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

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

 

The scope of the invention

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

The level of technology

The introduction of labels in 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 area, including the tagging of radioisotopes, dyes, fluorescent labels, chemiluminescent labels, and the like, with different properties and optimal applications. However, there is a continuous interest in the development of new labels. Of particular interest is the development of new protein labels, including fluorescent protein tag.

Green fluorescent protein (GFP), its mutants and homologues, widely known today because of their intensive use as fluorescent markers in vivo in Biomedicine examined in detail Lippincott-Schwartz and Patterson in Science (2003) 300 (5616):87-91). GFP from hydromedusa Aequorea aequorea (synonym of A. victoria), found Johnson et ai. J Cell Comp Physiol. (1962), 60:85-104, was discovered as part of the bioluminescent system of the Medusa, where GFP played the role of a secondary source that converts blue light from fotobanka aquarina in green light. Then, similar proteins were isolated from several is the space of a few bioluminescent coelenterates, including hydroid jellyfishPhialidium gregariumsea pensRenilla(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 show a green fuorescence, (radiation when 497-509 nm) and acted as secondary sources in bioluminescence. Fluorescent proteins have also been isolated from species Physalia and their N-terminal amino acid sequences were determined. (WO 03/017937).

cDNA encoding GFPA. victoria,was cloned Prasher et al. (Gene (1992), 111(2):229-33). It turned out that this gene may be heterologic 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 provides the opportunity for the use of GFP in cell biology as a genetically encoded fluorescent tags.

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

Conducted many studies to improve the Oia properties of GFP and GFP production-reagents, suitable and optimized for a range of research purposes. Developed new variants of GFP, such as DNA gumanitarnogo GFP, the protein product of which 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 humanitarianly protein is an "enhanced green fluorescent protein" (EGFP). Other mutations to GFP resulted in variants, radiant blue, blue-green and yellow-green light. However, despite the great usefulness of GFP, other fluorescent proteins with properties similar to or different from GFP, would be useful in this area. In particular, the benefits of new fluorescent proteins include opportunities fluorescence resonance energy transfer (FRET), which is based on the new spectra and best suitability for a wide range of excitation. In 1999, the GFP homologues have been cloned from abilitiesand species ofAnthozoa(Matz et al., Nature Biotechnol. (1999), 17: 969-973). This discovery showed that these proteins are not necessary components of bioluminescence mechanism. Retrieved fromAnthozoaGFP-shodya proteins showed a large spectral diversity, including blue-green, green, yellow, red fluorescent proteins and violet-blue effluorescence the chromoproteins (CPs) (Matz et al., Bioessays (2002), 24(10):953-959).

Chapters the initial lack retrieved from AnthozoaGFP-shodeh proteins is a strong oligomerization, which preclude 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 purpose is to provide a new Monomeric fluorescent proteins of different colors, as well as DNA encoding them, which does not have the disadvantages of known GFP.

View Hydrozoa is a potential source of such proteins. In addition to GFP fromAequorea victoriaand GFP homologues from other species ofAequorealike very close homologues of GFP fromAequorea macrodactyla(GenBank accession numbers AF435427-AF435433) andAequorea coerulescens(Gurskaya et al., Biochem J. (2003), 373(Pt 2): 403-408), no other genes encoding fluorescent proteins from Hydrozoa, up to this time was not cloned, although some of them have been characterized at the protein level for a very long time. Cloning and mutagenesis of fluorescent proteins from other sources not related to Aequorea Hydrozoa, is a promising way to generate new fluorescent labels with improved properties.

The invention

The present invention provides a molecule of nucleic acids encoding fluorescent or homebake and their mutants, and derivatives thereof. The specified nucleic acid may be isolated, synthesized by the or be present in their artificial environment.

In some embodiments the nucleic acid of the present invention are selected 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 derivatives thereof.

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 a 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 the same or identical to the above. Nucleic acid sequence that are different from sequences of nucleic acids, due to the degeneracy of the genetic code or gibridizatsii 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, or homologues, derivatives or mutants, or directed the and fused proteins, including proteins of the present invention.

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

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

Another embodiment provides methods of obtaining a chromogenic and/or fluorescent proteins, including protein expression in the appropriate cell host and isolation of protein from it. This method includes (a) providing the nucleic acid molecule of the present invention that encodes a fluorescent or chromo-protein, which is connected with the corresponding sequences regulating expression, (b) the protein expression of the indicated molecules of nucleic acid, and (C) isolation of the protein, essentially free from other proteins.

Also provided are antibodies specific to proteins of the present invention or their fragments.

Additionally, provided cells are the hosts, stable cell lines, transgenic alive is fair and transgenic plants, containing nucleic acids, vectors or expression cassettes of the present invention.

In another embodiment are provided with the oligonucleotides or probes comprising a nucleotide sequence capable of hybridizing to the claimed nucleic acids.

It also provides methods that use chromo - or fluorescent protein of the present invention or encoding its nucleic acid.

In the preferred embodiment is provided by way of the introduction of labels in biological molecules, where the method includes linking a specified biological molecules with the protein of the present invention.

In another preferred embodiment provides a method of introducing a label into the cell, where the method includes producing the protein of the present invention in the cell.

In another preferred embodiment is provided by way of the introduction of labels in the organelle of the cell, where the method includes obtaining the protein of the present invention, merged with appropriate intracellular signal localization in the cell.

In yet another preferred embodiment provides a method for the analysis of biological molecules, cells or organelles of the cell, where the method includes the detection of the fluorescent signal from the protein of the present invention.

In another prepost is enforced embodiment provides a method for the analysis of biological molecules cells or organelles of the cell, where the method includes the expression of the nucleic acid molecules of the present invention in the cell.

Additionally provides kits comprising nucleic acids or vectors or expression cassettes containing these nucleic acid or protein of the present invention.

Brief description of figures

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

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

Figure 3 shows the spectra of the excitation radiation for protein phiYFP-M1G1 (A) and phiYFP-M1C1 (B).

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

Figure 5 shows the spectra of the excitation radiation in wild-type hydr1GFP.

6 shows the absorption spectrum for wild-type hm2CP.

Fig.7 shows the spectra of the excitation radiation in wild-type hm2CP.

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

Detailed description of the invention

As used here, the term "fluoresce the s protein" or "fluoroprotein" means a protein, which is fluorescent; for example, it can be low, medium or intense fluorescence upon irradiation with light of appropriate excitation wavelength. The fluorescent property of these proteins represents a property that is a result of the interaction of two or more amino acid residues of the protein, and not one amino acid residue. Also fluorescent proteins of the present invention do not include proteins that exhibit fluorescence only from residues 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, low or afluorescent. As used here, the terms "chromoprotein" and "fluorescent protein" does not include luciferase, such as Renilla luciferase.

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

As used here, the term "EGFP" refers to a mutant variant of GFP with two aminokislot the e replacement: F64L and S65T (Heim et al., Nature 373 (1995), 663-664).

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

As used here, the term "fragment" is intended to include, for example, alternative playerwindow or truncated, or otherwise split a molecule of nucleic acid or protein.

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

As used here, the term "mutant" refers to a protein, opened in the present invention, in which one or more amino acids are added and/or substituted and/or deleted (deleterow) and/or inserted (insertion) in the N-terminal 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 which encodes a mutant protein. In addition, the term "mutant" here refers to any variant that is shorter or longer than the protein or nucleic acid.

As used here, "a homologue or homology" is the term ISP is lisovanim in this field to describe the connectivity of nucleotide or peptide sequence to another nucleotide or peptide sequence, which is determined by the degree of identity and/or similarity between the compared sequences.

To summarize the above, the present invention is directed to nucleic acid molecules encoding fluorescent and homebake and mutants, variants and derivatives thereof, as well as proteins and peptides encoded by these nucleic acids. Molecules of nucleic acids and proteins of interest are isolated from a species, not related toAequorea Hydrozoa. Target proteins of interest include yellow fluorescent protein phiYFP, fromPhialidium sp.green fluorescent protein hydr1GFP of hydroid jellyfish 1 (hydromedusa 1) of the suborderAnthomedusaeand purple chromoprotein, hm2CP of hydroid jellyfish 2 (hydromedusa 2) suborderAnthomedusae. Also proteins of interest are those which are essentially similar or derivative, or homologue, or mutants of the above-mentioned specific proteins. Also provided are fragments of nucleic acids and peptides, encoded by them, as well as antibodies specific to the proteins and peptides of the invention. In addition, provided cells are the hosts, stable cell lines and transgenic organisms comprising the above nucleic acid molecules. The claimed compositions of proteins and nucleic acids are the COI is the whether in a number of different applications and methods, in particular, for use in the labelling of proteins. In the end, are provided kits for use in such methods and applications.

Molecules of nucleic acids

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

The invention also encompasses nucleic acids which are homologous essentially similar, identical, derivatives or mimetics of nucleic acids encoding proteins or protein fragments of the present invention. The claimed nucleic acids are present in an environment other than their natural environment; for example, they are allocated are present in enriched stake is the operation, present or downregulation ofin vitroor in the cell or in the body, other than their naturally occurring environment environment.

Specific nucleic acid molecules of interest are molecules that encode the following chromium/fluoroprotein of Hydrozoa (and their homologues/derivatives/mutants): yellow fluorescent protein phiYFP fromPhialidium sp.green fluorescent protein, hydr1GFP of hydroid jellyfish 1 of the suborderAnthomedusaeand purple chromoprotein, hm2CP of hydroid jellyfish 2 of the suborderAnthomedusae. Each of these specific types of molecules of the nucleic acid of interest, now individually disclosed in more detail.

phiYFP

Molecules of nucleic acids encoding fluorescent/chromoproteins can be separated from the body of the classHydrozoapreferably squadHydroidamore preferably suborderLeptomedusaemore preferably from the familyCampanulariidaeand even more preferably from the genusPhialidium. In a particularly preferred embodiment the nucleic acid molecule isolated fromPhialidiumsp.encodes a specific protein, called PhiYFP. Homologues/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 more below the detail in the experimental part, also of interest. Bred the coding sequence of the cDNA of wild-type PhiYFP shown in SEQ ID NO: 01.

hydr1GFP

The nucleic acid molecules encoding f fluorescent/chromoproteins can be separated from the body of the classHydrozoapreferably squadHydroidamore preferably suborderAnthomedusae. Specific protein encoded by such a nucleic acid molecule, called hydr1GFP (i.e. anmGFP1). Homologues/mutants/derivatives of this protein are also of interest. Deduced sequence of the cDNA encoding wild-type for hydr1GFP shown in SEQ ID NO: 11.

hm2CP

Molecules of nucleic acids encoding fluorescent/chromoproteins can be separated from the body of the classHydrozoapreferably squadHydroidamore preferably suborderAnthomedusae. Specific protein encoded by such a nucleic acid molecule, called hm2CP (i.e. anm2CP). Homologues/mutants of this protein, such as S3-2 red fluorescent mutant hm2CP described below in more detail in the experimental part, is also of interest. Deduced sequence of the cDNA encoding wild-type for hm2CP shown in SEQ ID NO: 13.

Homologues of the above-described nucleic acid molecules are also of interest. The source of homologous nucleic acids can is to be any kind of plant or animal or 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 the corresponding homologues in nucleotide or amino acid level of at least about 40%, preferably approximately 50%, 55%, 60%, 65%, 70% or more, including 75%, 80%, 85%, 90% and 95% or more. A reference sequence will usually be at least approximately 60 nucleotides, often at least approximately 80 nucleotides, and may extend to the complete sequence is compared. The sequence identity is calculated from the reference sequence. Algorithms for sequence analysis are known in this field, such as BLAST, described in Altschul et al., J. Mol. Biol., 215, pp. 403-10 (1990) (for example, using the default settings, i.e., with parameters w=4 and T=17).

Homologues are identified by any of a variety of ways. The cDNA fragment of the present invention can be used as a hybridization probe against a cDNA library from the target organism, using conditions of low stringency. The probe can be a great fragment or one or more short degenerate primer. Nucleic acid having the similarity of the village is egovernance, detected by hybridization under low stringency, for example, at 50°and 6×SSC (0.9m NaCl /0,09m vehicle crew sodium citrate) followed by washing at 55×1×SSC (01,15M NaCl/of 0.015 M sodium citrate). The sequence identity can be determined by hybridization under conditions of high stringency, for example, at 50°With or higher and 0.1×SSC (15 mm sodium chloride/ 1.5 mm sodium citrate). Nucleic acids having a region that is essentially identical to the submitted sequences, e.g. allelic variants, genetically altered versions of nucleic acid, etc. that are associated with the submitted sequences under high stringency hybridization. Using probes, in particular labeled probes of DNA sequences, one can distinguish homologous or similar genes.

Also provided are nucleic acids that hybridize with the above-described nucleic acids in harsh conditions, preferably under conditions of high stringency (i.e., complementary to the previously described nucleic acids). An example of hybridization in harsh environments is at 50°With or higher and 0.1×SSC (15 mm sodium chloride/ 1.5 mm sodium citrate). Another example of hybridization under conditions of high rigidity is incubation overnight at 42°50% is aStore of formamide, 5×SSC (150 mm NaCl, 15 mm trinacria citrate), 50 mm sodium phosphate (pH 7,6), 5 × denhardt's solution, 10% dextran sulfate, and 20 microgram/ml denatured, cut DNA semen salmon, pre-washing in 0.1×SSC at approximately 65°C. Other conditions of high stringency hybridization are known in this field and can also be used to identify nucleic acids of the invention.

Also provided are nucleic acids encoding the variants, mutants or derivatives of the proteins of the invention. Mutants or derivatives can be obtained on the matrix nucleic acid selected from the above-described nucleic acids, by modification, deletion or addition of one or more nucleotides in a matrix of a sequence, or combinations thereof, to obtain a variant of the matrix nucleic acid. Modifications, additions or deletions can be performed by any means 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, random permutation, oligonucleotide-directed mutagenesis, PCR with the Assembly, a pair of PCR mutagenesis, mutagenesis in vivo, cassette mutagenesis, recursive ensemble mutagenesis, exponential multiple mutagenesis, site-specific the definition mutagenesis, non-specific mutagenesis, gene reassemblage, gene site-saturating mutagenesis (GSSM), synthetic reassemblage with legacies (SLR), or a combination. Modifications, additions or deletions can be made by a method comprising recombination, recursive recombination sequence, phosphothioate-modified mutagenesis DNA mutagenesis on uracil-containing matrix, mutagenesis with a double-pass point restoration for misalignment mutagenesis, mutagenesis of strain, deficient restorations, chemical mutagenesis, radioaktivnyi mutagenesis, dilatational mutagenesis, restriction-selective mutagenesis, restriction mutagenesis with cleaning, the synthesis of artificial genes, multiple mutagenesis, creating multiple chimeric nucleic acids, and combinations thereof. In some embodiments fluorescent proteins encoded by mutant or derivative of the nucleic acids have the same fluorescent properties of the fluorescent protein as wild type. In other embodiments, the mutant or derivative of the nucleic acids encode fluorescent proteins with altered spectral properties, as described here in more detail for mutants phiYFP-Y1, phiYFP-M1, phiYFP-M1G1, phiYFP-MS, S3-2.

In addition, also provided degenerate variants of the nucleic acids that encode b is the CTL of the present invention. Degenerate variants of the nucleic acids include replacement of codons of the nucleic acid to other codons encoding the same amino acids. In particular, degenerate variants of the nucleic acids are created to increase the expression in the cell host. In this embodiment, the codons of the nucleic acids, which are not preferable or are less preferred in the genes in the cell host, replaced by codons that are overly represented in coding sequences in genes in the cell host, where these replaced codons encode the same amino acid. Especial interest is the humanized version of the nucleic acids of the present invention. As used here, the term "humanitarianly" refers to the changes made in the nucleic acid sequence to optimize codons for expression of the protein in mammalian cells (Yang et al., Nucleic Acids Research (1996) 24: 4592-4593). Cm. also U.S. Patent No. 5795737, which describes the humanization of proteins, the disclosure of which is here incorporated by reference.

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

The genomic sequence of interest may include nucleic acid present between the initiating codon and the termination codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome. The genomic sequence of interest may additionally include 5'- and 3'-noncoding region, located in the Mature mRNA, as well as specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc. including flanking genomic DNA approximately 1 TPN, but it is possible and more, the 5'- or 3'-end of the transcribed region.

The nucleic acid molecules of the invention may encode all or part of the claimed protein. Two - and single-stranded fragments can be derived from the DNA sequence by chemically synthesizing oligonucleotides in accordance with standard methods, restriction enzyme digesta, PCR amplification, etc. basically, the DNA fragments will have a size of at least about 15 nucleotides, usually at least p is blithedale 18 nucleotides or about 25 nucleotides, and can be at least about 50 nucleotides. In some embodiments of the claimed nucleic acid molecules may have a size of about 100, about 200, about 300, about 400, about 500, about 600, about 700 nucleotides or more. The claimed nucleic acids may encode fragments of the claimed protein or total protein; for example, the claimed nucleic acid can encode a polypeptide of approximately 25 amino acids, about 50, about 75, about 100, about 125, about 150, about 200 amino acids up to the full length 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 typically "recombinant", i.e. flanked by one or more nucleotides with which it is usually not bound to the chromosome, found in nature in its natural organism, the host.

Nucleic acids of the present invention, for example, having the sequence SEQ ID NO: 01, 03, 05, 07, 09, 11, 13, 15, 17, 19 or 21, the corresponding cDNA, genes of full length and design can be obtained artificially in accordance with plural is om of different methods, well-known experts in this field. Suitable structures of nucleic acids are purified using standard methods of recombinant DNA, as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY, and according to the instructions, for example, in United States Dept. of HHS, National Institute of Health (NIH) Guidelines for Recombinant DNA Research.

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

Also provided vector and other structures of nucleic acid containing the claimed nucleic acid. Appropriate vectors include viral and non-viral vectors, plasmids, Comedy, phages, etc., preferably plasmids, and are used for cloning, amplification, expression, transfer, etc., the nucleic acid sequence of the present invention in a suitable host. The selection of the appropriate vector is clear for a skilled person skilled in the art, and many such vectors are available commercially. For preparation of design partial nucleic acid or full length usually inserted into the vector by attaching DNA ligase to the dissolved enzymes restriction site in the vector. Alternatively, as Adelina nucleotide sequence may be inserted by homologous recombination in vivo, typically, homologous accession plots to vector on the flanks of the desired nucleotide sequence. Homologous areas are legirovaniem oligonucleotides or polymerase chain reaction using primers, including, for example, as homologous parts, and a part of the desired nucleotide sequence.

Also provides expression cassettes or systems usedinter aliato obtain the claimed chromogenic or fluorescent protein or protein insertion or removal of a replica of the claimed nucleic acid molecules. The expression cassette may exist as an extrachromosomal element or may be incorporated into the genome of cells resulting from the introduction of the specified expression cassette into the cell. For the expression of the gene product encoded by a nucleic acid of the invention is expressed in any expression system, including, for example, bacterial systems, yeast, insect, amphibian or mammal. In the expression vector of the specified nucleic acid is operatively linked with a regulatory sequence, which may include promoters, enhancers, terminators, operators, repressor substances and inductors. Methods of obtaining the expression cassette or systems capable of expression of the desired product, known to unificirovannykh in this field.

Cell lines that stably Express the proteins of the present invention, can be selected by methods known in the art (for example, cotransfected with breeding marker such as dhfr, gpt, neomycin, hygromycin, making it possible to identify and highlight tropicabana cells that contain a gene incorporated into the genome).

The above-described expression systems can be used in prokaryotic or eukaryotic hosts. To obtain protein can be used in cell host, such asE. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or stand higher organism such as vertebrates, e.g. COS 7 cells, HEK 293, CHO, oocytesXenopusetc.

If you are using any of the above a host cell or appropriate cell hosts or organisms for replication and/or expression of nucleic acids of the invention, the resulting replicated nucleic acid is expressed protein or polypeptide are within the claims of the invention as a product of the host cell or organism. The product can be selected in a suitable way known in this field.

Also of interest to the promoter sequence of the genomic sequences of the present invention, where the sequence 5'-end sections can the be utilized for promoter elements, including enhancer binding sites, which, for example, provide for the regulation of gene expression in cells/tissues, where downregulation of gene declared proteins.

Also provided small DNA fragments of the claimed nucleic acids that are used as primers for PCR, hybridization screening of samples, etc. Large DNA fragments used to obtain the encoded polypeptides, as previously described. However, for use in geometric amplification reactions, such as geometric PCR, uses a pair of small DNA fragments, i.e. primers. The exact composition of the primer sequences is not critical for the invention, but for most applications the primers will gibridizatsiya with the stated sequence in harsh conditions, as is well known in this field. It is preferable to choose a pair of primers that will give the product of amplification of at least about 50 nucleotides, preferably at least about 100 nucleotides, and may extend to the complete sequence of the nucleic acid. Algorithms for the selection of primer sequences commonly known and available in commercial software packages. The amplification primers hybridize with complementary strands of DNA and will premirovat towards each other.

Molecules nuclein the second acid of the present invention can also be used to determine gene expression in the biological sample. The method, which investigates the cells for the presence of specific nucleotide sequences, such as genomic DNA or RNA, is well developed in this area. Briefly, DNA or mRNA extracted from the sample cell. the mRNA may be amplified RT-PCR using reverse transcriptase inhibitor for the formation of a complementary strand of DNA with advanced polymerase chain reaction amplification using primers specific for said DNA sequences. Alternatively, an mRNA sample is separated by gel-electrophoresis, transferred to a suitable carrier, such as nitrocellulose, nylon, etc. and then probing the fragment of the claimed DNA as a probe. Can also be used in other ways, such as tests of linkage of the oligonucleotide, hybridizationin situand hybridization of DNA probes attached to a solid chip. Detection of mRNA, hybridizing with the stated sequence, indicative of gene expression in the sample.

The claimed nucleic acids, including flanking sites of the promoter and the coding sections, can metrovacesa in a variety of ways known in this field to obtain a purposeful changes in the strength of the promoter or change the sequence of the encoded protein or properties of the encoded protein, including fluorescent St. istwa of the encoded protein.

In many embodiments the nucleic acid found in the form ofAequoreanot included in the scope of the invention. In some embodiments, the homolog of the GFP and the nucleic acid encoding it, fromAequorea victoria Aequorea macrodactylaandAequorea coeridscensnot included in the scope of the claimed invention.

Proteins

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

In many embodiments of the claimed proteins have a maximum absorption in the range from approximately 300 to 700, typically 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 another wavelength, while the excitation spectra of the claimed proteins are usually approximately from 300 to 700 nm. The claimed proteins generally have a maximum extinction coefficient, which is priblizitel is but from 25,000 to 150,000, and usually from about 45,000 to of 129,000. The size of the claimed protein is usually in the range from approximately 150 to 300 amino acids, and typically from about 200 to 300 amino acid residues and in General they have a molecular weight in the range from about 15 to 35 kDa, usually about from 17.5 to 32.5 kDa.

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

In some embodiments of the claimed proteins are placed quickly after expression in a cell host. Under fast putting understood that the proteins reach their tertiary structure, which ensures their chromo - or fluorescent property through a short period of time. In these embodiments, the proteins are placed within a period of time, which in General does not exceed about 3 days, usually no more than about 2 days, and often does not exceed about 1 day.

Specifically the proteins, of interest are chromium/fluoroprotein (and homologs, mutants, and their derivatives) of the species, not related to Aequorea Hydrozoa: phiYFP fromPhialidiumsp., green fluorescent protein hydr1GFP of hydroid jellyfish 1 (hydromedusa 1) of the suborderAnthomedusaeand purple chromoprotein, hm2CP of hydroid jellyfish 2 (hydromedusa 2) suborderAnthomedusae. Each of these specific types of polypeptide patterns of interest, hereinafter will be described individually in more detail.

phiYFP (and its derivatives/mutants)

Proteins of this embodiment has a maximum light absorption in the range from approximately 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, whereas the emission maximum is usually in the range from approximately 400 nm to 650 nm, and more often from about 450 to 600 nm, while in many incarnations radiation spectra are in the range of from about 470 to 550 nm, for example, from 505 to 515 or 520 to 530 nm, or from 530 to 540 nm. The size of the declared proteins typically ranges from about 200 to 250, usually from about 210 to 240 amino acid residues, and generally have a molecular weight in the range of about from 20 to 30, usually from about 22,50 up of 27.50 kDa. Specific interest in many voploscheni the x is phiYFP, which has the amino acid sequence shown in SEQ ID NO: 02. Also of interest are the mutants and derivatives of this sequence, for example, phiYFP-Y1, phiYFP-M1, phiYFP-M0, phiYFP-M1G1 and phiYFP-M1C1 as shown in SEQ ID No: 04, 06, 08, 18 and 20, respectively.

hydr1GFP (and its derivatives/mutants)

In many embodiments of the claimed proteins have a maximum absorption of light in the range 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, whereas the radiation spectra of the claimed proteins are typically in the range of 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 claimed protein is usually in the range of about 200 to 300 amino acids, and typically from about 220 to 290 amino acid residues, and generally have a molecular weight in the range from approximately 25 to 35 kDa, usually about from 26.5 to 32.5 kDa. Specific interest in many embodiments is a wild type fluorescent protein hydr1GFP, which has the amino acid sequence shown in SEQ ID No: 12, mutants and its derivatives.

hm2CP (and its mutants)

In many embodiments of the claimed proteins have a maximum absorption of light within AP is sustained fashion from 350 to 650, usually from about 450 to 600, and more often from about 490 to 595 nm, for example, 560 to 590 nm, while the absorption spectra of the claimed proteins are typically in the range of about 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 claimed protein is usually in the range of about 200 to 250, usually from about 210 to 240 amino acid residues, and in General they have a molecular weight in the range of about from 20 to 30 kDa, usually from about 22,50 up of 27.50 kDa. Specific interest in many embodiments is hm2CP (anm2CP), which has the amino acid sequence shown in SEQ ID No: 14. Also of interest is a mutant of this sequence, such as red fluorescent protein S3-2, etc. as shown, for example, in SEQ ID NO: 16.

Also provided homologs or proteins, the sequence of which differs from the above mentioned specific amino acid sequences claimed in the invention, that is, SEQ ID NO: 02, 04, 06, 08, 10, 12, 14, 16, 18, 20 or 22. Under the homolog is meant a protein having at least about 55%, typically at least about 60% and often at least about 65% amino acid sequence identity compared the amino acid sequences of SEQ ID NO: 02, 04, 06, 08, 10, 12, 14, 16, 18, 20 or 22 as determined using MegAlign, DNAstar clustal algorithm as described in D.G. Higgins and P.M. Sharp, "Fast and Sensitive multiple Sequence Alignments on a Microcomputer," CABIOS, 5 pp. 151-3 (1989) (with 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, for example 70%, 75%, 80%, 85%, 90% (for example, 92%, 93%, 94%) or higher, for example 95%, 96%, 97%, 98%, 99%, 99,5%, especially for the sequence of amino acids that provide a functional region of the protein.

Also provided are proteins that are essentially identical to the wild-type protein, where essentially identical means that the protein has the amino acid sequence identity to the sequence of the wild-type protein at least about 60%, usually at least about 65% and often at least about 70%, and in some specimens 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 occurring in nature. Mutants and derivatives can preserve the biological properties of proteins wild-type (e.g., naturally occurring), or may have biological properties that differ from the proteins di the CSO type. The term "biological property" of the proteins of the present invention include, but without limitation, the spectral properties such as the maximum absorption, emission maximum, the maximum extinction coefficient, brightness (for example, as compared with the wild-type protein or other reference protein such as green fluorescent protein (GFP) from A.victoria), and the like; biochemical properties, such as stability in vivo and/or in vitro (e.g., half-life); the speed of maturation, propensity for aggregation and propensity for oligomerization, and other such properties. Mutations include substitution of one amino acid, deletion or insertion of one or more amino acids, truncations or extensions of the N-end truncations extensions C-Terminus, etc.

Mutants and derivatives can be obtained using standard molecular biology techniques, as described in detail in the section "nucleic acid molecule" above. Here are several of the mutants. The examples provide General techniques and the use of standard methods, so that professionals skilled in this field can easily get a large number of additional mutants and to test whether altered biological (such as biochemical, spectral, etc) property. For example, fluorescence intensity can be measured using a spectrophotometer peresichnyi the excitation wavelengths.

Derivatives can also be obtained using standard methods and include changes using RNA, chemical modification, modification after translation after transcription, etc. for Example, derivatives can be obtained by methods such as altered phosphorylation or glycosylation, or acetylation, or limitirovanie, or different types of cleavage during maturation, etc.

The proteins claimed in the invention, which are naturally occurring proteins that are present in the environment, which is not normal for them, for example, they are separated from the normal environment. For example, provided a purified protein, where "purified" means that the protein is present in the mixture, which is essentially not containing achromogenes or fluorescent proteins of interest, where "essentially does not contain" means that less than 90%, usually less than 60% and more often less than 50% of the content of the mixture is achromogenes or fluorescent protein or mutants. Proteins of the present invention may also be present in the selected shape, which means that the protein is essentially not containing other proteins and other biological molecules found in natural surroundings, such as oligosaccharides, nucleic acids and fragments thereof, and the like, where the term "essentially does not contain" in this case means, less than 70%, usually less than 60% and more often less than 50% of the composition containing the isolated protein, represents some other biological molecule found in nature. In some embodiments, the proteins are present in a substantially purified form, where "essentially purified form" means purified to at least 95%, usually at least 97% and often at least 99%.

Also provided are fragments of proteins occurring in nature, as well as mutants and derivatives of the proteins described above. Biologically active fragments and/or fragments corresponding to functional domains, etc. represent a private interest. Fragments of interest are polypeptides, which 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 the size of 300 amino acids or more, but usually will not exceed about 250 amino acids, where the fragment will have a stretch of amino acids that is identical with the stated protein size of at least about 25 amino acids, usually at least about 45 amino acids, and in many embodiments at least about 50 amino acids. Some is plewniak the claimed polypeptides are approximately 25 amino acids, about 50, about 75, about 100, about 125, about 150, about 200, or about 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 protein of the wild type.

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

It also provides fusion proteins comprising a protein of the present invention, or its fragments, fused, for example, a degenerate sequence, the sequence vnutri emochnoy localization (for example, nuclear localization signal, proximally targeting signal sequence targeting the Golgi apparatus, consistency, targeting the mitochondria and so on), signal peptide, or any protein or polypeptide of interest. Proteins mergers may include, for example, fluoro/chromalox, as claimed in the invention, the polypeptide and the second polypeptide (the"merger partner")connected to the N-Terminus and/or C-Terminus fluoro/homopolyamide. Partners of the merger include, but are not limited to, polypeptides that can bind antibodies specific to the merge partner (e.g., epitope tags), antibodies or their binding fragments, polypeptides that provide a catalytic function or cause cell response, ligands or receptors or their mimetics, etc. In such proteins merge in the General case, the merge partner is not naturally associated with fluoro/chromalloy part of a fusion protein and is usually fluoro/chromolume not of the form Hydrozoa, as claimed in the invention, or a derivative and/or fragment; that is, they are not detected in the form of Hydrozoa.

Also provided are antibodies that are specifically associated with fluorescent or chromoprotein of the present invention. Appropriate antibodies can be obtained using methods known in this field. For example, Policl the national antibodies can be obtained as described (Harlow and Lane Antibodies: A Laboratory Manual, (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), and monoclonal antibodies may be obtained as described in (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')2and Fab are also of interest.

Transgenic

Nucleic acids of the present invention can be used to obtain transgenic organisms or site specific gene modifications in cell lines. Transgenic cells, as claimed in the invention contain one or more nucleic acids, as claimed in the present invention, as a transgene. For the purposes of the invention any acceptable a host cell can be used, including prokaryotic (e.g.,Escherichia coli,Streptomycessp.,Bacillus subtilis,Lactobacillus acidophilusand so on) or eukaryotic cell hosts. Transgenic organism, as claimed in the invention may be prokaryotic or eukaryotic organism, including bacteria, cyanobacteria, fungi, plants and animals, in which one or more cells of the organism contain heterogeneous nucleic acid, as claimed in the invention, is introduced through human intervention, such transgenic methods, which is known in this field.

The selected nucleic acid of the present invention can be introduced into the host by methods known in this field, such as infection, transfectional, transformation or transconjugate. Ways of transfer of a molecule of nucleic acid (i.e., DNA) in such organisms are widely known and are provided in references such as Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3ndEd., (2001) Cold Spring Harbor Press, Cold Spring Harbor, NY).

In one embodiment, the transgenic organism can be prokaryotic organism. Methods of transformation of prokaryotic hosts are well described in the art (for example, 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 can be fungi, such as yeast. Yeast is widely used as media for the expression of heterogeneous gene (for example, see Goodey et al Yeast biotechnology, D R Berry et al, eds, (1987), Allen and Unwin, London, pp 401-429) and King et al Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, Blackie, Glasgow (1989) pp 107-133). Several types of yeast vectors are available, including integrative vectors, which require recombination with the genome of the host for their support, and Autonomous replicated plasmid vectors.

Another host organism is an animal. Transgenic animals can be obtained transgenic ways the AMI, known in this area and are provided in the 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., L.C. Anderson, F.M. Loew, F.W. Quimby (Eds), American Medical Association, the 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 design of nucleic acid randomly included in the genome. Vectors for sustainable inclusion include plasmids, retroviruses and other animal viruses, YAC, etc

Nucleic acids can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by deliberate genetic manipulation, such as microinjection or infection with recombinant virus or recombinant virus vector, etc. the Term "genetic manipulation" does not include classical breeding or in vitro fertilization, and preferably is aimed at the introduction of recombinant nucleic acid molecules. These nucleic acid molecules can be incorporated into a chromosome or they can be extrachromosomal replicating DNA.

Design DNA for homologous recombination will be content shall be at least part of the nucleic acid of the present invention, where the gene has the desired genetic modificato(s) and includes a region of homology to the target locus. The DNA structures for arbitrary switching is not necessarily contain a region of homology with the mediator recombination. Can easily be included markers for positive and negative selection. Methods for producing cells having the target gene modification via homologous combination known in the field. For different ways transfection of mammalian cells, see Keown et al., Meth. Enzymol. (1990) 185:527-537.

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

The transgenic animal can be any animal, non-human, including mammalian, non-human, such as mouse, rat, bird or amphibian, etc. and used in a functional study, screening drugs, etc. are Typical examples of ISOE is isawanya transgenic animals include those described below.

Can also be obtained from transgenic plants. Methods for producing transgenic plant cells and plants are described in U.S. patents№№ 5767367; 5750870; 5739409; 5689049; 5689045; 5674731; 5656466; 5633155; 5629470; 5595896; 5576198; 5538879; 5484956; disclosure of which is incorporated here by reference. Methods of obtaining transgenic plants are also considered in Plant Biochemistry and Molecular Biology (eds. Lea and Leegood, John Wiley & Sons) (1993) pp. 275-295 and in Plant Biotechnology and Transgenic Plants (eds. Oksman-Caldentey and Barz), (2002) 719 p.

For example, embryogenic explants containing somatic cells, can be used to obtain transgenic host. After collecting the cells or tissue exogenous DNA of interest is introduced into plant cells, known for such introduction of a number of different ways. If you have dedicated protoplasts there is the possibility for insertion through DNA-mediated gene transfer protocols, including the incubation of the protoplasts with the selected DNA, such as plasmids containing target exogenous sequence encoding in the presence of polyvalent cations (e.g., PEG or PLO); or electroporation of protoplasts in the presence of a selected DNA comprising a target exogenous sequence. The protoplasts, which successfully incorporated the exogenous DNA is then selected, grown in the callus, and ultimately to a transgenic plant in which ontake with suitable quantities and relations stimulating factors, such as auxins and cytokines.

Other appropriate methods of obtaining plants can be used, such as the use of "gene gun", or Agrobacterium-mediated transformation, which is available for qualified professionals in this field.

Applications

Fluorescent proteins of the present invention (as described above components, as claimed in the invention), find use in several different applications. For example, they can be used in the methods of the introduction of labels, in the analysis or detection of biological molecules, cells or organelles of the cell. Typical uses for each of these types of proteins will be described below, and applications described herein are only illustrative and are in no way intended to limit the applicability of the proteins of the present invention according to this description.

In the preferred embodiment related to the method of introduction of labels in a biological molecule, a cell or a cell organelle, the claimed proteins find use as labelsin vivo(or molecule-reporters) in the analysis of cellular and molecular biology. The analyses of interest include, but are not limited to, analyses of gene expression, protein localization and co-localization, protein-protein interactions, interactions of the protein and nucleic acid, interactions, nucleic acid - nucleic acid cellular localization and localization of organelles and cell interactions cell organelles, etc. Fluorescent proteins of the present invention find application as a label biomolecules or label cell organelles in living and fixed cells; as markers in the cell or organelle merge, as markers of integration cells or organelles, as a marker of transfection (e.g., as a label for the selection of transfected cells containing the expression vector encoding at least one fluorescent protein of the invention), as a probe real-time operating at ecologicaleconomic concentrations, etc.

In addition, the claimed proteins can be used in the method of analysis of biological molecules. For example, they are used to identify and/or measure the expression of a target protein or polypeptide in a biological material. This method comprises: i) introducing into the cell the nucleic acid molecule containing the nucleotide sequence encoding a fluorescent protein of the present invention, where this nucleic acid molecule operatively linked and is under the control sequences that regulate the expression that controls the expression of the specified target protein or polypeptide; (ii) the expression of the mentioned n Cleanaway acid under appropriate conditions; and (iii) detecting fluorescent radiation fluorescent protein as a tool for measuring the expression of the protein of interest.

In particular, the claimed proteins are used for identifying and/or measuring the expression and/or localization of the target protein or polypeptide in a biological material. This method comprises: i) introducing into the cell the nucleic acid molecule containing the nucleotide sequence encoding a fluorescent protein of the present invention, where this nucleic acid molecule is fused with a sequence that encodes a target protein or polypeptide and operatively connected with and is under control sequence that controls the expression of the specified target protein; (ii) culturing cells under conditions suitable for expression of the target protein; and (iii) detecting fluorescent radiation fluorescent protein as a way of measuring the expression of a target protein.

Target applications include use of the claimed proteins in ways fluorescence resonance energy transfer (FRET). In these methods, the claimed proteins serve as donors and/or acceptors in combination with a second fluorescent protein or dye, such as other fluorescent protein, as claimed in the invention, or a fluorescent protein as described in Matz e al., Nature Biotechnology 17:969-973 (1999); green fluorescent protein from Aequorea victoria or fluorescent mutant, for example, as described in U.S. patent N 6066476; 6020192; 5985577; 5976796; 5968750; 5968738; 5958713; 5919445; 5874304, disclosure of which is incorporated here by reference; other fluorescent dyes such as coumarin and its derivatives, 7-amino-4-methylcoumarin and aminocoumarin; dyes for living tissues; cascade blue; or fluorescein and its derivatives, such as fluoresceinisothiocyanate and Oregon green; rodinovym dyes such as Texas red, tetramethylrhodamine, eosine and erythrosine; cyan dyes, such as Cy3 and Cy5; macrocyclic chelates lanthanoide ions such as quantum dye; and chemiluminescent dyes, such as luciferase, including the ones described in U.S. patents№№ 5843746; 5700673; 5674713; 5618722; 5418155; 5330906; 5229285; 5221623; 5182202; disclosure of which is incorporated here by reference.

Some examples of that in FRET-assays are declared fluorescent proteins include, but are not limited to, detection of protein-protein interactions, such as in twohybrid the system of mammals, transcription factor dimerization, membrane protein multimerization, multibillion complexation; as biosensor for many different events, where the peptide or protein covalently bind FRE fluorescent combination, including the claimed fluorescent proteins, and the bound peptide or protein is, for example, protease-specific substrate is mediated by caspase cleavage, the peptide undergoes a conformational change upon receipt of the signal, which increases or decreases FRET, such as PKA regulatory domain (camp-sensor), the site of phosphorylation (e.g., where is the site of phosphorylation of the peptide, or peptide has binding specificity of phosphorylated/dephosphorylating domain of another protein, or peptide has the Ca2+binding domain. In addition, the application of fluorescence resonance energy transfer, or FRET, in which proteins of the present invention find use include, but are not limited to, are described in U.S. patents№№ 6008373; 5998146; 5981200; 5945526; 5945283; 5911952; 5869255; 5866336; 5863727; 5728528; 5707804; 5688648; 5439797, disclosure of which is incorporated here by reference.

Fluorescent proteins of the present invention find application in the detection of the influence of the test material on the regulation of expression and/or translocation of one or more target proteins in the cell. Alternatively, they are used in the method of detecting the expression of the target protein and the simultaneous activity of sequences regulating expression, in response to the test material. Fluorescent proteins which are also used in the method of comparing the activity of two or more sequences regulating expression in the cell in response to the test material. Such methods can be performed in the presence and in the absence of the test material, whose influence on the way that should be measured.

Fluorescent proteins of the present invention also find use in applications including automated screening matrix of cells expressing fluorescent group-reporters, using microscopic display and electronic analysis. Screening can be used to open a medicine and in the field of functional genomics, where the claimed proteins are used as markers of whole cells to detect changes in multi-cellular transformation and movement, for example, the formation of multicellular tubules (the formation of blood vessel) by endothelial cells, moving cells through the system Fluoroblok Insert (Becton Dickinson Co.), wound healing or regrowth of a neuron. Screening can also be used, if the proteins of the present invention are used as tokens, are merged with peptides (such as the target sequence) or proteins that detect changes in intracellular localization as an indicator of cellular activity, for example, in signal transduction, such as factors kinase activity and translocation of transcription when the stimuli for the production of the AI. Examples include protein kinase C, protein kinase A, the transcription factor NFkB and NFAT; cellular cyclic proteins, such as cyclin A, cyclin B1 and cyclin E; cleavage by the protease with sequential movement of the split substrate; phospholipids with markers for intracellular structures such as the endoplasmic network, Golgi apparatus, mitochondria, peroxisomes, nucleus, nucleoli, plasma membrane, histones, endosome, complementary mechanism or microtubules.

Proteins of the present invention can also be used in screening high content to detect joint localization of other merged fluorescent protein marker localization as indicators of movements of intracellular fluorescent proteins/peptides or only as tokens. Examples of applications, including automated screening of sets of cells in which the claimed fluorescent proteins are used, include U.S. patent No. 5989835; as well as WO 00/17624, WO 00/26408, WO 00/17643 and WO 00/03246, disclosure of which is incorporated here by reference.

Fluorescent proteins of the present invention also find use in screening tests with high performance. The claimed fluorescent proteins are stable proteins with half-lives greater than 24 hours. Also provided is destabilized variants sawlani the fluorescent protein with a reduced half-lives, which can be used as reporters of transcription for the opening of the medicinal product. For example, a protein, as claimed in the invention, may be merged with the proposed proteolytic signal sequence derived from a protein with a shorter half-life, such as PEST-sequence of the gene interdiscursivity mouse, Boxing decomposition cycline B1 of mouse or ubiquitin, etc. To describe destabilized proteins and vectors that can be used to achieve the same, see for example, U.S. patent N 6130313, the disclosure of which is incorporated here by reference. Promoters in signal transduction can be detected using destabilized variants of the claimed fluorescent proteins for screening drugs, 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 a second messenger detectors by merging stated Belov with specific domains, such as PKC-gamma Ca binding domain of PKC-gamma DAG binding domain, SH2 domain, or SH3 domain, etc.

Secreted forms of the claimed proteins, which in turn can be used in several different applications, can be obtained by merging secreted leading sequences of the claimed protein.

Declared white and also find use in applying for sorting cells, activated fluorescence (FACS). In such applications the claimed fluorescent protein is used for labeling cell population, and the resulting labeled population of cells is then subjected to a sorting device for sorting cells activated by fluorescence, as is well known in this field. Ways FACS described in U.S. patent No. 5968738 and 5804387; the disclosure of which is incorporated here by reference.

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

Additional application of the proteins of the present invention include use as markers for introduction into cells or animals in the calibration in quantitative measurements; as markers or reporters in the oxygen biosensor devices for monitoring cell viability; as markers or tags for animals, Pets, toys, food, etc.

Declared fluo asterousia proteins also find use in the analysis of proteasome cleavage. For example, inactivated by cleavage of fluorescent assays can be performed using the claimed proteins, where the claimed proteins prepared for inclusion in processorspecific sequence cleavage, without destroying the fluorescent properties of the protein. The cleavage of a fluorescent protein activated protease fluorescence decreases sharply due to the destruction of the functional chromophore. Alternatively, activated by cleavage, the fluorescence can be applied using Belov of the present invention, when receiving proteins containing additional spacer elements sequence near/or inside of the chromophore. This option greatly reduces its fluorescence activity, because part of the functional chromophore separated by a spacer. The spacer is enclosed in the frame of two identical specific proteinopathies sites of cleavage. The splitting of the activated protease spacer carved least two residual "subunit" fluorescent protein could be re-reassemblage to obtain a functional fluorescent protein. Both of the above applications could be developed in the analysis for a number of different types of proteases, such as caspase and others.

The claimed proteins can also be used in the analysis fordetermination of phospholipid composition in biological membranes. For example, fusion proteins target proteins (or any other type of covalent or non-covalent modification of the requested proteins)that allow binding with specific phospholipids for localization/visualization in model phospholipid distribution in biological membranes, while allowing colocalization membrane proteins in specific phospholipid lesions can be associated with the claimed protein. For example, the PH domain of GRP1 has a high affinity for phosphatidyl-inititatives (PIP3), but not to PIP2. Protein fusion between the PH domain of GRP1 and declared proteins can be created for specific labeling of PIP3-rich areas in biological membranes.

The claimed fluorescent proteins also find use as biosensors in prokaryotic and eukaryotic cells, such as Ca2+ion indicator; pH indicator; indicator phosphorylation; or other indicator, such as magnesium, sodium, potassium, chloride and halide ions. Methods of using fluorescent proteins as biosensors also include those described in U.S. patent No. 5972638; 5824485 and 5650135 (as well as the references cited there), the disclosure of which is incorporated here by reference.

Antibodies claimed in the invention described above, also find use in many applications, including differe zirovanii declared proteins from other fluorescent proteins.

Sets

Also provided in accordance with the present invention, kits for use in implementing one or more of the above applications. In preferred embodiments, the kits can be used for labeling biological molecules. Sets usually include a protein of the invention or nucleic acid, its encoding, preferably with elements for the expression of the claimed Belov, for example, the design, such as a vector comprising nucleic acid encoding the claimed protein. The invention also includes means for obtaining such components sets. The method may include cDNA from jellyfish Hydrozoa and a pair of oligonucleotide primers to obtain nucleic acid, as claimed in the invention, for example, by PCR, or such means may include many fragments of nucleic acid, which when legirovanii can give nucleic acid encoding a fluorescent protein of the present invention, etc. kit Components are usually present in the corresponding storage medium, such as a buffered solution, typically in a suitable container. It also sets may contain antibodies specific for the indicated protein. In some embodiments of the kit include many different vectors, each of which encodes savlon the th protein, where the vectors are designed for expression in different environments and/or under different conditions, for example, constitutive expression, where the vector includes a strong promoter for expression in mammalian cells or vector with weak promoter, multiple cloning sites for selective insertion of promoter and non-standard expression, etc.

In addition to the above-described components of the claimed kits will further include instructions for carrying out the proposed methods. These instructions may be present in the claimed kits in various forms in the amount of one or more of which may be present in sets.

The following examples are offered as illustrative, but not restrictive.

EXAMPLES

Example 1

Cloning, sequencing and receiving recombining protein phiYFP

Bright yellow fluorescence in hydromedusaPhialidium Sp. (Cnidaria; Hydrozoa; Hydroida; Leptomedusae; Campanulariidae) was detected using a fluorescent microscope. To find the protein responsible for the fluorescence in the Medusa, was chosen strategy based on the expression screening of cDNA library in theE. coli. Amplificatoare cDNA samples were prepared using the kit for the amplification of cDNA SMART (Clontech) and cloned into PCR-Script vector (Stratagene). Approx the tion 10 5recombinant clones were skanirovaniya visually using a fluorescent stereoscopic microscope. Two fluorescent clone encoding the same yellow fluorescent proteins, were found and were named phiYFP. Nucleic acid and amino acid sequences for phiYFP shown in SEQ NO: 01, 02, and 23. Comparison of phiYFP with GFPA. victoriashown in figure 1. As it turned out, phiYFP more similar to GFP (50% identity)than with fluorescent proteins derived from coral.

To facilitate purification of protein coding section phiYFP gene was cloned in expressing vector pQE30 (Qiagen) so that the recombinant protein contained six-his-tag tag at its N end. After expression in E. coli, phiYFP protein was purified by metal-affinity resin TALON (Clontech). The spectra of the excitation radiation for phiYFP had peaks at 525 nm and 537 nm (figa), respectively. In contrast to wild-type GFP from A. victoria new protein has only one peak absorption-radiation, probably in accordance with deprotonirovannym state of the chromophore.

Example 2

Mutagenesis PhiYFP

The sequence PhiYFP encoding nucleic acid, was obtained as described above in Example 1. The applicant was modified nonspecific mutagenesis encoding wild-type protein. Nonspecific mutagenesis phiYFP led to a brighter Mutan is a, named phiYFP-Y1, with a slightly modified spectrum of the excitation radiation. This mutant contained three amino acid substitutions, in particular S2P, E174G, I201M (SEQ ID NO: 03, 04, and 24). phiYFP-Yl showed the brightness at 1.5 to 2 times higher than wild type phiYFP with from the visual comparison of colonies of E. coli expressing these fluorescent proteins. In addition, phiYFP-Yl demonstrates the spectrum of radiation, a bit is shifted into the red area, with a peak at 542 nm (see figv).

Proteins as phiYFP and phiYFP-Yl, was found to represent dimers. This was shown by protein gel electrophoresis is not heated protein samples (see Baird et al., above, 2000). Under these conditions, these FPs moved as yellow fluorescent band at approximately 50 kDa. Gel-filtration tests showed dimeric state phiYFP and phiYFP-Yl. Purified protein samples (˜1 mg/ml) were loaded on a column of Sephadex-100 (0,7×60 cm) and loirevalley solution of 50 mm phosphate buffer (pH 7.0) and 100 mm NaCl. EGFP, HcRedl and DsRed2 (Clontech) was used as monomer, dimer and tetramer standards, respectively.

Site-directed mutagenesis was used to increase the degree of monomeres option phiYFP-Y1. It was introduced six amino acid substitutions, in particular V103N, M166R, Y198N, T202S, TC, V221K. This mutant phiYFP-MO bore 9 substitutions: S2P, V103N, M166R, E174G, Y198N, I201M, T202S, TC, V221K (SEQ ID NOs: 05, 06, and 25). phiYFP-MO shows the low protein folding and low brightness, when it was expressed in E. coll. Its spectra excitation radiation is focused in the blue region compared to the parent mutant (maxima at 517 and 529 nm, respectively; figs). phiYFP-MO was more Monomeric protein according to gel-filtration tests.

To improve phiYFP-MO, the applicant used the non-specific mutagenesis. Diversity PCR Random Mutagenesis kit (CLONTECH) was used under conditions optimal for 5-6 mutations per 1000 BP colonies of E. coli expressing mutant proteins were visually skanirovaniya fluorescent stereoscopic microscope (SZX-12 (Olympus). The most striking clone with the spectrum, obviously shifted into the red area (compared to the original phiYFP-MO), was characterized further. This mutant, designated as phiYFP-M1, contained the following amino acid substitutions: E88D, V103N, MS, E174G, I201M, T202S, TC, V221K (SEQ ID NOs: 07, 08, and 26). The spectra of the excitation radiation for this protein had peaks at 524 and 539 nm, respectively, similar to the peaks phiYFP wild-type (figure 2). Purified phiYFP-M1 had a molar extinction coefficient 130000 M-1cm-1and fluorescent quantum yield of 0.40. When determining the molar extinction coefficient, the applicant relied on the assessment of the concentration of the Mature chromophore. Protein was denaturirovannah equal volume of alkali 2M NaOH. Under these conditions, GFP-like chromophore absorbs at 446 nm, and the molar ratios is NT extinction is 44000 M -1cm-1(Ward, W. W. Properties of the coelentrate green-fluorescent protein, in Bioluminescence and Chemiluminescence. Academic Press (1981), 235-242). Were measured absorption spectra of native and denatured by alkali phiYFP-M1. The molar extinction coefficient for the native state of the protein were assessed based on the absorption denaturirovannogo protein. To determine the quantum yield of fluorescence phiYFP-M1 was compared with equally absorbing EGFP (quantum yield of 0.60 ((Patterson et al., J. Cell. Sci. (2001), 114: 837-838)). phiYFP-M1 was slobodkina protein according to gel-filtration tests.

To increase expression in mammalian cells the applicant synthesized "humanitarian" version of phiYFP-M1 using codons optimized for mammals (SEQ ID NOs: 09, 10 and 27). "Humanitariannet" version phiYFP-M1 was subjected to site-directed and non-specific mutagenesis to obtain versions of the protein, emitting light in the green and blue areas. Were obtained mutant fluorescent proteins with green and blue fluorescence. Green mutant gumanitarnogo phiYFP-M1, named phiYFP-M1G1, contains the following amino acid substitutions compared to the phiYFP-M1) T65S, L148Q, Y203T, K231T, T232A (SEQ ID NO: 17, 18, and 31). The blue mutant gumanitarnogo phiYFP-M1, named phiYFP-M1C1, contains the following amino acid substitutions compared to the phiYFP-M1): L6Q, T65S, Y66W, N124K, C147Y, L148Q, Y203T, V224L (SEQ ID NO: 19, 20, and 32). The spectrum of the excitation radiation for this protein is shown in figa, B.

Example 3

Cloning, sequencing and receiving recombining protein hydr1GFP

Bright green fluorescence was detected in hydromedusa 1 (length of approximately 1 mm, figure 4) of the suborderAnthomedusae(Cnidaria, Hydrozoa, Anthomedusae) using a fluorescent microscope. To search for the gene responsible for the fluorescence in this Medusa was implemented strategy based on the expression screening of cDNA library in theE. coli. Amplification of cDNA samples was performed using a set of SMART cDNA (Clontech) and cloned in the vector PCR-Script (Stratagene). Approximately 105recombinant clones were visually skanirovaniya using fluorescent stereoscopic microscope. Three fluorescent clones were identified, where each coded the same green fluorescent protein, which is named hydr1GFP. Nucleotide and amino acid sequence for this protein is shown in SEQ ID NO: 11, 12, and 28. Comparison hydr1GFP with GFPA. victoriashown in figure 1. hydr1GFP, as it turns out, has more similarities with GFP (37% identity)than with fluorescent protein from coral.

To facilitate purification of protein coding section hydr1GFP gene was cloned in expressing vector pQE30 (Qiagen) so that the recombinant protein contained six-his-tag tag at its N end. After expression in E. coli, hydr1GFP protein was cisen using metal-affinity resin TALON (Clontech). The spectra of the excitation radiation for phiYFP was filmed at 474 nm and 494 nm (figure 5), respectively. In contrast to wild-type GFP from A. victoria new protein has only one peak absorption-radiation, probably in accordance with deprotonirovannym state of the chromophore.

Example 4

Cloning, sequencing and receiving recombining protein hm2CP

Bright green fluorescence was detected in a small hydromedusae 2 of the suborderAnthomedusae(Cnidaria, Hydrozoa, Anthomedusae) using a fluorescent microscope. To search for FP from this jellyfish the applicant has chosen a strategy based on the expression screening of cDNA library in theE. coli. Amplification of cDNA samples was performed using a set of SMART cDNA (Clontech) and cloned in the vector PCR-Script (Stratagene). Approximately 105recombinant clones were visually skanirovaniya using fluorescent stereoscopic microscope or the naked eye. Unexpectedly, the applicant has not detected fluorescent clones. Instead, it was identified violet effluorescence CF (hm2CP). Nucleotide and amino acid sequence of this protein is shown in SEQ ID NO: 13, 14, and 29. Comparison hm2CP with GFP is shown in figure 1. hm2CP, as it turns out, is a relatively distant homologue of GFP (up to 24% identity).

To facilitate protein purification encoding Uch the flow hm2CP was cloned in expressing vector pQE30 (Qiagen), to the recombinant protein contained six-his-tag tag at its N end. After expression inE. coli, hm2CP protein was purified by metal-affinity resin TALON (Clontech). Absorption spectrum of purified hm2CP had a unique maximum at 568 nm (6). Can be detected very weak red fluorescence (excitation maxima at 569 and 597 nm, respectively) hm2CP (Fig.7).

Example 5

Mutagenesis hm2CP

The sequence of a nucleic acid encoding a hm2CP, was obtained as described above in Example 4. To obtain fluorescent mutants hm2CP applicants have used non-specific mutagenesis. Set Diversity PCR Random Mutagenesis (Clontech) was used for nonspecific mutagenesis hm2CP in optimal conditions for 5-6 mutations per 1000 BP Colonies of E. coli expressing mutant proteins were visually skanirovaniya fluorescent stereoscopic microscope (SZX-12 (Olympus). The most striking options were selected and subjected to another cycle non-specific mutagenesis. Complete four cycles of mutagenesis has led to a vibrant and fast-maturing red fluorescent mutant, designated as S3-2. Compared with the original chromoproteins S3-2 had a 13 amino acid substitutions, in particular D24G, I30V, K73R, T91S, II18V, K136R, T145N, S154P, SA, Y162F, L181M, V199T, I201T (SEQ ID NO: 15, 16, and 30). Spectra of excitation and emission for this mutant had maxima at y 611 nm, respectively (Fig). Red fluorescent protein S3-2 has slobodkina nature, shows the data of gel filtration. To increase expression in mammalian cells applicants synthesized "humanitarian" version of S3-2 using codons optimized for mammals (SEQ ID NO: 21, 22, and 33).

Example 6

Preparation of polyclonal antibodies

Coding areas nucleic acid red fluorescent protein S3-2 and yellow fluorescent protein Phi-YFP-M1, obtained as described above in examples 2 and 5, respectively, were cloned in expressing vector pQE30 (Qiagen) so that the recombinant protein contained six-his-tag tag at N-end. After expression in E. Coli, hm2CP was purified by metal-affinity resin TALON (Clontech) in denaturing conditions. Rabbits were immunized and additionally were treated four times with monthly periods recombinant polypeptide DSN, emulsified in Freund's adjuvant. Ten or 11 days after each additional processing in animals was taken blood. The polyclonal anticavity was tested on recombinant protein using ELISA and Western immunoblotting.

Example 7

Tagging mammalian cells using protein PhiYFP and S3-2.

For fluorescent labeling of eukaryotic cells humanized version proteins phiYF-M1 and S3-2, obtained as described above in Examples 2 and 5, respectively, were cloned into the vector pEGFP-C1 (CLONTECH) between Agel and BglII restriction sites (instead of the EGFP-coding section). We used the following cell lines: renal epithelial cells human T, fibroblasts mouse embryos ZTZ, subcutaneous mouse fibroblasts L929, renal epithelial cells from African green monkey Vero and fibroblasts from the kidneys of African green monkeys COS1. Cells were transliterowany using LipofectAMINE reagent (Invitrogen) and were tested in 20 hours after transfection. Fluorescent microscope (Olympus CK40, equipped with a CCD camera (DP-50, Olympus)was used to remove images of the cells. Expression of phiYFP-M1 or S3-2 in different cell lines resulted in a bright yellow or red signals without aggregation. Fluorescence was clearly detected 24 hours after transfection. Toxicity to cells was not observed.

Example 8

The tagging of the protein and analysis of protein localization using protein PhiYFP and S3-2.

Humanized version of phiYFP-M1 (containing additional replacement N21D, TA, KT) and S3-2 proteins, obtained as described above in Examples 2 and 5, respectively, were fused with the cytoplasmic beta-actin person. Transfection of renal epithelial cells human T plasmids expressing phiYFP-M1 or 33-2-mehanny the merged design, resulted in bright fluorescence, which gave a picture very similar to that of mergers with EGFP.

Humanitariannet version phiYFP-M1 (containing additional replacement N21D, TA, CT) was further merged with the nucleolar protein, fibrillarin. Transfection of renal epithelial cells human T a plasmid expressing phiYFP-M1-labeled fused design resulted in bright fluorescence with the structure, the corresponding expected.

Example 9

Staining of mitochondria using PhiYFP

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

Example 10

The labelling of the Golgi apparatus using PhiYFP

Coding sequences humanized version of phiYFP-M1, obtained as described above in Example 2 were fused to the sequence coding 81 amino acid at the N end of beta 1,4-galactosyltransferase person (GT; Watzele & Berger (1990) Nucleic Acids. Res. 18:7174). This region of the beta-1,4-GT human is and contains a signal peptide binding to the membrane, which aims fused proteins on transblucency 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 kidney epithelial 293T human cells with plasmids expressing phiYFP-M1-labeled fused design resulted in fluorescent tagging trenabolone part of the Golgi apparatus in cells.

Example 11

Tagging of peroxisomes using PhiYFP

Coding sequences humanized version of phiYFP-M1, obtained as described above in Example 2, were merged with peroxisomal targeting signal 1 (PTS1). The PTS1 sequence encoding a Tripeptide SKL, which directs the fusion protein to the matrix of peroxisomes (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 kidney epithelial 293T human cells with plasmids expressing phiYFP-M1-labeled fused design resulted in fluorescent tagging peroxisome.

Example 12

The tagging engine using PhiYFP

Coding sequences humanized version of phiYFP-M1, obtained as described above in Example 2, were merged with three copies of a nuclear localization signal (NLS) large T-antigen of the virus in monkeys 40, at its C-end (Kalderon et al. Cell (1984) 39: 499-509; Lanford et al. Cell (1986) 46: 575-582). Transfection of the human p is targeted epithelial 293T cells with plasmids, expressing phiYFP-M1-labeled fused design, led to the fluorescent staining of nuclei.

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

1. The selected nucleic acid molecule which encodes a fluorescent protein is selected from the group consisting of:

(a) NUS is einevoll acid, which encodes a fluorescent protein characterized by the 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 which hybridizes in stringent conditions with the nucleic acid (a) or (b)above;

(d) a nucleic acid which 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. Cloning vector comprising the nucleic acid molecule according to claim 1 and elements for replication of the vector in the cell host.

3. Expressing a vector comprising the nucleic acid molecule according to claim 1 and elements for expression of the indicated nucleic acid in the cell-master.

4. Expressing cassette containing the nucleic acid according to claim 1 under the control of regulatory elements necessary for expression of the nucleic acid in the cell-master, to whom I am being integrated into the host cell genome or by introduction into the cell in the form of extrachromosomal element, capable of expression of the fluorescent protein encoded by the nucleic acid according to claim 1.

5. Cell, producing fluorescent protein encoded by the nucleic acid according to claim 1, containing the expression cassette according to claim 4 in the form of an extrachromosomal element or integrated into the genome of this cell.

6. A stable cell line producing fluorescent protein encoded by the nucleic acid according to claim 1, containing the expression cassette according to claim 4 in the form of an extrachromosomal element or integrated into the genome of this cell.

7. A method of obtaining a fluorescent protein encoded by the nucleic acid according to claim 1, comprising (a) providing the nucleic acid molecule according to claim 1, operatively linked to suitable regulatory expression elements, (b) the expression of a fluorescent protein from a specified nucleic acid molecule, and (C) isolation of the protein, essentially not containing other proteins.

8. Selected fluorescent protein is selected from the group consisting of:

(a) a protein characterized by the 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 the nucleic acid molecule, essentially corresponding to nucleotide the sequence shown in SEQ ID N: 1, 3, 5, 7, 9, 11, 15, 17, 19 or 21;

(C) a protein which has at least 95% sequence identity with the amino acid sequence (a) or (b)above.

9. Protein fusions, capable of fluorescence, comprising the protein of claim 8 and a merge partner, representing a peptide or polypeptide, characterized by the sequence of intracellular localization, or signal peptide.

10. Set for labeling biological molecules containing a nucleic acid according to claim 1, containing either the vector according to claim 2, expressing or containing cassette according to claim 3, or a protein according to claim 7 and instructions for labeling biological molecules.

11. The method of labeling biological molecules, including the specified binding biological molecules with the protein of claim 8.

12. The method of labeling cells, including the production of the protein of claim 8 in the cell.

13. The method of labeling organelles of cells, including the production of the protein of claim 8, flushed with a suitable intracellular signal localization in the cell.

Claims 1 to 13 in the part of SEQ ID NO: 1, 2, 3, 4 have priority of 12.11.2002 (filing date No. 60425570 in the U.S. Patent Office), part of SEQ ID NO: 5, 6, 7, 8, 9 have priority from 21.04.2003 (filing date No. 60464258 in the U.S. Patent Office), part of SEQ ID NO: 11, 12 have priority from 27.11.2002 (filing date No. 60429795 in the U.S. Patent Office), frequent in the SEQ ID NO: 15, 16 have priority on 20.06.2003 (filing date No. 60480080 in the U.S. Patent Office), part of SEQ ID NO: 17, 18, 19, 20 have priority 05.11.2003 (on the date of filing of application PCT/RU03/00474).



 

Same patents:

FIELD: biology, medicine.

SUBSTANCE: invention relates to nano-size composite material for DNA/RNA adsorption and desorption in form of nano-particles comprising of 80-99.5 mass % of tin oxide (SnO2) with tetragonal crystal lattice and 20-0.5 mass % of substance selected from group containing Fe, Fe2O3, Fe3O4 or mixture thereof. Said material makes it possible to increase specific capacity of DNA/RNA adsorption and desorption at desired magnetization of composite material, in particular being necessary for magnetic deposition.

EFFECT: new nano-size composite material for DNA/RNA adsorption and desorption.

1 tbl

FIELD: medicine, in particular oncology and hematology.

SUBSTANCE: claimed method includes determination of blood cell composition wherein absolute lymphocytes and CD34-lymphocytes in peripheral blood and percent ratio of CD34-cells to lymphocytes amount is calculated according to the next equation: CD34 % = CD34 abs./lymphocytes abs. x 100 %, wherein CD34 % is relative amount of CD34-lymphocytes; CD34 abs.; lymphocytes abs. is absolute lymphocyte amount. When patient treated by chemotherapy has CD34-lymphocyte content from to 0-7 %, infective complication will not be observed, and when said value is 7.1 % or more, there is danger of generalized infection.

EFFECT: earlier and more perfect determination of chemotherapy infective complications in patients.

3 ex, 1 tbl

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: one should isolate neutrophilic leukocytes, form immune complexes, measure background luminescence of incubation medium (A), add neutrophils, measure the level of spontaneous chemiluminescence (B), introduce immune complex and detect the value of stimulated chemiluminescence (C), calculate the coefficient of stimulation (CS) and at CS≥0.3 it is possible to diagnose allergic reaction. The innovation provides higher accuracy and specificity in detecting the presence of allergic sensitization to certain antigen, the chance for screening serial assay, excludes additional body sensitization associated with introducing allergens in case of skin sample.

EFFECT: higher accuracy of allergodiagnostics.

1 cl, 5 ex, 6 tbl

FIELD: medicine, microbiology.

SUBSTANCE: method involves preparing smears from clinical material taken in inspected patients and carrying out analysis for the presence chlamydia, mycoplasma and ureaplasma in smears by method of fluorescent antibodies. The total amount of cells damaged by these pathogens is determined. In 2-7 days from the treatment onset smears are taken again and analysis of clinical material is repeated by the same method. The high sensitivity to antibiotic in this microorganism is estimated in detection <5% of epithelial cells in smear containing bright-green granules of the corresponding agent, and the prescribed treatment course with the same antibiotic is continued. The low sensitivity to antibiotic is estimated in detection ≥5% of above indicated specifically luminescent granules of antigen, and in this case a medicinal preparation is replaced. Method provides carrying out determining the effectiveness of antibiotics effect for the first days after their prescription and to correct their prescription operatively. Method is simple and doesn't require the preliminary isolation of the pathogen culture.

EFFECT: improved method for express-control.

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The invention relates to medicine, in particular to obstetrics, and can be used for prediction of preeclampsia in women undergoing severe preeclampsia

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FIELD: chemistry.

SUBSTANCE: sorbent, with at least two different groups, which are capable of selective bonding with a substrate, consists of stages (i)-(ii): (i) definition of at least two groups, capable of bonding with a sorbent made from synthetic or natural first substrate, (ii) respectively, depositing of at least two different groups, capable of bonding the second synthetic or natural substrate, to one corresponding carrier, thereby forming at least one sorbent. The groups are the same as those groups on stage (i) or are groups, which are complementary them, and the second substrate on stage (ii) is the same as the first substrate corresponding to stage (i) or different from it. The groups are such that the component of Gibbs energy of individual groups in noncovalent bonds with the second substrate yield a negative value of Gibbs energy ΔG, so that there is stronger bonding, which leads to improvement of selectivity of separation relative to at least one substance, which should separate.

EFFECT: obtaining a sorbent capable of selecting a substrate.

24 cl, 13 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, in particular to urology and can be used for objective estimation of acute gestational pyelonephritis severity. For method realisation the following blood parameters are tested: content of erythrocytes, neutrophils, lymphocytes, glucose, urea, potassium, creatinine, albumin/globulin factor. Additionally urine is tested for proteinuria, pyuria, hematuria, considering clinical symptoms and morphometry of renal pelvis. Obtained values are compared to developed scale of clinical symptoms, and appropriate points are assigned. If total score is 0 to 32 points, slight acute gestational pyelonephritis is diagnosed. Total score exceeding 32 shows severe pyelonephritis. Declared method is available and can be used in any immunologic laboratory.

EFFECT: higher diagnostic accuracy of gestational pyelonephritis severity.

2 dwg, 3 tbl

FIELD: medicine.

SUBSTANCE: carry out a highly effective liquid chromatography of water methanol sample, using Serum of venous blood in this purpose. Define presence of characteristic complexes of absorption chromatographic peaks with certain volumes of retention. Depending on the diagnosed characteristic complex differentiate organ localization of the malignant neoplasm centres, including in sexual system, nephros, bladder, and also an adenoid tissue, and organ localization of somatic nonmalignant diseases.

EFFECT: use of the method allows performing screening diagnostics of malignant neoplasm and somatic nonmalignant diseases.

21 ex

FIELD: medicine.

SUBSTANCE: invention claims a method of T-cell epitop chartering for a given albumen or its fragment by using peripheral blood mononuclear cells (PBMC). The invention also concerns T-cell epitop identification in therapeutic albumens. In addition, the invention concerns a combined approach to application of epitop chartering according to identification of MHC class II ligands detected by the claimed chartering method, and construction of similar sequence with less amount of such ligands.

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17 cl, 3 dwg, 3 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine area, namely to gastroenterology and can be used in clinical practice. The way includes clinical and laboratory inspection of the patient. Deposit of the admixed saliva is analysed laboratorial and an activity indicator of ornithine decarboxylase (ODC) is defined. At value of an indicator of ODC activity of a saliva from 0.0010 to 0.0080 nkat/ml diagnose a Gee's disease of serious severity level, at value of an indicator of activity ODC from 0.0081 to 0.0145 nkat/ml - a moderate severity level Gee's disease, at value of ODC activity from 0.0146 to 0.0410 nkat/ml - a Gee's disease of easy severity level.

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1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: course and, length of disease, seasonality, age, focus localisation, form of disease, and gender are determined. Then, according to the table, the corresponding standard intense indicants (SII), risk factor weight numbers (FWN) are defined, risk prognostic coefficients (R) being defined as product of PIL by FWN. The risk of recidivation is calculated as quotient between summarised prognostic coefficients and weight number to define degree of recidivation risk. Iif the value of the said coefficient totals to 0.5-1.2 then no risk of erysipelas recidivation is defined, if the value totals to 1.3-1.9 then there is average risk of erysipelas recidivation - special care patient census; if R totals to 2.0-2.6 there is a high probability of erysipelas recidivation.

EFFECT: increased diagnosis accuracy and improved cure rates and patient's better quality of life.

3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention belongs to medicine, biology and biotechnology and concerns a method of human genotype definition on the basis of interleukin gene 6, position 174(G/C) polymorphism. The method involves use of allele-specific primers with polymerase chain reaction result registration directly in the course of reaction with the help of fluorescent-labeled samples, the reaction mix including primers for a site with desirable replacement, different for each allele, and primer with sample common to each couple of alleles.

EFFECT: streamlining of the method.

1 dwg

FIELD: medicine.

SUBSTANCE: claimed method belongs to medicine, biology and biotechnology fields and concerns definition of human genotype on the basis of polymorphism in interleukin-1-alpha gene in position 889(C/T). The method involves use of allele-specific primers with polymerase chain reaction directly in the course of reaction with the help of fluorescent marked samples, the reaction mix including primers for a site with desirable replacement, different for each allele, and primer with sample common to each couple of alleles.

EFFECT: streamlining of method.

2 cl, 1 dwg

FIELD: medicine; ophthalmology.

SUBSTANCE: essence of the invention is glycosaminoglycans content determination in patient's blood plasma in acute period of eye lesion in Reiter's syndrome. If glycosaminoglycans level in blood plasma is less than 16 mcg/ml, then recurrent course of disease can be forecasted. If glycosaminoglycans concentration in blood plasma is higher than 21.5 mcg/ml, course of disease can be estimated as favourable, without relapses.

EFFECT: improvement of clinical course frequency prognosis in eye lesion and early stage prognostics of eye disease clinical course.

3 ex

FIELD: medicine.

SUBSTANCE: invention can be applied to angiopathy development risk diagnostics in arterial hypertension (AH) cases with metabolic syndrome (MS). Essentially, thrombocyte aggregative activity with collagen, antithrombin III level, euglobuline lysis time, and nitrate level in patient blood are determined, before and during temporal venous occlusion. Then vascular wall antiaggregatory activity index (VWAAI), vascular antithrombin III synthesis index (VASI), vascular wall fibrinolytic activity index (VWFAI), and nitrate index (NI) are calculated. After this, vascular antithrombotic index (VATI) by formula: VATI = (VWAAI + VASI + VWFAI + NI)/4 is calculated. If the value achieves 2.27 and more, angiopathy development risk is eliminated; if 2.26-2.10, the patients are included in angiopathy development risk group; if 2.09 and less, angiopathy develops.

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2 tbl, 3 ex, 1 dwg

FIELD: medicine.

SUBSTANCE: invention is referred to medical genetics and can be used at revealing of predisposition to course of Alzheimer's disease (AD). As a result of the analysis of DNA samples of patients with DA on presence of polymorphisms in promotor area (in positions (-1082), (-819) and (-592)) gene IL-10 the genotypes bound to low production IL-10, which frequency of occurrence at patients authentically above, than at healthy controls have been defined. It is offered to use the fact of presence at the subject of the genotype concerning specified group, as an adverse prognostic sign at predisposition definition to DA. For the purpose of rising of reliability of the forecast it is offered to carry out an additional analysis of the DNA sample on presence of polymorphisms in genes IL-6, Aro-E and IL-1 for which connection with development DA was earlier established.

EFFECT: obtaining of an effective way of determination of predisposition to course of an Alzheimer's disease.

3 cl, 14 dwg, 9 tbl, 3 ex

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