Researchers from Moscow’s Shemykin-Ovchinnikov Institute of Bioorganic Chemistry and Russian biotech firm Evrogen have identified what they say is the brightest monomeric red fluorescent protein engineered so far. The protein, dubbed TagRFP, is characterized by complete chromophore maturation, prolonged fluorescence lifetime, and high pH-stability.
These properties led the researchers to conclude that TagRFP would make an excellent tag for protein-localization studies and fluorescence resonance energy-transfer applications.
TagRFP has excitation/emission peaks at 555/584 nm, the researchers said. It also has a 2.2-fold higher fluorescence quantum yield and a higher molar extinction coefficient compared to mCherry, a mutant monomeric variant of the red fluorescent protein DsRed.
Their work appears in the July 2007 issue of Nature Methods.
When the authors compared the maturation rate for TagRFP at 37°C to that for mCherry, they found a half-time of 40 minutes for mCherry and 100 minutes for TagRFP. They said that this maturation rate is unlikely to hinder the use of TagRFP in most applications.
TagRFP has very high pH stability, with a pKa of less than 4.0, the researchers said. This stability makes the signal nearly independent of cellular pH changes and allows the use of TagRFP within acidic organelles.
The investigators found that the time to bleach 50 percent of the TagRFP fluorescence emission intensity was similar to that for widely used fluorescent proteins such as EYFP, Clontech’s Cerulean, mPlum, or mCherry.
They also found that TagRFP displayed a biphasic rate of photobleaching, unlike most other fluorescent proteins, which display simple monophasic bleaching. TagRFP’s photobleaching comprised a fast initial phase over the first 100 seconds and a second phase that was similar to or slower than that of the other fluorescent proteins tested.
Transfections of Phoenix Eco and HeLa cells with a vector driving TagRFP expression without any fusion or localization signals resulted in bright red fluorescence 8 hours and 12 hours after transfection, respectively. The authors said that they did not observe any visible aggregates or non-specific localization at those time points.
They also said that Phoenix Eco cells expressing TagRFP still contained no visible aggregates of the protein five days after transfection.
The researchers reported that expression of both β-actin and α-tubulin fusions with TagRFP resulted in clear and accurate labeling of the actin filaments and microtubules. They also found that cells expressing TagRFP had an average fluorescence lifetime of 2.2 to 2.3 ns, but cells expressing mCherry had a fluorescence lifetime of 1.4 ns.
The high quantum yield and a prolonged fluorescence lifetime make TagRFP a prospective FRET donor for far-red acceptors, the investigators said. In addition, the high extinction coefficient of TagRFP makes it a FRET acceptor for the green fluorescent proteins.
The researchers said that in the orange, red, and far-red parts of the spectrum, naturally occurring fluorescent proteins are dimeric or tetrameric. However, this property results in mistargeting and aggregation of fused constructs, rendering these proteins inappropriate for use as fusion tags to study the localization, interaction, and motility of proteins of interest.
‘Fruit’ of Their Labor
According to the investigators, much effort has been focused on developing mutant monomeric proteins, such as the mFruits, mPlum and mCherry. Unfortunately, these variants are significantly less bright than DsRed.
The authors feel that a better solution to this problem is to develop bright monomeric fluorescent proteins, starting with naturally occurring fluorescent proteins that have a reduced tendency to oligomerize, so they require minimal modification for monomerization.
The researchers had previously cloned a red GFP-like protein, named eqFP578, from the sea anemone Entacmaea quadricolor. EqFP578 has excitation/emission peaks at 552/578 nm, a molar extinction coefficient of 102,000 M-1 cm-1, and a fluorescence quantum yield of 0.54.
“A better solution …is to develop bright monomeric fluorescent proteins … that have a reduced tendency to oligomerize.”
Unfortunately, wild-type eqFP578 has a relatively slow maturation rate at 37°C, so the authors optimized the protein using a random mutagenesis approach. The resulting protein, named TurboRFP, is a dimeric red fluorescent protein with excitation/emission peaks at 553/574 nm, a high pH-stability (a pKa of 4.4), fast maturation at 37°C, and high brightness.
Starting from TurboRFP, the researchers developed a monomeric red fluorescent protein, the final variant of which was TagRFP.
TurboRFP and TagRFP are marketed by Evrogen. The cost is €400 ($553) for 20 µg of each protein.
In an e-mail, Sergey Lukyanov, head of the laboratory of molecular technologies for biology and medicine at the Institute and a co-author of the paper, told CBA News this week that Evrogen plans to expand its line of fluorescent proteins by adding blue and far-red FPs. Work is already underway on these proteins, he said.
The company has started to develop advanced fluorescent protein-based tools, such as the genetically encoded photosensitizer KillerRed, the intercellular H2O2 sensor HyPer, and the high dynamic range calcium sensor Case12, said Lukyanov.
He added that Evrogen‘s product line increased more than twice during last year, and an increase in staff and expansion of facilities is planned for the beginning of next year to keep up with R&D developments.
Lukynaov said that a new product family or service will be added to the company’s catalogue each quarter. Most of the new offerings will be presented in the new company catalogue, scheduled for release in September.