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A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

Abstract

We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.

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Figure 1: Fluorescence imaging of mNeonGreen (mNG) fusion vectors.

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NCBI Reference Sequence

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NCBI Reference Sequence

Protein Data Bank

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Acknowledgements

We thank S.H. Haddock for the use of his lab's Shimadzu ultra-high-performance liquid chromatography instrument, W.R. Francis for help in compiling data, and S.G. Gilbert, C.K. Malik and R.A. Evans for their helpful contributions.

Author information

Authors and Affiliations

Authors

Contributions

M.I. cloned the original gene encoding LanYFP from B. lanceolatum, performed initial characterization of the protein, and identified the substitutions I118K (dimerizing) and N174T (folding enhancement). G.G.L. performed the majority of library construction, E. coli expression experiments, and protein purification. A.C., J.W. and Y.N. performed initial cloning and library construction for screening dimeric variants. R.N.D. performed FLIM-FRET experiments. J.R.A. performed single-molecule superresolution imaging experiments. M.W.D., P.J.C., M.A.B. and B.R.S. constructed mammalian expression and fusion plasmids, performed fixed- and live-cell imaging and FRET experiments, and helped write the manuscript. J.W. contributed to writing and editing the manuscript and supported the project. N.C.S. designed and planned the project, performed library design and screening, optical characterization and size-exclusion experiments, and wrote the manuscript.

Corresponding author

Correspondence to Nathan C Shaner.

Ethics declarations

Competing interests

M.I. is affiliated with Innoventus (Uppsala, Sweden), which administers patent licensing for the original LanYFP clone. N.C.S., A.C., Y.N. and J.W. are affiliated with Allele Biotechnology and Pharmaceuticals, Inc., which holds an exclusive license to LanYFP from Innoventus. Allele has filed for patent protection of mNeonGreen. The Scintillon Institute is a nonprofit research institute funded in part by Allele.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–16, Supplementary Tables 1–3, Supplementary Discussion (PDF 43094 kb)

Supplementary Data 1

PDB structure file of I-TASSER and RosettaDock generated model of LanYFP A/B dimer. (TXT 619 kb)

Supplementary Data 2

PDB structure file of I-TASSER and RosettaDock generated model of LanYFP A/C dimer. (TXT 618 kb)

Supplementary Table 4

Primers used in this study. (XLS 19 kb)

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Shaner, N., Lambert, G., Chammas, A. et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 10, 407–409 (2013). https://doi.org/10.1038/nmeth.2413

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  • DOI: https://doi.org/10.1038/nmeth.2413

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