The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection

Chapman, S., Faulkner, C., Kaiserli, E. , Garcia-Mata, C., Savenkov, E.I., Roberts, A.G., Oparka, K.J. and Christie, J.M. (2008) The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection. Proceedings of the National Academy of Sciences of the United States of America, 105(50), pp. 20038-20043. (doi:10.1073/pnas.0807551105)

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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 (≈10 kDa) flavin-based alternative to GFP (≈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 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

Item Type:Articles
Keywords:fluorescence imaging, molecular evolution, photoreceptor, LOV domain
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Kaiserli, Dr Eirini and Christie, Professor John
Authors: Chapman, S., Faulkner, C., Kaiserli, E., Garcia-Mata, C., Savenkov, E.I., Roberts, A.G., Oparka, K.J., and Christie, J.M.
Subjects:Q Science > QH Natural history > QH301 Biology
Q Science > QK Botany
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Proceedings of the National Academy of Sciences of the United States of America
ISSN:0027-8424
ISSN (Online):1091-6490
Published Online:05 December 2008

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