Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 42 (5), 386-396

Site-Specific Labeling of Proteins Using Unnatural Amino Acids


Site-Specific Labeling of Proteins Using Unnatural Amino Acids

Kyung Jin Lee et al. Mol Cells.


Labeling of a protein with a specific dye or tag at defined positions is a critical step in tracing the subtle behavior of the protein and assessing its cellular function. Over the last decade, many strategies have been developed to achieve selective labeling of proteins in living cells. In particular, the site-specific unnatural amino acid (UAA) incorporation technique has gained increasing attention since it enables attachment of various organic probes to a specific position of a protein in a more precise way. In this review, we describe how the UAA incorporation technique has expanded our ability to achieve site-specific labeling and visualization of target proteins for functional analyses in live cells.

Keywords: bioorthogonal; click chemistry; genetic code expansion; site-specific labeling; unnatural amino acid.

Conflict of interest statement

Disclosure of potential conflicts of interest

The authors declare no conflict of interest.


Fig. 1
Fig. 1. Unnatural amino acids used for site-specific protein labeling
1: 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), 2: (S)-1-carboxy-3-(7-hydroxy-2-oxo-2H-chromen-4-yl)propan-1-aminium (CouAA), 3: 3-(5-(dimethylamino)naphthalene-1-sulfonamide) propanoic acid (Dansylalanine), 4: Nɛ-p-azidobenzyloxycarbonyl lysine (PABK), 5: Propargyl-L-lysine (PrK), 6: Nɛ-(1-methylcycloprop-2-enecarboxamido) lysine (CpK), 7: Nɛ-acryllysine (AcrK), 8: Nɛ-(cyclooct-2-yn-1-yloxy)carbonyl)L-lysine (CoK), 9: bicyclo[6.1.0]non-4-yn-9-ylmethanol lysine (BCNK), 10: trans-cyclooct-2-ene lysine (2′-TCOK), 11: trans-cyclooct-4-ene lysine (4′-TCOK), 12: dioxo-TCO lysine (DOTCOK), 13: 3-(2-cyclobutene-1-yl)propanoic acid (CbK), 14: Nɛ-5-norbornene-2-yloxycarbonyl-L-lysine (NBOK), 15: cyclooctyne lysine (SCOK), 16: 5-norbornen-2-ol tyrosine (NOR), 17: cyclooct-2-ynol tyrosine (COY), 18: (E)-2-(cyclooct-4-en-1-yloxyl)ethanol tyrosine (DS1/2), 19: azidohomoalanine (AHA), 20: homopropargylglycine (HPG), 21: azidonorleucine (ANL), 22: Nɛ-2-azideoethyloxycarbonyl-L-lysine (NEAK).
Fig. 2
Fig. 2. Bioorthogonal reactions for labeling of a protein of interest
CuAAC, copper-catalyzed azide alkyne cycloaddition; Photoclick cycloaddition; SPAAC, strain-promoted azide alkyne cycloaddition; IEDDA, inverse electron-demand Diels–Alder cycloadditions.
Fig. 3
Fig. 3. Promising applications of UAA-based protein-labeling scheme
(A) Study of the conformational dynamics of sigle influenza hemagglutinin (HA), (B) high resolution proteomic analysis in diverse organisms, and (C) in vivo PET imaging and pharmacokinetics studies.

Similar articles

See all similar articles


    1. Adams S.R., Campbell R.E., Gross L.A., Martin B.R., Walkup G.K., Yao Y., Llopis J., Tsien R.Y. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J Am Chem Soc. 2002;124:6063–6076. doi: 10.1021/ja017687n. - DOI - PubMed
    1. Agard N.J., Prescher J.A., Bertozzi C.R. A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J Am Chem Soc. 2004;126:15046–15047. doi: 10.1021/ja044996f. - DOI - PubMed
    1. Alamudi S.H., Satapathy R., Kim J., Su D., Ren H., Das R., Hu L., Alvarado-Martínez E., Lee J.Y., Hoppmann C., et al. Development of background-free tame fluorescent probes for intracellular live cell imaging. Nat Commun. 2016;7:11964. doi: 10.1038/ncomms11964. - DOI - PMC - PubMed
    1. Ambrogelly A., Palioura S., Söll D. Natural expansion of the genetic code. Nat Chem Biol. 2007;3:29–35. doi: 10.1038/nchembio847. - DOI - PubMed
    1. Baskin J.M., Prescher J.A., Laughlin S.T., Agard N.J., Chang P.V., Miller I.A., Lo A., Codelli J.A., Bertozzi C.R. Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci U S A. 2007;104:16793–16797. doi: 10.1073/pnas.0707090104. - DOI - PMC - PubMed