Specific covalent labeling of recombinant protein molecules inside live cells

Science

Volumn 281, Issue 5374, 1998, Pages 269-272

  Griffin, B Albert ^a,c   Adams, Stephen R ^a   Tsien, Roger Y ^b  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b HOWARD HUGHES MEDICAL INSTITUTE   (United States)

^c Aurora Biosciences Corporation   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

RECOMBINANT PROTEIN;

ARTICLE; CHEMICAL LABELING; FLUORESCENCE; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN STRUCTURE; STRUCTURE ANALYSIS;

AMINO ACID SEQUENCE; CALMODULIN; CELL MEMBRANE PERMEABILITY; CELL SURVIVAL; CYSTEINE; ENERGY TRANSFER; ETHYLENE GLYCOL; FLUORESCEINS; FLUORESCENCE; FLUORESCENT DYES; GREEN FLUORESCENT PROTEINS; HELA CELLS; HUMANS; JURKAT CELLS; LIGANDS; LUMINESCENT PROTEINS; MOLECULAR SEQUENCE DATA; ORGANOMETALLIC COMPOUNDS; PEPTIDES; PROTEIN STRUCTURE, SECONDARY; RECOMBINANT PROTEINS; SPECTROMETRY, FLUORESCENCE; TRANSFECTION;

EID: 0032503999     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.281.5374.269     Document Type: Article

References (23)

1. G. T. Hermanson, Bioconjugate Techniques (Academic Press, San Diego, CA, 1996).
2. R. Y. Tsien, Annu. Rev. Biochem. 67, 509 (1998).
3. M. Chalfie and S. R. Kain, Eds., GFP: Green Fluorescent Protein Strategies and Applications (Wiley, New York, 1998).
4. J. L. Webb, Enzyme and Metabolic Inhibitors (Academic Press, New York, 1966) vol. III, pp. 595-819.
5. E. Kalef and C. Gitler, Methods Enzymol. 233, 395 (1994).
6. V. P. Whittaker, Biochem. J. 41, 56 (1947).
7. E. C. Webb and R. Van Heyningen, ibid., p. 74.
8. 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.
9. G. Merutka, W. Shalongo, E. Stellwagen, Biochemistry 30, 4245 (1991).
10. B. A. Griffin, thesis, University of California, San Diego (1998).
11. W. G. Lowe and C. S. Hamilton, J. Am. Chem. Soc. 57, 1081 (1935).
12. C. Spang, F. T. Edelmann, M. Noltemeyer, H. W. Roesky, Chem. Ber. 122, 1247 (1989).
13. R. Heim and R. Y. Tsien, Curr. Biol. 6, 178 (1996).
14. A. Miyawaki, et al., Nature 388, 882 (1997).
15. M. Ormö et al., Science 273, 1392 (1996).
16. F. Denizot and R. Lang, J. Immunol. Methods 89, 271 (1986).
17. G. Zlokarnik et al., Science 279, 84 (1998).
18. C. F. Ford, I. Suominen, C. E. Glatz, Protein Expr. Purif. 2, 95 (1991).
19. G. R. Crabtree and S. L. Schreiber, Trends Biochem. Sci. 21, 418 (1996).
20. -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.
21. 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′- GCCGAATTCTTAGGCCCTGGCGCAGCACTCCCTGCAGCAGGCCTCCCTGGCGGCGGCCTCGGCCTTGTACAGCTCGTCCA TGCCG-3′. The resulting gene was inserted into the pcDNAB 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.
22. 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).
23. 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.