Isolated nucleic acid coding fluorescent biosensor, expression cassette, cell, producing fluorescent biosensor, isolated fluorescent biosensor

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biology and chemistry and deals with isolated nucleic acid, coding fluorescent protein with biosensor properties, expression cassettes, providing expression of said fluorescent protein, cells, producing said protein, and peculiarly fluorescent protein with biosensor properties. Obtained fluorescent protein has amino acid sequence, given in SEQ ID NO:4, and intended for changing NAD+/NADH ratio inside cells by increasing signal with displacement of NAD+/NADH ratio towards decrease of NADH concentration.

EFFECT: claimed invention makes it possible to carry out analysis of processes in cell in real time mode.

4 cl, 6 dwg, 4 ex

 

The present invention relates primarily to the field of biology and chemistry and can be used, in particular, when creating biosensors for detection of adenine dinucleotide, constructed on the basis of fluorescent proteins.

Fluorescent proteins are a family of GFP (Green Fluorescent Protein, GFP), including the GFP from the jellyfish Aequorea victoria (avGFP), its mutants and homologues, today widely known for their extensive use as fluorescent markers in vivo in biomedical research that examined in detail Lippincott-Schwartz and Patterson (Science, 2003, 300(5616):87-91) and Chudakov et al. (J. Physiol Rev., 2010, Jul; 90(3):1103-63).

Fluorescent proteins capable of fluorescence when exposed to light of a suitable wavelength. The fluorescent properties of these proteins due to the interaction of two or more amino acid residues that form the chromophore, and not by the fluorescence of any single amino acid residue.

GFP of hydromedusa Aequorea aequorea (synonym of A. victoria) was described by Johnson et al. 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 green light (J. Cell Comp Physiol, 1962, 60:85-104). cDNA encoding A. victoria GFP was cloned Prasher et al. (Gene, 1992, 111(2):229-33). It turned out that this gene may be heterologic expressed in almost any body thanks to the unique ability of GFP to form an autocatalytic chromophore (Chalfie et al., Science 263, 1994, p.802). This information has opened up broad prospects 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; 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).

Conducted numerous studies to improve properties of avGFP (Aequorea victoria GFP) and to obtain variants of GFP, suitable and optimized for various research purposes. Optimization of the genetic code avGFP (codon usage) to improve expression in mammalian cells ("humanitarianly GFP, Haas, et al., Current Biology, 1996, 6: 315-324; Yang, et al., Nucleic Acids Research, 1996, 24: 4592-4593). Received various GFP mutants, including enhanced green fluorescent protein" (EGFP), which has two amino acid substitutions: F64L and S65T (Heim et al., Nature 373, 1995, R). Other mutants are blue, blue and yellow-green spectral variants of avGFP and contain substitutions of amino acid residues that form the chromophore, and/or residues that form the environment of the chromophore.

In 1999, the GFP homologues have been cloned from nubilipennis the different species of Anthozoa (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 from Anthozoa have greater spectral diversity and include cyan, green, yellow, red fluorescent proteins and violet-blue afluorescent the chromoproteins (CPs) (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 the family of GFP-like proteins includes hundreds of fluorescent and colored GFP homologues. The similarity of these proteins with GFP varies from 80-90% to less than 25% identity of amino acid sequence.

The obtained crystal structure of avGFP wild-type, GFP S65T mutant and the number of GFP homologues (Ormo et al. Science, 1996, 273: 1392-1395; Wall et al. Nat Stmct Biol, 2000, 7: 1133-1138; Yarbrough et al. Proc Nati 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, p.202; Quillin et al. Biochemistry, 2005, 44: 5774-5787). It was postulated that all members of the family have a common 3D structure (GFP-like domain), representing the so-called "barrel" of the 11 beta-layers, forming a compact counter-parallel structure within which is located an alpha helix containing the chromophore. The chromophore is formed inside the GFP-like house is and by oxidative cyclization of three conservative amino acid residues in the Central region of alpha-helix (Cody et al., Biochemistry, 1993, 32, p.1212). The provisions of the amino acid residues that form the chromophore, corresponds Ser65-Tyr66-Gly67 region avGFP. These amino acid residues can be easily identified from any GFP-like protein by aligning its sequence with the sequence avGFP.

Fluorescent proteins are a unique family of structurally related proteins, which are able to form an autocatalytic chromophore without the involvement of external substrates or cofactors. Under the action of the inducing light chromophore produces fluorescence, easy detektiruya using modern laboratory equipment (spectrofluorimeter, fluorescent microscope, fluorescent-activated cell sorters, tablet fluorimeter).

The process is autocatalytic formation of the chromophore proteins with different spectral properties are described in detail in several articles and includes several chemical reactions (Heim et. al. Proc Natl Acad Sci USA. 1994, 91:12501-12504; Ormo et al. Science 1996, 273:1392-1395; Yang et al. Nat Biotechnol. 1996, 14:1246-1251; Brejc et al. J. Proc Natl Acad Sci USA. 1997; 94: 2306-2311; Palm et al. Nat Struct Biol. 1997, 4:361-365; Gurskaya et al., BMC Biochem. 2001, 2:6; Gross et al. Proc Natl Acad Sci USA. 2000, 97:11990-11995; Wall et al. Nat Struct Biol. 2000, 7:1133-1138; Yarbrough et al., J. Proc Natl Acad Sci USA. 2001, 98:462-467; A.A. Pakhomov and Martynov V.I. Chem. Biol. 2008, 15, 755-764; Quillinet et al. Biochemistry. 2005, 44, p.5774; Yampolsky et al. Biochemistry, 2005, 44, p.5788; Shu et al. Biochemistry. 2006, 45, p.9639; Kikuchi et al. Biochemistry. 2008, 47, p.1157; Yampolsky et al., Biochemistry, 2009, 48 (33), p.8077).

GFP-like proteins are widely used to create genetically-encoded biosensors. The study of intracellular processes using such genetically encoded biosensors are becoming more popular, as only such sensors can provide information about the change of the investigated parameter directly in the live system. Such biosensors are in demand both in fundamental studies of the signaling pathways of the body, and when testing toxic and drugs on model cell lines or organisms. In comparison with chemical and physical methods of registration of biologically active substances by biosensors that require exogenously added dyes, substrates or cofactors, genetically encoded the nanobiosensors belong to the class of reagent-free and reusable sensors.

Biosensors based on fluorescent proteins are chimeric proteins, which include sensory domain - protein, protein domain or polypeptide sensitive to changes in certain parameters of cells, such as changes in the concentration of any ion or molecule (calcium ions, hydrogen peroxide, hydrogen ions and so on). As the signal part of the biosensor using GFP-like proteins or their variants subjected to circularly is permutatio (Suslova and Chudakov, Trends Biotechnol. 2005, 23(12), 605-13; Griesbeck, Curr Opin NeurobioL, 2004, v.14(5), p.636; Bunt and Wouters, Int Rev CytoL, 2004, v.237, p.205).

Creating peratrovich GFP-like proteins is necessary to increase the mobility of the chromophore environment and, consequently, for the greater lability of the spectral properties of the protein. Circular permutation of fluorescent proteins described Topell S. and R. Glockshuber (Methods in Molecular Biology. 2002, 183, p.31). For example, for a circular permutation avGFP in its primary structure is made gap in the area between 144 and 149 amino acids, the native N - and C-ends operatively combined using a polypeptide linker. New N - and C-ends are in the immediate vicinity of the chromophore and may affect its microenvironment. Circular permutation is performed on the level of nucleic acids by direct fusion to the 3'and 5'- end a nucleotide sequence that encodes a fluorescent protein, and make a gap in the sequence between codons, encoding the new N - and C-terminal amino acids. Methods for obtaining such structures are well known to specialists in this field.

In the circular permutation KFB acquired the ability to respond to a conformational change in the field of new N and With all the changes in the fluorescence spectrum (sensors using conventional fluorescent proteins were insensitive).

Effective the the General biosensors based on kpfb, obtained from avGFP, was demonstrated on the example of the sensor for calcium ions (Nagai et al., Proc Natl Acad Sci USA. 2001, 98(6), p.197) and hydrogen peroxide (Belousov et al., Nature Method. 2006, 4, p.281).

One of the most important molecules in the cell, for detecting which demanded genetically-encoded biosensors, nicotinamide adenine dinucleotide (figure 1). According to the chemical structure of this molecule is a dinucleotide. It is built of nicotinamide and adenine, connected by a chain consisting of two residues of D-ribose and two residues of phosphoric acid. Nicotinamide adenine dinucleotide (NAD), a coenzyme present in all living cells, is included in the enzyme group dehydrogenases that catalyze oxidation-reduction reactions; performs the function of a carrier of electrons and hydrogen, which takes from oxidizable substances. The reduced form (NADH) are able to transfer them to other substances.

Oxidized and reduced forms of adenine dinucleotide are differences in absorption spectra of (birch and Korovkin. Biological chemistry. Publishing House "Science", Moscow, 1990). NAD+ has a narrow absorption band with a maximum at 260 nm due to the presence of adenine in its structure. The reduced forms of coenzyme a second absorption band with a maximum at 340 nm, this is due to ice what noonien one double bond in nicotinamide complex of coenzyme during the restoration.

Nicotinamide adenine dinucleotide is involved in regulation of many important processes in cells, such as regulation of intracellular CA2+the regulation of gene expression. Known for his participation in the processes of aging and cell death. Important cellular parameter is the ratio of concentrations of the oxidized and reduced forms of adenine dinucleotide (NAD+/NADH). NAD+/NADH index reflects the redox and the overall metabolic status of the cell. In the cytoplasm the value of this parameter is strictly regulated and can vary from 700 to 1, while in the mitochondria, the range of variation is from 8 to 1 (Weihai Y. Frontiers in Bioscience. 2006, 11, 3129-3148).

Currently there are several approaches for the determination of NAD+ and NADH in cells and tissues. In particular, the use of enzymatic systems based on enzymes, which are used as cofactor NADH and NAD+ (Liu et al., Reviews in Fluorescence, 2006, 107-124). As an example, we can point system based on lactate dehydrogenase, involved the conversion of pyruvate to lactate. It is shown that changes in the system of correlation of pyruvate/lactate is proportional to the change in the ratio of NAD+/NADH. In addition, because of NADH in contrast to NAD+ has a fluorescence with a maximum at 460 nm, using alcoholdehydrogenase and ethanol as substrate, can nabludaetsa change in the intensity of fluorescence in the system. The main drawback of these methods is that their use is possible only on biological extracts.

Methods for the registration of changes in the ratio of NAD+/NADH in cells in real-time until recently had not been developed. Recently, there have been several variants of biosensors based on fluorescent proteins for a change in the ratio of NAD+/NADH in cells in real time.

As a sensor domain in these biosensors used NADH-binding bacterial protein Rex (Brekasis and Paget, EMBO J. 2003 22, p.4856). Protein Rex is an important regulator of transcription of components of the respiratory chain in response to changes in intracellular NAD+/NADH index in gram-positive bacteria (Brekasis and Paget, EMBO J. 2003, 22, p.4856; Corda and Girolamo, Embo J. 2003, 22, 1953-8).

Proteins Rex representatives from various bacteria have a significant degree of similarity, about 30% of their sequence is strictly conservative. For all of them postulated the same mechanism (Krystle et al., Molecular Cell. 2010, 38, p.563).

The known crystal structure of the protein T-Rex (from Thermus thermophilus), as well as some other members of the family. T-Rex is a dimer, each subunit consists of two domains connected by a short linker of five amino acids. N-terminal domain (amino acids 1-113) attributed the AET DNA the main role in linking DNA plays alpha-3 loop, the C-terminal domain (120-211 amino acids) is responsible for the binding of nucleotides and represents the motive of Rossman (Wang et al., Molecular Microbiology. 2008, 69(2), p.466). Dimeric state is stabilized due to the alpha-helical C-terminal domain, in particular alpha-8 helix, which is located in the space between the two domains, plays an important role in this. Molecules of NADH are associated with each C-terminal domain near the interface responsible for the formation of dimer (Patschkowski et al., In Bacterial Stress Responses, G.Storz and R.Hengge-Aronis, eds., Washington, DC: ASM Press, 2000, p.61). This leads to conformational changes that are transmitted to the movable linker between domains. In the N-domain dimer rotates with the formation of compact closed state, which are unable to bind DNA (Wang et al., Molecular Microbiology 2008, 69(2), p.466).

Thus, homodimeric Rex can be in three States (Wang et al., Molecular Microbiology. 2008, 69(2), p.466). At low concentration of free nucleotides protein may be in a transitional state is part of its molecules is in a free state, and some in a weak complex with DNA. In the presence of oxygen, when the index of NAD+/NADH has a high value, Rex forms a strong complex with the DNA molecule of NAD+, acting as a repressor of transcription of some genes (cydABC, rexhemACD other). If NAD+/NADH ratio is reduced in conditions of lack of oxygen, Rex binds NADH, which leads to conformational changes in the protein structure and the subsequent release of the protein from the complex with DNA. This activates the expression of genes whose products allow cells to survive in anoxic conditions.

There are several known ways of sensors for registration of NAD+/NADH on the basis of the Rex protein and cpYFP-fluorescent protein, subjected to circular permutation:

- biosensor FRex created on the basis of protein B-Rex (Zhao et al., Cell Metab. 2011, 14(4):555-566). The composition of the biosensor is exposed circular permutation fluorescent protein cpYFP, operatively associated with the N-Terminus with one full-subunit protein B-Rex, and with the end with the nucleotide-binding domain of the second subunit of the B-Rex. Thus, the sensor consists of three protein parts and is presented in figure 2;

- biosensor PEREDOX created on the basis of protein T-Rex (Hung et al., Cell Metab. 2011, Oct 5; 14(4):545-554). The composition of the biosensor is exposed circular permutation fluorescent protein cpYFP, quickly built between two subunits of the protein T-Rex (figure 2).

Also proposed is a variant chimeric protein PEREDOX-mCherry, which PEREDOX quickly merged with the red fluorescent protein mCherry, the fluorescence of which is to normalize the signal of the biosensor.

The resulting sensors allow high JV is cifically to register a change in the ratio of NAD+/NADH in cells in real time. However, all of these biosensors respond with an increase in peak fluorescence excitation at increasing concentrations in the environment of reduced forms of adenine dinucleotide. Thus, these biosensors are applicable in cases when in the course of biological process there is an increase in the concentration of reduced forms of adenine dinucleotide, however, not convenient for the registration of the ratio of NAD+/NADH in cases where the oxidation of adenine dinucleotide.

In addition, these biosensors have significant size (more than 560 amino acids) and complex multidomain structure. It can be difficult to create chimeric proteins based on them for direction of biosensors in certain cell compartments.

The task of the invention is the development of new biosensors for registration of NAD+/NADH in cells in real time, especially with lower molecular weight and are able to respond by increasing signal with increasing concentration of the oxidized form of adenine dinucleotide.

The problem is solved, we offer:

- selected nucleic acid that encodes a fluorescent biosensor for measuring changes in the ratio of NAD+/NADH inside the cells, the amino acid sequence of which is shown in SE ID NO:4, where specified biosensor responds by increasing signal at the offset ratio of NAD+/NADH in the direction of decreasing concentration of NADH;

- cassette expression, which, when integrated into the host cell genome or by introduction into the cell in the form of extrachromosomal element capable of expression of a fluorescent biosensor for measuring changes in the ratio of NAD+/NADH inside the cell and contains nucleic acid according to claim 1 under the control of regulatory elements necessary for expression of the nucleic acid in the cell-master.

- cell, producing biosensor encoded by a nucleic acid according to claim 1, containing the expression cassette according to claim 2 in the form of an extrachromosomal element or integrated into the genome of this cell.

- selected fluorescent biosensor for measuring changes in the ratio of NAD+/NADH inside the cells, encoded by the nucleic acid according to claim 1, where the specified biosensor responds by increasing signal at the offset ratio of NAD+/NADH towards the reduction of the concentration of NADH.

The present invention provides the selected nucleic acid molecule encoding a fluorescent biosensor for registration of the change of the ratio of NAD+/NADH, a signal which increases with displacement ratio of NAD+/NADH increase in the concentration of the oxidized form nicotinamide is disingenuity NAD+ and therefore decreases with increase in the concentration of NADH.

In some embodiments the nucleic acid of the invention encodes a fluorescent biosensor for registration of the change of the ratio of NAD+/NADH inside the cells. In some embodiments the biosensor responds by increasing signal with increasing concentration of the oxidized form of adenine dinucleotide (NAD+), that is, when the shift in the ratio of NAD+/NADH in favor of NAD+.

In some embodiments, the biosensor of the present invention consists of a single protein molecule T-Rex (SEQ ID NO:1), which quickly built protein molecule cpYFP (SEQ ID NO:2). In some embodiments, the biosensor of the present invention has the amino acid sequence shown in SEQ ID NO:4 (T-Rex-116-117-cpYFP-7). In some embodiments the nucleic acid of the present invention has the nucleotide sequence shown in SEQ ID NO:12. Molecules of nucleic acids that differ from the nucleotide sequences due to degeneracy of the genetic code, are also included in the scope of the invention.

In other embodiments, also provided are vectors comprising the nucleic acid of the invention.

In addition, the present invention provides expression cassettes comprising a nucleic acid of the invention and the regulatory elements, the mu is necessary for the expression of nucleic acids in the selected cell host. And also provided with 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 biosensors of the present invention, which are encoded by nucleic acids listed above. In addition, provided the set containing nucleic acids and/or vectors and/or expression cassettes comprising these nucleic acids of the invention.

Brief description of figures

The figure 1 presents the chemical structure of reduced (NADH) and oxidized (NAD+) forms of adenine dinucleotide and their mutual transition.

The figure 2 illustrates the structure of a known biosensors FRex and PEREDOX and biosensors T-Rex-116-117-cpYFP and T-Rex-79-80-cpYFP of the present invention.

The figure 3 presents the scheme of the coupled enzyme system.

The figure 4 shows the spectra of excitation and emission T-Rex-79-80-cpYFP-34.

The figure 5 shows the excitation spectrum T-Rex-116-117-cpYFP-7.

Figure 6 illustrates the dynamics of change in the spectrum of the sample containing T-Rex-116-117-cpYFP-7 by the addition of NADH.

The implementation of the invention

For a more complete disclosure of the above mentioned characteristics of the invention below present the Leno detailed description of the invention, briefly formulated 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 scope of the invention, which may allow other equally effective embodiments.

As indicated above, the present invention is directed to molecules of nucleic acids that encode biosensor for registration of the change of the ratio of NAD+/NADH, a signal which increases with displacement ratio of NAD+/NADH in the direction of decreasing concentration of NADH and increase the concentration of NAD+.

Nucleic acids of the present invention obtained by using recombinant technology. In preferred embodiments of the nucleic acids of the invention encode a protein having the amino acid sequence shown in SEQ ID NO:4. Also provided are vectors and expression cassettes comprising a nucleic acid of the present invention. In addition, provided the cells, stable cell lines, transgenic animals and transgenic plants comprising the nucleic acids, vectors or expression cassettes of the invention.

These nucleic what iSlate used in many different applications and methods in particular, for monitoring changes in the concentration of oxidized and reduced forms of adenine dinucleotide within cells. Finally, there are provided kits for use in such methods and applications.

Definition

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

As used here, the term "fluorescent protein" means a protein belonging to the family of GFP-like proteins, which 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 the fluorescent protein represents a property that is a result of the chromophore formed by autocatalytic cyclization of three or more amino acid residues in the polypeptide chain. As such fluorescent proteins of the invention do not include proteins that have fluorescence at the expense of individual fluorescent residues, such as tryptophan, tyrosine and phenylalanine. The term "fluorescent protein" refers to a fluorescent proteins GFP-family subjected to circular permutation.

As used here, the term "avGFP" otnositsa green fluorescent protein from jellyfish Aequorea victoria, including options avGFP, known from the prior art, designed to provide greater fluorescence intensity or fluorescence in other color regions. The sequence of the wild-type avGFP was disclosed Prasheretal. (Gene. 1992, 111: 229-33).

As used here, the term "fluorescent protein subjected to circular permutation", or "cpfb"refers to a protein derived from a fluorescent protein (e.g. from avGFP) using genetically engineered modification of nucleic acid, which resulted in the C - and N-ends of the original fluorescent protein are efficiently merged, and the new C - and N-ends are formed near the chromophore. Circular permutation does not affect the formation of the "barrel" GFP-like domain and the formation of active (capable of fluorescence) of the chromophore. Circular permutation leads to the fact that KFB acquires the ability to change the spectral characteristics when the conformational changes of protein domains or polypeptides, operatively fused to its C - and N-ends.

As used here, the term "cpYFP" refers to KFB on the basis of avGFP, the amino acid sequence of which is shown in SEQ ID NO:2.

As used here, the term "isolated" or "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 the nature of the circumstances. 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 (delegated), 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 vkluchaut, which have, for example, at least 85% identity, at least 90% identity, at least 95% identity, or at least 96%, 97%, 98% or 99% identity. Two sequences that are identical to one another, also essentially similar.

The percentage of 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., 1990, 215, p.403). 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 proteins" or "essentially similar proteins" refers to proteins that have amino acid sequences identical, at least 85%, usually identical to 90% or more, often identical, at least 95% or more (for example, 96% or more, 97% or more, 98% or more, 99% or more, 100%). The length of the homologous amino acid consequently the values of the "similar" proteins may be, at least 100 amino acid residues, usually at least 200 amino acid residues, or 300 amino acid residues.

In some embodiments, the term "similar proteins" or "essentially similar proteins" refers to proteins that have the amino acid sequence of the whole protein, identical, at least 85%, as a rule, identical to 90% or more, often identical, at least 95% or more (for example, 96% or more, 97% or more, 98% or more, 99% or more, 100%).

As used here, the term "functional" means that the nucleotide or amino acid sequence can function for specified tests or tasks. The term "functional"is used to describe a biosensor of the present invention, means that it changes the spectral characteristics when the change in the ratio of NAD+/NADH in the environment.

As used here, the term "environment" in relation to the biosensor refers to any medium in which this biosensor can function. For the selected protein it can be any buffer solution in which the sensor retains its functionality. For a protein expressed in the cell is the cytoplasm or cell compartment, in which the biosensor is localized.

As used here, "biochemical properties related to protein folding (collapsing) the speed of ripening, the speed of recovery after the reaction with the test substance, 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 can be defined as the amount of any quantitative fluorescent properties, such as intensity of fluorescence at a particular wavelength, or an integral fluorescence across the entire spectrum of emission.

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 Uwe is Chennai solubility, not necessarily have a reduced ability to oligomerization.

As used here, "oligomerization" refers to the propensity or ability of the expressed 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 under physiological conditions, and is relatively stable under these conditions. The reference to "the ability of proteins to oligomerizate means that proteins can form dimers, trimmers, tetramer or similar complexes in the special conditions. Typically, fluorescent proteins have the ability to oligomerization in physiological conditions. 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 this field.

The reference nucleotide sequence "encoding" polypeptide, means that the nucleotide sequence in the translation and transcription of mRNA is produced by the polypeptide. This may be specified as the coding circuit, Ident is CNA 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.

The term "operatively linked", "promptly built-in" or similar when describing the structure of the biosensor or chimeric proteins based on it refers to polypeptide sequences that are in physical and functional connection with one another. In the most preferred embodiments of the basic 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, protein T-Rex, part of the biosensor of the present invention retains the ability to bind adenine dinucleotide, and KBF - ability to fluorescence. In the case when the biosensor of the present invention stitched with interest signal intracellular localization of the chimeric protein retains the ability to react to a change in the ratio of NAD+/NADH, but is localized in a specific cellular compartment. As it is obvious for l is the God of specialist in the field of technology the nucleotide sequence encoding the chimeric protein, including "operatively linked" components (proteins, polypeptides, linker sequences, protein domains, and so on), consist of fragments encoding these components, where these fragments are covalently linked in such a way that during the translation and transcription of the nucleotide sequence is produced by a full-sized chimeric protein. In other words, the fragments are connected so that the connection no "breakthrough" reading frame and stop codons.

As used here, the term "NAD+/NADH" means the ratio of the concentrations of oxidized (NAD+) and reduced (NADH) forms of adenine dinucleotide in the environment. Formula dinucleotide presented in figure 1. As used here, the term "biosensor signal" means detective the change in the spectral characteristics of the biosensor in response to a change in the ratio of NAD+/NADH.

Molecules of nucleic acids

The present invention provides a nucleic acid molecule encoding a fluorescent biosensor for registration of the change of the ratio of NAD+/NADH, a signal which increases with increase in the concentration of the oxidized form of the dinucleotide. As here used, the nucleic acid molecule is supposedly the Kula DNA 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 structure of a biosensor is shown schematically in figure 2. Biosensor is a chimeric protein consisting of a single dual-domain subunit protein T-Rex from Thermus aquaticus (SEQ ID NO:1), which quickly built subjected to circular permutation protein cpYFP (SEQ ID NO:2). In preferred embodiments cpYFP operatively mounted between 70 and 120 amino acids of the protein T-Rex, for example, between 79 and 80 amino acids, or between 116 and 117 amino acids. In preferred embodiments the sequence of cpYFP operatively connected to the sequences of fragments of the T-Rex using amino acid linker sequences 1 and 2, as shown in figure 2. In preferred embodiments the linker sequence 1 consists of three amino acids (e.g., operatively linked amino acids S, a and G), and the linker sequence 2 consists of two amino acids (e.g., operatively linked amino acids G and T).

Methods of producing such chimeric proteins are well known to professionals in this field who. For example, part of the nucleic acid encoding the various elements (e.g., fragments of the amino acid sequence of the T-Rex from 1 to 116 amino acids and 117 of 211 amino acids, a linker sequence, protein cpYFP), can be built in a certain order in polylinker vector in such a way that between the various parts will not stop codons in the reading frame and will not sbec reading frames. Alternatively, the desired nucleotide sequence can be assembled from fragments using a DNA ligase or PCR with primers containing a part of the complementary terminal sequences of connected fragments.

The nucleic acid molecule encoding a fluorescent biosensor, can be synthesized from suitable nucleosidases. This method is based on the well-known in this field protocols. For example, the availability of information about the amino acid sequence or nucleotide sequence enables to make the selected nucleic acid molecule of the present invention using oligonucleotide synthesis. If information about the sequence of amino acids can be synthesized several nucleic acids that differ from each other due to the degeneracy of the genetic code. Methods selection of codons to require the constituent host are well-known in this field. Synthetic oligonucleotides can be prepared using phosphoramidite method, and the resulting constructs 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 invention can be synthesized following stages: several smaller fragments with the necessary complementarity, which contain the appropriate ends, capable of cohesion with neighboring fragment. Adjacent slices can be sewn using a DNA ligase or a method based on PCR. Nucleic acid encoding a biosensor may be any of many known methods.

In addition, also provided degenerate variants of the nucleic acids that encode the biosensor 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 voplosheniya nucleic acid, 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 versions of the nucleic acids of the present invention. As used here, the term "humanitarianly" refers to the changes made in the nucleic acid sequence to optimize codons for expression of the protein in mammalian cells (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.

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 biosensor of the present invention and is capable, under the right conditions (e.g., physiological intracellular conditions) to be used for expression of the protein in the cell host. 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 cf is de, different from the environment in which they are in natural conditions, such as they are selected, presented in a larger quantity, or are expressed in systems in vitro or in cells or organisms other than those in which they are found in natural conditions.

The claimed nucleic acids can be isolated and obtained essentially in purified form. Essentially purified form means that the nucleic acids are at least about 50% pure, usually at least about 90% pure and are typically "recombinant", i.e. flanked by one or more nucleotides with which it is usually not linked to the chromosome, found in nature in its natural organism, the host.

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 substitutions can be made in vitro using various technologies mutagenesis or is Holocene organisms owners, in specific breeding conditions that induce or select for these changes. Such specially obtained sequences can be called "mutants" or "derivatives" of the source sequence.

Mutant or derivative of the nucleic acid can be obtained on the matrix nucleic acid selected from the above-described nucleic acids, by modification, deletion or addition of one or more nucleotides in a matrix of a sequence, or combinations thereof to obtain a variant of the matrix nucleic acid. Modifications, additions or deletions can be performed by any means known in the art (see, for example, Gustin et al., Biotechniques. 1993, 14: 22; Barany, Gene. 1985, 37: 111-123; 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, oligonucleotide-directed mutagenesis, PCR with the Assembly, a pair of PCR mutagenesis, mutagenesis in 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 performed in the methods of the Ohm, 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 mutagenesis, restriction-selective mutagenesis, restriction mutagenesis with cleaning, the synthesis of artificial genes, multiple mutagenesis, creating multiple chimeric nucleic acids, and combinations thereof.

Also provided are nucleic acids encoding chimeric proteins consisting of a biosensor of the present invention and a signal of a specific subcellular localization. Such chimeric proteins can be obtained by operative connection of the nucleic acid of the present invention, the coding of the biosensor, and a nucleic acid that encodes a signal subcellular localization. Methods of obtaining such nucleic acid are well known to specialists in this field.

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 a plasmid, and used to to the onirovaniya, amplification, expression, transfer, etc., the nucleic acid sequence of the invention in a suitable host. The selection of the appropriate vector is understandable to a qualified specialist in this field, and there are many such commercially available vectors. For preparation of design full-sized nucleic acid or part thereof is usually embedded in the vector by attaching DNA ligase to the dissolved enzymes restriction site in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination in vivo, usually joining homologous sites to vector on the flanks of the desired nucleotide sequence. Homologous areas add 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 biosensors or chimeric 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 kassotakis in the cell.

In the cassette of the expression of specified nucleic acid is operatively linked with a regulatory sequence, which may include promoters, enhancers, terminators, operators, repressor substances and inductors and provides the initiation of the reading of RNA (transcription) in the cell host. In the cassette for the expression of a nucleic acid of the invention can also be associated with signals termination of transcription, functional in the cell host. Methods of production of expression cassettes or systems for the expression of the desired product is known for a specialist who is skilled in this area. The above-described expression systems can be used in prokaryotic or eukaryotic hosts. 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 transfetsirovannyh cells that contain a gene incorporated into the genome).

The protein product encoded by the nucleic acid of the invention can be obtained by expression in any expression system, including, for example, a bacterial system, the yeast system, cells, insects, amphibians or cells mlekopitayushchie is. For example, to obtain the 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 of the invention, the resulting replicated nucleic acid is expressed protein or polypeptide are within the claims of the invention as a product of the host cell or organism. The product can be selected in a suitable way known in this field.

Proteins

Nucleic acid according to the invention encodes a fluorescent biosensor for registration of the change of the ratio of NAD+/NADH, a signal which increases with increase in the concentration of the oxidized form of the dinucleotide. Specific biosensors of interest include biosensors having the amino acid sequence of SEQ ID No:4, SEQ ID No: 6, SEQ ID No: 8 and SEQ ID NO:10, the properties of which are described in detail in the experimental part, infra.

Declared biosensor has the ability to detectable fluorescence, which can be registered with visualisering, spectrophotometry, spectrofluorimetry, fluorescence microscopy, using FACS or other accepted method for registration of fluorescence. Declared biosensor has a maximum (peak) of the fluorescence emission in the range from 500 nm to 550 nm, for example, 520 nm, and two peaks of fluorescence excitation; the first in the range 400-450 nm (e.g., 420 nm) and the second in the range 470-510 nm (e.g., 490 nm). In advantageous embodiments, the first peak excitation is weaker than the second. In advantageous embodiments the first peak of the excitation can be measured using spectrophotometry, spectrofluorimetry, fluorescence microscopy, using FACS or other accepted method for registration of fluorescence.

Declared biosensor changes the intensity of fluorescence when excited by light with a wavelength of 490 nm, and the fluorescence intensity when excited by light with a wavelength of 420 nm remains unchanged. For purposes of the present invention the signal of the biosensor is the ratio of the fluorescence intensity when excited by light with a wavelength of 490 nm to the fluorescence intensity when excited by light with a wavelength of 420 nm. This parameter does not depend on the concentration of the biosensor in the environment.

In advantageous embodiments, the intensity of fluorescence stated biosensor when the excitation light with the length of any 490 nm decreases with the increase in the concentration of NADH in the environment, accompanied equimolecular increasing concentration in the environment of NAD+. Thus, by increasing the concentration of NAD+ in the environment is an increase in signal of the biosensor.

Check signal of the biosensor can be carried out using spectrophotometry, spectrofluorimetry, fluorescence microscopy, using FACS or other accepted method for registration of fluorescence.

Declared biosensor has high sensitivity and is capable of detecting a change in the ratio of NAD+/NADH in the range from 0.1 to 700, typically in the range from 400 to 0.1.

The sensitivity of the biosensor can be measured in cell lysate containing biosensor, in coupled enzymatic system, schematically shown in figure 3. In this system during the enzymatic reaction of glucose by the action of hexokinase is transformed into glucose-6-phosphate, which is the substrate for the dehydrogenase, is used as the cofactor NAD+. Thus, it is possible to simultaneously check the dynamics of changes in the ratio of NAD+/NADH using enzymatic systems and signal changes of the biosensor that is present in the environment.

Declared biosensor has a relatively small size and consists of 400-500 amino acids, usually the length of the biosensor 450-470 amino acids, for example 462 amino acids (including the th first methionine).

Also provided functional biosensors, which is essentially the same as the above biosensors, where essentially the same means that these proteins have an amino acid sequence identical to the sequence of SEQ ID Nos:4, 6, 8 or 10, at least 85% identity, typically at least 90% and often at least 95% (e.g., 95% or higher; 96% or higher, 97% or higher; 98% and above: 99% or greater or 100% identity to the sequence).

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, the intensity of fluorescence can be measured using an under different excitation wavelengths.

The biosensors of the invention are present in an environment other than their natural environment; for example, they recombinantly. Proteins of the invention can be selected, which means that the protein is essentially free from other proteins and other biological m is of the 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 is a certain 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%.

Declared biosensors can 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.

It also provides fusion proteins comprising the biosensor of the present invention, fused with a sequence of subcellular localization (e.g., signal localization in the nucleus, in peroxi the max, the Golgi apparatus, mitochondria, and so on). A polypeptide that provides a specific subcellular localization, may be operatively attached to the N-end and/or the end of the biosensor. The subcellular localization signals is well known to specialists in this field and are described, for example, Nakai K. (Advances in Protein Chemistry. 2000, Vol.54, p.277).

Transformants Nucleic acid of the present invention is used to obtain transformants, 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 nucleotide sequence that is new acid of the invention can be introduced in a host of ways, known in this field, such as 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. 2001, 3nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY).

In one embodiment the transgenic organism can be prokaryotic organism. Methods of transformation of prokaryotic hosts are well described in the art (for example, see Sambrook et al. Molecular Cloning: A Laboratory Manual, 1989, 2nd edition; 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, Alien and Unwin, London, p.401 and King et al Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, Blackie, Glasgow. 1989, p.107). 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. In another embodiment of the transgenic organisms can be animals.

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 Laoratory Handbook. (2003), 2nd edition, San Diego: Academic Press; Gersenstein and Vintersten, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed, (2002), Nagy A. (Ed), Cold Spring Harbor Laboratory; Blau et al. Laboratory Animal Medicine, 2nd Ed., (2002) Fox J.G., Anderson L.C., Loew P.M., 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. Alternatively, the design of the nucleic acids 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 in vitro fertilization, and preferably is aimed at the introduction of recombinant nucleic acid molecules. These nucleic acid molecules can be incorporated into the chromosome, or they can be extrachromosomal replicating DNA. Design DNA for homologous recombination will comprise at least part of the nucleic acid of the present invention, where the gene has the desired GE is micescu 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 mammalian cells (Keown et al., Meth. Enzymol. 1990, 185:527-537).

The transgenic animal can be any animal, non-human, including mammalian, non-human (such as a mouse, rat, bird or amphibian, etc. and used in a functional study, screening drugs, etc. can Also be obtained from transgenic plants. Methods for producing transgenic plant cells and plants are described in U.S. patents№№5767367; 5750870; 5739409; 5689049; 5689045; 5674731; 5656466; 5633155; 5629470; 5595896; 5576198; 5538879; 5484956; disclosure of which is incorporated here by reference. Methods of obtaining transgenic plants are also considered in Plant Biochemistry and Molecular Biology (eds. Lea and Leegood, John Wiley & Sons) 1993, p.275 in Plant Biotechnology and Transgenic Plants (eds. Oksman-Caldentey and Barz), (2002) p.719) 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 the plant cell is, while 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. Can be used with other suitable methods of obtaining plants that are available for qualified professionals in this field, such as the use of "gene gun", or Agrobacterium-mediated transformation.

Applications

The biosensors of the invention are genetically encoded fluorescent proteins, changing the spectral properties in the presence of hydrogen peroxide. They can be used for registration of the change of the ratio of NAD+/NADH inside the cells, especially during the processes, which are Paden is e concentrations of NADH and accordingly an increase in the concentration of NAD+.

For the implementation of the application must be received by nucleic acid encoding a biosensor. The receipt of such structures is obvious to any expert in this field. The resulting design should be built into the expression cassette or vector)that provides temporary or permanent expression of this nucleic acid in the cells of the host. The vector or expression cassette may contain elements that provide targeted delivery of the design in the required cells, or in the composition of the particles, providing targeted delivery. After transfection of the cells with the vector and the time required for developments in the cells of the expression product, can be carried out registration of the change in the ratio of NAD+/NADH inside the cells.

The biosensors of the present invention can be made with signals of different intracellular localization direction of biosensors in certain cellular compartments and registering vibrations of NAD+/NADH in these cell compartments.

Sets

Also provided in accordance with the present invention, kits for use in the implementation of the above applications. In preferred embodiments, the kits typically include a nucleic acid encoding a biosensor, preferably with elements for expression of the biosensor in the cell-the household is ine. For example, the kits can include a vector comprising nucleic acid encoding declared biosensor. Kit components are usually present in the corresponding storage medium, such as water or buffer solution, typically in a suitable container. In some embodiments the kit includes many different vectors, each of which encodes the claimed protein, where the vectors are designed for expression in different environments and/or under different conditions, for example, constitutive expression, where the vector includes a strong promoter for expression in mammalian cells or vector with weak promoter, multiple cloning sites for selective insertion of promoter and non-standard expression, etc.

In addition to the above-described components of the claimed kits will further include instructions for carrying out the proposed methods. These instructions may be present in the claimed kits in various forms (for example, in printed form or on electronic media in the form of text and/or graphics file) in the amount of one or more.

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

Examples of specific implementation of the present invention

Example 1

Receiving options biosensor

Nucleic acid, to yuushuu protein T-Rex from Thermus aquaticus, was obtained by PCR from genomic DNA of Thermus thermophilus using gene-specific primers and cloned in pQE-30 (Qiagen) no standard technology.

Nucleic acid encoding a cpYFP, synthesized from the appropriate nucleosidases and used for embedding in the sequence of the nucleic acid T-Rex frame read before the triplet encoding the following amino acid residues: 78, 79, 99, 100, 116, 117, 126, 130, 133, 136, 140. The respective DNA fragments T-Rex and cpYFP amplified with gene-specific primers, purified by electrophoresis in 1% agarose gel and combined into the whole design method "overlap extention" PCR as described Wurch et al. (Methods in Molecular Biology. 2004, 12 (9), p.653). Amplification was carried out on the unit PTC-100 Thermal Cycler (MJ Reserch). Each reaction sample also contained primers (0.5 µm), an equimolar mixture of dNTP (0.5 µm), matrix DNA (10-100 ng), Tersus polymerase (Evrogen). To bring the mixture to the desired volume of used water purification milliQ. Use the following mode of amplification: denaturation 95°C - 12 sec; annealing 65°C for 4 min; elongation 72°C - 1 min, 10 cycles Poland. Amplification of the full design was carried out by 15 cycles of PCR with end primers. Use the following mode of amplification: denaturation 95°C - 15 sec; annealing 60°C - 20 sec; elongation 72°C - 1 min Construct cloned in pQE-30 (Qiagen) according to the standard technology is used for transfection of cells of E. coli. On the first stage of selection was assessed by the presence of fluorescence brightness of colonies of E. coli that had been transformed by each of the obtained genetic constructs. In addition, estimated time of appearance of fluorescence in these colonies, which gave information about the speed of maturation of different versions of the sensor in bacterial cells.

The optimal selection of clones expressing the protein of the sensor was performed using fluorescent binocular microscope Olympus US SZX12. Visually assessed the presence of fluorescence and brightness of individual colonies of E. coli upon excitation in the blue and violet regions of the spectrum. Take into account the speed of appearance of fluorescence of individual colonies. The selected clones were transferred to a new Cup and pokasivali at 37°C for 10-12 hours.

For most clones containing different versions of the chimeric proteins, the fluorescence intensity was weak. When integrating cpYFP between the positions 117-118, 126-127, 130-131 protein T-Rex fluorescence clones expressing such versions of the chimeric proteins was not observed. More intense fluorescence compared to other possessed clones that expressed protein constructs, where cpYFP was integrated between the positions 79-80, 99-100 and 116-117. These versions were used in further testing.

The next stage was estimated sensitivity of the preferred options is isensor to the change in the ratio of NAD+/NADH. This recombinant protein was purified from cells using metal affinity chromatography resin TALON (Clontech, USA) and evaluated the magnitude of change in the fluorescence spectrum, depending on the availability in the environment of NADH and NAD+. Measurement of spectral characteristics was performed on a fluorescence spectrophotometer Varian Cary Eclipse. For this, cells that are descendants of the one selected at the first stage of the clone, in a small number were selected and suspended in 1 ml of PBS buffer in a ditch for fluorimetry. Next cell in the contributed surplus of NAD+ (250 nm protein 100 μm NAD+), after the change of the sensor signal in the same sample gradually increased the concentration of NADH to 25, 50, 100, 250 and 500 nm. Compared the responses of different versions of the sensor to the appearance of small amounts of NADH in the system against the background of excess NAD+. Thus, the selected version of the sensor is most sensitive to NADH.

To determine the concentration of protein denaturation was performed on the sample by adding NaOH to a final concentration of 1M. After this sample was removed absorption spectrum in the range from 350 to 500 nm on the spectrophotometer Varian Saga 100 Bio. Under these conditions, the chromophore biosensor absorbs at 450 nm (molar extinction coefficient = 40,000 M-1cM-1). Knowing the optical density and the molar extinction coefficient of chromophore, op is delali the protein concentration according to the formula as the product of molar extinction coefficient and optical density.

Spectra of fluorescence excitation of all tested proteins were submitted to a well-defined deprotonated form of the chromophore (maximum excitation - 490-500 nm), while the protonated form (maximum excitation at 420 nm) was expressed weaker.

Biosensors T-Rex-79-SO-cpYFP (SEQ ID NO:7, 8) and T-Rex-116-117-cpYFP (SEQ ID NO:9, 10) react even to small changes in the concentration of NADH in the system. For example, at a concentration of 250 nm in the sample T-Rex-79-80-the cpYFP fluorescence intensity when excited by light with a wavelength at 500 nm was 520 optical unit, at concentrations in the same sample of 25 nm NADH intensity decreased to 400 O.% and continued to decrease with further increase in the concentration of NADH. The intensity in the region of 420 nm remained unchanged.

Excitation spectrum T-Rex-99-100-cpYFP changed very little even when excess amounts of NADH in the sample. So T-Rex-99-100-cpYFP was excluded from further testing.

Example 2

Random mutagenesis by T-Rex-79-SO-cpYFP and T-Rex-116-117-cpYFP

The main drawback of the received versions of the biosensors was slow maturation of proteins. Fluorescence of bacterial clones that were transformed with the respective plasmids versions, was observed only on the second day.

To speed the ripening of nucleic acid biosensors T-Rex-79-SO-cpYFP and T-Rex-116-117-cpYFP, obtained as described in example 1, was subjected to random mutagenesis by using a set of random mutagenesis (Clontech) using methods supplied by the manufacturer. In further selected clones by the presence of bright fluorescence and time of appearance of fluorescence.

We selected 65 clones protein T-Rex-79-80-cpYFP, which is its mutant forms. Proteins were isolated and tested for the sensitivity of the fluorescence spectrum to NAD+ and NADH. From all selected mutant versions of only one (T-Rex-79-80-cpYFP-34) retained its sensitivity to NAD+ and NADH, identical to the original version of T-Rex-79-80-cpYFP. Compared to the original protein this version contained the replacement L169P, Y175N and D313G. All three mutations are located in the sequence cpYFP, while the structure of the T-Rex was not changed. Figure 4 shows the spectra of excitation and emission T-Rex-79-80-cpYFP-34. Sequences of nucleotides and amino acids of this protein is shown in SEQ ID No:5 and 6, respectively.

We selected 40 clones protein T-Rex-116-117-cpYFP, which is its mutant forms. Proteins were isolated and tested for the sensitivity of the fluorescence spectrum to NAD+ and NADH. On the basis of the test was selected version of T-Rex-116-117-cpYFP-7, which have the highest sensitivity to NAD+ and NADH. Compared to the original protein this version contained the replacement D350G. Figure 5 shows the spectra of excitation and emission T-Rex-116-117-cpYFP-7. Sequence well is Leonidov and amino acids of this protein is shown in SEQ ID No:3 and 4 respectively. Protein T-Rex-116-117-cpYFP-7 differs from the T-Rex-79-80-cpYFP-34 on the spectrum of excitation and has a more pronounced protonated form of the chromophore (maximum excitation by light of wavelength 420 nm). Typical dynamics of change of the spectrum of the sample containing T-Rex-116-117-cpYFP-7, by adding NADH is shown in Fig.6.

Example 3

The change in the spectral characteristics of T-Rex-116-117-cpYFP-7 and T-Rex-79-80-cpYFP-34 in coupled enzymatic system.

Biosensors T-Rex-116-117-cpYFP-7 and T-Rex-79-80-cpYFP-34, nucleic acids which were obtained as described in example 2, were isolated from bacteria as described in example 1, and used to test their sensitivity to the change in the ratio of NAD+/NADH in a coupled enzymatic system, shown in figure 3.

To do this in a spectrophotometric cuvette made of the following components of the reaction medium: 30 mm Tris-HCl buffer, 10 mm glucose, 15 mm MgCl2, 2.5 mm ATP (AppliChem), 1 mm NAD+ (Sigma). The amount of protein in the different samples was different because the sensor signal should not depend on its concentration. Next, the system was made of glucose-6-phosphate dehydrogenase, were incubated for 3-5 minutes, then started the reaction by addition of hexokinase. The increase of NADH in the system was detected by fluorescence at 340 nm. Thus we determined the dependence of the sensor signal (the ratio of the intensities 420/490 on the spectrum of excitation) from NAD+/ON THE H ratio in the system at a given time (the spectra were taken every 30 seconds). For both options, the intensity at 340 nm, which corresponds to the increase in the share of NADH in the system and reducing NAD+, was accompanied by a decrease in the intensity at 500 nm, which corresponds to the deprotonated form of the chromophore sensor. In the region of 420 nm almost no changes. Both showed high sensitivity to the change in the ratio of NAD+/NADH in the environment.

Example 4

Expression in mammalian cells.

Vectors based on the pQE-30, containing nucleic acid for T-Rex-116-117-cpYFP-7 and T-Rex-79-80-cpYFP-34, were obtained as described in examples 1 and 2. For the implementation of perchlorovinyl obtained constructs from the vector pQE30 in vector pCleGFP (Clontech, USA) a nucleic acid fragments containing the coding region of the biosensor, amplified using end gene-specific primers containing restriction sites for perchlorovinyl and polymerase Tersus (Evrogen). The resulting fragments were purified using the phenol-chloroform extraction and ligated in pCleGFP, preliminary, subject to the restriction on similar sites, in place of the EGFP.

Cells HeLa Kyoto seated in the slides (µ-Slide VI, Ibidi) in DMEM containing 10% inactivated fetal serum (PAA Laboratories) from 5-7 thousand cells on one track, transferrable appropriate vector and cultured for standartmetrologia.

Before microscopy environment all slides were replaced with Hanks solution without sodium bicarbonate. To visualize the fluorescence of transfected cells were used fluorescent microscope (Leica DMI 6000 Century Microscopy of cells was carried out at 37°C, detection of fluorescence was performed on the two channels correspond to the two peaks of excitation of the chromophore cpYFP (420 nm and 490-500 nm), emission 510 nm. For a channel corresponding to the protonated form of the chromophore cpYFP (420 nm)was used filters EFW Excitation: 427/10 (CFP), Emission: BP 542/27 (YFP). For deprotonated forms of the chromophore cpYFP(500 nm) filters Excitation: BP 504/12 (YFP); Emission: BP 542/27 (YFP). Used the lens NSH P2 ApoLambda blue 63*1,4 Oil. Both biosensor expressibility in HeLa cells Kyoto and kept the functionality that has been confirmed through testing of biosensors in the cell lysate. For testing used a coupled enzymatic system described in example 2.

All publications and patents 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 according to the invention, and should not be interpreted as recognition of any such publication about what atiom of the present invention.

1. The selected nucleic acid encoding a fluorescent protein with properties of a biosensor for measuring changes in the ratio of NAD+/NADH inside the cells, the amino acid sequence of which is shown in SEQ ID NO:4, where the specified fluorescent protein with properties of the biosensor responds by increasing signal at the offset ratio of NAD+/NADH in the direction of decreasing concentration of NADH.

2. The expression cassette, which, when integrated into the host cell genome or by introduction into the cell as part of extrachromosomal element capable of expression of the fluorescent protein with properties of a biosensor for measuring changes in the ratio of NAD+/NADH inside the cell and contains nucleic acid according to claim 1 under the control of regulatory elements necessary for expression of the nucleic acid in the cell-master.

3. Cell, producing a fluorescent protein with properties of the biosensor encoded by a nucleic acid according to claim 1, containing the expression cassette according to claim 2 comprising extrachromosomal element or integrated into the genome of this cell.

4. Selected fluorescent protein with properties of a biosensor for measuring changes in the ratio of NAD+/NADH inside the cells, encoded by the nucleic acid according to claim 1, where the specified fluorescent protein with properties of the biosensor responds by increasing signal when the placing of the ratio of NAD+/NADH in the direction of decreasing concentration of NADH.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Described is molecule of chimeric nucleic acid of porcine circovirus (PCV2Gen-1Rep), which includes molecule of nucleic acid, coding porcine circovirus of type II (PCV2), which contains sequence of nucleic acid, coding protein Rep of porcine circovirus of type 1 (PCV1). Chimeric molecule of nucleic acid is constructed by replacement of gene Rep ORF1 PCV2 with gene Rep ORF1 PCV1. Invention also includes biologically functional plasmid or viral vector, which contain unique molecules of chimeric nucleic acids, suitable host cells, transformed by plasmid or vector, infectious chimeric porcine circoviruses, which produce suitable host cells, method of obtaining immunogenic polypeptide product with application of novel chimera, viral vaccines, protecting pig against viral infection or syndrome of postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of protecting pigs against viral infection or postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of obtaining unique chimera PCV2Gen-1Rep and the like. Invention can be applied in veterinary.

EFFECT: invention additionally includes novel method of increasing level of replication and PCV2 titre in cell culture.

21 cl, 2 dwg, 6 ex

Fused rage proteins // 2513695

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biochemistry. Claimed is fused protein for treating diseases, mediated by advanced glycation end products (AGE), consisting of a fragment of a version of human receptor of advanced glycation end products (RAGE), which has two point mutations H217R and R221H, and a fragment of constant domain of human immunoglobulin IgG4, joined with linker if necessary. In addition, considered are: nucleic acid and recombinant host cell for obtaining fused protein, as well as pharmaceutical composition for treatment of AGE-mediated diseases, which contain fused protein.

EFFECT: invention ensures lower aggregation of fused protein.

13 cl, 19 dwg, 3 ex, 9 tbl

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biotechnology, in particular to novel peptide analogue of insulin-like growth factor-1 (IGF-1), which contains amino acid substitution of methionine in position 59 on Asn, Leu, Nle, Ile, Arg, A6c, Glu, Trp or Tyr, as well as other additional substitutions, inserts and deletions. Said peptide or its pharmaceutically acceptable salt is used in composition of pharmaceutical composition for treatment of IGF-1-mediated diseases, as well as in method of treating dwarfism.

EFFECT: invention makes it possible to obtain IGF-1 analogue-agonist, possessing higher biological activity with respect to native IGF-1.

17 cl, 2 tbl

FIELD: medicine.

SUBSTANCE: present group of inventions relates to biotechnology. What is presented is a humanised anti-CD79b antibody and its antigen-binding fragment produced of murine antibody MA79b and CD79b having a substantially analogous binding affinity thereto. A polynucleotide, a vector, a host cell and a method for producing the anti-CD79b antibody according to the invention; immunoconjugates, compositions and methods for cell growth inhibition, a method of treating an individual suffering cancer, a method of treating a proliferative disease and tumour in a mammal, a method for B-cell proliferation inhibition; a method for detecting the presence of CD79b in a sample and method for binding the antibody to the CD79b expressing cell are also disclosed.

EFFECT: given invention can find further application in therapy of the CD79b associated diseases.

86 cl, 20 tbl, 9 ex, 51 dwg

Anti-mif antibodies // 2509777

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and immunology. Invention discloses a monoclonal antibody and its antigen-binding parts which specifically bind the C-end or central part of the macrophage migration inhibitory factor (MIF). The anti-MIF antibody and its antigen-binding part further inhibit biological function of the human MIF. The invention also describes an isolated heavy and light chain of immunoglobulins obtained from anti-MIF antibodies, and molecules of nucleic acids which encode such immunoglobulins.

EFFECT: disclosed is a method of identifying anti-MIF antibodies, pharmaceutical compositions containing said antibodies and a method of using said antibodies and compositions for treating diseases associated with MIF.

22 cl, 14 dwg, 16 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to creation of recombinant plasmids providing expression of poly-epitopic tumour-associated antigens in dendritic cells capable of stimulation of specific cytocidal cells, and it may be used in medicine. Recombinant plasmid DNA pCI-UB-POLYEPI contains 11 epitopes of tumour-associated antigens of colorectal cancer, its size is 6 355 n. p. and it expresses the following amino acid sequence: DYKDDDDK-LLGVGTFVV-ADRIW-GLKAGVIAV-AAYARY-VLAFGLLLA-ADRIW-YQLDPKFITSI-AAYARY-IMIGVLVGV-ADRIW-YLSGADLNL-AAYARY-CGIQNSVSA-AAYARY-LLLLTVLTV-ADRIW-QYIKANSKFIGlTEL-ANIY-SIINFEKL-ARY-SASFDGWATVSVIAL-ARY-SERVRTYWIIIELKHKARE-ARY-IQNDTGFYTLHVIKSDLVNEE. Mature dendritic cells obtained by adding to immature dendritic cells of pro-inflammatory TNF-α (tumour necrosis factor) cytokine are transfected by constructed plasmid DNA pCl-UB-POLYEPI thus activating them. Then activated dendritic cells are cultured together with peripheral mononuclear blood cells of people sick with colorectal cancer for generation of antigen-specific antitumour cytocidal cells.

EFFECT: invention allows efficient generation of antigen-specific cytocidal cell with antitumour activity in vitro, required for immune response by the 1-st type T-helper to colorectal cancer antigens.

2 cl, 1 dwg, 4 ex

Anti-axl antibodies // 2506276

FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed are monoclonal antibodies which bind to the extracellular domain of receptor tyrosine kinase AXL and which at least partially inhibit AXL activity, as well as antigen-binding fragments. Also provided is an isolated nucleic acid molecule, a host cell and a method of producing a monoclonal antibody and an antigen-binding fragment thereof, as well as use of the monoclonal antibody or antigen-binding fragment thereof to produce a drug, pharmaceutical compositions, a method of diagnosing and a method of preventing or treating a condition associated with expression, overexpression and/or hyperactivity of AXL.

EFFECT: invention can be used in therapy and diagnosis of diseases associated with AXL.

23 cl, 20 dwg, 24 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically binds heparin-binding EGF-like growth factor (HB-EGF) and its antigen-binding fragment. Invention describes a nucleic acid molecule, an expressing vector, a host cell and a method for obtaining an antibody or its antigen-binding fragment, as well as use of antibody or its antigen-binding fragment for obtaining pharmaceutical composition for diagnostics, prevention or treatment of hyperproliferation disease, methods and sets for diagnostics and prevention or treatment of the state associated with HB-EGF expression. This invention can be further found in therapy of diseases determined with or related to HB-EGF expression.

EFFECT: improving efficiency of composition and treatment method.

34 cl, 43 dwg, 28 ex, 12 tbl

FIELD: biotechnologies.

SUBSTANCE: invention describes polynucleotide, expression vector, host cell and production method of humanised antibody together with their use, as well as medical preparation against rheumatoid arthritis, prophylaxis or treatment method of rheumatoid arthritis and use of humanised antibody at production of pharmaceutical preparation for prophylaxis or treatment of rheumatoid arthritis. This invention can be used in therapy of human diseases associated with α9 integrin.

EFFECT: improved activity and thermal stability.

14 cl, 6 dwg, 6 tbl, 11 ex

Organic compounds // 2502802

FIELD: biotechnologies.

SUBSTANCE: invention refers to eucariotic vector for expression of target recombinant product in a mammal cell and to its use, to a mammal cell for production of target recombinant product and to a method for its production, a method of a mammal cell selection and a method for obtaining a target recombinant product. Vector includes the first polynucleotide coding a functional folate receptor bound to a membrane as a selective marker and the second polynucleotide coding the target product that is expressed in a recombinant manner. Target product represents a pharmaceutically active, therapeutically active or diagnostic polypeptide. Functional folate receptor bound to the membrane and target product are expressed from the above expression vector. Sampling system is based on introduction of a gene of exogenic functional folate receptor bound to the membrane to a mammal cell.

EFFECT: invention allows effective selection of transformed cells and high yield of target product.

26 cl, 3 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Described is molecule of chimeric nucleic acid of porcine circovirus (PCV2Gen-1Rep), which includes molecule of nucleic acid, coding porcine circovirus of type II (PCV2), which contains sequence of nucleic acid, coding protein Rep of porcine circovirus of type 1 (PCV1). Chimeric molecule of nucleic acid is constructed by replacement of gene Rep ORF1 PCV2 with gene Rep ORF1 PCV1. Invention also includes biologically functional plasmid or viral vector, which contain unique molecules of chimeric nucleic acids, suitable host cells, transformed by plasmid or vector, infectious chimeric porcine circoviruses, which produce suitable host cells, method of obtaining immunogenic polypeptide product with application of novel chimera, viral vaccines, protecting pig against viral infection or syndrome of postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of protecting pigs against viral infection or postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of obtaining unique chimera PCV2Gen-1Rep and the like. Invention can be applied in veterinary.

EFFECT: invention additionally includes novel method of increasing level of replication and PCV2 titre in cell culture.

21 cl, 2 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Claimed is separated chimeric polynucleotide for amplification of production of heterologous protein of interest, which contains polynucleotide sequence of promoter SigA or SigH, functionally connected with polynucleotide, coding protein YmaH, with chimeric polynucleotide connecting sequence, which by, at least, 90% is identical to SEQ ID NO: 1, 2, 3 or 13. Also described are: expression vector, containing claimed nucleotide structure, and host cell Bacillus for production of heterologous protein of interest, which contains said vector. Claimed is method of obtaining modified Bacillus cell, including transformation of host cell of Bacillus-producent of heterologous protein of interest with said vector; and growing said modified cell in optimal conditions. Described is method of obtaining protein of interest in modified Bacillus cell, where method includes cultivation of said host cell; and growing said modified Bacillus cell in optimal conditions. Also described is method of amplification of expression of heterologous protein from Bacillus of interest includes obtaining said modified Bacillus cell; growing modified Bacillus cell in optimal conditions; and expression of said protein of interest in modified Bacillus cell, where expression of said heterologous protein of interest in modified Bacillus cell is amplified in comparison with expression of said protein of interest in said parent Bacillus host-cell.

EFFECT: invention makes it possible to increase output of target protein due to superexpression of protein YmaH.

30 cl, 4 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed is binding protein for binding one or more targets, which contains four polypeptide chains forming four functional antigen-binding sites. Four polypeptide chains contain VD1-(X1)n-VD2-C-(X2)n. VD1 stands for first variable domain of heavy chain, VD2 stands for second variable domain of heavy chain, C stands for CH1 domain, X1 stands for polypeptide linker, on condition that it is not constant domain, and X2 stands for Fc-region, and n equals 0 or 1. Two polypeptide chains contain VD1-(X1)n-VD2-C. VD1 stands for first variable domain of light chain, VD2 stands for second variable domain of light chain, C stands for CL domain, X1 stands for linker, on condition that it is not constant domain; and n equals 0 or 1. Conjugate of binding protein with visualising detecting cytotoxic or therapeutic agent is described. Disclosed are: nucleic acids (NA), coding polypeptide chains, as well as expressing vectors, vectors for replication, host cells which contain them, and method of obtaining antibody applying cells. Described is pharmaceutical composition for treatment or preventing target-associated disease or disorder based on binding protein. Method of treatment by introduction of binding protein is described.

EFFECT: application of invention provides new format (DVD-Ig) of antigen-binding molecules, which in the same dosage possess higher activity with respect to target than respective full-size antibodies, which can be applied in medicine for prevention and treatment of various diseases.

45 cl, 27 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a recombinant cell of Ralstonia eutropha, designed to obtain 2-hydroxyisobutyric acid. The cell is transformed by a plasmid with the sequence SEQ ID NO: 2.

EFFECT: cell bearing said plasmid produces 2-hydroxyisobutyric acid in concentration of up to 0,72 mmol/kg.

4 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to immunology. Described are antibodies against VEGF, one on which contains complementary regions with amino acid sequences SEQ ID NO:1, 2, 3, 4, 6 and 7, another contains complementary regions with amino acid sequences SEQ ID NO:1, 2, 3, 5, 6 and 7, disclosed in description. Also described are polynucleotides, coding said antibodies; espression vectors, containing said polynucleotides, and host cells, intended for obtaining antibodies in accordance with the claimed invention. Claimed is method of obtaining antibodies against VEGF, which includes expression of vector in host cell and separation of antibody. Disclosed is method of obtaining immunocongugate of antibody against VEGF, which includes conjugation of antibody with drug or cytotoxic agent. Described is method of VEGF identification, which includes identification of complex VEGF-antibody against VEGF in biological sample. In addition, described are compositions for treatment of VEGF-associated disease, one of which contains efficient quantity of antibody against VEGF, and another - efficient quantity of polynucleotide, coding said antibody. Also disclosed are methods of: 1) treating tumour, cancer or VEGF-associated cell proliferative disease; 2) inhibition if angiogenesis in subject and 3) inhibition of vascular permeability; consisting in introduction to subject of efficient quantity of antibody against VEGF in accordance with claimed invention.

EFFECT: invention makes it possible to obtain antibodies against VEGF and apply them for treatment of VEGF-associated diseases.

41 cl, 16 dwg, 2 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly a method for specific collection of DNA molecules (DNA aptamers) with high affinity for a recombinant protein target. Said method involves synthesis of a single polypeptide chain of a recombinant protein containing a fragment of glutathione S-transferase, a protein target, a peptide sequence split by the B. Anthracis lethal factor, a peptide which is biotinylated in vitro under the action of an E.coli biotin-ligase enzyme, binding the obtained recombinant polypeptide with an oligonucleotide library and immobilising the protein on paramagnetic particles bearing glutathione, washing the paramagnetic particles with the immobilised polypeptide from unbound oligonucleotides in a liquid stream, splitting the protein target with the bound DNA aptamers from the surface of paramagnetic particles with the B. anthracis lethal factor, separating and amplifying the DNA sequence with affinity to the recombinant protein target in a polymerase chain reaction and obtaining a set of single-chain DNA aptamers that are specific to the protein target.

EFFECT: invention provides efficient production of DNA aptamers with high affinity for recombinant protein targets.

4 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers immunology and medicine. What is presented is an antibody for the recovery of the central nervous system, comprising an antigen-binding site that specifically binds to human Nogo A polypeptide or human NiG described by (SEQ ID NO: 2 and 3, respectively, presented in the description), wherein the antigen-binding site comprises: CDR-H1-6A3 (SEQ ID NO:8), CDR-H2-6A3 (SEQ ID NO:9) and CDR-H3-6A3 (SEQ ID NO:10); and CDR-L1-6A3 (SEQ ID NO:11), CDR-L2-6A3 (SEQ ID NO:12) and CDR-L3-6A3 (SEQ ID NO:13). There are also described a polynucleotide coding the above antibody; an expression vector comprising the above polynucleotide; and a host cell specified in bacterium, yeast or mammalian cell line comprising myeloma, hybridoma, or immortalised B-cell for producing the antibody according to the present invention. A pharmaceutical composition for the CNS recovery comprising an effective amount of the above antibody mixed with at least one acceptable carrier or solvent is also described. Using the polynucleotide, the expression vector or the host cell for the above pharmaceutical composition is also described. The invention enables producing the human Nogo A or NiG antibody effective in treating CNS injuries.

EFFECT: what is presented is a method for producing the above antibody involving the polynucleotide or vector expression in the host cell.

16 cl, 11 dwg, 9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry and discloses a polypeptide having antimicrobial activity, which includes an amino acid sequence having at least 70% identity with an amino acid sequence corresponding to positions 1-21 of SEQ ID NO:2. The invention also relates to structures of nucleic acids, vectors and host cells which include a polynucleotide which encodes the polypeptide according to the invention, as well as a method of producing such a polypeptide and use of the polypeptide to destroy microbe cells.

EFFECT: invention widens the range of antimicrobial polypeptides.

18 cl, 2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biotechnology, in particular to novel peptide analogue of insulin-like growth factor-1 (IGF-1), which contains amino acid substitution of methionine in position 59 on Asn, Leu, Nle, Ile, Arg, A6c, Glu, Trp or Tyr, as well as other additional substitutions, inserts and deletions. Said peptide or its pharmaceutically acceptable salt is used in composition of pharmaceutical composition for treatment of IGF-1-mediated diseases, as well as in method of treating dwarfism.

EFFECT: invention makes it possible to obtain IGF-1 analogue-agonist, possessing higher biological activity with respect to native IGF-1.

17 cl, 2 tbl

FIELD: medicine.

SUBSTANCE: present group of inventions relates to biotechnology. What is presented is a humanised anti-CD79b antibody and its antigen-binding fragment produced of murine antibody MA79b and CD79b having a substantially analogous binding affinity thereto. A polynucleotide, a vector, a host cell and a method for producing the anti-CD79b antibody according to the invention; immunoconjugates, compositions and methods for cell growth inhibition, a method of treating an individual suffering cancer, a method of treating a proliferative disease and tumour in a mammal, a method for B-cell proliferation inhibition; a method for detecting the presence of CD79b in a sample and method for binding the antibody to the CD79b expressing cell are also disclosed.

EFFECT: given invention can find further application in therapy of the CD79b associated diseases.

86 cl, 20 tbl, 9 ex, 51 dwg

Fused rage proteins // 2513695

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biochemistry. Claimed is fused protein for treating diseases, mediated by advanced glycation end products (AGE), consisting of a fragment of a version of human receptor of advanced glycation end products (RAGE), which has two point mutations H217R and R221H, and a fragment of constant domain of human immunoglobulin IgG4, joined with linker if necessary. In addition, considered are: nucleic acid and recombinant host cell for obtaining fused protein, as well as pharmaceutical composition for treatment of AGE-mediated diseases, which contain fused protein.

EFFECT: invention ensures lower aggregation of fused protein.

13 cl, 19 dwg, 3 ex, 9 tbl

Up!