Expanding the Genetic Code for Photoclick Chemistry in E. coli, Mammalian Cells, and A. thaliana

Abstract

Bioorthogonal chemical reactions together with techniques to expand the genetic code have provided exciting new means for protein labeling and visualization in living systems, [1] as well as for optimizing the efficacy of therapeutic proteins. [2] Toward these goals, amino acids with small bioorthogonal functional groups, such as azide, alkyne, or cyclopropene moieties, [3] as well as larger reactive bioorthogonal groups, [4] such as cyclooctyne, norbornene, trans-cyclooctene, aryltetrazole, or aryltetrazine, have been site-specifically incorporated into proteins, allowing for selective conjugation of biophysical probes through azide-alkyne click chemistry (AAC), tetrazole-alkene photoclick chemistry (TAP), and reverse-electron demand Diels-Alder reactions. [5] The main advantages of the photoclick reaction (Supporting Information, Scheme S1) are: 1) its fast rate (up to 50 m À1 s À1 ); 2) that spatiotemporal control is initiated by a photo-induced reaction; 3) that the photoclick reaction is fluorogenic, allowing for high-contrast fluorescence imaging without tedious washing steps. In previous studies, we reported the site-specific incorporation of p-(2-tetrazole)phenylalanine (p-Tpa) [4a] and N-e-(1-methylcycloprop-2-enecarboxamido)lysine (CpK) [3c] in E. coli and mammalian cells. Subsequent photoirradiation of labeled proteins with UV light facilitates selective conjugation with dimethyl fumarate or diaryltetrazole, respectively.By expanding the genetic code and introducing photoclick chemistry to plants, important problems in plant chemical biology can be addressed, [6] such as photosynthesis and stress response, which can only be studied at the organismal level. Recently, expansion of the genetic code has been used to optimize therapeutic proteins produced in bacteria and mammalian cells. [2] Because plants offer an attractive alternative to microbial fermentation and animal cell cultures for high-yield production of recombinant proteins on an agricultural scale, [8] expanding the genetic code in plants would be useful for producing recombinant therapeutic proteins and enzymes with enhanced properties, better safety, and lower costs. [8] To fully realize the potential of photoclick reaction for tracking fast cellular processes, it is desirable that the unnatural amino acid (UAA) used has a small functional group such that there is minimal perturbation of the target protein, and a very brief exposure to long-wavelength UV light or violet-blue light is used to drive the photoclick reaction to minimize damage to cells and plants. Herein, we addressed these issues by genetically incorporating N-eacryllysine (AcrK, Figure 1 A), in response to an amber stop codon (TAG) in bacterial cells, mammalian cells, and plants. This new strategy was then used to efficiently label proteins both in vitro and in vivo. In comparison to lysine, AcrK has only four extra non-hydrogen atoms, which is significantly less than other UAAs. [3, 4] Replacing one lysine residue with AcrK should cause only minimal perturbation to ...

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

“…ND, no data. mammalian culture cells, and plants (58)(59)(60)(61)(62). Similar translational switches can also be constructed using these pairs and can be easily implemented with applicable organisms.…”

Playing with the Molecules of Life

“…24, 42, 43 These latter pairs are especially advantageous as they allow aaRSs to be evolved in E. coli prior to transfer of the machinery to more diverse eukaryotic hosts. Utilizing these orthogonal pairs, ncAAs have been encoded in B. cereus, 89 P. pastoris, 90 C. elegans, 91, 92 D. melanogaster, 93 A. thaliana, 94 Zebrafish embryos, 95 and the mouse. 96–99 More recently, it has been demonstrated that a native Trp aaRS/tRNA pair in E. coli can be functionally replaced with a counterpart from yeast, and the liberated Trp pair can be used to encode ncAAs in bacteria.…”

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

“…We have demonstrated that a genetically incorporated 36 allowed selectively turn-on fluorescent labeling of proteins in living cells. [42, 94] Two photocrosslinking NCAAs 23 and 37 have been genetically encoded using native and engineered PylRS-tRNA Pyl pairs and applied successfully to covalently crosslink associate proteins. Chen et al has pushed the technique further in using 37 to successfully profile in vivo substrates of a major acid-protection chaperone, HdeA, in E. coli periplasm.…”