Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications


The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems1. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37°C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl)3. For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl are desired. Here we describe the development of an improved version of YFP named “Venus”. Venus contains a novel mutation, F46L, which at 37°C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Comparative characterization of the fluorescence properties of YFP variants.
Figure 2: Application of Venus for observation of vesicle secretion and Ca2+ imaging.


  1. 1

    Tsien, R.Y. The green fluorescent protein. Ann. Rev. Biochem. 67, 509–544 (1998).

    CAS  Article  Google Scholar 

  2. 2

    Crameri, A., Whitehorn, E.A., Tate, E. & Stemmer, W.P.C. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol. 14, 315–319 (1996).

    CAS  Article  Google Scholar 

  3. 3

    Jayaraman, S., Haggie, P., Wachter, R.M., Remington, S.J. & Verkman, A.S. Mechanism and cellular applications of a green fluorescent protein–based halide sensor. J. Biol. Chem. 275, 6047–6050 (2000).

    CAS  Article  Google Scholar 

  4. 4

    Nagai, T, Sawano, A., Park, E.S. & Miyawaki, A. Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc. Natl. Acad. Sci. USA 98, 3197–3202 (2001).

    CAS  Article  Google Scholar 

  5. 5

    Sawano, A. & Miyawaki, A. Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Res. 28, e78 (2000).

    CAS  Article  Google Scholar 

  6. 6

    Reid, B.G. & Flynn, G.C. Chromophore formation in green fluorescent protein. Biochemistry 36, 6786–6791 (1997).

    CAS  Article  Google Scholar 

  7. 7

    Miyawaki, A. & Tsien, R.Y. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 327, 472–500 (2000).

    CAS  Article  Google Scholar 

  8. 8

    Heikal, A.A., Hess, S.T., Baird, G.S., Tsien, R.Y. & Webb, W.W. Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). Proc. Natl. Acad. Sci. USA 97, 11996–12001 (2000).

    CAS  Article  Google Scholar 

  9. 9

    Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. & Tsien, R.Y. Reducing the environmental sensitivity of yellow fluorescent protein. J. Biol. Chem. 276, 29188–29194 (2001).

    CAS  Article  Google Scholar 

  10. 10

    Miyawaki, A., Griesbeck, O., Heim, R. & Tsien, R.Y. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc. Natl. Acad. Sci. USA 96, 2135–2140 (1999).

    CAS  Article  Google Scholar 

  11. 11

    Lang, T. et al. Ca2+-triggered peptide secretion in single cells imaged with green fluorescent protein and evanescent-wave microscopy. Neuron 18, 857–863 (1997).

    CAS  Article  Google Scholar 

  12. 12

    Tsuboi, T., Zhao, C., Terakawa, S. & Rutter, G.A. Simultaneous evanescent wave imaging of insulin vesicle membrane and cargo during a single exocytotic event. Curr. Biol. 10, 1307–1310 (2000).

    CAS  Article  Google Scholar 

  13. 13

    Pouli, A., Kennedy, H., George Schofield, J. & Rutter, G. Insulin targeting to the regulated secretory pathway after fusion with green fluorescent protein and firefly luciferase. Biochem. J. 331, 669–675 (1998).

    CAS  Article  Google Scholar 

  14. 14

    Angleson, J.K. & Betz, W.J. Monitoring secretion in real time: capacitance, amperometry and fluorescence compared. Trends Neurosci. 20, 281–187(1997).

    CAS  Article  Google Scholar 

  15. 15

    Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    CAS  Article  Google Scholar 

  16. 16

    Cadwell, R.C. & Joyce, G.F. Mutagenic PCR. PCR Methods Appl. 3, S136–S140 (1994).

    CAS  Article  Google Scholar 

Download references


The plasmid construct pEGFP-N1-NPY was provided by W. Almers. We are grateful to W. Almers and R.Y. Tsien for valuable comments, M. Yamamoto-Hino, Y. Kawano, K. Shimizu, T. Miyata, and K. Nakamura for technical advice, and H. Kuramochi for technical assistance. This work was partly supported by grants from CREST (the Japan Science and Technology Corporation) to A.M., the Japanese Ministry of Education, Science and Culture to A.M., Special Postdoctoral Researcher Program of RIKEN to T.N., and President's Special Research Grant of RIKEN to T.N.

Author information



Corresponding author

Correspondence to Atsushi Miyawaki.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Nagai, T., Ibata, K., Park, E. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20, 87–90 (2002).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing