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

T. Nagai, K. Ibata, E. S. Park, M. Kubota, K. Mikoshiba, A. Miyawaki

Research output: Contribution to journalArticle

1946 Citations (Scopus)

Abstract

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems. 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 (CI-)3. For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and CI- 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 CI-. 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.

Original languageEnglish
Pages (from-to)87-90
Number of pages4
JournalNature Biotechnology
Volume20
Issue number1
DOIs
Publication statusPublished - 2002
Externally publishedYes

Fingerprint

Venus
Green Fluorescent Proteins
Proteins
Chlorides
Ions
Mutation
Mutagenesis
Fluorescence Resonance Energy Transfer
Neuropeptide Y
PC12 Cells
Biological systems
Chromophores
Acidosis
Organelles
Labeling
Brain
Fusion reactions
Visualization
Fluorescence
Oxidation

ASJC Scopus subject areas

  • Microbiology

Cite this

A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. / Nagai, T.; Ibata, K.; Park, E. S.; Kubota, M.; Mikoshiba, K.; Miyawaki, A.

In: Nature Biotechnology, Vol. 20, No. 1, 2002, p. 87-90.

Research output: Contribution to journalArticle

Nagai, T. ; Ibata, K. ; Park, E. S. ; Kubota, M. ; Mikoshiba, K. ; Miyawaki, A. / A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. In: Nature Biotechnology. 2002 ; Vol. 20, No. 1. pp. 87-90.
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