A facile system for encoding unnatural amino acids in mammalian cells

Abstract

A shuttle system has been developed to genetically encode unnatural amino acids in mammalian cells using aminoacyl-tRNA synthetases (aaRSs) evolved in E. coli. A pyrrolysyl-tRNA synthetase (PylRS) mutant was evolved in E. coli that selectively aminoacylates a cognate nonsense suppressor tRNA with a photocaged lysine derivative. Transfer of this orthogonal tRNA-aaRS pair into mammalian cells made possible the selective incorporation of this unnatural amino acid into proteins.

Figure 1

a) Northern blot analysis of tRNA charging in E. coli. The uncharged ${\mathrm{tRNA}}_{\mathrm{CUA}}^{\mathrm{Pyl}}$ band and the charged ${\mathrm{tRNA}}_{\mathrm{CUA}}^{\mathrm{Pyl}}$ band are indicated by arrows. ${\mathrm{tRNA}}_{\mathrm{CUA}}^{\mathrm{Pyl}}$ is only charged in the presence of both PylRS and Cyc. b) Western blot analysis of protein expression in mammalian cells. The full length mutant His-RBP4 is only expressed when CHO cells harboring both MmPylRS and tRNA plasmids were grown with 5 mM Cyc.

Figure 2

Evolution of a MmPylRS-${\mathrm{tRNA}}_{\mathrm{CUA}}^{\mathrm{Pyl}}$ pair that encodes ONBK in E. coli. a) Plate assay showing that NBK-1 and NBK-2 are able to survive up to 120 μg ml⁻¹ Cm challenges when supplemented with 1 mM ONBK. b) Genetic incorporation of ONBK into GFP protein in E. coli analyzed by SDS-PAGE. The expressed full length GFP proteins were purified by Ni²⁺-NTA chromatography and stained with coomassie blue. c) ESI-MS analysis of purified GFP149ONBK protein produced by NBK-1- ${\mathrm{tRNA}}_{\mathrm{CUA}}^{\mathrm{Pyl}}$ . The major peak (mass: 27,915 Da) corresponds to the full length GFP149ONBK; the minor peak (mass: 27,782 Da) corresponds to the same protein with the N-terminal Met posttranslationally cleaved (GFP149ONBK-M).

Figure 3

Shuttling the evolved synthetase into mammalian cells. a) Expression of EGFP37TAG protein using the NBK-1-${\mathrm{tRNA}}_{\mathrm{CUA}}^{\mathrm{Pyl}}$ pair in HEK293 cells in the presence of 1mM ONBK. The top pictures show the fluorescence images of cells and the bottom pictures show cells illuminated with visible light. b) ESI-MS analysis of purified EGFP37ONBK protein from CHO cells. Inset shows the deconvoluted spectrum of EGFP37ONBK. c) ESI-MS analysis of EGFP37ONBK after photolysis. EGFP37ONBK protein at a final concentration of 100 μM was irradiated (365 nm) for 20 min.

Scheme 1

The structures of pyrrolysine (Pyl), the pyrrolysine analogue: N^ε-cyclopentyloxycarbonyl-L-lysine (Cyc), and the photocaged lysine: o-nitrobenzyl-oxycarbonyl-N^ε-L-lysine (ONBK).