Novel fluorescent proteins from entacmaea quadricolor and method of obtaining said proteins

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention relates to a novel fluorescent protein from Entacmaea quadricolor and its functional mutants. The invention discloses nucleic acids which code the said protein, a vector which contains the said nucleic acids, a transgenic cell which carries the vector and a method of obtaining the said fluorescent proteins from transgenic cells. The composition of the said proteins and nucleic acids can be used in various applications and methods, particularly for labelling biomolecules, cells or cell organelles. The disclosed protein and nucleotide sequences can be used for testing activity of promoters under various conditions.

EFFECT: obtaining proteins with primarily red or far-red fluorescence.

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References to related applications

In this application claims the priority of provisional patent application U.S. serial number 60/761807, filed January 25, 2006, which is incorporated into this description by reference.

Background of invention

The scope of the invention

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

The level of technology

Fluorescent proteins, including green fluorescent protein (Green Fluorescent Protein, GFP), its mutants and homologues, today widely known for their extensive use as fluorescent markersin vivoin biomedical research, which examined in detail Lippincott-Schwartz and Patterson in Science (2003) 300(5616): 87-91.

Fluorescent proteins are proteins that are capable of fluorescence upon irradiation with light of suitable wavelength. The fluorescent properties of these proteins due to the interaction of two or more amino acid residues, but not the fluorescence of any single amino acid residue.

GFP of hydromedusaAequorea aequorea(synonym ofA. victoria) was described by Johnson et al. J Cell Comp Physiol. (1962), 60: 85-104, as part of the bioluminescent system of the Medusa, where GFP plays the secondary role of emmiter that converts blue light from fotobanka aquarina in C the green strip light. cDNA encodingA. victoriaGFP 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 chromophore (Chalfie et al., Science 263 (1994), 802-805). This information provides the opportunity for the use of GFP in cell biology as a genetically encoded fluorescent tags.

GFP has been used in a wide range of applications, including the study of gene expression and the 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 tool to visualize the intracellular distribution of organelles (Rizzuto et al., Curr. Biology (1995), 5: 635-642), for visualization of protein transport along the secretory pathway (Kaether and Gerdes, FEBS Letters (1995), 369: 267-271).

There have been numerous studies to improve properties of GFP and to obtain GFP-reagents suitable and optimized for various research purposes. Developed new version 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, a mutant variant of GFP, is an "enhanced green fluorescent protein" (EGFP), which has two amino acid is replacement: F64L and S65T (Heim et al., Nature 373 (1995), 663-664). Other mutants are blue, cyan and yellow-green spectral variants of GFP.

However, despite the wide use of GFP, other fluorescent proteins with properties similar to or different from GFP, would be useful in this area, in particular, proteins with new spectra or greater fluorescence intensity. In 1999, the GFP homologues have been cloned from non-bioluminescent species ofAnthozoa(Matz et al., Nature Biotechnol. (1999), 17: 969-973). This discovery demonstrated that these proteins are not necessarily component of a bioluminescent system. GFP-like proteins fromAnthozoahad a great spectral diversity, including cyan, green, yellow, red fluorescent proteins and violet-blue effluorescence the chromoproteins (CP) (Matz et al., Bioessays (2002), 24(10): 953-959). Further cDNA GFP-like proteins have been cloned from a number of hydroid jellyfish and copepods (Shagin et al., Mol Biol Evol. (2004), 21(5): 841-850). Today GFP-like proteins include more than 120 fluorescent and colored GFP homologues. The similarity of these proteins with GFP varies from 80-90% to less than 25% identity on amino acid sequence.

Crystal structure of GFP wild-type and GFP S65T mutant was resolved and showed that the tertiary structure of GFP is a barrel (Ormo et al., 1996, Science 273: 1392-1395; Yang, et al., 1996, Nature Biotech 14: 1246-1251). This barrel comp is it out of beta layers, forming a compact counter-parallel structure within which is located an alpha helix containing the chromophore. It was confirmed previously made assumption (Cody et al., Biochemistry (1993) 32, 1212-1218)that the chromophore is formed by the oxidative cyclization of three conservative amino acid residues. All tested GFP-like proteins have the same beta-barrel, as GFP (Ormo et al. Science (1996) 273: 1392-1395; Wall et al. Nat Struct Biol (2000), 7: 1133-1138; Yarbrough et al. Proc Natl Acad Sci USA (2001) 98: 462-467; Prescott et al. Structure (Camb) (2003), 11: 275-284; Petersen et al. J Biol Chem (2003), 278: 44626-44631; Wilmann et al. J Biol Chem (2005), 280: 2401-2404; Remington et al. Biochemistry (2005), 44, 202-212; Quillin et al. Biochemistry (2005), 44: 5774-5787).

The usefulness of fluorescent proteins as tools in molecular biology makes demand for other fluorescent proteins, or other improved properties compared to known fluorescent proteins. Thus, demand getting new fluorescent proteins that exhibit properties not available from the well-known fluorescent proteins and their coding DNA.

The invention

The present invention provides the selected nucleic acid molecule encoding a new fluorescent protein fromEntacmaea quadricolor(EqFP578) and its functional mutants, i.e. EqFP578-like proteins. In some embodiments of the specified nucleic acid may be selected is from Entacmaea quadricolor. In some embodiments the nucleic acid of the present invention obtained by the methods of genetic engineering.

In some embodiments the selected nucleic acid of the present invention encode a fluorescent protein of the wild type of theEntacmaea quadricoloras in SEQ ID NO: 02 (EqFP578). An example of the nucleotide sequence shown in SEQ ID NO: 01.

In some embodiments provides a selected nucleic acid that has a sequence specific hybridization with a part or a complementary chain of SEQ ID NO: 01.

In some embodiments the nucleic acid of the present invention encode a fluorescent protein that contains the amino acid sequence, which is essentially similar or identical to the sequence of the protein EqFP578 (SEQ ID NO: 02). Accordingly, the selected nucleotide sequence encoding natural allelic variants of SEQ ID NO: 01, also included in the scope of the present invention.

In some embodiments of these nucleic acids encode a functional mutants EqFP578, which are essentially similar or altered biochemical or spectral characteristics compared to wild type EqFP578.

In some embodiments the selected nucleic acid of the present invention encode mutant proteins, catalysterror amino acid sequence, essentially the same as the sequence of EqFP578 (SEQ ID NO: 02), and differ from EqFP578 (SEQ ID NO: 02) at least one amino acid substitution.

In preferred embodiments of the specified amino acid substitution selected from the group consisting of R32G and S131P; specified mutant fluorescent protein has an improved folding at 37°C compared to EqFP578. In preferred embodiments of the indicated mutant contains both replacement.

In preferred embodiments of the indicated mutant also contains other foldingbike mutations, for example, selected from the group consisting of E36G, K42R, F53V, K67R, T68A, L79F, I93V, F110L, N112D, I115L, R138L, G152S, H157R, Y169H, H171I, C172A, F174L, K188R, H193Y, M216V, K220R and R231K. These mutations improve the folding of the protein and the rate of maturation of the chromophore, resulting in a more rapid formation of fluorescent signalin vivo.

In other preferred embodiments the selected nucleic acid of the present invention encode obtained using genetic engineering techniques fluorescent protein having a modified N - and/or C-terminal part outside of the chromophore domain EqFP578, where this protein shows reduced ability to aggregation compared to wild type EqFP578. In preferred embodiments, the modified N-end contains replacement K6T, and C-terminal - replacement R231S. In some embodiments, a modified N-end also contains additional the additional amino acid sequence, selected from the group consisting of MGEY and MGED.

In some embodiments the selected nucleic acid of the present invention encode obtained using genetic engineering techniques functional fluorescent protein, which contains at least one amino acid substitution selected from the group consisting of R155E, Q159D, S173N, F192V, F194Y where the specified functional fluorescent protein has a reduced ability to oligomerization compared to wild type EqFP578. In preferred embodiments of the specified functional fluorescent protein also contains a replacement N122R, which even more reduces the tendency of the protein to oligomerization. In preferred embodiments of the specified functional fluorescent protein contains the replacement and also contains one or more substitutions selected from the group consisting of E36G, K42R, F53V, K67R, T68A, L79F, I93V, F110L, N112D, I115L, R138L, G152S, H157R, Y169H, H171I, C172A, F174L, K188R, H193Y, M216V, K220R and R231K where these replacements improve coagulation (folding) of the protein and increase the fluorescence intensityin vivo.

In some embodiments the selected nucleic acid of the present invention encode obtained using genetic engineering techniques functional fluorescent protein, which contains at least one amino acid substitution selected from the group consisting of H197R, S158G, N143S, N143H,N143F and N143Y, where specified functional fluorescent protein has an altered emission spectra and fluorescence excitation than the corresponding wild-type protein.

Examples of nucleic acid molecules of the present invention, which encode obtained using genetic engineering techniques of functional mutants EqFP578 include SEQ ID NO: 3, 5, 7, 9, 11, 13 and 15 or encode SEQ ID NO: 4, 6, 8, 10, 12, 14 and 16.

Molecules of nucleic acids that differ from the nucleotide sequences due to degeneracy of the genetic code or hybridize with them, are also included in the scope 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 selected cell host. In addition, it also provides cells, stable cell lines, transgenic animals and transgenic plants comprising the nucleic acids, vectors or expression cassettes of the present invention.

In other embodiments are provided with functional fluorescent proteins of the present invention, which are encoded by nucleic acids is specified above.

In some embodiments the functional fluorescent proteins of the present invention include amino acid sequence, which is essentially the same or identical to the sequence of the fluorescent protein of the wild type of theEntacmaea quadricolorhaving SEQ ID NO: 02 (EqFP578). Proteins of interest include wild type EqFP578 and its mutants, which have essentially the same or altered biochemical and/or spectral properties compared to wild type EqFP578.

In preferred embodiments of the specified amino acid substitution selected from the group consisting of R32G and S131P; specified mutant fluorescent protein has an improved folding at 37°C compared to EqFP578. In preferred embodiments of the indicated mutant contains both replacement.

In preferred embodiments of the indicated mutant also contains other foldingbike mutations, for example, selected from the group consisting of E36G, K42R, F53V, K67R, T68A, L79F, I93V, F110L, N112D, I115L, R138L, G152S, H157R, Y169H, H171I, C172A, F174L, K188R, H193Y, M216V, K220R and R231K. These mutations improve coagulation (folding) protein and the rate of maturation of the chromophore, resulting in a more rapid formation of fluorescent signalin vivo.

In other preferred embodiments obtained using genetic engineering techniques fluorescent protein has a modified N - and/or C-terminal part outside of the chromophore is about domain EqFP578, while this protein shows reduced ability to aggregation compared to wild type EqFP578. In preferred embodiments, the modified N-end contains replacement K6T, and C-terminal - replacement R231S. In some embodiments, a modified N-end also contains an additional amino acid sequence selected from the group consisting of MGEY and MGED (SEQ ID NO: 17 and 18, respectively).

In some embodiments obtained using genetic engineering techniques functional fluorescent protein of the present invention contains at least one amino acid substitution selected from the group consisting of R155E, Q159D, S173N and F192V, with specified functional fluorescent protein has a reduced ability to oligomerization compared to wild type EqFP578. In preferred embodiments of the specified functional fluorescent protein also contains a replacement N122R, which even more reduces the tendency of the protein to oligomerization. In preferred embodiments of the specified functional fluorescent protein contains the replacement and also contains one or more substitutions selected from the group consisting of E36G, K42R, F53V, K67R, T68A, L79F, I93V, F110L, N112D, I115L, R138L, G152S, H157R, Y169H, H171I, C172A, F174L, K188R, H193Y, M216V, K220R and R231K where these replacements improve coagulation (folding) of the protein and increase the intensity fluorescence> in vivo.

In some embodiments obtained using genetic engineering techniques functional fluorescent protein of the present invention contains at least one amino acid substitution selected from the group consisting of H197R, S158G, N143S, N143H, N143F and N143Y, with specified functional fluorescent protein has an altered emission spectra and fluorescence excitation than the corresponding wild-type protein.

Examples of fluorescent proteins of interest include SEQ ID NO: 02, 4, 6, 8, 10, 12, 14 and 16.

In addition, provided the set containing nucleic acids or vectors or expression cassettes comprising these nucleic acids of the present invention.

Also provided are antibodies that specifically bind proteins of the present invention or fragments thereof.

Brief description of figures

For a more complete disclosure of the above mentioned characteristics of the present invention below is a detailed description of the invention summarized above in reference to embodiments, some of which are illustrated additional figures. It should be noted that the appended figures illustrate only typical embodiments of the present invention and, therefore, should not be interpreted as limiting the amount of the image is to be placed, which may allow other equally effective embodiments.

Figure 1 shows the multiple alignment of the wild type EqFP578 and its mutants.

Figure 2 illustrates the excitation spectra (line 1) and emission (line 2) fluorescence of wild type EqFP578.

Figure 3 illustrates the spectra of excitation (line 1) and emission (line 2) fluorescence mutant EqFP578m1.

Figure 4 illustrates the spectra of excitation (line 1) and emission (line 2) fluorescence of mutant M1-602.

Figure 5 illustrates the excitation spectra (line 1) and emission (line 2) fluorescence of mutant M1-637.

6 illustrates excitation spectra (line 1) and emission (line 2) fluorescence of mutant M1-mono1.

Fig.7 illustrates the excitation spectra (line 1) and emission (line 2) fluorescence mutant nrM181-5 (before photoconversion).

Detailed description of the invention

To summarize the above, the present invention is directed to molecules of nucleic acids that encode fluorescent protein EqFP578 fromEntacmaea quadricolorand its mutants, as well as the proteins encoded by these nucleic acids. Molecules of nucleic acids of interest are isolated fromEntacmaea quadricoloror obtained by genetic engineering methods. Proteins of interest include fluorescent proteins having the amino acid sequence shown SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 and 16. Also of interest are proteins that are similar in substance, or mutants to these proteins. Also provided cell hosts, stable cell lines and transgenic organisms containing these molecules are nucleic acids. Also provided are antibodies specific to proteins of the present invention.

These protein and nucleotide compositions are applied in many different applications and methods, in particular, in applications labeling of cells, cell organelles or proteins. The protein and nucleotide compositions used in the methods of testing of promoter activity in various conditions. Finally, there are provided kits for use in such methods and applications.

Definitions

Various terms relating to the biological molecules of the present invention, are used above and also in the description and in the claims.

As used here, the term "fluorescent protein" means a protein that has the ability to fluorescence; 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 chromophore formed by Avoca aliciously cyclization of two or more amino acid residues in the polypeptide chain. As such fluorescent proteins of the present invention do not include proteins that have fluorescence at the expense of individual fluorescent residues, such as tryptophan, tyrosine and phenylalanine.

As used here, the term "GFP" refers to a green fluorescent protein fromAequorea victoriaincluding GFP variants 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. (1992, Gene 111: 229-33).

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

The term "humanitarianly" refers to changes in the nucleotide sequence of the fluorescent protein, made for optimization of the genetic code codons for expression in mammalian cells (Yang et al., 1996, Nucleic Acids Research 24: 4592-4593).

As used here, the term “EqFP578” refers to nucleic acid and a fluorescent protein of the wild type of theEntacmaea quadricolorwho has the nucleotide and amino acid sequence shown in SEQ ID NO: 1 and 2, respectively.

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 the cell are in estestvennyh conditions.

As used here, the term "mutant" or "derivative" 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, "homology" is the term used to describe the relationship of nucleotide sequences or amino acid sequences with other sequences of nucleotides or amino acids, which is determined by the degree of identity and/or similarity between the compared sequences.

As used here, an amino acid or nucleotide sequence essentially similar” or “essentially the same”as the reference sequence, if the amino acid or nucleotide sequences have at least 85% identity with the specified sequence within selected for comparison region. Thus, essentially the same as the sequence on the look to those which have, for example, at least 85% identity, at least 90% identity, at least 95% identity or at least 99% identity. Two sequences that are identical to one another, also essentially similar. For the purposes of the present invention the length of the compared sequences of fluorescent proteins will generally at least 160 amino acids, preferably at least 200 amino acids. For nucleic acids, the length of comparison sequences will generally at least 480 nucleotides; preferably at least 600 nucleotides.

The sequence identity is determined based on 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 the purposes of the present invention, comparison of nucleotide and amino acid sequences produced using the Blast software package provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast) using contains gaps alignment with default parameters, can be used to determine the level of identity and similarity between the nucleotide sequences and amino acid sequences.

As used here, the term "similar to the fluorescent proteins" refers to fluorescent proteins, which have essentially the same amino acid sequence when compared with the reference fluorescent protein. Basically, such a fluorescent protein when compared with the reference sequence of the fluorescent protein has a long sequence of length at least 160 amino acids that has at least 85% identity with the reference fluorescent protein.

As used here, the term "EqFP578-like protein" refers to wild type EqFP578 with SEQ ID NO: 2 and functional mutants, such as those that have the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 and 16. As used here, the term "EqFP578-like nucleic acid" refers to nucleic acid that encodes EqFP578-like protein (e.g., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 and 15). As used here, EqFP578-like protein contains the amino acid sequence, which is essentially similar or identical to the sequence of EqFP578 (SEQ ID NO: 02). The term "EqFP578-like protein" and "EqFP578-like nucleic acid" also refers to a shortened and elongated variants EqFP578 or its mutants and coded their nucleic acids.

As used here, the term "functional" means that the nucleotide or amino acid sequence can function for the specified test the project or task. The term "functional"is used to describe fluorescent protein means that the protein is suitable for use spectra of excitation and emission of fluorescence (i.e. has a detectable fluorescence).

As used here, "biochemical properties related to protein folding (collapsing), and rate of maturation, time-life, ability to aggregation, the ability to oligomerization, pH and temperature stability, and other properties.

As used here, "fluorescent properties" or “spectral properties" refers to the molar extinction coefficient at an appropriate wavelength, quantum yield of fluorescence, the spectral shape of the fluorescence excitation or emission spectrum, the wavelength corresponding to the maximum fluorescence excitation, and the wavelength corresponding to the maximum emission, the ratio of amplitude of excitation of fluorescence at two different wavelengths, the ratio of the amplitude of the emission at two different wavelengths, the lifetime of the excited state and the anisotropy of the optical properties. The measured difference in any of these properties between wild type EqFP578 and its mutants applicable. The measured difference can be defined as the amount of any quantitative fluorescent properties, for example, the intensity fluoresc is ncii at a certain wavelength or integral fluorescence across the entire spectrum of emission.

As used here, "the rate of maturation" refers to the speed of formation of Mature fluorescent protein (i.e. fluorescent protein, capable of producing fluorescence) after the broadcast. The rate of maturation can be characterized by the half-period of maturation. It was shown that the maturation of the fluorescent protein comprises two stages: (i) folding of the protein, which means the formation of protein beta-layers with a Central alpha-helix-containing amino acids that will form the chromophore. This stage is usually characterized by a rate constant of approximately 10(-2)s(-1)or a half-period of from several seconds to tens of seconds; (ii) the maturation of the chromophore when the protein chain undergoes cyclization and dehydration. This stage is usually characterized by a rate constant of approximately 10(-4)s(-1)or half length in a few minutes. Thus, this slower phase is rate-limiting in the maturation of green fluorescent protein (Reid BG, Flynn GC. Biochemistry. 1997 V. 36(22), PP. 6786-6791).

As used here, "aggregation" refers to the propensity or ability of the expressed protein to form insoluble precipitates (aggregates). "Aggregation" should be distinguished from "oligomerization". In particular, mutants with reduced ability to aggregation, for example, with increased rastvorimost the Yu, not necessarily have a reduced ability to oligomerization (i.e. turn tetramer in dimers or monomers or dimers to monomers).

As used here, "oligomerization" refers to the tendency or ability expressioning protein to form complexes (oligomers) as a result of the specific interaction between two or more polypeptides. Specified specific interaction observed in special conditions, for example, in physiological conditions, and is relatively stable under these conditions. The reference to "the ability of proteins to oligomerizate means that proteins can form dimers, trimers, tetramer or similar complexes in the special conditions. Typically, fluorescent proteins have the ability to oligomerization in physiological conditions, although, as described here, fluorescent proteins can also be oligomerizate when other, for example, pH than pH under physiological conditions. The conditions under which fluorescent proteins form oligomers or show a propensity for oligomerization, can be determined using well known methods such as gel filtration, or other means known in the field.

The term "functionally linked" or similar when describing fused protein refers to a polypeptide sequences that are in the physical is tion and functional connection with one another. In the most preferred embodiments the functions of the polypeptide components of the chimeric molecule is not changed in comparison with the functional properties of the selected polypeptide components. For example, the fluorescent protein of the present invention may be merged with interest by merger partner. In this case, the protein retains the fluorescent properties of the fluorescent protein, and interest polypeptide retains its original biological activity. In some embodiments of the present invention the activity as a fluorescent protein, and the protein of interest can be lowered as compared with the activity of the isolated proteins. Such fused proteins also find use in the present invention.

As used here, the term "specifically hybridizes" refers to the Association between two single-stranded molecules of nucleic acids or sufficiently complementary sequences that permit such hybridization in a predefined conditions usually used in this field (sometimes used the term "essentially complementary").

The reference nucleotide sequence encoding the polypeptide means that the nucleotide sequence in the translation and transcription of mRNA Ave is dotiruetsja this polypeptide. This may be specified as the coding circuit that is identical to the mRNA and commonly used in the list of sequences and the complementary circuit, which is used as a template for transcription. As is obvious to any expert in the art, the term also includes any degenerate nucleotide sequences encoding the same amino acid sequence. The nucleotide sequence encoding the polypeptide, include sequences containing introns.

As used here, the numbering of amino acid residues and substitutions corresponds to the numbering of amino acid residues in the sequence of the wild type EqFP578 (SEQ ID NO: 2). For the mutant proteins, the position of amino acid residue or replacement can be determined using the protein alignment (figure 1).

Molecules of nucleic acids

The present invention provides a molecule of nucleic acids encoding fluorescent protein EqFP578, having amino acid sequence SEQ ID NO: 2 and its mutants. Molecules of nucleic acids encoding a shorter or longer versions of EqFP578 and its mutants are also within the present invention.

Specific nucleic acid molecules of interest include molecules encoding these fluorescent proteins: red fluo scanty protein from Entacmaea quadricolorhaving the sequence of SEQ ID NO: 2; and its mutants with optimized properties: folding at 37°C, reduced ability to aggregation and/or oligomerization, and the modified spectral characteristics. Amino acid sequences of these mutants are shown in SEQ ID NO: 4, 6, 8, 10, 12, 14 and 16. Examples of specific mutant nucleic acid of the present invention shown in SEQ ID NO: 3, 5, 7, 9, 11, 13 and 15.

Each of these specific types of molecules of the nucleic acid of interest is disclosed in more detail below in the experimental part.

As here used, the nucleic acid molecule is a DNA molecule, such as genomic DNA or cDNA molecule, or an RNA molecule such as a molecule of mRNA. As used here, the term “cDNA” refers to nucleic acids that have the placement of sequence elements found in native Mature mRNA species, where sequence elements are exons and 5' and 3' non-coding region.

The nucleic acid molecule encoding a fluorescent protein that can be synthesized from suitable nucleosidase or isolated from biological sources. Both methods are based on well-known in this field protocols. For example, the availability of sequence information linakis is from (for example, SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16) or information on a nucleotide sequence (such as SEQ ID NO: 3, 5, 7, 9, 11, 13 or 15) provides the ability to get the selected nucleic acid molecule of the present invention using oligonucleotide synthesis. In the case of sequence information of several amino acids nucleic acids that differ from each other due to the degeneracy of the genetic code, can be synthesized. Methods selection of codons for the desired host are well-known in this field.

Synthetic oligonucleotides can be obtained by using phosphoramidite method, and the resulting construction can be purified using methods well known in the field, such as high performance liquid chromatography (HPLC) or other methods 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 described in, for example, United States Dept. of HHS, National Institute of Health (NIH) Guidelines for Recombinant DNA Research. Long double-stranded DNA molecules of the present invention can be synthesized following stages: several smaller fragments with the necessary complementarity, which contain the appropriate ends, capable of cohesion with neighboring fragment can be. Adjacent slices can be sewn using a DNA ligase or meth is Yes, PCR-based.

Molecules of nucleic acids encoding fluorescent proteins of the present invention can also be cloned from biological sources of typeCnidariapreferably from the classAnthozoa, more preferably from a subclass ofZoanthariamore preferably from the orderActiniariaand even more preferably from the familyActiniidaefor example, fromEntacmaea quadricolor.

In some embodiments, the nucleic acid molecule of the present invention is a DNA (or cDNA) molecule containing an open reading frame which encodes a fluorescent protein fromEntacmaea quadricoloraccording to the present invention and is capable, under the right conditions (e.g., physiological intracellular conditions) to be used for ekspressirovali fluorescent protein according to the present invention. The present invention also encompasses nucleic acids which are homologous, is essentially the same as, identical to, or derived from nucleic acids encoding the proteins of the present invention. These nucleic acids are in an environment different from the environment in which they are in natural conditions, for example, they are selected, presented in a larger quantity, or are expressed in the systemin vitroor in cells or organisms in environments which, different from that in which they are in natural conditions.

Changes or differences in nucleotide sequence between vysokoshirotnymi nucleotide sequences may represent nucleotide substitutions in the sequences that occur during the normal replication or duplication. Other substitutions can be designed and inserted into the sequence for a particular purpose, such as changing the codons of certain amino acids or nucleotide sequence of the regulatory region. Such special replacement can be madein vitrowith the use of various technologies mutagenesis or received organisms hosts that are in a specific selection conditions that induce or select for these changes. Such specially obtained sequences can be called "mutants" or "derivatives" of the source sequence.

Nucleic acid encoding a EqFP578-like polypeptide amino acid sequence which is a mutant, derivative, variant or allelic variant of the sequence shown in SEQ ID NO: 2, is also provided by the present invention. Nucleic acid encoding such a polypeptide can have more than 60% homology with the coding sequence shown is SEQ ID NO: 1, more than about 70% homology, more than about 80% homology, more than about 90% homology or greater than about 95% homology.

Nucleic acid encoding such a polypeptide or fragment may be any of many known methods. The cDNA fragment of the present invention can be used as a hybridization probe against a cDNA library of target organism in conditions of high stringency. The probe can be a great fragment or one or more short degenerate primers. Nucleic acids having sequence similarity can be detected by hybridization under conditions of high stringency, for example, at 50°C or higher (e.g. 60°C or 65°C), 50% of formamide, of 0.1×SSC (15 mm sodium chloride/1.5 mm sodium citrate), 0.1% of SDS. Nucleic acids having a region is essentially identical with the reference sequence, for example, allelic variants, genetically modified variants of nucleic acids, etc. that are associated with the reference sequence in the hybridization conditions of high stringency. Using probes, in particular labeled probes their DNA sequence, it is possible to allocate such nucleotide sequence.

Mutant or derivative of the nucleic acid can be obtained on the matrix nucleic acid selected from the above-described well Leonovich 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; 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 (error-prone PCR, shuffling (shuffling, oligonucleotide-directed mutagenesis, PCR with the Assembly, a pair of PCR mutagenesis, mutagenesisin vivo, cassette mutagenesis, recursive multiple mutagenesis, exponential multiple mutagenesis, site-specific mutagenesis, random mutagenesis, gene reassemblage (gene ' s reassembly), gene site-saturating mutagenesis (GSSM), synthetic reconstruction with legirovaniem (SLR), or a combination. Modifications, additions or deletions can be made by a method comprising recombination, recursive recombination sequences, phosphothioate-modified mutagenesis DNA mutagenesis on brazilterramar matrix mutagenesis with a double-pass point restoration for misalignment mutagenesis, mutagenesis of strain, deficient restorations, chemical mutagenesis, radioactive mutagenesis, dilatational Mut is the Genesis, 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 or biochemical properties of fluorescent protein as wild type. In other embodiments, a mutant or derivative of the nucleic acids encode fluorescent proteins with altered properties.

In addition, also provided degenerate variants of the nucleic acids that encode proteins 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 of the host cell, is replaced by codons that are abundantly represented in coding sequences in genes in the cell host, where these replaced codons encode the same amino acid. Of particular interest are humanized version nekleenov the x 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 (human) (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. Examples of degenerate variants of interest are described in more detail in the experimental part below.

Nucleic acids encoding the shorter and longer versions of EqFP578 and its mutants are also included in the scope of the present invention. As used here, these options proteins contain the amino acid sequence of EqFP578-like protein with modified C-, N - or both ends. In extended versions of the C - or N-terminal protein may contain additional amino acid residues. In shortened versions of one or more (usually up to 8, usually up to 7 and preferably up to 5) amino acid residues may be deleted from the sequence or replaced with any other amino acid residues. Such modifications do not change essentially the fluorescent properties of proteins, but can facilitate protein folding in the cell-master, reduce the ability to aggregate or to modulate other biochemical properties of proteins, for example, the half life. In some the embodiments, these modifications do not alter the biochemical properties of the protein. All kinds of modifications and mutations, above, are carried out at the level of nucleic acids.

Molecules of nucleic acids according to 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, enzymatic restriction, PCR amplification, etc. basically, the DNA fragments will have a size of at least about 15 nucleotides, usually at least about 18 nucleotides, or about 25 nucleotides, and can be at least about 50 nucleotides. In some embodiments 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 proteins or full-size proteins; 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 shall be selected 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 linked to the chromosome, found in nature in its natural organism, the host.

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. Suitable vectors include viral and non-viral vectors, plasmids, Comedy, phages, etc., preferably plasmids, and are used for cloning, amplification, expression, transfer, etc. nucleic acid sequence according to 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 full-sized nucleic acid or its part is usually inserted into the vector by attaching DNA ligase to the dissolved enzymes restriction site in the vector. Viola is rnative desired nucleotide sequence can be inserted by homologous recombination in vivotypically attach homologous sites 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 system, used inter alia for specified chromogenic or fluorescent proteins or fused proteins based on them or to replicate the claimed nucleic acid molecules. The expression cassette may exist as an extrachromosomal element or may be incorporated into the genome of 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 according to the invention is expressed in any expression system, including, for example, bacterial systems, yeast, insect, amphibian, or mammalian cells. 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 expression cassettes or systems for the expression of the desired product is known with what Elliston, qualified 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, co-transfection with breeding marker such as dhfr, gpt, neomycin, hygromycin that makes possible the detection and isolation of 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 as E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or cells of a higher organism such as vertebrates, e.g. COS 7 cells, SOME 293, Cho, Xenopus oocytes, etc.

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 according to 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 provided short DNA fragments of the claimed nucleic acids that are used as primers for PCR, a hybrid of the struc screening probes, etc. Long 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 short DNA fragments, i.e. primers. The exact sequence of the primer is not critical for the invention, but for most applications the primers will gibridizatsiya with the stated sequence under conditions of stringency 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 sequences of primers generally known and available in commercial software packages. Primers for the amplification hybridize with complementary strands of DNA and will be satraplatin counter amplification reaction.

The nucleic acid molecule 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, isolated DNA and mRNA from the sample cell. the mRNA may be amplified RT-PCR using reverse transcriptase inhibitor for the formation of a complementary strand of DNA, followed by amplification using the polymerase chain reaction using primers specific for said DNA sequences. Alternative mRNA sample is separated using gel electrophoresis, transferred to a suitable medium, for example, nitrocellulose, nylon, etc. and then test fragment declared as the DNA sample. You can also use other methods, such as analyses of staple oligonucleotides, in situ hybridization and hybridization of DNA probes immobilized on a solid chip. Detection of mRNA for hybridization with the stated sequence, indicates gene expression in the sample.

Proteins

Also provided in accordance with the claimed invention fluorescent proteins, derivatives and mutants of them, including a full-sized proteins and their parts or fragments. Also provided options natural protein fromEntacmaea quadricolor(SEQ ID NO: 2), where such variants are proteins that are homologous or substantially similar to a natural protein, and mutants of the natural protein, as described in more detail below.

The claimed proteins similar fluorescent screens, i.e. have the ability to detectable fluorescence. In many embodiments of the claimed protein region is give red or far-red fluorescence, that is, they have the maximum fluorescence excitation in the range from about 450 nm to 700 nm, usually from about 470 nm to 650 nm, and most often from about 500 to 600 nm, for example, from 550 to 595 nm, whereas the maximum emission of the claimed protein is in the range from about 530 to 700 nm, usually from about 550 nm to 670 nm, and most often from about 560 to 650 nm, for example, from 574 to 637 nm. In other embodiments of the claimed proteins have a maximum fluorescence excitation in the range of from about 350 to 500 nm, usually from about 370 nm to 450 nm, and most often from about 390 to 420 nm, for example, 400 nm, while the maximum emission of the claimed protein is in the range from approximately 400 to 530 nm, usually from about 420 nm to 510 nm, for example, 470 nm.

The claimed proteins usually have a maximum extinction coefficient in the range from about 30,000 to 150,000, and usually from about 6000 to 120000, for example, from 90000 to 120000.

The claimed proteins typically vary in length from about 150 to 300 amino acids, and typically from about 200 to 300 amino acid residues, and usually have a molecular weight in the range from approximately 15 to 35 kDa, usually about from 17.5 to 32.5 kDa.

In some embodiments of the claimed proteins are bright, where bright is understood that the fluorescent proteins may be detected by conventional methods (e.g., visual screening, spectrophotometry, spectrofluorimetry, fluorescence microscopy, FACS instruments and so on). The brightness of the specific fluorescence of fluorescent proteins is determined by their quantum yield multiplied by the maximum coefficient of xtinction and divided by 1000. In some embodiments of the claimed proteins possess the brightness of the fluorescence in the range of from about 10 to 90, usually from about 40 to 80, and most often from about 50 to 75.

In some embodiments of the claimed proteins photoactivated, Fotogalerie or photoperiodically, i.e. they change their fluorescent properties when exposed to light of a certain wavelength and intensity. For example, they exhibit a blue fluorescence (emission of approximately 440-500 nm) before irradiation and red fluorescence (emission of approximately 540-650 nm) after irradiation with UV light (for example, about 400 nm). In some embodiments, these proteins may have a red fluorescence before irradiation, which decreases under the action of irradiation (e.g. UV or blue light). In other embodiments, these proteins can be afluorescent (chromogenic) up to, but fluorescence after irradiation with UV or blue light.

In some embodiments of the claimed proteins are placed quickly after expression in a cell host. Under fast time-the eye of the stacking means, what proteins reach their tertiary structure, which provides them with fluorescent property, within a short period of time. In these embodiments, the proteins are placed within a period of time, which in General does not exceed about 48 hours, usually no more than about 24 hours, and often does not exceed about 12 hours (e.g., half-laying may be 3 hours).

Specific proteins of interest include red fluorescent protein EqFP578 fromEntacmaea quadricolorof typeCnidariapreferably from the classAnthozoa, more preferably from a subclass ofZoanthariamore preferably from the orderActiniariaand more preferably from the familyActiniidae(SEQ ID NO: 2); and its functional mutants.

Mutants can save properties of proteins wild-type (e.g., natural) or may have biological properties that are different from the forms of the proteins of the wild type. The term “biological properties of the proteins of the present invention include, but are not limited to, spectral properties, such as maximum fluorescence excitation maximum emission maximum extinction coefficient, brightness (for example, when compared to the reference protein) and the like; biochemical properties, such asin vivoand/orin vitrostability (e.g., half); the rate of maturation, the tendency to agregats the and and the propensity for oligomerization and other similar properties (when compared to the reference protein). Mutations include single amino acid substitutions, deletions and insertions of one or several amino acids, shortening or lengthening of the N-end shortening or lengthening of the C-Terminus and the like.

Mutants can be obtained using standard molecular biology techniques, as described in detail in the section "nucleic acid molecule" above. 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 (e.g., biochemical, spectral etc) property. For example, fluorescence intensity can be measured using a spectrophotometer at different excitation wavelengths.

Interest proteins can also be modified using standard techniques, including RNA editing, chemical modification, modification after translation after transcription, etc. for Example, derivatives of proteins of interest can be obtained by methods such as altered phosphorylation or glycosylation, or acetylation, or limitirovanie, or different types of cleavage during maturation, etc.

In some embodiments, these mutants contain at least one and inoculate replacement which improves folding (folding) of protein at 37°C, where this substitution selected from the group consisting of R32G and S131P. In preferred embodiments of the indicated mutant contains both replacement.

In some embodiments, these mutants also contain at least one or more amino acid substitutions selected from the group consisting of E36G, K42R, F53V, K67R, T68A, L79F, I93V, F110L, N112D, I115L, R138L, G152S, H157R, Y169H, H171I, C172A, F174L, K188R, H193Y, M216V, K220R and R231K where these foldingbike replacement reinforce collapsing (folding) protein and chromophore maturation at 37°C.

In some embodiments, these mutants have modified N - and/or C-terminal part and contain at least one substitution of K6T and R231S or both, where these substitutions reduce the ability of the mutant to aggregation compared with the corresponding wild-type protein.

In some embodiments of the indicated mutant is elongated variant EqFP578 containing additional N-terminal amino acid sequence selected from the group consisting of MGEY (SEQ ID NO: 17) or MGED (SEQ ID NO: 18)where the specified amino acid sequence reduces the ability of the mutant to aggregation.

In some embodiments of interest functional fluorescent mutant contains at least one amino acid substitution selected from the group consisting of R155E, Q159D, S173N, F192V and F194Y, where is shown the mutant has a reduced ability to oligomerization compared to wild type EqFP578. In the preferred embodiment of the specified functional fluorescent protein also contains a replacement N122R, which even more reduces the ability of the protein to oligomerization. In the preferred embodiment of the indicated mutant contains the replacement and also contains one or more substitutions selected from the group consisting of E36G, K42R, F53V, K67R, T68A, L79F, I93V, F110L, N112D, I115L, R138L, G152S, H157R, Y169H, H171I, C172A, F174L, K188R, H193Y, M216V, K220R and R231K where these replacements improve folding and fluorescence intensity of the proteinin vivo.

In some embodiments obtained using genetic engineering techniques functional fluorescent protein of the present invention contains at least one amino acid substitution selected from the group consisting of H197R, S158G, N143S, N143H, N143F or N143Y where the specified functional fluorescent protein has a modified spectra of excitation and emission of fluorescence compared to wild-type protein.

Specific mutants of interest include obtained using genetic engineering techniques functional fluorescent proteins, including amino acid composition SEQ ID NO: 4, 6, 8, 10, 12, 14 and 16, and are discussed in more detail in the experimental part below.

Also provided are proteins that are essentially similar to the above specific proteins, where "essentially the same" means that b is the CTL have the amino acid sequence, the identical sequence of the original protein, at least about 85% identity, typically at least about 90% and often at least about 95%, for example, 95%, 96%, 97%, 98%, 99% or 100% sequence identity).

Proteins of the present invention are present in an environment other than their natural environment; for example, they are isolated from the natural environment or recombinantly. Proteins of the present invention can be selected, which means that the protein is essentially free from other proteins and other biological molecules present in the natural environment, such as oligosaccharides, nucleic acids and their fragments, etc. where the term “essentially free” means that less than 70%, usually less than 60% and more often less than 50% of the composition containing the isolated protein, are some other biological molecules than 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. are of particular 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. In some embodiments of 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 properties 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, e.g. the, fromEntacmaea quadricoloras was described above. The claimed proteins can also be obtained by artificial means, for example, by expression of a recombinant nucleic acid coding sequence of the protein of interest, in an appropriate host, as described above. For protein purification may be used any conventional method, where the appropriate methods for 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, displacement chromatography, gel electrophoresis, affinity chromatography, etc.

Also provided is fused proteins comprising a protein of the present invention, or its fragments, fused, for example, with a sequence, providing a rapid degradation sequence subcellular localization (e.g., signal localization in the nucleus, signal localization in peroxisome, signal localization in the Golgi apparatus, signal localization in mitochondria and so on), signal peptide, or any protein or polypeptide of interest. Slit proteins may include, for example, claimed in the invention fluorescent protein and a second polypeptide (the“merger partner”)fused in frame to the N-terminal and/or C-end of the fluorescent protein. Partners of the merger include, but are not limited to, polypeptides that mo is ut to 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 merger, the General partner of the merger is not naturally linked to a fluorescent protein part of the fused protein and is not usually a fluorescent protein fromEntacmaea quadricoloraccording to the present invention; that is, it is not in organismsEntacmaea quadricolor.

Also provided are antibodies that are specifically associated with the fluorescent protein of the present invention. Suitable antibodies may be obtained using methods known in this field. For example, polyclonal antibodies may 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')2 and Fab, are also of interest.

Transformants

Nucleic acids of the present invention can be used to obtain transformer is s, including 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, Streptomyces sp., Bacillus subtilis, Lactobacillus acidophilus, etc.) 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 ways as technology transgenes, which are known in this field.

The selected nucleic acid according to the present invention can be introduced into the host by methods known in this field, for example, by infection, transfection, 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, 3nd Ed., (2001) Cold Spring Harbor Press, Cold Spring Harbor, NY).

In one embodiment transgenic shall organism can be a 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 of the transgenic organisms 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 can replicate autonomously plasmid vectors.

Another host organism is an animal. Transgenic animals can be produced by transgenic methods known in this field 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 changes of the endogenous locus. An alternative design, the function of nucleic acids is included randomly 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 inseminationin vitroand preferably is aimed at the introduction of recombinant nucleic acid molecules. These nucleic acid molecules can be incorporated into the chromosome, or they may be extrachromosomal replicating DNA.

Design DNA for homologous recombination will contain at least a portion of a nucleic acid of the present invention, where the gene has the desired genetic modification(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 cells is to mammals, 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 the presence of factors inhibiting leukemia (LIF). Transformed ES or embryonic stem cells can be used to produce transgenic animals, using the appropriate method described in this field.

The transgenic animal can be any animal, non-human, including mammalian, non-human (e.g., mouse, rat, bird or amphibian, etc. and used in a functional study, screening drugs, etc. are Typical examples of 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 into this description 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 tissues of the 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 purified DNA, such as plasmid containing the target exogenous sequence of interest, 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 callus and, ultimately, in the transgenic plant by contact with suitable quantities and relations are stimulating factors, such as auxins and cytokines.

Other suitable 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 well as other op the sled above components, claimed in the invention) are used in several different applications. For example, they can be used in the methods of tagging, 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 a method for labeling biological molecules, cells or organelles of the cell, the claimed proteins find use as labelsin vivo(or molecule-reporters) in the analyses of molecular and cell biology. Methods of interest include, but are not limited to, analysis of gene expression, protein localization and co-localization analysis of protein-protein interactions, interactions between protein and nucleic acid interactions, nucleic acid with nucleic acid analysis of the cellular localization and localization of organelles cells, analysis of 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 merge cells or organelles, as markers of integration tile and 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 according to the invention), as a sample, working in real time at near physiological concentrations, etc.

In addition, the claimed proteins can be used in the methods of analysis of gene expression (for example, the activity of the promoter). In other words, they are used for detecting and/or measuring the level of expression of interest protein or polypeptide in a biological material. This method includes: (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 functionally linked and is under the control of a regulatory sequence that controls expression of the target protein or polypeptide of interest; (ii) the expression of the aforementioned nucleic acid under appropriate conditions; and (iii) determining the fluorescence emission of the fluorescent protein as a measure of the expression level of the protein of interest.

In particular, the claimed proteins are used to detect and/or measure the level of expression and/or localization-what about the interest of a protein or polypeptide in a biological material. This method includes: (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 protein of interest or polypeptide, and is functionally connected and is under the control of a regulatory sequence that controls expression of the target protein or polypeptide of interest; (ii) culturing cells under conditions suitable for expression of the protein of interest; and iii) determining the fluorescence emission of the fluorescent protein as a way of measuring the level of expression/localization of the protein of interest.

Target applications include use of the claimed proteins in the methods of 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, having the appropriate spectra excitation/emission fluorescence; 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 is th; rodinovym dyes such as Texas red, tetramethylrhodamine, eosine and erythrosine; and cyan dyes such as SS3 and Su; 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 into this description by reference.

Some examples of that in FRET applications use the claimed fluorescent proteins include, but are not limited to, detection of protein-protein interactions in the two-hybrid system, mammalian, such as dimerization of the transcription factor, multimerization membrane proteins, education multialkali complexes, functioning as part of biosensors for a variety of different events, where the peptide or protein covalently bind FRET fluorescent combination, including the claimed fluorescent proteins, and the bound peptide or protein, representing, for example, a specific substrate for cleavage by caspase; the peptide undergoes a conformational change upon receiving the signal that increases or decreases FRET, such as PKA regulatory domain (camp-sensor), the site of phosphorylation (e.g., the site of phosphorylation of the peptide, or peptide, the method is first to specifically bind phosphorylated/dephosphorylated domain of another protein), or peptide, having Sa2+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 into this description by reference.

Fluorescent proteins of the present invention find use in methods for the detection of the influence of the test substance on the regulation of the 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 substance. Fluorescent proteins also find use in the methods of comparing the activity of two or more sequences regulating expression in the cell in response to the test substance. Such methods can be performed in the presence and in the absence of the test substance, whose influence on the process should be measured.

Fluorescent proteins of the present invention also find use in applications including automatic screening of a multitude of cells expressing fluorescent reporter group, with the use of what Finance microscopic display and electronic analysis. Screening can be used for discovery of drugs and functional genomics, where the claimed proteins are used as markers of whole cells to detect changes in multi-cellular reorganization and migration, for example, in the formation of multicellular tubules (the formation of blood vessel) by endothelial cells, migration of cells through the system Fluoroblok Insert (Becton Dickinson Co.), the 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 kinases and transcription factors during stimulation. Examples include protein kinase C, protein kinase a, the transcription factors NFkB and NFAT; cell cycle 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 and what to use 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 automatic screening of multiple cells in which the claimed fluorescent proteins are used, include U.S. patent No. 5989835 and applications WO 0017624, WO 00/26408, WO 00/17643 and WO 00/03246, the disclosure of which is incorporated into this description by reference.

Fluorescent proteins of the present invention also find application in high-performance screening tests. The claimed fluorescent proteins are stable proteins with half-lives greater than 24 hours. Also provided is destabilized variants of the claimed fluorescent proteins with 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 sequence, inducing degradation cycline B1 of mouse, or ubiquitin, etc. To describe destabilized proteins and vectors that can be used to achieve the same, see, for example, paten the U.S. No. 6130313, disclosure of which is incorporated into this description by reference. Using destabilized variants of the claimed fluorescent proteins for screening of drugs can be detected promoters in signal transduction, such as, for example, API, NFAT, NFkB, Smad, STAT, p53, E2F, Rb, myc, CRE, ER, GR and TRE etc.

The claimed proteins can be used as a secondary messenger detectors, by merging stated Belov with specific domains, such as RCC-gamma Sa-binding domain, RKS-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 the fusion leader sequence with the stated proteins.

The claimed proteins also find use in fluorescent-activated cell sorting (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 into this description by reference.

The claimed proteins also find use as Matki> in vivoin transgenic animals. For example, expression of the claimed protein can be related to 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 into this description 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.

The claimed fluorescent proteins also find use in the analysis of proteasome cleavage. For example, analyses of inactivated by cleavage of fluorescence can be carried out with the use of the claimed proteins, where the claimed proteins contain protease-specific sequence cleavage. The cleavage of a fluorescent protein activated protease fluorescence decreases sharply due to the destruction of the functionality is a high chromophore. The above analysis can be prepared for different types of proteases, such as caspase and others.

The claimed proteins can also be used in assays to determine the phospholipid composition in biological membranes. For example, target proteins that allow binding with specific phospholipids for localization/visualization in model phospholipid distribution in biological membranes, along with allowing colocalization membrane proteins in specific phospholipid lesions may be associated with the claimed proteins fused proteins (or any other form covalent or non-covalent modification of the requested proteins).

The claimed fluorescent proteins also find use as biosensors, sources of circularly peratrovich fluorescent proteins and biosensors of them. Methods of manufacture and use of circularly peratrovich fluorescent proteins described in Nagai et al., Proc Natl Acad Sci USA, 2001, V. 98(6), pp. 3197-3202, Nagai et al., Proc Natl Acad Sci USA, 2004, V. 101(29), pp. 10554-10559, Filippin et al., J. Biol Chem., 2003, V. 278(40), pp. 39224-34 and U.S. patent No. 6469154 and 6699687, the disclosure of which is incorporated into this description by reference. Biosensors, such as CA2+ion indicator, pH indicator, the indicator phosphorylation, or other indicator of enzymatic activity or the indicator ions, such as the magician is s, sodium, potassium, chloride and halide ions can be used in prokaryotic and eukaryotic cells. 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 into this description by reference.

Antibodies, as claimed in the invention described above, also find use in numerous applications, including differentiation of the claimed 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 according to the invention or nucleic acid, its encoding, preferably with elements for the expression of the claimed proteins, for example, the design, such as a vector comprising nucleic acid encoding the claimed protein.

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

EXAMPLES

Example 1

Cloning of cDNA encoding a red fluorescent protein fromEntacmaea quadricolor

To search for fluorescent who CSOs protein of Entacmaea quadricolorwas used strategy for screening cDNA expression libraries inE. coli. A small piece (about 1 mm in length) tentaclesEntacmaea quadricolor(Eukaryota; Metazoa; Cnidaria; Anthozoa; Zoantharia; Actiniaria; Nynantheae; Actiniidae; Entacmaea), which had the red fluorescence, was used to prepare total RNA. Total RNA was isolated using an assay kit NucleoSpin RNA II kit (Clontech). Amplificatory sample cDNA was prepared using assay kit SMART cDNA amplification kit (Clontech) and cloned into PCR-Script vector (Stratagene). Approximately 5×104recombinant clones were proskanirovanno visually using a fluorescent stereomicroscope. The result was identified new red fluorescent protein named EqFP578 (or eqFP578, SEQ ID NO: 1 and 2). Fluorescent protein had about 76% identity of amino acid sequences with other fluorescent protein Entacmaea quadricolor (GenBank Id AAN05449) and 64% amino acid sequence identity with afluorescent red protein asCP562 fromAnemonia sulcata also(GenBank Id AAG41206).

Example 2

Characterization of EqFP578 (SEQ ID NO: 1, 2)

The coding sequence of the nucleic acid EqFP578 was obtained as described in example 1, and expressing cloned in the vector pQE30 (Qiagen), so that the recombinant protein contained six his-tag residues at N-end. After expre is in these E. colithe protein was purified by metal-affinity resin TALON (Clontech) and characterized.

Protein has a peak excitation/emission fluorescence at 552 and 578 nm, respectively (figure 2). The purified protein has a molar extinction coefficient 102000 M-1cm-1and quantum fluorescence yield of 0.54. To determine the molar extinction coefficient, the concentration of the Mature chromophore was determined. Protein was are denatured under alkaline conditions in an equal volume of 2M NaOH. In these conditions, the DsRed-like chromophores (including the chromophore EqFP578) are converted into the GFP-like chromophores that absorb at 446 nm with a molar extinction coefficient of 44,000 M-1cm-1(Ward, W. W., Bioluminescence and Chemiluminescence (1981), Academic Press, 235-242). The absorption spectra of native and denatured EqFP578 was measured. The molar extinction coefficient for the native protein was calculated based on the absorption of denatured protein. To determine the quantum yield of fluorescence EqFP578 compared with fluorescence DsRed2 in number with the same absorption and quantum yield of 0.55.

According to the results of gel filtration was shown that EqFP578 is a dimeric protein. Purified protein (~1 mg/ml) was applied on a column with Sephadex-100 (0,7×60 cm) and washed with 50 mm phosphate buffer (pH 7.0)containing 100 mm NaCl. EGFP, HcRed1 and DsRed2 (Clontech) were used as standards monomial is REGO, dimer and tetramer protein, respectively. Additionally, the tendency to form tetramers was detected when purified recombinant EqFP578 used for gel filtration in a concentration of approximately 10 mg/ml

Example 3

Obtaining mutants EqFP578

In each case, for undirected mutagenesis used set of reagents Diversity PCR Random Mutagenesis kit (CLONTECH) in optimal conditions to obtain 5-6 mutations per 1000 BP

The coding sequence of the nucleic acids of the wild type EqFP578 was obtained as described in example 1. To increase expression in mammalian cells, the authors used the following strategy: 1 - random mutagenesis was used to search protein with an increased rate of maturation and styling; 2 - encoding sequence was "humanitarian"; 3 additional random mutagenesis was undertaken to further increase the speed of maturation and brightness of protein inE. coliat 37°C. the result was obtained mutant EqFP578 called EqFP578m1 (SEQ ID NO: 3, 4), with a genetic code that is optimized for expression in mammalian cells and contains six amino acid substitutions compared to the wild type EqFP578: R32G, T68A, L79F, L110F, S131P and L138R.

Recombinant EqFP578m1 was prepared, purified and characterized as described in example 2 for wild type EqFP578. Fluorescent t the VA mutant similar, but not identical to the properties of the wild type EqFP578. It has a peak excitation/emission fluorescence at 553 and 574 nm, respectively (figure 3). The purified protein has a molar extinction coefficient 104000 M-1cm-1and quantum yield of fluorescence of 0.65. As wild type EqFP578, EqFP578m1 - dimer.

Although EqFP578m1 not aggregates, as expressed byin vivo, it forms aggregates ofin vitroaccording to gel electrophoresis. This property potentially limits the applicability of protein to obtain stable cell lines and transgenic animals. To reduce the ability to aggregation of nucleic acid encoding the protein was subjected to additional modifications, which increased the local negative charge (i.e. the concentration of negatively-charged amino acid residues) at the C - and N - ends of the protein. In particular, the amino acids at the C - and N - ends were replaced to reduce local positive charge, for example, by using site-directed mutagenesis were made replacement K6T and R231S. Furthermore, additional amino acid sequence (MGEY) was added to the N-end of the protein. Random mutagenesis of the obtained nucleic acid led to additional fadingaway mutations K188R, which provides more rapid appearance of the fluorescent signalin vivoas was shown by expression of the protein is the strain XL-1Blue E. coli. Nucleotide and amino acid sequences neuregelung mutant, named M1-NA, shown in SEQ ID NO: 5, 6, respectively.

Recombinant M1-NA was prepared, purified and characterized as described in example 2 for wild type EqFP578. According to the results of gel filtration M1-NA is a dimeric protein at a concentration of 1 mg/ml of the Fluorescent properties of M1-NA are the same as those EqFP578m1. M1-NA - bright fluorescent protein, characterized by peaks in the excitation/emission fluorescence at 553/574 nm. Quantum yield of fluorescence M1-NA =0,67, molar extinction coefficient (at 553 nm) =92000 M(-1)cm(-1). According to the results of gel electrophoresis M1-NA does not form aggregatesin vitro. He also does not form any visible aggregates for expression in eukaryotic cells, as was shown for HeLa and 293T cell lines.

Also, using random mutagenesis was obtained option EqFP578m1 (M1-602), having shifted to the red region of the spectra of the excitation/emission fluorescence. Compared to EqFP578m1 he has a replacement N143S, providing a shift in the red region of the spectra of excitation/emission fluorescence. Random mutagenesis has also brought replace F110L, I115L, R138L, G152S and F174L, which improved the folding and maturation of the chromophore, resulting in a more rapid appearance of the fluorescent signalin vivoas it was shown what about when the expression in strain XL-1Blue E. coli. As for M1-NA, N - and C-ends of the M1-602 were modified by the replacement K6T and R231S and add additional N-terminal amino acid sequence (MGED). Nucleotide and amino acid sequences M1-602 protein shown in SEQ ID NO: 7, 8, respectively.

Recombinant M1-602 was prepared, purified and characterized as described in example 2 for wild type EqFP578. Its characteristics are: the peak excitation/emission fluorescence - when 574/602 nm (see the spectra in figure 4). Quantum yield of fluorescence M1-602 =0,35, molar extinction coefficient (at 574 nm) =74400 M(-1)cm(-1). According to the results of gel filtration M1-602 is a dimeric protein at a concentration of 1 mg/ml

A mutant with even more biased krasnogolovy region spectra excitation/emission fluorescence, named M1-637, was obtained by site directed mutagenesis of residues H197 and G158 based on the nucleic acid that encodes a protein M1-602. Compared to M1-602 M1-637 contains replacement H197R and G158S that a shift in the fluorescence spectrum in the far-red region of the spectrum. Nucleotide and amino acid sequences M1-637 protein shown in SEQ ID NO: 9, 10, respectively.

Recombinant M1-637 was prepared, purified and characterized as described in example 2 for wild type EqFP578. M1-637 - far-red fluorescent protein, characterized the clinical peaks of excitation/emission fluorescence at 591/637 nm (see spectra figure 5). According to the results of gel filtration, M1-637 is a dimeric protein at a concentration of 1 mg/ml

Due to the excellent characteristics of EqFP578 and its mutants described above are of interest to create a bright Monomeric red fluorescent protein with useful properties. The availability of the crystal structure of eqFP611 (Petersen et al., 2003, J Biol Chem v. 278 pp. 44626-44631), a close homologue of eqFP578, allowed the authors to identify amino acid residues responsible for dimerization and weak tetramerization eqFP578 and its mutants. A parallel site-directed mutagenesis of several important amino acid residues responsible for dimerization through the so-called “secondary” or “hydrophilic” interface, i.e. R155E, Q159D, S173N, F192V and F194Y, was carried out on the nucleic acid that encodes a protein EqFP578m1. At the same time to optimize the folding and maturation of Monomeric protein was used random mutagenesis of the whole coding sequence of the protein, with the key positions of the secondary interface were recorded primers. For minor inhibition of dimerization via the ' primary' (“hydrophobic”) interface was also added to replace N122R, which has been described as blocking effective tetramerization eqFP611 (Wiedenmann et al., 2005, J Biomed Opt v. 10: p. 14003).

Bad maturing red fluorescent B. the POC, obtained after the first round of mutagenesis was verified by gel-filtration, and it was shown that it is a monomer. It showed that the introduced mutations reduced dimerization. Further, several additional rounds of random mutagenesis were carried out to optimize coagulation (folding) protein and its maturation at 37°C. After each round, the library was skanirovali in the expressionE. coliusing a fluorescent stereomicroscope Olympus SZX-12, with a TRITC filter set. From 10 to 20 of the brightest clones were selected and verified by sequencing. Only those protein variants that contained key replacement in the secondary interface, were selected for further work. 5-10 selected variants compared the quantum yield of fluorescence, the molar extinction coefficient and photostability. The completion of the maturation of the red chromophore was checked by measuring the absorption spectra. The final version, M1-mono1, was Monomeric red fluorescent protein with a peak excitation/emission fluorescence at 555/584 nm (6). Nucleotide and amino acid sequences M1-mono1 protein shown in SEQ ID NO: 11, 12, respectively.

In addition, two cyanide protein was obtained on the basis of M1-mono1. Nucleic acid encoding M1-mono1, was used for site-directed and subsequent what about the random mutagenesis. The resulting plasmids encoding mutant fluorescent proteins were transliterowany inE. Coliand the most striking cyanide options were selected. First, nrM181-5, contains replacement N143H and R67K that lead to cyan fluorescence of the chromophore and the protein ability to photoconversion to a red fluorescent form. Compared to M1-mono1 nrM181-5 also contains replacement R42K, C172A, V216M and R220K that enhance the maturation of the chromophore and the protein folding. Nucleotide and amino acid sequences nrM181-5 protein shown in SEQ ID NO: 13, 14, respectively.

Recombinant nrM181-5 was prepared, purified and characterized as described in example 2 for wild type EqFP578. nrM181-5 is a cyan fluorescent protein, characterized by peaks in the excitation/emission fluorescence at 400/470 nm (see spectra figure 7). According to the results of gel filtration nrM181-5 is a Monomeric protein at a concentration of 1 mg/ml Protein capable of photoconversion in the red fluorescent form in response to irradiation with violet light.

The second protein Cyan-2-1 contains replacement N143F and H197Y that lead to cyan fluorescent protein chromophore. Cyan-2-1 also includes replacement E36G and F53V that improve coagulation and maturation of the chromophore. Nucleotide and amino acid sequence of Cyan-2-1 protein shown in SEQ ID NO: 15, 16, respectively.

Ryoko is pinentry Cyan-2-1 was prepared, purified and characterized as described in example 2 for wild type EqFP578. Cyan-2-1 represents a cyan fluorescent protein, characterized by peaks in the excitation/emission fluorescence at 400/470 nm. According to the results of gel filtration Cyan-2-1 is a Monomeric protein at a concentration of 1 mg/ml Protein is not capable of photoconversion in the red fluorescent form in response to irradiation with violet light.

Example 4

Preparation of polyclonal antibodies

The coding region of the nucleic acid EqFP578m1 was obtained as described in example 3, and cloned in the expression vector pQE30 (Qiagen), so that the recombinant protein contained six his-tag residues at N-end. After expression inE. colithe protein was purified by metal-affinity resin TALON (Clontech) under denaturing conditions. Rabbits were immunized and additionally were treated four times at monthly periods recombinant polypeptides, emulsified in Freund's adjuvant. After 10 or 11 days after each treatment the blood was collected animals. The polyclonal anticavity was tested on recombinant protein using ELISA and Western immunoblotting.

Example 5

Staining of cells and organelles mammals using mutants EqFP578

For fluorescent labeling of eukaryotic cells encoding placentas the activity EqFP578m1, M1-NA, M1-602, M1-637, M1-mono1, nrM181-5 and Cyan-2-1, obtained as described above in example 3, were cloned in pEGFP-N1 vector (Clontech) between the restriction sites AgeI and BglII (instead of the coding sequence of EGFP). Cell lines HeLa, 293T and Phoenix were temporarily transliterowany received vectors by using LipofectAMINE reagent (Invitrogen) and tested in 20 hours after transfection. For visualization of cells used fluorescent microscope (Olympus CK40, equipped with a CCD camera (DP-50, Olympus). In each case, expression of the protein in all cell lines resulted in bright fluorescence without visible aggregation. Fluorescence is easily detected after 20 hours after transfection.

To test proteins chimeric constructs with subcellular localization signals, signal localization in the mitochondria (MTS) VIII subunit of cytochrome C oxidase person was cloned in N1-vectors, obtained as described above. Transfection of HeLa cells resulted in efficient protein translocation into the mitochondria of the cells of the host. In each case, the bright fluorescence is easily detected 24 hours after transfection. The visible protein aggregation was not observed.

Example 7

Tagging of proteins with mutant EqFP578

Coding sequences EqFP578m1, M1-NA, M1-602, M1-637, M1-mono1, nrM181-5 and Cyan-2-1, obtained as described above in example 3, were cloned in pEGFP-1 vector (Clontech) to replace the coding sequence of EGFP. Coding sequences partners merge (cytoplasmic beta-actin man, fibrillarin and protein bid) were functionally associated with the sequences of the mutants EqFP578, above, by cloning sequences partners merge in polylinker C-vectors in frame with the coding sequences of fluorescent proteins. The vectors were transliterowany HeLa cells. In each case, the distribution of fluorescence was consistent with the expected pattern of distribution partner mergers in the cell. Bright fluorescence was clearly visible 24 hours after transfection.

However, in the case EqFP578m1, M1-NA, M1-602, M1-637, abnormal distribution of the fluorescent signal was observed in the cells of the host, when as a merge partner used alpha-tubulin. On the other hand, fused proteins, including M1-mono1, nrM181-5 and Cyan-2-1 and alpha-tubulin showed the expected distribution of the fluorescent signal, confirming the Monomeric nature of these proteins.

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 not Dol is but be construed as recognition of any such publication, the prototype of the present invention.

1. The selected nucleic acid encoding a functional fluorescent protein selected from the group consisting of:
(a) a nucleic acid that encodes a protein characterized by the amino acid sequence essentially corresponding to the amino acid sequence represented in any of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 and 16; or
(b) a nucleic acid represented by any of SEQ ID NO: I, 3, 5, 7,9,11, 13 and 15; or
(c) a nucleic acid that encodes a protein having a sequence that is at least 90% identical to the amino acid sequence of (a)described above.

2. Vector replication, comprising a nucleic acid molecule according to claim 1 and the elements of the replication of the vector in the cell-master required for amplification of the vector containing a nucleic acid according to claim 1, in the cell-master.

3. The expression cassette containing (a) blastomycete transcription functional in the cell-host; (b) nucleic acid according to claim 1; and (C) the area of transcription termination functional in the cell host, which is 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.

4. The cell or its progeny, producing a fluorescent protein encoded by the nucleic acid according to claim 1, containing expression cluster according to claim 4 in the form of an extrachromosomal element or integrated into the genome of this cell.

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

6. Selected functional fluorescent protein, which is encoded by a nucleic acid according to claim 1, where the specified protein selected from the group consisting of:
(a) a protein characterized by the amino acid sequence essentially corresponding to the amino acid sequence represented in any of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 and 16; or
(b) a protein having a sequence that is at least 90% identical to the amino acid sequence of (a)described above;

7. The way the floor is placed fluorescent protein, encoded by a nucleic acid according to claim 1, comprising (a) obtaining nucleic acid molecules essentially consisting of a nucleic acid molecule according to claim 1, operatively linked to suitable regulatory elements, (b) the expression of a fluorescent protein with a specified nucleic acid molecule, and (C) the allocation of a target fluorescent protein, essentially, not with other proteins.



 

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FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to biotechnology. In particular, the invention relates to an Escherichia coli BL21 (pVEGF-A165) strain and can be used to produce a vascular endothelial growth factor - GST-VEGF-A165 protein. A novel Escherichia coli BL21 (pVEGF-A165) cell strain is obtained, which is transformed by the pGEX-VEGF-A165 plasmid. This strain produces a recombinant GST-VEGF-A165 protein.

EFFECT: invention enables to obtain a Escherichia coli BL21 (pVEGF-A165) strain which is stably transformed by plasmid which codes VEGF, and which secrete this factor in extracellular space when cultured in vitro.

3 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: recombinant virus containing human gene p53, its application, manufacture method and pharmaceutical composition are offered.

EFFECT: invention can be used for gene therapy in human malignant neoplasm treatment and prevention.

8 cl, 14 dwg, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to bioengineering. In particular, the invention relates to method of obtaining recombinant mutant horse cytochrome c. This method is realised by introduction of K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K mutations through site-directed mutagenesis into the horse cytochrome c gene which is contained in pBPCYCS/3 plasmid DNA. Further, the Escherichia coli JM-109 strain of the obtained recombinant plasmid DNA is transformed and the target protein is expressed and introduced through cation-exchange and adsorption chromatography.

EFFECT: invention enables use of recombinant mutant horse cytochrome c as a test system for measuring the rate of generation of superoxide in membrane preparations.

3 dwg, 5 ex

FIELD: biotechnologies.

SUBSTANCE: method is based on assessment of ACTN3 genotype, where genotype 577RR is positively related to sprinter or power abilities; genotype 577XX is negatively related to sprinter or power abilities; genotype 577XX is positively related to stamina; genotype 577RX is positively related to sprinter or power abilities; and genotype 577RX is negatively related to stamina in women.

EFFECT: method makes it possible to select type of sports or competitions for an individual, for instance speed-power type of sports or type of sports that requires stamina, and to develop more optimal training regime for a single sportsman.

1 dwg, 6 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: gene of human vascular endothelial growth factor (VEGF) optimised for expression in mammal cells is presented.

EFFECT: due to introduction of VEGFopt gene into pC4W-VEGFopt plasmid, the invention allows improving the yield of an end product in 1,5 times in comparison with the technology of pC4W-hVEGF165 plasmid containing a natural gene.

3 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention concerns technology of genetically engineered constructs to be applied in cell-based and gene therapy. Gene of human hepatocyte growth factor optimised for expression in mammal cells is presented.

EFFECT: invention allows doubling the production of hepatocyte growth factor in comparison with a human natural gene.

3 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: there is offered application of group of survival-improving polypeptide cone cells originated from rod cells and designated as RDCF, and also coding molecules of nucleic acid to prepare medicines, particularly pharmaceutical compositions used to treat retinal dystrophy. Methods for preparing RDCF by recombinant DNA technologies, and required aids, as well as preparation of antibodies distinguishing said polypeptides are described.

EFFECT: improved clinical effectivenesses.

12 cl, 19 dwg, 1 ex

FIELD: microbiology.

SUBSTANCE: invention represents plasmid vector for transfer of DNA, which comprises sequence coding various fragments of oncoprotein p185neu, which are able to induce immune response in respect to tumors, which hyperexpress p185neu. Invention is also related to pharmaceutical composition on the basis of vector for prophylactics or treatment of patients with risk of development of p185neu-positive tumors, or patients with primary tumors, metastases or relapses of p185neu-positive tumors.

EFFECT: invention makes it possible to increase efficiency of prophylactics or treatment of patients with risk of development of p185neu-positive tumors, or patients with primary tumors, metastases or relapses of p185neu-positive tumors.

10 cl, 14 dwg, 2 tbl, 2 ex

FIELD: microbiology.

SUBSTANCE: glia cells are extracted, seeded, and cell is cultivated to produce monomolecular layer. Then cells are extracted and washed. Total RNA is extracted from them, reverse transcription is carried out, as well as amplification of produced DNA. Genes expression is assessed under action of tested compound.

EFFECT: invention makes it possible to accelerate screening of compounds, to search for new compounds with highest activity, which are potential neuroprotectors.

4 cl, 4 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: there is described method for preparing a polymorphous region of gene PAR1 which can contain T to C replacement in position 3090 and/or A to C replacement in position 3329 of the polynucleotide sequence of wild type gene (NM_001992) with applying a pair of specific primers, and also the method for observing said region prepared of a DNA-containing biological sample for the presence or absence of the specified replacements. There are offered complete sets of the components application of which provides both amplification of the polymorphous region of gene PAR1 under the invention, and if needed, further analysis for genetic modifications in positions 3090 and/or 3329.

EFFECT: higher accuracy of estimating risk of cardiovascular diseases.

7 cl, 17 dwg, 1 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and the DNA version related to adult hypolactasia. The substance of invention includes a nucleic acid molecule containing the 5'-end part of the intestinal digestive lactase-phlorizin hydrolase (LPH) gene which participates or serves as an indicator of adult hypolactasia. The said nucleic acid molecule is selected from a group consisting of: (a) nucleic acid molecules having the SEQ ID N0:1 sequence or containing it, where the SEQ ID NO:1 sequence and (b) nucleic acid molecules having the SEQ ID NO:2 sequence or containing it, (c) nucleic acid molecules consisting of at least 20 nucleotides whose complementary strand is hybridised in strict conditions with the nucleic acid molecule at point (a) or (b), where the said polynucleotide/or nucleic acid molecule contains a cytosine residue in a position corresponding to position -13910 in the 5'-direction from the LPH gene; and (d) nucleic acid molecules consisting of at least 20 nucleotides whose complementary strand is hybridised in strict conditions with the nucleic acid molecule at point (a) or (b), where the said polynucleotide/nucleic acid molecule contains a guanine residue in a position corresponding to position -22018 in the 5'-direction from the LPH gene.

EFFECT: design of a method of testing presence or predisposition to adult hypolactasia, which is based on SNP analysis, contained in the said nucleic acid molecule.

65 cl, 7 ex, 8 tbl, 9 dwg

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