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Expression and Detection of Green Fluorescent Protein (GFP)

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Book cover Recombinant Protein Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 63))

Abstract

Light is produced from several bioluminescent species through the action of green fluorescent proteins (GFPs) (reviewed in ref. 1). One such GFP from the jellyfish Aequorea victoria fluoresces following the transfer of energy from the Ca2+-activated photoprotein aequorin(2,3). This energy transfer mechanism is radiationless, and proceeds via the interaction of these two proteins in photogenic cells located at the base of the jellyfish. Expression of GFP in vivo does not alter the spectral properties of the protein (4). Full-length GFP appears to be required for fluorescence, however the chromophore responsible for light absorption is located within a hexapeptide at positions 64–69 (5). This region of GFP contains a Ser65-dehydro Tyr66-Gly67 cyclic tripeptide which functions as the minimal chromophore.

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References

  1. Prasher, D. C. (1995) Using GFP to see the light. Trends Genet. 11, 320–323.

    Article  PubMed  CAS  Google Scholar 

  2. Inouye, S. and Tsuji, F. I. (1994) Aequorea green fluorescent protein expression of the gene and fluorescent characteristics of the recombinant protein. FEBS Lett. 341, 277–280.

    Article  PubMed  CAS  Google Scholar 

  3. Ward, W. W. (1979) Energy transfer processes in bioluminescence, in Photochemical and Photobiological Reviews (Smith, K. C., ed.), Plenum, New York, pp. 1–57.

    Google Scholar 

  4. Chalfie, M. Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.

    Article  PubMed  CAS  Google Scholar 

  5. Cody, C. W., Prasher, D. C., Westler, W. M., Prendergast, F. G., and Ward, W. W. (1993) Chemical structure of the hexapeptide chromophore of Aequorea green-fluorescent protein. Biochemistry 32, 1212–1218.

    Article  PubMed  CAS  Google Scholar 

  6. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., and Cormier, M. J. (1992) Primary structure of the Aequorea victoria green fluorescent protein. Gene 111, 229–233.

    Article  PubMed  CAS  Google Scholar 

  7. Coxon, A. and Bestor, T. H. (1995) Proteins that glow in green and blue. Chem. Biol. 2, 119–121.

    Article  PubMed  CAS  Google Scholar 

  8. Cubitt, A. B., Heim, R., Adams, S. R., Boyd, A. E., Gross, L. A., and Tsien, R. Y. (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem 20, 448–455.

    Article  CAS  Google Scholar 

  9. Stearns, T. (1995) The green revolution. Current Biol. 5, 262–264.

    Article  CAS  Google Scholar 

  10. Kaether, C. and Gerdes, H.-H. (1995) Visualization of protein transport along the secretory pathway using green fluorescent protein. FEBS Lett. 369, 267–271.

    Article  PubMed  CAS  Google Scholar 

  11. Pines, J. (1995) GFP in mammalian cells. Trends Genet. 11, 326,327.

    Article  PubMed  CAS  Google Scholar 

  12. Flach, J., Bossie, M., Vogel, J., Corbett, A., Jinks, T., Willins, D. A., and Silver, P. A. (1994) A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm. Mol. Cell. Biol. 14, 8399–8407.

    PubMed  CAS  Google Scholar 

  13. Baulcombe, D. C., Chapman, S., and Santa Cruz, S. (1995) Jellyfish green fluorescent protein as a reporter for virus infections. Plant J. 7, 1045–1053.

    Article  PubMed  CAS  Google Scholar 

  14. Kain, S. R., Adams, M., Kondepudi, A., Yang, T. T., Ward, W. W., and Kitts, P. (1995) The green fluorescent protein as a reporter of gene expression and protein localization. BioTechniques 19, 650–655.

    PubMed  CAS  Google Scholar 

  15. Amsterdam, A. and Hopkins, N. (1995) Transient and transgenic expression of green fluorescent protein (GFP) in living zebrafish embryos CLONTECHniques. X(3), 30.

    Google Scholar 

  16. Ikawa, M., Kominami, K., Yoshimura, Y., Tanaka, K., Nishimune, Y., and Okabe, M. (1995) Green fluorescent protein as a marker in transgenic mice. Devel. Growth Differ. 37, 455–459.

    Article  CAS  Google Scholar 

  17. Yeh, E., Gustafson, K., and Bouhanne, G. L. (1995) Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila Proc. Natl. Acad. Sci. USA 92, 7036–7040.

    Article  CAS  Google Scholar 

  18. Davis, D. F., Ward, W. W., and Cutler, M. W. (1995) Post-translational chromophore formation in recombinant GFP from E. coli requires oxygen: Proceedings of the 8th International Symposium on Bioluminescence and Chemiluminescence. (in press).

    Google Scholar 

  19. Heim, R., Prasher, D. C., and Tsien, R. Y. (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91, 12,501–12,504.

    Article  PubMed  CAS  Google Scholar 

  20. Ward, W. W., Cody, C. W., Hart, R. C., and Cormier, M. J. (1980) Spectrophotometric identity of the energy transfer chromophores in Renilla and Aequorea green-fluorescent proteins. Photochem. Photobiol. 31, 611–615.

    Article  CAS  Google Scholar 

  21. Delagrave, S., Hawtin, R. E., Silva, C. M., Yang, M. M., and Youvan, D. C.(1995) Red-shifted excitation mutants of the green fluorescent protein. Bio/Technology 13, 151–154.

    Article  PubMed  CAS  Google Scholar 

  22. Yang, T. T., Kain, S. R., Kitts, P., Kondepudi, A., Yang, M. M., and Youvan, D. C. (1996) Dual color microscopic imagery of cells expressing the green fluorescent protein and a red-shifted variant. Gene (in press).

    Google Scholar 

  23. Heim, R., Cubitt, A. B., and Tsien, R. Y. (1995) Improved green fluorescence. Nature 373, 663,664.

    Article  PubMed  CAS  Google Scholar 

  24. Ehrig, T., O’Kane, D. J., and Prendergast, F. G. (1995) Green fluorescent protein mutants with altered excitation spectra. FEBS Lett. 367, 163–166.

    Article  PubMed  CAS  Google Scholar 

  25. Wang, S. and Hazelrigg, T. (1994) Implications for bed mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature 369, 400–403.

    Article  PubMed  CAS  Google Scholar 

  26. Inouye, S. and Tsuji, F. I. (1994) Evidence for redox forms of the Aequorea green fluorescent protein. FEBS Lett. 351, 211–214.

    Article  PubMed  CAS  Google Scholar 

  27. Bokman, S. H., and Ward, W. W. (1981) Renaturation of Aequorea green-fluorescent protein. Biochem. Biophys. Res. Comm. 101, 1372–1380.

    Article  PubMed  CAS  Google Scholar 

  28. Robart, F. D., and Ward, W. W. (1990) Solvent perturbations of Aequorea green fluorescent protein. Photochem. Photobiol. 51, 92s.

    Google Scholar 

  29. Roth, A. (1985) Purification and protease susceptibility of the green-fluorescent protein of Aequorea victoria with a note on Halistra ura. Ph.D. thesis, Rutgers University, New Brunswick, NJ.

    Google Scholar 

  30. Ward, W. W. (1981) Properties of the Coelenterate green-fluorescent proteins, in Bioluminescence and Chemiluminescence, Basic Chemistry and Analytical Applications (DeLuca, M., and McElroy, W. D., ed.), Academic, New York, pp. 235–242.

    Google Scholar 

  31. Ward, W. W. and Bokman, S. H. (1982) Reversible denaturation of Aequorea green-fluorescent protein, physical separation and characterization of the renatured protein. Biochemistry 21, 4535–4540.

    Article  PubMed  CAS  Google Scholar 

  32. Surpin, M. A. and Ward, W. W. (1989) Reversible denaturation of Aequorea green fluorescent protein—thiol requirement. Photochem. Photobiol. 49, WPM–B2.

    Google Scholar 

  33. Lim, C. R., Kimata, Y., Oka, M., Nomaguchi, K., and Kohno, K. (1995) Thermosensitivity of green fluorescent protein fluorescence utilized to reveal novel nuclear-like compartments in a mutant nucleoporin Nsp1. J. Biochem. 118, 13–17.

    PubMed  CAS  Google Scholar 

  34. Buikema, W. (1995) Oral presentation at Fluorescent Proteins and Applications Meeting, Palo Alto, CA, March 6–7.

    Google Scholar 

  35. Hodgkinson, S. (1995) GFP in Dictyostelium. Trends Genet. 11, 327,328.

    Article  PubMed  CAS  Google Scholar 

  36. Burlage, R., Yang, Z., and Mehlhorn, T. (1995) Fluorescent microorganisms are detected in bacterial transport experiments. Poster presentation at Fluorescent Proteins and Applications Meeting, Palo Alto, CA, March 6–7.

    Google Scholar 

  37. Kitts, P., Adams, M., Kondepudi, A., Gallagher, D., and Kain, S. (1995) Green fluorescent protein (GFP): a novel reporter for monitoring gene expression in living organisms. CLONTECHniques X(1), 1–3.

    Google Scholar 

  38. Yu, J. and van den Engh, G. (1995) Flow-sort and growth of single bacterial cells transformed with cosmid and plasmid vectors that include the gene for green-fluorescent protein as a visible marker. Abstracts of papers presented at the 1995 meeting on “Genome Mapping and Sequencing,” Cold Spring Harbor, NY, p. 293.

    Google Scholar 

  39. Jacobi, C. A., Roggenkamp, A., Rakin, A., and Heesemann, J. (1995) Yersinia enterocolitica Yop E-GFP hybrid protein expression in vitro and in vivo. Poster presentation at Fluorescent Proteins and Applications Meeting, Palo Alto, CA, March 6–7.

    Google Scholar 

  40. Riles, L., Waddle, J., and Johnston, M. (1995) GFP as a reporter of gene expression in yeast. Poster presentation at Fluorescent Proteins and Applications Meeting, Palo Alto, CA, March 6–7.

    Google Scholar 

  41. Schlenstedt, G., Saavedra, C., Loeb, J. D. J., Cole, C. N., and Silver, P. A. (1995) The GTP-bound form of the yeast Ran/TC4 homologue blocks nuclear protein import and appearance of poly(a)+ RNA in the cytoplasm. Proc. Natl. Acad. Sci. USA 92, 225–229.

    Article  PubMed  CAS  Google Scholar 

  42. Nabeshima, K., Kurooka, H., Takeuchi, M., Kinoshita, K., Nakaseko, Y., and Yanagida, M. (1995) p93dis, which is required for sister chromatid separation, is a novel microtubule and spindle pole body-associating protein phosphorylated at the Cdc2 target sites. Genes Devel. 9, 1572–1585.

    Article  PubMed  CAS  Google Scholar 

  43. Sengupta, P., Colbert, H. A., and Bargmann, C. I. (1994) The C elegans gene odr7 encodes an olfactory-specific member of the nuclear receptor superfamily. Cell 79, 971–980.

    Article  PubMed  CAS  Google Scholar 

  44. Treinin, M. and Chalfie, M. (1995) A mutated acetylcholine receptor subunit causes neuronal degeneration in C. elegans. Neuron. 14, 871–877.

    Article  CAS  Google Scholar 

  45. Barthmaier, P. and Fyrberg, E. (1995) Monitoring development and pathology of Drosophila indirect flight muscles using green fluorescent protein. Devel. Biol. 169, 770–774.

    Article  CAS  Google Scholar 

  46. Brand, A. (1995) GFP in Drosophila: Trends Genet. 11, 324,325.

    PubMed  CAS  Google Scholar 

  47. Marshall, J., Molloy, R., Moss, G. W. J., Howe, J. R., and Hughes, T. E. (1995) The jellyfish green fluorescent protein, a new tool for studying ion channel expression and function, Neuron 14, 211–215.

    Article  PubMed  CAS  Google Scholar 

  48. Olson, K. R., McIntosh, J. R., and Olmsted, J. B. (1995) Analysis of MAP4 function in living cells using green fluorescent protein (GFP) chimeras. J. Cell Biochem. 130, 639–650.

    CAS  Google Scholar 

  49. Lo, D. C., McAllister, A. K., and Katz, L. C. (1994) Neuronal transfection in brain slices using particle-mediated gene transfer. Neuron 13, 1263–1268.

    Article  PubMed  CAS  Google Scholar 

  50. Plautz, J. D., Day, R. N., Dailey, G. M., Welsh, S. B., Hall, J. C., Halpain, S., and Kay, S. A. (1996) Green fluorescent protein and its derivatives as a versatile marker for gene expression in living Drosophila melanogaster, plant and mammalian cells. Gene 173, 83–87.

    Article  PubMed  CAS  Google Scholar 

  51. Yu, K. L. and Dong, K. W. (1995) Application of luciferase and green fluorescent protein as reporter for analysis of human gonadotropin-releasing hormone gene promoters. Poster presentation at Fluorescent Proteins and Applications Meeting, Palo Alto, CA, March 6–7.

    Google Scholar 

  52. Tannahill, D., Bray, S., and Harris, W. A. (1995) A Drosophila E(sp1) gene is “neurogenic” in Xenopus: A green fluorescent protein study. Devel. Biol. 168, 694–697.

    Article  CAS  Google Scholar 

  53. Wu, G.-I., Zou, D.-J., Koothan, T., and Cline, H. T. (1995) Infection of frog neurons with vaccinia virus permits in vivo expression of foreign proteins. Neuron 14, 681–684.

    Article  PubMed  CAS  Google Scholar 

  54. Amsterdam, A., Lin, S., and Hopkins, N. (1995) The Aequorea victorla green fluorescent protein can be used as a reporter in live zebrafish embryos. Devel. Biol. 171, 123–129.

    Article  CAS  Google Scholar 

  55. Haseloff, J. and Amos, B. (1995) GFP in plants. Trends Genet 11, 328,329.

    Article  PubMed  CAS  Google Scholar 

  56. Niedz, R. P., Sussman, M. R., and Satterlee, J. S. (1995) Green fluorescent protein: an in vivo reporter of plant gene expression. Plant Cell Reports 14, 403–406.

    Article  CAS  Google Scholar 

  57. Galbraith, D. W., Sheen, J., Lambert, G. M., and Grebenok, R. J. (1995) Flow cytometric analysts of transgene expression in higher plants: green fluorescent protein. Methods Cell Biol. 50, 1–12.

    Google Scholar 

  58. Hu, W. and Chein, C.-L. (1995) Expression of Aequorea green fluorescent protein in plant cells. FEBS Lett. 369, 331.

    Article  PubMed  CAS  Google Scholar 

  59. Tormakangas, K., Ritala, A., and Teeri, T. H. (1995) Expression of GFP in plant cells. Poster presentation, at Fluorescent Proteins and Applications Meeting, Palo Alto, CA, March 6–7.

    Google Scholar 

  60. Beachy, R. (1995) Oral presentation at Fluorescent Proteins and Applications Meeting, Palo Alto, CA, March 6–7.

    Google Scholar 

  61. Chen, C. and Okayama, H. (1988) Calcium phosphate-mediated gene transfer, a highly efficient transfection system for stably transforming cells with plasmid DNA. BioTechniques 6, 632–638.

    PubMed  CAS  Google Scholar 

  62. Sambrook, J., Fritsch, E. F., and Mamatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  63. Cheng, L. and Kam, S. (1995) Analysis of GFP and RSGFP expression in mammalian cells by flow cytometry. CLONTECHniques X(4), 20.

    Google Scholar 

  64. Ropp, J. D., Donahue, C. J., Wolfgang-Kinball, D., Hooley, J. J., Chin, J. Y. W., Hoffman, R. A., Cuthbertson, R. A., and Bauer, K. D. (1995) Aequorea green fluorescent protein (GFP) analysis by flow cytometry. Cytometry 21, 309–317.

    Article  PubMed  CAS  Google Scholar 

  65. Harlow, E. and Lane, E. (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  66. Kain, S. R., Mai, K., and Sinai, P, (1994) Human multiple tissue western blots, a new immunological tool for the analysis of tissue-specific protein expression. BioTechniques 17, 982–987.

    PubMed  CAS  Google Scholar 

  67. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4._Nature 227, 680–685.

    Article  PubMed  CAS  Google Scholar 

  68. O’Farrell, P. H. (1970) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4111.

    Google Scholar 

  69. Scopes, R. K. (1987) Protein Purification, Principles and Practice, 2nd ed., Springer-Verlag, New York.

    Google Scholar 

  70. Bjerrum, O. J., and Heegaard, N. H. H., eds. (1988) CRC Handbook of Immunoblotting of Proteins CRC, Boca Raton, FL.

    Google Scholar 

  71. Towbin, H., Staehelm, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4356.

    Article  PubMed  CAS  Google Scholar 

  72. Kozak, M. (1987) An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids 15, 8125–8148.

    Article  CAS  Google Scholar 

  73. Silver, P. (1995) Oral presentation at Fluorescent Proteins and Applications Meeting, Palo Alto, CA, March 6–7.

    Google Scholar 

  74. Cox, J. (1995) Oral presentation at Fluorescent Proteins and Applications Meeting, Palo Alto, CA, March 6–7.

    Google Scholar 

  75. Rizzuto, R., Brini, M., Pizzo, P., Murgia, M., and Pozzan, T. (1995) Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Current Biol. 5, 635–642.

    Article  CAS  Google Scholar 

  76. Bian, J., Lin, X., and Tang, J. (1995) Nuclear localization of HIV-1 matrix protein P17, The use of A Victoria GFP in protein tagging and tracing. FASEB J. 9, A1279#132.

    Google Scholar 

  77. Sullivan, K. F., Hahn, K., and Shelby, R. D. (1995) Bioluminescent labeling of human centromere DNA sequences in vivo. Poster presentation at Fluorescent Proteins and Applications Meeting, Palo Alto, CA, March 6–7.

    Google Scholar 

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Kain, S.R., Kitts, P. (1997). Expression and Detection of Green Fluorescent Protein (GFP). In: Tuan, R.S. (eds) Recombinant Protein Protocols. Methods in Molecular Biology™, vol 63. Humana Press. https://doi.org/10.1385/0-89603-481-X:305

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  • DOI: https://doi.org/10.1385/0-89603-481-X:305

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