RS/tRNA Foundational Publication Support
Kaya, Emine, Milan Vrabel, Christian Deiml, Stefan Prill, Viviana Fluxa, and Thomas Carell. (2012) 2012. “A Genetically Encoded Norbornene Amino Acid For The Mild And Selective Modification Of Proteins In A Copper-Free Click Reaction.”. Angewandte Chemie (International Ed. In English) 51 (18): 4466-9. doi:10.1002/anie.201109252.
RS/tRNA Protocols and Structural Information
Schneider, Sabine, Michael J Gattner, Milan Vrabel, Veronika Flügel, Verónica López-Carrillo, Stefan Prill, and Thomas Carell. (2013) 2013. “Structural Insights Into Incorporation Of Norbornene Amino Acids For Click Modification Of Proteins.”. Chembiochem : A European Journal Of Chemical Biology 14 (16): 2114-8. doi:10.1002/cbic.201300435.
RS/tRNA Usage Publications
Scheidler, Christopher M, Milan Vrabel, and Sabine Schneider. (2020) 2020. “Genetic Code Expansion, Protein Expression, And Protein Functionalization In .”. Acs Synthetic Biology 9 (3): 486-493. doi:10.1021/acssynbio.9b00458.
RS/tRNA Pair Development Year
2012
ncAA(s) Incorporated
Norbornene-methoxycarbonyl-Lysine
ncAA Structure (png, jpg, jpeg)
ncAA Utility
Useful for click-chemistry with nitrile imines and tetrazines
RS Organism of Origin
Parent RS
RS Mutations
Y306G
Y384F
I405R
Y384F
I405R
tRNA Organism of Origin
Parent tRNA
tRNA Anticodon
CUA
RS/tRNA Availability
n/a
Used in what cell line?
RS/tRNA Additional Notes
also called MmPylRS-norb
RS selection based on incorporation into YFP. ncAA incorporated into human polymerase kappa (residue 103) produced at 2 mg/L, confirmed by MS, and reactive with nitrile imine and tetrazine in click or photoclick reactions.
In structure paper showed incorporates both endo- and exo- isomers similarly (~9 mg/L into carbonic anhydrase site 121), and incorporates cyclopropyl-norbornene less well (3 mg/L).
In 2020 paper used in B subtilis with 1 mM ncAA (to produce GFP(site 26) protein at ~20 mg/L confirmed by MS. ncAA also put into an antibody fragment (2 mg/L secreted compared with 15 mg/L for the wild type fragment) using a PG10(Amber-Sec) strain with secretion system genes having amber stop codons changed to keep that system functional. The ncAA-Ab fragments reacted well with tetrazines. GCE machinery was stably integrated into genome of B subtilis K07 at the amyE-locus.
RS selection based on incorporation into YFP. ncAA incorporated into human polymerase kappa (residue 103) produced at 2 mg/L, confirmed by MS, and reactive with nitrile imine and tetrazine in click or photoclick reactions.
In structure paper showed incorporates both endo- and exo- isomers similarly (~9 mg/L into carbonic anhydrase site 121), and incorporates cyclopropyl-norbornene less well (3 mg/L).
In 2020 paper used in B subtilis with 1 mM ncAA (to produce GFP(site 26) protein at ~20 mg/L confirmed by MS. ncAA also put into an antibody fragment (2 mg/L secreted compared with 15 mg/L for the wild type fragment) using a PG10(Amber-Sec) strain with secretion system genes having amber stop codons changed to keep that system functional. The ncAA-Ab fragments reacted well with tetrazines. GCE machinery was stably integrated into genome of B subtilis K07 at the amyE-locus.