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: The mutagen MNNG introduces many DNA replication errors, thereby facilitating the reversion of the hAGT/UAG145 to hAGT/C145 codons which would bypass the EF-Tu selection. As will be shown in the next paragraph, the hAGT/A137/U145 variant is fully active, while the hAGT/A137/C145 protein is inactive. Therefore, the hAGT/A137/UAG145 construct is used for the selection of the EF-Tu library with UTu components in E. coli (ΔselA ΔselB Δada Δogt T7RNAp). To fully select the diverse library, a total of five MNNG treatments with a higher drug dose (20 μg/ml) was necessary. From ≈109 plated cells, 15–30 colonies grew, likely representing EF-Tu variants favoring the most efficient Sec-incorporation. The coding region of EF-Tu variants from 12 colonies were sequenced, resulting in one false-positive clone with an internal amber-stop codon at position 98. From the remaining 11 clones, all contained an R67, nine contained W98 and 10 R274, indicating these residues as being conserved. At position 217, there is a slight preference for Lys, whereas position 216 remained randomized (Figure 3). Position 98 was chosen to provide the compensatory space needed by R274; however, Trp is the bulkiest of the 20 standard aa. This contradiction can be explained with the assumption that W98 is flipped out of EF-Tu's aa binding pocket—like the Ile in the case of the Sec-specific elongation factor SelB (Figure 2). One clone with R98 and P274 was isolated. Here, the positive side chain of Arg reaches into the EF-Tu aa binding pocket from position 98 with space compensation provided by the small Pro at position 274.
Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.