FusionRed

Red fluorescent protein FusionRed

- Superior performance in fusions
- Low cytotoxicity
- Fast maturation, high pH-stability and photostability
- Proven suitability to generate stably transfected cell lines
- Recommended for protein labeling and long-term experiments

FusionRed is a red fluorescent protein characterized by improved performance in fusions and low toxicity [Shemiakina et al., 2012]. FusionRed lacks the residual tendency of other monomeric RFPs to dimerize at high concentration and behaves as a pure monomer at concentrations up to 10 mg/ml in HPLC analysis. Such "supermonomeric" properties ensure superior efficiency of FusionRed in protein labeling applications, especially in the cells with high expression level.

Similarly to parental mKate2, FusionRed demonstrates fast maturation rate, high pH-stability and photostability, and significantly lower cytotoxicity than widely used mCherry [Shaner et al., 2004] and mRuby [Kredel et al., 2009].

FusionRed is mainly intended for protein labeling and long-term experiments including generation of transgenic animals.

Main properties

FusionRed spectra

FusionRed normalized excitation (thin line) and emission (thick line) spectra.

Spectra viewer tool
Download FusionRed spectra (xls)

CHARACTERISTIC
* Brightness is a product of extinction coefficient and quantum yield, divided by 1000.
** Purified recombinant protein behaves as a pure monomer at concentrations of 10 mg/ml as verified by high-performance liquid chromatography (HPLC)
*** Time to bleach 50% of fluorescent signal brightness.
Molecular weight, kDa26
Polypeptide length, aa232
Fluorescence colorred
Excitation maximum, nm580
Emission maximum, nm608
Quantum yield0.19
Extinction coefficient, M-1cm-194 500
Brightness*18.0
Brightness, % of EGFP53
pKa4.6
Structuresupermonomer**
Aggregationno
Maturation half-time, min130
Photostability, widefield***150
Photostability, confocal***176
Cell toxicitynot observed
Possible limitationssuperior performance in fusions, low cytotoxicity

HPLC analysis of FusionRed in comparison with selected fluorescent proteins loaded in a high concentration.

FusionRed – orange line, mKate2 – black line, mNeptune – gray line. Proteins were loaded at a concentration of 10 mg/ml. While HPLC demonstrates reversible dimerization of mKate2 and reveals a dimeric character for the mKate derivative, far-red fluorescent protein mNeptune [Lin et al., 2009], FusionRed behaves as a pure monomer. Data from Shemiakina et al., 2012

Recommended filter sets and antibodies

FusionRed can be recognized using Anti-tRFP antibody (Cat.# AB233-AB234) available from Evrogen.

FusionRed can be detected using TRITC filter set or similar. Recommended Omega Optical filter sets are QMAX-Red and XF102-2.

Performance and use

FusionRed can be easily expressed and detected in a wide range of organisms. Mammalian cells transiently transfected with FusionRed expression vectors produce bright fluorescence in 10-12 hours after transfection. FusionRed performance in fusions has been demonstrated in a number of models.

FusionRed use for protein labeling in mammalian cells.

Scale bars represent 10 μm. Data from Shemiakina et al., 2012

Labeling efficiency testing

The performance of FusionRed and mCherry was directly compared in HeLa CCL2 cells for the following targets: connexin-43, endosomes, vinculin, and the Golgi complex. In this experiment, the cells were transfected with each of the four target fusions, fixed, and then compared for localization efficiency, which was determined by calculating the percentage of properly expressing cells versus the total number of transfected cells. FusionRed demonstrated clear advantage in all four fusions. Data from Shemiakina et al., 2012

Cytotoxicity testing in HeLa cells

The cytotoxicity of FusionRed relative to selected red fluorescent proteins and EGFP was evaluated in the following experiment: HeLa cells were transfected with appropriate vectors encoding EGFP or one of the following red fluorescent proteins: mRuby, FusionRed, mKate2 or mCherry. Next, the EGFP-expressing cells were mixed with those expressing one of the RFPs, resulting in 4 separate cell mixtures: EGFP and mRuby, EGFP and FusionRed, EGFP and mKate2, and EGFP and mCherry. 48 hours after transfection, the green-to-red cell ratios were calculated utilizing flow cytometry and each of the cell mixtures were then plated into 3 plates. After additional 92 hour incubation, the green-to-red cell ratios were recalculated. Because only living cells were counted for this experiment, the difference between the ratios before and after the incubation can be assumed to accurately reflect RFP toxicity versus EGFP. mRuby exhibited a more than 10-fold higher cytotoxicity level compared to EGFP, while the remaining RFPs were almost as cytotoxic as EGFP in this experiment. Data from Shemiakina et al., 2012

Cytotoxicity testing in Xenopus laevis tadpoles
The eye formation is a complex multistage process that depends on many mechanisms, any disturbance of which may result in an abnormal phenotype. Here eye development was used as a sensitive readout of possible abnormal course of embryogenesis caused by expression of EGFP, FusionRed, and mCherry fusions. In these experiments, actin and vimentin fusions of EGFP and FusionRed were only slightly more toxic than the uninjected controls, while expression of mCherry fusions led to notable decrease of the average eye size. Moreover, in many cases (80% of tadpoles with the reduced eye size), the reduced eye phenotype was accompanied by varying degree of an unclosed optic fissure, indicating an abnormal eye development.

Stage 41 Xenopus laevis tadpoles expressing fluorescent protein fusions. Top panels: white light; bottom panels: fluorescence. Data from Shemiakina et al., 2012

Left graph: Mean values of eye size of Xenopus laevis embryos injected with plasmids expressing different fluorescent protein fusions.

Right graph: Percentage of reduced eyes with unclosed optic fissure.

Data from Shemiakina et al., 2012

Long-term expression
The excellent suitability of FusionRed for long-term experiments was proved both in experiments with stably transfected cell lines and in transgenic animals. In addition to its low cytotoxicity, FusionRed does not show abnormal lysosomal localization typical for many fluorescent proteins in long-term expression.

Being expressed in HeLa cells, both mKate2 and FusionRed remain evenly localized in cytoplasm after 96 hours of cultivation, while mCherry and mRuby show abnormal lysosomal localization. Data from Shemiakina et al., 2012


Available variants and fusions
VariantDescriptionRelated vectorCat.#Click for image
Humanized FusionRed FusionRed codon usage is optimized for high expression in mammalian cells [Haas et al., 1996], but it can be successfully expressed in many other heterological systems. pFusionRed-C FP411
pFusionRed-N FP412
pFusionRed-B FP438
FusionRed-actin fusion Human β-actin is fused to the FusionRed C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of β-actin in living cells. pFusionRed-actin FP413
FusionRed-annexin fusion Human annexin is fused to the FusionRed C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of annexin in living cells. pFusionRed-annexin FP414
FusionRed-CD151 fusion Human CD151 is fused to the FusionRed C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of CD151 in living cells. pFusionRed-CD151 FP415
FusionRed-Cx26 fusion Rat connexin 26 is fused to the FusionRed N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of connexin 26 in living cells. pFusionRed-Cx26 FP416
FusionRed-Cx43 fusion Rat connexin 43 is fused to the FusionRed N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of connexin 43 in living cells. pFusionRed-Cx43 FP417
FusionRed-f-mem fusion 20 amino acid farnesylation signal from c-Ha-Ras is fused to the FusionRed C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of plasma membrane. pFusionRed-f-mem FP418
FusionRed-Golgi fusion Golgi targeting sequence (fragment of human β-1,4-galactosyltransferase) is fused to the FusionRed N-terminus. When expressed in mammalian cells, this variant provides red fluorescent labeling of Golgi apparatus. pFusionRed-Golgi FP419
FusionRed-ER fusion Signal sequence of calreticulin is fused to the FusionRed N-terminus and the endoplasmic reticulum retention sequence KDEL is fused to the FusionRed C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of the lumen of the endoplasmic reticulum. pFusionRed-ER FP420
FusionRed-H2B fusion Human histone H2B is fused to the FusionRed N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of histone H2B in living cells. pFusionRed-H2B FP421
FusionRed-laminB1 fusion Human lamin B1 is fused to the FusionRed C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of lamin B1 in living cells. pFusionRed-laminB1 FP422
FusionRed-vimentin fusion Human vimentin is fused to the FusionRed N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of vimentin in living cells. pFusionRed-vimentin FP423
FusionRed-vinculin fusion Human vinculin is fused to the FusionRed N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of vinculin in living cells. pFusionRed-vinculin FP424
FusionRed-zyxin fusion Human zyxin is fused to the FusionRed N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of zyxin in living cells. pFusionRed-zyxin FP425
FusionRed-actinin fusion Human α-actinin is fused to the FusionRed N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of α-actinin in living cells. pFusionRed-actinin FP426
FusionRed-endo fusion Human RhoB GTPase is fused to the FusionRed C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of vesicles of the endocytic pathway. pFusionRed-endo FP427
FusionRed-MAP4 fusion Microtubule binding domain of mouse microtubule-associated protein 4 (MAP4) is fused to the FusionRed C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of microtubules in living cells. pFusionRed-MAP4 FP428
FusionRed-paxillin fusion Chicken paxillin is fused to the FusionRed N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of paxillin in living cells. pFusionRed-paxillin FP429
FusionRed-PDHA1 fusion Human pyruvate dehydrogenase alpha 1 (PDHA1) is fused to the FusionRed N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of PDHA1 in living cells. pFusionRed-PDHA1 FP430
FusionRed-Rab5a fusion Human Ras-related protein Rab-5A is fused to the FusionRed C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of Rab-5A in living cells. pFusionRed-Rab5a FP431
FusionRed-talin fusion Mouse talin-1 protein is fused to the FusionRed N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of talin in living cells. pFusionRed-talin FP432
FusionRed-tubulin fusion Human α-tubulin is fused to the FusionRed C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of α-tubulin in living cells. pFusionRed-tubulin FP433
FusionRed-VE-cadherin fusion Human VE-cadherin is fused to the FusionRed N-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of VE-cadherin in living cells. pFusionRed-cadherin FP434
FusionRed-CDC42 fusion CDC42 is fused to the FusionRed C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of CDC42 in living cells. pFusionRed-CDC42 FP435
FusionRed-myosin fusion Myosin regulatory light polypeptide 9 is fused to the FusionRed C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of myosin in living cells. pFusionRed-myosin FP436
FusionRed-PCNA fusion Proliferating cell nuclear antigen (PCNA) is fused to the FusionRed C-terminus. When expressed in mammalian cells, this fusion provides red fluorescent labeling of proliferating cell nuclear antigen (PCNA) in living cells. pFusionRed-PCNA FP437


References:

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