Phi-Yellow fluorescent proteins

- Bright yellow fluorescence
- Proven suitability to generate stably transfected cell lines
- Destabilized variant is available

PhiYFP and PhiYFP-m are the mutants of a natural yellow fluorescent protein from Phialidium sp. (Cnidaria; Hydrozoa; Hydroida; Leptomedusae; Campanulariidae) [Shagin et al., 2004] and previous versions of TurboYFP. Excitation/emission maxima of Phi-Yellow proteins are at 525 and 537 nm, respectively. They exhibit less brightness and maturation rate than TurboYFP, but are more suitable for generation of stably transfected cell lines. The emission wavelength of these proteins is ideally positioned between those of green and red fluorescent proteins, allowing easy separation of these fluorescent tags by flow cytometry using common channels of detection and a single laser excitation line.

Main properties

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

CHARACTERISTIC PhiYFP PhiYFP-m *Brightness is a product of extinction coefficient and quantum yield, divided by 1000. Molecular weight, kDa 26.8 26.8 Polypeptide length, aa 234 234 Fluorescence color yellow yellow Excitation maximum, nm 525 525 Emission maximum, nm 537 537 Quantum yield 0.40 0.39 Extinction coefficient, M-1cm-1 130 000 124 000 Brightness* 52.0 48.4 Brightness, % of EGFP 158 147 pKa 6.0 6.0 Structure weak dimer weak dimer Aggregation no no Maturation rate at 37°C fast fast Photostability high high Cell toxicity not observed not observed Main advantages bright and fast-maturing yellow fluorescent proteins suitable for generation of stably transfected cell lines Possible limitations weak dimers with limited applicability for fusions generation; PhiYFP can not be used for generation of fusions to its C-terminus

Recommended filter sets and antibodies

The proteins can be recognized using Anti-PhiYFP (Cat.# AB601-AB602) and Anti-PhiYFP(d) (Cat.# AB603-AB604) antibodies available from Evrogen.

Recommended filter sets and antibodies

Phi-Yellow proteins can be detected using Omega Optical filter set XF104-3 or Chroma Technology Corp. filter set 42003 ("ZsYellow1").

Performance and use

Phi-Yellow proteins can be easily expressed and detected in a wide range of organisms, from bacteria to mammals. Transient transfection of mammalian cell lines with these proteins results in bright yellow fluorescent signals without visible aggregation. Fluorescence is clearly detected within 12 hrs after transfection.

Fluorescent microscopy of transiently transfected mammalian cells expressing Phi-Yellow proteins.

Suitability of Phi-Yellow proteins to generate stably transfected cells has been proven by Marinpharm company. Variuos cell lines are commercially available.

		Fluorescent microscopy of stably transfected mammalian cells expressing PhiYFP in cytosol. (A) M3 mouse melanoma; (B) T-406 human glioma; (C) PC-12 rat phaeochromocytoma cells; (D) PC-12 cells after the addition of nerve growth factor; (E) Walker 256 rat tumour cells; (F) BC3H1 cells; (G) T24 human bladder carcinoma cells; (H) T24 cells expressing destabilized variant PhiYFP-m-dest1. Images were kindly provided by Dr. Christian Petzelt (Marinpharm).

Despite dimerization capacity, Phi-Yellow proteins demonstrate successful performance in fusions with subcellular localization signals and many cellular proteins. However, we recommend that you use TagFPs for protein labeling applications. Please see section "Protein Localization Tags" to select a reporter for such purposes.
Important note: PhiYFP allows generation of fusions to its N-terminus, whereas PhiYFP-m is optimized to generate fusions to its C-terminus. PhiYFP can not be used to generate C-terminal fusions.

Phi-Yellow proteins can be used in multicolor labeling applications with cyan, green, red, and far-red fluorescent dyes.

	Fluorescent microscopy of stably transfected mammalian cells expressing Phi-Yellow-tagged fusions. (A-B) Mitochondria-targeted PhiYFP in (A) 3T3 mouse fibroblasts; (B) PtK rat kangaroo cells; (C) T24 human bladder carcinoma cells expressing peroxisome-targeted PhiYFP-m; (D) PhiYFP-m fusion with β-actin in PtK rat kangaroo cells. Images were kindly provided by Dr. Christian Petzelt (Marinpharm).

Available variants and fusions

PhiYFP: PhiYFP 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. PhiYFP allows generation of fusions to its N-terminus but unsuited to generate C-terminal fusions.

PhiYFP-m variant: PhiYFP-m variant is a mutant of PhiYFP. It is suitable for fusion generation to its C-terminus.

PhiYFP-mito fusion: A mitochondrial targeting sequence (MTS) is linked to the PhiYFP 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 green fluorescent labeling of mitochondria.

PhiYFP-m-peroxi fusion: Peroximal targeting signal [Gould et al., 1989] encoding tripeptide SKL was fused to the 3' end of PhiYFP-m sequence. This tripeptide targets the fusion protein to the matrix of peroxisomes.

Destabilized PhiYFP-m variant (PhiYFP-m-dest1): PhiYFP-m-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.
PhiYFP-m-dest1 retains spectral properties of the initial protein, but has shorter half-lives (approximately 2 hrs) as measured by the analysis of fluorescence intensity of cells treated with a protein synthesis inhibitor, cycloheximide. Because of rapid turnover, PhiYFP-m-dest1 can be used to measure changes in gene expression.

Fluorescence intensities of cells expressing PhiYFP-m-dest1 rapidly decrease in response to cycloheximide (CHX). Mammalian cells expressing PhiYFP-mP-dest1 under the control of CMV promoter were treated with CHX. After 1.5 hours CHX treatment, fluorescence intensity of cells was greatly reduced.