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Engineering the elongation factor Tu for efficient selenoprotein synthesis.

Haruna K, Alkazemi MH, Liu Y, Söll D, Englert M - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.

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Overview of the Sec-specific genetic selection system. (A) O6-methylguanosine within DNA and human AGT with the active site residue at position 145 indicated as ‘X’. Whereas the hAGT C145 and U145 variants allow DNA repair, the hAGT S145 protein is inactive. The chemical structure of MNNG, which causes the conversion of guanosine to O6-methylguanosine in DNA, is shown. (B) E. coli­ ΔselA, ΔselB, Δada, Δogt, T7RNAp cells were co-transformed with the UTu components and the hAGT C145, UAG145 and S145 variants. After three selection rounds with 10 μg/ml MNNG pulses and 2 h of recovery in-between, the indicated 10−3, 10−4 and 10−5 dilutions of cell suspensions were plated on LB agar to check for growth after 24 h.
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Figure 1: Overview of the Sec-specific genetic selection system. (A) O6-methylguanosine within DNA and human AGT with the active site residue at position 145 indicated as ‘X’. Whereas the hAGT C145 and U145 variants allow DNA repair, the hAGT S145 protein is inactive. The chemical structure of MNNG, which causes the conversion of guanosine to O6-methylguanosine in DNA, is shown. (B) E. coli­ ΔselA, ΔselB, Δada, Δogt, T7RNAp cells were co-transformed with the UTu components and the hAGT C145, UAG145 and S145 variants. After three selection rounds with 10 μg/ml MNNG pulses and 2 h of recovery in-between, the indicated 10−3, 10−4 and 10−5 dilutions of cell suspensions were plated on LB agar to check for growth after 24 h.

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.


Engineering the elongation factor Tu for efficient selenoprotein synthesis.

Haruna K, Alkazemi MH, Liu Y, Söll D, Englert M - Nucleic Acids Res. (2014)

Overview of the Sec-specific genetic selection system. (A) O6-methylguanosine within DNA and human AGT with the active site residue at position 145 indicated as ‘X’. Whereas the hAGT C145 and U145 variants allow DNA repair, the hAGT S145 protein is inactive. The chemical structure of MNNG, which causes the conversion of guanosine to O6-methylguanosine in DNA, is shown. (B) E. coli­ ΔselA, ΔselB, Δada, Δogt, T7RNAp cells were co-transformed with the UTu components and the hAGT C145, UAG145 and S145 variants. After three selection rounds with 10 μg/ml MNNG pulses and 2 h of recovery in-between, the indicated 10−3, 10−4 and 10−5 dilutions of cell suspensions were plated on LB agar to check for growth after 24 h.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150793&req=5

Figure 1: Overview of the Sec-specific genetic selection system. (A) O6-methylguanosine within DNA and human AGT with the active site residue at position 145 indicated as ‘X’. Whereas the hAGT C145 and U145 variants allow DNA repair, the hAGT S145 protein is inactive. The chemical structure of MNNG, which causes the conversion of guanosine to O6-methylguanosine in DNA, is shown. (B) E. coli­ ΔselA, ΔselB, Δada, Δogt, T7RNAp cells were co-transformed with the UTu components and the hAGT C145, UAG145 and S145 variants. After three selection rounds with 10 μg/ml MNNG pulses and 2 h of recovery in-between, the indicated 10−3, 10−4 and 10−5 dilutions of cell suspensions were plated on LB agar to check for growth after 24 h.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.

Show MeSH
Related in: MedlinePlus