RS/tRNA Foundational Publication Support
Xiao, Han, Francis B Peters, Peng-Yu Yang, Sean Reed, Johnathan R Chittuluru, and Peter G Schultz. (2014) 2014. “Genetic Incorporation Of Histidine Derivatives Using An Engineered Pyrrolysyl-Trna Synthetase.”. Acs Chemical Biology 9 (5): 1092-6. doi:10.1021/cb500032c.
RS/tRNA Pair Development Year
2014
ncAA(s) Incorporated
3-methyl-DL-histidine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
n/a
beta-(3-Pyridyl)-DL-alanine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
n/a
3-(2-furyl)alanine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
n/a
3-(2-Thienyl)alanine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
n/a
D-2-(5-BromoThienyl)-alanine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
n/a
RS Organism of Origin
Parent RS
RS Mutations
L270I
Y271F
L274G
C313F
Y349F
Y271F
L274G
C313F
Y349F
tRNA Organism of Origin
Parent tRNA
tRNA Anticodon
CUA
Multiple tRNAs?
Methanosarcina mazei pyrrolysyl tRNA (MmtRNAPyl) was utilized in place of the Methanosarcina barkeri when incorporating into HEK293T cells.
RS/tRNA Availability
N/A
RS/tRNA Additional Notes
In DH10B cells, pLeiG-GFP-Asp134TAG was co-transformed with the PylHRS/tRNApyl pair both in the presence and absence of 1mM 3-methyl-DL-histidine. Full length protein expression only occuring in the presence of ncAA, with a yield of 9.3mg/L protein in rich media and 7.1mg/L yield in minimal media.
Histidine analogues beta-(3-Pyridyl)-DL-alanine, 3-(2-furyl)alanine, 3-(2-Thienyl)alanine, D-2-(5-BromoThienyl)-alanine were also tested using pLeiG-GFP-Asp134TAG, PylHRS in DH10B cells and minimal media. in the presence of 1mM of each ncAA, full length protein yields of 4.0, 3.0, 4.2, and 12.0mg/L were achieved respectively. No yield was obtained in the absence. Additionally, in rich media, 1mM 3-(2-Thienyl)alanine and D-2-(5-BromoThienyl)-alanine supplements yielded 5.1 and 16.8mg/L full-length protein respectively.
In HEK293T cells, PylHRS using the alternative tRNA MmtRNApyl was co-transfected alongside pAcBac2·tR2-EGFP in the presence or absence of histidine analogues 3-methyl-DL-histidine, beta-(3-Pyridyl)-DL-alanine, 3-(2-furyl)alanine, 3-(2-Thienyl)alanine, and D-2-(5-BromoThienyl)-alanine. Full-length EGFP expression was observed in the presence of 3-methyl-DL-histidine and 3-(2-Thienyl)alanine, with yields of 4 μg/10^7 cells and 3 μg/10^7 cells, respectively. Incorporation confirmed by ESI-MS, SDS-PAGE and MS/MS analysis.
Histidine analogues beta-(3-Pyridyl)-DL-alanine, 3-(2-furyl)alanine, 3-(2-Thienyl)alanine, D-2-(5-BromoThienyl)-alanine were also tested using pLeiG-GFP-Asp134TAG, PylHRS in DH10B cells and minimal media. in the presence of 1mM of each ncAA, full length protein yields of 4.0, 3.0, 4.2, and 12.0mg/L were achieved respectively. No yield was obtained in the absence. Additionally, in rich media, 1mM 3-(2-Thienyl)alanine and D-2-(5-BromoThienyl)-alanine supplements yielded 5.1 and 16.8mg/L full-length protein respectively.
In HEK293T cells, PylHRS using the alternative tRNA MmtRNApyl was co-transfected alongside pAcBac2·tR2-EGFP in the presence or absence of histidine analogues 3-methyl-DL-histidine, beta-(3-Pyridyl)-DL-alanine, 3-(2-furyl)alanine, 3-(2-Thienyl)alanine, and D-2-(5-BromoThienyl)-alanine. Full-length EGFP expression was observed in the presence of 3-methyl-DL-histidine and 3-(2-Thienyl)alanine, with yields of 4 μg/10^7 cells and 3 μg/10^7 cells, respectively. Incorporation confirmed by ESI-MS, SDS-PAGE and MS/MS analysis.