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Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion |
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| Authors: | Ilka U. Heinemann,Alexis J. Rovner,Hans R. AerniSvetlana Rogulina,Laura ChengWilliam Olds,Jonathan T. FischerDieter Sö ll,Farren J. Isaacs,Jesse Rinehart |
| Affiliation: | a Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8144, USA b Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8230, USA c Department of Cellular & Molecular Physiology, Yale University, New Haven, CT 06520-8026, USA d Department of Chemistry, Yale University, New Haven, CT 06520-8107, USA e Systems Biology Institute, Yale University, West Haven, CT 06516-7388, USA |
| Abstract: | Genetically encoded phosphoserine incorporation programmed by the UAG codon was achieved by addition of engineered elongation factor and an archaeal aminoacyl-tRNA synthetase to the normal Escherichia coli translation machinery (Park et al., 2011) Science 333, 1151) [2]. However, protein yield suffers from expression of the orthogonal phosphoserine translation system and competition with release factor 1 (RF-1). In a strain lacking RF-1, phosphoserine phosphatase, and where seven UAG codons residing in essential genes were converted to UAA, phosphoserine incorporation into GFP and WNK4 was significantly elevated, but with an accompanying loss in cellular fitness and viability. |
| Keywords: | GFP, green fluorescent protein RF-1, release factor-1 (prfA) RF-2, release factor-2 (prfB) SepRS, phosphoseryl-tRNA synthetase WNK4, serine/threonine-protein kinase WNK4 Sep, phosphoserine MAGE, multiplex automated genome engineering |
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