RRC ID 49219
Author Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A.
Title A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.
Journal Nat. Biotechnol.
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 degrees 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-). 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 degrees 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.
Volume 20(1)
Pages 87-90
Published 2002-1
DOI 10.1038/nbt0102-87
PII nbt0102-87
PMID 11753368
MeSH Animals Bacterial Proteins / genetics* Bacterial Proteins / metabolism* Cerebellum / metabolism Gene Transfer Techniques Genetic Techniques* Genetic Vectors Kinetics Luminescent Proteins / genetics* Luminescent Proteins / metabolism* Mice Microscopy, Confocal Mutagenesis Mutation PC12 Cells Rats Scyphozoa Time Factors Transfection
IF 35.724
Times Cited 1630
WOS Category BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Resource
DNA material Venus/pCS2 (RDB15116) pNPY-Venus-N1 (RDB15118) NPY-Venus/pEF-BOS (RDB15119) YC2.12/pCS2 (RDB15132) mVenus/pRSETB (RDB15117).