RS/tRNA Foundational Publication Support
Mahdavi, Alborz, Graham D Hamblin, Granton A Jindal, John D Bagert, Cathy Dong, Michael J Sweredoski, Sonja Hess, Erin M Schuman, and David A Tirrell. (2016) 2016. “Engineered Aminoacyl-Trna Synthetase For Cell-Selective Analysis Of Mammalian Protein Synthesis.”. Journal Of The American Chemical Society 138 (13): 4278-81. doi:10.1021/jacs.5b08980.
RS/tRNA Pair Development Year
2013
ncAA(s) Incorporated
Azidonorleucine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
Biorthogonal Ligation, Click Chemistry
RS Organism of Origin
Parent RS
RS Mutations
L274G
tRNA Organism of Origin
Parent tRNA
tRNA Anticodon
UAC
RS/tRNA Availability
Can be obtained from AddGene Plasmid #63177
RS/tRNA Additional Notes
Is generated to tag mammalian cell proteins with Anl. Showed robust Anl incorportation across cell line (CHO, COS7, HeLa) detected by in-gel fluoresence labelling after TAMRA labelling. Selectivity testing by addition of Met while holding Anl concentration at 1.5mM, showed after detection with TAMRA in-gel fluoresence that the K[A]/K[M] value of 0.25. Showing that it activates Met 4-fold faster than Anl at equimolar concentration
In Mouse cell (CHO), testing with co-expressing the RS and mCherry at 1.5mM Anl for 6 hours showed fluoresence exclusively in the mCherry-expressing cell, showing cell-selective incorporation of Anl. Proteomic analysis with azadibenzo-cycloctyne-functionalized beads to capture Anl-labelled proteins, a total 996 protien were captured while 786 of these protien weren't found in the Met-treated controls. This showed a 19-fold more acquisition in spectral counts.
In HeLa cells, analysis of the proteome of HeLa cells expressing L274GMmMetRS identified 161 sites of Anl incorporation, 150 (93%) at internal Met positions.
In Mouse cell (CHO), testing with co-expressing the RS and mCherry at 1.5mM Anl for 6 hours showed fluoresence exclusively in the mCherry-expressing cell, showing cell-selective incorporation of Anl. Proteomic analysis with azadibenzo-cycloctyne-functionalized beads to capture Anl-labelled proteins, a total 996 protien were captured while 786 of these protien weren't found in the Met-treated controls. This showed a 19-fold more acquisition in spectral counts.
In HeLa cells, analysis of the proteome of HeLa cells expressing L274GMmMetRS identified 161 sites of Anl incorporation, 150 (93%) at internal Met positions.