A Genetically Encoded ε‐N‐Methyl Lysine in Mammalian Cells

Probing Protein–Protein Interactions with a Genetically Encoded Photo‐crosslinking Amino Acid

“…A single colony was used to inoculate 2YT medium [25 mL, containing l-arabinose (0.2 %), tetracycline (20 mg mL À1 ), and kanamycin(50 mg mL Protein expression and purification from HEK 293T cells: The mammalian expression vector pCMV has been described previously. [30,31] In brief, the plasmid contains a copy of the synthetase gene driven by a CMV promoter, and a copy of the tRNA gene under the control of a human U6 promoter ( Figure S2). The new plasmid pCMV-AbK was created by replacing the synthetase in the previous pCMV-NBK plasmid.…”

“…The new plasmid pCMV-AbK was created by replacing the synthetase in the previous pCMV-NBK plasmid. [30,31] HEK 293T cells were grown in DMEM supplemented with 10 % fetal bovine serum (FBS). Cells at 70 % confluency were transfected with mixtures of the corresponding plasmids by using Lipofectamine 2000 (Invitrogen).…”

“…[30][31][32][33] We postulated that the wt-mbPylRS/tRNA Pyl pair could be subjected to directed evolution to optimize its amber suppression efficiency in the presence of AbK. We therefore used a mbPylRS library randomized at residues Leu270, Tyr271, Leu274, and Cys313 (and an additional Tyr349Phe mutation) [31] to select synthetases that can efficiently charge AbK to tRNA Pyl . A series of positive and negative selections were performed in E. coli strain DH10B as previously described.…”

Posttranslational Modification of Proteins Incorporating Nonnatural Amino Acids

“…An MbPylRS library was developed by randomizing the residues Leu270, Tyr271, Leu274, Cys313, and an additional Tyr349Phe [105] to obtain a mutant that could effectively charge AbK (Table 12.2). A positive and negative selection was performed in E. coli as before.…”

A Modified Epigenetics Toolbox to Study Histone Modifications on the Nucleosome Core

“…[33,34] The development of similar strategies to introduce methylated lysines at specific positions in recombinant proteins has recently been reported as well. [35][36][37] This genetic code expansion approach is expected to provide a rich source for defined nucleosome substrates with a range of native modifications. This should allow a detailed analysis of the kinetic parameters of Dot1 and other enzymes that act on nucleosome core residues.…”