Genetically Encoded Norbornene Directs Site-specific Cellular Protein Labelling Via a Rapid Bioorthogonal Reaction

Overview

Journal Nat Chem

Specialty Chemistry

Date 2012 Mar 23

PMID 22437715

Citations 192

Authors

Kathrin Lang

Lloyd Davis

Jessica Torres-Kolbus

Chungjung Chou

Alexander Deiters

Jason W Chin

Affiliations

Soon will be listed here.

Abstract

The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNA(CUA) pair in Escherichia coli and mammalian cells. We developed a series of tetrazine-based probes that exhibit 'turn-on' fluorescence on their rapid reaction with norbornenes. We demonstrate that the labelling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions. We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labelling in vitro and on mammalian cells, and demonstrate the rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface.

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References

Halo T, Appelbaum J, Hobert E, Balkin D, Schepartz A . Selective recognition of protein tetraserine motifs with a cell-permeable, pro-fluorescent bis-boronic acid. J Am Chem Soc. 2008; 131(2):438-9. PMC: 2659559. DOI: 10.1021/ja807872s. View

Li X, Fekner T, Ottesen J, Chan M . A pyrrolysine analogue for site-specific protein ubiquitination. Angew Chem Int Ed Engl. 2009; 48(48):9184-7. DOI: 10.1002/anie.200904472. View

Shaner N, Steinbach P, Tsien R . A guide to choosing fluorescent proteins. Nat Methods. 2005; 2(12):905-9. DOI: 10.1038/nmeth819. View

Hinner M, Johnsson K . How to obtain labeled proteins and what to do with them. Curr Opin Biotechnol. 2010; 21(6):766-76. DOI: 10.1016/j.copbio.2010.09.011. View

Popp M, Antos J, Grotenbreg G, Spooner E, Ploegh H . Sortagging: a versatile method for protein labeling. Nat Chem Biol. 2007; 3(11):707-8. DOI: 10.1038/nchembio.2007.31. View

Wang L, Zhang Z, Brock A, Schultz P . Addition of the keto functional group to the genetic code of Escherichia coli. Proc Natl Acad Sci U S A. 2003; 100(1):56-61. PMC: 140882. DOI: 10.1073/pnas.0234824100. View

Chin J, Cropp T, Anderson J, Mukherji M, Zhang Z, Schultz P . An expanded eukaryotic genetic code. Science. 2003; 301(5635):964-7. DOI: 10.1126/science.1084772. View

Fernandez-Suarez M, Baruah H, Martinez-Hernandez L, Xie K, Baskin J, Bertozzi C . Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nat Biotechnol. 2007; 25(12):1483-7. PMC: 2654346. DOI: 10.1038/nbt1355. View

Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D . Green fluorescent protein as a marker for gene expression. Science. 1994; 263(5148):802-5. DOI: 10.1126/science.8303295. View

10.

Blackman M, Royzen M, Fox J . Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels-Alder reactivity. J Am Chem Soc. 2008; 130(41):13518-9. PMC: 2653060. DOI: 10.1021/ja8053805. View

11.

Wang J, Zhang W, Song W, Wang Y, Yu Z, Li J . A biosynthetic route to photoclick chemistry on proteins. J Am Chem Soc. 2010; 132(42):14812-8. PMC: 2965590. DOI: 10.1021/ja104350y. View

12.

Yin J, Straight P, McLoughlin S, Zhou Z, Lin A, Golan D . Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc Natl Acad Sci U S A. 2005; 102(44):15815-20. PMC: 1276090. DOI: 10.1073/pnas.0507705102. View

13.

Devaraj N, Weissleder R, Hilderbrand S . Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjug Chem. 2008; 19(12):2297-9. PMC: 2677645. DOI: 10.1021/bc8004446. View

14.

Chin J, Santoro S, Martin A, King D, Wang L, Schultz P . Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. J Am Chem Soc. 2002; 124(31):9026-7. DOI: 10.1021/ja027007w. View

15.

Devaraj N, Weissleder R . Biomedical applications of tetrazine cycloadditions. Acc Chem Res. 2011; 44(9):816-27. PMC: 3166440. DOI: 10.1021/ar200037t. View

16.

Fekner T, Li X, Lee M, Chan M . A pyrrolysine analogue for protein click chemistry. Angew Chem Int Ed Engl. 2009; 48(9):1633-5. DOI: 10.1002/anie.200805420. View

17.

Nguyen D, Elliott T, Holt M, Muir T, Chin J . Genetically encoded 1,2-aminothiols facilitate rapid and site-specific protein labeling via a bio-orthogonal cyanobenzothiazole condensation. J Am Chem Soc. 2011; 133(30):11418-21. DOI: 10.1021/ja203111c. View

18.

Devaraj N, Hilderbrand S, Upadhyay R, Mazitschek R, Weissleder R . Bioorthogonal turn-on probes for imaging small molecules inside living cells. Angew Chem Int Ed Engl. 2010; 49(16):2869-72. PMC: 3433403. DOI: 10.1002/anie.200906120. View

19.

Deiters A, Cropp T, Mukherji M, Chin J, Anderson J, Schultz P . Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae. J Am Chem Soc. 2003; 125(39):11782-3. DOI: 10.1021/ja0370037. View

20.

Neumann H, Peak-Chew S, Chin J . Genetically encoding N(epsilon)-acetyllysine in recombinant proteins. Nat Chem Biol. 2008; 4(4):232-4. DOI: 10.1038/nchembio.73. View