RS/tRNA Foundational Publication Support
Chen, Peng R, Dan Groff, Jiantao Guo, Weijia Ou, Susan Cellitti, Bernhard H Geierstanger, and Peter G Schultz. (2009) 2009. “A Facile System For Encoding Unnatural Amino Acids In Mammalian Cells.”. Angewandte Chemie (International Ed. In English) 48 (22): 4052-5. doi:10.1002/anie.200900683.
RS/tRNA Pair Development Year
2010
ncAA(s) Incorporated
o-Nitrobenzyl oxycarbonyl-Nε-L lysine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
n/a
RS Organism of Origin
Parent RS
RS Mutations
Y306M
L309A
C348A
Y384F
L309A
C348A
Y384F
tRNA Organism of Origin
Parent tRNA
tRNA Anticodon
CUA
RS/tRNA Availability
N/A
RS/tRNA Additional Notes
In BL21 cells, NBK-1 was co-transformed alongside pBAD-GFP149TAG both with and without 1mM o-Nitrobenzyl oxycarbonyl-Nε-L lysine (ONBK) supplement. SDS-page and ESI-MS then confirmed that full length protein was only expressed in the presence of 1mM ONBK, yielding approxamitely 10 mg/L protein.
In Hek293 cells, plasmid containing NBK-1 was co-transfected alongside EGFP37TAG, cells were then permitted to grow both with and without 1mM ONBK for 36 hours before being studied by fluorescence microscopy. Fluorescence microscopy showed protein expression only in the presence of ONBK. ESI-MS was performed as well, which confirmed the results of fluoresence microscopy, and confirmed the presence of intact photocaged Lys.
In Hek293 cells, plasmid containing NBK-1 was co-transfected alongside EGFP37TAG, cells were then permitted to grow both with and without 1mM ONBK for 36 hours before being studied by fluorescence microscopy. Fluorescence microscopy showed protein expression only in the presence of ONBK. ESI-MS was performed as well, which confirmed the results of fluoresence microscopy, and confirmed the presence of intact photocaged Lys.