RRC ID 4033
著者 Chapman S, Faulkner C, Kaiserli E, Garcia-Mata C, Savenkov EI, Roberts AG, Oparka KJ, Christie JM.
タイトル The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection.
ジャーナル Proc Natl Acad Sci U S A
Abstract Fluorescent proteins (FPs) based on green fluorescent protein (GFP) are widely used throughout cell biology to study protein dynamics, and have extensive use as reporters of virus infection and spread. However, FP-tagging of viruses is limited by the constraints of viral genome size resulting in FP loss through recombination events. To overcome this, we have engineered a smaller ( approximately 10 kDa) flavin-based alternative to GFP ( approximately 25 kDa) derived from the light, oxygen or voltage-sensing (LOV) domain of the plant blue light receptor, phototropin. Molecular evolution and Tobacco mosaic virus (TMV)-based expression screening produced LOV variants with improved fluorescence and photostability in planta. One variant in particular, designated iLOV, possessed photophysical properties that made it ideally suited as a reporter of subcellular protein localization in both plant and mammalian cells. Moreover, iLOV fluorescence was found to recover spontaneously after photobleaching and displayed an intrinsic photochemistry conferring advantages over GFP-based FPs. When expressed either as a cytosolic protein or as a viral protein fusion, iLOV functioned as a superior reporter to GFP for monitoring local and systemic infections of plant RNA viruses. iLOV, therefore, offers greater utility in FP-tagging of viral gene products and represents a viable alternative where functional protein expression is limited by steric constraints or genome size.
巻・号 105(50)
ページ 20038-43
公開日 2008-12-16
DOI 10.1073/pnas.0807551105
PII 0807551105
PMID 19060199
PMC PMC2604982
MeSH Animals Cryptochromes Directed Molecular Evolution Flavins / chemistry Flavoproteins / analysis* Flavoproteins / genetics Flavoproteins / metabolism Flavoproteins / radiation effects Fluorescence Genes, Reporter Genetic Engineering Green Fluorescent Proteins / analysis Green Fluorescent Proteins / genetics Green Fluorescent Proteins / metabolism Green Fluorescent Proteins / radiation effects Luminescent Proteins / analysis* Luminescent Proteins / genetics Luminescent Proteins / metabolism Luminescent Proteins / radiation effects Microscopy, Confocal Microscopy, Fluorescence Oxygen / metabolism Photobleaching Plant Viruses / genetics Plant Viruses / metabolism Plant Viruses / physiology* Plants / virology* Recombinant Fusion Proteins Tobacco Mosaic Virus / genetics Tobacco Mosaic Virus / physiology Viral Proteins / analysis* Viral Proteins / genetics Viral Proteins / metabolism Viral Proteins / radiation effects
IF 9.412
引用数 155
WOS 分野 BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
シロイヌナズナ / 植物培養細胞・遺伝子 pda18798