A Facile System for Encoding Unnatural Amino Acids in Mammalian Cells

Abstract

Ein Shuttle‐System zur Verwendung von in E. coli selektierten Aminoacyl‐tRNA‐Synthetasen (aaRSs) für den Einbau von nichtnatürlichen Aminosäuren in Säugerzellen wurde entwickelt. Eine in E. coli selektierte Mutante der Pyrrolysyl‐tRNA‐Synthetase (PylRS) aminoacyliert selektiv eine Nonsense‐Suppressor‐tRNA mit einem photoaktivierbaren Lysinderivat. Durch Übertragung dieses orthogonalen tRNA‐aaRS‐Paares in Säugerzellen gelang es, diese nichtnatürliche Aminosäure selektiv in Proteine einzubauen.

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 .…”

“…[35] To test whether AbKRS engineered in E. coli can be directly adapted to encode AbK in mammalian cells, a pCMV-AbK plasmid was constructed to express both AbKRS and tRNA Pyl (Figure S2). [30] HEK (human embryonic kidney) 293T cells were transfected with pCMV-AbK (in which tRNA Pyl and AbKRS are expressed under the U6 and CMV promoters, respectively) and pEGFP-Tyr39TAG (His 6 -tagged enhanced green fluorescent protein gene with a TAG mutation at residue 39). Transiently transfected cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) in the absence or presence of 1 mm AbK for 48 h. EGFP fluorescence was imaged with an inverted fluorescence microscope, and was only observed when AbK was added to the culture medium (Figure 3 A).…”

Genetisch kodierte kupferfreie Klick‐Chemie

“…Die hohe FRET-Effizienz dieser Population ist in Einklang mit der Kristallstruktur von GFP, [22] [23] Andere modifizierte Cyclooctine, wie Dibenzocyclooctine, [24] könnten hingegen wegen ihrer Größe die Synthetase und/oder die Translationsmaschinerie des Wirtes vor beträchtliche Herausforderungen stellen. Da pylRS von M. mazei bekanntermaßen auch orthogonal in einer Vielzahl an eukaryotischen Organismen [19,25] …”