Method of screening gamma-secretase inhibitors

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a method of identifying γ-secretase and its inhibitors and can be used in medicine when searching for active compounds for treating Alzheimer's disease. A genetic structure is formed, which codes fused protein, which contains a signal peptide and amino acid sequence GAIIGLMVGGVVIATVIVITLVML. In the obtained fused protein, except the GAIIGLMVGGVVIATVIVITLVML sequence, all sites acting as a signal for endo- or exocytosis, and/or protease splitting site are excluded.

EFFECT: invention allows for highly effective identification of γ-secretase or substances which inhibit its activity by reducing background signal and increasing specificity of the signal.

41 cl, 4 dwg, 17 ex

 

The present invention relates to improved methods for determining the activity of γ-secretase, the individual stages of such methods and the application of the above methods. This invention is based on the provisions of the application WO 00/34511 A2, which is incorporated in this description by reference.

Alzheimer's disease (AD) is a neurodegenerative brain disease, which is accompanied at the cellular level by the massive loss of neurons in the extremities and the brain. In the affected areas of the brain at the molecular level can be detected protein deposits, called plaques, which are a major feature of Alzheimer's disease. Protein, the most common in these plaques, is a peptide that contains 40-42 amino acids, called β-peptide. β-peptide is a cleavage product of a much larger protein containing 695-770 amino acids, the so-called amyloid protein precursor (APP).

The RDA is an integral transmembrane protein, which first crosses the double layer of lipid. Thus, a large part of this protein is extracellular, while the shorter C-terminal domain facing cytosol (figure 1). β-peptide shaded dark grey in figure 1. About two-thirds of β-peptide is in uncle the internal domain, and about one third is located in the transmembrane domain of APP.

In addition to the membrane of the RDAs, it is possible to detect the secreted form of amyloid protein precursor, which consists of a large ectodomain the RDA and the RDA is defined assec(“secreted by the RDA”). ARRsecformed from the RDA in the proteolytic cleavage mediated α-secretases. Proteolytic cleavage occurs at the site of the amino acid sequence of the RDA, which is the amino acid sequence β-peptide (after amino acid residue 16 β-peptide). Proteolysis of APP by the α-secretase excludes education β-peptide.

Thus, β-peptide can be formed from the RDA only as a result of alternative processing. Theoretically proved that such processing involves two other proteases, the first protease, referred to as β-secretases that it N-end β-peptide in the RDA, and a second protease, referred to as γ-secretases that releases With the end β-peptide (Kang, J. et al., Nature, 325, 733) (figure 1).

Secretase (α-secretase, β-secretase, γ-secretase) are of great interest, in particular, in connection with studies of Alzheimer's disease, for example, to identify secrets or factors involved in the regulation of the activity of secrets and education β-peptide (Wolfe, M.S. (2001), J. Med. Chem., 44(13), 2039-2060). Inhibition of β-secretase and, in particular,γ-secretase reduces the production β-peptide, on the other hand, activation of α-secretase can increase the processing of APP in se and simultaneously reduce the formation of β-peptide. Transgenic organism C. elegans, which was discovered in the course of such studies, as described in the application at the German patent DE No. 19849073 A1.

There is considerable evidence that β-peptide (β) plays an important role in causing Alzheimer's disease. Among other things, theoretically grounded neurotoxicity β-fibrils in cell culture (Yankner, B.A. et al., (1990) Proc. Natl. Acad. Sci. USA, 87, 9020). In subjects with down syndrome, a symptom of which is the presence of additional copies of the gene encoding the RDA, neuropathology characteristic of Alzheimer's disease, occurs even at the age of 30 years. It is assumed that overexpression of APP leads to increased transformation in β-peptide (Rumble, B., et al., (1989), N. Engl. J. Med., 320, 1446).

The most significant indicator of the Central role β-peptide, apparently, are familial forms of Alzheimer's disease. Detected mutations of the gene RDAs near the sites of cleavage of the β - and γ-secretases or mutation of two other AD-associated genes (presenilin), in which cell cultures significantly increase the production β-peptide (Scheuner, D. et al., (1996), Nature Medicine, 2, 864).

There is considerable evidence that during the processing of APP is first cleaved with the formation of β-peptide under the action of the m β-secretase and then serves as a substrate for γ-secretase. Therefore, γ-secretase plays an important role in the formation β-peptide (Wolfe, M.S. (2001), loc. cit).

As a rule, β-peptide is difficult to detect, as it becomes only a small number of RDA (Simons, M., et al., Neurosci(1996) 1;16(3):899-908). In addition, β-peptide is a very small fragment with a length of about 4 kDa, which is characterized by a significant tendency to autoaggregate due to hydrophobic nature. So β-peptide easily deposited under physiological conditions (Hilbich, C., et al., (1991) J. Mol. Biol., 218, 149) and precipitated forms can not be detected.

β-peptide found in eukaryotic cells immunobiological methods, such as ELISA, immunoprecipitation and Western blotting (Suzuki, N. et al., Science 1994, 27, 264(5163) 1336; Haass, C. et al., (1992) Nature, 359, 322). In addition, Wolff and others (1999) described the in vitro assays designed to determine the activity of γ-secretase from purified membrane fractions containing PS1 (presenilin 1). These methods require time-consuming, as they include the stage of incubation with the appropriate antibodies, stage lysis of cells derived from acceptable cell culture, or the use of model organisms (e.g., C. elegans). These methods are not suitable for execution in an automated analysis system, for example, for high-throughput screening to identify the connection is to be made which specifically inhibit or reduce the activity of γ-secretase. Partly because of the fact that the activity of γ-secretase depends on the Assembly of proteins (Mattson, (2003) Nature 422, 385), and, as it was known up to now, it is only active in the complex environment of membrane lipid.

The activity of γ-secretase can be detected by the method described in the application WO 00/34511A2, which is designed for determining the activity of γ-secretase and detection of γ-secretase by identifying β-peptide. In the method described in the application WO 00/34511A2 used a transgene that encodes a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO:1) as the enzymatic site of the target γ-secretase, signal peptide (SP) at the 5'-end, the promoter and optionally additional coding and/or noncoding nucleotide sequence that is introduced into the cell for expression of the indicated fused protein.

As a result, specific cleavage of the fused protein γ-secretases present in the cell formed by the first partial protein containing the amino acid sequence GAIIGLMVGGVV (SEQ ID NO:2), and the second partial protein containing the amino acid sequence VIVITLVML (SEQ ID NO:3). The above first and/or second partial protein detect, for example, using a suitable reporter, for example, gene-reporter, AK is everemore released activator of transcription associated with the first and/or second incomplete protein.

Given the known problems associated with the detection β-peptide, the present invention is an improvement of the method described in the application WO 00/34511A2, for example, by attenuation of the background signal and/or increase the specificity of the signal to improve the signal-to - noise ratio when performing analysis according to the invention.

Very surprising is the fact that the signal-to - noise in the manner described in the application WO 00/34511A2, can be improved by reducing non-specific release of the first and/or second partial protein due to non-specific activity of the protease. A similar result, for example, is achieved by matching the peptide according to the application WO 00/34511A2 by removing/deleting all other sequences/fragments of the sites of cleavage by a protease and/or internalisers sequences, in addition to the site of cleavage of the γ-secretases. Therefore, the present invention relates to an improved method for determining the activity of γ-secretase and detection of a protein having the activity of γ-secretase.

Specific embodiments of this invention relate to methods of identifying γ-secretase, cDNA that encodes a γ-secretase, subunit protein γ-secretase or γ-secretase-like proteases and ways to identify farmaceuticas and active compounds, which can be modulated, for example, to reduce or inhibit the activity of a protein having the activity of γ-secretase. Such substances are of special interest if they are pharmaceutically acceptable and suitable for the treatment of Alzheimer's disease.

The present invention relates to a method of detecting γ-secretase, which is that:

1) use a transgene that encodes a protein containing:

a) a first nucleotide sequence encoding a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO:1);

b) a second nucleotide sequence encoding a signal peptide at the 5'-end of the first nucleotide sequence;

(C) promoter and

d) optionally additional coding and/or noncoding nucleotide sequence;

2) this trashed injected into the cell and Express a fused protein;

3) protein break down within the amino acid sequence of SEQ ID NO:1 γ secretases present in the cell, resulting in a first partial protein containing the amino acid sequence GAIIGLMVGGVV (SEQ ID NO:2), and the second partial protein containing the amino acid sequence VIVITLVML (SEQ ID NO:3); and

4) find the first incomplete protein and/or the second incomplete protein, provided that with the exception of SEQ IDNO:1 specified protein no peptide, acting as a signal for endo - or exocytosis, and/or site of cleavage by the protease.

When implementing this method, the specified protein preferably does not contain any (i.e. no peptide addition SEQ ID NO:1) peptide, which acts as a signal for endo - or exocytosis, and the site of cleavage by the protease with the exception of SEQ ID NO:1.

This invention relates also to a method of detecting the activity of γ-secretase, which is that:

1) get/use a transgene encoding a protein containing the following elements:

a) a first nucleotide sequence encoding a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO:1);

b) a second nucleotide sequence encoding a signal peptide at the 5'-end of the first nucleotide sequence;

(C) promoter and

d) optionally additional coding and/or noncoding nucleotide sequence;

2) this trashed injected into the cell and Express a fused protein;

3) protein break down within the amino acid sequence of SEQ ID NO:1 γ secretases present in the cell, resulting in a first partial protein containing the amino acid sequence GAIIGLMVGGVV (SEQ ID NO:2), and the second partial protein containing amino acid is the selected VIVITLVML (SEQ ID NO:3); and

4) determine the number of the second incomplete protein and the activity of γ-secretase based on the amount formed of the second incomplete protein, provided that with the exception of SEQ ID NO:1 specified protein no peptide that acts as a signal for endo - or exocytosis, and/or site of cleavage by the protease.

The methods according to the invention (“analysis of the screening method β-peptide”, “analysis of γ-secretase”) suitable for detection in vivo γ-secretase (protein having the activity of γ-secretase) or activity of γ-secretase that makes possible universal application of these methods even, for example, when performing high-performance methods of screening (“HTS”). In these methods lack the above-mentioned disadvantages of the known detection methods, in particular, labour-intensive stages of separation and detection, and significantly improved specific signal activity of γ-secretase. A more specific signal is achieved by a significant attenuation of the background signal and eliminate or reduce the release of the first and second incomplete proteins under the action of nonspecific proteases.

An important element methods according to the invention is that the cleavage of a C-terminal fragment of APP γ-protease into two fragments, namely the first incomplete protein containing linakis is now a sequence GAIIGLMVGGVV (SEQ ID NO:2), and the second partial protein containing the amino acid sequence VIVITLVML (SEQ ID NO:3), there is a diffusion of the second partial protein containing the amino acid sequence VIVITLVML (SEQ ID NO:3), into the cytosol of the cell (figure 2). The specified second incomplete protein, which can be easily detected in the cytosol of cells, for example, in the form of a fused protein with factor activation of transcription (TAF) using gene-reporter, serves as a tool for detecting the presence of γ-secretase or quantitative determination of the activity of γ-secretase. The site of cleavage of the γ-secretases is located in the transmembrane domain of APP (Kang, J. et al., (1987) Nature, 325, 733). Specified transmembrane domain of APP has the amino acid sequence GAIIGLMVGGVV40IA42TVIVITLVML. γ-Secretase breaks down the website after V40, A42or T43. β-Peptide produced by eukaryotic cells in culture cells, secreted into the supernatant medium.

Using acceptable reporter system (e.g., AF and the corresponding gene-reporter) the selection of the second incomplete protein can activate the expression of reporter protein that can be found in eukaryotic cells. In the detection of reporter protein can be demonstrated that there was a cleavage of APP γ secretases. Thus, it is possible to produce qualitative and/elekrichestvom the determination of the activity of γ-secretase.

The constituent elements of this method can be described in more detail as follows:

The first nucleotide sequence encodes the amyloid protein precursor (APP) or part thereof, containing SEQ ID NO:1, and indicated by the RDA or part does not contain any other peptide fragments acting as a signal for endo - or exocytosis, and/or sites of cleavage by the protease. Specified the first nucleotide sequence preferably encodes a protein containing an amino acid sequence comprising SEQ ID NO:1, for example SEQ ID NO:6 or SEQ ID NO:14. In other embodiments of the invention, the first nucleotide sequence encodes processionary or modified by the RDA, for example, the resulting siteprovides mutagenesis, in order to avoid encoding the peptide fragment that acts as a signal for endo - or exocytosis, and/or site of cleavage by the protease, in addition to SEQ ID NO:1. In another embodiment of the invention specified by the RDA or part thereof, specified encoded first nucleotide sequence is a protein selected from the RDA man or mouse (for example, APLP1 or APLP2).

The second nucleotide sequence preferably encodes any acceptable signal peptide (SP). The signal peptide contains, for example, the signal p is tidy, corresponding to SEQ ID NO:5 (signal peptide ARR human), SEQ ID NO:12 (SUC2 signal peptide of yeast, “SP2”), SEQ ID NO:13 (signal peptide VM, “SP3”), or any other signal peptide, for example, described in the publication Heijne et al., Nucl. Acids Res. (1986), 14(11) 4683-4690.

As a promoter you can use either regulated or constitutive promoter. This promoter can be used, for example, for expression in mammalian cells, C. elegans, yeast or Drosophila. Appropriate promoters for mammalian cells are, for example, CMV, HSV TK, SV40, LTRS (all are manufactured by the company Clontech, Heidelberg, Germany) and RSV (e.g., Invitrogen™ life technologies, NV Leek, Netherlands). Promoters that can be used for expression in C. elegans include, for example, unc-119, unc-54, hsp16-2, goa-1 and sl-12. For expression in yeast are suitable the ADH1 promoter (constitutive) (Vlckova et al. (1994) Gene, 25(5), 472-4), GAL1 (conditionally inducible) (Selleck et al. (1987) Nature 325, 173-7), MET3 (conditional) (Cherest et al. (1987) Mol. Gen. Genet. 210, 307-13) and MET (for example, Kerjan et al. (1986) Necleic Acids Res. 14(20), 7861-71). For expression in Drosophila can be used, for example, promoters MT (metallothionein), As or Ds47 (all are manufactured by the company Invitrogen™ life technologies).

When implementing this method, it is preferable to use eukaryotic cells, e.g. human cells, or cells other than human, for example, cells of the monkey, hamster, mouse, Drosophila is, striped Pertini or yeast. For example, you can use either HeLa, HEK293, H4, SH-SY5Y, H9, Cos, CHO, N2A, SL-2 or Saccharomyces cerevisiae. In a specific embodiment of the invention it is possible to use cells of C. elegans. These cells may belong to a transgenic animal other than human. In a specific embodiment of the invention the transgenic cells may belong transgenic organism C. elegans. In particular, this invention relates to methods, the implementation of which can be used by yeast cells, for example, from strain MaV203 (Invitrogen™ life technologies, Rockville, MD, USA) or EGY48 (OriGene Technologies, Inc. Rockville, MD, USA).

The transgene encodes a protein, which consists of incomplete proteins encoded by the first and the second nucleotide sequence and, if necessary, additional nucleotide sequences. Thus, the protein contains the first incomplete protein and the second incomplete protein, and optionally an additional incomplete protein. However, it is important that protein did not contain peptide fragments acting as a signal for endo - or exocytosis, and/or sites of cleavage by the protease with the exception of SEQ ID NO:1. The sites of cleavage by a protease known to experts in the field of databases proteases, for example, MEROPS (Rawling et al. (2002) MEROPS: the protease database, Nucleic Acids Res. 30, 343-346).

Protein is about the present invention preferably does not contain the cleavage site by the protease, which is the site of cleavage by caspase, for example, (IVL)ExD, in particular, VEVA, VEVD, and in another embodiment the invention, the protein of the present invention does not contain a signal peptide for endo - or exocytosis, which is a signal for internalization of APP, for example, NpxY or Di-leucine, in particular, NPTY.

In one specific embodiment of the invention fused protein has the amino acid sequence of SEQ ID NO:14. In addition to SEQ ID NO:1 specified protein does not contain any other (one or more) of the peptide fragments acting as a signal for endo - or exocytosis (for example, signal the internalization of the RDA), and/or sites of cleavage by a protease (e.g., caspase).

In particular, when implementing this method, you can use the transgene having a nucleotide sequence corresponding to SEQ ID NO:15 (SPC55GV TAG). In particularly preferred embodiments, the implementation of this method, the transgene is introduced into a vector. This particular variant of the invention, also referred to as SP-C55-Gal4-VP16 (i.e. SPC55GV). In this case Express a fused protein containing the signal peptide of the RDA fragment s RDAs, GAL4 and VP16. This protein, located in the transmembrane domain, split in the fragment s, and the second is an incomplete protein that is part of a fused protein that contains one part fragm the NTA s, GAL4 and VP16, find using reporter plasmids.

In addition to transgenic constructs SPC55GV other reporter construct, in which the domain that activates transcription, for example, may be inserted between the transmembrane domain and the cytosolic domain SPC55 or label (e.g., MYC, FLAG) at the N - and C-end and between the transmembrane and cytosolic domain SPC55.

Additional encoding nucleotide sequence can encode, for example, a protein that can be used to detect the second incomplete protein. Additional encoding nucleotide sequence may be located at the 3'-end of the first nucleotide sequence. Additional encoding nucleotide sequence encodes, for example, chimeric protein, or other protein, created from a number of domains, for example, a protein containing the DNA-binding domain and a domain that activates transcription. In a specific embodiment of the invention additional encoding nucleotide sequence encodes a protein that comprises the GAL4-binding domain and a domain that activates transcription, VP16 (GAL4-VP16, “GV”), and more incomplete protein preferably has the amino acid sequence of SEQ ID NO:7. In yeast cells, more incomplete protein may also contain LexA-binding domain (for example the EP, LexA-VP16). This additional incomplete protein is particularly suitable for implementing methods using cells of yeast strain EGY48.

In particular, the present invention relates to methods of using cells, cotransfection reporter plasmid. Reporter plasmid contains a gene-reporter under the control of a regulated promoter. For example, gene-reporter can encode GFP and its derivatives, for example, EGFP (enhanced green fluorescent protein), EBFP, EYFP, d2EGFP, GFPuv or luciferase (e.g., Promega, Mannheim, Germany), CAT (e.g., Promega), SEAP (e.g., Clontech), βGal (e.g., Clontech), fluorescent protein coral reefs (RCFP, Clontech) or inducers of apoptosis, for example, Fas, TNF-R1 death domain cells and homologues (Tartaglia et al. (1993) Cell 74, 845-53), ced3, ced4, ced9. As a regulated promoter reporter plasmid may contain a minimal promoter, for example, GAL4-binding site in combination with the minimal promoter of HIV CD4 promoter or promoter mec7. The selection of an appropriate regulated promoter depends on the domain that activates transcription.

A particular variant embodiment of the invention relates to a method using yeast cells. Alternatively, yeast expressing vector pDBTrp (Invitrogen™ life technologies, the Netherlands, catalog No. 10835023), which impose the promoter METH-25 (SEQ ID NO:10),), which contains a large number of other expressing vectors with different promoters (for example, the inducible GAL1 promoter, a constitutive active promoter ADH1) and different breeding markers (ADE, LEU, TRP, HIS, LYS, PHE).

A particular variant embodiment of the invention relates to the use of yeast cells containing the GAL4 or LexA-inducible genes that are stably integrated into their genome or are outside the chromosome. In these embodiments of the invention are preferably used yeast strains MaV203 (Invitrogen™ life technologies Inc., Rockville, MD, USA) or EGY48 (OriGene Technologies, Inc., Rockville, MD, USA).

Concrete option implementation of the methods according to the invention refers to the use of cells, which additionally transfected another recombinant vector. Cells used in these embodiments of the invention, it is preferable not have endogenous γ-secretase or do not have the activity of endogenous γ-secretase and therefore cannot be detected by the above methods. Such cells can be transformed optional vector containing the nucleotide sequence, preferably cDNA, which encodes the γ-secretase, subunit protein γ-secretase or γ-secretase-like proteinase. For example, you can use the cDNA library. This variant implementation of the method according to the present invention can among other things be used for the ID is γ-secretase, subunit protein γ-secretase or γ-secretase-like proteases, and also cDNA that encodes a γ-secretase, subunit protein γ-secretase or γ-secretase-like proteinase. CDNA library, which can be scanned to search for γ-secretase, subunit protein γ-secretase or γ-secretase-like proteases can be obtained from cells or tissue of any organism, for example, b-cells, neurons, glycidol, hippocampus, whole brain, placenta, kidney. cDNA preferably derived from cells of vertebrates (such as hamster, rat, mouse, dog, monkey, human), in particular from cells or tissues.

In the case of cells without transfection does not have activity of γ-secretase, but after transfection the cDNA library are beginning to show the activity of γ-secretase, we can conclude that the cDNA present in such cells, encodes γ-secretase, subunit protein γ-secretase or γ-secretase-like proteinase. Specified cDNA can be distinguished by known methods from cells possessing such activity, and then to explore the known methods.

The present invention relates to a transgene that encodes a protein and contains the following elements:

a) a first nucleotide sequence encoding a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITVML (SEQ ID NO:1);

b) a second nucleotide sequence encoding a signal peptide at the 5'-end of the first nucleotide sequence;

(C) promoter and

d) at least one additional nucleotide sequence from the 3'-end of the first nucleotide sequence, which encodes the DNA-binding domain and a domain that activates transcription;

moreover, besides SEQ ID NO:1 specified protein does not contain peptides, acting as a signal for endo - or exocytosis, and/or sites of cleavage by the protease.

The first nucleotide sequence preferably encodes the RDA or its part, which in addition to SEQ ID NO:1 does not contain other peptide fragments acting as a signal for endo - or exocytosis, and/or site of cleavage by the protease.

The transgene, for example, may have the nucleotide sequence of SEQ ID NO:15.

The transgene may be acceptable vector, such as pcDNA 3.1+ or pDBTrp.

Another variant embodiment of the invention relates to a method of application of the transgene and/or vector of the present invention for producing transgenic cells with the indicated transgenic cell is not necessarily introduced into the organism other than human, suitable for use as a reporter of the organism in vivo. For example, the indicated transgene and/or the vector can be used for the floor is to be placed transgenic organism C. elegans. In another embodiment of the invention the specified transgene and/or the vector used to produce transgenic yeast cells such as S. cerevisiae.

The present invention relates also to a method for producing a transgenic organism other than human, for example, transgenic organism C. elegans, in accordance with which specified the transgene and/or a vector containing the indicated transgene, microinjection in the gonads of the body, for example, C. elegans. The present invention relates also to the cell containing the transgene according to the invention, the transgenic organism C. elegans that contain the transgene. The present invention relates also to the cell, in particular a yeast cell containing a transgene according to the invention, preferably in an acceptable vector. The present invention further relates to cells, preferably yeast cells containing the transgene according to the invention and the cDNA library, which can be the object library of expressed cDNA sequences (cDNA library).

The present invention relates to the use of these transgenic or recombinant cells, preferably cells of yeast or C. elegans, in the method of determining or identifying γ-secretase, cDNA that encodes a γ-secretase, cDNA that encodes a subunit protein γ-secret is s, cDNA encoding a γ-secretase-like proteinase, or activity of γ-secretase, subunit protein γ-secretase or γ-secretase-like proteases, the use of these cells in the method of identifying inhibitors of the activity of γ-secretase (γ-secretase, subunit protein γ-secretase or γ-secretase-like proteases) and to method of using said cells.

The present invention particularly relates to methods of identifying substances (effectors)that modulate (i.e. inhibit, reduce, enhance or modify) the activity of γ-secretase, subunit protein γ-secretase or γ-secretase-like proteases, which include the following stages:

1) obtaining the transgenic organism other than human, for example, transgenic organism C. elegans or Saccharomyces cerevisiae, or transgenic cells, and

specified transgenic organism other than human or transgenic cells contain a transgene of the present invention;

transgenic organism other than human or transgenic cell further contains a reporter plasmid that has a binding site of a protein, a minimal promoter and a gene-reporter; and

if necessary, cDNA encoding a γ-secretase, subunit protein γ-secretase or γ-secretase-like proteinase;

this transgenic organizations is m, other than human or transgenic cell expresses the transgene and, if necessary, γ-secretase, subunit protein γ-secretase or γ-secretase-like protease, encoded cDNA;

2) cultivating a transgenic organism other than human or transgenic cells with the test substance to be researching;

3) determination of the number of the second incomplete protein.

The present invention relates also to a method of identifying effectors γ secretase, subunit protein γ-secretase or γ-secretase-like proteases, which is that:

1) get and use the transgene according to the invention;

2) the indicated transgene, reporter plasmid and, if necessary, cDNA encoding a γ-secretase, subunit protein γ-secretase or γ-secretase-like protease, is introduced into the host cell genome and protein, encoded by the specified transgene and, if necessary, γ-secretase, subunit protein γ-secretase or γ-secretase-like protease, encoded cDNA Express in the presence of the analyte;

3) protein

a) break down within the amino acid sequence of SEQ ID NO:1 γ secretases present in the cell, resulting in a

b) formed by the first partial protein containing the amino acid sequence GAIIGLMVGGVV (SEQID NO:2), and the second partial protein containing the amino acid sequence VIVITLVML (SEQ ID NO:3); and

4) produce qualitative or quantitative determination of the above-mentioned second incomplete protein.

The present invention relates also to methods of identifying substances inhibiting the activity of γ-secretase, subunit protein γ-secretase or γ-secretase-like proteases, the implementation of which a transgene encoding a protein containing the signal peptide and SEQ ID NO:1, Express in the presence of the analyte and reporterrors plasmids and determine the effect of said substance on the amount of the formed second partial protein containing the amino acid sequence VIVITLVML (SEQ ID NO:3).

The present invention also relates to inhibitors of γ-secretase, subunit protein γ-secretase or γ-secretase-like proteases identified by the methods according to the invention.

These methods can be used together with the system s-Gal4-VP16 (i.e. fused protein containing s, GAL4 and VP16, or nucleic acid encoding the corresponding protein):

1) identification and definition (qualitative and/or quantitative) activity of γ-secretase, subunit protein γ-secretase or γ-secretase-like proteinase;

2) identification of γ-secretase, subjudice what about the protein γ-secretase or γ-secretase-like proteases in different tissues, cells and organisms or species, and to identify and highlight the corresponding cDNA encoding γ-secretase, subunit protein γ-secretase or γ-secretase-like protease, and subsequent use of these cDNA;

3) screening in vivo, for example, yeast cells such as Saccharomyces cerevisiae), C. elegans or cell culture, which allows to determine the activity of γ-secretase, subunit protein γ-secretase or γ-secretase-like proteases without the use of immunobiological methods:

4) identification and study of substances, for example, pharmacologically active compounds for modulation of the enzymatic or biological activity of γ-secretase, subunit protein γ-secretase or γ-secretase-like proteases, such as effectors (inhibitors, activators, modulators) γ secretase, subunit protein γ-secretase or γ-secretase-like proteases. In particular, this method can be used to perform high-throughput screening (HTS). Through the use of systems analysis method HTS to identify substances that can be used for the treatment of Alzheimer's disease and/or prophylactic treatment;

5) research or in the case of Alzheimer's disease more in-depth study of mutated RDA or its parts or functions of the membrane about the EPHA;

6) replacement described fused proteins/transgenes, for example, s in SP-C55-Gal4-VP16, other fragments according to the invention and studies using these methods γ secretase, subunit protein γ-secretase or γ-secretase-like proteases, their activity and the possibility of regulating such activity.

Another variant of implementation of the present invention relates to pharmaceutical compositions containing pharmaceutically active compound, the inhibitory activity of γ-secretase, subunit protein γ-secretase or γ-secretase-like proteases, which have been identified by the method according to the invention.

Another variant of implementation of the present invention relates to a method for producing a pharmaceutical composition, which includes the implementation of the method according to the invention and a drug containing the above identified pharmaceutically active compound.

Another variant of implementation of the present invention relates to a method for producing a pharmaceutical composition, which comprises (a) a method according to the invention and b) mixing the identified pharmaceutically active compounds with pharmaceutically inert inorganic and/or organic fillers.

Another variant of implementation of the present invention applies the test set to detect the activity of γ-secretase, subunit protein γ-secretase or γ-secretase-like proteases, which includes a transgene, the vector or the cell according to the invention.

Figure 1: figure 1 shows the amyloid protein precursor (isoform ARR and isoforms ARR or ARR) and the cleavage products by secretases.

Figure 2: schematic representation of the principle underlying the methods according to the invention: the site of cleavage of the β-secretases at the N-Terminus; the site of cleavage of the γ-secretases in the transmembrane region; C100 = C100 fragment of amyloid protein precursor (APP); GAL4-VP16 = DNA-binding domain that activates transcription domain (including DNA-binding domain and activator of transcription), which is associated with the domain binding protein, DNA reporter plasmids.

Figure 3: design of expressing plasmids SP-C100-GAL4-VP16: AA = amino acids; restriction sites SacI cleavage, Hind III and Kpn I, showing the location of the cleavage site in the plasmid.

Figure 4: expressing plasmid pDBTrp-MET25-SP-C100-GAL4-VP16: design expressing plasmids designed for expression of the transgene in yeast.

The following examples illustrate the present invention without limiting its scope.

Examples

Example 1. The creation of expressing plasmids pcDNA3.1+containing SP-C100-GAL4/VP16

This plasmid encodes the signal peptide (S) of the RDA, which merged with the 100 C-terminal amino acid residues of APP (C100). The remains C100 start at the N-Terminus β-peptide and ending With the end of the RDAs. The indicated plasmid should be further split γ secretases to highlight β-peptide.

GAL4/VP16 (SEQ ID NO:7) was merged with the end of the SP-C100 (SEQ ID NO:6).

GAL4/VP16 contains the first 147 amino acid residues of the yeast activator GAL4 transcription and 78 C-terminal amino acid residues VP16, an activator of transcription of herpes simplex virus. As a fused protein fragment of GAL4 begins to function as DNA binding, while the fragment VP16 activates transcription (Sadowski et al., 1988).

pcDNA3.1+ (Invitrogen™ life technologies, The Netherlands, catalog No. V79020) serves as a plasmid vector.

Example 2. The creation of the reporter plasmid pGL2-MRG5-EGFP

Reporter plasmid pGL2-MRG5 to mammalian cells is pGL2 (Promega), in which the DNA fragment of pMRG5 (Ikeda et al., 1998), which contains five DNA binding of GAL4 sites at the top of the Central promoter of human immunodeficiency virus (HIV) (Kretzschmar et al., 1994), insert at the top from gene-luciferase reporter pGL2. For easier detection in cell culture gene-reporter luciferase was replaced with the gene for EGFP (enhanced green fluorescent protein)obtained from the vector pEGFP-N1 (Clontech Laboratories, Heidelberg).

Example 3. Cotransfected neuroblastoma cell people the century

Cells human neuroblastoma SH-SY5Y (ATSS CRL-2266) were cotranslational both plasmids of examples 1 and 2 and then examined under a microscope in the light with a wavelength of 480 nm, which was the excitation of EGFP. Thus, it was possible to detect EGFP-expressing cells, with a strong green fluorescence. To ensure that the green fluorescence is specifically dependent on the expression of EGFP reporter plasmid, cells SH-SY5Y was transferrable only reporter plasmid pGL2-MRG5-EGFP. In these cells was not detected by green fluorescence. The expression of activated GAL4-VP16, which was supposed to proteolytic selection GAL4/VP16 from-end SP-C100-GAL4/VP16.

Example 4. Using system C100-Gal4/VP16 to detect cDNA that encodes the activity of γ-secretase, cDNA libraries

SP-C100-Gal4/VP16 cloned in yeast expressing the vector pDBTrp (Invitrogen™ life technologies, The Netherlands, catalog No. 10835023) under control of the promoter MET, replacing part of the vector pDBTrp containing the ADH promoter and domains GAL4DB (located between the CYH2 gene and multiple cloning sites), a DNA fragment containing the promoter MET of RME (Mumberg et al., 1994), up from SP-C100-Gal4-VP16. This design transformed yeast strain MaV203 (Invitrogen™ life technologies). Strain MaV203 genetically modified and contains three GAL4-induces the selected gene-reporter (URA3, HIS3, lacZ), which is stably integrated into the genome (Vidal et al., 1996). Proteolytic domain highlighting GAL4/VP16 in strain MaV203 of SP-C100-Gal4-VP16 caused the activation of the read URA3 and HIS3, due to which the growth occurs on tablets without uracil or histidine.

Expression of cDNA protein SP-C100-Gal4-VP16 in the strain MaV203 caused so insignificant activity reporters that this is functionally active in vivo analysis system suitable for screening and detection of cDNA expression γ secretase in the cDNA library.

Example 5. Identification of γ-secretase by screening the cDNA library of human cells

Recombinant yeast strain MaV203 in example 4 was used for screening the cDNA library of human cells (ATSC 87286; American type culture collection, Manassas, VA, U.S.A.; Elledge et al., 1991) with the aim of finding a cDNA that encodes a protein with the activity of γ-secretase. Alternatively, for screening cDNA that encodes a γ-secretase or protein having γ-secretase-like activity, you can use a cDNA library from human hippocampus introduced into yeast expressing vectors R-MET (Mumberg et al., 1994) or R-ADH1 (Mumberg et al., 1995).

Example 6. Cloning SP2-C100 and SP2-C100-GAL4/VP16

The coding region of the signal peptide of human SP-C100-GAL4/VP16 (described in example 1) was replaced with the signal peptide selected from the SUC2 gene of yeast (SP2; SEQ ID NO:12), financial p the result which was obtained design, coding SP2-C100-GAL4/VP16 (SEQ ID NO:19).

SP2-C100 created, amplificare the coding region of the Mature form C100 (without the signal sequence, compare SEQ ID NO:4) using the 5'-terminal priming, which included the coding sequence for the signal peptide SUC2 (SEQ ID NO:12), and the 3'end of the seed corresponding to the natural termination codon (Kang et al., (1987)). To facilitate replacement of the signal peptide priming IN (SEQ ID NO:23) and IN (SEQ ID NO:24) were created so that the resulting PCR product contained an additional NheI site, linking the coding region of the signal peptide and Mature peptide.

Protein SP2-C100-GAL4/VP16 was obtained by EcoRI cleavage, which was carved C100 fragment of SP2-C100 and replaced by the fragment C100-GAL4/VP16.

These fragments were cloned in yeast expressing the vector pDBTrp (Invitrogen™ life technologies, The Netherlands, catalog No. 10835023), containing the promoter METH described in example 4.

Example 7. Cloning SP2-C-GAL4/VP16-100

To obtain design SP2-C-GAL4/VP16-100 (SEQ ID NO:17) was carried out with three independent PCR reaction using the following starters:

1. Using SP2-C100 as a matrix, using a nucleating EN (SEQ ID NO:25) and IN (SEQ ID NO:26) amplified ectodomain and transmembrane domain C100 so that the product of the PCR contained the 3'-end flanking region, which overlaps the coding region of the GAL4/VP16.

2. Using seed IN (SEQ ID NO:27), IN (SEQ ID NO:28) and GAL4/VP16 as a matrix DNA, performed the PCR reaction for amplification of the coding region 5'- and 3'-end flanking regions corresponding to each side of the SP2-C100.

3. the 3'end segment SP2-C100, encoding the cytoplasmic domain C100, amplified using nucleating EN (SEQ ID NO:29) and IN (SEQ ID NO:30), resulting in 5'-end was covered by the coding region of the GAL4/VP16. The resulting PCR products (about 200 BP, 720 BP and 100 BP) was purified and used to perform the last PCR in the presence of nucleating IN and IN corresponding to the 5'- and 3'-ends of the SP2-C100. The PCR product of the last length of about 100 BP cloned in grozeva expressing the vector obtained from pDBTrp (Invitrogen™ life technologies, The Netherlands, catalog No. 10835023), which contained the promoter METH described in example 4.

Example 8. Cloning SP3-C100, SP3-C100-GAL4/VP16 and SP3-C-GAL4/VP16-100

To create three plasmid vectors designed for the expression of SP3-C100, SP2-C100-GAL4/VP16 (SEQ ID NO:21) and SP3-C-GAL4/VP16-100 (SEQ ID NO:32) in cellular systems mammals, C100, C100-GAL4/VP16 or-GAL4/VP16-100 was subclinically from yeast expressing the vectors in example 6 or 7 in expressing the vector pRc/CMV for mammalian cells (Invitrogen, catalog No. V75020)containing the coding region for the signal peptide VM (SP3, SEQ IDNO:13). Coding region C100, C100-GAL4/VP16 or-GAL4/VP16-100 ligated in reading frame with the coding region SP3 on a single restriction site NheI described in example 6.

Example 9. The increased expression of C100-GAL4/VP16 in yeast

Quantitative determination of the expression level of different designs in crude lysates from transformed yeast showed that the expression of SP-C100-GAL4/VP16 was very low compared with lysates from yeast cells transformed with vectors encoding fusion with the signal peptide yeast SUC2. For example, expression of SP2-C100-GAL4/VP16 caused a strong expression of specific bands of the expected size. However, by the method of Western blot turns could also discover a band with higher electrophoretic mobility, indicating nonspecific destruction of the recombinant protein in yeast. The stability of the protein was increased in the case of C-GAL4/VP16-100 (see below) with the introduction of the domain of the GAL4/VP16 in the frame read in C100 near the site of cleavage of the γ-secretases. Both fused protein was characterized by levels of expression comparable with the design, coding C100 without GAL4/VP16, suggesting that two different merger between C100 and GAL4/VP16 did not prevent expression of the protein.

Example 10. Improved background signal in yeast

Increased expression of C100-GAL4/VP16 due to replacement signal peptide is consistent with a strong increase in non-specific activation of reporter systems URA3, HIS3 and lacZ in yeast strain V203. As in yeast lacking the activity of γ-secretase, such effect, apparently, was the result of nonspecific processing C100-GAL4/VP16 cellular proteases and select the active GAL4/VP16.

If you move your domain GAL4/VP16 closer to the site of cleavage of the γ-secretases was essentially completely eliminated nonspecific proteolytic cleavage detected in C100-GAL4/VP16 on the site between transmembrane domain C100 and aminocom.com domain of GAL4/VP16.

The splitting of different designs were tested in strain V203 by research GAL4/VP16-dependent activation of reporter systems.

The transformation strain MaV203 using SP-C100-GAL/VP16 was obtained phenotype Ura+, His-(compare example 4). Increased levels of expression due to the replacement of the signal peptide SP peptide SP2 (with the formation of SP2-C100-GAL4/VP16) caused a strong activation of all indicators to a level similar detected in the positive control sample, and strain MaV203 constitutively expressed reprezentirovanii (full-size) GAL4 protein (encoded by plasmid pCL1; Clontech Laboratories).

In contrast MaV203 cells expressing SP2-C-GAL4/VP16-100 at a level comparable with SP2-C100-GAL4/VP16, was created phenotype Ura-, His-, which was also formed by strain MaV203, transformed a blank control, etc) the rum.

Therefore, SP2-C-GAL4/VP16-100 can be highly expressed in yeast, but is still characterized by very low nonspecific activation of the GAL4-dependent reporter. High expression of protein SP2-C-GAL4/VP16-100 in combination with a weak background signal of non-specific cleavage/activation of the reporter is a distinctive feature of the capture system with optimized signal-to - noise.

Example 11. Processing protein SP3-C-GAL4/VP16-100 γ secretases in mammalian cells

To confirm that the protein SP3-C-GAL4/VP16-100, expressed in mammalian cells, correctly procession γ secretases, protein SP3-C-GAL4/VP16-100 was transferrable in mammalian cells, which was found to Express endogenous γ-secretase (Haass et al., 1992). For expression in mammalian cells, signal peptide in SP2-C-GAL4/VP16-100 was replaced analogously to example 8 signal peptide mammals, protein isolated from the basal membrane VM, which is known for high level of expression (SP3; SEQ ID NO:13). The processing of γ-secretases controlled by quantification of secretion β into the culture medium. Secreted β found by the method of multi-layer ELISA using monoclonal antibodies A and biotinylated antibody 4G8 (Senetek PLC, Napa, California, USA; cf. Kim et al., 1990), respectively, as the IMM is Britanny and identifying antibodies.

After transfection SP3-C100 was observed eight-fold increase in secretion β compared to the empty control vector. Cells transfected SP3-C100-GAL4/VP16 or SP3-C-GAL4/VP16-100, secretively similar amount of β, from which it follows that either C-terminal or kolomanbrunnen the fusion of GAL4/VP16 does not affect the proteolytic processing of γ-secretases.

Example 12. Activation of transcription of GAL4/VP16-dependent gene reporter protein WITH GAL4/VP16-100, expressed in mammalian cells

Processing C100-GAL4/VP16 and GAL4/VP16-100 γ secretases causes release of polypeptide containing the GAL4/VP16 and additional amino acids, of the flanking parts of the C100. Proteins SP3-C100-GAL4/VP16 and SP3-C-GAL4/VP16-100 was cotranslationally reporter plasmid pGL2-MRG5-EGFP for mammalian cells (Ikeda et al., 1998), described in example 2, which contains five DNA binding of GAL4 sites at the top of the Central promoter of human immunodeficiency virus (HIV) and cDNA coding GFP. Cotransfected pGL2-MRG5-EGFP in the GAL4/VP16-containing fused proteins in both cases, caused the appearance of GFP-positive cells.

Example 13. The creation of expressing plasmids SP3-C55-GAL4/VP16 for mammalian cells

Constructs containing the sequence C100 RDAs and GAL4/VP16, include the site of cleavage of the γ-secretases and the site of cleavage of caspase-like proteases. In order to avoid splitting SP3-C100-GAL4/VP16 was carrying ecifically the caspase-like protease between the authentic website γ secretase and the domain of GAL4/VP16 emitting GAL4/VP16 and activation of the reporter system in mammalian cells, removed in SP3-C100-GAL4/VP16 C-terminal segment C100, including 45 amino acids and encodes the cytoplasmic domain of APP. The removal of the 45 C-terminal amino acids in the C100 also allows you to eliminate the “signal peptide internalization near-end of the RDA, which directs the endocytosis of APP after introduction into the plasma membrane.

Thus, expressing plasmid SP3-C55-GAL4/VP16 for mammalian cell contains a signal peptide VM (SEQ ID NO:13), 55 N-terminal amino acid residues (s; SEQ ID NO:6) ARR-C100 and GAL4/VP16. S begins at the N-Terminus β-peptide and ends at the transmembrane domain of APP. In the protein SP3-C55-GAL4/VP16 there is only the site of cleavage of the γ-secretases. S includes the site of cleavage of the γ-secretases and should be split γ secretases to highlight β-peptide and GAL4/VP16. In addition, since endocytosis signal peptide internalization is not in SP3-C55-GAL4/VP16, only catalyzed γ-secretases splitting s-GAL4/VP16 associated with the plasma membrane, induces the release of β-peptide and activation of the GAL4/VP16-dependent transcription of the reporter system.

This expressing plasmid was obtained from vector SP3-C-GAL4/VP16-100 by introducing a termination codon (TAG) after a sequence of GAL4/VP16. This operation is performed by replacing a fragment of HpaI-ClaI protein SP3-C-GAL4/VP16-100, a DNA fragment obtained by PCR using the-W SP3-C-GAL4/VP16-100 as a matrix DNA, 5'end of the top seed from a single HpaI site in the GAL4/VP16 and 3'-terminal priming (5'-CCATCGATTTTCTAACCCCCACCGTA-3'; SEQ ID NO:31), which introduces a termination codon TAG (underlined) and a ClaI restriction site at the C-end of the open reading frame GAL4/VP16.

Example 14. Stably transfected cells NC

Cells NEC (line embryonic cells of the human kidney, ATSS)) cotranslational the plasmid SP3-C55-GAL4/VP16 and reporter plasmid-based luciferase PGL2-MRG5 (described in example 2). Then selected lines resistant cells, and the cells were incubated with 400 μg/ml of geneticin (GibcoBRL), selected clones resistant to neomycin, and explored in relation to sustainable expression of SP3-C55-GAL4/VP16 and luciferase.

Example 15. Transient transfection of cells NC

Cells NEK was cotranslationally vector SP3-C55-GAL4/VP16 (0,03 µg) and reporter-based vector pGL2 luciferase-MRG5 (1 µg) 12 advance tablets.

Connection DPAT (Elan Pharmaceuticals; Dovey et al., 2001) and L-685458 (Merck Pharmaceuticals; Shearman et al., 2000), known as inhibitors of γ-secretase, regardless of the dose inhibited the luciferase activity and the production of β and had the value of the IC50equal to 14 nm, respectively (DAPT) and 19 nm (L-685458).

Quantification of luciferase activity produced by means of a set for analysis of luciferase Bright-Glo (Promega). Quantitative determi the bookmark β in cell medium produced by the method of ELISA, described in example 11, using antibodies 4G8 and E (company Senetek). These antibodies are specific for amino acids 17-24 (4G8) and 1-17 (6E10) β-peptide.

In experiments on the time of transfection β also identified methods thus and Western blot turns. Both methods was detected lane 4 kDa, corresponding β-peptide.

Example 16. Pharmacological study of the stably transfected cells NC

The cell clone NECK, stably expressing SP3-C55-GAL4/VP16 and design MRG5 luciferase identified in accordance with example 14. This cell clone was used to investigate the response of the analyzed system stably transfected mammalian cell on DAPT (Elan Pharm.; Dovey et al., 2001) and L-685458 (Merck Pharm.; Shearman et al., 2000). Both compounds inhibited the luciferase activity (24 hour treatment) regardless of the dose, when values of the IC50equal to 230 nm, respectively (DAPT) and 130 nm (L-685458).

Example 17. Identification of inhibitors of γ-secretase

To identify inhibitors of γ-secretase double-stably transfected cells NEC (see example 14) were incubated in 96-advance tablets in the presence of one or more of the compounds under study (for example, screening libraries of compounds) considering the analysis of concentration equal to 10 μm or n the same and after 24 hours was determined by luciferase activity. Quantification of luciferase activity can be performed using the test kit luciferase (Promega), set for analysis of luciferase Bright-Glo (Promega) or any other method of quantitative determination of luciferase. The decrease in luciferase activity reflects the reduction in the activity of γ-secretase.

Links

Dovey et al. (2001) J. Neurochem. 76, 173.

Elledge et al. (1991) Proc. Natl. Acad. Sci. USA 88. 1731.

Estus et al. (1992) Science 255, 726.

Haass et al. (1992) Nature 359. 322.

Heijne et al. (1986) Nucl. Acids Res. 14(11), 4683-4690.

Hilbich et al. (1991) J. Mol. Biol. 218, 149.

Ikeda et al. (1998) Mol. Cell. Biol. 18, 10.

Kang et al. (1987) Nature 325, 733.

Kim et al. (1990) Neurosci Res. Comm. 7, 113.

Kretzschmar et al. (1994) Mol. Cell. Biol. 14. 3927.

Maruyama et al. (1994) Biochem. Biophys. Res. Commun. 202, 1517.

Mattson (2003) Nature 422, 385.

Mumberg et al. (1994) Nucl. Acids Res. 22, 5767.

Mumberg et al. (1995) Gene 156, 119.

Rumble et al. (1989), N. Engl. J. Med. 320, 1446.

Sadowski et al. (1988) Nature 335, 563.

Scheuner et al. (1996), Nature Medicine 2, 864.

Shearman et al. (2000) Biochemistry 39, 8698.

Simons et al. (1996) J. Neurosci. 16(3), 899-908.

Suzuki et al. (1994) Science 264(5163), 1336-1340.

Vidal et al. (1996) Proc. Natl. Acad. Sci. USA 93, 10315.

Yankner et al.(1990) Proc. Natl. Acad. Sci. USA 87, 9020.

For General work with recombinant DNA:

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

To work with yeast (transformation DNA):

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. (1994) Current Protocols in Molecular Biology, pp. 13.7.1-13.7.2. Grene Publishing Associates/Wiley-Interscience, New York.

To work with the organism C. elegans (transgenic organisms):

Mello, C., and Fire, A. (1995) DNA transformation. In: Epstein, H.F. and Shakes. D.C., ed. Caenorhabditis elegans: Modern Biological Analysis of an Organism. Methods in Cell Biology, Vol. 48. Academic Press, San Diego, CA, pp. 451-482.

SEQ ID NO:1 (fragment RDAs)

SEQ ID NO:2 (fragment of APP (first incomplete protein))

SEQ ID NO:3 (fragment of APP (second incomplete protein))

SEQ ID NO:4 (fragment C100)

SEQ ID NO:5 (signal peptide RDAs person (SP))

SEQ ID NO:6 (fragment C)

SEQ ID NO:7 (GAL4-VP16)

SEQ ID NO:8 (SP-C100-GAL4-VP16)

SEQ ID NO:9 (RDAs person)

SEQ ID NO:10 (Recombinant plasmid pDBTrp-MET25-SP-C100-GAL4-VP16)

SEQ ID NO:11 (protein SP-C100-GAL4-VP16)

SEQ ID NO:12 (signal peptide gene SUC2 yeast (SP2))

SEQ ID NO:13 (signal peptide protein b is the basal membrane mammals VM (SP3))

SEQ ID NO:14 (SP-C55-GAL4-VP16)

SEQ ID NO:15 (SP-C55-GAL4/VP16-TAG)

SEQ ID NO:16 (Recombinant plasmid pDBTrp-MET25-SP-C-Gal4/VP16-100)

SEQ ID NO:17 (SP2-C-GAL4/VP16-100)

SEQ ID NO:18 (SP2-C-GAL4/VP16-100)

SEQ ID NO:19 (SP2-C100-GAL4/VP16)

SEQ ID NO:20 (S2-C100-GAL4/VP16)

SEQ ID NO:21 (SP3-C100-GAL4/VP16)

SEQ ID NO:22 (SP3-C100-GAL4/VP16)

SEQ ID NO:23 (Seed IN)

SEQ ID NO:24 (Seed IN)

SEQ ID NO:25 (the Seed IN)

SEQ ID NO:26 (Seed IN)

SEQ ID NO:27 (Seed IN)

SEQ ID NO:28 (Seed IN)

SEQ ID NO:29 (the Seed IN)

SEQ ID NO:30 (Seed IN)

SEQ ID NO:31 (3'-terminal priming s)

SEQ ID NO:32 (Plasmid SP3-C-GAL4/VP16-100)

1. Method intracellular detection of active γ-secretase, which is that
A. use a genetic construct that encodes a protein that contains
a) a first nucleotide sequence encoding a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO:1);
b) a second nucleotide sequence encoding a signal peptide at the 5'-end of the first nucleotide sequence;
(c) promoter and
d) if necessary, additional coding and/or non-coding nucleotide sequence;
Century found genetic construct is introduced into the cell, and it expressives gene that encodes a protein, which
i) is cleaved in the activity of γ-secretase with the formation of the first incomplete protein containing the amino acid sequence GAIIGLMVGGVV (SEQ ID NO: 2), and the second partial protein containing the amino acid sequence VIVITLVML (SEQ ID NO: 3);
ii) does not split in case of absence in the cell active γ-secretase;
C. education the first incomplete protein and/or the second partial protein determi is elaut presence in the cell active γ-secretase;
and which is characterized by the fact that genetic construct encoding a protein selected taking into account the fact that with the exception of SEQ ID NO:1 in the fused protein excluded all sites, acting as a signal for endo - or exocytosis, and/or site of cleavage by the protease.

2. Method intracellular detection of active γ-secretase, which is that
A. use a genetic construct encoding a protein that contains
a) a first nucleotide sequence encoding a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO:1);
b) a second nucleotide sequence encoding a signal peptide at the 5'-end of the first nucleotide sequence;
(c) promoter and
d) if necessary, additional coding and/or non-coding nucleotide sequence;
Century found genetic construct is introduced into the cell, and it expressives gene that encodes a protein, which
i) is cleaved in the activity of γ-secretase with the formation of the first incomplete protein containing the amino acid sequence GAIIGLMVGGVV (SEQ ID NO:2), and the second partial protein containing the amino acid sequence VIVITLVML (SEQ ID NO:3);
ii) does not split in case of absence in the cell active γ-secretase;
C. in the presence of a second incomplete protein is the cytosol of cells, determine the presence in the cell active γ-secretase;
and which is characterized by the fact that genetic construct encoding a protein selected taking into account the fact that with the exception of SEQ ID NO:1 in the fused protein excluded all sites, acting as a signal for endo - or exocytosis, and/or site of cleavage by the protease.

3. The method according to any one of claims 1 and 2, in which the first nucleotide sequence encodes a protein resulting from the modification of the amino acid sequence of SEQ ID NO:4.

4. The method according to any one of claims 1 and 2, where the second nucleotide sequence encodes a signal peptide ARR human (SEQ ID NO:5), SUC2 (SEQ ID NO:12) or VM (SEQ ID NO:13).

5. The method according to claim 4, in which the signal peptide has the amino acid sequence of SEQ ID NO:5.

6. The method according to any one of claims 1 and 2, where the promoter is the promoter used for expression in mammalian cells, yeast and Drosophila.

7. The method according to claim 6, where the promoter is a CMV promoter, HSV TK, RSV, SV40, LTRS, unc119, unc54, hsp16-2, G0A1, sel-12, ADH1, GAL1, MET3, MET25, MT, Ac5 or Ds47.

8. The method according to any one of claims 1 and 2, where the cell is a eukaryotic cell.

9. The method of claim 8, where the cell is a human cell.

10. The method of claim 8, where the cell is a cell other than human.

11. The method according to claim 10, where the cell is a HeLa cell, 293, H4, SH-SY5Y, H9, Cos, CHO, 2A, SL2 or a yeast cell.

12. The method according to claim 11, where the cell is a Saccharomyces cerevisiae cell.

13. The method according to any one of claims 1, 2, 5, 7, 9-12, where the protein contains the amino acid sequence of SEQ ID NO:6.

14. The method according to item 13, where additional coding nucleotide sequence is at the 3'-end of the first nucleotide sequence.

15. The method according to 14, where additional encoding nucleotide sequence encodes a protein that is expressed in the form of a gene encoding a protein with the first incomplete protein and the second incomplete protein, and can be used to determine the second incomplete protein.

16. The method according to clause 15, where the additional encoding nucleotide sequence encodes a protein containing a DNA-binding domain and a domain that activates transcription.

17. The method according to clause 16, where additional encoding nucleotide sequence encodes a protein consisting of the GAL4-binding domain and a domain that activates transcription of a fragment of VP16 (GAL4-VP16).

18. The method according to any one of claims 1 and 2, where the cell cotransfection reporter plasmid containing a gene-reporter under the control of a regulated promoter.

19. The method according to p, where the regulated promoter is activated by the domain that activates transcription.

20. The method according to claim 19, where the reporter plasmid encodes the gene-reporter for EGFP (at Lenogo green fluorescent protein), Ura 3, His 3 or Lac Z, and regulated promoter contains GAL4-binding sites and a minimal promoter HIV.

21. The method according to any one of claims 1, 2, 5, 7, 9-12, 14-17, 19-20, where the genetic construct contains a nucleotide sequence encoding SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22.

22. The method according to item 21, where the genetic construct is a vector.

23. The method according to item 22, where the recombinant vector is a pcDNA 3.1+.

24. The method according to any one of claims 1, 2, 5, 7, 9-12, 14-17, 19-20, 22-23, characterized in that the activity of endogenous γ-secretase in the cell is not detected.

25. The method according to paragraph 24, where cell cotransfection using cDNA library.

26. The method according A.25, in which cDNA derived from human tissues or tissue, non-human or from human cells or cells other than human is present in the cDNA library.

27. Genetic structure providing in the cell the expression of a gene encoding a fused protein comprising a protein that contains the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO:1), a signal peptide, a DNA-binding domain and a domain that activates transcription, containing
a) a first nucleotide sequence encoding a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO:1);
b) a second nucleotide sequence encoding a signal peptide at the 5'-end of the first nucleotide sequence;
(c) promoter and
d) at least one additional nucleotide sequence from the 3'-end of the first nucleotide sequence, which encodes the DNA-binding domain and a domain that activates transcription
wherein the genetic construct encoding a protein selected taking into account the fact that, with the exception of SEQ ID NO:1, in that the slit protein excluded all sites, acting as a signal for endo - or exocytosis and/or site of cleavage by the protease.

28. The genetic construct according to item 27, where the first nucleotide sequence encodes the RDA or part of the RDA.

29. Genetic design on one or both PP and 28, which has a nucleotide sequence encoding SEQ ID NO:14.

30. Vector providing in the cell the expression of a gene encoding a fused protein comprising a protein that contains the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO:1), a signal peptide, a DNA-binding domain and a domain that activates transcription, and containing the genetic construct according to any or several p-29.

31. The vector according to item 30, which is a vector pDBTrp or pcDNA 3.1+.

32. A method of obtaining a transgenic cells that synthesize the protein, including a protein that contains the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO:1), a signal peptide, a DNA with anywaysi domain and domain activating transcription for determining the activity of γ-secretase, according to which the cell transferout vector on one or both PP or 31.

33. The cell containing the genetic construct according to one or more p-29 providing in the cell the expression of a gene encoding a fused protein comprising a protein that contains the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO:1), a signal peptide, a DNA-binding domain and a domain that activates transcription.

34. Yeast cell containing a genetic construct according to one or more p-29 providing in the cell the expression of a gene encoding a fused protein comprising a protein that contains the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO:1), a signal peptide, a DNA-binding domain and a domain that activates transcription.

35. The use of cell 34 to identify a cDNA that encodes a γ-secretase, subunit protein γ-secretase or γ-secretation the proteinase.

36. A method for the identification of cDNA γ secretase, subunit protein γ-secretase or γ-secretstore proteinase, which is that
a) receive a cell of clause 34, which transform the method according to PP and 26; and
b) identify the cell which formed the second partial protein containing the amino acid sequence VIVITLVML(SEQ ID NO:3) in accordance with the method according to any of claim 2 to 26;
c) isolated from the indicated cell cDNA encoding a γ-secretase.

37. The use of cells 34 in the method of identifying inhibitors of the activity of γ-secretase, subunit protein γ-secretase or γ-secretstore proteases.

38. The method of identifying substances inhibiting the activity of γ-secretase, subunit protein γ-secretase or γ-secretstore proteases, which includes the following stages:
A. obtaining a transgenic organism other than human or transgenic cells that contain genetic construct that contains the following elements:
a) a first nucleotide sequence encoding a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO:1);
b) a second nucleotide sequence encoding a signal peptide at the 5'-end of the first nucleotide sequence;
(c) promoter and
d) if necessary, additional non-coding and/or coding nucleotide sequence;
reporter plasmid with the binding site of the protein, a minimal promoter, a gene-reporter and, if necessary, cDNA encoding a γ-secretase, subunit protein γ-secretase or γ-secretation the proteinase,
this transgenic organism other than human or transgenic cell expresses the genetic structure and, when necessary, is Timoti, γ-secretase, subunit protein γ-secretase or γ-secretation the protease encoded by the indicated cDNA;
Century, the cultivation of transgenic organism other than human or transgenic cells with the test substance and
C. determining the amount of the second incomplete protein with the indicated reporter plasmids, where the release of the second incomplete protein activates expression of the indicated reporter gene, which is found in transgenic organism other than human or transgenic cell, non-human, and the number of the second incomplete protein is determined in the presence and absence of the analyte;
wherein the genetic construct encoding a protein selected taking into account the fact that with the exception of SEQ ID NO:1 in the fused protein excluded all sites, acting as a signal for endo - or exocytosis, and/or site of cleavage by the protease.

39. The method of identifying substances inhibiting the activity of γ-secretase, subunit protein γ-secretase or γ-secretstore proteinase, which is that
A. use a genetic construct that contains the following elements:
a) a first nucleotide sequence encoding a protein containing the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO:1);
b) a second nucleotide sequence, encoding a signal peptide at the 5'-end of the first nucleotide sequence;
(c) promoter and
d) optionally additional coding and/or non-coding nucleotide sequence;
Century found the genetic construction of reporter plasmid, which contains the binding site of the protein, a minimal promoter and a reporter gene, and, optionally, cDNA encoding a γ-secretase, subunit protein γ-secretase or γ-secretation the proteinase, is injected into the cell, and it is synthesized protein encoded by the specified genetic design, and, if necessary, γ-secretase, subunit protein γ-secretase or γ-secretation the protease encoded by the indicated cDNA in the presence of the analyte;
C. protein
a) cleaved within the amino acid sequence of SEQ ID NO:1 γ secretases, subunit protein γ-secretase or γ-secretstore the proteinase present in the cell, if the test substance is an inhibitor of the activity of γ-secretase, subunit protein γ-secretase or γ-secretstore proteases, resulting in a first partial protein containing the amino acid sequence GAIIGLMVGGVV (SEQ ID NO:2), and the second partial protein containing the amino acid sequence VIVITLVML (SEQ ID NO:3)or
b) does not split in Utri amino acid sequence of SEQ ID NO:1 γ secretases, subunit protein γ-secretase or γ-secretstore the proteinase present in the cell, if the test substance is an inhibitor of the activity of γ-secretase, subunit protein γ-secretase or γ-secretstore proteases, resulting not formed a detectable quantity of the first and/or second incomplete protein;
D. determine the number of the second incomplete protein with the indicated reporter plasmids, where the release of the second incomplete protein activates expression of the indicated reporter gene, which is found in transgenic organism, and the number of the second incomplete protein is determined in the presence and absence of the analyte;
wherein the genetic construct encoding a protein selected taking into account the fact that with the exception of SEQ ID NO:1 in the fused protein excluded all sites, acting as a signal for endo - or exocytosis, and/or site of cleavage by the protease.

40. The method of identifying substances inhibiting the activity of γ-secretase, subunit protein γ-secretase or γ-secretstore proteases, in which the genetic construct that encodes a protein containing a signal peptide and SEQ ID NO:1, Express in the presence of the analyte of interest and determine the effect of this substance on the number which has formed the second incomplete protein containing the amino acid sequence VIVITLVML (SEQ ID NO:3), and the number of the second incomplete protein is determined in the presence and absence of the analyte.

41. The test set to detect the activity of γ-secretase, subunit protein γ-secretase or γ-secretstore proteases, which includes genetic construct according to any one of p-29, vector according to any one of p-31 or cell of clause 34.



 

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FIELD: production methods.

SUBSTANCE: method is based on the capability of defibrotide to increase the fermentation activity of plasmin and foresee the stages: a) making the contact in reactional area defibrotide, plasmin and substrate specific for plasmin which, because of reaction, provides the defined product b) the definition of the amount of obtained product in temporary points.

EFFECT: invention allows to define the biological activity of defibrotide in comparison with standard etalon with height accuracy and big repeatability.

9 cl, 6 dwg, 4 tbl, 1 ex

FIELD: medicine, oncology, molecular pharmacology.

SUBSTANCE: invention relates to a method and set for identifying the individual subjected to risk for arising in it the vascular and cancer disease. Method involves stages for the quantitative determination of the analyte concentration, i. e. pepsinogen I (PGI), in serum sample taken in the personal individual; comparison of the analyte concentration determined by the proposed method with a method-specific boundary value for this analyte; determination of the homocysteine concentration in a serum sample taken in this individual. The set comprises the combination of separate components that are necessary for the quantitative determination of the PGI concentration. Method provides the early detection of the possibility for arising the vascular and cancer disease in the patient.

EFFECT: improved method for assay.

4 cl

The invention relates to medicine, in particular to the creation of a new pharmaceutical compositions containing collagenase microbial origin

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The invention relates to biotechnology and can be used for the development of anti-NPC therapeutic agents

The invention relates to the field of Bioorganic chemistry, namely to a new 6-(moselhotel-L-prolyl - L-arginyl)aminonaphthalene-1-isobutylamino formula:

< / BR>
as a substrate for the determination of thrombin

The invention relates to physico-chemical biology and biotechnology, namely, biological chemistry, molecular biology, Bioorganicheskaya chemistry, and can be used by scientific institutions in the study of the properties of proteins and enzymes
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FIELD: medicine.

SUBSTANCE: invention concerns medicine and Fc-erythropoietin fused protein with improved pharmacokinetics. Invention claims novel sialylated Fc-EPO fused proteins preferably including modification pair in Fc part, as well as in EPO part, showing improved pharmacokinetics. Particularly, Fc-EPO proteins have longer half-life in blood serum and higher efficiency in vivo. Fc-EPO fused proteins synthesised in BHK cells show much longer half-life in blood serum and higher efficiency in vivo than similar Fc-EPO fused proteins obtained in other cell lines, such as NS/0 cells.

EFFECT: improved pharmacokinetic properties of erythropoietin.

23 cl, 14 ex, 6 tbl, 11 dwg

Fused proteins il-7 // 2369616

FIELD: medicine.

SUBSTANCE: there is provided fused protein, including immunoglobulin chain and the molecule IL-7 or its fragment, displaying activity typical to IL-7, that is modified in comparison with IL-7 of wild type, where modification in IL-7 represents amino-acid residues in positions 70 and 91, that are glycated, and amino-acid residue in the position 116 is nonglycated. There is proposed pharmaceutical composition, containing described protein. There is provided the method fro production of described fused protein, including: host cell transformation by DNA, coding specified fused protein IL-7, host cell culture and collection of fused protein IL-7.

EFFECT: invention can be applied for treatment of disorders, accompanied by immune deficiencies.

27 cl, 16 dwg, 1 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: according to the invention there is provided protein fused with albumine comprising two or more tandem-oriented polypeptides GLP-1 or its fragments containing (7-36 A8G)), nucleic acid molecules coding albumine proteins according to the investigation as well as vectors containing the specified nucleic acids, host cells transformed by vectors comprising the specified nucleic acids, methods of albumine protein preparation according to the invention and their application methods. Moreover, the present invention refers to pharmaceutical composition containing fused proteins of albumine, treatment of diseases, disorders and conditions using fused proteins of albumine according to the invention.

EFFECT: improved therapeutic polypeptide stability.

34 cl, 81 ex, 11 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicines, particularly to the use of chimeric peptide VP-22_p16INK4a for epithelial and mesenchymal malignant neoplasms treatment. The claimed chimeric peptide VP-22_p16INK4a contains two amino acid sequences. The first sequence comprises inhibitor of cycline kinases as active fragment p16INK4a as therapeutic agent and the second sequence comprises peptide VP22 of herpes simplex virus as carrier agent to deliver cycline kinase inhibitor into target cells.

EFFECT: enlarging the application range of medicine.

9 dwg

FIELD: medicine.

SUBSTANCE: there is offered method for identification and/or verification of inhibitors of receptor tyrosine kinases that involves application of a new test system which represents a yeast host cell containing an expression vector including a nucleic acid sequence that encodes fused protein essentially consisting of a complete cytoplasmic part of analysed receptor tyrosine kinase and the dimerisation domain and, if necessary, in addition including anchoring sequence for fused protein in a membrane wherein expression of fused protein conduces to termination of cell proliferation. The method provides production of specified host cells being in contact with a candidate compound and identification of inhibitors of tested tyrosine kinase activity as a result of cultivation on the assumption that inhibition of tyrosine kinase activity with a candidate compound causes restoration of proliferation process.

EFFECT: prospected application of the invention is related to development of selective therapeutic, including anticancer, agents.

13 cl, 5 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: proposed is a recombinant single-strand trispecific antibody for treating tumours which express CEA. The said antibody consists of a series of three antibody fragments: anti-CEA-scFv, anti-CD3-scFv and VH CD28-antibody, linked by two intermediate linkers (intermediate linker Fc and intermediate linker HSA). If necessary, a c-myc-mark or (His)6-mark can be added at the C-end. Described is DNA, which codes the antibody, expression vector based on it and E.coli cell, containing the vector.

EFFECT: use of the invention is more beneficial in clinical use compared to bispecific antibodies and known trispecific antibodies, makes easier clearing and expression of an antibody, which can further be used in treating CEA-mediated tumours.

10 cl, 21 dwg, 11 ex

FIELD: medicine.

SUBSTANCE: hybrid protein - human insulin precursor consists of N-end fragment of human gamma-interferon connected through peptide linker with amino acid sequence of human proinsulin. Recombinant human insulin is obtained by cultivation of Escherichia coli JM109/pHINS11 strain-producer, carrying plasmid pHINS11, isolation of inclusion bodies and their dissolving in buffer which contains urea and dithiotreitole. Then hybrid protein re-naturation, sedimentation of admixture compounds, purification of re-naturated hybrid protein by ion-exchanging chromatography, combined fermentative hydrolysis of hybrid protein with tripsin and carbopeptidase B are carried out. At the last stage insulin purification with cation-exchanging chromatography and method of highly efficient reverse phase liquid chromatography are carried out.

EFFECT: simplification of obtaining highly purified recombinant human insulin and increase of its output.

6 cl, 1 dwg, 4 tbl, 5 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: current invention relates to the field of biotechnology and immunology. Proposed is an antibody, specific to the human ED-B. Antibody specified is a molecule in the form of either dimerizated mini-immunoglobulin or IgG1, whose variable region comes from the antibody L19. In case the mini-immunoglobulin variable region L19 is merged with εS2-CH4, then as in the case IgG1, the variable region L19 is merged with the constant domain of IgG1. Conjugates of antibodies with radioisotopes have been discovered. Described is the coding nucleic acid, carrying its host cell, capable of producing antibodies, and method of obtaining antibodies from cells. Discovered is a method of determining the degree of bonding of antibodies, also compositions based on antibodies. Described is the use of antibodies for preparing medicine for treating either damage related to angiogenesis, or for treating tumours. Utilisation of the invention provides antibodies, which possess high accumulating capacity to tumours, improved capability to bonding with radioactive labels and unexpectedly retains immunoreactivity in the plasma, in comparison to scFv L19. Antibody specified can be used in diagnostics and treatment of tumours.

EFFECT: obtaining antibodies which can be used in diagnostics and treatment of tumours.

22 cl, 13 dwg, 8 tbl

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to biotechnology and can be used in biomedicine for producing hyaluronan. Proposal is given of a method of producing hyaluronan, involving culturing Bacillus host cells in conditions, suitable for obtaining hyaluronan, and extraction of the target product from the culture medium. The Bacillus host cell contains a genetic structure, comprising a promoter, functionally active in the given cell, and encoding a region, consisting of a nucleotide sequence, encoding streptococcal hyaluronansynthase (hasA); sequence encoding UDP-glucose-6-dehydrogenase Bacillus (tuaD) or a similar enzyme of streptococcal origin (hasB), and a sequence, encoding bacterial or streptococcal UDP-glucose pyrophosphorylase (gtaB and hasC respectively). Use of the invented method provides for production of considerable quantities of hyaluronan with good examined, nonpathogenic cellular system.

EFFECT: obtaining considerable of hyaluronan with good examined, nonpathogenic cellular system.

15 cl, 45 dwg, 2 tbl, 20 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to genetic engineering, more specifically to chimeric polypeptides, containing an antagonist of growth hormone receptor. The invention can be used in medicine. The binding domain of the growth hormone is modified by substituting glycine amino acid residue in position 120 and is further modified in site 1, where at least one amino acid residue is substituted, which increases affinity of the growth hormone to its binding domain on the growth hormone receptor. The amino acid residue is then conjugated with the ligand-binding domain of the growth hormone receptor, through a peptide linker.

EFFECT: obtaining a highly effective antagonist of the growth hormone receptor with longer half-life, reduced immunogenesity and nontoxicity, compared to known mutant forms.

35 cl, 16 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: vitamin K dependent protein is made by separating a cultivated eukaryotic cell that contains an expressing vector that contains a nucleic acid molecule coding vitamin K dependent protein and associated sequences regulating expression. The associated sequences contain the first promoter and the nucleic acid molecule coding gamma-glutamylcarboxylase, and the second promoter. The first promoter represents a pre-early promoter of human cytomegalovirus (hCMV), and the second promoter is a pre-early promoter SV40. Herewith the expressing relation of vitamin K dependent protein and gamma-glutamylcarboxylase is 10:1 to 250:1.

EFFECT: invention allows for making gamma-carboxylated vitamin K dependent protein in production quantities.

29 cl, 5 dwg, 6 tbl, 7 ex

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