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. 1997 Nov;73(5):2782–2790. doi: 10.1016/S0006-3495(97)78307-3

Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy.

G H Patterson 1, S M Knobel 1, W D Sharif 1, S R Kain 1, D W Piston 1
PMCID: PMC1181180  PMID: 9370472

Abstract

We have investigated properties relevant to quantitative imaging in living cells of five green fluorescent protein (GFP) variants that have been used extensively or are potentially useful. We measured the extinction coefficients, quantum yields, pH effects, photobleaching effects, and temperature-dependent chromophore formation of wtGFP, alphaGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F). Absorbance and fluorescence spectroscopy showed little difference between the extinction coefficients and quantum yields of wtGFP and alphaGFP. In contrast, S65T and EGFP extinction coefficients made them both approximately 6-fold brighter than wtGFP when excited at 488 nm, and EBFP absorbed more strongly than the wtGFP when excited in the near-UV wavelength region, although it had a much lower quantum efficiency. When excited at 488 nm, the GFPs were all more resistant to photobleaching than fluorescein. However, the wtGFP and alphaGFP photobleaching patterns showed initial increases in fluorescence emission caused by photoconversion of the protein chromophore. The wtGFP fluorescence decreased more quickly when excited at 395 nm than 488 nm, but it was still more photostable than the EBFP when excited at this wavelength. The wtGFP and alphaGFP were quite stable over a broad pH range, but fluorescence of the other variants decreased rapidly below pH 7. When expressed in bacteria, chromophore formation in wtGFP and S65T was found to be less efficient at 37 degrees C than at 28 degrees C, but the other three variants showed little differences between 37 degrees C and 28 degrees C. In conclusion, no single GFP variant is ideal for every application, but each one offers advantages and disadvantages for quantitative imaging in living cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802–805. doi: 10.1126/science.8303295. [DOI] [PubMed] [Google Scholar]
  2. Chattoraj M., King B. A., Bublitz G. U., Boxer S. G. Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8362–8367. doi: 10.1073/pnas.93.16.8362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chiu W., Niwa Y., Zeng W., Hirano T., Kobayashi H., Sheen J. Engineered GFP as a vital reporter in plants. Curr Biol. 1996 Mar 1;6(3):325–330. doi: 10.1016/s0960-9822(02)00483-9. [DOI] [PubMed] [Google Scholar]
  4. Cormack B. P., Bertram G., Egerton M., Gow N. A., Falkow S., Brown A. J. Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans. Microbiology. 1997 Feb;143(Pt 2):303–311. doi: 10.1099/00221287-143-2-303. [DOI] [PubMed] [Google Scholar]
  5. Cormack B. P., Valdivia R. H., Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996;173(1 Spec No):33–38. doi: 10.1016/0378-1119(95)00685-0. [DOI] [PubMed] [Google Scholar]
  6. Crameri A., Whitehorn E. A., Tate E., Stemmer W. P. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol. 1996 Mar;14(3):315–319. doi: 10.1038/nbt0396-315. [DOI] [PubMed] [Google Scholar]
  7. Cubitt A. B., Heim R., Adams S. R., Boyd A. E., Gross L. A., Tsien R. Y. Understanding, improving and using green fluorescent proteins. Trends Biochem Sci. 1995 Nov;20(11):448–455. doi: 10.1016/s0968-0004(00)89099-4. [DOI] [PubMed] [Google Scholar]
  8. Gerdes H. H., Kaether C. Green fluorescent protein: applications in cell biology. FEBS Lett. 1996 Jun 24;389(1):44–47. doi: 10.1016/0014-5793(96)00586-8. [DOI] [PubMed] [Google Scholar]
  9. Haas J., Park E. C., Seed B. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr Biol. 1996 Mar 1;6(3):315–324. doi: 10.1016/s0960-9822(02)00482-7. [DOI] [PubMed] [Google Scholar]
  10. Heim R., Cubitt A. B., Tsien R. Y. Improved green fluorescence. Nature. 1995 Feb 23;373(6516):663–664. doi: 10.1038/373663b0. [DOI] [PubMed] [Google Scholar]
  11. Heim R., Prasher D. C., Tsien R. Y. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12501–12504. doi: 10.1073/pnas.91.26.12501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heim R., Tsien R. Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol. 1996 Feb 1;6(2):178–182. doi: 10.1016/s0960-9822(02)00450-5. [DOI] [PubMed] [Google Scholar]
  13. Inouye S., Tsuji F. I. Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein. FEBS Lett. 1994 Mar 21;341(2-3):277–280. doi: 10.1016/0014-5793(94)80472-9. [DOI] [PubMed] [Google Scholar]
  14. Kaether C., Gerdes H. H. Visualization of protein transport along the secretory pathway using green fluorescent protein. FEBS Lett. 1995 Aug 7;369(2-3):267–271. doi: 10.1016/0014-5793(95)00765-2. [DOI] [PubMed] [Google Scholar]
  15. Luby-Phelps K., Taylor D. L., Lanni F. Probing the structure of cytoplasm. J Cell Biol. 1986 Jun;102(6):2015–2022. doi: 10.1083/jcb.102.6.2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mellman I., Fuchs R., Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. doi: 10.1146/annurev.bi.55.070186.003311. [DOI] [PubMed] [Google Scholar]
  17. Mitra R. D., Silva C. M., Youvan D. C. Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein. Gene. 1996;173(1 Spec No):13–17. doi: 10.1016/0378-1119(95)00768-7. [DOI] [PubMed] [Google Scholar]
  18. Morise H., Shimomura O., Johnson F. H., Winant J. Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry. 1974 Jun 4;13(12):2656–2662. doi: 10.1021/bi00709a028. [DOI] [PubMed] [Google Scholar]
  19. Niswender K. D., Blackman S. M., Rohde L., Magnuson M. A., Piston D. W. Quantitative imaging of green fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits. J Microsc. 1995 Nov;180(Pt 2):109–116. doi: 10.1111/j.1365-2818.1995.tb03665.x. [DOI] [PubMed] [Google Scholar]
  20. Ogawa H., Inouye S., Tsuji F. I., Yasuda K., Umesono K. Localization, trafficking, and temperature-dependence of the Aequorea green fluorescent protein in cultured vertebrate cells. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11899–11903. doi: 10.1073/pnas.92.25.11899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ormö M., Cubitt A. B., Kallio K., Gross L. A., Tsien R. Y., Remington S. J. Crystal structure of the Aequorea victoria green fluorescent protein. Science. 1996 Sep 6;273(5280):1392–1395. doi: 10.1126/science.273.5280.1392. [DOI] [PubMed] [Google Scholar]
  22. Prasher D. C., Eckenrode V. K., Ward W. W., Prendergast F. G., Cormier M. J. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992 Feb 15;111(2):229–233. doi: 10.1016/0378-1119(92)90691-h. [DOI] [PubMed] [Google Scholar]
  23. Rizzuto R., Brini M., De Giorgi F., Rossi R., Heim R., Tsien R. Y., Pozzan T. Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo. Curr Biol. 1996 Feb 1;6(2):183–188. doi: 10.1016/s0960-9822(02)00451-7. [DOI] [PubMed] [Google Scholar]
  24. Siemering K. R., Golbik R., Sever R., Haseloff J. Mutations that suppress the thermosensitivity of green fluorescent protein. Curr Biol. 1996 Dec 1;6(12):1653–1663. doi: 10.1016/s0960-9822(02)70789-6. [DOI] [PubMed] [Google Scholar]
  25. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  26. Xu C., Zipfel W., Shear J. B., Williams R. M., Webb W. W. Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10763–10768. doi: 10.1073/pnas.93.20.10763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yang T. T., Cheng L., Kain S. R. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res. 1996 Nov 15;24(22):4592–4593. doi: 10.1093/nar/24.22.4592. [DOI] [PMC free article] [PubMed] [Google Scholar]

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