RU2258531C1 - Protein eliciting property of intramembrane aspartate protease, method for its preparing - Google Patents

Abstract

FIELD: biology, biochemistry, science-practical pharmacology, molecular biology.

SUBSTANCE: invention relates to isolated DNA fragment with indicated nucleotide sequence encoding protein that elicits property of intramembrane endoprotease (IMPAS), and to a method for preparing this protein. Invention provides the possibility for development of preparations used for diagnosis of diseases associated with proteolysis of intracellular receptors. Invention provides expanding assortment of agents useful in treatment of different neurological, cardiovascular and other diseases associated with the disturbed proteolysis of proteins.

EFFECT: improved preparing method, valuable medicinal properties of protein.

3 cl, 4 dwg, 1 tbl, 3 ex

Description

The invention relates to the field of molecular biology, scientific and practical pharmacology and biochemistry and relates to new drugs useful in violation of the endoproteolysis of proteins. Protein endoproteolysis is a universal mechanism involved in many cellular processes, hereditary diseases, cell death (necrosis and apoptosis), regenerative processes and the response of cells and the body to infections, cancer cell transformation, neurodegeneration and stress factors. Recently, two previously unknown homologous genes have been discovered: presenilin 1 and presenilin 2 (PS1 and PS2) (Sherrington and Rogaev et al., Nature, 1995; Rogaev et al., Nature, 1995; Rogaev et al., Genomics, 1997), - coding proteins with multiple transmembrane domains, mutations in which lead to the development of an early familial form of Alzheimer's disease, abnormal proteolysis of the APP protein (precursor of β-amyloid), accumulation of insoluble complexes of amyloid plaques in the brain of patients. Mutations in the PS1 and PS2 genes enhanced endoproteolysis (γ-secretase activity) in the APP transmembrane domain (Borchelt et al., 1996; Duff et al., 1996; Lemere et al., 1996; Citron et al., 1997). The first step was taken in the discovery of genes encoding an amino acid sequence similar to presenilins without determining their functions (Grigorenko et al., 2002). The absence of any homology between PS1 and PS2 with known proteases or protease sites, the unprecedented nature of a possible process of endoproteolysis within membranes (not described in eukaryotes) did not allow us to draw an unambiguous conclusion about whether presenilins are γ-secretases with intrammembrane endoprotease activity, or data proteins with multiple transmembrane domains are cofactors necessary for endoproteolysis. Further detection of IMPAS (IMP) genes encoding IMPAS (IMP) proteins with a structure similar to presenilins and also having an intrammembrane (i.e., the proteolysis site is located within the hydrophobic domain of the predicted localization in the membrane structures of the substrate cell) with proteolytic activity, allows us to distinguish a special class of aspartate intram Membrane endoproteases and to study the universal molecular genetic mechanisms of intram Membrane proteolysis in mammals and other eukaryotes.

The following properties determine similar sequences of aspartate transmembrane proteins of different families (presenilins, IMPAS and prepilin peptidases of type IV):

1) transmembrane structural organization (i.e. the presence of multiple hydrophobic, membrane-penetrating domains);

2) the presence of evolutionarily conservative aspartate residues within the membrane domains;

3) the presence of at least three conservative functionally critical sites, which we have defined as:

a) D - GxG D - P xL (broad definition for aspartate intrabranch proteins of different families);

b) ^Y / _F D ^v / _I F ^W / _F V FX3 - 6LG ^I / _F G D ^I / _V 66 P GxxxxxxxR ^Y / _F D - Q P ALLY LV (narrow definition for human IMPAS proteins (hIMP1-hIMP5), possessing 10-92% homology for the full sequence of the protein),

where x is any amino acid, - is the amino acid site between the sites; isolation of groups of similar amino acids: 1-DN, 2-EQ, 3-ST, 4-KR, 5-FYW, 6-LIVM.

The technical result of the claimed invention is the expansion of the arsenal of tools that can be useful in the treatment of various neurological, cardiovascular and other diseases associated with impaired protein proteolysis.

Constructs were obtained in expressing eukaryotic vectors containing human IMPAS genes - hIMP1-hIMP5. The constructs were used to obtain stable and transiently transfected cultures of a number of mammalian cells. Functional analysis of IMPAS genes and proteins included in vitro and in vivo analysis. The in vitro model showed that IMPAS proteins are involved in intram Membrane proteolytic cleavage, which is regulated by inhibitors within membrane proteolysis, as well as mutations in invariant amino acids - potential catalytic sites of IMPAS proteins or sites that regulate proteolysis. In vitro, in the nematode roundworm model, the participation of IMPAS genes in embryonic and postembryonic development and possible lipid-lipoprotein-dependent metabolic processes or cellular signals of metabolic pathways associated with lipid metabolism have been shown.

The invention is illustrated by the following examples.

Example 1. The sequence, design and properties of IMPAS genes.

1) Obtaining and cloning full-sized copies of human cDNA of the IMPAS family.

We have cloned and analyzed five IMPAS human genes:

(1) hIMP1 gene, isoform 1 (SEQ NO1) encoding the protein SEQ NO8;

(2) the hIMP1 gene, isoform 2 (SEQ NO2) encoding the protein SEQ NO9;

(3) hIMP1 gene, isoform 3 (SEQ NO3) encoding the protein SEQ NO10;

(4) hIMP2 gene (SEQ NO4) encoding the protein SEQ NO11;

(5) hIMP3 gene (SEQ NO5) encoding the protein SEQ NO12;

(6) hIMP4 gene (SEQ NO6) encoding the protein SEQ NO13;

(7) hIMP5 gene (SEQ NO7) encoding the protein SEQ NO14.

The following primers were used to clone full-size copies of cDNA of human IMPAS genes:

The nucleotide sequences of alternative hIMP1 gene transcripts (isoforms 1-3) were determined by sequencing cloned RT-PCR products from human hippocampus (Clontech, cDNA). PCR products for cloning full-size copies of human hIMP2-hIMP4 cDNA genes were obtained from human blood cDNA. Human genomic DNA was used to clone the hIMP5 gene.

For cloning, the vectors pcDNA3, pcDNA4 / myc-His B and pcDNA6 / V5-HisA (Invitrogen) used for expression in mammalian cells were used. The obtained PCR fragments and the vector for cloning were restriction with the corresponding endonucleases, ligated among themselves according to the following schemes and sequenced (Fig. 1A-G):

(1) hIMP1 (isoform 1):

1) PCR product (with human blood leukocyte cDNA):

20.1.2 dir - 20.3 rev, restricted to EcoRI, XhoI;

cloned into pcRINA restriction by EcoRI, XhoI

2) PCR product (with plasmid matrix 1)):

20.1 dir — pcRINA4B-20 EcoRI-restricted; XhoI was cloned into the pcRINA4 / myc-HisB vector EcoRI-restricted Xhol.

3) PCR product (with plasmid matrix 1)):

20.1.2dir - R-XHO20, restricted by EcoRI, XhoI was cloned into the pcDNA6 / V5-HisA vector, restricted by EcoRI, XhoI.

(2, 3) hIMP1 (isoforms 2, 3):

PCR products (with human blood leukocyte cDNA):

20.1 dir - 20.3 rev, restricted to EcoRI, XhoI;

cloned into pcRINA restriction by EcoRI, XhoI

(4) hIMP2

1) PCR products (with human blood leukocyte cDNA):

12.1 dir - 12.2 rev, restricted to EcoRI, SphI;

12.3 dir - 12.3 rev, restricted to SphI, XhoI;

cloned into pcRINA restriction by EcoRI, XhoI

2) PCR product (with plasmid matrix 1)):

12NotIdir-12XbaIrev, NotI-restricted, XbaI was cloned into the pcDNA4 / myc-HisB vector, NotI-restricted, XbaI.

(5) hIMP3

1) PCR products (with human blood leukocyte cDNA):

15.1 dir - 15.2 rev, restricted to NotI, Bsp 1407I;

15.3 dir - 15.3 rev, restricted to Bsp1407I, XbaI;

cloned into the pcDNA3 vector restricted by NotI, XbaI.

2) PCR product (with plasmid matrix 1)):

15.1 dir-pc4B-15, NotI-restricted, XhoI was cloned into the pcDNA4 / myc-HisB, NotI-restricted, XhoI.

3) PCR product (with plasmid matrix 1)):

15.1 dir - pc6A-15, NotI-restricted, XhoI was cloned into the pcDNA6 / V5-HisA NotI-restricted, XhoI vector.

(6) hIMP4

1) PCR product (c to human blood leukocyte DNA):

19.1 dir - 19.2 rev was cloned into the pGEM-T easy vector (Promega).

2) PCR product (with human blood leukocyte cDNA):

19.4 dir - 19.4 rev was cloned into the pGEM-T easy vector (Promega).

3) EcoRI-MluI fragment from 1);

PCR product (with human blood leukocyte cDNA): 19.3 dir - 19.3 rev, restricted by MluI, BamHI;

BamHI-NotI fragment of 2);

cloned into pcDNA3 vector restricted by EcoRI, NotI.

4) EcoRI-MluI fragment from 1);

PCR product (with plasmid matrix 3)): 19.3 dir - pc4B-19, restricted to MluI, XhoI;

cloned into pcDNA4 / myc-HisB vector restricted by EcoRI, XhoI.

5) EcoRI-MluI fragment from 1);

PCR product (with plasmid matrix 3)): 19.3 dir - pc6A-19, restricted by MluI, XhoI;

cloned into pcDNA6 / V5-HisA vector restricted by EcoRI, XhoI.

(7) hIMP5

PCR product (with human genomic DNA):

17 NotIdir-17XbaIrev, NotI-restricted, XbaI was cloned into the pcDNA4 / myc-HisB NotI-restricted, XbaI vector.

Processing of plasmid DNA with restriction endonucleases was carried out in buffers recommended by the manufacturer (MBI Fermentas) for 3 hours at a temperature of 37 ° C. The enzyme was inactivated by heating to 65 ° C for 15 min or the restriction mixture was purified on QIAGEN columns according to the recommendations of the manufacturer. The ligation reaction was carried out using phage T4 DNA ligase (MBI Fermentas) in 1x ligase buffer (MBI Fermentas) overnight at 4 ° C. A tube with 50 μl of competent cells of E. coli, strain XL-Blue, stored at -70 ° C, was incubated in an ice bath until completely thawed. A ligase mixture was added to thawed cells and the cells were incubated in an ice bath for 30 minutes. Next, a temperature shock was performed by immersing cells in a 42 ° C water bath for 60 sec and then immediately into ice for 2 min. 500 ml of LB medium was added and the culture was grown for one hour at 37 ° C. The cell suspension was scattered on plates with agarized LB medium containing a selection antibiotic ampicillin at a concentration of 100 μg / ml. The resistance gene for this antibiotic was located in the plasmid vector, which was used for cloning and contained in the ligase mixture. Cups were placed in a thermostat at 37 ° C overnight.

The next day, the clones were checked for insertion by PCR with appropriate primers. 3-4 clones, positive for PPR analysis, were individually seeded in tubes with liquid LB medium containing 100 μg / ml ampicillin. Tubes were incubated at 37 ° C with aeration overnight. Plasmid DNA from E. coli cells was isolated using the QIAprep Spin Miniprep Kit (QIAGEN) according to the manufacturer's instructions. Insert size was confirmed by restriction to cloning sites.

The electrophoretic separation of restriction fragments was carried out in horizontal 1% agarose gels in 1x TAE buffer for 1 hour at a voltage of 100V. Ethidium bromide was added to the gel to a concentration of 0.5 μg / ml. The magnitude of the linear DNA fragments was determined by comparison with marker strips of DNA of a known size obtained by restriction of phage λ with PstI restriction enzyme. The cloned sequence was sequenced.

2) Properties of human IMPAS genes (study of expression at the level of transcription).

A wide representation of polyA + RNA hIMP1-hIMP4 in different human tissues, increased expression in the pancreas and decreased in the muscles and tissues of the heart compared with brain cells and other tissues was shown (Fig. 2).

HIMP5 polyA + RNA is predominantly present in the pancreas. High variability of spliced hIMP1 isoforms was detected in the hippocampus of the human brain. The three most represented isoforms are cloned into the plasmid vectors pcDNA3, pcDNA4 / myc-His B and pcDNA6 / V5-HisA (Invitrogen). The expression of hIMP1-hIMP4 genes has been identified in blood leukocytes and brain cells of patients with Alzheimer's disease (AD), schizophrenia and healthy people. Reduced hIMP1 expression was detected in the neocortex of patients with AD.

A cDNA panel from various human tissues and organs (Clontech) was used as a template for PCR.

The following primer pairs were used for PCR analysis of gene expression:

Example 2

Getting protein. Transformed mammalian cell cultures expressing hIMP1-hIMP5 proteins.

For protein expression, the obtained recombinant constructs with IMPAS genes in the vectors pcDNA3, pcDNA4 / myc-His B and pcDNA6 / V5-HisA (Invitrogen) were used to obtain stable and transiently transfected cultures of a number of mammalian cells (CHO cells, Chinese hamster ovaries, PC 12 pheochromocytoma of rat and human embryonic kidney cells HEK293).

We have shown that:

1) The hIMP1-hIMP5 proteins are found in the cell both in the form of individual full-sized proteins (30-80 kDa) and as part of high-molecular complexes (from 80 kDa and more than 150 kDa). Figure 3 illustrates the exogenous and endogenous expression of IMPAS proteins in mammalian cells by the example of hIMP1, hIMP4, hIMP5 proteins. Figure 3A shows Western hybridization with antibodies to the V5 epitope on lysates of Chinese hamster CHO ovary cells, 3B - PC 12 cells (lane 1) and HEK293 human embryonic kidney cells (lane 3), transfected with a plasmid containing the hIMP1 gene cDNA ( isoform 1) in the pcDNA6 / V5-His A vector (Invitrogen) (controls — FIG. 3A, lane 3; FIG. 3B, lanes 2, 4). Fig. 3B illustrates the expression of hIMP4 protein by Western hybridization with antibodies to the c-myc epitope on HEK293 cell lysates transfected with a plasmid containing hIMP4 in the pcDNA4 / myc-HisB vector. FIG. 3G shows Western hybridization with antibodies to the c-myc epitope on HEK293 cell lysates transfected with a plasmid containing hIMP5 in the pcDNA4 / myc-HisB vector (lane 3 control). Endogenous expression of hIMP1 protein in mammalian cells (PC 12, H4, HEK283, brain cells) is detected using antibodies to the N-terminal portion of the hIMP1 gene (Fig. 3D).

2) The high molecular weight forms of IMPAS proteins are homodimeric complexes that are SDS-resistant under electrophoresis. To prove the existence of IMPAS proteins in the form of homodimeric complexes, cotransfection of mammalian cell cultures with plasmids expressing the hIMP1 protein fused to c-myc and V5 epitopes was performed. It was shown that hIMP1-V5 protein can be immunoprecipitated using antibodies to c-myc and, conversely, hIMP1-c-myc protein using antibodies to V5. Figure 3E shows Western hybridization with antibodies to the V5 epitope on lysates of mammalian cells transfected with plasmids containing hIMP1 in the pcDNA4 / myc-HisB and pcDNA6 / V5-HisA vectors (1 - immunoprecipitate with antibodies to the V5 epitope, 2 - cell lysate, 3 — immunoprecipitate with antibodies to the c-myc epitope, 4 — control serum.) hIMP1-c-myc protein is immunoprecipitated using antibodies on V5 (lane 3).

3) Exogenous expression of hIMP1-hIMP5, more than 10 times higher than the level of endogenous expression of these genes in mammalian cells, did not have a pronounced toxic effect, because stably and transiently transfected cells survived and proliferated.

PC 12 rat pheochromocytoma cells were grown at 37 ° C and 5% CO ₂ on RPMI-1640 medium (ICN, USA) supplemented with 15% fetal calf serum (ICN, USA) and 80 μg / ml gentamicin. CHO cells, Chinese hamster ovaries, human kidney cells HEK293 were cultured on DMEM medium (GibcoBRL, USA) containing 10% fetal bovine serum (GibcoBRL, USA) and 100 units. penicillin / streptomycin act.

Transfection of mammalian cells was carried out using the LipofectAMINE PLUS kit (Gibco BRL), according to the method of the manufacturer. After 24-48 hours, the cells were lysed (to study transient expression) or a selection antibiotic (zeocin 0.1 μg / ml or blasticidin 5 μg / ml) was added to the cell culture to obtain stably transfected lines. To preserve the obtained lines, the cells were frozen. Cells were removed from the surface of the plate or vial in serum and 7% DMSO (Serva) was added, after which 1 ml aliquots of the cells were cooled in the refrigerator at + 4 ° C, then transferred to -70 ° C in kelvinator and finally to liquid nitrogen .

To isolate the total protein, the transformed cells were washed twice with cold PBS buffer before lysis, lysed in a modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM sodium chloride, 1 mm EDTA, 1 tablet of a “cocktail” of Complete, Mini, EDTA-free inhibitors per 10 ml of buffer (Roche) for one hour at 4 ° C, centrifuged at> 10,000 rpm for 15 minutes at 4 ° C. The supernatant was transferred to a new tube, protein concentration was determined and used for gel application. Electrophoresis was performed on 6-10% SDS polyacrylamide gels. After electrophoretic separation, the proteins were transferred onto a PVDF membrane. To detect proteins on the blot, an ECL reagent kit (Amersham Pharmacia Biotech), primary antibodies to the N-terminal part of PS1 (ac. 1-92), and antibodies to the first 17 ac. The N-terminal portion of the hIMP1 gene, commercial antibodies to V5 epitopes, c-myc, and the corresponding secondary antibodies.

Example 3

Properties of IMPAS Proteins:

1) The property of IMPAS proteins as aspartate intram Membrane endoproteases has been shown by the example of binding and intram Membrane proteolytic cleavage of the PS1 substrate hIMP1 protein.

It has been shown that

1) hIMP1 firmly binds to the full-sized unprocessed form of PS1 (PSIholo) and the C-terminal processed fragment of PS1 (PS1 CTF). Fig. 4A shows the immunoprecipitation with antibodies (I.P. AB) to hIMPI (to the N-terminal portion of hIMP1 or to the C-terminal epitope of c-myc) of the full-length unprocessed form of PS1 (PS1 holo) and the C-terminal processed fragment of PS1 (PS1 CTF). In reverse experiments, hIMP1 binds to antibodies to the N-terminal portion of presenilin 1 (PS1 NTF). PI1, PI2 - control sera.

2) Full-sized PS1 proteins, upon cotransfection with wild-type hIMP1, undergo proteolytic cleavage with the formation of proteolysis products (Fig. 4B, 1 — full-length PS1 protein (PS1 holo); 2 — proteolytic cleavage product), the proteolysis site is located in the X hydrophobic domain PS 1 wild-type man. FIG. 4B shows Western hybridization with antibodies to the N-terminal portion of PS1 (NTF PS1) on HEK293 cell lysates transfected with various forms of PS1 and hIMP1 or PS1 and pcDNA4 (control vector).

3) To prove that IMPAS genes are aspartic proteases:

a) mutations were introduced into the catalytically active sites of hIMP1 - aspartate residues (D219A, D265A), as well as into the conserved proline residue (P317L). These mutations inhibited the proteolytic cleavage of PS1 by hIMP1 protein (FIG. 4B);

b) A mutation at the non-catalytically active site of hIMP1 G264A did not alter the proteolytic activity of hIMPI against PS1 substrate (Fig. 4B);

c) The well-known inhibitor of intrabranch aspartic proteases L-685,458 stopped the proteolytic cleavage of PS1 hIMP1 protein. DMSO was added to control cells (without L-685,458), which was used to dilute the inhibitor (Fig. 4B).

HEK293 cells were transfected with constructs containing various forms of the hIMP1 and PS1 genes, lysed after 48 hours and applied to electrophoresis under the conditions described above. L-685,458 inhibitor was used at a concentration of 7.5 μm / μl and was added to the medium when changing medium after transfection was added for 24 hours. Western hybridization was performed with antibodies to the N-terminal portion of PS1.

To study the binding of hIMP1 and PS1 proteins, cotransfection of hIMP1 was carried out in the pcDNA4 / myc-HisB and PS1 plasmid vector. Cell lysates were immunoprecipitated with various antibodies to hIMP1 (to the N-terminal part of hIMP1 or to the C-terminal epitope of hIMP1 c-myc) or antibodies to the N-terminal part of PS1 using ProteinA-Sepharose CL-4B according to the protocol of the manufacturer (Amersham Pharmacia) . Immunoprecipitates were used for Western hybridization with antibodies to PS1 and hIMP1.

2) The participation of IMPAS proteins in embryonic development and metabolic pathways associated with lipid metabolism.

To determine the functions of the IMPAS genes and the proteins encoded by them in the whole organism, the hIMP1 gene ortholog was inactivated in Caenorhabditis elegans roundworm and the phenotypes studied. Inhibition of the hIMP1 (imp-2) ortholog gene caused embryonic death, stunted growth of larvae, impaired coordination of movement, and death of worms at all stages of morphogenesis as a result of impaired molting processes. (Table 1). The observed changes phenocopied the developmental defects of C. elegans with cholesterol deficiency in the medium, as well as with the inhibition of a number of genes associated with lipid metabolism or steroid-dependent signal transduction. So, we observed the most similar phenotype in parallel experiments on the inactivation of the megalin / gp330 gene homolog in the nematode (lrp-1) (Table 1).

IMPAS activity was inhibited by the interference of double-stranded RNA (dsRNAi, dsRNAi) by microinjection of double-stranded RNA (dsRNA) into the worm body cavity, and also using dsRNA producing E. coli cells as food. The cultivation of the worms was carried out according to a standard technique (Brenner, 1974). Constructs containing imp-2 (yk671a5) cDNA as well as lrp-1 (yk260f8) genes in the pBluescriptSK (-) vector were obtained from Y. Kohara (Genome Biology Lab, National Institute of Genetics, Mishima). Injection dsRNA was synthesized using the Ambion MEGAscript® T7 Transcription Kit reagent kit according to the manufacturer's method. Purified PCR product of the imp-2 gene cDNA, with a T7 primer sequence at both ends, was used as a template. The resulting dsRNA was diluted to the desired concentration (1-5 μg / μl) and injected into the body cavity of the worm. 4-6 hours after the injection, the worms were individually transferred to fresh cups. Over the next two to three days, an analysis of F1 offspring was performed.

For dsRNAs using dsRNA producing E. coli cells as food, imp-2 cDNA was cloned at EcoRI, XhoI sites into vector L4440. This vector contains two T7 promoters located on different DNA chains and at different ends of the polylinker sequence, which makes it possible to use the L4440 vector, with the gene cloned into it, for producing dsRNA in E. coli cells. The PCR product corresponding to the lrp-1 DNA fragment obtained on the yk260f8 matrix with the primers gpRNAdir-TTTTGGATCC-GCCGTACTTGCTCTCCATTC and gpRNArev-TTTTCTCGAG-CCATCCAATCGACATTTTCC was cloned into the L44H vector at the X44I site at 4444 E. coli HT115 cells, transformed with constructs imp-2 / L444Q and lrp-11 L4440, were grown in 1 L of LB medium containing 100 μg / ml ampicillin and 20 μg / ml tetracycline to a density of OD (600) ~ 1. Then the bacteria were centrifuged at 4 ° C, 7.000-10.000 rpm for 15 minutes The precipitate was dissolved in 50 ml of LB medium containing 60 μg / ml ampicillin, 7% DMSO, packaged in 5 ml and stored aliquots at -70 ° C. For use in the experiment, a fresh aliquot was taken each time, thawed at room temperature, 100 μl IPTG (200 mg / ml), 200 μl ampicillin (50 mg / ml) were added and bacteria were seeded onto agar plates. A synchronized worm population at stage L1 was placed on plates with E. coli producing dsRNA. The phenotype of worms and F1 offspring were analyzed for the next 2-3 days. The studied worms were placed on agar glass in M9 buffer and covered with coverslip. The phenotype was analyzed on a microscope with Nomarsky optics at a magnification of x 1000-2000.

It was shown that IMPAS proteins are necessary for the normal development of eukaryotes and are involved in important metabolic pathways associated with lipid metabolism and membrane plasticity.

Thus, recombinant vector systems containing IMPAS genes, a protein encoded by them, purified or synthesized antibodies, peptides or polypeptides can be used to study new proteolytic mechanisms in vivo and in vitro, to develop diagnostics and treatment of diseases associated with proteolysis of intracellular receptors or proteins with hydrophobic domains, accumulation of sparingly soluble peptides containing hydrophobic sites (for example, with Alzheimer's disease) or protein proteolysis products that affect lipid metabolism men, structural properties of membranes, signal transmission between cells, for example, in neuropsychiatric, cardiovascular diseases and cancer.

List of references

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Claims (3)

1. The selected DNA fragment encoding a protein having the property of aspartate intrammembrane endoprotease, wherein the DNA fragment has a nucleotide sequence selected from the group of SEQ ID NO1-NO7. 2. A protein having the property of aspartic intram Membrane endoprotease encoded by the DNA fragment according to claim 1 and characterized by an amino acid sequence selected from the group of SEQ ID NO8-NO14. 3. The method for producing a protein according to claim 2, comprising cultivating host mammalian cells containing recombinant DNA with a fragment according to claim 1, under conditions that allow the expression of said DNA and the isolation of an expressed protein having the properties of aspartate intramembrane endoprotease.

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