A Facile Method for Producing Selenocysteine-Containing Proteins

Abstract

Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo-tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNA^Ser . Ser-allo-tRNA was converted into Sec-allo-tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec-allo-tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDH_H with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo-tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo-tRNA^UTu system offers a new selenoprotein engineering platform.

Keywords: genetic code expansion; protein engineering; selenocysteine; selenoproteins; synthetic biology.

Figure 1

Translating the UAG stop codon as serine and selenocysteine. A) Cloverleaf structures of E. coli supD tRNA^Ser and allo-tRNA^UTu1. B) FDH_H expression in E. coli ΔselABC ΔfdhF (ME6) cells at 25 °C with allo-tRNA^UTu1, with or without Aeromonas salmonicida (As) SelA, and with reporter fdhF variant genes. Am =amber UAG codon.

Figure 2

Se metabolism engineering. A) Putative pathways of selenium transfer to As SelA in engineered E. coli carrying pSecUAG-ADT. B) FDH_H expression in ME6 cells at 25 °C. C) Intact protein mass spectrometry of the human GPx1(Ser49 and Sec49) mixture obtained from ME6 cells carrying pSecUAG-ADT. The calculated masses are 23133 Da for GPx1(Ser49) and 23193 Da for GPx1(Sec49). In many cases, putative peaks for mono-oxidized (+16 Da), di-oxidized (+32 Da), and a slightly larger (+42–47 Da) GPx1 were observed. Sec incorporation is estimated to be 55%.

Figure 3

Engineering allo-tRNA structures. A) Cloverleaf structures of two allo-tRNA variants. Introduced mutations are indicated with red letters. B) Glutathione peroxidase (GPx) activities of GPx1 produced with allo-tRNA^UTu1D and allo-tRNA^UTu2D in ME6 cells at 25°C. Each bar represents the average of three independent experiments using different E. coli colonies. C) FDH_H expression in ME68z cells at 25 °C. As SelA was expressed with a strong promoter (++++) or a weak promoter (+). Allo-tRNAs^UTu were expressed with the indicated promoters (PargW >PselC).

Figure 4

Engineering of A. salmonicida SelA. A) The amino acid residues involved in the fixation of the SelA N-terminal domain in the crystal structure of Aquifex aeolicus SelA and Thermoanaerobacter tengcongensis tRNA^Sec (PDB ID: 3w1k). B) Screening for highly active As SelA variants by an NMC-A β-lactamase reporter assay in E. coli C321.ΔA.opt ΔselAB. Serial dilutions of cells expressing wildtype or mutant SelA were spotted on ampicillin-containing agar plates and incubated at 30°C. C) FDH_H expression at 30 °C in ME6 fdhF(5 UAG codons) cells with pSecUAG-D-allo-tRNA^UTu1D and pMWcat-AsSelA-(GUG) expressing wildtype or mutant SelA. Sodium selenite was added to a final concentration of 5 μM. D) FDH_H expression level in ME68z fdhF (5 UAG codons and ΔSECIS) cells at 37°C was highest when both allo-tRNA^UTu2D and SelA^Evol were expressed at a moderate level (arabinose (ara) 0.01% and “++”, respectively).

Figure 5

Optimizing the Sec-encoding system in ME68z cells. A) SDS-PAGE of purified GPx1 proteins produced with pSecUAG-Evol 1 and 2. Arabinose concentrations were 0.01% (++) or 0.001% (+). The supernatant and flow-through fractions were also applied to the gel. Unexpectedly, SelA^Evol was also purified by nickel chromatography. B) Intact protein mass spectrometry of the rightmost sample of panel (A) produced by using pSecUAG-Evol2. Sec incorporation is estimated to be 84%. The peak of 23237.601 might derive from GPx1(Sec49) with a Cys-to-Sec substitution at any of the five Cys positions. C) Glutathione peroxidase (GPx) activities of GPx1 produced with SelA^Evol variants. Each bar represents the average of three independent experiments using different E. coli colonies. D) The cleavage patterns of the MXB variants produced with As SelA variants plus SufS(C364A). In vitro intein cleavage was achieved with 100 mM dithiothreitol at room temperature over 16 h.