Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins

Lang, Kathrin; Chin, Jason W.

doi:10.1021/cr400355w

Cited by 945 publications

(976 citation statements)

References 378 publications

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“…Further development of the conformational sensors will include application of unnatural amino acids (UAAs) mutagenesis and bioorthogonal chemistry, which allow cotranslational residue-specific incorporation of UAAs and chemoselective labeling of small GTPases in the cell (48).…”

Section: Discussionmentioning

confidence: 99%

Spatiotemporal imaging of small GTPases activity in live cells

Voss

Krüger

Koch

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Ras-like small GTPases function as molecular switches and regulate diverse cellular events. To examine the dynamics of signaling requires spatiotemporal visualization of their activity in the cell. Current small GTPase sensors rely on specific effector domains that are available for only a small number of GTPases and compete for endogenous regulator/effector binding. Here, we describe versatile conformational sensors for GTPase activity (COSGAs) based on the conserved GTPase fold. Conformational changes upon GDP/GTP exchange were directly observed in solution, on beads, and in live cells by Förster resonance energy transfer (FRET). The COSGAs allow for monitoring of Rab1 and K-Ras activity in live cells using fluorescence lifetime imaging microscopy. We found that Rab1 is largely active in the cytoplasm and inactive at the Golgi, suggesting that the Golgi serves as the terminal of the Rab1 functional cycle. K-Ras displays polarized activity at the plasma membrane, with less activity at the edge of the cell and membrane ruffles.

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Section: Discussionmentioning

confidence: 99%

Spatiotemporal imaging of small GTPases activity in live cells

Voss

Krüger

Koch

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Such noncanonical amino acids (NCAAs) have diverse applications in chemical biology (8), serve as intermediates in the synthesis of natural products (9,10), and are privileged scaffolds for the development of pharmaceuticals (11). Despite its natural ability to produce these desirable compounds, TrpS has enjoyed only limited application (7).…”

mentioning

confidence: 99%

Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

Buller

Brinkmann‐Chen

Romney

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

146

257

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Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope.protein engineering | allostery | noncanonical amino acid | PLP

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“…Orthogonal aminoacyl‐tRNA synthetase/tRNA pairs that enable the incorporation of diverse ncAAs have been discovered,4 and the site‐specific incorporation of ncAAs bearing bioorthogonal reactive groups provides an attractive, and potentially general, strategy for generating ADCs 5. However, the ncAAs that have been incorporated into antibodies so far, which contain ketones or azides, are not optimal for bioconjugation.…”

mentioning

confidence: 99%

Rapid and Efficient Generation of Stable Antibody–Drug Conjugates via an Encoded Cyclopropene and an Inverse‐Electron‐Demand Diels–Alder Reaction

2018

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Homogeneous antibody–drug conjugates (ADCs), generated by site‐specific toxin linkage, show improved therapeutic indices with respect to traditional ADCs. However, current methods to produce site‐specific conjugates suffer from low protein expression, slow reaction kinetics, and low yields, or are limited to particular conjugation sites. Here we describe high yielding expression systems that efficiently incorporate a cyclopropene derivative of lysine (CypK) into antibodies through genetic‐code expansion. We express trastuzumab bearing CypK and conjugate tetrazine derivatives to the antibody. We show that the dihydropyridazine linkage resulting from the conjugation reaction is stable in serum, and generate an ADC bearing monomethyl auristatin E that selectively kills cells expressing a high level of HER2. Our results demonstrate that CypK is a minimal bioorthogonal handle for the rapid production of stable therapeutic protein conjugates.

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Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins

Cited by 945 publications

References 378 publications

Spatiotemporal imaging of small GTPases activity in live cells

Spatiotemporal imaging of small GTPases activity in live cells

Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

Rapid and Efficient Generation of Stable Antibody–Drug Conjugates via an Encoded Cyclopropene and an Inverse‐Electron‐Demand Diels–Alder Reaction

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