RS/tRNA Foundational Publication Support
Bryson, David I., Chenguang Fan, Li-Tao Guo, Corwin Miller, Dieter Söll, and David R. Liu. (dec) 2017. “Continuous Directed Evolution Of Aminoacyl-Trna Synthetases”. Nature Chemical Biology 13: 1253-1260. doi:10.1038/nchembio.2474.
RS/tRNA Pair Development Year
2018
ncAA(s) Incorporated
Nε-(tert-Butoxycarbonyl)-L-lysine (BocLys)
ncAA Structure (png, jpg, jpeg)
ncAA Utility
Extra-‐chromosomal array (low stability), biolistic bombardment. Scattered in different places in animal.
RS Organism of Origin
Parent RS
RS Mutations
V31I
T56P
H62Y
A100E (IPYE)
T56P
H62Y
A100E (IPYE)
tRNA Organism of Origin
Parent tRNA
tRNA Anticodon
CUA
RS/tRNA Availability
Can be purchased from AddGene Plasmid #99222
RS/tRNA Additional Notes
The chimeric pyrrolysyl-tRNA synthetase (chPylRS) used in this study is derived from Methanosarcina species, combining residues 1–149 of M. barkeri PylRS (MbPylRS) with residues 185–454 of M. mazei PylRS (MmPylRS). This design enhanced solubility and provided higher specific activity for L-pyrrolysine than MmPylRS alone. To evolve improved catalytic properties BocK was used, a non-canonical lysine analog, during phage-assisted continuous evolution (PACE). The evolved variant, chPylRS(IPYE), acquired four activating mutations (V31I, T56P, H62Y, A100E) localized in the N-terminal tRNA-binding domain, not the active site.
In BL21(DE3),
chPylRS(IPYE) enabled efficient BocK incorporation at up to three positions in sfGFP, increasing expression levels up to 4-fold. Kinetic analysis revealed an 8.7× increase in kcat, a 5.7× decrease in KM for tRNAPyl, and a 45-fold improvement in catalytic efficiency.
In BL21(DE3),
chPylRS(IPYE) enabled efficient BocK incorporation at up to three positions in sfGFP, increasing expression levels up to 4-fold. Kinetic analysis revealed an 8.7× increase in kcat, a 5.7× decrease in KM for tRNAPyl, and a 45-fold improvement in catalytic efficiency.