Red fluorescent protein TurboRFP

- Superbright red (orange) fluorescence
- Fast maturation
- Destabilized version is available
- Recommended for gene expression analysis, cell and organelle labeling

TurboRFP is a red fluorescent protein (excitation/emission maxima are 553 and 574 nm, respectively) derived from sea anemone Entacmaea quadricolor [Merzlyak et al., 2007]. Possessing high photostability and pH stability, TurboRFP is more than twice brighter than DsRed2. Fast TurboRFP maturation makes it clearly detectable in mammalian cells as early as within 8-10 hrs after transfection. TurboRFP is mainly intended for applications where fast appearance of bright fluorescence is crucial. It is specially recommended for cell and organelle labeling and tracking the promoter activity. Destabilized TurboRFP variant allows accurate analysis of rapid and/or transient events in gene regulation.

Main properties

TurboRFP normalized excitation (thin line) and emission (thick line) spectra. Download TurboRFP spectra (xls)

CHARACTERISTIC *Brightness is a product of extinction coefficient and quantum yield, divided by 1000. Molecular weight, kDa 26.1 Polypeptide length, aa 231 Fluorescence color red (orange) Excitation maximum, nm 553 Emission maximum, nm 574 Quantum yield 0.67 Extinction coefficient, M-1cm-1 92 000 Brightness* 61.6 Brightness, % of EGFP 187 pKa 4.4 Structure dimer Aggregation no Maturation rate at 37°C super fast Photostability high Cell toxicity not observed Main advantages superbright and fast-maturing red fluorescent protein Possible limitations dimer, limited applicability for fusions generation

Recommended filter sets and antibodies

TurboRFP can be recognized using Anti-tRFP antibody (Cat.# AB231-AB232) available from Evrogen.

Recommended Omega Optical filter sets are QMAX-Yellow, XF108-2, XF101-2, and XF111-2. TurboRFP can also be detected using TRITC filter set or similar.

Performance and use

TurboRFP can be expressed and detected in a wide range of organisms. Mammalian cells transiently transfected with TurboRFP expression vectors give bright fluorescent signals within 8-10 hrs after transfection. No cell toxic effects and visible protein aggregation are observed.

Despite its dimeric structure, TurboRFP demonstrates successful performance in fusions with subcellular localization signals and many cellular proteins. However, we recommend that you use specially optimized protein localization TagFPs to select a reporter for such purposes.

TurboRFP can be used in multicolor labeling applications with cyan, green, yellow, and far-red fluorescent dyes.

		TurboRFP use for cell and organelle labeling. Fluorescent microscopy of mammalian cells expressing cytoplasmic TurboRFP and TurboRFP fusion with mitochondrial targeting signal. Images made from HeLa cells 24 hrs after transfection.

TurboRFP maturation kinetics: TurboRFP matures noticeably faster than other red fluorescent proteins. To compare maturation of TurboRFP and DsRed-related proteins, HeLa cells were transiently transfected with mammalian expression vectors comprising TurboRFP, DsRed2, or DsRed-Express fluorescent proteins under the control of CMV promoter. The DNA concentrations were equalized before transfection. Cells were photographed using fluorescent microscope after different periods of cultivation. Faster appearance of bright fluorescence was detected in the case of TurboRFP. In addition, unlike DsRed-related proteins, no abnormal Golgi-like localization of TurboRFP was observed within 7 days after transfection.

Fluorescent microscopy of mammalian cells expressing TurboRFP, DsRed2, and DsRed-Express. TurboRFP gives the brightest signal 22 hrs after transfection; DsRed2 and DsRed-Express show abnormal Golgi-like localization 7 days after transfection, whereas TurboRFP localizes evenly in cytosol.

Available variants and fusions

TurboRFP 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.

TurboRFP-mito fusion: A mitochondrial targeting sequence (MTS) is linked to the TurboRFP N-terminus. MTS was derived from the subunit VIII of human cytochrome C oxidase [Rizzuto et al., 1989; Rizzuto et al., 1995]. When expressed in mammalian cells, this variant provides red fluorescent labeling of mitochondria.

Destabilized TurboRFP variant (TurboRFP-dest1): TurboRFP-dest1 is produced by fusing the initial protein with PEST amino acid sequence encoded by region 422-461 of mouse ornithine decarboxylase gene [Li et al., 1998]. This sequence targets the protein to degradation and enables a rapid protein turnover. TurboRFP-dest1 retains spectral properties of the initial protein, but has shorter half-life (approximately 1-2 hrs) as measured by the analysis of fluorescence intensity of cells treated with a protein synthesis inhibitor, cycloheximide. Because of rapid turnover, TurboRFP-dest1 can be used to measure changes in gene expression.