Modified green fluorescent proteins and methods for using same Number:7,417,131 from the United States Patent and Trademark Office (PTO) owispatent The present invention provides nucleic acid molecules encoding mutant fluorescent proteins as well as proteins encoded by these nucleic acids. In addition, host-cells, stable cell lines and transgenic organisms comprising the above-referenced nucleic acid molecules are provided. The subject protein and nucleic acid compositions find use in a variety of different applications and methods, particularly for labeling of biomolecules, cells, or cell organelles.<H fluorescent, proteins, Modified, green, , 7417131.html, Patent, pto, 7417131

Title: Modified green fluorescent proteins and methods for using same

Abstract: The present invention provides nucleic acid molecules encoding mutant fluorescent proteins as well as proteins encoded by these nucleic acids. In addition, host-cells, stable cell lines and transgenic organisms comprising the above-referenced nucleic acid molecules are provided. The subject protein and nucleic acid compositions find use in a variety of different applications and methods, particularly for labeling of biomolecules, cells, or cell organelles.

Patent Number: 7,417,131 Issued on 08/26/2008 to Lukyanov

Inventors: Lukyanov; Sergey A. (Moscow, RU)
Assignee: Evrogen Joint Stock Company (Moscow, RU)

Appl. No.: 11/580,348

Filed: October 13, 2006

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
60733429	Nov., 2005

Current U.S. Class: 536/23.1 ; 435/252.3; 435/320.1; 435/69.1

Current International Class: C12P 21/06 (20060101); C07H 21/02 (20060101); C12N 15/00 (20060101); C12N 9/20 (20060101)

Field of Search: 530/350 435/69.1,252.3,320.1,320.5 536/23.2,23.1

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Primary Examiner: Monshipouri; Maryam
Attorney, Agent or Firm: Patterson & Sheridan, L.L.P.

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. provisional patent application Ser. No. 60/733,429, filed Nov. 4, 2005, which is herein incorporated by reference.

Claims

What is claimed is:

1. An isolated nucleic acid, comprising: a nucleic acid sequence encoding a genetically engineered fluorescent protein that is substantially identical to and has at least 97% identity with the amino acid sequence selected from the group consisting of SEQ ID NO:18.

2. The nucleic acid of claim 1, wherein the amino acid sequence of the genetically engineered fluorescent protein comprises one or more amino acid substitutions selected from the group consisting of K3G, E6D, T9A, P58T, F99L, F99H, M128K, M128E, I136M, Y151H, N144S, K162E, K156M, T214A, G228C, G228S, and K238R.

3. The nucleic acid of claim 1, wherein the genetically engineered fluorescent protein has an amino acid sequence that is selected from the group consisting of SEQ ID NO:18.

4. A vector comprising the nucleic acid of claim 2.

5. A vector comprising the nucleic acid of claim 1.

6. A vector comprising the nucleic acid of claim 3.

7. An expression cassette comprising: (a) a transcriptional initiation region that is functional in an expression host; (b) the nucleic acid according to claim 2; and (c) and a transcriptional termination region functional in said expression host.

8. An expression cassette comprising: (a) a transcriptional initiation region that is functional in an expression host; (b) the nucleic acid according to claim 1; and (c) a transcriptional termination region functional in said expression host.

9. An expression cassette comprising: (a) a transcriptional initiation region that is functional in an expression host; (b) the nucleic acid according to claim 3; and (c) and a transcriptional termination region functional in said expression host.

10. An host cell or progeny thereof, comprising the expression cassette according to claim 7 as part of an extrachromosomal element or integrated into the genome of a host cell as a result of introduction of said expression cassette into said host cell.

11. An host cell or progeny thereof, comprising the expression cassette according to claim 8 as part of an extrachromosomal element or integrated into the genome of a host cell as a result of introduction of said expression cassette into said host cell.

12. An host cell or progeny thereof, comprising the expression cassette according to claim 9 as part of an extrachromosomal element or integrated into the genome of a host cell as a result of introduction of said expression cassette into said host cell.

13. A transgenic cell, or progeny thereof, comprising the nucleic acid according to claim 2.

14. A transgenic cell, or progeny thereof, comprising the nucleic acid according to claim 1.

15. A transgenic cell, or progeny thereof, comprising the nucleic acid according to claim 3.

16. A kit comprising at least one nucleic acid according to claim 2.

17. An isolated nucleic acid, comprising: a nucleic acid sequence encoding a genetically engineered fluorescent protein with the amino acid sequence of SEQ ID NO: 18.

Description

FIELD OF THE INVENTION

This invention relates generally to the field of biology and chemistry. More particularly, the invention is directed to fluorescent proteins.

BACKGROUND OF THE INVENTION

Green Fluorescent Protein (GFP) from the hydromedusa Aequorea victoria (synonym A. A.), described by Johnson et al. in J Cell Comp Physiol. (1962), 60:85-104, was found as a part of bioluminescent system of the jellyfish where GFP played the role of a secondary emitter transforming blue light from the photoprotein aequorin into green light.

cDNA encoding A. victoria GFP was cloned by Prasher et al. (Gene, 1992, V. 111 (2), pp. 229-233). It turned out that this gene can be heterologically expressed in practically any organism due to unique ability of GFP to form a fluorophore by itself (Chalfie et al., Gene (1992), 111(2):229-233). This finding opens broad perspectives for use of GFP in cell biology as a genetically encoded fluorescent label.

A great deal of research is being performed to improve the properties of GFP and to produce GFP reagents useful and optimized for a variety of research purposes. New versions of GFP have been developed, such as a "humanized" GFP DNA, the protein product of which has increased synthesis in mammalian cells (Haas, et al., Current Biology 1996, V. 6, pp. 315-324; Yang, et al., Nucleic Acids Research 1996, V. 24, pp. 4592-4593). One such humanized protein is "enhanced green fluorescent protein" (EGFP). Other mutations to GFP have resulted in blue-, cyan- and yellow-green light emitting versions. Also, GFP variants with improved folding and cellular fluorescence under incubation at 37.degree. C. have been obtained. Useful A. victoria GFP mutants are described in detail in U.S. Pat. Nos. 5,491,084, 5,625,048, 5,777,079, 5,804,387, 6,090,919, 5,874,304, 5,968,750, 6,020,192, 6,027,881, 6,046,925, 6,054,321, 6,066,476, 6,096,865, 6,146,826, 6,414,119, 6,638,732, 6,699,687, 6,803,188, 6,077,707, 6,124,128, 6,172,188, 6,818,443, 6,194,548, 6,265,548, 6,319,669, 6,403,374, 6,593,135, 6,800,733, 6,780,975, 6,852,849, and 6,919,186.

GFP homologs from different species including Anthozoa and Arthropoda were isolated (Matz et al., Nature Biotechnol. 1999, V. 17, pp. 969-973; Shagin et al., Mol Biol Evol. 2004, V. 21(5), pp. 841-850). A number of biological and biomedical applications of these proteins are discussed in detail by Lippincott-Schwartz and Patterson in Science, 2003, V. 300(5616), pp. 87-91. Also, close homologues of A. victoria GFP were isolated from other jellyfishes the of Aequorea genus including A. macrodactyla green fluorescent protein, GFPxm (Xia et al., Mar Biotechnol 2002, V. 4(2), pp. 155-62) and A. coerulescens GFP-like protein, AcGFPL (Gurskaya et al., Biochem J. (2003), 373(Pt 2): 403-408).

A. macrodactyla GFPxm shares 83% identity with A. victoria GFP. Wild type GFPxm is not useful as a fluorescent marker in cell-based assays because of a low maturation speed at 37.degree. C. Modification of GFPxm to optimize its maturation speed at temperatures of 35-39.degree. C. provide a means for detecting the reporter in mammalian cells at lower levels of expression and/or increased sensitivity relative to wild type GFPxm. This greatly improves the usefulness of the GFPxm in studying cellular functions in living cells.

SUMMARY OF THE INVENTION

This invention provides functional engineered fluorescent proteins with increased maturation speed at a temperature of 20.degree. C. or above compared to wild type A. macrodactyla green fluorescent protein (GFPxm), wherein said functional engineered fluorescent proteins are substantially identical to the amino acid sequence of A. macrodactyla green fluorescent protein (GFPxm) (SEQ ID NO:2) and comprise a F220L amino acid substitution.

In a preferred embodiment, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a functional fluorescent protein whose amino acid sequence is substantially similar to the amino acid sequence of A. macrodactyla green fluorescent protein (GFPxm) (SEQ ID NO:2) and differs from SEQ ID NO:2 by at least an amino acid substitution F220L. Said functional fluorescent protein has an increased maturation speed at a temperature of 20.degree. C. or above as compared with GFPxm.

In a preferred embodiment, a nucleic acid molecule of the present invention encodes a fluorescent protein that also comprises additional amino acid substitutions selected from the group consisting of K3G, E6D, T9A, P58T, F99L, F99H, M128K, M128E, I136M, Y151H, N144S, K162E, K156M, T214A, G228C, G228S, and K238R, wherein said functional fluorescent protein has increased maturation speed at a temperature of 20.degree. C. or above compared to wild-type A. macrodactyla GFPxm.

In preferred embodiments, a nucleic acid molecule of the present invention encodes a functional fluorescent protein that is substantially similar to the amino acid sequence of GFPxm and comprises additional one or more amino acid substitution(s) that alter its fluorescent properties and/or optimize folding, as shown for example in SEQ ID NOs: 18-24.

In another preferred embodiment, this invention provides a functional mutant fluorescent protein whose amino acid sequence is substantially similar to the amino acid sequence of A. macrodactyla GFPxm (SEQ ID NO:2) and which differs from SEQ ID NO:2 by at least an amino acid substitution F220L. Said functional mutant fluorescent protein has an improved maturation speed at a temperature of 20.degree. C. or above as compared with GFPxm. Examples of mutant fluorescent proteins having amino acid compositions selected from the group consisting of SEQ ID NOS 4-24 are also provided, wherein said mutant fluorescent proteins have an improved maturation speed at a temperature of 20.degree. C. or above as compared with GFPxm.

In yet other embodiments there are provided vectors comprising a nucleic acid of the present invention. In addition, the present invention provides an expression cassette comprising a nucleic acid of the present invention and regulatory elements necessary for expression of the nucleic acid in the cell.

Additionally, host cells, stable cell lines, transgenic animals and transgenic plants comprising nucleic acids, vectors or expression cassettes of the present invention are provided.

Additionally, kits comprising nucleic acids or vectors or expression cassettes harboring said nucleic acids, or protein of the present invention are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the normalized excitation (line 1) and emission (line 2) spectra of GFPxm fluorescent protein.

FIG. 2 illustrates the normalized excitation (line 1) and emission (line 2) spectra of Mut 2 fluorescent protein.

FIG. 3 illustrates the normalized excitation (line 1) and emission (line 2) spectra of Mut-g9 fluorescent protein.

FIG. 4 shows the relative brightness of E. coli colonies expressing GFPxm, Mut 2, or Mut-g9 fluorescent protein after growth at different temperatures. Temperature conditions and incubation time are indicated at the bottom of histogram. All data are normalized to the brightness of Mut-g9 expressing colonies after 36 hours growth at 20.degree. C.

FIG. 5 shows curves of fluorescence growth of E. coli colonies expressing GFPxm (line 1), Mut 2 (line 2), or Mut-g9 (line 3) during 6 hours after induction.

FIG. 6 illustrates the normalized excitation (line 1) and emission (line 2) spectra of tagGFP.

FIG. 7A illustrates the normalized excitation (line 1) and emission (line 2) spectra of tagCFP.

FIG. 7B illustrates the normalized excitation (line 1) and emission (line 2) spectra of tagYFP1.

DETAILED DESCRIPTION

As used herein the term "fluorescent protein" means a protein that is fluorescent; e.g., it may exhibit low, medium or intense fluorescence upon irradiation with light of the appropriate excitation wavelength. The fluorescent characteristic of fluorescent protein is one that arises from the fluorophore wherein the fluorophore results from autocatalytic cyclization of two or more amino acid residues in the polypeptide backbone. As such, the fluorescent proteins of the present invention do not include proteins that exhibit fluorescence only from residues that act by themselves as intrinsic fluors, i.e., tryptophan, tyrosine and phenylalanine.

As used herein, "fluorescent property" refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation spectrum or emission spectrum, the excitation wavelength maximum and emission wavelength maximum, the ratio of excitation amplitudes at two different wavelengths, the ratio of emission amplitudes at two different wavelengths, the excited state lifetime, or the fluorescence anisotropy. A measurable difference in any one of these properties between wild-type GFPxm and the mutant form is useful. A measurable difference can be determined as the amount of any quantitative fluorescent property, e.g., the amount of fluorescence at a particular wavelength, or the integral of fluorescence over the emission spectrum.

As used herein, "maturation rate" or "maturation speed" refers to the rate of mature fluorescent protein formation (i.e., a fluorescent protein capable of producing fluorescence) after translation. Maturation rate can be characterized with a half-time of maturation. It has been discovered that maturation of fluorescent protein includes two steps: (i) Protein folding that means formation of a protein beta-barrel with a central alpha-helix containing amino acids that will form chromophore. This step is commonly characterized with a rate constant of about 10.sup.(-2)s.sup.(-1) or half-time from several seconds to tens of seconds; (ii) Chromophore maturation, that is protein backbone cyclization and dehydration. This stage is commonly characterized with a rate constant of about 10.sup.(-4)s.sup.(-1) or half-time about several minutes. Therefore, this slower step is the limiting step in green fluorescent protein maturation (Reid B G, Flynn G C. Biochemistry. 1997 V. 36(22), PP. 6786-6791).

As used herein, the term "GFP" refers to the green fluorescent protein from A. victoria, including prior art versions of GFP engineered to provide greater fluorescence or fluoresce in different colors. The sequence of wild type GFP has been disclosed in Prasher et al., Gene 111 (1992), 229-33.

As used herein, the term "GFPxm" refers to the wild type green fluorescent protein from A. macrodactyla.

As used herein the term "isolated" means a molecule or a cell that is an environment different from that in which the molecule or the cell naturally occurs.

Reference to a nucleotide sequence "encoding" a polypeptide means that the sequence, upon transcription and translation of mRNA, produces the polypeptide. This includes both the coding strand, whose nucleotide sequence is identical to mRNA and whose sequence is usually provided in the sequence listing, as well as its complementary strand, which is used as the template for transcription. As any person skilled in the art recognizes, this also includes all degenerate nucleotide sequences encoding the same amino acid sequence. Nucleotide sequences encoding a polypeptide include sequences containing introns.

As used herein the term "mutant" refers to a protein disclosed in the present invention, in which one or more amino acids are added and/or substituted and/or deleted and/or inserted at the N-terminus, and/or the C-terminus, and/or within the native amino acid sequences of the proteins of the present invention. As used herein the term "mutant" refers to a nucleic acid molecule that encodes a mutant protein. Moreover, the term "mutant" refers to any shorter or longer version of the protein or nucleic acid herein.

As used herein, "homologue or homology" is a term used in the art to describe the relatedness of a nucleotide or peptide sequence to another nucleotide or peptide sequence, which is determined by the degree of identity and/or similarity between said sequences compared.

As used herein, an amino acid sequence or a nucleotide sequence is "substantially identical" to a reference sequence if the amino acid sequence or nucleotide sequence has at least 90% sequence identity (e.g. 90%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity) with the reference sequence over a given comparison window. As used herein, an amino acid sequence or a nucleotide sequence is "substantially similar" to a reference sequence if the amino acid sequence or nucleotide sequence has at least 80% sequence identity (e.g. 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity) with the reference sequence over a given comparison window. Sequence identity is calculated based on a reference sequence. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al., J. Mol. Biol., 215, pp. 403-10 (1990).

As summarized above the present invention is directed to nucleic acid molecules comprising nucleotide sequences that encode mutant fluorescent proteins, as well as proteins encoded by these nucleic acids. Proteins of interest are substantially identical to the wild type A. macrodactyla green fluorescent protein GFPxm (SEQ ID NO:2) and comprise at least an amino acid substitution F220L. Said mutants are functional fluorescent proteins having an improved maturation speed at a temperature of 20.degree. C. or above as compared with GFPxm.

In one embodiment, said mutant comprises only a F220L substitution. Inventors of the present invention have discovered that the F220L substitution results in measurable increase of maturation rate of the GFPxm at a temperature of 20.degree. C. or above as compared with wild-type GFPxm. Inventors of the present invention have further discovered that the F220L substitution alters fluorescent properties of the protein as compared with A. macrodactyla GFPxm.

In another preferred embodiment, said mutant also comprises additional amino acid substitutions that further increase maturation rate of the protein at a temperature of 20.degree. C. or above, e.g. mutant having amino acid sequence selected from the group consisting of SEQ ID NOS 6, 8, 10, 12, 14, 16, and 18 is provided.

Above-noted mutations in GFPxm may be combined with mutations that further increase folding, reduce oligomerization or influence the spectral properties of GFPxm and its mutants, as shown for example in SEQ ID NOs: 18-24.

In yet other embodiments there are provided vectors comprising a nucleic acid of the present invention. In addition, the present invention provides an expression cassette comprising a nucleic acid of the present invention and regulatory elements necessary for expression of the nucleic acid in the cell.

Also of interest are proteins and nucleic acids that are substantially similar to, or derivatives, or homologues, or mutants of, the above-referenced specific proteins and nucleic acids. In addition, host-cells, stable cell lines and transgenic organisms comprising above-referenced nucleic acid molecules are provided. The subject protein and nucleic acid compositions find use in a variety of different applications and methods, particularly cell and protein labeling applications. Finally, kits for use in such methods and applications are provided.

Nucleic Acid Molecules

The present invention provides nucleic acid molecules comprising nucleotide sequences that encode mutant fluorescent proteins that are substantially identical to the wild type A. macrodactyla green fluorescent protein GFPxm (SEQ ID NO:2) and comprise at least an amino acid substitution F220L.

A nucleic acid molecule as used herein is a DNA molecule, such as genomic DNA molecules or cDNA molecules, or an RNA molecule, such as mRNA molecules.

In particular, said nucleic acid molecules are DNA molecules comprising an open reading frame that encodes a fluorescent protein of the invention. The subject nucleic acids are present in an environment other than their natural environment; e.g., they are isolated, present in enriched amounts, or are present or expressed in vitro or in a cell or organism other than their naturally occurring environment. In a preferred embodiment, nucleic acid molecules of the present invention are engineered, i.e. obtained from a naturally occurring protein, e.g. wild type A. macrodactyla green fluorescent protein GFPxm, by means of modifications.

The modifications, as well as additions or deletions can be introduced by any method known in the art (see for example Gustin et al., Biotechniques (1993) 14: 22; Barany, Gene (1985) 37: 111-123; and Colicelli et al., Mol. Gen. Genet. (1985) 199:537-539, Sambrook et al., Molecular Cloning: A Laboratory Manual, (1989), CSH Press, pp. 15.3-15.108) including error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-directed mutagenesis, random mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a combination thereof. The modifications, additions or deletions may be also introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation or a combination thereof.

Specific nucleic acid molecules of interest comprise nucleotide sequences that encode following fluorescent proteins: Mut 2 (SEQ ID NO 4); Mut 235 (SEQ ID NO 6); Mut 235-1 (SEQ ID NO 8); Mut 235-2 (SEQ ID NO 10); Mut 235-4 (SEQ ID NO 12); Mut-g9 (SEQ ID NO 14); Mut 235-4G6 (SEQ ID NO 16). Also of interest are nucleic acid molecules comprising nucleic acid sequences that encode Mut-g9 mutants, tagGFP (also called macGFP, SEQ ID NO: 18), tagCFP (SEQ ID NO:20), tagYFP1 (SEQ ID NO: 22) and tagYFP2 (SEQ ID NO:24), wherein fluorescent properties of these mutants are altered as compared with Mut-g9 protein.

Examples of nucleotide sequences that encode the foregoing proteins are shown in SEQ ID NOS 3-23.

Each of these particular types of nucleic acid molecules of interest is discussed in greater detail individually in the "Examples" section infra.

Also provided are nucleic acids that hybridize to the above-described nucleic acids under stringent conditions, preferably under high stringency conditions (i.e., complements of the previously-described nucleic acids). An example of stringent conditions is hybridization at 50.degree. C. or higher and 0.1.times.SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another example of high stringency hybridization conditions is overnight incubation at 42.degree. C. in a solution of 50% formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5.times.Denhardt's solution, 10% destran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm DNA, followed by washing in 0.1.times.SSC at about 65.degree. C. Other high stringency hybridization conditions are known in the art and may also be used to identify nucleic acids of the invention.

In addition, degenerate variants of the nucleic acids that encode the proteins of the present invention are also provided. Degenerate variants of nucleic acids comprise replacements of the codons of the nucleic acid with other codons encoding the same amino acids. In particular, degenerate variants of the nucleic acids are generated to increase its expression in a host cell. In this embodiment, codons of the nucleic acid that are non-preferred or a less preferred in genes in the host cell are replaced with the codons over-represented in coding sequences in genes in the host cell, wherein said replaced codons encode the same amino acid. In a preferred embodiment, nucleic acids of the present invention are humanized. As used herein, the term "humanized" refers to changes made to the nucleic acid sequence to optimize the codons for expression of the protein in mammalian (human) cells (Yang et al., Nucleic Acids Research (1996) 24: 4592-4593). See also U.S. Pat. No. 5,795,737 which describes humanization of proteins.

The nucleic acids of the present invention, the corresponding cDNAs, full-length genes and constructs can be generated synthetically by a number of different protocols known to those of skill in the art. Appropriate nucleic acid constructs are purified using standard recombinant DNA techniques as described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd Ed., (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY, and under regulations described in, e.g., United States Dept. of HHS, National Institute of Health (NIH) Guidelines for Recombinant DNA Research.

It has been found that fluorescent proteins can be genetically fused to other target proteins and used as markers to identify the location and amount of the target protein produced. Accordingly, this invention provides nucleic acids encoding fusion proteins that comprise a fluorescent protein and additional amino acid sequences. Such sequences can be, for example, up to about 15, up to about 100, up to about 200 or up to about 1000 amino acids long. The fusion proteins possess the ability to fluoresce that is determined by a fluorescent protein portion.

Also provided are vector and other nucleic acid constructs comprising the subject nucleic acids. Suitable vectors include viral and non-viral vectors, plasmids, cosmids, phages, etc., preferably plasmids, and used for cloning, amplifying, expressing, transferring etc. of the nucleic acid sequence of the present invention in the appropriate host. The choice of appropriate vector is well within the skill of the art, and many such vectors are available commercially. To prepare the constructs, the partial or full-length nucleic acid is inserted into a vector typically by means of DNA ligase attachment to a cleaved restriction enzyme site in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination in vivo, typically by attaching regions of homology to the vector on the flanks of the desired nucleotide sequence. Regions of homology are added by ligation of oligonucleotides, or by polymerase chain reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence, for example.

Also provided are expression cassettes or systems used inter alia for the production of the subject fluorescent proteins or fusion proteins thereof or for replication of the subject nucleic acid molecules. The expression cassette may exist as an extrachromosomal element or may be integrated into the genome of the cell as a result of introduction of said expression cassette into the cell. For expression, the gene product encoded by the nucleic acid of the invention is expressed in any convenient expression system, including, for example, bacterial, yeast, insect, amphibian, or mammalian systems. In the expression vector, a subject nucleic acid is operably linked to a regulatory sequence that can include promoters, enhancers, terminators, operators, repressors and inducers. Methods for preparing expression cassettes or systems capable of expressing the desired product are known for a person skilled in the art.

Cell lines, which stably express the proteins of present invention, can be selected by the methods known in the art (e.g. the co-transfection with a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of the transfixed cells that contain the gene integrated into a genome).

The above-described expression systems may be used in prokaryotic or eukaryotic hosts. Host-cells such as E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or cells of a higher organism such as vertebrates, e.g., COS 7 cells, HEK 293, CHO, Xenopus oocytes, etc., may be used for production of the protein.

When any of the above-referenced host cells, or other appropriate host cells or organisms are used to replicate and/or express the nucleic acids of the invention, the resulting replicated nucleic acid, expressed protein or polypeptide is within the scope of the invention as a product of the host cell or organism. The product may be recovered by an appropriate means known in the art.

Proteins

Also provided by the subject invention are functional mutant fluorescent proteins whose amino acid sequences are substantially identical to the amino acid sequence of A. macrodactyla GFPxm (SEQ ID NO:2) and which differ from SEQ ID NO:2 by at least an amino acid substitution F220L. Said functional mutant fluorescent proteins have an improved maturation speed at a temperature of 20.degree. C. or above as compare with GFPxm.

In a preferred embodiment, a fluorescent protein of the present invention comprise only a F220L substitution as compared with SEQ ID NO:2 and has increased maturation rate as compared with A. macrodactyla GFPxm. In a preferred embodiment, this fluorescent protein also has altered fluorescent properties as compared with A. macrodactyla GFPxm.

In another preferred embodiment, the F220L substitution is combined with other mutations to improve the properties of the protein. For example, different combinations of amino acid substitutions selected from the group consisting of K3G, E6D, T9A, P58T, F99L, F99H, M128K, M128E, I136M, Y151H, N144S, K162E, K156M, T214A, G228C, G228S, and K238R further increase protein maturation speed at a temperature of 20.degree. C. or above as shown in the "Example" section.

In many embodiments, the subject proteins have an absorbance maximum ranging from about 300 to 700 nm, usually from about 350 to 650 nm and more usually from about 400 to 600 nm. The subject proteins are fluorescent proteins, by which is meant that they can be excited at one wavelength of light following which they will emit light at another wavelength. The excitation spectra of the subject proteins typically ranges from about 300 to 700 nm. The subject proteins generally have a maximum extinction coefficient that ranges from about 25,000 to 150,000 and usually from about 45,000 to 129,000. The subject proteins typically range in length from about 150 to 300 amino acids and usually from about 200 to 300 amino acid residues, and generally have a molecular weight ranging from about 15 to 35 kDa, usually from about 17.5 to 32.5 kDa.

In certain embodiments, the subject proteins are bright, where by bright is meant that the protein fluorescence can be detected by common methods (e.g., visual screening, spectrophotometry, spectrofluorometry, fluorescent microscopy, by FACS machines, etc.) Fluorescence brightness of particular fluorescent proteins is determined by its quantum yield multiplied by maximal extinction coefficient.

In certain embodiments, the subject proteins has an increased maturation speed at a temperature of 20.degree. C. or above as compared with GFPxm. Maturation speed can be estimated by the time required for proteins to achieve their tertiary structure that gives rise to their fluorescent quality in a certain period of time. In other words, maturation speed of a fluorescent protein can be estimated by fluorescence intensity of host cells expressing subject protein after certain period of time after host cell transfection with an expression construct capable of expressing said fluorescent protein.

In certain embodiments, the subject proteins have an increased maturation speed at a temperature of 20.degree. C. or above, preferably of 30.degree. C. or above, most preferably at a temperature ranging from 35.degree. C. to 39.degree. C., e.g. at 37.degree. C. It is well known that many cells, including mammalian cells, are incubated at approximately 37.degree. C. in order to secure optimal and/or physiologically relevant growth. Cell lines originating from different organisms or tissues may have different relevant temperatures ranging from about 35.degree. C. for fibroblasts to about 38.degree. C.-39.degree. C. for mouse beta-cells.

For example, to compare the maturation speeds of fluorescent proteins at different temperatures, the following approach can be used: host cells (e.g. bacterial cells, preferably E. coli cells) are transfected with an expression vector encoding a fluorescent protein under the control of a suitable promoter. In a certain embodiment, fluorescent protein expression starts up immediately after transfection (when a constitutive promoter is used, or due to the leakage of an inducible promoter). In another embodiment, fluorescent protein expression is induced by the method well-known in the art. Host cells and grown on petri dish at 20, 30 or 37.degree. C. for certain periods of time (e.g., 36, 24 and 12 hours after start of fluorescent protein expression) fluorescence of E. coli colonies is detected by the common methods (e.g., visual screening, spectrophotometry, spectrofluorometry, fluorescent microscopy, by FACS machines, etc.) and brightness of its fluorescence is calculated.

Specific proteins of interest are mutant green fluorescent proteins: Mut 2 (SEQ ID NO 4); Mut 235 (SEQ ID NO 6); Mut 235-1 (SEQ ID NO 8); Mut 235-2 (SEQ ID NO 10); Mut 235-4 (SEQ ID NO 12); Mut-g9 (SEQ ID NO 14); and Mut 235-4G6 (SEQ ID NO 16). Specific proteins of interest have a maturation speed at a temperature of 20.degree. C. or above higher than GFPxm protein.

Specific proteins of interest are discussed in greater detail individually in the "Examples" section infra.

Proteins that are substantially similar or substantially identical to the specific amino acid sequences of the subject invention, i.e., SEQ ID NOs: 4-16 are also provided. Sequence identity is calculated based on a reference sequence as determined using MegAlign, DNAstar clustal algorithm as described in D. G. Higgins and P. M. Sharp, "Fast and Sensitive multiple Sequence Alignments on a Microcomputer," CABIOS, 5 pp. 151-3 (1989) (using parameters ktuple 1, gap penalty 3, window 5 and diagonals saved 5). In many embodiments, amino acid sequences of interest have much higher sequence identity e.g., 93%, 95%, 97%, 99%, 100%, particularly for the sequence of the amino acids that provide the functional regions of the protein.

Proteins that are mutants of the above-described proteins are also provided. Mutants may retain biological properties of the source proteins, or may have biological properties which differ from the wild type proteins. The term "biological property" of the proteins of the present invention refers to, but is not limited to, fluorescent properties; biochemical properties, such as in vivo and/or in vitro stability (e.g., half-life); maturation speed, aggregation tendency and oligomerization tendency and other such properties. Mutations include single amino acid changes, deletions or insertions of one or more amino acids, N-terminal truncations or extensions, C-terminal truncations or extensions and the like.

Mutants can be generated using standard techniques of molecular biology as described in details in the section "Nucleic acid molecules" above. Mutants described herein includes. (1) a mutant of the Mut-g9 with enhanced fluorescent properties comprising substitutions I167T, F223S, S65C, and F64L as compared with Mut-g9 (SEQ ID NO:14). Said mutant also possesses increased maturation speed as compared with GFPxm and Mut-9 proteins. The amino acid sequence of this mutant named tagGFP (also macGFP) is shown in SEQ ID NO: 18; (2) a mutant of the tagGFP with cyan-shift in fluorescence spectra that comprises C65A, Y66W, L99H, I123V, K128E, D129G, F145A, N146I, H148D, V163A, T167I, T203C, T205S, C227Y substitutions as compared with tagGFP. The amino acid sequence of this mutant named tagCFP is shown in SEQ ID NO: 20; (3) a mutant of the tagGFP with yellow-shift in fluorescence spectra that comprises C65T, 168V, E76K, M153T, F224V, C228S and T203Y substitutions as compared with tagGFP. The amino acid sequence of this mutant named tagYFP is shown in SEQ ID NO: 22.

Given the guidance provided in the Examples, and using standard techniques, those skilled in the art can readily generate a wide variety of additional mutants and test whether a biological (e.g. biochemical, spectral, etc.) property has been altered. For example, fluorescence intensity can be measured using a spectrophotometer at various excitation wavelengths.

The proteins of the present invention are present in the isolated form, by which is meant that the protein is substantially free of other proteins and other naturally-occurring biological molecules, such as oligosaccharides, nucleic acids and fragments thereof, and the like, where the term "substantially free" in this instance means that less than 70%, usually less than 60% and more usually less than 50% of the composition containing the isolated protein is some other natural occurring biological molecule. In certain embodiments, the proteins are present in substantially purified form, where by "substantially purified form" means at least 95%, usually at least 97% and more usually at least 99% pure.

In a preferred embodiment, the subject proteins are synthetically produced, e.g. by expressing a recombinant nucleic acid coding sequence encoding the protein of interest in a suitable host, as described above. Any convenient protein purification procedures may be employed, where suitable protein purification methodologies are described in the Guide to Protein Purification, (Deuthser ed.) (Academic Press, 1990). For example, a lysate may be prepared from the original source and purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, and the like.

Also provided are fusion proteins comprising a protein of the present invention, or functional fragments thereof, fused, for example, to a degradation sequence, a sequence of subcellular localization (e.g. nuclear localization signal, peroximal targeting signal, Golgi apparatus targeting sequence, mitochondrial targeting sequence, etc.), a signal peptide, or any protein or polypeptide of interest. Fusion proteins may comprise for example, a fluorescent protein of subject invention and a second polypeptide ("the fusion partner") fused in-frame at the N-terminus and/or C-terminus of the fluorescent protein. Fusion partners include, but are not limited to, polypeptides that can bind antibodies specific to the fusion partner (e.g., epitope tags), antibodies or binding fragments thereof, polypeptides that provide a catalytic function or induce a cellular response, ligands or receptors or mimetics thereof, and the like.

Also provided are antibodies that bind specifically to the fluorescent proteins of the present invention. Suitable antibodies may be produced using the techniques known in the art. For example, polyclonal antibodies may be obtained as described in (Harlow and Lane Antibodies: A Laboratory Manual, (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) and monoclonal antibodies may be obtained as described in (Goding Monoclonal Antibodies: Principles and Pr