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A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein

Nature Methods volume 13, pages 763–769 (2016)Cite this article

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A Corrigendum to this article was published on 29 September 2016

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Abstract

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation–emission peaks, a large extinction coefficient (180,000 M−1cm−1) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.

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Figure 1: Allophycocyanin, chromophore structures, and smURFP mutations.
Figure 2: smURFP + BV-purified protein, spectra, and comparison of APCα and BPH FPs expressed in E. coli and smURFP + BV expressed in vivo.
Figure 3: Increasing chromophore concentration within cells increases fluorescence.
Figure 4: smURFP expressed in vivo and smURFP fusions in mammalian cells.
Figure 5: Time-lapse microscopy of FR and NIR FUCCI expressed in HEK293A cells.

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  • 16 September 2016

    In the version of this article initially published, Roger Y. Tsien was listed as the corresponding author. On account of his sad demise shortly after publication, Erik A. Rodriguez has been added as corresponding author. This has been updated in the HTML and PDF versions of the article.

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Acknowledgements

National Institute of General Medical Sciences Postdoctoral Fellowship F32GM089114 supported the project (E.A.R.). US National Institutes of Health grants GM086197 (R.Y.T.), NS027177 (R.Y.T.), NS090590 (J.Y.L.), and the Howard Hughes Medical Institute (R.Y.T.) supported the project. We thank P. Steinbach for measuring photostability, Q. Xiong for FACS, S. Adams for advice and experimental expertise, and P. Arcaira for help with mouse experiments. We thank M. Lin (Departments of Pediatrics and Bioengineering, Stanford, Stanford, CA) for iRFP713 vectors, M. Davidson (Addgene) for fusion vectors, and A. Miyawaki (Laboratory for Cell Function Dynamics, RIKEN Brain Science Institute, Wako, Japan) for FUCCI vectors. This paper is dedicated to the memory of Roger Tsien, who passed away shortly after it was published.

Author information

Author notes

  1. Erik A Rodriguez and Geraldine N Tran: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology, University of California, San Diego, La Jolla, California, USA

    Erik A Rodriguez, Jessica L Crisp & Roger Y Tsien

  2. School of Medicine, University of California, San Francisco, San Francisco, California, USA

    Geraldine N Tran

  3. Howard Hughes Medical Institute, La Jolla, California, USA

    Larry A Gross & Roger Y Tsien

  4. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA

    Xiaokun Shu

  5. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California, USA

    Xiaokun Shu

  6. School of Medicine, University of Tasmania, Hobart, Tasmania, Australia

    John Y Lin

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Contributions

E.A.R. and G.N.T. created bacterial expression plasmids, evolved and developed smURFP and APCαFPs in E. coli, and characterized properties. E.A.R. prepared mammalian plasmids, made the smURFP homology model, created TDsmURFP, performed BV incorporation rates, performed mammalian cell experiments (HO-1, different chromophores, protein stability, and FP photobleaching), created FR and NIR FUCCI, and performed fluorescence imaging in vitro and in vivo. J.Y.L. transduced neurons. E.A.R. and J.Y.L. created virus and stable HT1080 cells for mouse models. J.Y.L. and J.L.C. injected animals with cancer cells and chromophores. J.L.C. prepared plasma. E.A.R., G.N.T., and J.Y.L. purified PCB and analyzed data. L.A.G. performed MS. X.S. chose the Trichodesmium APCα gene and oversaw the first three rounds of evolution of APCα + PCB. R.Y.T. and E.A.R. oversaw the design and analysis of the experiments. All authors contributed to writing and discussion.

Corresponding authors

Correspondence to Erik A Rodriguez or Roger Y Tsien.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–20 and Supplementary Tables 1–4 (PDF 28027 kb)

Time-lapse microscopy of mAG-hGem(1/110) and smURFP-hCdtI(30/120) FUCCI expressed in HEK293A cells.

Video is full field, 40X objective view of Supplementary Fig. 20 and total time is 70 h. mAG-hGem(1/110) and smURFP-hCdtI(30/120) fluorescence are shown in green and red, respectively. Green is EX / EM = 495(10) / 535(25) nm and red is EX / EX = 628(40) / 680(30) nm. EX is excitation and EM is emission. (MOV 7537 kb)

Time-lapse microscopy of FR and NIR FUCCI expressed in HEK293A cells.

Video is full field, 40X objective view of Fig. 5 and total time is 49 h. IFP2.0-hGem(1/110) and smURFP-hCdtI(30/120) fluorescence are shown in green and red, respectively. Green is EX / EM = 665(45) / 725(50) nm and red is EX / EX = 628(40) / 680(30) nm. EX is excitation and EM is emission. (MOV 5254 kb)

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Rodriguez, E., Tran, G., Gross, L. et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat Methods 13, 763–769 (2016). https://doi.org/10.1038/nmeth.3935

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