title: Engineering the elongation factor Tu for efficient selenoprotein synthesis.
abstract: Selenocysteine (Sec) is naturally co-translationally incorporated into proteins by recoding the UGA opal codon with a specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element). We have recently developed a SECIS-free selenoprotein synthesis system that site-specifically--using the UAG amber codon--inserts Sec depending on the elongation factor Tu (EF-Tu). Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis. A Sec-specific selection system was established by expression of human protein O(6)-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon. The formed hAGT selenoprotein repairs the DNA damage caused by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine, and thereby enables Escherichia coli to grow in the presence of this mutagen. An EF-Tu library was created in which codons specifying the amino acid binding pocket were randomized. 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.
authors: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors: Ken-ichi
authors: K
authors: Haruna
authors: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors: Muhammad H
authors: MH
authors: Alkazemi
authors: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors: Yuchen
authors: Y
authors: Liu
authors: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA Department of Chemistry, Yale University, New Haven, CT 06520-8114, USA markus.englert@yale.edu.
authors: Dieter
authors: D
authors: Söll
authors: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA markus.englert@yale.edu.
authors: Markus
authors: M
authors: Englert
mesh_terms: D001216:Asparagine / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis; D015202:Protein Engineering; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism; D017279:Selenocysteine / Q000378:metabolism*; D051140:Selenoproteins / Q000096:biosynthesis*
chemical_list: D001426:Bacterial Proteins; D012345:RNA, Transfer, Amino Acid-Specific; C061878:SelB protein, Bacteria; D051140:Selenoproteins; C036928:tRNA, selenocysteine-; D017279:Selenocysteine; D001216:Asparagine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010444:Peptide Elongation Factor Tu; D003545:Cysteine