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: We tested our UTu system for Sec incorporation with four different proteins. The first two examples are of intein-mediated protein splicing. The first case was the control plasmid pMXB10 of the New England Biolabs IMPACT system. In this plasmid the coding region of the maltose-binding protein (MBP) is fused to the Mxe gyrase intein–chitin binding domain. Upon incubation of the full-length 70 kDa protein with DTT, the intein active site C384 cleaves the adjacent peptide bond to produce two fragments of MBP (42 kDa) and intein–chitin binding protein (CBP, 28 kDa). A similar cleavage is seen by the U384-containing protein; no significant cleavage activity has been seen for MXB S384 (Figure 5A). Judged by the cleavage pattern Cys and Sec are equally active.
Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.