RS/tRNA Foundational Publication Support
Kim, Chan Hyuk, Mingchao Kang, Hak Joong Kim, Abhishek Chatterjee, and Peter G Schultz. (2012) 2012. “Site-Specific Incorporation Of Ε-N-Crotonyllysine Into Histones.”. Angewandte Chemie (International Ed. In English) 51 (29): 7246-9. doi:10.1002/anie.201203349.
RS/tRNA Pair Development Year
2013
ncAA(s) Incorporated
Nε-Crotonyl-L-lysine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
Natural PTM
RS Organism of Origin
Parent RS
RS Mutations
L274A
C313F
T349F
C313F
T349F
tRNA Organism of Origin
Parent tRNA
tRNA Anticodon
CUA
RS/tRNA Availability
N/A
RS/tRNA Additional Notes
Kcr-RS was cotransformed into DH10B cells grown in a LB medium supplemented with 5mM Nε-Crotonyl-L-lysine (Kcr) and 20mM nicotinamide was added after 1mM IPTG induction at OD600 0.8, resulting in 10mg/L yield of mutant GFP after being left overnight. Results were confirmed by ESI-MS.
In BL21(DE3) cells, site-specifically crotonylated human histone subunit 2B (H2B) was co-transformed into the cell alongside Kcr-RS and supplemented with and without 5mM Kcr and 20mM nicotinamide following 1mM IPTG induction at OD600 0.8. Cells were left overnight before the yield of full length H2B was found to be 2mg/L only in the presence of Kcr via SDS-PAGE analysis. Western blotting and ESI-MS confirmed these results.
In Hek293T cells, pEGFP-Tyr39TAG was cotransfected with a pCMV-Kcr plasmid bearing Kcr-RS/tRNApyl into the cell, which was grown within a DMEM media supplemented with 10% BVS with and without Kcr. Fluorescence microscopy revealed bright green fluorescence only in the presence of Kcr after 48 hours, incorporation was confirmed by ESI-MS.
In BL21(DE3) cells, site-specifically crotonylated human histone subunit 2B (H2B) was co-transformed into the cell alongside Kcr-RS and supplemented with and without 5mM Kcr and 20mM nicotinamide following 1mM IPTG induction at OD600 0.8. Cells were left overnight before the yield of full length H2B was found to be 2mg/L only in the presence of Kcr via SDS-PAGE analysis. Western blotting and ESI-MS confirmed these results.
In Hek293T cells, pEGFP-Tyr39TAG was cotransfected with a pCMV-Kcr plasmid bearing Kcr-RS/tRNApyl into the cell, which was grown within a DMEM media supplemented with 10% BVS with and without Kcr. Fluorescence microscopy revealed bright green fluorescence only in the presence of Kcr after 48 hours, incorporation was confirmed by ESI-MS.