Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Science 10 July 1998:
Vol. 281. no. 5374, pp. 269 - 272
DOI: 10.1126/science.281.5374.269
Prev | Table of Contents | Next

Reports

Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells

B. Albert Griffin, * Stephen R. Adams, Roger Y. Tsien dagger

Recombinant proteins containing four cysteines at the i, i + 1, i + 4, and i + 5 positions of an alpha  helix were fluorescently labeled in living cells by extracellular administration of 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein. This designed small ligand is membrane-permeant and nonfluorescent until it binds with high affinity and specificity to the tetracysteine domain. Such in situ labeling adds much less mass than does green fluorescent protein and offers greater versatility in attachment sites as well as potential spectroscopic and chemical properties. This system provides a recipe for slightly modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently stained by small nonfluorescent dye molecules added from outside the cells.

B. A. Griffin, Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093-0647, USA. S. R. Adams, Department of Pharmacology, University of California San Diego, La Jolla, CA 92093-0647, USA. R. Y. Tsien, Department of Pharmacology, Department of Chemistry and Biochemistry, and Howard Hughes Medical Institute, University of California San Diego, La Jolla, CA 92093-0647, USA.
*   Present address: Aurora Biosciences, 11010 Torreyana Road, San Diego, CA 92121, USA.

dagger    To whom correspondence should be addressed.

Read the Full Text


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Advanced Correlative Light/Electron Microscopy: Current Methods and New Developments Using Tokuyasu Cryosections.
K. Cortese, A. Diaspro, and C. Tacchetti (2009)
J. Histochem. Cytochem. 57, 1103-1112
   Abstract »    Full Text »    PDF »
Glycoprotein-Dependent Acidification of Vesicular Stomatitis Virus Enhances Release of Matrix Protein.
C. E. Mire, D. Dube, S. E. Delos, J. M. White, and M. A. Whitt (2009)
J. Virol. 83, 12139-12150
   Abstract »    Full Text »    PDF »
Biarsenical Labeling of Tetracysteine-Tagged Proteins for Tracking Existing and Newly Synthesized Pools of Proteins.
A. K. Pattnaik and D. Panda (2009)
CSH Protocols 2009, pdb.prot5343
   Abstract »    Full Text »
A small functional intramolecular region of NodD was identified by mutation.
B. Hou, F. Li, X. Yang, and G. Hong (2009)
Acta Biochim Biophys Sin 41, 822-830
   Abstract »    Full Text »    PDF »
Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells.
S. C. Das, D. Panda, D. Nayak, and A. K. Pattnaik (2009)
J. Virol. 83, 2611-2622
   Abstract »    Full Text »    PDF »
Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag.
P. Wu, W. Shui, B. L. Carlson, N. Hu, D. Rabuka, J. Lee, and C. R. Bertozzi (2009)
PNAS 106, 3000-3005
   Abstract »    Full Text »    PDF »
NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations.
T. A. Jennings, S. G. Mackintosh, M. K. Harrison, D. Sikora, B. Sikora, B. Dave, A. J. Tackett, C. E. Cameron, and K. D. Raney (2009)
J. Biol. Chem. 284, 4806-4814
   Abstract »    Full Text »    PDF »
Specific Biarsenical Labeling of Cell Surface Proteins Allows Fluorescent- and Biotin-tagging of Amyloid Precursor Protein and Prion Proteins.
Y. Taguchi, Z.-D. Shi, B. Ruddy, D. W. Dorward, L. Greene, and G. S. Baron (2009)
Mol. Biol. Cell 20, 233-244
   Abstract »    Full Text »    PDF »
Azatryptophans endow proteins with intrinsic blue fluorescence.
S. Lepthien, M. G. Hoesl, L. Merkel, and N. Budisa (2008)
PNAS 105, 16095-16100
   Abstract »    Full Text »    PDF »
Polar secretion of proteins via the Xcp type II secretion system in Pseudomonas aeruginosa.
F. Senf, J. Tommassen, and M. Koster (2008)
Microbiology 154, 3025-3032
   Abstract »    Full Text »    PDF »
Recombineering-mediated tagging of Drosophila genomic constructs for in vivo localization and acute protein inactivation.
K. J. T. Venken, J. Kasprowicz, S. Kuenen, J. Yan, B. A. Hassan, and P. Verstreken (2008)
Nucleic Acids Res. 36, e114
   Abstract »    Full Text »    PDF »
Inactivation of clathrin heavy chain inhibits synaptic recycling but allows bulk membrane uptake.
J. Kasprowicz, S. Kuenen, K. Miskiewicz, R. L.P. Habets, L. Smitz, and P. Verstreken (2008)
J. Cell Biol. 182, 1007-1016
   Abstract »    Full Text »    PDF »
Physical and Functional Interactions of Monoubiquitylated Transactivators with the Proteasome.
C. T. Archer, L. Burdine, B. Liu, A. Ferdous, S. A. Johnston, and T. Kodadek (2008)
J. Biol. Chem. 283, 21789-21798
   Abstract »    Full Text »    PDF »
Selective Labeling of Proteins with Chemical Probes in Living Cells.
M. Z. Lin and L. Wang (2008)
Physiology 23, 131-141
   Abstract »    Full Text »    PDF »
Rational Optimization and Imaging In Vivo of a Genetically Encoded Optical Voltage Reporter.
L. Sjulson and G. Miesenbock (2008)
J. Neurosci. 28, 5582-5593
   Abstract »    Full Text »    PDF »
A hCXCR1 transgenic mouse model containing a conditional color-switching system for imaging of hCXCL8/IL-8 functions in vivo.
L. Zheng, C.-n. Njauw, and M. Martins-Green (2007)
J. Leukoc. Biol. 82, 1247-1256
   Abstract »    Full Text »    PDF »
From the Cover: Copper-free click chemistry for dynamic in vivo imaging.
J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang, I. A. Miller, A. Lo, J. A. Codelli, and C. R. Bertozzi (2007)
PNAS 104, 16793-16797
   Abstract »    Full Text »    PDF »
Characterization of Three Classes of Membrane Proteins Involved in Fungal Azole Resistance by Functional Hyperexpression in Saccharomyces cerevisiae.
E. Lamping, B. C. Monk, K. Niimi, A. R. Holmes, S. Tsao, K. Tanabe, M. Niimi, Y. Uehara, and R. D. Cannon (2007)
Eukaryot. Cell 6, 1150-1165
   Abstract »    Full Text »    PDF »
Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit.
O. P. Ernst, V. Gramse, M. Kolbe, K. P. Hofmann, and M. Heck (2007)
PNAS 104, 10859-10864
   Abstract »    Full Text »    PDF »
Structure and Conformational Changes in the C-terminal Domain of the beta2-Adrenoceptor: INSIGHTS FROM FLUORESCENCE RESONANCE ENERGY TRANSFER STUDIES.
S. Granier, S. Kim, A. M. Shafer, V. R. P. Ratnala, J. J. Fung, R. N. Zare, and B. Kobilka (2007)
J. Biol. Chem. 282, 13895-13905
   Abstract »    Full Text »    PDF »
Biochemical and Functional Analyses of the Human Toll-like Receptor 3 Ectodomain.
C. T. Ranjith-Kumar, W. Miller, J. Xiong, W. K. Russell, R. Lamb, J. Santos, K. E. Duffy, L. Cleveland, M. Park, K. Bhardwaj, et al. (2007)
J. Biol. Chem. 282, 7668-7678
   Abstract »    Full Text »    PDF »
A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure.
C. T. Hauser and R. Y. Tsien (2007)
PNAS 104, 3693-3697
   Abstract »    Full Text »    PDF »
Caspase-1-dependent processing of pro-interleukin-1beta is cytosolic and precedes cell death.
D. Brough and N. J. Rothwell (2007)
J. Cell Sci. 120, 772-781
   Abstract »    Full Text »    PDF »
The Amino-Conserved Domain of Human Cytomegalovirus UL80a Proteins Is Required for Key Interactions during Early Stages of Capsid Formation and Virus Production.
A. N. Loveland, N. L. Nguyen, E. J. Brignole, and W. Gibson (2007)
J. Virol. 81, 620-628
   Abstract »    Full Text »    PDF »
Golgi twins in late mitosis revealed by genetically encoded tags for live cell imaging and correlated electron microscopy.
G. M. Gaietta, B. N. G. Giepmans, T. J. Deerinck, W. B. Smith, L. Ngan, J. Llopis, S. R. Adams, R. Y. Tsien, and M. H. Ellisman (2006)
PNAS 103, 17777-17782
   Abstract »    Full Text »    PDF »
Isolation of a High-Affinity Functional Protein Complex between OmcA and MtrC: Two Outer Membrane Decaheme c-Type Cytochromes of Shewanella oneidensis MR-1.
L. Shi, B. Chen, Z. Wang, D. A. Elias, M. U. Mayer, Y. A. Gorby, S. Ni, B. H. Lower, D. W. Kennedy, D. S. Wunschel, et al. (2006)
J. Bacteriol. 188, 4705-4714
   Abstract »    Full Text »    PDF »
A genetically encoded fluorescent amino acid.
D. Summerer, S. Chen, N. Wu, A. Deiters, J. W. Chin, and P. G. Schultz (2006)
PNAS 103, 9785-9789
   Abstract »    Full Text »    PDF »
Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT).
D. C. Dieterich, A. J. Link, J. Graumann, D. A. Tirrell, and E. M. Schuman (2006)
PNAS 103, 9482-9487
   Abstract »    Full Text »    PDF »
The fluorescent toolbox for assessing protein location and function..
B. N. G. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien (2006)
Science 312, 217-224
   Abstract »    Full Text »    PDF »
Patch Fluorometry: Shedding New Light on Ion Channels.
J. Zheng (2006)
Physiology 21, 6-12
   Abstract »    Full Text »    PDF »
Flashy Science: Controlling Neural Function with Light.
S. M. Thompson, J. P. Y. Kao, R. H. Kramer, K. E. Poskanzer, R. A. Silver, D. Digregorio, and S. S.-H. Wang (2005)
J. Neurosci. 25, 10358-10365
   Full Text »    PDF »
Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase.
J. Yin, P. D. Straight, S. M. McLoughlin, Z. Zhou, A. J. Lin, D. E. Golan, N. L. Kelleher, R. Kolter, and C. T. Walsh (2005)
PNAS 102, 15815-15820
   Abstract »    Full Text »    PDF »
The Endogenous CXXC Motif Governs the Cadmium Sensitivity of the Renal Na+/Glucose Co-Transporter.
X. Xia, G. Wang, Y. Peng, M.-G. Tu, J. Jen, and H. Fang (2005)
J. Am. Soc. Nephrol. 16, 1257-1265
   Abstract »    Full Text »    PDF »
Dynamic Fluorescent Imaging of Human Immunodeficiency Virus Type 1 Gag in Live Cells by Biarsenical Labeling.
L. Rudner, S. Nydegger, L. V. Coren, K. Nagashima, M. Thali, and D. E. Ott (2005)
J. Virol. 79, 4055-4065
   Abstract »    Full Text »    PDF »
The Salmonella enterica Serovar Typhimurium-Encoded Type III Secretion Systems Can Translocate Chlamydia trachomatis Proteins into the Cytosol of Host Cells.
T. D. Ho and M. N. Starnbach (2005)
Infect. Immun. 73, 905-911
   Abstract »    Full Text »    PDF »
Short Tetracysteine Tags to {beta}-Tubulin Demonstrate the Significance of Small Labels for Live Cell Imaging.
M. Andresen, R. Schmitz-Salue, and S. Jakobs (2004)
Mol. Biol. Cell 15, 5616-5622
   Abstract »    Full Text »    PDF »
Labeling of fusion proteins with synthetic fluorophores in live cells.
A. Keppler, H. Pick, C. Arrivoli, H. Vogel, and K. Johnsson (2004)
PNAS 101, 9955-9959
   Abstract »    Full Text »    PDF »
A general approach for chemical labeling and rapid, spatially controlled protein inactivation.
K. M. Marks, P. D. Braun, and G. P. Nolan (2004)
PNAS 101, 9982-9987
   Abstract »    Full Text »    PDF »
Thermodynamic and structural characterization of 2'-nitrogen-modified RNA duplexes.
J. W. Pham, I. Radhakrishnan, and E. J. Sontheimer (2004)
Nucleic Acids Res. 32, 3446-3455
   Abstract »    Full Text »    PDF »
Binding of SycH Chaperone to YscM1 and YscM2 Activates Effector yop Expression in Yersinia enterocolitica.
E. D. Cambronne, J. A. Sorg, and O. Schneewind (2004)
J. Bacteriol. 186, 829-841
   Abstract »    Full Text »    PDF »
In vivo oligomerization and raft localization of Ebola virus protein VP40 during vesicular budding.
R. G. Panchal, G. Ruthel, T. A. Kenny, G. H. Kallstrom, D. Lane, S. S. Badie, L. Li, S. Bavari, and M. J. Aman (2003)
PNAS 100, 15936-15941
   Abstract »    Full Text »    PDF »
Myosin Va and microtubule-based motors are required for fast axonal retrograde transport of tetanus toxin in motor neurons.
G. Lalli, S. Gschmeissner, and G. Schiavo (2003)
J. Cell Sci. 116, 4639-4650
   Abstract »    Full Text »    PDF »
Multimodal optical imaging.
C. Lawler, W. A. Suk, B. R. Pitt, C. M. St. Croix, and S. C. Watkins (2003)
Am J Physiol Lung Cell Mol Physiol 285, L269-L280
   Abstract »    Full Text »    PDF »
Light Microscopy Techniques for Live Cell Imaging.
D. J. Stephens and V. J. Allan (2003)
Science 300, 82-86
   Abstract »    Full Text »    PDF »
Addition of the keto functional group to the genetic code of Escherichiacoli.
L. Wang, Z. Zhang, A. Brock, and P. G. Schultz (2003)
PNAS 100, 56-61
   Abstract »    Full Text »    PDF »
Pulse-Chase in the Light Microscope.
D. Goodenough and F. Postma (2002)
Mol. Interv. 2, 132-134
   Abstract »    Full Text »    PDF »
Multicolor and Electron Microscopic Imaging of Connexin Trafficking.
G. Gaietta, T. J. Deerinck, S. R. Adams, J. Bouwer, O. Tour, D. W. Laird, G. E. Sosinsky, R. Y. Tsien, and M. H. Ellisman (2002)
Science 296, 503-507
   Abstract »    Full Text »    PDF »
Fluorescent Histochemical Techniques for Analysis of Intracellular Signaling.
M. P. Oksvold, E. Skarpen, J. Widerberg, and H. S. Huitfeldt (2002)
J. Histochem. Cytochem. 50, 289-303
   Abstract »    Full Text »    PDF »
Chloride Concentration in Endosomes Measured Using a Ratioable Fluorescent Cl- Indicator. EVIDENCE FOR CHLORIDE ACCUMULATION DURING ACIDIFICATION.
N. D. Sonawane, J. R. Thiagarajah, and A. S. Verkman (2002)
J. Biol. Chem. 277, 5506-5513
   Abstract »    Full Text »    PDF »
The Biotin Switch Method for the Detection of S-Nitrosylated Proteins.
S. R. Jaffrey and S. H. Snyder (2001)
Sci. STKE 2001, pl1
   Abstract »    Full Text »    PDF »
GFP imaging: methodology and application to investigate cellular compartmentation in plants.
M. R. Hanson and R. H. Kohler (2001)
J. Exp. Bot. 52, 529-539
   Abstract »    Full Text »    PDF »
Cell Surface Engineering by a Modified Staudinger Reaction.
E. Saxon and C. R. Bertozzi (2000)
Science 287, 2007-2010
   Abstract »    Full Text »
Detoxification of Arsenic by Phytochelatins in Plants.
M. E.V. Schmoger, M. Oven, and E. Grill (2000)
Plant Physiology 122, 793-802
   Abstract »    Full Text »    PDF »
Mammalian cell lines expressing functional RNA polymerase II tagged with the green fluorescent protein.
K Sugaya, M Vigneron, and P. Cook (2000)
J. Cell Sci. 113, 2679-2683
   Abstract »    PDF »
Visualizing Protein-Protein Interactions in the Nucleus of the Living Cell.
R. N. Day, S. K. Nordeen, and Y. Wan (1999)
Mol. Endocrinol. 13, 517-526
   Full Text »
Receptor-mediated Targeting of Fluorescent Probes in Living Cells.
J. Farinas and A. S. Verkman (1999)
J. Biol. Chem. 274, 7603-7606
   Abstract »    Full Text »    PDF »
Mechanisms of Calcium Influx into Hippocampal Spines: Heterogeneity among Spines, Coincidence Detection by NMDA Receptors, and Optical Quantal Analysis.
R. Yuste, A. Majewska, S. S. Cash, and W. Denk (1999)
J. Neurosci. 19, 1976-1987
   Abstract »    Full Text »    PDF »
Direct Imaging of DNA in Living Cells Reveals the Dynamics of Chromosome Formation.
E. M.M. Manders, H. Kimura, and P. R. Cook (1999)
J. Cell Biol. 144, 813-822
   Abstract »    Full Text »    PDF »
Aldol sensors for the rapid generation of tunable fluorescence by antibody catalysis.
B. List, C. F. Barbas III, and R. A. Lerner (1998)
PNAS 95, 15351-15355
   Abstract »    Full Text »    PDF »
Inhibition of NF-kappa B Activation by Arsenite through Reaction with a Critical Cysteine in the Activation Loop of Ikappa B Kinase.
P. Kapahi, T. Takahashi, G. Natoli, S. R. Adams, Y. Chen, R. Y. Tsien, and M. Karin (2000)
J. Biol. Chem. 275, 36062-36066
   Abstract »    Full Text »    PDF »
Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation.
K. L. Kiick, E. Saxon, D. A. Tirrell, and C. R. Bertozzi (2002)
PNAS 99, 19-24
   Abstract »    Full Text »    PDF »
Analysis of retrograde transport in motor neurons reveals common endocytic carriers for tetanus toxin and neurotrophin receptor p75NTR.
G. Lalli and G. Schiavo (2002)
J. Cell Biol. 156, 233-240
   Abstract »    Full Text »    PDF »


To Advertise     Find Products

Science. ISSN 0036-8075 (print), 1095-9203 (online)