DOI: 10.1126/science.281.5374.269. Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells

Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells

10.1126/science.281.5374.269

Crossref journal-article

American Association for the Advancement of Science (AAAS)

Science (221)

Abstract

Recombinant proteins containing four cysteines at the i , i + 1, i + 4, and i + 5 positions of an α 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.

Bibliography

Griffin, B. A., Adams, S. R., & Tsien, R. Y. (1998). Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells. Science, 281(5374), 269â272.

Authors 3

B. Albert Griffin (first)
Stephen R. Adams (additional)
Roger Y. Tsien (additional)

References 23 Referenced 1,397

G. T. Hermanson Bioconjugate Techniques (Academic Press San Diego CA 1996). (10.1016/B978-012342335-1/50012-9)
10.1146/annurev.biochem.67.1.509
M. Chalfie and S. R. Kain Eds. GFP: Green Fluorescent Protein Strategies and Applications (Wiley New York 1998).
J. L. Webb Enzyme and Metabolic Inhibitors (Academic Press New York 1966) vol. III pp. 595–819.
Kalef E., Gitler C., Methods Enzymol. 233, 395 (1994). (10.1016/S0076-6879(94)33046-8) / Methods Enzymol. by Kalef E. (1994)
Whittaker V. P., Biochem. J. 41, 56 (1947). (10.1042/bj0410056) / Biochem. J. by Whittaker V. P. (1947)
E. C. Webb and R. Van Heyningen ibid. p. 74.
Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; E Glu; R Arg; W Trp; and X any amino acid.
Merutka G., Shalongo W., Stellwagen E., Biochemistry 30, 4245 (1991). (10.1021/bi00231a020) / Biochemistry by Merutka G. (1991)
B. A. Griffin thesis University of California San Diego (1998).
Lowe W. G., Hamilton C. S., J. Am. Chem. Soc. 57, 1081 (1935). (10.1021/ja01309a035) / J. Am. Chem. Soc. by Lowe W. G. (1935)
Spang C., Edelmann F. T., Noltemeyer M., Roesky H. W., Chem. Ber. 122, 1247 (1989). (10.1002/cber.19891220706) / Chem. Ber. by Spang C. (1989)
10.1016/S0960-9822(02)00450-5
10.1038/42264
10.1126/science.273.5280.1392
Denizot F., Lang R., J. Immunol. Methods 89, 271 (1986). (10.1016/0022-1759(86)90368-6) / J. Immunol. Methods by Denizot F. (1986)
Zlokarnik G., et al., Science 279, 84 (1998). (10.1126/science.279.5347.84) / Science by Zlokarnik G. (1998)
Ford C. F., Suominen I., Glatz C. E., Protein Expr. Purif. 2, 95 (1991). (10.1016/1046-5928(91)90057-P) / Protein Expr. Purif. by Ford C. F. (1991)
Crabtree G. R., Schreiber S. L., Trends Biochem. Sci. 21, 418 (1996). (10.1016/S0968-0004(96)20027-1) / Trends Biochem. Sci. by Crabtree G. R. (1996)
FLASH-EDT 2 was synthesized by reacting 4′ 5′- bis(acetoxymercuri)fluorescein (fluorescein mercuric acetate) with 20 equivalents of arsenic trichloride (caution: highly toxic!) and 8 equivalents of di-isopropylethylamine in dry N -methylpyrrolidinone with palladium acetate as a catalyst. After 3 hours at room temperature the reaction was quenched in pH 7 buffer with excess 1 2-ethanedithiol. The product was extracted into chloroform and chromatographed on silica gel with a 1:9 mixture of ethyl acetate and toluene. Trituration with ethanol gave an orange-red solid in 37% yield. 1 H nuclear magnetic resonance (at 200 MHz in CDCl 3 ) peaks were as follows: δ2.3 (s OH) 3.57 (m -SCH 2 CH 2 S-) 6.60 (d H-2') 6.69 (d H-1') 7.19 (d H-7) 7.66 (m H-5 6) 8.03 (d H-4). Electrospray mass spectroscopy in negative ion mode indicated a monoisotopic mass for the –1 ion of 663.0 daltons (theoretical value 662.85). The extinction coefficient was 4.1 × 10 4 M –1 cm –1 at 507.5 nm at pH 7. The peptide Ac-Trp-Glu-Ala-Ala-Ala-Arg-Glu-Ala-Cys-Cys-Arg-Glu-Cys-Cys-Ala-Arg-Ala-amide was prepared by standard solid-phase methods and purified by n -butanol/water countercurrent chromatography.
ECFP ( 14 ) is Aequorea victoria GFP with mammalian codons and the following additional mutations: K26R F64L S65T Y66W N146I M153T V163A and N164H ( 8 ). A gene encoding a fusion of the peptide Ala-Glu-Ala-Ala-Ala-Arg-Glu-Ala-Cys-Cys-Arg-Glu-Cys-Cys-Ala-Arg-Ala to the COOH-terminus of ECFP was created with the following primer in a polymerase chain reaction (PCR): 5′-GCCGAATTCTTAGGCCCTGGCGCAGCACTCCCTGCAGCAGGCCTCCCTGGCGGCGGCCTCGGCCTTGTACAGCTCGTCCATGCCG-3′. The resulting gene was inserted into the pcDNA3 vector (Invitrogen) at Hind III and Eco RI restriction sites. After amplification in DH5 bacteria it was transfected into HeLa cells with Lipofectin (Gibco-BRL). The gene for Xenopus calmodulin was mutated to encode cysteines at residues 6 7 10 and 11 by PCR with the following primer: 5′-CGCGGATCCGCCACCATGCATGACCAACTGACATGCTGCCAGATTTGCTGCTTCAAAGAAGCCTTCTCATTATTC-3′ and inserted into pcDNA3 as described above.
Images of cells at room temperature were acquired with a cooled charge-coupled device camera (Photometrics Tucson AZ) controlled by Metafluor software (Universal Imaging West Chester PA).
Supported by NIH (grant NS27177 to R.Y.T. and a traineeship to B.A.G. from grant T32 CA09523) and the Howard Hughes Medical Institute. R.Y.T. is a consultant for Aurora Biosciences Corp. We thank L. A. Gross for mass spectra A. Miyawaki for help with molecular biology and T. Knapp for help with toxicity testing.

Dates

Type	When
Created	23 years, 1 month ago (July 27, 2002, 1:37 a.m.)
Deposited	1 year, 7 months ago (Jan. 12, 2024, 5:24 p.m.)
Indexed	2 weeks, 5 days ago (Aug. 7, 2025, 4:39 p.m.)
Issued	27 years, 1 month ago (July 10, 1998)
Published	27 years, 1 month ago (July 10, 1998)
Published Print	27 years, 1 month ago (July 10, 1998)

Funders 0

None

BibTeX

@article{Griffin_1998, title={Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells}, volume={281}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.281.5374.269}, DOI={10.1126/science.281.5374.269}, number={5374}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Griffin, B. Albert and Adams, Stephen R. and Tsien, Roger Y.}, year={1998}, month=jul, pages={269–272} }

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