Photoprotein with increased bioluminescence

FIELD: medicine; pharmacology.

SUBSTANCE: it is obtained a chimerous photo protein (photin), presented by amino-acid sequence of obeline protein, which part (from the rest in position 50 to the rest in position 94) is replaced by a homologous site of amino-acid sequence of clitine protein (the rests 53-97). The method of obtaining new chimerous photo protein by the method of recombinant DNA and vectors applied to it and cells is described. It is offered to use photo protein under the invention as the calcium indicator in various test systems in vitro and in vivo.

EFFECT: increased level of bioluminescence in comparison with natural protein.

11 cl, 6 dwg, 2 tbl, 3 ex

 

This invention relates to a chimeric fotobase with high luminescent activity, its use as an indicator of calcium in the systems of reporter genes in cell-based assays for the detection and measurement of intracellular calcium.

The level of technology

Bioluminescence is the ability of living organisms to emit visible light through systems chemiluminescent reactions. For bioluminescent reactions requires three main components: luciferin, luciferase and molecular oxygen. However, some reactions may also require other components, including cations (Ca++and Mg++) and cofactors (ATP, NAD(p)H). Luciferase are enzymes that catalyze the oxidation of the substrate, luciferin, and give an unstable intermediate product. Light is emitted when an unstable intermediate product decays to its ground state, forming oxyluciferin. There are many different unrelated types of luciferin, although many species of at least seven phylogenetic types use the same luciferin called coelenterazine, which contains the loop formed by three amino acids (2 tyrosine and phenylalanine). Some animals (e.g., jellyfish) system luciferin/luciferase can be extracted in the form of stability in the CSO "fotobanka", which emits light when the binding of calcium. Pocobelli differ from Lucifers the fact that they are stable oxidized intermediate complexes luciferase and luciferin. Pocobelli present in many marine coelenterates and allow these organisms to emit light for a variety of purposes, including breeding, nutrition and protection [1]. While bacteria emit light continuously, many other organisms luminescence occurs in the form of flashes with a duration of typically 0.1-1 sec. It requires quick on/off of the enzymatic reaction and the presence of reagents connected properly and ready for rapid mobilization. The coelenterates flash-induced calcium flux. Sites fotobelov to bind calcium homologous calmoduline. In the presence of calcium pocobelli emit visible light through an intramolecular reaction. There are many luminescent organisms, but to date only seven fotobelov, namely classically [2, 3], acorin [4-6], mitotropin (SYN. galistin) [7, 8], Klitin (SYN. palidin) [8, 9], obelin[2, 6, 10, 11], Mnemiopsis [12, 13] and beromen [12, 13]. All these proteins are complexes apolka, chromophore of imidazopyridine (coelenterazine) and oxygen. Their structures are highly conserved, the OS is especially in the field, containing three binding site of calcium (EF-hands). These patterns EF-hands are typical for a family of calcium-binding proteins. Photoblog emits light when interacting with calcium, which is closely associated with a pocket of EF-hands. The reaction represents an event in a single cycle and leads to the release of CO2and light emission in the blue region (λmax= 470 nm). The term "fotoblog" defines associated with luciferine polypeptide which is capable of luminescence, whereas "apopotosis" is used to indicate protein without luciferin.

The most investigated Fotoalbom is equalin isolated from Aequorea victoria [14], and obelin isolated from Obelia longissima [15]. When the binding of Ca++equalin undergoes a conformational change, by itself, turning into oxygenase (luciferase), which then catalyzes the oxidation coelenterazine through communication of molecular oxygen. Blue fluorescent protein consists of Coelenterata, which is the oxidation product coelenterazine, ecovalence associated with apothecom. Photoblog can be regenerated from abovetable by incubation with coelenterazine, molecular oxygen, EDTA and 2-mercaptoethanol or dithiothreitol. As coelenterazine is common fluorescent substrate is, used by fotoalbumi aquarium, Micromine, klitina and abelina, the response of the light emission is probably the same in the case of these four fotobelov [16]. The recent receipt of the primary structure and crystallographic data aquarine and obeline gave more information about their functioning. Native obelin of hydroid Obelia longissima is a single-chain protein of 195 amino acid residues (a/K) with an approximate molecular mass of 20 KD, which contains ecovalence associated chromophore group coelenterazine. Analysis of primary structures litina shows that it contains 189, and belongs to the family fotobelov. However, the Ca++-binding photoblog hydroids differs from other Ca++-binding proteins such as calmodulin and troponin C, a relatively high content of cysteine residues, histidine, tryptophan, Proline and tyrosine.

Studies of the structure and function of obelin the luminescent protein described by Bondar VS et al., Biochemistry (2001), 66(9): 1014-8, Vysotski ES et al., (2003), 42(20): 6013-24 and L. Deng et al., FEBS Lett. (2001), 506(3): 281-5. In particular, in two recent publications described bioluminescent and emission properties of the mutant obelin the luminescent protein W92F.

Pocobelli widely used in the method of reporter genes to monitor events in the cell associated with signal transduction and gene expression.

Studies is the investigation of cellular events and their regulation requires sensitive, non-invasive analytical methods. Pocobelli and in General bioluminescence are a great reporter systems, as they have virtually no background in contrast to fluorescent systems.

Pocobelli expressed in mammalian cells for monitoring changes in calcium in response to various stimuli. Intracellular calcium concentration can be measured by adding the cofactor coelenterazine to mammalian cells expressing photoblog, and further registration of emission of photons, which is an indicator of intracellular calcium concentration.

Description of the invention

It was found that when chimerical protein obelin the luminescent protein (apollina) through the replacement area located between the first two binding sites of calcium, the corresponding region fotobanka selected from litina, aquaria, classicline, mitotropin, Mnemiopsis and banovina, get new photoblog with increased bioluminescence.

Used in this sense obelin and can refer to any of fotobelov isolated from various species of Obelia, including Obelia longissima and Obelia geniculata [17]. The base of the nucleotide and amino acid sequences of obelin the luminescent protein is listed in SEQ ID NO: 1 and 2, respectively. Basic amino acid and nucleotide sequence litina, mitotropin and aquarina on peyrovani in GenBank with access numbers Q08121, P39047, AAA27720 and accordingly L13247, L31623, L29571, whereas sequence classicline, Mnemiopsis and Berolina described in references 2, 3, 12, and 13.

"The corresponding region or fragment" used in this description means any amino acid sequence in the selected fotobanka, coinciding with the sequence of obelin the luminescent protein under appropriate alignments, with the exception of at least 1, preferably at least 5, more preferably at least 10 amino acid residues that are not conservative in their respective proteins (obelin and selected photoblog), with the specified region or fragment preferably comprises residues 42-122, more preferably residues 50-95 that relate to the sequence of obelin the luminescent protein.

According to a preferred variant of the invention, the chimeric protein is produced by replacing residues 50 to 94 amino acid sequence of obelin the luminescent protein fragment sequence litina extending from residue 53 to residue 97. Such photoblog, which is named "fotin", has the amino acid sequence of SEQ ID NO: 3.

Chimeric proteins proposed in the invention can be further modified by deletion, addition or substitution of one or more amino acid residues, provided that about the Ile activity fotobanka in relation to the emission of light and reactivity in terms of calcium is maintained or increased. Especially preferred are replacement provisions 55, 66, 67, 73, 74, 75, 78, 83, 84, 87, 89 and 94, which are obelin the luminescent protein sequence.

As shown in vitro, fotin produces intense bioluminescence in response to stimulation by calcium, which is usually higher than the bioluminescence observed in the case of natural fotobelov.

To obtain chimeric fotobelov prepare the construction of recombinant DNA, the carrier part of the coding sequences of obelin the luminescent protein and selected other fotobanka other than obelin the luminescent protein, using conventional genetic engineering, the resulting chimeric product is inserted into the vector, Express in a suitable host and then isolate and purify. For example, cDNA encoding obelin and other photoblog, it is possible to amplify in PCR or constructed in vitro with synthetic oligonucleotides, and the products can recombine using the appropriate restriction sites, naturally occurring or artificially introduced into the oligonucleotides used for amplification or for designing in vitro. Expressing the vector may contain, in addition to recombinant constructs, the promoter, the binding site of the ribosome, an initiation codon, a stop codon or a consensus site enhancers of transcription. The vector may also contain breeding marker for you is the population of master cells, containing design DNA. Suitable vectors are, for example, plasmids, yeast or bacteria, bacteriophages, viruses, retroviruses or DNA. Vectors carrying recombinant construct may be introduced into the host by using conventional techniques. Cells-owners can be a prokaryotic or eukaryotic cells, such as cells, bacteria, yeast or mammals. The preferred hosts for expression/products fotobanka are mammalian cells, including epithelial cells or lymphocytic origin, such as Hek-293, CHO-K1, HepG2 and HL-60. These cells can Express apopotosis in the cytoplasm [18], in mitochondria adding to fotobase guide in mitochondria sequence or in any other compartment cells [19-21]. Alternatively, you can use other cells, non-mammalian cells, such as cells, bacteria or fungi. After selecting the type of host cell genetic designs can be entered by means of precipitation with calcium phosphate, electroporation, or other conventional methods. After transfection, the cells are grown in suitable environments and subjected to screening for appropriate activities. Photoblog proposed in the invention, it is possible to isolate and purify by conventional methods such as extraction, precipitation, chromate is graphy, affinity chromatography, electrophoresis.

Point mutations can be introduced in the chimeric product by extending overlapping sites by PCR.

To get fotin, NdeI/MunI fragment of the gene obelin the luminescent protein was replaced by a fragment of 135 nucleotides corresponding to the coding sequence litina within nucleotides from 156 to 291.

In the following aspect the invention is directed to a nucleic acid molecule encoding a chimeric protein disclosed in this description. Encoding DNA sequence can be modified on the basis of the degeneracy of the genetic code, for example, the change of use of codons for improved expression in heterologous systems.

In a preferred embodiment of the invention, the DNA sequence encoding a protein fotin selected from SEQ ID nos: 4 and 5.

To test whether the chimeric product functional, carried out experiments on transcription and translation in vitro in a cell-free system broadcast wheat germ in the presence coelenterazine. In these experiments, the formation of active fotobanka register, testing luminescence mix for broadcast after the addition of calcium ions. In order to compare the measured luminescence with the amount of protein produced, the reaction of the broadcast is carried out in the presence of35 S-methionine. The amount of newly synthesized polypeptide is determined by measuring the incorporation35S-methionine in insoluble in trichloroacetic acid fraction. The results of these experiments demonstrate the sensitivity and efficiency of photinus in generating bioluminescence in response to stimulation by calcium ions.

In the following embodiment, the invention offer chimeric proteins are used as indicators of calcium, especially for measurement of intracellular calcium concentration. In a typical analysis add cofactor, such as coelenterazine, mammalian cells expressing photoblog, and the light emission register by methods known in this field, for example, by using commercially available luminometer.

Cells expressing photoblog and the receptor involved in the mobilization of intracellular calcium, can be used to test the molecules of the candidates in relation to their impact on the modulation of the receptor. Chimeric photoblog according to the invention can be used in many cell-based functional assays that use measurement of intracellular calcium in order to evaluate the activity of proteins, in particular, receptors associated with G-protein (GPCR)and ion channels in the cytoplasmic membrane. Although a large and fast the first increase in intracellular calcium concentration after stimulation of the GPCR and ion channels can be identified by various reporters, such as sensitive calcium fluorescent dyes, the use of bioluminescent fotobelov [22] preferably, as their background luminescence is practically absent, in contrast to fluorescent dyes; in addition, the measurement of calcium using fotobelov, in addition to the production of fast signals, gives a high signal-to-noise ratio with a wide range of reception sensitivity [22, 23]. Cell-based functional tests usually consist of adding the appropriate agonist to the culture of cells expressing both GPCR or ion channels, and fotoblog, and then determining any changes in the calcium concentration, for example, increase the content of calcium, induced either by the rapid influx from the extracellular environment, or release from intracellular stores (18), by measuring the activity of fotobanka.

The use of cells that Express how fotin, and the receptor involved in the modulation of intracellular calcium concentration, provides an effective system for screening compounds for their effect on the release of intracellular calcium. High-performance screening tests can also be performed using fotobanka according to the invention as a reporter system. The sensitivity of the system, and is also a high signal-to-noise allows the use of small volumes for analysis.

In the next version of fotoblog according to the invention kongugiruut with a molecule representing analytical, diagnostic or therapeutic interest, and conjugated product is used in a competitive solid-phase immunoassay to determine the amount of a given molecule in biological samples. For example, photoblog can be chemically anywhereman with hormonal proteins and used in solid-phase immunoassay with specific hormone antibodies to determine the hormone levels in saliva [24]. In yet another variant get merged product between Fotoalbom according to the invention, and other (poly)peptide, such as a hormone, antigen peptide, a light or heavy chain immunoglobulin, avidin, streptavidin or protein A, known methods of genetic engineering, which is described in the patent US6087476 (which is incorporated in this description by reference), and use in immunodiagnostic methods or visualization.

The following examples illustrate the invention in more detail.

Description of the drawings

Figure 1. Induced calcium bioluminescence newly synthesized fotobelov. Pocobelli broadcasted in a cell-free system of wheat germ in the presence coelenterazine. All aliquots of the mixture for broadcast were incubated for 2 h in the dark at 30°C. the reaction is mesh added CaCl 250 mm and registered luminescence for 10 seconds.

Figure 2. Broadcast DNA fotobelov in the presence coelenterazine measuring luminescence upon dilution of the reaction mixture. AQ: acorin, PH: fotin, OB: obelin.

Figure 3. (A) dose-Dependent ATP light emission during stimulation of endogenous ATP receptor expressed by the cell CHO-K1, transtitional DNA Photina. Transfection of cells, the collection and expression of the photinus was carried out as described in example 1. Cells were treated with two different concentrations of ATP. (B) Cells CHO-K1, transfetsirovannyh DNA obelin the luminescent protein, was used as a positive control. Luminescence was expressed in RLU (relative light unit).

Figure 4. Dose-dependent activity curve Photina in cells CHO-K1 transfected as endothelin receptor A and Fotina. The receptor is activated by injection of endothelin concentration of 100 and 500 nm.

Figure 5. Dose-dependent curve derived from cells expressing photoblog fotin and receptor vanilloid 1 rats (VR1). Specific agonist capsaicin used in concentrations of 10 and 50 microns.

EXAMPLES

1. Transcription/translation in vitro DNA fotobanka

Broadcast fotobelov carried out in a cell-free system of wheat germ (set TNT, Promega), following the General instructions of the supplier. For each reaction mixture for transcripts and/broadcast in vitro used about 2 μg DNA. Volume for broadcast (50 μl) contained 25 μl of wheat germ extract, 2 μl of reaction buffer, a mixture of amino acids, except methionine, Rnasin and coelenterazine (40 μm), T7 polymerase. The mixture also contained35S-methionine (specific activity 1000 CI/mmol).35S-methionine was used to determine the amount of fotobanka synthesized in vitro. With this purpose, 5 µl of each sample mixture for broadcast besieged THU on ice for 30 min, filtered, washed with cold 5% THU, methanol, dried and placed in vials for counting.

The number of photinus and obelin the luminescent protein, obtained in a cell-free system, is shown in table I.

Table I

The incorporation of radioactive label in the products broadcast fotobelov
Added DNAObelin (1 µg)Fotin (1 µg)Equalin (1 µg)
35's account after 90-min broadcast (imp./min)8169829013049

2. Analysis fotobanka

5 and 10 ál of the mixture to broadcast directly mixed with 95-90 μl of PBS solution in a 96-hole tablet, which was set up in a luminometer (Berthold). To start the light emission by photobeam in the hole were injected with 50 μl of a solution (50 mm CaCl2and register is uminescence within 10 seconds.

The results of translation in vitro DNA Photina, obelin the luminescent protein and aquarina shown in figure 1. Measuring the luminescence obtained by dilution of the reaction mixtures for broadcast fotobanka in vitro, shown in figure 2. Comparison of data luminescence with a score of THU-insoluble fractions obtained during the broadcast with different amounts of DNA Photina and obelin the luminescent protein, is shown in table II (the measured luminescence proportional to the amount of newly synthesized fotobanka).

Table II

The content of the radioactively labeled fotobanka and its luminescence in the mixture for broadcast
ObelinFotin
DNA (ng)Account in THU (imp./min/μl)Luminescence (RLU/ml)Account in THU (imp./min/μl)Luminescence (RLU/ml)
25027223589442927990308
12523962719522094858046
631030139241807339348
315794197050288326
1626513482196 30617

3. Examples of cell-based functional assays

3.1.Expressing fotin clones obtained by transfection of cells CHO-K1 (materials and methods). Two days after transfection cells were treated with trypsin and were diluted 10 or 100 times. After cells were grown to a well-isolated colonies, colonies were transferred to new cups and conducted breeding on the basis of their functional response (fluorescent signal) at different concentrations of ATP, which is known to stimulate endogenous receptor CHO and increases the concentration of Ca++in the cytoplasm. At the end of each experiment the cells were literally by perfusion with a solution containing Triton X-100 and CaCl2. Active photoblog reconstructed, incubare cells with 10 μm coelenterazine diluted in PBS containing 2 mm calcium in the dark at 37°C in an atmosphere with 5% CO2within 3 hours. For measurement of the light emission cells were literally in the presence of calcium and record the emitted luminescence. The number of photons emitted within 10 seconds, integrated with a luminometer and visualized on the screen. Cells transfetsirovannyh empty plasmid or nitrostilbene (data not shown), did not increase the emission of photons. In order to detect changes in calcium concentrations, were injected with 10, 50 and 100 μm And the f and determined the kinetics of the response to calcium. The obtained curve is shown in figure 3 (A). The cell line expressing photoblog obelin, was used as a positive control, figure 3 (B).

3.2.Created a line of CHO cells expressing the receptor for endothelin A and fotin. Upon stimulation with an agonist of this receptor induces an increase in intracellular calcium concentration, which is measured by luminescence Photina. Cells were cultured as monolayer in DMEM/F12 containing 10% fetal calf serum (FBS)in 96-well pad. On the day of the experiment, culture medium was removed and cells were incubated in the presence of 10 μm coelenterazine in PBS for at least 3 hours. The peptide endothelin was diluted in PBS at a concentration of 100 and 500 nm. About 50 ál of endothelin were injected with in each well and measured the response. The emitted light is immediately recorded in a time period of 30 seconds. Dose-dependent response to endothelin is shown in figure 4.

3.3.Line of CHO cells expressing how apoptin and the capsaicin receptor VR1 - permeable to calcium ion channel were grown to confluence on 80-95% in vials for tissue culture and collected when processed by trypsin. The cells were distributed at 10,000 cells/well in 96-well white plates in the growth medium, and incubated overnight at 37°C in a humidified incubator with 5% CO2. For the experiments on Liu is inessence to the cells was added coelenterazine 10 μm for 3 hours at 37° C, 5% CO2. The calcium response was stimulated by addition of 10 and 50 µm capsaicin in each well. For the kinetics of fluorescent bursts followed, using a Labsystem Luminoskan Ascent, which performs injection of reagents and registration of emission of light from each individual wells. Adding capsaicin caused a rapid and transient fluorescence signal within 30 seconds with a peak level occurring approximately at the point 10 seconds (figure 5).

MATERIALS AND METHODS

Reagents

Restriction enzymes were purchased from New England Biolabs and used according to the supplier's instructions. Rnasin and set TNT for transcription and translation in vitro were from Promega (Madison, WI). Polymerase Pfu Turbo, reagents for PCR and competent cells of E. coli XL-1Blue and BL21 were from Stratagene (La Jolla, CA). Oligonucleotides were purchased from Primm (Milan). Coelenterazine was from Prolume Ltd. (Pittsburg, PA). All other chemicals were from standard sources and were pure for analysis or the best quality.

PCR Assembly for the synthesis of DNA sequences obelin the luminescent protein in one stage

For PCR Assembly requires four stages: synthesis of oligonucleotides, the Assembly of the gene, gene amplification and cloning. The authors synthesized 30 oligonucleotides with a length of 40 nucleotides, which together encode both strands of the gene sequence obelin the luminescent protein. Overlapping complementary oligonucleic the species was 20 nucleotides. Equal amounts taken from each solution 30 oligonucleotides were combined to a final concentration of approximately 250 μm mixed oligonucleotides before 250-fold dilution in 20 μl mixture for PCR Geneamp XL (Perkin Elmer). The amplification process was carried out in three stages. The first stage was conducted with the joint oligonucleotides as follows: 40°C for 2 min, then add polymerase, 72°C for 10 s, then 40 cycles (94°C for 15 s, 40°C for 30 sec and 72°C for 10 sec). The reaction mixture was diluted three times with fresh mixture for PCR and Taq polymerase. The second stage consisted of 25 cycles (94°C for 15 s, 40°C for 30 sec and 72°C for 45 sec). The reaction mixture was again diluted three times in a complete mixture for PCR. Conditions for the third stage consisted of 20 cycles (94°C for 15 s, 40°C for 30 sec and 72°C for 70 sec). The reaction products were analyzed by electrophoresis in 1% agarose gel and the fragments were cloned in the vector pcrBlunt for further stages of cloning. The plasmid was named pcr-OB. The accuracy of the PCR reactions was confirmed by dideoxy-sequencing.

Construction of the chimeric protein

Four pairs of oligonucleotides were designed based on the gene sequence litina. The area between the EF-hand I and EF-hand II was in the bran to chimerical gene. Oligonucleotide primers consisted of the following:

Annealed oligonucleotides were cloned into the unique NdeI/MunI-sites in the vector pcr-OB. A plasmid containing the chimeric gene product, called pcr-Photin. DNA Photina was further subcloned into the vector pcDNA3, which contains the T7 promoter.

PCR-based change of use of codons

The method of lengthening the overlapping areas on the basis of PCR were used to produce mutant obelin the luminescent protein. Designed six pairs of primers with ten different tochkovymi mutations, which create changes in the use of ten different codons. In order to amplify the DNA fragments used for lengthening overlapping fragments was carried out by PCR reaction with 2.5 units of Pfu polymerase, 50 ng of template DNA, 250 μm of each dNTP, and 50 pmol of each primer in a total volume of 100 μl. The parameters used cycle consisted of 94°C for 1 min, 45°C for 1 min and 72°C for 1 min 30 sec during the first 10 cycles, followed by 20 cycles with the annealing temperature 50°C. Site-specific mutations were confirmed by DNA sequencing performed in Primm (Milan). All molecular methods can be carried out and, using standard protocols.

Cell culture CHO

All cells were cultured in standard wet conditions at 37°C and 5% CO2. The CHO cells were maintained in DMEM/F12 + FBS 10% + pins./strept. + G418 0.5 mg/ml + 1.6 mm pyruvate + NaHCO30,2%, all reagents from Life Technologies. Transfection of DNA was performed when cells were grown on plates until confluence of 70-80%.

LINKS

1. Kendall, J.M., and Badminton, M.N. (1998).Aequorea victoriabioluminescence moves into an exciting new era. Trends Biotechnology. 16(5):216-24.

2. Campbell, A. K., Hallet, R. A., Daw, M.E., Ryall, R.C., Hart and Herring, P.J. (1981). Application of the photoprotein obelin to the measurement of free Ca++in cells. In Bioluminescence and Chemiluminescence, basic Chemistry and Analytical applications (Edited by M.A. deLuca and W. D. McElroy), pp. 601-607. Academy Press, New York.

3. Herring, P.J. (1979) Some features of the bioluminescence of the radiolarianThalassicola sp.Mar. Biol. 53, 213-216.

4. Shimomura, O., Johnson F.H., and Saiga, Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan,Aequorea.J. Cell. Comp. Physiol. 59, 223-239.

5. Shimomura, O., Johnson F.H., and Saiga, Y (1963) Further data on the bioluminescent protein, aequorin. J. Cell. Comp. Physiol. 62, 1-8.

6. Morin, J.G. and J.W. Hastings (1971) Biochemistry of the bioluminescence of colonial hydroids and other coelenterates. J. Cell. Physiol. 77, 305-311.

7. Shimomura, O., Johnson, F.H. and Saiga, Y. (1963) Extraction and properties of halistaurin, a bioluminescent protein from the hydromedusanHalistaura.J. Cell. Physiol. 62, 9-15.

8. Shimomura, O., and Shimomura, A. (1985) Halistaurin, phialidin and modified forms of aequorin as Ca++indicator in biological systems. Biochem. J. 228, 745-749.

9. Levine, L.D., and Ward, W.W. (1982) Isolation and characterization of a photoprotein "phialidin" and a spectrally unique green-fluorescent protein from the biluminescent jellyfish Phialidium gregarium.Comp. Biochem. Physiol. 72B, 77-85.

10. Morin, J.G. and Hastings (1971) Energy transfer in a bioluminescent system. J. Cell. Physiol. 77, 313-318.

11. Campbell, A.K. (1974) Extraction, partial purification and properties of obelin the calcium-activated protein from the hydroidObelia geniculata.Biochem. J. 143, 411-418.

12. Ward, W.W. and Selinger (1974) Extraction and purification of calcium-activated photoprotein from the ctenophoresMnemiopsis sp.andBern ovata.Biochemistry 13, 1491-1499.

13. Ward, W.W. and H.H. Seliger (1974) Properties of mnemiopsin, and berovin, calcium-activated photoproteins from the ctenophoresMnemiopsis sp. and Beroë ovata.Biochemistry 13, 1500-1510.

14. Johnson, F.H. and Shimomura, O. (1978) Introduction to the bioluminescence of Medusa, with special reference to the photoprotein aequorin. Methods Enzymol. 57, 271-291.

15. Illarionov BA, Bondar VS, Illarionova VA, Vysotski ES. Sequence of the cDNA encoding the Ca++-activated photoprotein obelin from the hydroid polypObelia longissima.Gene. 1995 14; 153(2):273-4.

16. Blinks, J. R., Weir, G. W., Hess, P. and Prendergast, F.G. (1982). Measurement of Ca++concentrations in living cells. Prog. Biophys. Mol. Biol. 40, 1-114.

17. Markova SV, Vysotski ES, Blinks JR, Burakova LP, Wang BC, Lee J., (2002) Obelin from the bioluminescent marine hydroid Obelia geniculata: cloning, expression, and comparison of some properties with those of other Ca2+-regulated photoproteins. Biochemistry. 2002 Mar 19; 41(7):2227-36.

18. Button D, Brownstein M. (1993) Aequorin-expressing mammalian cell lines used to report Ca++mobilization. Cell Calcium. Oct; 14(9):663-71.

19. Mattheakis, L., and Ohler, L.D. (2002) Seeing the light: Calcium imaging in cells for drug discovery. Drug Discovery Today S, 15-19.

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1. Chimeric apopotosis characterized by the amino acid sequence corresponding to the protein sequence of obelin the luminescent protein, a fragment from residue in position 50 to the residue at position 94 is replaced by a fragment of the amino acid sequence of the protein litina from the residue at position 53 to the residue at position 97, and has increased in comparison with abelina level of bioluminescence.

2. Chimeric apopotosis according to claim 1, having amino acid sequence SEQ ID NO:3.

3. Protein consisting of the amino acid sequence of the chimeric abovetable according to any one of claims 1 to 2, and the amino acid sequence of auxiliary polypeptide.

4. An isolated nucleic acid molecule encoding a chimeric apopotosis, which is characterized by nucleotide sequence corresponding to the amino acid sequence of chimeric protein according to any one of claims 1 and 2.

5. An isolated nucleic acid molecule according to claim 4, having the nucleotide sequence of SEQ ID NO:4 or SEQ ID NO:5.

Frienemies the chimeric abovetable according to any one of claims 1 to 2 in combination with the substrate luciferine for detection of calcium ions.

7. The use according to claim 6 for the quantitative determination of calcium ions.

8. The use according to claim 7 for determining the intracellular concentration of calcium.

9. A host cell containing the nucleic acid molecule according to claim 4 or 5, and expressing the chimeric apopotosis.

10. A host cell according to claim 9, selected from the cells of bacteria, yeast, fungi, plants, insects and mammals.

11. The method of obtaining the chimeric abovetable, comprising growing a host cell according to claim 9 or 10, under conditions suitable for expression of fotobanka, and removing the expressed protein.



 

Same patents:

FIELD: biochemistry, analytical biochemistry.

SUBSTANCE: reagent for quantitative determination of adenosine 5'-triphosphate (ATP) by bioluminescent method comprises luciferase isolated from recombinant E. coli cells comprising plasmid pLR with native firefly luciferase gene, or luciferase isolated from recombinant E. coli cells comprising plasmid with m-pLR with the Luciola migrelica mutant luciferase gene wherein histidine residue at the position 433 is replaced for tyrosine residue. Also, the reagent comprises magnesium sulfate, mixture of tris-(hydroxymethyl)-aminomethane and acetic acid as a buffer, mixture of ethylenediaminetetraacetate sodium, dithiothreitol, bovine serum albumin and trehalose as stabilizing agents and water. In the assay method of adenosine 5'-triphosphate the reagent provides enhancing sensitivity and reproducibility in determination of ATP and reducing consumption of enzyme due to increase of specific activity of the reagent. The reagent provides carrying out determination of adenosine 5'-triphosphate by bioluminescence intensity at 560-570 nm in using firefly Luceola migrelica native luciferase, and by bioluminescence intensity at 606-610 nm in using luciferase wherein histidine residue at the position 433 is replaced for tyrosine residue.

EFFECT: improved assay method, valuable properties of reagent.

1 dwg, 1 tbl, 4 ex

FIELD: biochemistry, analytical biochemistry.

SUBSTANCE: reagent for determination of adenosine 5'-triphosphate (ATP) represents an aqueous solution of firefly luciferase containing luciferin, magnesium sulfate, tris-(hydroxymethylaminomethane), acetic acid, ethylenediaminetetraacetate sodium, dithiothreitol, bovine serum albumin and a stabilizing agent taken among the group including polybrene, polyvinylpyrrolidone, N-phthalylchitosan and sucrose. Using the invention provides enhancing precision and reproducibility of ATP assay. Invention can be used medicine, ecology, pharmacy and food industry.

EFFECT: improved assay method.

5 cl, 2 tbl, 3 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: method involves sowing out samples of mixed cultures in liquid selective media, determination of cells number accumulating in media during microorganisms growth by bioluminescent method and mathematical treatment of kinetic data of the growth of individual bacterial cultures, determination of the parent concentration of microorganisms relating to different taxonomic groups. Invention allows carrying out the simultaneous identification of bacterial cells relating to different taxonomic groups and presenting in mixed cultures simultaneously, and to enhance precision in determination of cells number in the broad concentration range and to reduce the total analysis time significantly. In industry mixed cultures are used widely in different branches of food industry (dairy, meat, brewing and others) and in other biotechnological processes (biological treatment of sewage waters, bioremediation of soils, producing methane from waste in different manufactures and others). Invention can be used for differentiated determination of microorganisms number in mixed cultures that are widely distributed in nature: in air, soil, ponds, as components of natural microflora in higher organisms and among contaminants causing injury of different objects.

EFFECT: improved method for determination.

1 tbl, 3 dwg, 5 ex

FIELD: biochemistry, in particular production of immobilized enzymes useful on chemistry, biochemistry, medicine, histology, microbiology, ecology and agriculture for bioluminescence analysis.

SUBSTANCE: target reagent is obtained by production of 3-5 % gel by boiling of starch suspension in phosphate buffer, gel cooling to 24-30°C, mixing of buffer solution of luminescent bacterium bienzyme system NADH:FMH-oxydoreductase-lucirerase with starch gel, dosage on lavsan film and drying at 4-10°C. According the invention components are introduced in the next order: trimyristin aldehyde, nicotinamidadeninedinucleotide, bienzyme system NADH:FMH-oxydoreductase-lucirerase, and flavin mononucleotide. Substrate solution of nicotinamidadenine dinucleotide, flavin mononucleotide, and trimyristin aldehyde are prepared in phosphate buffer with pH 6.8-7.0.

EFFECT: reagent of improved quality due to decreased quantity of reaction mixture components (from four to one), reduced analysis period by two times and increased measurement accuracy.

2 cl, 1 dwg, 4 ex

FIELD: gene and protein engineering for various luminescent assays.

SUBSTANCE: new luciferase mutant forms have been obtained. Said mutant forms have increased thermostability and optionally different emission wave length in contrast with respective wild type enzymes. In all disclosed muteins natural amino acid residue in position equivalent to 357-position in Photinus pyralis luciferase sequence is replaced with other residue, preferably with uncharged polar amino acid (in particular tyrosine) residue. Mutant luciferases of present invention are useful in various analytical systems as reporter agent.

EFFECT: Mutant luciferases with new properties.

22 cl, 15 dwg, 6 tbl, 12 ex

The invention relates to methods of analysis of toxic compounds and can be used in environmental monitoring

The invention relates to environmental and genetic toxicology

The invention relates to the field of biochemistry

The invention relates to a method of calibration of chemical analyses and, in particular, but not limited to this method of calibration tests Osobowych substances in analytical Microbiology, for example, the assay for adenosine 5'-triphosphate (ATP)

The invention relates to sanitation and medicine and can be used to determine the total microbial contamination or content coli-forms in food, biological fluids, swabs from process equipment using bioluminescence

FIELD: biology, gene engineering.

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

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

13 cl, 12 dwg,12 ex

FIELD: chemistry, biotechnology.

SUBSTANCE: invention relates to field of biotechnology and concerns obtaining factor VII protein by method of recombinant DNA. Recombinant plasmid DNA was constructed for expression of blood clotting factor VII in mammalian cells, which is product of ligating of fragment of cDNA of human factor VII gene, flanked by sites of restrictases Xhol and BamHI recognising, with large XhoI/BglII fragment of vector pEFZeo, including genes of resistance to ampicillin and zeocin. As result of BHK cell transformation with new recombinant plasmid, cell line BHK/F7 was obtained, which produces recombinant protein of factor VII with output of up to 40 mkg/ml.

EFFECT: obtaining cell line producing recombinant protein of factor VII.

2 cl, 4 dwg, 4 ex

FIELD: agriculture.

SUBSTANCE: development of a reliable means for the identification of the corresponding transformation event in the plant genome. As a result of agrobacterial transformation of the Lugovskoy potato with a genetic maker containing the gene cryIIIa, and the analysis of the resulting transgenic plants demonstrating resistance to the Colorado beetle, the transgenic line (transformation event) 1210 amk has been selected. In the genomic DNA of the plants belonging to this line a fragment has been identified and sequenced, related to the genetic maker insertion area and being a recombinant sequence, one part of which relates to the genetic maker inserted, and the other part relates to the flanking area of the Lugovskoy potato genomic DNA. It is proposed to use the identified recombinant sequence as an identifier of this unique transformation event.

EFFECT: obtaining transgenic potato that is resistant to Colorado beetle and complies with requirements of biological and food safety on basis of high yield Russian variety.

5 cl, 5 dwg, 7 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to genetic engineering. New interferon-binding proteins, which modulate activity of different interferon-α subtypes, as well as interferon-β activity, are obtained. Described is cloning of DNA fragment, which codes interferon-α/β, binding protein IFNAB-BPI, and expression of obtained DNA fragment in host-cells both with formation of respective polypeptide and in form of fused proteins. Practical application of obtained proteins as components of pharmaceutical compositions for inhibiting activity of IFN-α or IFN-β is suggested. Invention can be applied in medicine for inhibiting undesirable impact of IFN-α or IFN-β.

EFFECT: obtaining new interferon-binding proteins, which can find application in medicine for inhibiting undesirable impact of IFN-α or IFN-β.

13 cl, 10 dwg, 6 tbl, 17 ex

FIELD: medicine.

SUBSTANCE: invention relates to the field of biotechnology, in particular to genetic and cell engineering, and can be used in medicine. Suggested is method for obtaining cell culture for tissue engineering in region of ischemia, which involves co-cultivation in vitro of stromal cells from subcutaneous adipose tissue (SCAT) with cells of either total or enriched with CD31- or NG2-positive cells of cardiomyocite fraction (CMF), which is caried out at ratio of SCAT/CMF of 1:4 to 4:1 until microscopically determined capillary-like structures appear in culture. Cell culture with angiogenic phenotype induced in process of co-cultivation obtained by suggested method ensures effective local activation of angiogenesis when introduced into ischemised tissue.

EFFECT: obtaining cell culture, which ensures effective local activation of angiogenesis when introduced into ischemised tissue.

4 cl, 15 dwg, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and genetic engineering. Polypeptide with sequence SEQ ID NO:3 binds with androgen receptor and increases capability of androgen receptor to transactivate androgen-sensitive gene. Also claimed are nucleic acid encoding such polypeptide, and vector containing such nucleic acid. Group of inventions can be used for creation of transgenic animals, which express gene of protein acting in complex with androgen receptor, serving as models for elaboration of medications for cancer treatment.

EFFECT: increase in androgen receptor capability to transactivate androgen-sensitive gene.

24 cl

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering and biotechnology and can be used for labelling of proteins, cells and organisms. Substitutes added to nucleic acid Aequorea coerulescens, coding colourless GFP-like protein acGFP is considered as means of production of nucleic acid coding fluorescent protein. Expression cassette containing this nucleic acid under regulatory elements control provides in-cell biosynthesis of fluorescent protein. This genetic structure is used for production of transgenic organism and cell producing fluorescent protein, coded by specified nucleic acid. This fluorescent protein is applied in methods for labelling of cells, intracellular structure and protein, as well as for promotor transcriptional activity registration.

EFFECT: invention application ensures widen range of labelling means used in biochemistry, molecular biology and medical diagnostics.

11 cl, 34 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to delivery system for antigen as T-cell, containing, at least, one antigen and loading antigen-presenting cells with specified antigen. Application of T-cell containing at least one antigen is offered in medical product for antigen delivery to lymph node. Application of antigen associated with T-cell containing at least one other antigen causing immune response is offered in medical product for immune response monitoring against at least one other antigen. Application of T-cell containing at least one antigen is offered in medical product for antigen-presenting cells (APC) loading with antigen.

EFFECT: offered method of T-cell production for application under any item mentioned above, including T-cell isolation, T-cell activation, T-cell cultivation and antigen introduction to T-cell, where T-cell is transduced with antigen.

24 cl, 8 ex, 1 tbl, 7 dwg

FIELD: gene engineering.

SUBSTANCE: invention relates to plant gene engineering. Artificial promoter is a chimeric molecule of recombinant DNA and produces high expression level of DNA molecule combined with 3'-end when injected into plant cells. Basic genetic elements of chimeric molecules include promoter nucleus with consensus TATA-box and exon/intron/exon area and translation enhancer. All of these elements are structured artificially. Regulatory elements of transcription may be built into the given promoter in reverse direction for organo-specific, tissue-specific or specific stage of expression development. In order to increase levels of built artificial genetic elements translation/transcription, they may be functionally inserted between any promoter, which is active in cell or any DNA sequence.

EFFECT: increase of translation/transcription levels.

68 cl, 12 dwg, 7 tbl, 4 ex

FIELD: molecular biology, biotechnology, medicine, pharmaceutical industry.

SUBSTANCE: invention proposes RNA molecule able to target-specific interference of RNA and represents a double-stranded RNA molecule of size 19-22 nucleotides and having 3'-bulge consisting of 1-5 nucleotides This RNA is prepared by combining two chains of RNA and used for preparing a pharmaceutical composition. After incorporation into the multicellular eucaryotic organism this RNA molecule provides promotion to target-specific interference of RNA and results to decreasing level of expression of the gene-target or to knockout of the gene-target. Method for promotion of target-specific interference of RNA using this RNA molecule is used in assay and modulation of gene function. Cell containing endogenous nucleic acid-target, RNA molecule able for target-specific RNA interference and exogenous nucleic acid-target is used in analytical procedures. Using the invention provides silence of the gene-target mediated by target-specific interference of RNA.

EFFECT: valuable biological properties of RNA.

40 cl, 24 dwg, 3 ex

FIELD: medicine; pharmacology.

SUBSTANCE: variants of the combined protein which contain the extracellular domain of a human receptor of a hormone of growth and the domain which includes alarm sequence for joining glycosylphosphatidylynozyte (GPI) anchors are offered.

EFFECT: effective medical product for acromegalia and gigantism treatment.

8 cl, 16 dwg

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