Engineering the elongation factor Tu for efficient selenoprotein synthesis.
Bottom Line: Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis.Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library.The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.
Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.Show MeSH
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Mentions: To establish this system in E. coli, the two hAGT homologs ada and ogt were knocked out. The resulting cells were sensitive to the methylating agent MNNG. Any repair of MNNG-induced O6-methylguanine-DNA damage now requires the heterologous expression of hAGT variants. We tested the genetic selection system with the hAGT C145 (wild type) and S145, as well with the UTu-generated U145/S145 variants. Cells expressing hAGT, tRNAUTu and SelA were subjected to three selection rounds with a 2-h recovery period at 30°C between the MNNG pulses. Next, sequential dilutions were plated on LB agar to obtain the fraction of surviving E. coli cells (Figure 1). While the hAGT C145 actively protected E. coli, expression of the hAGT S145 variant did not lead to cell survival when plated from a 10−3 dilution. A UAG codon (position 145) in hAGT mRNA is translated by the UTu system creating a mixture of hAGT U145 and S145 proteins—as confirmed by mass spectrometry of the purified mixture (Supplementary Figure S1). Visible growth comparable to that of wild-type hAGT indicates the active protection afforded by the hAGT U145 variant. Hence, any component of the UTu system that raises the amount of hAGT U145 protein is in the selected population.
Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.