A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein

Abstract

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because less light is scattered, absorbed, or reemitted by endogenous biomolecules. A new class of FP was developed from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, named small Ultra-Red FP (smURFP), covalently attaches biliverdin (BV) 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 comparable photostability to eGFP. SmURFP has significantly increased BV incorporation rate and protein stability compared to the bacteriophytochrome (BPH) FPs. BV supply is limited by membrane permeability, so expression of heme oxygenase-1 with heme precursors increases fluorescence of BPH/APCα FPs. SmURFP (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence in situ comparable to FPs from jellyfish/coral. A far-red/near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.

Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling

“…3D). Meanwhile, it is reported that supplementation of Heme precursor, 5-aminolevulinic acid (ALA), and iron(ii) sulfate increases Biliverdin biosynthesis up to threefold (34). Contrary to our expectation, these treatments did not significantly increase PCB synthesis in either control HeLa or BVRA KO HeLa cells that expressed PHFF (Fig.…”

New imaging probes to track cell fate: reporter genes in stem cell research

“…Like BLI, light absorbance from hemoglobin is a problem, decreasing signal to noise ratio [2,68,85], hence fluorescent protein variants have been developed that emit at longer wavelength, with peak emission wavelength of 592nm, 635nm, 646nm and 675nm [86,–91]. Rodriguez et al developed a novel RFP from an allophycocyanin α-subunit (APCα), small ultra-red FP (smURFP), which covalently attaches a biliverdin (BV) chromophore without help from a lyase, and has a 670-nm excitation–emission peak [51]. Shu et al engineered IFP1.1, a bacterial phytochrome protein from Deinococcus radiodurans that incorporates biliverdin as the chromophore and fluoresces with peak emission at 708 nm that is well expressed in mammalian cells and in mice for whole-body imaging.…”

“…For example, the DMT1 reporter gene discussed in the MRI reporter gene section has been shown to be useful for tracking neural stem cells in a rat brain using a dual-modality PET and MRI manganese based imaging approach [55]. Another reporter gene that has been used in multimodal imaging is a triple fusion reporter consisting of firefly luciferase, a red or green fluorescent protein and a herpes simplex virus type 1 thymidine kinase (HSV1 TK), which allows one to track cell fate using bioluminescent, fluorescent, and PET imaging [51,56–58]. Another previously mentioned reporter gene, hNIS, can be used for multimodal imaging using PET and Cerenkov Luminescence Imaging.…”

New dimensions in intravital multi-photon imaging of immune reactions

“…Almost all of the genetically encoded fluorophores presently in use are derived from jellyfish (Aequorea victoria) or various corals (Discoma sp, Entacmacea quadricolor) 34,35 . These proteins native to aquatic invertebrates did not evolve to maximize their molecular brightness or photostability upon laser illumination in mammalian tissue, and thus all of the useful genetically encoded fluorophores have required much in vitro selection and optimization to make them more useful.…”