Renilla GFP mutants with increased fluorescent intensity and spectral shift Number:7,083,931 from the United States Patent and Trademark Office (PTO) owispatent The present invention provides a polynucleotides encoding mutants of green fluorescent protein from Renilla reniformis, including humanized sequences which permit enhanced expression of the encoded polypeptides in mammalian cells.<H fluorescent, intensity, Renilla, GFP, mut, 7083931.html, Patent, pto, 7083931

Title: Renilla GFP mutants with increased fluorescent intensity and spectral shift

Abstract: The present invention provides a polynucleotides encoding mutants of green fluorescent protein from Renilla reniformis, including humanized sequences which permit enhanced expression of the encoded polypeptides in mammalian cells.

Patent Number: 7,083,931 Issued on 08/01/2006 to Gurtu

Inventors: Gurtu; Vanessa Elaine (Carlsbad, CA)
Assignee: Stratagene California (La Jolla, CA)

Appl. No.: 10/815,337

Filed: April 1, 2004

Current U.S. Class: 435/6 ; 435/252.3; 435/254.11; 435/325; 435/69.1; 435/7.1; 530/350; 536/23.5

Current International Class: C12Q 1/68 (20060101); C07H 21/04 (20060101); C07K 14/435 (20060101); C12N 5/10 (20060101); C12P 21/02 (20060101); G01N 33/66 (20060101)

References Cited [Referenced By]

U.S. Patent Documents


5625048	April 1997	Tsien et al.
5777079	July 1998	Tsien et al.
5804387	September 1998	Cormack et al.
5874304	February 1999	Zolotukhin et al.
5968738	October 1999	Anderson et al.
5968750	October 1999	Zolotukhin et al.
6172188	January 2001	Thastrup et al.
6232107	May 2001	Bryan et al.
2002/0064842	May 2002	Sorge et al.
2002/0132318	September 2002	Zhao et al.

Foreign Patent Documents


WO 97/11094	Mar., 1997	WO
WO 97/20078	Jun., 1997	WO
WO 97/42320	Nov., 1997	WO
WO 98/06737	Feb., 1998	WO
WO 98/21355	May., 1998	WO
WO 01/64843	Sep., 2001	WO
WO 01/68824	Sep., 2001	WO
WO 02/048174	Jun., 2002	WO
WO 03/033650	Apr., 2003	WO

Other References

Chalfie, et al.; "Green Fluorescent Protein as a Marker for Gene Expression"; (1994); Science; 263: 802-805. cited by other .
Tsien; "The Green Fluorescent Protein"; (1998); Annu. Rev. Biochem.; 67: 509-544. cited by other .
Ward; "Energy Transfer Processes in Bioluminescence"; (1979); Photochem. Photobiol. Rev.; 4: 1-57. cited by other .
Ward, et al.; "Spectral Perturbations of the Aequorea Green-Fluorescent Protein"; (1982); Photochem. Photobiol.; 35: 803-808. cited by other .
Heim, et al., "Improved Green Fluorescence"; (1995); Nature; 373: 663-664. cited by other .
Chalfie; "Green Fluorescent Protein"; (1995); Photochemistry and Photobiology; 62(4): 651-656. cited by other .
Ehrig, et al.; "Green-fluorescent protein mutants with altered fluorescence excitation spectra"; (1995); FEBS Lett.; 367: 163-166. cited by other .
Surpin, et al.; "Development of Monoclonal Antibodies to Aequorea Green-Fluorescent Protein and their Cross Reaction with Renilla Green-Fluorescent Protein"; (1987); Photochem. Photobiol.; 45(Supp.): 95S. cited by other .
Delagrave, et al.; "Red-Shifted Excitation Mutants of the Green Fluorescent Protein"; (1995); Biotechnology; 13: 151-154. cited by other .
Yang, et al.; "Dual color microscopic imagery of cells expressing the green fluorescent protein and a red-shifted variant"; (1996); Gene; 173: 19-23. cited by other .
Ward and Cormier; "An Energy Transfer Protein in Coelenterate Bioluminescence"; The Journal of Biological Chemistry; (1979); 254 (3): 781-788. cited by other .
Copy of International Search Report dated Aug. 18, 2004. cited by other.

Primary Examiner: Wax; Robert A.
Attorney, Agent or Firm: Williams; Kathlinee M. Edward Angell Palmer & Dodge, LLP

Parent Case Text

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/460,432, filed on Apr. 4, 2003. The entire teachings of the above application are incorporated herein by reference.

Claims

What is claimed is:

1. A mutant Green Fluorescent Protein (GFP) from Renilla reniformis, selected from the group consisting of: (a) the amino acid sequence of mutant GM1; (b) the amino acid sequence of mutant GM2; (c) the amino acid sequence of mutant GM3; (d) the amino acid sequence of mutant GM4; (e) the amino acid sequence of mutant GM6; (f) the amino acid sequence of mutant T1; (g) the amino acid sequence of mutant T6; (h) the amino acid sequence of mutant T8; (i) the amino acid sequence of mutant T11; (j) the amino acid sequence of mutant T12; (k) the amino acid sequence of mutant T13; (l) the amino acid sequence of mutant T14; (m) the amino acid sequence of mutant T15; and (n) the amino acid sequence of mutant T17.

2. A polynucleotide encoding a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, selected from the group consisting of: (a) a polynucleotide encoding the amino acid sequence of mutant GM1; (b) a polynucleotide encoding the amino acid sequence of mutant GM2; (c) a polynucleotide encoding the amino acid sequence of mutant GM3; (d) a polynucleotide encoding the amino acid sequence of mutant GM4; (e) a polynucleotide encoding the amino acid sequence of mutant GM6; (f) a polynucleotide encoding the amino acid sequence of mutant T1; (g) a polynucleotide encoding the amino acid sequence of mutant T6; (h) a polynucleotide encoding the amino acid sequence of mutant T8; (i) a polynucleotide encoding the amino acid sequence of mutant T11; (j) a polynucleotide encoding the amino acid sequence of mutant T12; (k) a polynucleotide encoding the amino acid sequence of mutant T13; (l) a polynucleotide encoding the amino acid sequence of mutant T14; (m) a polynucleotide encoding the amino acid sequence of mutant T15; and (n) a polynucleotide encoding the amino acid sequence of mutant T17.

3. The polynucleotide of claim 2, said polynucleotide being humanized.

4. A vector comprising the polynucleotide of claim 3.

5. A host cell containing the vector of claim 4.

6. A mutant Green Fluorescent Protein (GFP) from Renilla reniformis, selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:34; (b) the amino acid sequence of SEQ ID NO:36; (c) the amino acid sequence of SEQ ID NO:38; (d) the amino acid sequence of SEQ ID NO:40; (e) the amino acid sequence of SEQ ID NO:42; (f) the amino acid sequence of SEQ ID NO:44; (g) the amino acid sequence of SEQ ID NO:46; (h) the amino acid sequence of SEQ ID NO:48; (i) the amino acid sequence of SEQ ID NO:50; (j) the amino acid sequence of SEQ ID NO:52; (k) the amino acid sequence of SEQ ID NO:54; (l) the amino acid sequence of SEQ ID NO:56; (m) the amino acid sequence of SEQ ID NO:58; and (n) the amino acid sequence of SEQ ID NO:60.

7. A polynucleotide encoding a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, selected from the group consisting of: (a) the polynucleotide sequence of SEQ ID NO:33; (b) the polynucleotide sequence of SEQ ID NO:35; (c) the polynucleotide sequence of SEQ ID NO:37; (d) the polynucleotide sequence of SEQ ID NO:39; (e) the polynucleotide sequence of SEQ ID NO:41; (f) the polynucleotide sequence of SEQ ID NO:43; (g) the polynucleotide sequence of SEQ ID NO:45; (h) the polynucleotide sequence of SEQ ID NO:47; (i) the polynucleotide sequence of SEQ ID NO:49; (j) the polynucleotide sequence of SEQ ID NO:51; (k) the polynucleotide sequence of SEQ ID NO:53; (l) the polynucleotide sequence of SEQ ID NO:55; (m) the polynucleotide sequence of SEQ ID NO:57; and (n) the polynucleotide sequence of SEQ ID NO:59.

8. The polynucleotide of claim 7, said polynucleotide being humanized.

9. A vector comprising the polynucleotide of claim 8.

10. A host cell containing the vector of claim 9.

11. A mutant Green Fluorescent Protein (GFP) from Renilla reniformis, selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:4; (b) the amino acid sequence of SEQ ID NO:6; (c) the amino acid sequence of SEQ ID NO:8; (d) the amino acid sequence of SEQ ID NO:10; (e) the amino acid sequence of SEQ ID NO:12; (f) the amino acid sequence of SEQ ID NO:14; (g) the amino acid sequence of SEQ ID NO:16; (h) the amino acid sequence of SEQ ID NO:18; (i) the amino acid sequence of SEQ ID NO:20; (j) the amino acid sequence of SEQ ID NO:22; (k) the amino acid sequence of SEQ ID NO:24; (l) the amino acid sequence of SEQ ID NO:26; (m) the amino acid sequence of SEQ ID NO:28; and (n) the amino acid sequence of SEQ ID NO:30.

12. A polynucleotide encoding a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, selected from the group consisting of: (a) the polynucleotide sequence of SEQ ID NO:3; (b) the polynucleotide sequence of SEQ ID NO:5; (c) the polynucleotide sequence of SEQ ID NO:7; (d) the polynucleotide sequence of SEQ ID NO:9; (e) the polynucleotide sequence of SEQ ID NO:11; (f) the polynucleotide sequence of SEQ ID NO:13; (g) the polynucleotide sequence of SEQ ID NO:15; (h) the polynucleotide sequence of SEQ ID NO:17; (i) the polynucleotide sequence of SEQ ID NO:19; (j) the polynucleotide sequence of SEQ ID NO:21; (k) the polynucleotide sequence of SEQ ID NO:23; (l) the polynucleotide sequence of SEQ ID NO:25; (m) the polynucleotide sequence of SEQ ID NO:27; and (n) the polynucleotide sequence of SEQ ID NO:29.

13. A vector comprising the polynucleotide of claim 12.

14. A host cell containing the vector of claim 13.

15. A method of producing mutant Renilla reniformis GFP comprising the steps of: (a) culturing a cell containing a recombinant vector comprising a wild type or humanized polynucleotide sequence encoding mutant Renilla reniformis GFP under conditions where the mutant Renilla reniformis GFP protein is expressed, wherein said polynucleotide sequence is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29; and (b) isolating said mutant Renilla reniformis GFP protein from said cell, wherein said mutant Renilla reniformis GFP has a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:30; thereby producing mutant Renilla reniformis GFP.

16. A method of producing a Renilla reniformis fusion protein, said method comprising the steps of: culturing a cell containing a polynucleotide sequence encoding said polypeptide of interest linked with a humanized polynucleotide encoding mutant Renilla reniformis GFP wherein said humanized polynucleotide is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29, and wherein the linked polynucleotide sequences are fused in frame, under conditions where the mutant Renilla reniformis GFP protein is expressed.

17. A method of determining the location of a polypeptide of interest in a cell, said method comprising determining the location of the fusion protein of claim 16.

18. A method of identifying a cell into which a recombinant vector has been introduced, said method comprising the steps of: (a) providing a cell containing a recombinant vector comprising a humanized polynucleotide which encodes mutant Renilla reniformis GFP, wherein said humanized polynucleotide is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29, and wherein said cell permits expression of said humanized polynucleotide; (b) illuminating said population with light within the excitation spectrum of mutant Renilla reniformis GFP; and (c) detecting fluorescence in the emission spectrum of mutant Renilla reniformis GEP in said population, where detection of fluorescence in the cell indicates that the recombinant vector has been introduced into the cell; thereby identifying a cell into which said recombinant vector has been introduced.

19. The method of claim 18, wherein said GFP is expressed as a fusion polypeptide.

20. The method of claim 18, wherein said GFP is expressed as a distinct polypeptide.

21. The method of claim 18, wherein said cells are identified by FACS analysis.

22. A method of detecting the activity of a transcriptional regulatory sequence, said method comprising the steps of: (a) culturing a cell containing a nucleic acid sequence comprising said transcriptional regulatory sequence operably linked to a humanized nucleic acid sequence encoding mutant Renilla reniformis GFP to form a reporter construct, under conditions where the mutant Renilla reniformis GFP is expressed, wherein said humanized nucleic acid sequence is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29; and (b) detecting mutant Renilla reniformis GFP fluorescence in said cell, wherein detection of fluorescence indicates activity of said transcriptional regulatory sequence; thereby detecting the activity of a transcriptional regulatory sequence.

23. A method of detecting the presence of a modulator of a transcriptional regulatory sequence, said method comprising the steps of: (a) culturing a cell containing a nucleic acid sequence comprising said transcriptional regulatory sequence operably linked to a humanized nucleic acid sequence encoding mutant Renilla reniformis GFP to form a reporter construct, under conditions where the mutant Renilla reniformis GFP is expressed, wherein said humanized nucleic acid sequence is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29; and (b) detecting mutant Renilla reniformis GFP fluorescence in said cell, wherein said fluorescence indicates the presence of said modulator; thereby detecting the presence of a modulator of a transcriptional regulatory sequence.

24. A method of screening for an inhibitor of a transcriptional regulatory sequence, said method comprising the steps of: (a) culturing a cell containing a nucleic acid sequence comprising said transcriptional regulatory sequence operably linked to a humanized nucleic acid sequence encoding mutant Renilla reniformis GFP to form a reporter construct, under conditions where the mutant Renilla reniformis GFP is expressed, wherein said humanized nucleic acid sequence is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29; (b) contacting said cell with a candidate inhibitor of said transcriptional regulatory sequence; and (c) detecting mutant Renilla reniformis GFP fluorescence in said cell, wherein a decrease in said fluorescence relative to that detected in the absence of said candidate inhibitor indicates that said candidate inhibitor inhibits the activity of said transcriptional regulatory sequence.

25. A method of producing a fluorescent molecular weight marker, said method comprising the steps of: (a) culturing a cell containing a humanized nucleic acid sequence encoding mutant Renilla reniformis GFP linked in frame to a nucleic acid sequence encoding a polypeptide of known relative molecular weight such that said linked molecules encode a fusion polypeptide, under conditions where the mutant Renilla reniformis GFP is expressed, wherein said humanized nucleic acid sequence is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29; (b) isolating said fusion polypeptide from said cell, wherein said fusion polypeptide is a relative molecular weight marker.

26. The method of claims 15, 16, 18 or 22 25, wherein said cell is a mammalian cell.

27. The method of claims 15, 16, 18 or 22 25, wherein said cell is a human cell.

28. A mutant Green Fluorescent Protein (GFP) from Renilla reniformis, wherein the mutation comprises an amino acid substitution at one or more of the following residues: (a) F43; (b) E120; (c) L101; and (d) Y103.

29. The mutant GFP of claim 28, wherein said mutation is E120G.

Description

BACKGROUND

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become an extremely useful tool for tracking and quantifying biological entities in the fields of biochemistry, molecular and cell biology, and medical diagnostics (Chalfie et al., 1994, Science 263:802 805; Tsien, 1998, Ann. Rev. Biochem. 67:509 544). There are no cofactors or substrates required for fluorescence, thus the protein can be used in a wide variety of organisms and cell types. GFP has been used as a reporter gene to study gene expression in vivo by insertion downstream of a test promoter. The protein has also been used to study the subcellular localization of a number of proteins by direct fusion of the test protein to GFP, and GFP has become the reporter of choice for monitoring the infection efficiency of viral vectors both in cell culture and in animals. In addition, a number of genetic modifications have been made to GFP resulting in variants for which spectral shifts correspond to changes in the cellular environment such as pH, ion flux, and the phosphorylation state of the cell. Perhaps the most promising role for GFP as a cellular indicator is its application to fluorescence resonance energy transfer (FRET) technology. FRET occurs with fluorophores for which the emission spectrum of one overlaps with the excitation spectrum of the second. When the fluorophores are brought into close proximity, excitation of the "donor" fluorophore results in emission from the "acceptor". Pairs of such fluorophores are thus useful for monitoring molecular interactions. Fluorescent proteins such as GFP are useful for analysis of protein:protein interactions in vivo or in vitro if their fluorescent emission and excitation spectra overlap to allow FRET. The donor and acceptor fluorescent proteins may be produced as fusions with the proteins one wishes to analyze for interactions. These types of applications of GFPs are particularly appealing for high throughput analyses, since the readout is direct and independent of subcellular localization.

Purified A. victoria GFP is a monomeric protein of about 27 kDa that absorbs blue light with excitation wavelength maximum of 395 nm, with a minor peak at 470 nm, and emits green fluorescence with an emission wavelength of about 510 nm and a minor peak near 540 nm (Ward et al., 1979, Photochem. Photobiol. Rev. 4:1 57). The excitation maximum of A. victoria GFP is not within the range of wavelengths of standard fluorescein detection optics. Further, the breadth of the excitation and emission spectra of the A. victoria GFP are not well suited for use in applications involving FRET. In order to be useful in FRET applications, the excitation and emission spectra of the fluorophores are preferably tall and narrow, rather than low and broad. There is a need in the art for GFP proteins that are amenable to the use of standard fluorescein excitation and detection optics. There is also a need in the art for GFP proteins with narrow, preferably non-overlapping spectral peaks.

The use of A. victoria GFP as a reporter for gene expression studies, while very popular, is hindered by relatively low quantum yield (the brightness of a fluorophore is determined as the product of the extinction coefficient and the fluorescence quantum yield). Generally, the A. victoria GFP coding sequences must be linked to a strong promoter, such as the CMV promoter or strong exogenous regulators such as the tetracycline transactivator system, in order to produce readily detectable signal. This makes it difficult to use GFP as a reporter for examining the activity of native promoters responsive to endogenous regulators. Higher intensity would obviously also increase the sensitivity of other applications of GFP technology. There is a need in the art for GFP proteins with higher quantum yield.

Another disadvantage of A. victoria GFP involves fluctuations in its spectral characteristics with changes in pH. At high pH (pH 11 12), the wild-type A. victoria GFP loses absorbance and excitation amplitude at 395 nm and gains amplitude at 470 nm (Ward et al., 1982, Photochem. Photobiol. 35:803 808). A. victoria fluorescence is also quenched at acid pH, with a pKa around 4.5. There is a need in the art for GFPs exhibiting fluorescence that is less sensitive to pH fluctuations.

Further, in order to be more useful in a broad range of applications, there is a need in the art for GFP proteins exhibiting increased stability of fluorescence characteristics relative to A. victoria GFP, with regard to organic solvents, detergents and proteases often used in biological studies. There is also a need in the art for GFP proteins that are more likely to be soluble in a wider range of cell types and less likely to interfere non-specifically with endogenous proteins.

A number of modifications to A. victoria GFP have been made with the aim of enhancing the usefulness of the protein. For example, modifications aimed at enhancing the brightness of the fluorescence emissions or the spectral characteristics of either the excitation or emission spectra or both have been made. It is noted that the stated aim of several of these modification approaches was to make an A. victoria GFP that is more similar to R. reniformis GFP in its excitation and emission spectra and fluorescence intensity.

Literature references relating to A. victoria mutants exhibiting altered fluorescence characteristics include, for example, the following. Heim et al. (1995, Nature 373:663 664) relates to mutations at S65 of A. victoria that enhance fluorescence intensity of the polypeptide. The S65T mutation to the A. victoria GFP is said to "ameliorate its main problems and bring its spectra much closer to that of Renilla".

A review by Chalfie (1995, Photochem. Photobiol. 62:651 656) notes that an S65T mutant of A. victoria, the most intensely fluorescent mutant of A. victoria known at the time, is not as intense as the R. reniformis GFP.

Further references relating to A. victoria mutants include, for example, Ehrig et al., 1995, FEBS Lett. 367:163 166); Surpin et al., 1987, Photochem. Photobiol. 45(Suppl):95S; Delagrave et al., 1995, BioTechnology 13:151 154; and Yang et al., 1996, Gene 173:19 23.

Patent and patent application references relating to A. victoria GFP and mutants thereof include the following. U.S. Pat. No. 5,874,304 discloses A. victoria GFP mutants said to alter spectral characteristics and fluorescence intensity of the polypeptide. U.S. Pat. No. 5,968,738 discloses A. victoria GFP mutants said to have altered spectral characteristics. One mutation, V163A, is said to result in increased fluorescence intensity. U.S. Pat. No. 5,804,387 discloses A. victoria mutants said to have increased fluorescence intensity, particularly in response to excitation with 488 nm laser light. U.S. Pat. No. 5,625,048 discloses A. victoria mutants said to have altered spectral characteristics as well as several mutants said to have increased fluorescence intensity. Related U.S. Pat. No. 5,777,079 discloses further combinations of mutations said to provide A. victoria GFP polypeptides with increased fluorescence intensity. International Patent Application (PCT) No. WO 98/21355 discloses A. victoria GFP mutants said to have increased fluorescence intensity, as do WO 97/20078, WO 97/42320 and WO 97/11094. PCT Application No. WO 98/06737 discloses mutants said to have altered spectral characteristics, several of which are said to have increased fluorescence intensity.

In addition to A. victoria, GFPs and other fluorescent proteins have been identified in a variety of other coelenterates and anthazoa. Other GFPs cloned include A. victoria (Prasher, 1992, Gene 111:229 233) and Renilla mulleri (WO 99/49019). A red-shifted fluorescent protein cloned from the coral Discosoma (Matz, M. V. et al., 1999, Nat. Biotechnol. 17:969 973) and named DsRed. Biochemical properties of DsRed and a mutant of DsRed are reported in Baird, G. S. et al. (2000, Proc. Natl. Acad. Sci. USA 97:11984 89).

The native R. reniformis protein was purified and characterized by Ward, W. et al. (J. Biol. Chem. 254 3:781 788) in 1979. The native protein was found to have a 5 fold greater extinction coefficient than native A. victoria GFP. In addition the R. reniformis GFP forms a non-dissociable homodimer, shows a broad range of pH stability, is resistant to organic solvents, detergents, and proteases, and has a narrow excitation and emission spectra. RT-PCR with gene specific primers has revealed the presence of two distinct isoforms of R. reniformis GFP (WO 01/68824) and (WO 01/64843).

SUMMARY OF THE INVENTION

Disclosed herein are the polynucleotide and polypeptide sequences for a series of mutants of R. reniformis GFP that display increased fluorescence intensity and/or alterations to the fluorescence spectra. Also disclosed are humanized versions of the polynucleotides encoding those mutants.

The invention features mutant Green Fluorescent Protein (GFP) sequences, and nucleic acids encoding them, and particularly humanized forms of the nucleic acids.

The invention also features a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, where the mutation includes an amino acid substitution in the Beta Strand 4 portion of the protein, relative to the wild-type form of the protein, and where the mutant GFP protein has one or more of the following characteristics: (a) enhanced emission intensity, relative to wild-type GFP protein from Renilla reniformis; (b) a narrower or broader emission spectrum, relative to wild-type GFP protein from Renilla reniformis; and (c) a shift in excitation or emission maxima, relative to wild-type GFP protein from Renilla reniformis; (d) a shift in maturation rate, relative to wild-type GFP protein from Renilla reniformis; and (e) exhibiting less quenching of fluorescence at acidic pH, relative to wild-type GFP protein from Renilla reniformis.

The invention also features a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, where the mutation includes an amino acid substitution in the loop region of the protein between Beta Strand 2 and Beta Strand 3, relative to the wild-type form of the protein, and where the mutant GFP protein has one or more of the following characteristics: (a) enhanced emission intensity, relative to wild-type GFP protein from Renilla reniformis; (b) a narrower or broader emission spectrum, relative to wild-type GFP protein from Renilla reniformis; and (c) a shift in excitation or emission maxima, relative to wild-type GFP protein from Renilla reniformis; (d) a shift in maturation rate, relative to wild-type GFP protein from Renilla reniformis; and (e) exhibiting less quenching of fluorescence at acidic pH, relative to wild-type GFP protein from Renilla reniformis.

The invention additionally features a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, where the mutation includes an amino acid substitution in the loop region of the protein between Beta Strand 5 and Beta Strand 6, relative to the wild-type form of the protein, and where the mutant GFP protein has one or more of the following characteristics: (a) enhanced emission intensity, relative to wild-type GFP protein from Renilla reniformis; (b) a narrower or broader emission spectrum, relative to wild-type GFP protein from Renilla reniformis; and (c) a shift in excitation or emission maxima, relative to wild-type GFP protein from Renilla reniformis; (d) a shift in maturation rate, relative to wild-type GFP protein from Renilla reniformis; and (e) exhibiting less quenching of fluorescence at acidic pH, relative to wild-type GFP protein from Renilla reniformis.

In another aspect, the invention features a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, where the mutation includes an amino acid substitution in the region of the protein extending from the beginning of Beta Strand 1 through the end of the loop region between Beta Strands 2 and 3, relative to the wild-type form of the protein, and where the mutant GFP protein has one or more of the following characteristics: (a) enhanced emission intensity, relative to wild-type GFP protein from Renilla reniformis; (b) a narrower or broader emission spectrum, relative to wild-type GFP protein from Renilla reniformis; and (c) a shift in excitation or emission maxima, relative to wild-type GFP protein from Renilla reniformis; (d) a shift in maturation rate, relative to wild-type GFP protein from Renilla reniformis; and (e) exhibiting less quenching of fluorescence at acidic pH, relative to wild-type GFP protein from Renilla reniformis.

The invention also features a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, where the mutation includes an amino acid substitution in the region of the protean extending from the beginning of Beta Strand 4 through the end of Beta Strand 6, relative to the wild-type form of the protein, and where the mutant GFP protein has one or more of the following characteristics: (a) enhanced emission intensity, relative to wild-type GFP protein from Renilla reniformis; (b) a narrower or broader emission spectrum, relative to wild-type GFP protein from Renilla reniformis; and (c) a shift in excitation or emission maxima, relative to wild-type GFP protein from Renilla reniformis; (d) a shift in maturation rate, relative to wild-type GFP protein from Renilla reniformis; and (e) exhibiting less quenching of fluorescence at acidic pH, relative to wild-type GFP protein from Renilla reniformis.

The invention also features a polynucleotide encoding the mutant Renilla reniformis Green Fluorescent Proteins (GFPs) as described above. The polynucleotide can be humanized. The polynucleotide can be in a vector, and the vector can be contained in a host cell.

The invention also features a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, selected from the group consisting of: (a) the amino acid sequence of mutant GM1; (b) the amino acid sequence of mutant GM2; (c) the amino acid sequence of mutant GM3; (d) the amino acid sequence of mutant GM4; (e) the amino acid sequence of mutant GM6; (f) the amino acid sequence of mutant T1; (g) the amino acid sequence of mutant T6; (h) the amino acid sequence of mutant T8; (i) the amino acid sequence of mutant T11; (j) the amino acid sequence of mutant T12; (k) the amino acid sequence of mutant T13; (1) the amino acid sequence of mutant T14; (m) the amino acid sequence of mutant T15; and (n) the amino acid sequence of mutant T17. The amino acid substitutions making up these mutants are described herein.

The invention also features a polynucleotide encoding a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, selected from the group consisting of: (a) a polynucleotide encoding the amino acid sequence of mutant GM1; (b) a polynucleotide encoding the amino acid sequence of mutant GM2; (c) a polynucleotide encoding the amino acid sequence of mutant GM3; (d) a polynucleotide encoding the amino acid sequence of mutant GM4; (e) a polynucleotide encoding the amino acid sequence of mutant GM6; (f) a polynucleotide encoding the amino acid sequence of mutant T1; (g) a polynucleotide encoding the amino acid sequence of mutant T6; (h) a polynucleotide encoding the amino acid sequence of mutant T8; (i) a polynucleotide encoding the amino acid sequence of mutant T11; (j) a polynucleotide encoding the amino acid sequence of mutant T12; (k) a polynucleotide encoding the amino acid sequence of mutant T13; (l) a polynucleotide encoding the amino acid sequence of mutant T14; (m) a polynucleotide encoding the amino acid sequence of mutant T15; and (n) a polynucleotide encoding the amino acid sequence of mutant T17. The polynucleotide can be humanized. The polynucleotide can be in a vector, and the vector can be contained in a host cell.

In an additional aspect, the invention features a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:34; (b) the amino acid sequence of SEQ ID NO:36; (c) the amino acid sequence of SEQ ID NO:38; (d) the amino acid sequence of SEQ ID NO:40; (e) the amino acid sequence of SEQ ID NO:42; (f) the amino acid sequence of SEQ ID NO:44; (g) the amino acid sequence of SEQ ID NO:46; (h) the amino acid sequence of SEQ ID NO:48; (i) the amino acid sequence of SEQ ID NO:50; (j) the amino acid sequence of SEQ ID NO:52; (k) the amino acid sequence of SEQ ID NO:54; (l) the amino acid sequence of SEQ ID NO:56; (m) the amino acid sequence of SEQ ID NO:58; and (n) the amino acid sequence of SEQ ID NO:60.

The invention also features a polynucleotide encoding a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, selected from the group consisting of: (a) the polynucleotide sequence of SEQ ID NO:33; (b) the polynucleotide sequence of SEQ ID NO:35; (c) the polynucleotide sequence of SEQ ID NO:37; (d) the polynucleotide sequence of SEQ ID NO:39; (e) the polynucleotide sequence of SEQ ID NO:41; (f) the polynucleotide sequence of SEQ ID NO:43; (g) the polynucleotide sequence of SEQ ID NO:45; (h) the polynucleotide sequence of SEQ ID NO:47; (i) the polynucleotide sequence of SEQ ID NO:49; (j) the polynucleotide sequence of SEQ ID NO:51; (k) the polynucleotide sequence of SEQ ID NO:53; (l) the polynucleotide sequence of SEQ ID NO:55; (m) the polynucleotide sequence of SEQ ID NO:57; and (n) the polynucleotide sequence of SEQ ID NO:59. The polynucleotide can be humanized. The polynucleotide can be in a vector, and the vector can be contained in a host cell.

The invention features a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:4; (b) the amino acid sequence of SEQ ID NO:6; (c) the amino acid sequence of SEQ ID NO:8; (d) the amino acid sequence of SEQ ID NO:10; (e) the amino acid sequence of SEQ ID NO:12; (f) the amino acid sequence of SEQ ID NO:14; (g) the amino acid sequence of SEQ ID NO:16; (h) the amino acid sequence of SEQ ID NO:18; (i) the amino acid sequence of SEQ ID NO:20; (j) the amino acid sequence of SEQ ID NO:22; (k) the amino acid sequence of SEQ ID NO:24; (l) the amino acid sequence of SEQ ID NO:26; (m) the amino acid sequence of SEQ ID NO:28; and (n) the amino acid sequence of SEQ ID NO:30.

Another feature of the invention is a polynucleotide encoding a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, selected from the group consisting of: (a) the polynucleotide sequence of SEQ ID NO:3; (b) the polynucleotide sequence of SEQ ID NO:5; (c) the polynucleotide sequence of SEQ ID NO:7; (d) the polynucleotide sequence of SEQ ID NO:9; (e) the polynucleotide sequence of SEQ ID NO:11; (f) the polynucleotide sequence of SEQ ID NO:13; (g) the polynucleotide sequence of SEQ ID NO:15; (h) the polynucleotide sequence of SEQ ID NO:17; (i) the polynucleotide sequence of SEQ ID NO:19; (j) the polynucleotide sequence of SEQ ID NO:21; (k) the polynucleotide sequence of SEQ ID NO:23; (l) the polynucleotide sequence of SEQ ID NO:25; (m) the polynucleotide sequence of SEQ ID NO:27; and (n) the polynucleotide sequence of SEQ ID NO:29. The polynucleotide can be in a vector, and the vector can be contained in a host cell.

The invention also features a method of producing mutant Renilla reniformis GFP, including the steps of: (a) culturing a cell containing a recombinant vector including a wild type or humanized polynucleotide sequence encoding mutant Renilla reniformis GFP under conditions where the mutant Renilla reniformis GFP protein is expressed; and (b) isolating the mutant Renilla reniformis GFP protein from the cell; thereby producing mutant Renilla reniformis GFP.

In another aspect, the invention features a method of producing a Renilla reniformis fusion protein, the method including the steps of: culturing a cell containing a polynucleotide sequence encoding the polypeptide of interest linked with a humanized polynucleotide encoding mutant Renilla reniformis GFP wherein the linked polynucleotide sequences are fused in frame, under conditions where the mutant Renilla reniformis GFP protein is expressed. A method of determining the location of a polypeptide of interest in a cell can use the production method described above.

An additional feature of the invention is a method of identifying a cell into which a recombinant vector has been introduced, the method including the steps of: (a) providing a cell containing a recombinant vector including a humanized polynucleotide which encodes mutant Renilla reniformis GFP, wherein the cell permits expression of the humanized polynucleotide; (b) illuminating the population with light within the excitation spectrum of mutant Renilla reniformis GFP; and (c) detecting fluorescence in the emission spectrum of mutant Renilla reniformis GFP in the population, where detection of fluorescence in the cell indicates that the recombinant vector has been introduced into the cell; thereby identifying a cell into which the recombinant vector has been introduced. In these methods, the GFP can be expressed as a fusion polypeptide, or a distinct polypeptide. The cells can be identified by FACS analysis.

Another feature of the invention is a method of detecting the activity of a transcriptional regulatory sequence, the method including the steps of: (a) culturing a cell containing a nucleic acid sequence including the transcriptional regulatory sequence operably linked to a humanized nucleic acid sequence encoding mutant Renilla reniformis GFP to form a reporter construct, under conditions where the mutant Renilla reniformis GFP is expressed; and (b) detecting mutant Renilla reniformis GFP fluorescence in the cell, wherein detection of fluorescence indicates activity of the transcriptional regulatory sequence; thereby detecting the activity of a transcriptional regulatory sequence.

The invention also features a method of detecting the presence of a modulator of a transcriptional regulatory sequence, the method including the steps of: (a) culturing a cell containing a nucleic acid sequence including the transcriptional regulatory sequence operably linked to a humanized nucleic acid sequence encoding mutant Renilla reniformis GFP to form a reporter construct, under conditions where the mutant Renilla reniformis GFP is expressed; and (b) detecting mutant Renilla reniformis GFP fluorescence in the cell, wherein the fluorescence indicates the presence of the modulator; thereby detecting the presence of a modulator of a transcriptional regulatory sequence.

The invention additionally features a method of screening for an inhibitor of a transcriptional regulatory sequence, the method including the steps of: (a) culturing a cell containing a nucleic acid sequence including the transcriptional regulatory sequence operably linked to a humanized nucleic acid sequence encoding mutant Renilla reniformis GFP to form a reporter construct, under conditions where the mutant Renilla reniformis GFP is expressed; (b) contacting the cell with a candidate inhibitor of the transcriptional regulatory sequence; and (c) detecting mutant Renilla reniformis GFP fluorescence in the cell, wherein a decrease in the fluorescence relative to that detected in the absence of the candidate inhibitor indicates that the candidate inhibitor inhibits the activity of the transcriptional regulatory sequence.

In another aspect, the invention features a method of producing a fluorescent molecular weight marker, the method including the steps of: (a) culturing a cell containing a humanized nucleic acid sequence encoding mutant Renilla reniformis GFP linked in frame to a nucleic acid sequence encoding a polypeptide of known relative molecular weight such that the linked molecules encode a fusion polypeptide, under conditions where the mutant Renilla reniformis GFP is expressed; (b) isolating the fusion polypeptide from the cell, wherein the fusion polypeptide is a relative molecular weight marker.

In the above methods, the cell can be a mammalian cell. The cell can also be a human cell. In the above methods, the mutant Renilla reniformis GFP can be selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:30. The nucleic acid sequence encoding mutant Renilla reniformis GFP can be selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29.

The invention additionally features a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, where the mutation comprises an amino acid substitution in one of the following regions of the protein, relative to the wild-type form of the protein: (a) the Beta Strand 4 region of the protein; (b) the loop region of the protein between Beta Strand 2 and Beta Strand 3; (c) the loop region of the protein between Beta Strand 5 and Beta Strand 6; (d) the region of the protein extending from the beginning of Beta Strand 1 through the end of the loop region between Beta Strands 2 and 3; and (e) the region of the protein extending from the beginning of Beta Strand 4 through the end of Beta Strand 6; and where the mutant GFP protein also has one or more of the following characteristics: (r) exhibiting less quenching over a broad pH range, relative to wild-type GFP protein from Renilla reniformis.; (s) exhibiting greater resistance to one or more of the following: proteases, solvents, detergents and chaotropic agents; and (t) exhibiting reduced tendency to oligomerize.

The invention also features a mutant Green Fluorescent Protein (GFP) from Renilla reniformis, wherein the mutation comprises an amino acid substitution at one or more of the following residues: (a) F43; (b) E120; (c) L101; and (d) Y103.

By "mutant GFP protein" is meant that the protein contains an amino acid substitution at one or more amino acid residues relative to the reference GFP protein, and that the resulting protein displays one or more of the following characteristics: (a) enhanced emission intensity, (b) a narrower emission spectrum, and/or (c) exhibiting less quenching of fluorescence at acidic pH, relative to the reference GFP protein. By "reference GFP protein" is meant the protein from which the mutant GFP was derived. For example, one can begin with a wild type GFP nucleic acid sequence, introduce one or more mutations that produce amino acid substitution(s), and produce a mutant GFP protein. One can also humanize the nucleic acid sequence encoding a GFP protein, and then introduce one or more mutations that produce amino acid substitution(s).

The mutant proteins as described herein also include those proteins that contain more than one of the amino acid substitutions as described here, or specific combinations of those amino acid substitutions, or one or more of those amino acid substitutions in combination with other amino acid substitutions. Some specific combinations of amino acid substitutions confer beneficial properties to the resulting mutant GFP. For instance, as shown herein, a mutant GFP containing the combination of E120G, F43L and R125H matures faster than wild type hrGFP at 37.degree. C., that is, it is brighter earlier at elevated incubation temperature.

The mutant proteins as described herein also include other amino acid substitutions made at the sites described herein.

The term "humanized GFP sequence" or "humanized mutant GFP sequence" refers to a polynucleotide coding sequence in which one or more codons of the polynucleotide have been altered to codons which are more preferred for expression in mammalian cells. Methods of humanizing proteins are well known in the art, and such a humanized GFP nucleic acid sequence is provided herein as SEQ ID NO:1. For example, in human genes the preferred codon for alanine is "GCC". The codon "GCG", which also codes for alanine, can therefore be changed to "GCC" to enhance expression of the overall protein in mammalian cells. Other codons can also be replaced, and preferred human codons and other changes to enhance protein expression in human and mammalian systems are discussed further below.

Preferably, the amount of fluorescent polypeptide expressed in a human cell from a humanized GFP polynucleotide sequence is at least two-fold greater, on either a mass or a fluorescence intensity scale per cell, than the amount expressed from an equal amount or number of copies of a wild type R. reniformis GFP polynucleotide.

As used herein, the term "humanized codon" means a codon, within a polynucleotide sequence encoding a non-human polypeptide, that has been changed to a codon that is more preferred for expression by human cells relative to that codon encoded by the non-human organism from which the non-human polypeptide is derived. Species-specific codon preferences stem in part from differences in the expression of tRNA molecules with the appropriate anticodon sequence. That is, one factor in the species-specific codon preference is the relationship between a codon and the amount of corresponding anticodon tRNA expressed.

By saying that a protein (e.g., a test protein, e.g., a mutant Renilla reniformis GFP) has "enhanced emission intensity", or "increased fluorescence intensity" or "increased brightness" relative to another protein (e.g., a reference GFP protein), means that the fluorescence intensity of the test protein is greater than that of the reference protein, that is, the mutant protein is "brighter" than the reference protein under a given set of conditions. Brightness is measured as the product of the molar extinction coefficient and quantum yield (see, e.g., the spectroscopic studies in Baird, G. S. et al., 2000, Proc. Natl. Acad. Sci. USA 97(22)11984 11989). For example, the brightness for wild-type A. victoria GFP is generally (9500)(0.8)=7600 units M.sup.-1 cm.sup.-1. For EGFP (Clontech, Palo Alto, Calif., USA), the brightness is (55000)(0.6)=40600 units M.sup.-1 cm.sup.-1.

For spectral analysis with pure proteins, the spectral analysis is performed as described in Example 4, below, using quantitated purified proteins. The fluorescence intensity divided by the amount of protein is calculated, and the values compared between those of hrGFP and the mutant protein. A mutant protein with greater than 1-fold higher value over the wild type hrGFP is considered "brighter".

The cells expressing the various wild-type and mutant proteins can also be assayed by FACS analysis, and the mean value calculated for each, as described in Example 7, below. A mutant protein with greater than 1-fold higher value over the wild type hrGFP is considered, "brighter".

Preferably, the fluorescence intensity of the test protein is at least twice that of the wild-type GFP protein (i.e., 15200), more preferably, at least three times (i.e., 22800), and most preferably, at least four times (i.e., 30400) that of the wild type GFP protein.

By saying that a protein (e.g., a test protein, e.g., a mutant Renilla reniformis GFP) has "narrower emission spectrum" relative to another protein (e.g., a reference protein, e.g., wild-type Renilla reniformis GFP), means that the emission spectrum of the test protein is narrower than that of the reference protein, that is, that the spectrum for the test has narrower shoulders than the spectrum for the reference protein. "Narrower shoulders" refers to the wavelength maximum .+-.75 nm, preferably the wavelength maximum .+-.50 nm, and most preferably the wavelength maximum .+-.25 nm.

By saying that a protein (e.g., a test protein, e.g., a mutant Renilla reniformis GFP) "exhibits less quenching of fluorescence at acidic pH" relative to another protein (e.g., a reference protein, e.g., wild-type Renilla reniformis GFP), means that, under a given set of acidic conditions, the fluorescence intensity of the test protein exhibits less of a decrease than that of the reference protein. By saying that a protein (e.g., a test protein, e.g., a mutant Renilla reniformis GFP) "exhibits less quenching over a broad pH range" relative to another protein (e.g., a reference protein, e.g., wild-type Renilla reniformis GFP), means that, as the pH of the test protein's immediate environment deviates from neutral, the fluorescence intensity of the test protein exhibits less of a decrease than that of the reference protein. "Less quenching" in this regard means that a decrease in fluorescence intensity of 100% would be completely quenched, a decrease of 50% would be less quenced, a decrease of 10% would beneven less quenched, and most preferably, a decrease of 0% would be no quenching. Preferably, such a protein exhibits less quenching over a broad pH range, maintaining its general intensity over a more broad pH range relative to the wild-type hrGFP.

The mutant proteins as described herein can also exhibit greater resistance to proteases (e.g., proteinase K, trypsin, chymotrypsin, papain, pronase), solvents (e.g., ethanol, methanol, acetonitride), detergents (e.g., SDS, Tween 20, Trition X-100), and/or chaotropic agents (e.g., 8M urea, 4M guanidine HCl). By "exhibits greater resistance" to these agents, it is meant that the protein tends to function more normally relative to the reference protein under those same conditions, e.g., preferably there is no substantial decrease in intensity of the protein, or change in excitation or emission maxima.

The mutant proteins as described herein can also show reduced tendency to oligomerize, that is, a monomer being more preferred than a dimer, which would be more preferred than a trimer), as determined by analytical gradient ultracentrifugation and native protein gels.

The mutant protein can also exhibit a shift in in vivo maturation time relative to the wild-type version of the protein, as determined by examination of transfected cells by fluorescence microscopy. Maturation at 36 hours post-transfection is preferred, maturation at 24 hours post-transfection is more preferred, and maturation at 12 hours or less post-transfection is most preferred.

The term "variant thereof" when used in reference to an R. reniformis GFP means that the amino acid sequence bears one or more residue differences relative to the wild type R. reniformis GFP sequence and has at least the same, preferably improved (as described herein) biological activity (fluorescence intensity) of the wild type polypeptide.

As used herein, the term "increased fluorescence intensity" or "increased brightness" refers to fluorescence intensity or brightness that is greater than that exhibited by wild-type R. reniformis GFP under a given set of conditions. Generally, an increase in fluorescence intensity or brightness means that fluorescence of a variant is at least 5% or more, and preferably 10%, 20%, 50%, 75%, 100% or more, up to even 5 times, 10 times, 20 times, 50 times or 100 times or more intense or bright than wild-type R. reniformis GFP under a given set of conditions.

Assays can also be performed to determine color shift of the mutant proteins. A spectral analysis can be performed (e.g., as described in Example 4, below). Bacterial colonies expressing the hrGFP proteins and the mutant proteins can be observed with filters and various lens combinations (e.g., as described in Example 2, below), to determine the different color emitted by the mutant protein. Mammalian cells expressing the hrGFP proteins and the mutant proteins can be observed under a fluorescent microscope equipped with different fluorescent filter cubes (Omega Optical) to determine if the mutant emits a different color relative to the green of standard hrGFP (e.g., SEQ ID NO:2). If the emission maximum for a given mutant protein is 21 nm or greater than the emission spectrum of the wild type hrGFP, then the mutant protein is color-shifted to the red side of the spectrum. If the emission maximum for a given mutant protein is 29 nm or less than the emission spectrum of the wild type hrGFP, then the mutant protein is color-shifted to the blue side of the spectrum.

As used herein, the term "fused heterologous polypeptide domain" refers to an amino acid sequence of two or more amino acids fused in frame to R. reniformis GFP. A fused heterologous domain may be linked to the N or C terminus of the R. reniformis GFP polypeptide.

As used herein, the term "fused to the amino-terminal end" refers to the linkage of a polypeptide sequence to the amino terminus of another polypeptide. The linkage may be direct or may be mediated by a short (e.g., about 2 20 amino acids) linker peptide.

As used herein, the term "fused to the carboxy-terminal end" refers to the linkage of a polypeptide sequence to the carboxyl terminus of another polypeptide. The linkage may be direct or may be mediated by a linker peptide.

As used herein, the term "linker sequence" refers to a short (e.g., about 1 20 amino acids) sequence of amino acids that is not part of the sequence of either of two polypeptides being joined. A linker sequence is attached on its amino-terminal end to one polypeptide or polypeptide domain and on its carboxyl-terminal end to another polypeptide or polypeptide domain.

As used herein, the term "excitation spectrum" refers to the wavelength or wavelengths of light that, when absorbed by a fluorescent polypeptide molecule of the invention, causes fluorescent emission by that molecule.

As used herein, the term "emission spectrum" refers to the wavelength or wavelengths of light emitted by a fluorescent polypeptide.

As used herein, the terms "distinguishable" or "detectably distinct" mean that standard filter sets allow either the excitation of one form of a polypeptide without excitation of another given polypeptide, or similarly, that standard filter sets allow the distinction of the emission from one polypeptide form from the emission spectrum of another. Generally, distinguishable or detectably distinct excitation or emission spectra have peaks that vary by more than 1 nm, and preferably vary by more than 2, 3, 4, 5, 10 or more nm.

As used herein, the term "fusion polypeptide" refers to a polypeptide that is comprised of two or more amino acid sequences, from two or more proteins that are not found linked in nature, that are physically linked by a peptide bond. As used herein, only one protein which comprises a "fusion polypeptide" of the present invention is a fluorescent protein.

As used herein, the term "emission spectrum overlaps the excitation spectrum" means that light emitted by one fluorescent polypeptide is of a wavelength or wavelengths that causes excitation and emission by another fluorescent polypeptide.

As used herein, the term "population of cells" refers to a plurality of cells, preferably, but not necessarily of same type or strain.

As used herein, the term "FACS analysis" refers to the method of sorting cells, fluorescence activated cell sorting, wherein cells are stained with or express one or more fluorescent markers. In this method, cells are passed through an apparatus that excites and detects fluorescence from the marker(s). Upon detection of fluorescence in a given portion of the spectrum by a cell, the FACS apparatus allows the separation of that cell from those not expressing that fluorescence spectrum.

As used herein, the term "operably linked" means that a given coding sequence is joined to a given transcriptional regulatory sequence such that transcription of the coding sequence occurs and is regulated by the regulatory sequence.

As used herein, the term "reporter construct" refers to a polynucleotide construct encoding a detectable molecule, linked to a transcriptional regulatory sequence conferring regulated transcription upon the polynucleotide encoding the detectable molecule. A detectable molecule is preferably an R. reniformis GFP.

As used herein, the term "responsive to the presence of a modulator" means that a given transcriptional regulatory sequence is either turned on or turned off in the presence of a given compound. As used herein, gene expression is "turned on" when the polypeptide encoded by the gene sequence (e.g., a GFP polypeptide) is detectable over background, or alternatively, when the polypeptide is detectable in an increased amount over the amount detected in the absence of a given modulator compound. In this context, "increased amount" means at least 10%, preferably 20%, 50%, 75%, 100% or more, up to even 5 times, 10 times, 20 times, 50 times, or 100 times or more higher than background detection, with background detection being the amount of signal observed in the absence of the modulator compound.

As used herein, the term "modulator of a transcriptional regulatory sequence" refers to a compound or chemical moiety that causes a change in the level of expression from a transcriptional regulatory sequence. Preferably, the change is detectable as an increase or decrease in the detection of a reporter molecule or reporter molecule activity, with at least 10%, 20%, 50%, 75%, 100%, or even 5 times, 10 times, 20 times, 50 times or 100 times or more increased or decreased level of reporter signal relative to the absence of a given modulator.

As used herein the term "inhibitor of a transcriptional regulatory sequence" refers to a compound or chemical moiety that causes a decrease in the amount of a reporter molecule or reporter molecule activity expressed from a given transcriptional regulatory sequence. As used herein, the term "decrease" when used in reference to the detection of a reporter mole