# Patent

## Title
Green fluorescent protein (GFP) peptides from Rhacostoma

## Abstract

The present technology is directed to the nucleic acid molecule encoding novel fluorescent proteins, in particular, green fluorescent proteins (GFPs), such as those that may be isolated from an organism of genus Rhacostoma, as well as compositions comprising the same and methods for analyzing a physiologically active substance in a cell wherein the fluorescent proteins are expressed in the cell.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national stage of International Application No. PCT/US14/30345, filed Mar. 17, 2014, which claims benefit of U.S. Provisional Application No. 61/799,464, filed Mar. 15, 2013. The entire contents of each and every foregoing application are hereby incorporated herein by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 30, 2014, is named 22900011.003_SL.txt and is 23,318 bytes in size.


BACKGROUND
Green-fluorescent proteins (GFPs) have been found to be useful tools for genetically labeling proteins, enzymes, antibodies, cells, tissues, organs, and organisms. In addition, GFP is widely used as a brilliant and sensitive reporter in biochemical assays. GFP's desirability comes from its intrinsic fluorescence and the fact that GFP can be introduced genetically. GFP makes its own intrinsic chromophore (fluorophore) without any required enzymes or cofactors other than molecular oxygen. (Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992 Feb. 15;111(2):229-33; Chalfie M, Tu Y, Euskirchen G, Ward W W, Prasher D C. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb. 11;263(5148):802-5.)
The known GFPs have mostly originated from members of the phylum Cnidaria (which includes jellyfish, sea pansies, corals, sea pens, and hydroids). As biochemical markers, most of the cloned GFPs have specific niches (Shaner N C, Steinbach P A, Tsien R Y. A guide to choosing fluorescent proteins. Nat Methods. 2005 Dec.;2(12):905-9.). For example, as a group, they span the color range from blue to red in their fluorescence emission properties and they come as monomers, dimers, or tetramers. There are differences in photostability, pH sensitivity, extinction coefficient, and fluorescence quantum yield. Some express better than others in heterologous organisms and some seem ideally suited for certain instrument systems (for example, fluorescence activated cell sorting (FACS), confocal microscopy, argon ion laser excitation, fluorescence resonance energy transfer (FRET), and western blots). The abbreviation GFP is usually used to refer to the proteins isolated from, or cloned from, the jellyfish Aequorea victoria, or on occasion to the sea pansy or Renilla reniformis. 
To fit into a particular niche, physical and spectral properties of a given fluorescent protein (FP) can be altered by mutagenesis (selected reviews: Heim R, Tsien R Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol. 1996 Feb. 1;6(2):178-82; Shaner N C, Patterson G H, Davidson M W. Advances in fluorescent protein technology. J Cell Sci. 2007 Dec. 15;120(Pt 24):4247-60.). But, the degree of alteration possible seems to be related to the starting amino acid structure of the “parent” protein. Sometimes this intrinsic sequence is called the “scaffold.” It is proposed herein that GFPs with very different inherent scaffolds could be genetically modified in ways that other GFPs (having different scaffolds) cannot be modified. Random or directed mutagenesis of a truly novel GFP has been developed herein.
SUMMARY OF THE DISCLOSED TECHNOLOGY
In certain embodiments, the present technology is directed to novel fluorescent proteins, for example, green fluorescent proteins (GFPs) such as those that are isolated from Rhacostoma, a jellyfish; as well as compounds comprising such fluorescent proteins.
In other embodiments, the present technology is directed to cloning of the novel fluorescent proteins, as well as mutants and methods for preparing and using the same in research, diagnostic and therapeutic applications.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the DNA sequence of a fluorescent protein developed as described herein, according to certain embodiments, and indicated as SEQ ID NO: 1.
FIG. 2 shows a protein sequence of a fluorescent protein developed as described herein, according to other embodiments. This is SEQ ID NO: 2 and is deduced from SEQ ID NO: 1.
FIG. 3 shows an excitation spectrum of a fluorescent protein of SEQ ID NO: 2. Extract from E. coli expressing Rhacostoma GFP was diluted in 10 mM Tris pH 8.0 buffer to give an absorbance of 0.05 at 466 nm. A fluorescence excitation spectrum was collected using a Gilford Fluoro IV spectrofluorometer. The emission monochromator was set to 505 nm. Fluorescence was recorded in arbitrary units.
FIG. 4 shows an emission spectrum of a fluorescent protein of SEQ ID NO: 2. Extract from E. coli expressing Rhacostoma GFP was diluted in 10 mM Tris pH 8.0 buffer to give an absorbance of 0.05 at 466 nm. A fluorescence emission spectrum was collect using a Gilford Fluoro IV spectrofluorometer. The excitation monochromator was set to 466 nm. Fluorescence was recorded in arbitrary units.
FIG. 5 shows an absorption spectrum of partially purified fluorescent protein from sequence 2. The fluorescent protein was diluted eight-fold into 10 mM Tris pH 8.0 buffer and the absorption spectrum was collected on a Cary 100 spectrophotometer.
FIG. 6 shows an excitation spectrum of partially purified fluorescent protein from sequence 2. Protein was diluted in 10 mM Tris pH 8.0 buffer to give an absorbance of 0.059 at 466 nm. A fluorescence excitation spectrum was collect using a Gilford Fluoro IV spectrofluorometer. The emission monochromator was set to 505 nm. Fluorescence was recorded in arbitrary units.
FIG. 7 shows an emission spectrum of partially purified fluorescent protein from SEQ ID NO: 2. Protein was diluted in 10 mM Tris pH 8.0 buffer to give an absorbance of 0.059 at 466 nm. A fluorescence emission spectrum was collect using a Gilford Fluoro IV spectrofluorometer. The excitation monochromator was set to 450 nm. Fluorescence was recorded in arbitrary units.
FIG. 8 shows the size exclusion HPLC profile of partially purified fluorescent protein. Full scale is 0.1 AU at 277 nm.
FIG. 9 shows alignment of GFP (Aequorea victoria gi|1169893) (SEQ ID NO: 14) with fluorescent proteins, or GFP-like proteins, from Rhacostoma (seq 2) (SEQ ID NO: 2), coerulescens (Aequorea coerulescens gi|34421676) (SEQ ID NO: 15), magnificus (Aldersladia magnificus gi|183186849) (SEQ ID NO: 16), macrodactyla (Aequorea macrodactyla gi|18175254) (SEQ ID NO: 17), Obelia (Obelia sp. MH-2011 gi|342221051) (SEQ ID NO: 18), Clytia_gregaria (Clytia gregaria gi|298257355) (SEQ ID NO: 19), and Phialidium (Phialidium sp. SL-2003 gi|40365351) (SEQ ID NO: 20). Introduced gaps are shown by dots. Dark shading shows identity to the consensus sequence, light shading shows similarity to the consensus sequence. Sequences were aligned with ClustalX (Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., Higgins, D. G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics, 23:2947-2948).
FIG. 10 shows fluorescence emission spectra of the Rhacostoma FP (solid line) and Enhanced Green Fluorescent Protein (EGFP, Heim R, Cubitt A B, Tsien R Y. Improved green fluorescence. Nature. 1995 Feb. 23;373(6516):663-4) (dashed line). Rhacostoma FP and EGFP were diluted in 10 mM Tris pH 8.0 buffer to give an absorbance of 0.046 at 450 nm. A fluorescence emission spectrum was collected using a Gilford Fluoro IV spectrofluorometer with the excitation monochromator set to 450 nm. Fluorescence was recorded in arbitrary units.
FIG. 11 shows an excitation spectrum (dashed line) and emission spectrum (solid line) of Rhacostoma mutant protein V204Y/M221T. Washed E. coli cells were diluted into 10 mM Tris pH 8.0 buffer to an optical density of 0.1 at 600 nm. The fluorescence spectra were collected with a Gilford Fluoro IV spectrofluorometer with the PMT voltage set to 600. An emission wavelength of 530 nm was used for the excitation spectrum and an excitation of 460 nm was used for the emission spectrum. Escherichia coli cells expressing wild type Rhacostoma FP required 400 volts at the PMT to elicit a similar signal intensity, corresponding to an approximate 20 fold difference in intensity.
FIG. 12 shows the DNA sequence of the V204Y/M221T mutant developed as described herein, according to certain embodiments, and indicated as SEQ ID NO: 3.
FIG. 13 shows the protein sequence of the V204Y/M221T mutant developed as described herein, according to certain embodiments, and indicated as SEQ ID NO: 4.



DETAILED DESCRIPTION
The present technology generally relates to novel fluorescent proteins, such as green fluorescent proteins (GFPs), which have found to be useful markers for gene expression; as well as compounds comprising such green fluorescent proteins and methods of preparing and using the same. In particular, in certain embodiments, the present technology is directed to methods and processes for preparing monomers and dimers of such fluorescent proteins.
Herein, the nucleic acid sequences and the corresponding amino acid sequences of novel fluorescent proteins have been discovered and isolated. The novel fluorescent proteins may be derived from a previously uncharacterized jellyfish of the genus Rhacostoma, including Rhacostoma atlantica. In the GFPs derived in certain embodiments herein, the sequences were identified by PCR using degenerate primers corresponding to conserved amino acid regions of GFPs from other Leptomedusae. In certain embodiments, a resulting amino acid sequence has only a 55% identity with its closest match from a BLAST search. Expression in E. coli confirmed that the nucleic acid sequence encodes for a fluorescent protein.
Rhacostoma GFP has an amino acid scaffold that is different from any of its taxonomic relatives. While some GFPs differ from others by as few as 2 amino acids, the GFP in certain embodiments herein differs from its nearest neighbor by more than 100 amino acids (45% dissimilar in amino acid sequence). A sequence comparison is shown in FIG. 9.
In a non-limiting example, a specimen of Rhacostoma atlantica was collected off the coast of New Jersey. Total RNA was isolated with a MasterPure Complete DNA and RNA Purification Kit (Epicentre) and a cDNA library was synthesized and amplified with a SMARTer PCR cDNA synthesis kit (Clonetech). Degenerate PCR primers were designed based on conserved amino acid regions of GFPs from other Leptomedusae. PCR was performed on the cDNA library with the degenerate primers and a PCR primer incorporating a poly T sequence to prime on the 3′ end of the cDNA molecule. PCR products were ligated into a pGEM vector and transformed into E. coli. Plasmids were prepared from the E. coli, sequenced, and examined for GFP like sequences. The resulting 3′ fragment of the DNA corresponding to the novel fluorescent protein was then used to design a set of reverse primers. In order to provide an additional 5′ template, the cDNA library was ligated into a pGEM vector. PCR was performed on the ligation mixture using a primer from the 3′ end of the DNA corresponding to the novel fluorescent protein and a primer corresponding to vector sequence at the 5′ end of the incorporation site. The resulting 5′ fragment of the DNA corresponding to the novel fluorescent protein was then used to generate a new set of forward primers. The full length sequence was obtained by PCR on the original cDNA library using a set of primers from the DNA corresponding to the novel fluorescent protein obtained above. The PCR product was cloned into a pGEM vector and sequenced.
BLAST searches were performed with BLASTP (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402.) Sequence comparisons were performed with ClustalX (Larkin, M. A., Blackshields, G, Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., Higgins, D. G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics, 23:2947-2948.)
For protein expression, the cDNA was subcloned into a pBAD vector between the Nde I and Kpn I sites. The resulting vector was used to transform E. coli DH10B cells which were then selected on plates with carbenicillin and L-arabinose. A fluorescent colony was selected and grown overnight at 37C in LB media with carbenicillin. The overnight culture was diluted 24-fold into fresh LB media with carbenicillin and allowed to grow for 3 hours at 37 degrees C. The culture was then induced with the addition of 0.1% L-arabinose and allowed to grow for 22 hours at room temperature.
The fluorescent E. coli were harvested by centrifugation and the pellet resuspended in a 10 mM Tris pH 8.0 solution with 0.5 mg/ml lysozyme added. After repeated freeze-thaw cycles, the viscous supernatant was pulled through a series of small gauge needles to shear the DNA. Fluorescence excitation and emission spectra were collected and are shown in FIG. 3 and FIG. 4, respectively.
Purification
Whole E. coli cells, transformed with the gene for native Rhacostoma GFP, and grown in 500 ml of medium in 2.8 L Fernbach flasks, were collected by centrifugation in a refrigerated (4 C) Sorvall centrifuge at 10,000 rpm in 250 mL bottles. The supernatants were discarded and the cell pellets were removed and suspended in an aqueous solution of 1.6 M ammonium sulfate, buffered with 50 mM Tris-HCl at pH 8.0. This buffer also contained 0.02% sodium azide to kill the cells and 0.1% PMSF to block serine proteases from degrading the GFP.
The cell suspension was diluted in the above buffer to a sufficient extent for the next step, (three-phase partitioning), to be most effective. Each of sixteen 50-mL falcon tubes was filled to the 25 mL mark with this suspension. Then 25 mL of t-butanol was added. Each tube was shaken vigorously, by hand, for 60 seconds and then the samples were centrifuged at room temperature in the same Falcon tubes at 3700 rpm in a table-top swinging bucket centrifuge for 20 min. This step produced three distinct phases: (1) a clear, upper organic layer containing 5 mL of water that came from the aqueous ammonium sulfate solution ( ̃30 mL total volume); (2) a robust semi-solid disk (about 3 mm thick); and (3) a clear, green-fluorescent lower liquid phase ( ̃20 mL). The alcohol layer was aspirated away and the congealed, semi-solid disks were lifted out with a spatula. The lower phase was kept. This completes the first stage of three-phase partitioning (TPP).
Fresh t-butanol (30 mL) was added to the aqueous layer, again with vigorous shaking. Further dehydration of the aqueous layer occurred. After centrifugation, as performed in Stage I, there was again an alcohol layer at the top of the tubes ( ̃33 or 34 mL), a paper thin, intensely green disk below, and an aqueous layer below this ( ̃16 mL), almost devoid of GFP. Both liquid layers were aspirated away leaving the thin green disk adhering to the side wall of each Falcon tube. This completes Stage II of TPP.
Next, using a minimum volume of pH 8-buffered 1.6 M ammonium sulfate stock solution, the green disks were serially transferred into a single Falcon tube. Four to five rounds of washing with tiny volumes of stock solution were performed until the GFP was just barely dissolved. This solution was dispensed into 12 microfuge tubes that were then spun at 16,000 rpm for 10 min. This time, four phases appeared after centrifugation. From top to bottom, the following were observed: (1) a clear organic layer; (2) a thin, non-fluorescent white disk; (3) a brilliantly fluorescent, but clear, aqueous layer; and (4) a small pellet at the bottom of the tube. The fluorescent layer was pipetted out, taking care not to include any of the other three phases of material. This completes Stage III of TPP. An absorption spectrum showed the absorbance at 466 nm divided by the absorbance at 277 nm of about 0.87, with a recovery of 101.8 OD's at 466 nm.
The whole TPP sample was applied to a Phenyl Sepharose FF column (16×70 mm=14 mL) in 1.6 M ammonium sulfate buffered to pH 8.0 with TrisHCl (start buffer). After 225 mL of washing with start buffer (16 bed volumes) the absorbance at 277 nm of the final 15 mL fraction had dropped to 0.076 O.D. Units.
Following this extensive washing step, the ammonium sulfate concentration was dropped to 0.8 M causing the fluorescent protein to elute isocratically. Isocratic elution was continued until the OD 466 dropped to 0.032 (down 100-fold from the peak value of 3.31). After Phenyl Sepharose FF, only the earliest fractions and the terminally pulsed fractions (in dilute Tris buffer) had absorbance ratios significantly below 1.78. Those fractions were excluded from the pool. The absorption spectrum, shown in FIG. 5, has a ratio of absorbance at 466 nm to absorbance at 277 nm of 1.76. A portion was concentrated in a Millipore 10K cutoff spin filter, the fluorescence excitation and emission spectra shown in FIGS. 6 and 7, respectively. A sample of concentrated material was applied to a Phenomenex BioSep SEC-S2000 HPLC column that was run in a buffer of 50 mM sodium phosphate, 100 mM NaCl, 0.02% NaN3 at pH 7.5 (FIG. 8). The column profile shows the majority of the protein in one nearly symmetrical peak. The HPLC profile and the improvement in the A466/A277 ratio indicate a substantial purification of the protein.
The molar extinction coefficient of the fluorescent protein was determined by estimating the chromophore concentration under alkaline conditions. The protein sample was mixed with 1/100 volume of 10 M NaOH and the concentration calculated based on a molar excitation coefficient of 44,100 M−1 cm−1 at 446 nm for the alkaline-denatured chromophore. (Ward, W. W. 1981. Properties of the coelenterate green fluorescent proteins. In Bioluminescence and Chemiluminescence: Basic Chemistry and Analytical Applications (M. A. DeLuca and W. D. McElroy, eds.) pp. 225-234.)
To generate fluorescent color variants of Rhacostoma FP, we used a combination of site-directed and random mutagenesis. Mutation of threonine 203 to tyrosine in wt Aequorea GFP contributes, at least in part, to the formation of a yellow fluorescing protein (Tsien R Y. The green fluorescent protein. Annu Rev Biochem. 1998;67:509-44). The Phialidium yellow fluorescent protein (Shagin D A, Barsova E V, Yanushevich Y G, Fradkov A F, Lukyanov K A, Labas Y A, Semenova T N, Ugalde J A, Meyers A, Nunez J M, Widder E A, Lukyanov S A, Matz M V. GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Mol Biol Evol. 2004 May; 21(5):841-50. Epub 2004 Feb. 1), one of the closest related to Rhacostoma, also has a tyrosine in this position (FIG. 9). We mutated the homologous position in Rhacostoma GFP. Mutant V204Y was created by site-directed mutagenesis using a primer overlap extension method (Ho SN, Hunt HD, Horton RM, Pullen JK, Pease L R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51-9.). Phusion polymerase (NEB) was used and template DNA was removed by treatment with Dpn I. The resulting mutant was expressed in E. coli as described above, but was non-fluorescent or weakly fluorescent. It is likely that additional mutations would be necessary to accommodate the introduction of the tyrosine, so a random mutagenesis approach was employed to identify one or more mutations to restore fluorescence. The non-fluorescent mutant also provided an opportunity to easily select novel fluorescent mutants, as any non-mutated colonies would remain dark when viewed on the surface of a blue-light box. The V204Y mutant was subject to random mutagenesis using the GeneMorph II EZClone Domain Mutagenesis Kit (Agilent Technologies, Inc). The mutated plasmid was transformed into NEB 10 E. coli and grown on LB agar plates containing 100 micrograms/ml carbenicillin and 0.1% arabinose. Fluorescent colonies were grown as described above and a portion of the E. coli was resuspended in 10 mM Tris buffer pH 8.0. The fluorescence excitation and emission spectra of one such colony is shown in FIG. 11. The excitation peak of the mutant is now in an appropriate region for excitation with the 488 nm line of an argon laser and the emission of the mutated FP was red-shifted by about 8-9 nm. Sequencing revealed the random mutagenesis introduced a methionine to threonine substitution at position 221 (SEQ ID NO: 3 and SEQ ID NO: 4) yielding a double mutant, Rhacostoma V204Y M221T. The mutation was red-shifted compared to the wild type, but reduced in intensity or expression by twenty fold or more. Mutation V204Y M221T could serve as a template for subsequent rounds of random or directed mutagenesis.
Results/Description
1. We have identified the nucleic acid sequence and the corresponding amino acid sequence of a novel fluorescent protein similar that that found in Rhacostoma atlantica.
2. The novel fluorescent protein was successfully expressed in E. coli, demonstrating that the DNA sequence encodes a fluorescent protein. The fluorescence excitation and emission spectra of an E. coli extract expressing Rhacostoma GFP are shown in FIGS. 3 and 4, respectively.
3. Rhacostoma GFP has an absorption max at about 466 nm (FIG. 5).
4. The excitation spectrum of the wild type Rhacostoma GFP is red shifted compared to wild type GFP from Aequorea victoria. The extinction coefficient at 466 is 58,000 M−1 cm−1.
5. FIG. 10 shows the emission spectra of the Rhacostoma FP and EGFP excited at the same wavelength and optical density. EGFP is a mutant variant of GFP, having amino acid substitutions F46L and S65T (GFP numbering). (Heim R, Cubitt A B, Tsien R Y. Improved green fluorescence. Nature. 1995 Feb. 23;373(6516):663-4.) The quantum yield of the Rhacostoma FP was estimated to be 0.74 by comparison to EGFP at similar optical densities at 450 nm and using a value of 0.6 for the EGFP quantum yield (Clontech).
The emission spectrum of the Rhacostoma FP, with a peak in the 495-500 region (FIG. 4, FIG. 7, and FIG. 10), makes this protein a good candidate for FRET assays with fluorescent proteins such as YFP (Tsien R Y. The green fluorescent protein. Annu Rev Biochem. 1998; 67:509-44) and mCitrine (Griesbeck O, Baird G S, Campbell R E, Zacharias D A, Tsien R Y. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem. 2001 Aug 3;276(31):29188-94.). The slightly bluer Cyan Fluorescent proteins (CFPs) can be used as donors for the yellow fluorescent proteins (Day R N, Davidson M W. Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells. Bioessays. 2012 May; 34(5):341-50.). The CFP, based on Aequorea GFP has a tryptophan in the chromophore instead of a tyrosine, and the emission spectra can be very wide, about 60 nm at half height, where the emission spectra of Rhacostoma FP is narrow, 33 nm at half height. A narrow emission spectrum could be helpful in FRET assays where emission from the donor chromophore can be problematic. The Teal Fluorescent Protein (TFP) has excitation and emission properties in the same region (Ai H W, Henderson J N, Remington S J, Campbell R E. Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochem J. 2006 Dec 15;400(3):531-40.), but is derived from the Clavularia soft coral protein and has only 26% identity with Rhacostoma (based on alignment using the ClustalX program and comparing to gi110589865). However, it is interesting to note that the amino acid before tyrosine 69 (Rhacostoma numbering) in the chromophore is alanine for both the TFP and the Rhacostoma FP, not serine as in GFP (serine 65 in GFP). Thus, the Rhacostoma FP provides a new source for development and modification of proteins for use in this color range.
In most of the naturally occurring GFP's, the amino acid found in position 68 of the chromophore possesses an R-group capable of hydrogen bonding to another amino acid. Often that amino acid is a serine. In virtually all cases, a hydrogen bond connects serine 68 to the highly conserved glutamic acid in position 223 (refer to the Rhacostoma numbering in FIG. 9). The carboxyl group of glutamic acid 223 projects inward from a beta strand that lies on the perimeter of the beta can. So, in effect, serine (and thus the chromophore) is tethered to the outside rim of the beta can. Unless this beta strand is distorted, the hydrogen bond between serine 68 and glutamic acid 223 regulates the distance between the chromophore imidazolone oxygen and nearby electron withdrawing groups. In the current numbering system, those electron withdrawing groups come from glutamine 95 and arginine 97, both completely conserved in the pile-up diagram of FIG. 9. With a hydrogen bonding serine in position 68, the chromophore is pulled away from glutamine 95 and arginine 97 such that their electrostatic bonds with the chromophore imidazolone is weakened. This allows electrons to flow toward the tyrosine phenolic hydroxyl group, reducing overall electron delocalization and creating a blue spectral shift in excitation and emission. In Phialidium GFP (Shagin D A, Barsova E V, Yanushevich Y G Fradkov A F, Lukyanov K A, Labas Y A, Semenova T N, Ugalde J A, Meyers A, Nunez J M, Widder E A, Lukyanov S A, Matz M V. GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Mol Biol Evol. 2004 May; 21(5):841-50. Epub 2004 Feb. 1) and in the Aequorea mutant, EGFP, a threonine is substituted for serine in position 68. Now a hydrogen bond is made between the threonine hydroxyl and glutamic acid 223. This substitution provides more “slack” in the hydrogen bond tether, such that the chromophore slips closer to glutamine 95 and arginine 97. A pronounced spectral shift to the red is seen in both cases. (William W. Ward, Biochemical and Physical Properties of Green Fluorescent Protein, in: Green Fluorescent Protein, M. Chalfie and S. Kain, eds. Wiley-Liss 1998, pp. 45-75.)
But in Rhacostoma GFP, what is normally a hydrogen bonding amino acid in position 68 is, surprisingly, an alanine. Alanine cannot engage in hydrogen bonding. So, in the same way that tyrosine 68 in Phialidium GFP and EGFP permit “slack” in the tether, so does alanine in Rhacostoma GFP. So, it appears that the effect of alanine 68 mimics that of threonine 68 in Phialidium GFP and EGFP, thus the red shift in all three. Among the 8 GFP's in the FIG. 9 pile-up, there is one other exception—Obelia GFP (Aglyamova G V, Hunt M E, Modi C K, Matz M V. Multi-colored homologs of the green fluorescent protein from hydromedusa Obelia sp. Photochem Photobiol Sci. 2011 Aug; 10(8)). Obelia GFP has a cysteine in this critical position 68. While cysteine is capable of hydrogen bonding, the sulfur atom is larger than a carbon atom. Assuming that the cysteine is hydrogen bonding, the tether may not hold Obelia GFP's chromophore close to glutamic acid 223. Thus Obelia GFP also displays a red shift.
The new fluorescent proteins developed herein have many uses that include, but are not limited to, the following:
A. As a fluorescent molecular marker. The protein can be expressed in an organism or cell as a fusion protein with a second protein or under direct control of a gene regulator or promoter. The Rhacostoma protein is readily expressed in E. coli without the need to alter DNA triplet codes for any of the amino acids.
B. In fluorescence resonance energy transfer (FRET) methods. The fluorescent protein can be an energy donor or acceptor. The Rhacostoma fluorescent protein is well suited to FRET studies because of its relatively narrow fluorescence emission spectrum (as shown in FIG. 4). A narrow emission spectrum reduces interference with the fluorescence from FRET partners. The narrow fluorescence emission peak in the 495-500 nm region makes the Bii GFP a good candidate for FRET assays with the yellow fluorescent protein.
C. As a reporter molecule attached to a solid support or bead. For example, the protein can be attached to the plate or bead with a peptide linker containing a specific protease cleavage site. Cleavage with the specific protease can result in release of the subject fluorescent protein. The GFPs discussed herein can be used with a GFP-on-a-string protease assay, as discussed in U.S. Pat. No. 7,883,863.
D. Mutagenesis studies. The Rhacostoma GFP sequence has been, before the present, an unexplored template for modification. Parameters for exploration include monomer/dimer equilibrium, wavelength of excitation and emission, extinction coefficient, quantum yield, pH sensitivity, rate of maturation, turnover rate, and photostability. The desired property is largely dependent upon the application. For example, a FRET assay may require a blue fluorescence, while in vivo imaging is improved with a far red fluorescence.
Mutation studies have been explored. Some Rhacostoma mutations have been designed to generate a monomer protein and a protein with a green, yellow, or red emission. The wild type protein has been characterized with respect to its pH profile, expression level, stability, and monomer/dimer equilibrium. Some mutations have excitation spectra red shifted compared to the wild type protein, potentially overlapping more with the 488 nm argon laser line. Argon lasers are widely used in fluorescence instrumentation such as fluorescence-activated cell sorters. Fluorescent proteins can be useful in cell sorters, either as proteins expressed in the cell, or attached to antibodies or ligands that bind to the outside of the cell. Beads can also be used in cell sorters, using fluorescent proteins to identify the beads.
Additional PCR Details
The Advantage-HF 2 PCR Kit (Clontech Laboratories, Inc.) was used for amplifying DNA from the Rhacostoma cDNA library. The thermocycler used was a Minicycler from MJ Research. Unless otherwise stated, the primer concentration was 200 nM. A typical reaction was 94° C. for 90 seconds followed by 26 cycles of 30 seconds at 94° C., 30 seconds at 60° C., and 3 minutes at 72° C.
Primers used for Rhacostoma Cloning 1st set PCR reactions—Rhacostoma library as template









>smart_tail1



(SEQ ID NO: 5)



GTGGTATCAACGCAGAGTACTTTTTTTTTTTTT


 



>FP202-27 Leptomedusa_primer



(SEQ ID NO: 6)



HGGDRANNTHCCWGTWCCATGGBCWAC






Primer FP202-27 was used at 2uM. The reaction was run as above with 26 cycles. The product was then re-amplified for an additional 15 cycles.
2nd set PCR reactions—template was Rhacostoma library ligated into pGEM









>BGAL primer



(SEQ ID NO: 7)



TGACCATGATTACGCCAAGCTATTTAGGTG


 



>712



(SEQ ID NO: 8)



TGGTAGTGCAGTCATTCCACATCAACG


 



>642



(SEQ ID NO: 9)



CGGCAGTGATGTATTCCACCATCCTC






Nested PCR reactions. The first reaction used a primer combination of BGAL/712 followed by BGAL/642 primers. Each was run with 26 cycles.
3rd set PCR—Rhacostoma library as template









>13



(SEQ ID NO: 10)



CAGCGAGCGATACATCACACACACC


 



>28



(SEQ ID NO: 11)



CACACACACCAAGAACATTCAAAGTTTCC


 



>712



(SEQ ID NO: 9)



TGGTAGTGCAGTCATTCCACATCAACG






Nested PCR reaction 13/712 followed by 28/712, each at 25 cycles.
4th Set PCR reactions—subclone from pGEM into pBAD









>NDE_145



(SEQ ID NO: 12)



CAAACCCATATGAGCACTGGAAAGACTGG


 



>KPN_145



(SEQ ID NO: 13)



CGGGTACCTTATCCTCCTTTATTATAAGCAGTGTC






The reaction was run as above but with 21 cycles.

## Claims

1. A fusion protein comprising a fluorescent protein linked to a polypeptide, the fluorescent protein comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2.

2. The fusion protein of claim 1, wherein the fluorescent protein is a green fluorescent protein (GFP).

3. The fusion protein of claim 1, wherein the fluorescent protein is isolated from an organism of genus Rhacostoma.

4. A composition comprising the fusion protein of claim 1.

5. A monomer, dimer or tetramer of the fusion protein of claim 1.

6. The fusion protein of claim 1, wherein the fluorescent protein is encoded by a
nucleic acid molecule comprising
a nucleotide sequence that exhibits at least 80% sequence identity to the sequence of nucleotides set forth in SEQ ID NO: 1.

7. The fusion protein of claim 6, wherein the nucleic acid is optimized for expression in human cells, animal cells, bacterial cells, yeast cells, fungal cells, plant cells or insect cells.

8. The fusion protein of claim 6, wherein the nucleic acid is DNA or RNA.

9. A diagnostic composition comprising the fusion protein of claim 1.

10. A method comprising:
cleaving a fusion protein having a fluorescent protein linked to a polypeptide and having a protease cleavage site, the fluorescent protein including an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 2; and
expressing the fluorescent protein in a cell.