Research abstract
Technical Report abstract
Nature Biotechnology 20, 87 - 90 (2002)
doi:10.1038/nbt0102-87
A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications
Takeharu Nagai1, Keiji Ibata2, Eun Sun Park1, Mie Kubota3, Katsuhiko Mikoshiba2 & Atsushi Miyawaki1
Abstract
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.
- Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan.
- Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan.
- Laboratory for Molecular Dynamics of Mental Disorder, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan.
Correspondence to: Atsushi Miyawaki1 e-mail: matsushi@brain.riken.go.jp
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