Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome

By X. Shu, A. Royant, Michael Z. Lin, T. Aguilera, V. Lev-Ram, Paul A. Steinbach, Roger Tsien. Science, 2009

674 citations recorded by the source corpus.

Infrared Vision Proteins from jellyfish and corals that fluoresce in the visible wavelength range have revolutionized optical imaging of cells. However, these wavelengths are absorbed by hemoglobin, water, and lipids and the proteins are thus not appropriate for deep-tissue imaging. Now Shu et al. (p. 804) have engineered a bacteriophytochrome from Deinococcus radiodurans that incorporates biliverdin as the chromophore, to fluoresce with excitation and emission spectra of 648 and 708 nanometers, respectively. These infrared fluorescent proteins are expressed well in mammalian cells and mice, and can be used for whole-body imaging. An engineered infrared fluorescent protein derived from an extremophile bacterium gives a strong signal in mammalian cells. A bacteriophytochrome incorporating biliverdin has been engineered to generate strong infrared fluorescence in mammalian cells and whole mice. Visibly fluorescent proteins (FPs) from jellyfish and corals have revolutionized many areas of molecular and cell biology, but the use of FPs in intact animals, such as mice, has been handicapped by poor penetration of excitation light. We now show that a bacteriophytochrome from Deinococcus radiodurans, incorporating biliverdin as the chromophore, can be engineered into monomeric, infrared-fluorescent proteins (IFPs), with excitation and emission maxima of 684 and 708 nm, respectively; extinction coefficient >90,000 M−1 cm−1; and quantum yield of 0.07. IFPs express well in mammalian cells and mice and spontaneously incorporate biliverdin, which is ubiquitous as the initial intermediate in heme catabolism but has negligible fluorescence by itself. Because their wavelengths penetrate tissue well, IFPs are suitable for whole-body imaging. The IFPs developed here provide a scaffold for further engineering.

Subfields: Cellular and Molecular Neuroscience, Molecular Biology, Plant Science

View Paper: https://www.semanticscholar.org/paper/e0ec4b0fa4b3798148849a743d9497e6f9a85286

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Citations in the corpus

A Guide to Fluorescent Protein FRET Pairs (2016, background): “4, from the Deinococcus radiodurans bacteriophytochrome, utilizes Biliverdin IX α (BV), which is ubiquitous in mammalian cells, as a chromophore to produce ﬂuorescence [49].”
Molecular and cellular approaches for diversifying and extending optogenetics. (2010, background): “…that penetrates much more deeply into scattering biological tissues (as with the far-red utility recently demonstrated for fluorescent proteins) (Shu et al., 2009), and therefore will allow recruitment of larger volumes (Aravanis et al., 2007; Adamantidis et al., 2007; Gradinaru et al., 2009).”
The growing and glowing toolbox of fluorescent and photoactive proteins (2016, background): “4, which was engineered from a truncated DrBphP from Deinococcus radiodurans [13].”
Preclinical Whole-body Fluorescence Imaging: Review of Instruments, Methods and Applications (2009, positive): “Significant work is also devoted to the development of novel fluorescent proteins with spectral features allowing them to be used for deep tissue imaging [102-105].”
Fluorescent proteins and their applications in imaging living cells and tissues. (2010, background): “An alternative solution for semigenetically encoded infrared labeling on the basis of bacterial phytochromes was recently developed (396).”
Going deeper than microscopy: the optical imaging frontier in biology (2010, methodology): “This has occurred via the development of imaging methods that accurately account for the diffusive nature of photons beyond the 10 TMFP range and because of the availability of improved optical reporter”
A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms (2011, methodology): “4, which is readily bleached by (1)O2 (Figure S2) [21].”