RU2338785C2 - Fluorescing proteins and chromoproteins from kinds hydrozoa which are not concerning to aequorea, and methods of their obtaining - Google Patents

Abstract

FIELD: biology, gene engineering.

SUBSTANCE: invention can be used for marking of biological objects. The molecule of nucleic acid which codes the fluorescing protein chosen from fluorescing proteins of representatives of kind Phialidium sp. are both suborder Anthomedusae and fluorescing mutants of the specified proteins allocated. By means of the allocated nucleic acid are obtained cloning and expressing vectors, fluorescing protein, the protein of merge capable to fluorescence, and also the expressing cartridge. The cell and the stable cellular line, containing such express ionic cartridge, produce fluorescing fiber. The fluorescing protein, nucleic acid coding it and the express ionic genetic designs containing this nucleic acid, use in a set for marking of a biological molecule. Fluorescent protein is also used in methods of marking of a biological molecule, a cell or a cellular organella.

EFFECT: invention application allows dilating an arsenal of agents for marking of biological objects.

13 cl, 12 dwg,12 ex

Description

Field of Invention

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

State of the art

The introduction of labels into proteins, cells or organisms of interest plays an important role in many biochemical, molecular biological and medical diagnostic applications. Many different labels have been developed and used in this field, including labeling with radioisotopes, dyes, fluorescent labels, chemiluminescent labels, etc., with various properties and optimal applications. However, there is ongoing interest in developing new tags. Of particular interest is the development of new protein tags, including fluorescent protein tags.

Green fluorescent protein (GFP), its mutants and homologs, widely known today due to their intensive use as in vivo fluorescent markers in biomedicine, are discussed in detail by Lippincott-Schwartz and Patterson in Science (2003) 300 (5616): 87-91). GFP from Aequorea aequorea hydromedusa (synonym for A. victoria), discovered by Johnson et ai. at J Cell Comp Physiol. (1962), 60: 85-104, was discovered as part of a bioluminescent jellyfish system, where GFP played the role of a secondary source that converts blue light from an equorin photo protein to green light. Similar proteins were then isolated from several bioluminescent coelenterates, including the hydroid jellyfish Phialidium gregarium , marine feathers Renilla (class Anthozoa) and others (see Ward et al. In Photochem. Photobiol. (1982), 35: 803-808; Levine et al . in Comp. Biochem. Physiol. (1982), 72B: 77-85; Chalfie in Photochem. Photobiol. (1995), 62: 651-656). All these proteins exhibit green fuorescence, (radiation at 497-509 nm) and act as secondary sources in bioluminescence. Fluorescent proteins were also isolated from Physalia species and their N-terminal amino acid sequences were determined. (WO 03/017937).

cDNA encoding A. victoria GFP was cloned by Prasher et al. (Gene (1992), 111 (2): 229-33). It turned out that this gene can be heterologically expressed in almost any organism due to the unique ability of GFP to independently form a fluorophore (Chalfie et al., Gene (1992), 111 (2): 229-233). This information opens up wide prospects for the use of GFP in cell biology as a genetically encoded fluorescent label.

GFP has been widely used in various applications, including the study of gene expression and protein localization (Chalfie et al., Science 263 (1994), 802-805, and Heim et al. In Proc. Nat Acad. Sci. (1994), 91: 12501-12504), as a means for visualizing the intracellular distribution of organelles in cells (Rizzuto et al, Curr. Biology (1995), 5: 635-642), for visualizing protein transfer along the secretory pathway (Kaether and Gerdes, FEES Letters (1995) 369: 267-271).

Many studies have been conducted to improve the properties of GFP and to produce GFP reagents suitable and optimized for a number of research purposes. New GFP variants have been developed, such as humanized GFP DNA, whose protein product has increased synthesis in mammalian cells (Haas, et al., Current Biology (1996), 6: 315-324; Yang, et al., Nucleic Acids Research (1996), 24: 4592-4593). One such humanized protein is an "enhanced green fluorescent protein" (EGFP). Other mutations to GFP have led to variants emitting blue, blue-green and yellow-green light. However, despite the great usefulness of GFP, other fluorescent proteins with properties similar or different from GFP would be useful in this area. In particular, the benefits of the new fluorescent proteins include the possibility of fluorescence resonant energy transfer (FRET), the capabilities of which are based on new spectra and better suitability for a wide excitation spectrum. In 1999 GFP godu homologs were cloned from non-bioluminescent species Anthozoa (Matz et al, Nature Biotechnol ( 1999) 17:.. 969-973). This discovery showed that these proteins are not necessary components of the bioluminescent mechanism. Extracted from Anthozoa GFP-like proteins showed a large spectral diversity, including blue-green, green, yellow, red fluorescent proteins and violet-blue non-fluorescent chromoproteins (CPs) (Matz et al., Bioessays (2002), 24 (10): 953 -959).

A major drawback of GFP-like proteins recovered from Anthozoa is the strong oligomerization that prevents the use of these proteins in many applications (Lauf et al., FEBS Lett. (2001), 498: 11-15; Campbell et al., Proc. Natl. Acad. Sci. USA (2002), 99: 7877-7882; Mizuno et al., Biochemistry (2001), 40: 2502-2510). Accordingly, the goal is to provide new monomeric fluorescent proteins of various colors, as well as DNA encoding them, which do not have the disadvantages of the known GFP.

Hydrozoa is a potential source of such proteins. In addition to GFP from Aequorea victoria and GFP homologs from other Aequorea species, similar to very close GFP homologs from Aequorea macrodactyla (GenBank, accession numbers AF435427-AF435433) and Aequorea coerulescens (Gurskaya et al., Biochem J. (2003), 373 ): 403-408), no other genes encoding fluorescent proteins from Hydrozoa have been cloned to date, although some of them have been characterized at a protein level for a very long time. Cloning and mutagenesis of fluorescent proteins from sources other than Aequorea Hydrozoa is a promising way to produce new fluorescent labels with improved properties.

SUMMARY OF THE INVENTION

The present invention provides nucleic acid molecules encoding fluorescent or chromoproteins and their mutants, and their derivatives. The specified nucleic acid can be isolated, synthesized or present in its artificial environment.

In some embodiments, the nucleic acid of the present invention is isolated from a species not related to Aequorea Hydrozoa, including Phialidium sp. and two fluorescent jellyfish or hydroid jellyfish 1 and 2 (hydromedusa 1 and 2) of the suborder Anthomedusae, or mutants or their derivatives.

In some embodiments, the nucleic acid of the present invention encodes a protein that has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22. In some embodiments the nucleic acid encodes a homolog, mutant, derivative compound, mimetic or fragment of the specified protein.

In some embodiments, the nucleic acid of the present invention has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21, or which is homologous, essentially same or identical to those indicated. Nucleic acid sequences that differ from the presented nucleic acid sequences due to degeneracy of the genetic code or hybridizable with them are also included in the scope of the present invention.

In other embodiments, the invention is directed to proteins that are encoded by the claimed nucleic acids or essentially similar to them, or homologs, derivatives or their mutants, or directed to fusion proteins, including the proteins of the present invention.

Also provided are fragments of nucleic acids of the present invention and nucleic acids that hybridize under high stringency conditions with the nucleic acids of the present invention.

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

In yet another embodiment, methods are provided for producing a chromogenic and / or fluorescent protein, comprising expressing a protein in an appropriate host cell and isolating a protein from it. Said method comprises (a) providing a nucleic acid molecule of the present invention encoding a fluorescent or chromo protein coupled to appropriate expression control sequences, (b) expressing a protein from said nucleic acid molecule, and (c) isolating a protein substantially free of other proteins.

In addition, antibodies specific for the proteins of the present invention or fragments thereof are provided.

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

In yet another embodiment, oligonucleotides or probes are provided comprising nucleotide sequences capable of hybridization with the claimed nucleic acids.

Methods are also provided that utilize the chromo- or fluorescent protein of the present invention or a nucleic acid encoding it.

In a preferred embodiment, a method is provided for introducing labels into a biological molecule, wherein said method comprises binding said biological molecule to a protein of the present invention.

In another preferred embodiment, a method for introducing labels into a cell is provided, wherein said method comprises producing a protein of the present invention in a cell.

In another preferred embodiment, there is provided a method for introducing labels into an organelle of a cell, wherein said method comprises producing a protein of the present invention fused to a suitable intracellular localization signal in the cell.

In yet another preferred embodiment, a method for analyzing a biological molecule, cell or cell organelle is provided, wherein said method comprises detecting a fluorescent signal from a protein of the present invention.

In another preferred embodiment, there is provided a method for analyzing a biological molecule, cell or organelle of a cell, wherein said method comprises expressing a nucleic acid molecule of the present invention in a cell.

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

Brief Description of the Figures

Figure 1 illustrates the alignment of GFP, phiYFP, hydr1GFP and hm2CP amino acid sequences. Entered gaps are indicated by dots. Residues identical to the corresponding amino acids in GFP are represented by dashed lines.

Figure 2 shows the excitation spectra (dashed line) and radiation (solid line) for wild-type phiYFP (A) and its mutants: phiYFP-Y1 (B), phiYFP-M0 (C), and phiYFP-M1 (D).

FIG. 3 shows excitation-emission spectra for phiYFP-M1G1 (A) and phiYFP-M1C1 (B) proteins.

Figure 4 is a sketch of hydromedusa 1 (A) and hydromedusa 2 (B) of the suborder Anthomedusae .

5 shows excitation-emission spectra for wild-type hydr1GFP.

6 shows an absorption spectrum for wild-type hm2CP.

7 shows excitation-emission spectra for wild-type hm2CP.

Fig. 8 shows excitation-emission spectra for the red fluorescent S3-2 mutant of the hm2CP protein.

DETAILED DESCRIPTION OF THE INVENTION

As used here, the term "fluorescent protein" or "fluoroprotein" means a protein that is fluorescent; for example, it may exhibit low, medium, or intense fluorescence when exposed to light with a suitable wavelength for excitation. The fluorescent property of these proteins is a property that results from the interaction of two or more amino acid residues of a protein, and not a single amino acid residue. Also, the fluorescent proteins of the present invention do not include proteins that exhibit fluorescence only from residues that fluoresce themselves as internal fluorophores, i.e. tryptophan, tyrosine and phenylalanine.

As used here, the term "chromoprotein" or "chromogenic protein" means a colored protein, which may be fluorescent, weakly or non-fluorescent. As used here, the terms “chromoprotein” and “fluorescent protein” do not include luciferase, such as Renilla luciferase.

As used herein, the term “GFP” refers to the green fluorescent protein from Aequorea victoria, including GFP variants known in the art, designed to provide greater fluorescence or fluorescence in other color regions. The wild-type sequence of GFP was disclosed in Prasher et al., Gene 111 (1992), 229-33.

As used here, the term "EGFP" refers to a mutated variant of GFP having two amino acid substitutions: F64L and S65T (Heim et al., Nature 373 (1995), 663-664).

As used here, the term "isolated" means a molecule or cell that is in an environment other than the environment in which the molecule or cell is in vivo.

As used here, the term "fragment" is intended to include, for example, an alternatively spliced or truncated, or otherwise cleaved, nucleic acid or protein molecule.

As used here, the term "derivative" refers to a mutant or modified due to RNA, or a chemically modified or otherwise modified nucleic acid molecule or to a mutant, or a chemically modified or otherwise modified protein.

As used here, 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 removed (deleted) and / or inserted (inserted) at the N-terminus and / or C -end, and / or within the native amino acid sequences of the proteins of the present invention.

As used here, the term "mutant" refers to a nucleic acid molecule that encodes a mutant protein. In addition, the term “mutant” refers to any variant that is shorter or longer than a protein or nucleic acid.

As used herein, “homologue or homology” is a term used in the art to describe the linkage 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 compared sequences.

To summarize the above, the present invention is directed to nucleic acid molecules encoding fluorescent and chromoproteins and mutants, variants and their derivatives, as well as proteins and peptides encoded by these nucleic acids. Nucleic acid molecules and proteins of interest are isolated from non- Aequorea Hydrozoa species. Target proteins of interest include the yellow fluorescent protein, phiYFP, from Phialidium sp. , Green fluorescent protein hydr1GFP from cnidarian jellyfish 1 (hydromedusa 1) of the suborder Anthomedusae, and purple chromoprotein, hm2CP from cnidarian jellyfish 2 (hydromedusa 2) of the suborder Anthomedusae. Also, proteins of interest are those that are essentially similar or derivatives, or homologs, or mutants of the aforementioned specific proteins. Also provided are nucleic acid fragments and peptides encoded by them, as well as antibodies specific for the proteins and peptides of the invention. In addition, host cells, stable cell lines, and transgenic organisms including the aforementioned nucleic acid molecules are provided. The claimed protein and nucleic acid compositions find use in a number of different applications and methods, in particular for use in tagging proteins. As a result, kits are provided for use in such methods and applications.

Nucleic acid molecules

The present invention provides nucleic acid molecules encoding fluorescent / chromoproteins from a species of Hydrozoa other than the genus Aequorea, derivatives, mutants and homologues of these proteins, as well as fragments thereof. The nucleic acid molecule, as used here, is a DNA molecule, such as genomic DNA molecules or cDNA molecules, or RNA molecules, such as mRNA molecules. In particular, these nucleic acid molecules are cDNA molecules having an open reading frame that encodes a chromo / fluorescent protein of the invention from Hydrozoa or a fragment thereof and is capable, under suitable conditions, of providing expression as a fluorescent / chromoprotein or protein fragment ( peptide) according to the invention.

The invention also encompasses nucleic acids that are homologous, substantially similar, identical, derivatives or mimetics of nucleic acids encoding the proteins or protein fragments of the present invention. The claimed nucleic acids are present in an environment different from their natural environment; for example, they are isolated, present in enriched amounts, or are present or expressed in vitro either in a cell or in an organism other than their naturally occurring environment.

Specific nucleic acid molecules of interest are those that encode the following Hydrozoa chromo / fluoroproteins (and their homologs / derivatives / mutants): yellow fluorescent protein, phiYFP from Phialidium sp. , green fluorescent protein, hydr1GFP from hydroid jellyfish 1 from the suborder Anthomedusae , and purple chromoprotein, hm2CP from hydroid jellyfish 2 from the suborder Anthomedusae . Each of these specific types of nucleic acid molecules of interest is now individually disclosed in more detail.

phiYFP

Nucleic acid molecules encoding fluorescent / chromoproteins can be isolated from the Hydrozoa class organism, preferably the Hydroida order, more preferably the Leptomedusae suborder, more preferably from the Campanulariidae family, and even more preferably from the Phialidium genus. In a particularly preferred embodiment, the nucleic acid molecule isolated from Phialidium sp. encodes a specific protein called PhiYFP. Homologs / mutants / derivatives of this protein, such as phiYFP-Y1, phiYFP-M1, phiYFP-M0, phiYFP-M1G1 (i.e. phiYFP-G1 or phiGFPl), and phiYFP-MlCl (i.e. phiYFP-G1 or phiGFP1) described below in more detail in the experimental part, also of interest. The deduced wild-type cDNA coding sequence for PhiYFP is shown in SEQ ID NO: 01.

hydr1GFP

Nucleic acid molecules encoding fluorescent / chromoproteins can be isolated from the body of the Hydrozoa class, preferably the Hydroida order, more preferably the Anthomedusae suborder. A specific protein encoded by such a nucleic acid molecule is called hydr1GFP (i.e. anmGFP1). Homologs / mutants / derivatives of this protein are also of interest. The deduced wild-type cDNA coding sequence for hydr1GFP is shown in SEQ ID NO: 11.

hm2CP

Nucleic acid molecules encoding fluorescent / chromoproteins can be isolated from the body of the Hydrozoa class, preferably the Hydroida order, more preferably the Anthomedusae suborder. A specific protein encoded by such a nucleic acid molecule is called hm2CP (i.e., anm2CP). Homologs / mutants of this protein, such as the S3-2 red fluorescent mutant hm2CP, described below in more detail in the experimental part, are also of interest. The deduced wild-type cDNA coding sequence for hm2CP is shown in SEQ ID NO: 13.

Homologs of the above nucleic acid molecules are also of interest. The source of homologous nucleic acids may be any kind of plant or animal, or the sequence may be fully or partially synthetic, including nucleic acid mimetics. In some embodiments, the nucleic acid of the present invention has a sequence identity with corresponding homologues at nucleotide or amino acid levels of at least about 40% and, preferably, about 50%, 55%, 60%, 65%, 70% or more, including 75%, 80 %, 85%, 90% and 95% or more. The reference sequence will usually be at least about 60 nucleotides in size, more often at least about 80 nucleotides in size, and can extend to the entire sequence with which it is being compared. The sequence identity is calculated based on the 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) (for example, using the default settings, that is, with the parameters w = 4 and T = 17).

Homologists are identified in any of a variety of ways. A cDNA fragment of the present invention can be used as a hybridization probe against a cDNA library from a target organism using low stringency conditions. The probe may be a large fragment or one or more short degenerate primers. Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50 ° C and 6 × SSC (0.9 M sodium chloride / 0.09 M sodium citrate), followed by washing at 55 × C in 1 × SSC (01 15 M sodium chloride / 0.015 M sodium citrate). The sequence identity can be determined by hybridization under conditions of high stringency, for example, at 50 ° C. or higher and 0.1 × SSC (15 mM sodium chloride / 1.5 mM sodium citrate). Nucleic acids having a region substantially identical to the presented sequences, for example allelic variants, genetically modified nucleic acid variants, etc., bind to the presented sequences under conditions of high stringency of hybridization. Using probes, in particular labeled probes of DNA sequences, homologous or similar genes can be isolated.

Nucleic acids are also provided that hybridize with the above nucleic acids under stringent conditions, preferably under high stringency conditions (i.e., complementary to the previously described nucleic acids). An example of hybridization under stringent conditions is at 50 ° C. or higher and 0.1 × SSC (15 mM sodium chloride / 1.5 mM sodium citrate). Another example of hybridization under high stringency conditions is overnight incubation at 42 ° C in a 50% solution of formamide, 5 × SSC (150 mm NaCl, 15 mm trisodium citrate), 50 mm sodium phosphate (pH 7.6), 5 × solution Denhardt, 10% dextran sulfate, and 20 μg / ml denatured, DNA-cut salmon seminal fluid, pre-washed with 0.1 × SSC at approximately 65 ° C. Other high stringency hybridization conditions are known in the art and can also be used to determine the nucleic acids of the invention.

Nucleic acids encoding variants, mutants or derivatives of the proteins of the invention are also provided. Mutants or derivatives can be obtained on a matrix nucleic acid selected from the above nucleic acids by modifying, deletion or addition of one or more nucleotides in a matrix sequence or a combination thereof, to obtain a variant of the matrix nucleic acid. Modifications, additions or deletions can be performed 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, rearrangement, oligonucleotide- directed mutagenesis, assembly PCR, pairwise PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential multiple mutagenesis, site-specific mutagenesis, nonspecific mutagenesis, gene reassay mating, gene site-saturating mutagenesis (GSSM), artificial reassembly with ligation (SLR), or combinations thereof. Modifications, additions or deletions can also be carried out by a method including recombination, recursive sequence recombination, phosphotioate-modified DNA mutagenesis, uracil-containing mutagenesis, double-pass mutagenesis, mismatch-specific rediscover mutagenesis, mutagenesis of a recovery-deficient chemical mutagenesis, radioactive mutagenesis, deletion mutagenesis, restriction-selective mutagenesis, restriction mutagenesis with purification, synthesis with artificial genes, multiple mutagenesis, creation of multiple chimeric nucleic acids, and combinations thereof. In some embodiments, the fluorescent proteins encoded by the mutant or nucleic acid derivatives have the same fluorescent properties as the wild-type fluorescent protein. In other embodiments, the mutant or derivatives of nucleic acids encode fluorescent proteins with altered spectral properties, as described in more detail for phiYFP-Y1, phiYFP-M1, phiYFP-M1G1, phiYFP-M1C1, S3-2 mutants.

In addition, degenerate nucleic acid variants that encode the proteins of the present invention are also provided. Degenerate nucleic acid variants include substitutions of nucleic acid codons with other codons encoding the same amino acids. In particular, degenerate nucleic acid variants are created to increase expression in the host cell. In this embodiment, nucleic acid codons that are not preferred or less preferred in the genes in the host cell are replaced by codons that are overly represented in the coding sequences in the genes in the host cell, where said substituted codons encode the same amino acid. Of particular interest is the humanized version of the nucleic acids of the present invention. As used herein, the term “humanized” refers to substitutions made in a nucleic acid sequence to optimize codons for protein expression in mammalian cells (Yang et al., Nucleic Acids Research (1996) 24: 4592-4593). See also US Patent No. 5795737, which describes the humanization of proteins, the disclosure of which is incorporated herein by reference.

The term "cDNA", as used here, is intended to include nucleic acids that reflect the arrangement of sequence elements located in the native mature variety of mRNA, where sequence elements are exons and 5- 'and 3'-non-coding regions. Typically, a mRNA species has adjacent exons, with intermediate introns, which, if present, are removed by nuclear RNA splicing to create continuous encoding of the open reading frame protein.

The genomic sequence of interest may include a nucleic acid present between the initiation codon and the termination codon, as defined in the listed sequences, including all introns that are usually present on the native chromosome. The genomic sequence of interest may further include 5'- and 3'-untranslated regions located in mature mRNA, as well as specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including flanking genomic DNA approximately 1 kb, but possibly larger, at the 5 ′ or 3 ′ end of the transcribed region.

The nucleic acid molecules of the invention can encode all or part of the claimed proteins. Double and single chain fragments can be obtained from a DNA sequence by chemical synthesis of oligonucleotides in accordance with standard methods, restriction enzyme digestion, PCR amplification, etc. Basically, the DNA fragments will have a size of at least about 15 nucleotides, usually at least about 18 nucleotides or about 25 nucleotides, and can be at least about 50 nucleotides. In some embodiments, the claimed nucleic acid molecules may have a size of approximately 100, approximately 200, approximately 300, approximately 400, approximately 500, approximately 600, approximately 700 nucleotides or more. The claimed nucleic acids may encode fragments of the claimed proteins or complete proteins; for example, the claimed nucleic acids can encode polypeptides of approximately 25 amino acids, approximately 50, approximately 75, approximately 100, approximately 125, approximately 150, approximately 200 amino acids up to the full length of the protein.

The claimed nucleic acids can be isolated and obtained essentially in purified form. Essentially purified form means that the nucleic acids are at least about 50% pure, usually at least about 90% pure and are usually "recombinant", that is, flanked by one or more nucleotides that it does not normally bind to a chromosome found naturally in its natural host organism.

Nucleic acids of the present invention, for example, having the sequence of SEQ ID NO: 01, 03, 05, 07, 09, 11, 13, 15, 17, 19, or 21, corresponding cDNAs, genes of full length and design, can be obtained artificially in accordance with many different techniques known to those skilled in the art. Suitable nucleic acid constructs are purified using standard recombinant DNA methods as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ^nd Ed., (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY, and according to the instructions described, for example, in the United States Dept. of HHS, National Institute of Health (NIH) Guidelines for Recombinant DNA Research.

Also provided are nucleic acids that encode fusion proteins comprising the protein of the present invention, or fragments thereof, which are discussed in more detail below.

Also provided are a vector and other nucleic acid constructs containing the claimed nucleic acids. Suitable vectors include viral and non-viral vectors, plasmids, cosmids, phages, etc., preferably plasmids, and are used for cloning, amplification, expression, transfer, etc., of the nucleic acid sequence of the present invention in a suitable host. The selection of a suitable vector is understandable to a person skilled in the art, and many such vectors are commercially available. To prepare the construct, a partial or full length nucleic acid is usually inserted into the vector by attaching a DNA ligase to a restriction enzyme cleaved site in a vector. Alternatively, the desired nucleotide sequence can be inserted by in vivo homologous recombination, usually by attaching homologous regions to a vector on the flanks of the desired nucleotide sequence. Homologous regions are added by ligation of oligonucleotides or by polymerase chain reaction using primers including, for example, both homologous regions and part of the desired nucleotide sequence.

Also provided are expression cassettes or systems used inter alia to produce the claimed chromogenic or fluorescent proteins or their insertion proteins, or to replicate the claimed nucleic acid molecules. The expression cassette may exist as an extrachromosomal element or may be incorporated into the genome of a cell as a result of introducing 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 systems, yeast, insect, amphibian or mammal. In the expression vector, said nucleic acid is operably linked to a regulatory sequence, which may include promoters, enhancers, terminators, operators, repressors and inducers. Methods for preparing an expression cassette or systems capable of expressing a desired product are known to those skilled in the art.

Cell lines that stably express the proteins of the present invention can be selected by methods known in the art (e.g., cotransfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin, which makes it possible to identify and isolate transfected cells that contain the gene, included in the genome).

The above expression systems can be used in prokaryotic or eukaryotic hosts. To obtain the protein, 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, for example COS 7 cells, HEK 293, CHO, Xenopus oocytes, etc.

If any of the aforementioned host cells or other suitable 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 isolated by a suitable method known in the art.

Also of interest are the promoter sequences of the genomic sequences of the present invention, where the sequence of the 5'-terminal regions can be used for promoter elements, including enhancer binding sites, which, for example, provide for regulation of expression in cells / tissues where the gene of the claimed proteins is expressed.

Also provided are small DNA fragments of the claimed nucleic acids, which are used as primers for PCR, hybridization of screening samples, etc. Large fragments of DNA are used to obtain encoded polypeptides, as previously described. However, for use in geometric amplification reactions, such as geometric PCR, a pair of small DNA fragments, i.e. primers, are used. The exact composition of the primer sequences is not critical to the invention, but for most applications, the primers will hybridize to the claimed sequence under stringent conditions, as is known in the art. It is preferable to select a pair of primers that will give an amplification product of at least about 50 nucleotides, preferably at least about 100 nucleotides, and can extend to the entire nucleic acid sequence. Primer sequence selection algorithms are commonly known, and are available in commercial software packages. Amplification primers hybridize to complementary strands of DNA and will be primed towards each other.

The nucleic acid molecules of the present invention can also be used to determine gene expression in a biological sample. A method in which cells are examined for the presence of specific nucleotide sequences, such as genomic DNA or RNA, is well established in the art. Briefly, DNA or mRNA is isolated from a cell sample. mRNA can be amplified by RT-PCR using reverse transcriptase to form a complementary DNA strand, with a preliminary polymerase amplification chain reaction, using primers specific for the claimed DNA sequences. Alternatively, the mRNA sample is separated by gel electrophoresis, transferred to a suitable carrier, such as nitrocellulose, nylon, etc., and then probed with a fragment of the claimed DNA as a probe. Other methods may also be used, such as oligonucleotide crosslinking assays, in situ hybridization, and hybridization with DNA probes attached to a solid chip. Detection of mRNA hybridized with the claimed sequence is indicative of gene expression in the sample.

The claimed nucleic acids, including flanking regions of the promoter and coding regions, can be mutated by various methods known in the art to obtain targeted changes in the strength of the promoter or to change the sequence of the encoded protein or the properties of the encoded protein, including the fluorescent properties of the encoded protein.

In many embodiments, nucleic acids found in the form of Aequorea are not included in the scope of the invention. In some embodiments, the GFP homolog and nucleic acids encoding it from Aequorea victoria, Aequorea macrodactyla , and Aequorea coeridscens are not included in the scope of the claimed invention.

Squirrels

Also provided in accordance with the claimed invention are chromo- and fluorescent proteins from other species not related to Aequorea Hydrozoa, and their mutants, including full-length proteins, as well as parts or fragments thereof. Variations of a naturally occurring protein are also provided, where such variations are homologous or substantially similar to a naturally occurring protein and mutants that are naturally occurring proteins, as described in a more detailed description below.

In many embodiments, the claimed proteins have a maximum absorption in the range of from about 300 to 700, usually from about 350 to 650, and more often from about 400 to 600 nm. If the claimed proteins are fluorescent proteins, which means that they can be excited by light of one wavelength, after which they emit light of a different wavelength, while the excitation spectra of the declared proteins are usually from about 300 to 700 nm. The claimed proteins generally have a maximum extinction coefficient, which is from about 25,000 to 150,000 and usually from about 45,000 to 129,000. The size of the declared proteins is usually in the range of from about 150 to 300 amino acids and usually from about 200 to 300 amino acid residues and in general in the case, they have a molecular weight in the range of about 15 to 35 kDa, usually about 17.5 to 32.5 kDa.

In some embodiments, the claimed proteins are bright, where bright means that chromo- and fluorescent proteins can be detected by conventional methods (e.g., visual screening, spectrophotometry, spectrofluorimetry, fluorescence microscopy, FACS devices, etc.). Fluorescence specific fluorescent proteins is determined by their quantum yield multiplied by the maximum extinction coefficient. The brightness of chromoproteins can be expressed by its maximum extinction coefficient.

In some embodiments, the claimed proteins are inserted rapidly after expression in a host cell. By fast folding, it is understood that the proteins reach their tertiary structure, which ensures their chromium or fluorescent property, in a short period of time. In these embodiments, proteins are stacked over a period of time that generally does not exceed about 3 days, usually does not exceed about 2 days, and more often does not exceed about 1 day.

Specific proteins of interest are chromo / fluoroproteinami (and homologs, mutants, and derivatives thereof) from a species not related to Aequorea Hydrozoa: phiYFP from Phialidium sp, green protein fluoresces, hydr1GFP from cnidarian jellyfish 1 (hydromedusa 1) of the suborder Anthomedusae. and purple chromoprotein, hm2CP from hydroid jellyfish 2 (hydromedusa 2) of the suborder Anthomedusae . Each of these specific types of polypeptide structures of interest will hereinafter be examined individually in more detail.

phiYFP (and its derivatives / mutants)

The proteins of this embodiment have a maximum light absorption in the range of from about 350 to 550, usually from about 450 to 550, and often from about 435 to 540 nm, for example, from 515 to 530 nm, or from 480 to 490, while the maximum radiation is usually in a range from about 400 nm to 650 nm, and more often from about 450 to 600 nm, while in many embodiments, the emission spectra are in the range from about 470 to 550 nm, for example, from 505 to 515 or from 520 to 530 nm, or from 530 up to 540 nm. The size of the claimed proteins is typically from about 200 to 250, usually from about 210 to 240 amino acid residues, and generally have a molecular weight in the range of from about 20 to 30, usually from about 22.50 to 27.50 kDa. Of particular interest in many embodiments is phiYFP, which has the amino acid sequence shown in SEQ ID NO: 02. Mutants and derivatives of this sequence, for example, phiYFP-Y1, phiYFP-M1, phiYFP-M0, phiYFP-M1G1 and phiYFP- are also of interest. M1C1, as presented in SEQ ID No: 04, 06, 08, 18 and 20, respectively.

hydr1GFP (and its derivatives / mutants)

In many embodiments, the claimed proteins have a maximum light absorption in the range of from about 400 to 600 and more often from about 450 to 550 nm, and often from about 460 to 500 nm, for example, from 470 to 480 nm, while the emission spectra of the claimed proteins are usually found in the range of from about 450 to 650, usually from about 460 to 600 nm, and more often from about 480 to 550 nm, for example, from 480 to 500 nm and sometimes from 490 to 500 nm. The size of the declared proteins is usually in the range of from about 200 to 300 amino acids and usually from about 220 to 290 amino acid residues, and generally have a molecular weight in the range of about 25 to 35 kDa, usually about 26.5 to 32.5 kDa . Of particular interest in many embodiments is the wild type hydr1GFP fluorescent protein, which has the amino acid sequence shown in SEQ ID No: 12, mutants and its derivatives.

hm2CP (and its mutants)

In many embodiments, the claimed proteins have a maximum light absorption in the range of about 350 to 650, usually about 450 to 600, and more often, about 490 to 595 nm, for example, 560 to 590 nm, while the absorption spectra of the claimed proteins are usually in the range of about from 450 to 650, usually from about 500 to 640 nm, and more often from about 580 to 620 nm, for example, from 590 to 620 nm. The size of the declared proteins is usually in the range of about 200 to 250, usually about 210 to 240 amino acid residues, and in general they have a molecular weight in the range of about 20 to 30 kDa, usually about 22.50 to 27.50 kDa . Specific of interest in many embodiments is hm2CP (anm2CP), which has the amino acid sequence shown in SEQ ID No: 14. Also, a mutant of this sequence, for example, red fluorescent S3-2 protein, and the like, is presented as shown for example in SEQ ID NO: 16.

Homologs or proteins are also provided whose sequence differs from the above specific amino acid sequences claimed in the invention, i.e. SEQ ID NO: 02, 04, 06, 08, 10, 12, 14, 16, 18, 20 or 22. Under the homologue it is understood a protein having at least about 55%, usually at least about 60% and more often at least about 65% amino acid sequence identity with respect to the amino acid sequences of SEQ ID NO: 02, 04, 06, 08, 10, 12, 14, 16, 18, 20 or 22 as defined using By using MegAlign, the 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 the parameters: ktuple 1, gap penalty 3, window 5 and diagonals saved 5). In many embodiments, the homologues of interest have a much higher sequence identity, e.g. 70%, 75%, 80%, 85%, 90% (e.g. 92%, 93%, 94%) or higher, e.g. 95%, 96 %, 97%, 98%, 99%, 99.5%, especially for the sequence of amino acids that provide functional areas of the protein.

Also provided are proteins that are substantially identical to a wild-type protein, where substantially identical means that the protein has an amino acid sequence identity with the wild-type protein sequence of at least about 60%, usually at least about 65%, and more often at least at least about 70%, and in some samples, the identity may be much higher, for example 75%, 80%, 85%, 90%, 95% or higher.

Also provided are proteins that are derivatives or mutants of the above proteins found in nature. Mutants and derivatives may retain the biological properties of wild-type proteins (eg, those found in nature), or may have biological properties that are different from wild-type proteins. The term “biological property” of the proteins of the present invention refers, but is not limited to spectral properties, such as absorption maximum, radiation maximum, maximum extinction coefficient, brightness (for example, as compared to a wild-type protein or other reference protein, such as green fluorescent protein (GFP) from A.victoria), and the like; biochemical properties such as in vivo and / or in vitro stability (e.g., half-life); ripening rate, a tendency to aggregation and a tendency to oligomerization and other such properties. Mutations include substitutions of one amino acid, deletion or insertion of one or more amino acids, truncations or extensions of the N-terminus, truncations of extensions of the C-terminus, and the like.

Mutants and derivatives can be obtained using standard methods of molecular biology, as described in detail in the section "nucleic acid molecules" above. Several mutants are described here. The examples provide general techniques and the use of standard methods, so that specialists qualified in this field can easily obtain a large number of additional mutants and check whether the biological (e.g. biochemical, spectral, etc.) property has been changed. For example, fluorescence intensity can be measured using a spectrophotometer at various excitation wavelengths.

Derivatives can also be obtained using standard methods and include changes using RNA, chemical modifications, modifications after translation and after transcription, etc. For example, derivatives can be prepared by methods such as altered phosphorylation or glycosylation, or acetylation, or lipidation, or by various types of cleavage upon maturation, and the like.

The proteins of the invention, which are naturally occurring proteins, are present in an environment that is not common to them, for example, they are separated from the normal environment. For example, a purified protein is provided where “purified” means that the protein is present in a mixture that is substantially free of non-chromogenic or fluorescent proteins of interest, where “substantially free” means that less than 90%, usually less than 60% and more often less than 50% of the contents of the mixture are non-chromogenic or fluorescent proteins or their mutants. The proteins of the present invention may also be present in isolated form, which means that the protein is essentially free of other proteins and other biological molecules found in a natural environment, such as oligosaccharides, nucleic acids and fragments thereof, and the like, where the term “essentially free” in this case means that less than 70%, usually less than 60% and more often less than 50% of the composition containing the isolated protein is some other biological molecule found in nature. In some embodiments, the proteins are present in a substantially purified form, where “substantially purified form” means purified at least 95%, usually at least 97%, and more often at least 99%.

Fragments of proteins found in nature are also provided, as are the mutants and derivatives of the proteins described above. Biologically active fragments and / or fragments corresponding to functional domains, etc. are of private interest. Fragments of interest are polypeptides that typically have a size of at least about 30 amino acids, usually at least about 50 amino acids, preferably at least about 75 or 100 amino acids, and can be 300 amino acids or more, but usually will not exceed a size of approximately 250 amino acids, where the fragment will have a section of amino acids that is the same as the declared protein, at least about 25 amino acids in size, and usually at least ere about 45 amino acids, and at least about 50 amino acids in many embodiments. In some embodiments, the claimed polypeptides have a size of approximately 25 amino acids, approximately 50, approximately 75, approximately 100, approximately 125, approximately 150, approximately 200, or approximately 250 amino acids, up to the full size of the protein. In some embodiments, the protein fragment retains all or substantially all of the specific property of the wild-type protein.

The claimed proteins and polypeptides can be obtained from natural sources or artificially synthesized. For example, wild-type proteins can be obtained from biological sources that express proteins, such as Hydrozoa species, such as some of the above. The claimed proteins can also be obtained artificially, for example by expression of a recombinant nucleic acid encoding the sequence of a protein of interest in an appropriate host, as described above. Any conventional protein purification techniques can be applied where suitable protein purification methods are described in Guide to Protein Purification, (Deuthser ed.) (Academic Press, 1990). For example, a lysate can be prepared from a source and purified using HPLC, size exclusion chromatography, gel electrophoresis, affinity chromatography, and the like.

Fusion proteins including the protein of the present invention, or fragments thereof fused, for example, with a degenerate sequence, an intracellular localization sequence (e.g., a nuclear localization signal, a peroximal targeting signal, a Golgi targeting sequence, a mitochondrial targeting sequence, etc., are also provided. ), a signal peptide or any protein or polypeptide of interest. Fusion proteins may include, for example, the fluoro / chromoprotein of the invention, a polypeptide and a second polypeptide (“fusion partner”) linked within the N-terminus and / or C-terminus of the fluoro / chromopolypeptide. Fusion partners include, but are not limited to, polypeptides that can bind antibodies specific to the fusion partner (e.g., epitope tags), antibodies or their binding fragments, polypeptides that provide catalytic function or elicit a cellular response, ligands or receptors, or mimetics thereof, etc. In such fusion proteins, in general, the fusion partner does not naturally bind to the fluoro / chromoprotein portion of the fusion protein and is usually non-Hydrozoa fluoro / chromoproteins of the invention, or a derivative / fragment thereof; that is, they are not found in the form of Hydrozoa.

Antibodies that specifically bind to the fluorescent or chromoprotein of the present invention are also provided. Appropriate antibodies can be prepared using methods known in the art. For example, polyclonal antibodies can be prepared as described in (Harlow and Lane Antibodies: A Laboratory Manual, (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), and monoclonal antibodies can be prepared as described in (Coding Monoclonal Antibodies : Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology; 3rd edition, (1996) Academic Press). Chimeric antibodies, including humanized antibodies, as well as single chain antibodies and antibody fragments such as Fv, F (ab ') ₂ and Fab are also of interest.

Transgenics

The nucleic acids of the present invention can be used to produce transgenic organisms or site-specific gene changes in cell lines. The transgenic cells of the invention comprise one or more nucleic acids of the invention as a transgene. For the purposes of the invention, any suitable host cell can be used, including prokaryotic (e.g., Escherichia coli , Streptomyces sp., Bacillus subtilis , Lactobacillus acidophilus , etc.) or eukaryotic host cells. A transgenic organism of the invention may be a prokaryotic or eukaryotic organism, including bacteria, cyanobacteria, fungi, plants and animals in which one or more cells of the body contain a heterogeneous nucleic acid of the invention, introduced by human intervention, by such transgenic methods, which are known in the art.

The isolated nucleic acid of the present invention can be introduced into the host by methods known in the art, for example, infection, transfection, transformation or trans-conjugation. Methods for transferring a nucleic acid molecule (i.e., DNA) into such organisms are widely known and provided in references such as Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3 ^nd Ed., (2001) Cold Spring Harbor Press, Cold Spring Harbor, NY).

In one embodiment, the transgenic organism may be a prokaryotic organism. Methods for transforming prokaryotic hosts are well described in the art (e.g., see Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edition (1989) Cold Spring Harbor Laboratory Press and Ausubel et al., Current Protocols in Molecular Biology (1995) John Wiley & Sons, Inc).

In another embodiment, the transgenic organism may be fungi, for example, yeast. Yeast is widely used as a carrier for heterogeneous gene expression (e.g. see Goodey et al Yeast biotechnology, DR Berry et al, eds, (1987) Allen and Unwin, London, pp 401-429) and King et al Molecular and Cell Biology of Yeasts EF Walton and GT Yarronton, eds, Blackie, Glasgow (1989) pp 107-133). Several types of yeast vectors are available, including integrative vectors that require recombination with the host genome to support them, and plasmid vectors are autonomously replicated.

Another host organism is an animal. Transgenic animals can be obtained by transgenic methods known in the art and provided in references such as Pinkert, Transgenic Animal Technology: a Laboratory Handbook, 2nd edition (2203) San Diego: Academic Press; Gersenstein and Vintersten, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed, (2002) Nagy A. (Ed), Cold Spring Harbor Laboratory; Blau et al., Laboratory Animal Medicine, 2nd Ed., (2002) Fox J.G., Anderson L.C., Loew F.M., Quimby F.W. (Eds), American Medical Association, American Psychological Association; Gene Targeting: A Practical Approach by Alexandra L. Joyner (Ed.) Oxford University Press; 2nd edition (2000). For example, transgenic animals can be obtained by homologous recombination, where the endogenous locus is changed. Alternatively, the nucleic acid construct is randomly incorporated into the genome. Vectors for sustainable incorporation include plasmids, retroviruses and other animal viruses, YAC, and the like.

Nucleic acids can be introduced into a cell, directly or indirectly, by introducing into a cell precursor, by intentional genetic manipulation, such as microinjection or infection with a recombinant virus or recombinant viral vector and the like. The term “genetic manipulation” does not include classical cross-fertilization or in vitro fertilization, but is preferably directed to the introduction of recombinant nucleic acid molecules. These nucleic acid molecules may be included in the chromosome or they may be extrachromosomal replicating DNA.

DNA constructs for homologous recombination will contain at least a portion of the nucleic acid of the present invention, where the gene has the desired genetic modification (s) and includes homology regions with a target locus. DNA constructs for arbitrary incorporation do not necessarily contain a region of homology with a recombination mediator. Markers for positive and negative selection can easily be included. Methods for producing cells having targeted gene modifications through a homologous combination are known in the art. For various methods for transfecting mammalian cells, see Keown et al., Meth. Enzymol. (1990) 185: 527-537.

For embryonic stem (ES) cells, ES cell lines can be used, or embryonic cells can be obtained directly from a host, such as a mouse, rat, guinea pig, etc. Such cells are grown on a suitable fibroblast-feeding layer or grow in the presence of leukemia inhibition factor (LIF). Transformed ES or embryonic cells can be used to obtain transgenic animals using a suitable method described in this field.

Transgenic animals can be any non-human animals, including a non-human mammal (e.g., mouse, rat), bird or amphibian, etc., and are used in functional research, drug screening, and the like. Representative examples of the use of transgenic animals include those described below.

Transgenic plants can also be obtained. Methods for producing transgenic plant cells and plants are described in US patent No. 5767367; 5,750,870; 5,739,409; 5,689,049; 5,689,045; 5,674,731; 5656466; 5,633,155; 5,629,470; 5,595,896; 5,576,198; 5,538,879; 5,484,956; disclosures of which are incorporated herein by reference. Methods for producing transgenic plants are also discussed in Plant Biochemistry and Molecular Biology (eds. Lea and Leegood, John Wiley & Sons) (1993) pp. 275-295 and Plant Biotechnology and Transgenic Plants (eds. Oksman-Caldentey and Barz), (2002) 719 p.

For example, embryogenic explants containing somatic cells can be used to produce a transgenic host. After collecting cells or tissue, exogenous DNA of interest is introduced into the plant cells, and a number of different methods are known for such administration. In the presence of isolated protoplasts, it becomes possible to introduce through DNA-mediated gene transfer protocols, including incubating protoplasts with isolated DNA, such as plasmids, including the desired exogenous coding sequence in the presence of polyvalent cations (eg, PEG or PLO); or electroporation of protoplasts in the presence of isolated DNA, including the target exogenous sequence. Protoplasts that successfully incorporate exogenous DNA are then selected, grown in callus, and ultimately in a transgenic plant in contact with suitable amounts and ratios of stimulatory factors such as auxins and cytokines.

Other suitable plant production methods may be used, such as the use of a "gene gun" or Agrobacterium-mediated transformation, which are available to those skilled in the art.

Application methods

The fluorescent proteins of the present invention (like the other components described above, claimed in the invention) find use in a number of different applications. For example, they can be used in labeling methods, in the analysis or detection of a biological molecule, cell, or cell organelle. Typical uses for each of these types of proteins will be described below, the uses described herein are illustrative only and are not intended to limit the use of the proteins of the present invention as described herein.

In a preferred embodiment relating to a method for introducing labels into a biological molecule, cell or cell organelle, the claimed proteins find use as in vivo tags (or reporter molecules) in cell and molecular biology analyzes. Assays of interest include, but are not limited to, gene expression analyzes, protein localization and co-localization, protein-protein interactions, protein-nucleic acid interactions, nucleic acid-nucleic acid interactions, cell localization and localization of cell organelles and cell organelle interactions and etc. The fluorescent proteins of the present invention find use as biomolecule tags or cell organelle tags in living and attached cells; as markers in a cell or fusion organelles, as integration markers of a cell or organelles, as transfection markers (for example, as labels for selecting transfected cells containing expression vectors encoding at least one fluorescent protein of the invention), as a real-time probe working with near-physiological concentrations, etc.

In addition, the claimed proteins can be used in the method of analysis of a biological molecule. For example, they are used to identify and / or measure the expression of a target protein or polypeptide in a biological material. This method includes: i) introducing into the cell a nucleic acid molecule containing a nucleotide sequence encoding a fluorescent protein of the present invention, wherein said nucleic acid molecule is operably linked and controlled by an expression control sequence that controls the expression of said target protein or polypeptide; ii) expressing said nucleic acid under appropriate conditions; and iii) detecting the fluorescent radiation of the fluorescent protein as a means for measuring the expression of the protein of interest.

In particular, the claimed proteins are used to identify and / or measure the expression and / or localization of a target protein or polypeptide in a biological material. This method includes: i) introducing into the cell a nucleic acid molecule containing a nucleotide sequence encoding a fluorescent protein of the present invention, wherein said nucleic acid molecule is fused to a sequence encoding a target protein or polypeptide and is operably linked and controlled by a sequence that controls expression the specified target protein; ii) culturing the cell under conditions suitable for expression of the target protein; and iii) detecting the fluorescent radiation of the fluorescent protein as a method of measuring the expression of the target protein.

Targeted uses include the use of the claimed proteins in fluorescence resonant energy transfer (FRET) methods. In these methods, the claimed proteins serve as a donor and / or acceptor in combination with a second fluorescent protein or dye, for example, another fluorescent protein of the invention, or a fluorescent protein, as described in Matz et al., Nature Biotechnology 17: 969-973 (1999 ); a green fluorescent protein from Aequorea victoria or a fluorescent mutant thereof, for example, as described in US Pat. Nos. 6,064,476; 6020192; 5,985,577; 5,976,796; 5,968,750; 5,968,738; 5958713; 5,919,445; 5,874,304, the disclosures of which are incorporated herein by reference; other fluorescent dyes such as coumarin and its derivatives, 7-amino-4-methylcoumarin and aminocoumarin; dyes for living tissues; cascading blue; or fluorescein and its derivatives, such as fluorescein isothiocyanate and Oregon green; rhodamine dyes such as Texas Red, tetramethylrodamine, eosins and erythrosines; cyan dyes such as Cy3 and Cy5; macrocyclic chelates of lantonoid ions, such as a quantum dye; and chemiluminescent dyes, such as luciferase, including those described in US patent No. 5843746; 5,700,673; 5,674,713; 5,618,722; 5,418,155; 5,330,906; 5,229,285; 5,221,623; 5,182,202; disclosures of which are incorporated herein by reference.

Specific examples of the use of the claimed fluorescent proteins in FRET assays include, but are not limited to, the detection of protein-protein interactions, such as in the mammalian two-hybrid system, dimerization transcription factor, membrane protein multimization, multi-protein complexation; as a biosensor for many different events, where a peptide or protein covalently binds a FRET fluorescent combination, including the claimed fluorescent proteins, and the bound peptide or protein is, for example, a protease-specific substrate for caspase-mediated cleavage, a peptide that undergoes a conformational change after receiving a signal that increases or decreases FRET, such as the PKA regulatory domain (cAMP sensor), the phosphorylation site (for example, where the phosphorylation site is located in the peptide Or the peptide has binding specificity to phosphorylated / dephosphorylated domain of another protein), or the peptide has Ca ²⁺ binding domain. In addition, applications of fluorescence resonant energy transfer or FRET, in which the proteins of the present invention find use, include, but are not limited to, described in US patent No. 6008373; 5,998,146; 5,981,200; 5,945,526; 5,945,283; 5,911,952; 5,869,255; 5,866,336; 5,863,727; 5,728,528; 5,707,804; 5,688,648; 5,439,797, the disclosures of which are incorporated herein by reference.

The fluorescent proteins of the present invention find use in a method for detecting the influence of a test material on the regulation of expression and / or translocation of one or more target proteins in a cell. Alternatively, they find use in a method for detecting the expression of a target protein and the simultaneous activity of an expression control sequence in response to test material. Fluorescent proteins also find use in a method for comparing the activity of two or more sequences regulating expression in a cell in response to test material. Such methods can be performed in the presence and absence of the test material, the influence of which on the method should be measured.

The fluorescent proteins of the present invention also find application in applications including automated screening of a matrix of cells expressing fluorescent reporter groups using microscopic imaging and electronic analysis. Screening can be used to discover the drug and in the field of functional genomics, where the claimed proteins are used as markers of whole cells to detect changes in multicellular transformation and movement, for example, in the formation of multicellular tubules (formation of a blood vessel) by endothelial cells, the movement of cells through the Fluoroblok Insert system (Becton Dickinson Co.), wound healing, or the growth of a neuron. Screening can also be used if the proteins of the present invention are used as markers fused to peptides (such as target sequences) or proteins that show changes in intracellular localization as an indicator of cellular activity, for example, in signal transduction, such as kinase and translocation translocation factors with stimulation. Examples include protein kinase C, protein kinase A, transcription factor NFkB and NFAT; cyclic cellular proteins such as cyclin A, cyclin B1 and cyclin E; protease cleavage with sequential movement of the cleaved substrate; phospholipids with markers for intracellular structures, such as the endoplasmic reticulum, Golgi apparatus, mitochondria, peroxisomes, nucleus, nucleoli, plasma membrane, histones, endosomes, lysosomes or microtubes.

The proteins of the present invention can also be used in high-content screening to detect co-localization of other fluorescent fusion proteins with localization markers as indicators of intracellular fluorescent protein / peptide movements or as markers only. Examples of applications, including automated screening of multiple cells in which the claimed fluorescent proteins are used, include US patent No. 5989835; same as WO 00/17624, WO 00/26408, WO 00/17643 and WO 00/03246, the disclosures of which are incorporated herein by reference.

The fluorescent proteins of the present invention also find use in high-throughput screening assays. The claimed fluorescent proteins are stable proteins with half-lives of more than 24 hours. Also provided are destabilized variants of the claimed fluorescent proteins with reduced half-lives that can be used as transcription reporters for drug discovery. For example, a protein of the invention can be fused to a putative proteolytic signal sequence derived from a protein with a shorter half-life, such as a PEST sequence from a mouse ornithine decarboxylase gene, with box decomposition of cyclin B1 from mouse or ubiquitin, etc. . For a description of destabilized proteins and vectors that can be used to obtain the same, see, for example, US Pat. No. 6,130,313, the disclosure of which is incorporated herein by reference. Promoters in signal transduction pathways can be detected using destabilized variants of the claimed fluorescent proteins for drug screening, such as, for example, AP1, NFAT, NFkB, Smad, STAT, p53, E2F, Rb, myc, CRE, ER, GR and TRE etc.

The claimed proteins can be used as second messenger detectors by fusing the claimed whites with specific domains, such as the PKC gamma Ca binding domain, PKC gamma DAG binding domain, SH2 domain or SH3 domain, etc.

Secreted forms of the claimed proteins, which in turn can be used in a number of different applications, can be obtained by fusion of secreted leading sequences with the claimed protein.

The claimed proteins also find use in the sorting of fluorescence activated cells (FACS). In such applications, the claimed fluorescent protein is used to label a cell population, and the resulting labeled cell population is then sorted in a fluorescence activated cell sorting device, as is known in the art. FACS methods are described in US patent No. 5968738 and 5804387; disclosures of which are incorporated herein by reference.

The claimed proteins also find use as in vivo tags for transgenic animals. For example, expression of a protein of interest may be accompanied by tissue-specific promoters, where such methods are used in studies for gene therapy, such as testing the effectiveness of transgenic expression, among other applications. A typical application of fluorescent proteins in transgenic animals, which explains such applications, is in WO 00/02997, the disclosure of which is incorporated herein by reference.

Additional uses of the proteins of the present invention include use as markers for administration to cells or animals and when calibrated in quantitative measurements; as markers or reporters in oxygen biosensor devices for monitoring cell viability; as markers or tags for animals, pets, toys, food, and the like.

The claimed fluorescent proteins also find use in protease cleavage assays. For example, cleavage-inactivated fluorescence assays can be performed using the claimed proteins, where the claimed proteins are prepared for inclusion in a protease-specific cleavage sequence without compromising the fluorescent properties of the protein. When a fluorescent protein is cleaved by an activated protease, fluorescence decreases sharply due to the destruction of the functional chromophore. Alternatively, cleavage activated fluorescence can be used using the whites of the present invention when proteins are obtained containing an additional spacer sequence near / or within the chromophore. This option significantly reduces its fluorescence activity, because parts of the functional chromophore are separated by a spacer. The spacer is framed by two identical specific protease-specific cleavage sites. When cleaved by an activated protease, the spacer would be excised and the two residual “subunits” of the fluorescent protein could be reassembled to produce a functional fluorescent protein. Both of the applications described above could be developed in assays for a number of different types of proteases, such as caspases and others.

The claimed proteins can also be used in assays to determine the phospholipid composition in biological membranes. For example, fusion proteins of target proteins (or any other type of covalent or non-covalent modification of the claimed proteins) that allow binding to specific phospholipids for localization / visualization in a model of phospholipid distribution in biological membranes, whereas membrane-capable membrane proteins in specific phospholipid foci can be associated with the claimed proteins. For example, the PH domain of GRP1 has a high affinity for phosphatidyl-inositol triphosphate (PIP3), but not PIP2. Also, a fusion protein between the PH domain of GRP1 and the claimed proteins can be created for specific labeling of PIP3-enriched sites in biological membranes.

The claimed fluorescent proteins also find use as biosensors in prokaryotic and eukaryotic cells, such as a Ca ²⁺ ion indicator; pH indicator; phosphorylation indicator; or as an indicator of others, such as magnesium, sodium, potassium, chloride, and ion halide. Methods for using fluorescent proteins as biosensors also include those described in US Pat. Nos. 5,972,638; 5,824,485 and 5650135 (as well as the references cited there), the disclosure of which is incorporated herein by reference.

The antibodies of the invention described above also find use in a variety of applications, including differentiating the claimed proteins from other fluorescent proteins.

Sets

Kits are also provided in accordance with the present invention for use in carrying out one or more of the above applications. In preferred embodiments, kits can be used to label a biological molecule. Kits typically include an inventive protein or a nucleic acid encoding it, preferably with elements for expressing the claimed whites, for example, a construct such as a vector comprising a nucleic acid encoding the claimed protein. The invention also encompasses means for preparing such kit components. The method may include cDNA from a Hydrozoa jellyfish and a pair of oligonucleotide primers to produce a nucleic acid of the invention, for example, by PCR, or the means may include many nucleic acid fragments that, when ligated, can produce a nucleic acid encoding the fluorescent protein of the present invention , etc. The components of the kits are usually present in an appropriate storage medium, such as a buffered solution, usually in an appropriate container. Also, antibodies specific for the protein may be present in the kits. In separate embodiments, the kit includes many different vectors, each of which encodes a protein of interest, where the vectors are designed for expression in different environments and / or under different conditions, for example, for constitutive expression, where the vector includes a strong promoter for expression in mammalian cells or a vector with attenuated promoter with multiple cloned sites for selective insertion of the promoter and non-standard expression, etc.

In addition to the components described above, the claimed kits will further include instructions for implementing the claimed methods. These instructions may be present in the claimed kits in various forms in an amount of one or more that may be present in the kits.

The following examples are provided as illustrative but not restrictive.

EXAMPLES

Example 1

Cloning, sequencing and production of phiYFP recombinant protein

Bright yellow fluorescence in hydromedusa Phialidium Sp. (Cnidaria; Hydrozoa; Hydroida; Leptomedusae; Campanulariidae ) was detected using a fluorescence microscope. To find the protein responsible for fluorescence in this jellyfish, a strategy was chosen based on screening for the cDNA expression library in E. coli . Amplified cDNA samples were prepared using the SMART cDNA amplification kit (Clontech) and cloned into a PCR-Script vector (Stratagene). Approximately 10 ⁵ recombinant clones were screened visually using a fluorescence stereoscopic microscope. Two fluorescent clones encoding the same yellow fluorescent proteins were found and were named phiYFP. The nucleic acid and amino acid sequences for phiYFP are shown in SEQ NO: 01, 02 and 23. A comparison of phiYFP with A. victoria GFP is shown in FIG. It turned out that phiYFP is more similar to GFP (50% identity) than to fluorescent proteins from coral derivatives.

To facilitate protein purification, the coding region of the phiYFP gene was cloned into the pQE30 expression vector (Qiagen) so that the recombinant protein contained a six-histidine tag at its N-terminus. After expression in E. coli, the phiYFP protein was purified using a TALON metal-affinity resin (Clontech). Excitation-emission spectra for phiYFP had peaks at 525 nm and 537 nm (FIG. 2A), respectively. Unlike the wild-type GFP A. victoria, the new protein has only one absorption-radiation peak, probably in accordance with the deprotonated state of the chromophore.

Example 2

Mutagenesis PhiYFP

The nucleic acid encoding PhiYFP sequence was obtained as described above in Example 1. The applicant encoded a wild-type coding protein by nonspecific mutagenesis. The nonspecific phiYFP mutagenesis resulted in a brighter mutant called phiYFP-Y1, with a slightly altered excitation-radiation spectrum. This mutant contained three amino acid substitutions, in particular S2P, E174G, I201M (SEQ ID NO: 03, 04, and 24). phiYFP-Yl showed a brightness of 1.5 to 2 times higher than the wild-type phiYFP when bilingual visual comparison of E. coli colonies expressing these fluorescent proteins. In addition, phiYFP-Yl shows a radiation spectrum slightly shifted to the red region with a peak at 542 nm (see FIG. 2B).

Proteins of both phiYFP and phiYFP-Yl have been found to be dimers. This has been shown by protein gel electrophoresis of unheated protein samples (see Baird et al., Supra, 2000). Under these conditions, these FPs moved as a yellow fluorescent band of approximately 50 kDa. Gel filtration tests showed the dimeric state of phiYFP and phiYFP-Yl. Purified protein samples (~ 1 mg / ml) were loaded onto a Sephadex-100 column (0.7 × 60 cm) and eluted with a solution of 50 mM phosphate buffer (pH 7.0) and 100 mM NaCl. EGFP, HcRedl, and DsRed2 (Clontech) were used as monomeric, dimeric, and tetrameric standards, respectively.

Site-directed mutagenesis was used to increase the degree of monomerism of the phiYFP-Y1 variant. Six amino acid substitutions were introduced, in particular V103N, M166R, Y198N, T202S, T206K, V221K. Fully this phiYFP-MO mutant carried 9 substitutions: S2P, V103N, M166R, E174G, Y198N, I201M, T202S, T206K, V221K (SEQ ID NOs: 05, 06, and 25). phiYFP-MO showed low protein folding and low brightness when it was expressed in E. coll. Its excitation-emission spectra were shifted to the blue region compared to the maternal mutant (maxima at 517 and 529 nm, respectively; FIG. 2C). phiYFP-MO was a more monomeric protein according to gel filtration tests.

To improve phiYFP-MO, the applicant has applied nonspecific mutagenesis. The Diversity PCR Random Mutagenesis kit (CLONTECH) was used under optimal conditions for 5-6 mutations per 1000 bp. E. coli colonies expressing mutant proteins were visually screened with a SZX-12 fluorescence microscope (Olympus). The brightest clone with a spectrum obviously shifted to the red region (compared to the original phiYFP-MO) was further characterized. This mutant, designated phiYFP-M1, contained the following amino acid substitutions: E88D, V103N, M166C, E174G, I201M, T202S, T206K, V221K (SEQ ID NOs: 07, 08, and 26). The excitation-emission spectra for this protein had peaks at 524 and 539 nm, respectively, similarly to wild-type phiYFP peaks (FIG. 2). Purified phiYFP-M1 had a molar extinction coefficient of 130,000 M ^-1 cm ^-1 and a fluorescence quantum yield of 0.40. In determining the molar extinction coefficient, the applicant relied on an estimate of the concentration of the mature chromophore. The protein was denatured with an equal volume of alkali 2M NaOH. Under these conditions the GFP-like chromophore absorbs at 446 nm and its molar extinction coefficient is 44,000 M ^-1 cm ^{-1 (Ward, WW Properties of the} coelentrate green-fluorescent protein, in Bioluminescence and Chemiluminescence. Academic Press ( 1981), 235 -242). The absorption spectra of native and alkali denatured phiYFP-M1 were measured. The molar extinction coefficient for the native state of the protein was estimated based on the absorption of the denatured protein. To determine the quantum yield, phiYFP-M1 fluorescence was compared with equally absorbing EGFP (quantum yield 0.60 ((Patterson et al., J. Cell. Sci. (2001), 114: 837-838)). PhiYFP-M1 was weakly dimeric protein according to gel filtration tests.

To enhance expression in mammalian cells, the applicant synthesized a “humanized” version of phiYFP-M1 using codons optimized for mammals (SEQ ID NOs: 09, 10, and 27). The humanized version of phiYFP-M1 was subjected to site-directed and non-specific mutagenesis to produce protein versions that emit light in the green and blue regions. Mutant fluorescent proteins with green and blue fluorescence were obtained. The green humanized phiYFP-M1 mutant, called phiYFP-M1G1, contains the following amino acid substitutions (compared to phiYFP-M1) T65S, L148Q, Y203T, K231T, T232A (SEQ ID NO: 17, 18 and 31). The blue humanized phiYFP-M1 mutant called phiYFP-M1C1 contains the following amino acid substitutions (compared to phiYFP-M1): L6Q, T65S, Y66W, N124K, C147Y, L148Q, Y203T, V224L (SEQ ID NO: 19, 20 and 32 ) The excitation-emission spectra for this protein are shown in FIGS. 3A, B.

Example 3

Cloning, sequencing and production of the recombinant hydr1GFP protein

Bright green fluorescence was detected in hydromedusa 1 (approximately 1 mm long, FIG. 4) of the suborder Anthomedusae ( Cnidaria, Hydrozoa, Anthomedusae ) using a fluorescence microscope. To search for the gene responsible for fluorescence in this jellyfish, a strategy was implemented based on screening for the cDNA expression library in E. coli . Amplification of cDNA samples was carried out using the SMART cDNA kit (Clontech) and cloned into a PCR-Script vector (Stratagene). About 10 ⁵ recombinant clones were visually screened using a fluorescence stereoscopic microscope. Three fluorescent clones were identified, where each encoded the same green fluorescent protein, which is called hydr1GFP. The nucleotide and amino acid sequences for this protein are shown in SEQ ID NO: 11, 12 and 28. Comparison with hydr1GFP GFP A. victoria shown in Figure 1. hydr1GFP, as it turned out, is more similar to GFP (37% identity) than to fluorescent coral proteins.

To facilitate protein purification, the coding region of the hydr1GFP gene was cloned into the pQE30 expression vector (Qiagen) so that the recombinant protein contained a six-histidine tag at its N-terminus. After expression in E. coli, hydr1GFP protein was purified using TALON metal-affinity resin (Clontech). Excitation-emission spectra for phiYFP were recorded at 474 nm and 494 nm (Fig. 5), respectively. Unlike the wild-type GFP A. victoria, the new protein has only one absorption-radiation peak, probably in accordance with the deprotonated state of the chromophore.

Example 4

Cloning, sequencing and production of recombinant hm2CP protein

Bright green fluorescence was detected in minor hydromedusa 2 from the suborder Anthomedusae ( Cnidaria, Hydrozoa, Anthomedusae ) using a fluorescence microscope. To search for FP from this jellyfish, the applicant chose a strategy based on screening an E. coli cDNA expression library. Amplification of cDNA samples was performed using a SMART cDNA kit (Clontech) and cloned into a PCR-Script vector (Stratagene). About 10 ⁵ recombinant clones were visually screened using a fluorescence stereoscopic microscope or with the naked eye. Unexpectedly, the applicant did not find fluorescent clones. Instead, purple non-fluorescent CP (hm2CP) was identified. The nucleotide and amino acid sequences of this protein are shown in SEQ ID NO: 13, 14 and 29. A comparison of hm2CP with GFP is shown in FIG. hm2CP, as it turned out, is a relatively distant homologue of GFP (up to 24% identity).

To facilitate protein purification, the hm2CP coding region was cloned into the pQE30 expression vector (Qiagen) so that the recombinant protein contained a six-histidine tag at its N-terminus. After expression in E. coli , the hm2CP protein was purified using a TALON metal-affinity resin (Clontech). The absorption spectrum of purified hm2CP had a single maximum at 568 nm (Fig.6). Very weak red fluorescence (excitation maxima at 569 and 597 nm, respectively) hm2CP can be detected (Fig. 7).

Example 5

Mutagenesis hm2CP

The nucleic acid sequence encoding hm2CP was obtained as described above in Example 4. Applicants used nonspecific mutagenesis to obtain fluorescent mutants of hm2CP. The Diversity PCR Random Mutagenesis (Clontech) kit was used for non-specific hm2CP mutagenesis under conditions optimal for 5-6 mutations per 1000 bp. E. coli colonies expressing mutant proteins were visually screened with a SZX-12 fluorescence microscope (Olympus). The most striking variants were selected and subjected to another cycle of nonspecific mutagenesis. The full four cycles of mutagenesis resulted in a bright and rapidly ripening red fluorescent mutant, designated S3-2. Compared to the original chromoprotein, S3-2 had 13 amino acid substitutions, in particular D24G, I30V, K73R, T91S, II18V, K136R, T145N, S154P, C161A, Y162F, L181M, V199T, I201T (SEQ ID NO: 15, 16, and thirty). The excitation and emission spectra for this mutant had maxima at 585 and 611 nm, respectively (Fig. 8). The red fluorescent protein S3-2 has a weakly dimeric nature, shown by gel filtration data. To enhance expression in mammalian cells, applicants synthesized a “humanized” version of S3-2 using codons optimized for mammals (SEQ ID NO: 21, 22, and 33).

Example 6

Preparation of Polyclonal Antibodies

The coding regions of the nucleic acids of the red fluorescent protein S3-2 and the yellow fluorescent protein Phi-YFP-M1, obtained as described above in examples 2 and 5, respectively, were cloned into the expression vector pQE30 (Qiagen) so that the recombinant proteins contained a six-histidine tag at the N-end. After expression in E. Coli, hm2CP was purified using TALON metal-affinity resin (Clontech) under denaturing conditions. Rabbits were immunized and further treated four times with monthly periods of recombinant DSN polypeptide emulsified in Freund's adjuvant. Ten or 11 days after each additional treatment, animals were bled. Polyclonal antiserum was tested on recombinant protein using ELISA and Western immunoblotting.

Example 7

Labeling mammalian cells using PhiYFP and S3-2 proteins.

For fluorescence labeling of eukaryotic cells, humanized versions of the phiYFP-M1 and S3-2 proteins obtained as described above in Examples 2 and 5, respectively, were cloned into the pEGFP-C1 vector (CLONTECH) between the Agel and BglII restriction regions (instead of the EGFP-coding region) . The following cell lines were used: 293T human renal epithelial cells, ZT3 mouse embryo fibroblasts, L929 subcutaneous mouse fibroblasts, Vero African green monkey renal epithelial cells, and COS1 African green monkey kidney fibroblasts. Cells were transfected using LipofectAMINE reagent (Invitrogen) and were tested 20 hours after transfection. An Olympus CK40 fluorescence microscope equipped with a CCD camera (DP-50, Olympus) was used to capture cell images. Expression of phiYFP-M1 or S3-2 on different cell lines resulted in bright yellow or red signals without aggregation. Fluorescence was clearly detected 24 hours after transfection. Cell toxicity was not observed.

Example 8

Protein labeling and protein localization analysis using PhiYFP and S3-2 proteins.

Humanized versions of phiYFP-M1 (containing additional substitutions N21D, T170A, K221T) and S3-2 proteins obtained as described above in Examples 2 and 5, respectively, were fused with human cytoplasmic beta-actin. Transfection of human 293T renal epithelial cells with plasmids expressing phiYFP-M1 or 33-2-labeled fusion constructs resulted in bright fluorescence, which gave a picture very similar to that in fusions with EGFP.

The humanized version of phiYFP-M1 (containing additional substitutions N21D, T170A, K221T) was additionally fused to the nucleolar protein, fibrillarin. Transfection of human 293T renal epithelial cells with a plasmid expressing phiYFP-M1-labeled fusion constructs resulted in bright fluorescence with a structure as expected.

Example 9

Labeling mitochondria using PhiYFP

The coding sequences of the humanized version of phiYFP-M1 obtained as described above in Example 2 were fused to the mitochondrial target sequence (MTS) from the human cytochrome oxidase C subunit VIII. Transfection of human 293T renal epithelial cells with plasmids expressing the phiYFP-M1-MTS fusion construct resulted in efficient translocation of the protein into the mitochondria of the host cells. Fluorescence was clearly detected 24 hours after transfection.

Example 10

Labeling a Golgi apparatus using PhiYFP

The coding sequences of the humanized version of phiYFP-M1, obtained as described above in Example 2, were fused to the sequence coding for 81 amino acids at the N-terminus of human beta 1,4-galactosyltransferase (GT; Watzele & Berger (1990) Nucleic Acids. Res. 18 : 7174). This human beta-1,4-GT region contains a signal peptide binding to a membrane that targets fusion proteins to the trans-enveloped region of the Golgi apparatus (Llopis et al. Proc. Natl. Acad. Sci. USA (1998) 95: 6803-6808; Yamaguchi & Fukuda J. Biol. Chem. (1995) 270: 12170-12176; Gleeson et al. Glycoconjugate J. (1994) 11: 381-394). Transfection of 293T human renal epithelial cells with plasmids expressing the phiYFP-M1-labeled fusion construct led to fluorescent labeling of the trans-enveloped portion of the Golgi apparatus in cells.

Example 11

Labeling Peroxisome Using PhiYFP

The coding sequences of the humanized version of phiYFP-M1 obtained as described above in Example 2 were fused to peroxisomal targeting signal 1 (PTS1). The PTS1 sequence encoding the SKL tripeptide that targets the fusion protein to the peroxisome matrix (Gould et al. J. Biol. Chem. (1989) 108: 1657-1664; Gould et al. EMBO J. (1990) 9: 85-90; Monosov et al. J. Histo. Cytochem. (1996) 44: 581-589)). Transfection of 293T human renal epithelial cells with plasmids expressing the phiYFP-M1-labeled fusion construct resulted in fluorescent peroxisome labeling.

Example 12

Labeling the core using PhiYFP

The coding sequences of the humanized version of phiYFP-M1, obtained as described above in Example 2, were fused to three copies of the nuclear localization signal (NLS) of the large T-antigens of monkey virus 40, at its C-terminus (Kalderon et al. Cell (1984) 39 : 499-509; Lanford et al. Cell (1986) 46: 575-582). Transfection of 293T human renal epithelial cells with plasmids expressing the phiYFP-M1-labeled fusion construct resulted in fluorescent labeling of nuclei.

All publications and patent applications cited in the present description, are introduced into the present description by reference, as if each individual publication or patent application was specifically and separately introduced by reference. The citation of any publication is in accordance with the context and interpretation of the present invention and should not be construed as recognition of any such publication as a prototype of the present invention.

Claims (13)

1. An isolated nucleic acid molecule that encodes a fluorescent protein selected from the group consisting of: (a) a nucleic acid that encodes a fluorescent protein, characterized by an amino acid sequence essentially corresponding to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 12, 16, 18 or 20; (b) a nucleic acid characterized by a nucleotide sequence essentially corresponding to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19 or 21; (c) a nucleic acid that hybridizes under stringent conditions with the nucleic acid of (a) or (b) above; (d) a nucleic acid that encodes a protein that has at least 95% sequence identity with the amino acid sequence (a) above; (e) a nucleic acid that has at least 85% sequence identity with the nucleotide sequence of (b) above. 2. The cloning vector comprising the nucleic acid molecule according to claim 1 and elements for replicating the vector in the host cell. 3. An expression vector comprising a nucleic acid molecule according to claim 1 and elements for expressing said nucleic acid in a host cell. 4. The expression cassette containing the nucleic acid according to claim 1 under the control of regulatory elements necessary for expression of the nucleic acid in the host cell, which, when integrated into the genome of the cell or when introduced into the cell as an extrachromosomal element, is capable of expressing the fluorescent protein encoded nucleic acid according to claim 1. 5. A cell producing a fluorescent protein encoded by a nucleic acid according to claim 1, containing an expression cassette according to claim 4 in the form of an extrachromosomal element or an element integrated into the genome of this cell. 6. A stable cell line producing a fluorescent protein encoded by a nucleic acid according to claim 1, containing an expression cassette according to claim 4 in the form of an extrachromosomal element or an element integrated into the genome of this cell. 7. A method for producing a fluorescent protein encoded by a nucleic acid according to claim 1, comprising (a) providing a nucleic acid molecule according to claim 1, operably linked to suitable expression regulating elements, (b) expressing a fluorescent protein from said nucleic acid molecule, and ( c) isolating a protein substantially free of other proteins. 8. The selected fluorescent protein selected from the group consisting of: (a) a protein characterized by an amino acid sequence essentially corresponding to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 12, 16, 18 or 20; (b) a protein encoded by a nucleic acid molecule essentially corresponding to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 15, 17, 19, or 21; (c) a protein that has at least 95% sequence identity with the amino acid sequence of (a) or (b) above. 9. A fusion protein capable of fluorescence, consisting of the protein of claim 8 and a fusion partner, which is a peptide or polypeptide characterized by an intracellular localization sequence, or a signal peptide. 10. A kit for labeling a biological molecule, containing the nucleic acid according to claim 1, or containing the vector according to claim 2, or containing the expression cassette according to claim 3, or containing the protein according to claim 7 and instructions for labeling the biological molecule. 11. A method for labeling a biological molecule, comprising binding said biological molecule to a protein of claim 8. 12. A method for labeling a cell, comprising producing a protein of claim 8 in a cell. 13. A method for labeling a cell organelle, comprising producing the protein of claim 8, fused to a suitable intracellular localization signal in the cell. Claims 1-13 regarding SEQ ID NO: 1, 2, 3, 4 take priority on November 12, 2002 (by the date of filing of application No. 60425570 with the US Patent Office), regarding SEQ ID NO: 5, 6, 7, 8 , 9 have priority from 04/21/2003 (by the date of filing of application No. 60464258 to the US Patent Office), in part SEQ ID NO: 11, 12 have priority from 11/27/2002 (by the date of filing of application No. 60429795 to the US Patent Office), parts of SEQ ID NO: 15, 16 have priority from 06/20/2003 (according to the filing date of application No. 60480080 to the US Patent Office), in parts of SEQ ID NO: 17, 18, 19, 20 take priority from 05/05/2003 (by date of filing PCT / RU03 / 00474).

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