or Sec-substituted Grx1	Benzyl viologen assay for endogenous FDH, mass spectrometry of Grx1, enzyme activities of Grx1 and GPx1	≈50–65% (Sec-Grx1 vs. Ser-Grx1 or Sec-GPx1 vs. Ser-GPx1), double activity of Sec-Grx1 vs. Cys-Grx1, GPx1 ≈6 units/mg	Unclear, not quantified	Ser-mediated suppression	Aldag et al. (28)
SelB-mediated suppression of alternative codons using tRNASec variants with altered anticodon sequences	Endogenous FDH, human TrxR1	Benzyl viologen assay for endogenous activities of FDH and specific activities of purified FDH, mass spectrometry, specific activity of TrxR	FDH, 12–100% depending upon codon usage; TrxR ≈95–98%	FDH, up to 12-fold levels of endogenous wild type FDH; TrxR unclear, not quantified	Unclear, codon-dependent	Bröcker et al. (29)
Engineered tRNASec for EF-Tu-mediated Sec insertion at UAG, also tested in the RF1- depleted C321.ΔA host	Sec-substituted NMC-A β-lactamase, endogenous FDH, Sec-substituted DHFR, Sec-substituted azurin, human GPx1	Antibiotic resistance due to functional β-lactamase, benzyl viologen assay for endogenous FDH, mass spectrometry	Sec-DHFR, ≈100%, other selenoproteins produced unclear specificity	Unclear, not quantified	Ser-mediated suppression	Thyer et al. (32)
Further engineered tRNASec for EF-Tu-mediated Sec insertion at UAG with lower Ser-mediated suppression	Endogenous FDH, Sec-substituted Grx1	Benzyl viologen assay for endogenous FDH, specific activities of FDG and Sec-substituted Grx1, mass spectrometry	FDH, ≈70%; Grx1, ≈100%	Unclear, not quantified using expression in vivo; in cell-free systems ≈2–80 ng obtained per reaction depending upon construct	Ser-mediated suppression	Miller et al. (30)
UAG-directed Sec insertion in RF1-depleted host, with or without bacterial SECIS element	Rat and human TrxR isoenzymes, human GPx1	Enzyme activities and mass spectrometry	With SECIS, ≥95%. No SECIS, ≈30%. TrxR1 ≈35–40 units/mg. GPx1 ≈10 units/mg	With SECIS, ≈20 mg/liter. No SECIS, 1–3 mg/liter	UAG-directed truncation, Gln- and Lys-mediated suppression	This work