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Rhacostoma Green Fluorescent Protein (R-GFP)

Ursa BioScience is pleased to launch the highly novel Green Fluorescent Protein, Rhacostoma; which has exceptional and unique fluorescence properties, many superior to the classic Aequorea GFP. Rhacastoma is not available from anywhere else in the world today.

A novel Green Fluorescent Protein from the United States east coast jellyfish Rhacostoma atlantica has been cloned. This dimeric protein expresses very well in E.coli, so centrifuged bacterial pellets are intensely fluorescent.  Because the protein is maximally excited in the blue (466 nm excitation optimum), it appears exceptionally bright in room light.  In this respect, Rhacostoma GFP (R-GFP) resembles the very popular “enhanced GFP” (E-GFP), one of the earliest mutants of Aequorea GFP.  There is virtually no excitation in the 320 nm to 350 nm region of this new GFP.  At slightly alkaline pH (8.0), there is almost no excitation upon irradiation with the 365 nm mercury vapor line (found in standard long-wave UV lamps).  The light absorption at 365 nm is only 4% as great as it is at 466 nm.  The emission maximum is ≈ 505 nm.

Significant Opportunities of Rhacostoma GFP for the Life Sciences

Rhacostoma GFP has an amino acid sequence 45% different from its nearest phylogenetic neighbor. This is remarkable, as most GFP’s from closely related species have similar amino acid sequences.  So different is the amino acid sequence of Rhacostoma GFP, that this protein may well have a very useful protein sequence scaffold for generating new and versatile color variants.  Mutagenesis to alter excitation and emission spectra is currently underway. Several mutants with further red-shifted absorption maxima have already been found and will be released in the near-future. Rhacostoma GFP has temperature and pH sensitivities that could make it valuable in monitoring  intracellular conditions. The very low excitation in the 320-350 nm region of the spectrum might also serve as a negative control in some applications. Similar to Aequorea GFP, Rhacostoma GFP has two excitation peaks that vary in intensity as a function of pH.  A new, blue-shifted peak appears at pH values lower than 8.0. Based upon preliminary data, the absorption spectral shift in Rhacostoma GFP seems to be more pronounced (more sensitive to near neutral pH) than the corresponding pH shift in Aequorea GFP.  Precise measurements of these and other physical properties are underway.


Spectroscopic Data

Figure 1 compares the 3D excitation-emission maps for both the new and novel Rhacostoma GFP with that of the classical Aequorea GFP, plotted on the same scales. Unlike classical GFP, Rhacostoma shows much reduced to little excitation  < 400 nm.

Rhacastoma atlantica

Figure 1. (A) Excitation-Emission Map (EEM) recorded for wt-GFP (Aequoria) dissolved in Tris-EDTA (pH 7.6)  at + 21 C. The circles (white) indicates where fluorescence lifetimes have been recorded. (B) Data for Rhacostoma GFP.

 

Tables 1 and 2 show the fluorescence lifetimes recorded using time-correlated single photon counting (TCSPC), for both Rhacostoma and wt-GFP from Aequoria in Tris-EDTA, pH 7.6 at 21 C. While wt-GFP from Aequoria shows a lifetime  3.3 ± 0.2 ns, Rhacostoma is slightly shorter 2.85 ± 0.1 ns. The white circles in both Figures 1 and 2 show where the lifetimes were recorded.

Table 1 and 2

 

 

Figure 2 compares the 3D excitation-emission steady-state anisotropy maps for both the new and novel Rhacostoma GFP with that of the classical Aequorea GFP, plotted on the same scales. Similar to wt-gfp from Aequoria, Rhacostoma shows a very high anisotropy over its emission spectrum.

Rhacostoma atlantica

Figure 2. (A) Excitation-Emission Anisotropy Map (EEAM) recorded for wt-GFP (Aequorea) dissolved in Tris-EDTA (pH 7.6)  at + 21 C. (B) EEAM recorded for Rhacostoma GFP.

Note that the contour lines presebted in panels A and B are representative for the intensity values as presented in Figure 1, and does not reflect the anisotropy values that are color coded.

 

Figure 3 compares the emission intensity Vs Temperature of Rhacostoma GFP with that of the classical Aequorea GFP, plotted during a heating and cooling cycle, the protein being at each constant temperature for ≈ 15 mins. Interestingly, Rhacostoma shows a temperature dependence seldom seen with a great many of the other fluorescent proteins, offering significant opportunities for cellular environmental measurements.

Rhacostoma atlantica

Figure 1. (A) Stability of Rhacostoma in Tris-EDTA (pH 7.6) during heat and cool cycle. The excitation wavelength was 460 nm. First the sample is heated from +5 C to +60 C, then the sample is cooled back to + 5 C again. (B) Same experiment as in panel A but recorded for wt-GFP (Aequorea).

 

We are offering Rhacostoma GFP in both 50µg and 200µg quantities, 10 mM Tris buffer at pH 8.0, with < 0.02 % NaN3. Enquiries for larger quantities can be sent to This email address is being protected from spambots. You need JavaScript enabled to view it. .

 

 

Rhacostoma atlantica

Rhacostoma atlantica

Rhacostoma atlantica

Rhacostoma atlantica

GFP expressed in E. Coli.


Rhacastome GFP

GFP under room light.