WO1992013080A1 - Microsomal endopeptidase - Google Patents

Abstract

Unique microsomal endopeptidases are discussed. The endopeptidases are useful in enhancing the processing of proteins that are substrates for the endopeptidase in vivo and in vitro. The microsomal endopeptidase is characterized by (a) a molecular weight of 65,000 ± 3,000 daltons by SDS-PAGE under reducing conditions; (b) cleaves proteins C-terminal to a basic amino acid residue; and (c) retains at least 50 % activity at pH 7.0-10.0. The endopeptidases may also be used to cleave fusion proteins in vitro. DNA sequences encoding the endopeptidases and host cells transfected or transformed to express an endopeptidase are also disclosed.

Description

Description

MICROSOMAL ENDOPEPTIDASE

Technical Field

The present invention is directed generally toward the production of proteins, and more specifically, to the use of DNA sequences encoding an endopeptidase in the efficient processing of proteins.

Background of the Invention

Generally, proteins that transit the secretory apparatus in eukaryotes are initially synthesized as larger precursor polypeptides. In addition to signal peptide cleavage upon translocation into the endoplasmic recticulum (Blobel and Dobberstein, J. Cell Biol. 67:835- 851, 1975; von Heijne, J. Mol. Biol. 173.243-251. 1984), many polypeptides require further proteolytic processing for their full maturation and, in many cases, for their biological activity. Such processing steps include specific proteolytic cleavage, subunit polymerization, disulfide bond formation, post-translational or co- translational modification of certain amino acids, and glyσosylation. One group of proteins that undergo extensive post-translational processing are the vitamin K-dependent proteins, including factor VII, factor IX, factor X, prothrombin and protein C. These vitamin K-dependent proteins are synthesized in the liver as single chain precursors which are processed at their amino termini prior to their secretion into the blood stream. For example, factor X is synthesized with forty additional amino acid residues at its N-terminus. Proteolytic cleavage by a signal peptidase is believed to first remove the N-terminal hydrophobic signal peptide of 23 amino acids, and subsequent cleavage after the Arg residue at the -1 position results in the removal of a propeptide consisting of 17 amino acids. Frequently, as in the case of factor VII, factor IX, prothrombin, protein C and many other proteins, cleavage occurs at sites marked by paired basic amino acid residues, primarily Lys-Arg and Arg-Arg. A prototypic proprotein endopeptidase is the yeast enzyme KEX2. The KEX2 gene encodes an endopeptidase that functions late in the secretory pathway of S_. cerevisiae to cleave polypeptide chains of prepro- killer toxin and prepro-alpha-factor at the paired basic amino acid sequences Lys-Arg and Arg-Arg. In addition to its use in mediating the processing of proinsulin and proalbu in expressed in yeast (Thim et al., Proc. Natl. Acad. Sci. fUSA) 82-6766-6700, 1986; Sleep et al. , Biotechnology £.:42-46, 1990), the KEX2 gene product has also been used for the in vivo processing of protein C precursors (see Kumar et al., U.S. Serial No. 07/392,861; Foster et al., U.S. Serial No. 07/456,092 and Mulvihill et al., EP 319,944, herein incorporated by re erence) . More recently, the processing efficiency of pro¬ ven Willebrand factor (v F) at its natural cleavage site (Lys-Arg) has been increased through the use of another endopeptidase, the fur gene product (Wise et al., Proc. Natl. Acad. Sci. fUSAΪ 87:9378-9382_f 1990). In addition to the use of gene products, such as

KEX2 and fur, in the in vivo processing of protein precursors, certain cleaving enzymes have been used for the large scale in vitro conversion of prohormones. These prohormones, produced through bacterial cloning, are treated with a preparation of an enzyme having specificity for cleaving the prohor one only at the site of paired basic amino acid residues (see U.S. Serial No. 06/874,633, by Y. P. Loh) . Proteolytic enzymes also have a broad range of industrial uses in such fields as food processing, wine making, brewing and detergent formulation. Although a number of enzymes have been shown or have been proposed to be involved in the processing of proteins, there is a need in the art for additional endopeptidases that may be used to enhance the processing of proteins in vivo and in vitro. The present invention fulfills this need, and further provides other related advantages.

Summary of Invention Briefly stated, the present invention provides unique microsomal endopeptidases characterized by: a) a molecular weight of 65,000 ± 3,000 daltons by SDS-PAGE under reducing^' conditions; b) cleaves proteins C-terminal to a basic amino acid residue; and c) the retention of at least 50% activity at pH 7.0-10.0. In one embodiment, the endopeptidase exhibits maximum activity at pH 8.7, and exhibits essentially no activity at below pH 5.0.

The present invention also provides an isolated DNA molecule encoding an endopeptidase as described above. Such a DNA molecule is useful in enhancing the processing of proteins which are -substrates for the endopeptidase, including factor VII, factor IX, factor X, protein C, prothrombin, protein S, protein Z, bone gla protein, and tissue plasminogen activator (t-PA) . Expression vectors comprising such a DNA molecule, and host cells transfected or transformed to express an endopeptidase as described above are also disclosed.

Within another aspect of the present invention, a host cell transfected or transformed to express a first DNA sequence encoding a protein, and a second DNA sequence encoding a microsomal endopeptidase as described above is also provided. Within a preferred embodiment, the host cell is a mammalian cell.

Within a related aspect of the present invention, a method for producing a protein is provided, comprising (a) introducing into a host cell a DNA sequence encoding an endopeptidase as described above and a DNA sequence encoding a protein that is a substrate for the endopeptidase; (b) culturing the host cell under conditions which allow the DNA sequences to be expressed; and (c) isolating the protein from the host cell. 5 These and other aspects of the invention will become evident upon reference to the following detailed description and attached drawings.

Brief Description of the Drawings 10 Figure l diagrammatically illustrates a purification scheme for a microsomal endopeptidase of the present invention.

Figure 2 depicts selected peptide substrates (SEQ ID NOS: 1-5) for a microsomal endopeptidase of the 15 present invention.

Figure 3 graphically depicts the relative activity of a microsomal endopeptidase of the present invention at selected pH conditions.

Figure 4 graphically depicts the relative 20. reaction rates for peptides (SEQ ID NOS: 2, 6, 7) having different amino acids at the -4 position.

Figures 5A-5C depict the preliminary sequence analysis of a partial rabbit endopeptidase cDNA (SEQ ID NOS. 12 and 13) . Numbers to the left indicate nucleotide 5 position. X indicates an undetermined amino acid.

Detailed Description of the Invention

Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth 0 definitions of certain terms to be used hereinafter.

Transfection or Transformation: The process of introducing cloned DNA into host cells. Transfection refers to inserting DNA into mammalian cell lines. The process of inserting DNA into fungal and bacterial cells 5 is known as transformation. A number of transfection and transformation procedures are well known in the art. As noted above, many proteins require proteolytic processing for their full maturation and, in some cases, for biological activity. However, due to inefficient or improper processing by the host cell, often only low levels of protein or activity are achieved. In addition, recombinant proteins are sometimes produced as fusion proteins, requiring proteolytic processing subsequent to expression. The present invention, through the unique endopeptidases described herein, is useful in enhancing the processing of proteins in vivo and in vitro in these and other applications.

As diagrammatically illustrated in Figure 1, an endopeptidase of the present invention may be purified by homogenization of liver tissue or liver-derived cell lines, from rabbit,, mouse, rat, primate and other suitable sources. The homogenate is centrifuged to obtain a microsomal fraction, which is then extracted with a non- ionic detergent. It is preferred to utilize a non- aromatic detergent (i.e., a detergent that does not absorb at 280 n and interfere with protein detection) , such as Lubrol PX. This extract is applied to an anion-exchange chromatographic column, packed with a derivatized agarose medium, such as DEAE-Sepharose. Elution is effected using a linear salt gradient, and active fractions are pooled and dialyzed. Further purification is achieved through multiple chro atography steps, including affinity chromatography, ion exchange chromatography, and size- exclusion chromatography.

In a preferred embodiment, the microsomal fraction is applied to an affinity chromatography column loaded with a "dye agarose" medium such as reactive Green- 19 agarose. Elution from this column is effected by a step-wise salt gradient, and flow-through fractions are pooled. The pooled fractions are then subjected to affinity chromatography using Benzamidine Agarose (Sigma) , and active fractions are obtained during a step-wise salt gradient elution. These fractions are then pooled and dialyzed, followed by application of the pooled fraction to an additional step of anion-exchange chromatography. Active fractions are then pooled and applied to a hydroxylapatite column, and eluted with a linear salt gradient. The active fractions are pooled and loaded on a chromatography column packed with an agarose derivative that binds sulfur-containing compounds, such as an organomercurial-agarose column (Bio-Rad) . Elution is then effected with an appropriate buffer, e.g., buffer containing β-mercaptoethanol. Active fractions are pooled and concentrated, and the concentrated pool is applied to a chromatography column similar in function to the Sephacryl S-200 column (Pharmacia) . The purification procedure described herein results in about a 1400-fold purification of enzyme activity with a yield of 1% from the Lubrol extract of rabbit liver.

Genomic or cDNA sequences encoding the endopeptidase may be obtained from libraries prepared from cells and tissues according to known procedures. If partial clones are obtained, it is necessary to join them in proper reading frame to produce a full length clone, using such techniques as endonuclease cleavage, ligation and loopout mutagenesis. For instance, the isolated endopeptidase could be digested and fragments sequenced to provide information for probe - design. Using oligonucleotide probes derived from the endopeptidase amino acid sequence, generally of at least about fourteen nucleotides and up to twenty-five or more nucleotides in length, DNA sequences encoding the endopeptidase may be obtained. Thus the present invention includes DNA molecules comprising from about 14 to 2128 nucleotides of the sequence shown in Figure 5 (SEQ ID NO:12) from nucleotide 11 to 2138. A preferred cloning method makes use of the polymerase chain reaction (PCR) technology disclosed in U.S. Patent Nos. 4,683,195 and 4,683,202. Amino acid sequence information is used to generate sets of degenerate oligonucleotide primers. The primers are then used to amplify endopeptidase DNA sequences in the PCR reaction. If desired, PCR-generated clones may be used as probes to isolate cDNA or genomic clones. Preferred sources of mRNA for generating cDNA clones include liver tissue and liver-derived cell lines, such as the HepG2 cell line (ATCC HB 8065) . Suitable libraries may be prepared according to conventional methods or may be obtained from commercial suppliers (e.g., Stratagene Cloning Systems, La Jolla, CA) . Genomic and cDNA sequences encoding the endopeptidase may also be isolated using the expression cloning method of Young and Davis Proc. Natl. Acad. Sci. (USA. 8j)-_L194--1198, 1983. Briefly, using the expression vector λ gtll, foreign DNA is inserted into the β- galactosidase gene lacZ. and synthesis of hybrid proteins from a library of clones is promoted. Phage containing inserts are distinguished by their inability to produce blue plaques on lacZ-hosts on X-Gal plates. Screening of endopeptidase-producing clones is facilitated by the use of the amber mutation S100, which enables accumulation of large quantities of phage products in the absence of lysis. Production of detectable quantities of endopeptidase upon induction is further enhanced by using host strains that are defective in protein degradation (Ion mutants) . Efficient lysogeny is best obtained using hf1A (high frequency lysogeny) mutants of E. coli.

Examination of lysogens for endopeptidase production incorporates standard plating and nitrocellulose filter replica lifts. Filters are washed and treated with DNase to increase endopeptidase availability,, and incubated in BSA/buffer to reduce non¬ specific protein binding. Filters are incubated with IgG diluted in buffer, and then washed. Bound antibody is reacted with ¹²⁵I-labeled protein A, and filters are autoradiographed to detect signals corresponding to endopeptidase-producing colonies. Antibodies useful for screening "clones may be produced by conventional procedures of immunization and purification. Briefly, a purified or partially purified endopeptidase is administered to an animal such as a mouse, rat, rabbit or goat in an amount sufficient to cause an immune response. It is preferred to administer the endopeptidase in combination with an adjuvant, such as Freund's adjuvant, in order to enhance the immune response. Although a single injection of the endopeptidase may be sufficient to induce antibody production in the animal, it is generally preferred to administer a large initial injection followed by one or more booster injections over a period-of several weeks to several months. (See, e.g., Hurrell, J.G.R. (ed.), Monoclonal Hvbridoma Antibodies: Techniques and Application. CRC Press Inc., Boca Raton, FL, 1982, which is incorporated herein by reference.) Blood is then collected from the animal -and clotted, and antibodies are isolated from the serum using conventional techniques such as salt precipitation, ion exchange chromatography, affinity chromatography or high performance liquid chromatography.

For expression, a DNA sequence encoding the endopeptidase is inserted into a suitable expression vector, which in turn is used to transform or transfect appropriate host cells for expression. -Expression vectors for use in carrying out the present invention will comprise a promoter capable of directing the transcription of a cloned DNA and a transcriptional terminator, operably linked with the sequence encoding the endopeptidase so as to produce a continuously transcribable gene sequence which produces sequences in reading frame and continuously translated to produce the enzyme.

Host cells for use in practicing the present invention include mammalian, avian, plant, insect, bacterial and fungal cells, but preferably eukaryotic cells. Preferred eukaryotic cells include cultured mammalian cell lines (e.g., rodent or human cell lines) and fungal cells, including species of yeast (e.g., Saccharomvces spp., particularly S . cerevisiae.

Schizosaccharomyces spp. , or Kluyveromyces spp.) or filamentous fungi (e.g., Aspergillus spp., Neurosoora spp.). Methods for producing recombinant proteins in a variety of prokaryotic and eukaryotic host cells are generally known in the art. In general, a host cell line will be selected on the basis of its ability to produce the protein of interest at a high level or its ability to carry out at least some of the processing steps necessary for the biological activity of the protein. In this way, the number of cloned DNA sequences which must be transfected into the host cell line may be minimized and overall -yield of biologically active protein may be maximized.

Preferred cultured mammalian cells for use in the present invention include the COS-1 (ATCC CRL 1650) , BHK (ATCC CRL 10314) and BALB/c 3T3 (ATCC CRL 163) cell lines. In addition, a number of other mammalian cell lines may be used within the present invention, including 293 (ATCC CRL 1573), Rat Hep I (ATCC CRL 1600), Rat Hep II (ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC CCL 75.1), Human hepatoma (ATCC HTB-52) , Hep G2 (ATCC HB 8065), Mouse liver (ATCC CCL 29.1), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. fUSA^ 77:4216-4220. 1980).

Mammalian expression vectors for use in carrying out the present invention will include a promoter capable of directing the transcription of a cloned gene or cDNA. Preferred promoters include viral promoters and cellular promoters. Viral promoters include the immediate early cytomegalovirus promoter (Boshart et al.. Cell 41:521-530,. 1985), the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981), and the major late promoter from Adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2:1304- 1319, 1982). Cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al., U.S. Patent No. 4,579,821), a mouse V_κ promoter (Bergman et al., Proc. Natl. Acad. Sci. fUSA) £1:7041-7045, 1983; Grant et al., Nuc. Acids. Res. JL5_:5496, 1987) and a mouse Vg promoter (Loh et al.. Cell 22:85-93, 1983). Also contained in the expression vectors is a polyadenylation signal located downstream of the coding sequence of interest. Polyadenylation signals from SV40 (Kaufman and Sharp, ibid.) , the polyadenylation signal from the Adenovirus 5 E1B region and the human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res. .9:3719-3730, 1981) may be used. Vectors can also include enhancer sequences, such as the -SV40 enhancer and the mouse μ enhancer (Gillies, Cell 3_:717-728, 1983) . Expression vectors may also include sequences encoding -the adenovirus VA RNAs. Suitable vectors can be obtained from commercial sources (e.g., Stratagene, LaJolla, CA) .

Cloned DNA sequences may be introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al.. Cell 14.:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 2:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982) , or DEAE-dextran mediated transfection (Ausubel et al. (eds.), Current Protocols in Molecular Biology. John Wiley and Sons, Inc., NY, 1987), which are incorporated herein by reference. To identify cells that have stably integrated the cloned DNA, a selectable marker is generally introduced into the cells along with the gene or cDNA of interest. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker. Preferred amplifiable selectable markers are the DHFR gene and the neomycin resistance gene. Selectable markers are reviewed by Thilly (Mammalian Cell Technology. Butterworth Publishers, Stoneham, MA, which is incorporated herein by reference) . The choice of selectable markers is well within the level of ordinary skill in the art.

Selectable markers may be introduced into the cell on a separate vector at the same time as the endopeptidase sequence, or they may be introduced on the same vector. If on the same vector, the selectable marker and the endopeptidase sequence may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, Levinson and Si onsen, U.S. Patent No. 4,713,339). It may also be advantageous to add additional DNA, known as "carrier DNA" to the mixture which is introduced into the cells. Transfected mammalian cells are allowed to grow for a period of time, typically 1-2 days, to begin expressing the DNA sequence(s) of interest. Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable selectable marker the drug concentration may be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels. Cells expressing the introduced sequences are selected and screened for production of the protein of interest in the desired form or at the desired level. Cells which satisfy these criteria may then be cloned and scaled up for production.

Other eukaryotic cells, including yeast and filamentous fungi, may also be used within the present invention. These lower eukaryotic hosts provide certain advantages over mammalian cell lines, including ease and economy of culturing and existing industrial fermentation capacity. By manipulating cells as described herein, fungal cells and other eukaryotic cells capable of expressing cloned DNA sequences can be engineered to produce the endopeptidase. In addition, promoters, terminators and methods for introducing expression vectors encoding the endopeptidase into plant, avian and insect cells are well known in the art. The use of baculoviruses, for example, as vectors for expressing heterologous DNA sequences in insect cells has been reviewed by Atkinson et al. fPestic. Sci. 28:215-224, 1990) . The use of Agrobacterium rhizogenes as vectors for expressing genes in plant cells has been reviewed by Sinkar et al. (J. Biocsci. (Bangalore) 11:47-58, 1987). Host cells containing DNA constructs of the present invention are then cultured to express the DNA encoding the endopeptidase. The cells are cultured according to standard methods in a culture medium containing nutrients required for growth of the chosen host cells. A variety of suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals, as well as other components, e.g., growth factors or serum, that may be required by the particular host cells. The growth medium will generally select for cells containing the DNA construct(s) by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co- transfected with the DNA construct. The microsomal endopeptidase of the present invention is useful in a variety of environments, including the processing of proteins in vivo and in vitro, and the cleavage of fusion proteins in vitro. Within one embodiment, the endopeptidase is purified from recombinant cell cultures by separating media from cells, and purifying the protein from media essentially as described in Example 1. If protein is produced cytoplasmically, cells are lysed and protein is recovered from a cleared lysate. Within another embodiment, DNA sequences encoding an endopeptidase as described herein may be used to enhance the processing of proteins that are substrates for the endopeptidase. Such proteins will include a cleavage site with a basic amino acid at the -1 position, and will usually include a basic residue at positions -2 and/or -4. Potential substrates for the endopeptidase of the present invention include the propeptide cleavage sites of coagulation factors VII, IX and X, protein C, activated protein C, protein S, protein Z, bone gla protein, tissue plasminogen activator (t-PA) , and analogs and derivatives of these proteins, as well as the internal processing sites in factor X, protein C, complement C3, complement C4 and complement C5. DNA sequences encoding the endopeptidase and a protein that is a substrate for the endopeptidase may be introduced into a host cell on the same vector or on different vectors. Use of a single vector with one selectable marker minimizes problems which can result from marker instability. Genes or cDNAs on a vector may be controlled by separate promoters or by a single promoter. In the latter arrangement, which gives rise to a polycistronic message, the genes or cDNAs will be separated by translational stop and start signals. Two or more vectors may, of course, be used whenever co- expression of the endopeptidase and a substrate protein is desired. When using multiple vectors, it is preferred to first introduce the DNA sequence encoding the endopeptidase, select - for cells expressing it (as discussed above) , and then introduce the DNA sequence encoding the substrate protein.

The endopeptidase of the present invention may also be used to cleave fusion proteins from a solid support, such as an affinity column, and therefore aid in the purification of such proteins (see Hopp et al.. Bio/Technology 6.:1204, 1988) . Briefly, through use of an appropriate oligonucleotide (which encodes a substrate for the endopeptidase) that has been attached to a cDNA encoding the desired protein, a cleavable fusion protein product is generated. The fusion protein is then passed over an affinity column which is specific for the substrate marker encoded by the oligonucleotide. Suitable affinity columns include those having an antibody or binding protein specific for the substrate marker. The protein is then bound to the column through the substrate marker, and unbound material removed by washing. Subsequently, the desired protein may be obtained through treatment of the fusion protein with an endopeptidase as described herein, the endopeptidase cleaving the fusion protein at the appropriate site. Alternatively, the endopeptidase of the present invention may be used in any environment requiring cleavage of substrate proteins, including in vitro activation of clotting factors or industrial applications in which a substrate protein is to be degraded. The following examples are offered by way of illustration, not by way of limitation.

EXAMPLES Example 1

Enzyme Purification

Peptidase activity was assayed using synthetic peptides which mimic the processing site of prothrombin. The NH2""^termi^nal amino group of the peptide was acetylated. Each protein fraction and the peptide (15 nmol) were mixed in 0.1 M sodium borate, pH 9.0, 0.1 mM C0CI2, 0.1% Lubrol PX (Sigma Chemical Co., St. Louis, MO), and incubated at 37°C for 15 to 30 minutes. The reaction was stopped by addition of TCA. After the proteins were precipitated, fluorescamine (200 μl of a 0.3 mg/mL solution in acetone) was added to measure the amount of peptide hydrolyzed. Fluorescamine reacts with the new free amino group to produce a fluorescent chromophore under alkaline conditions. It can be quantitatively measured by comparing to a standard curve of the peptide NH2-A-N-S-F-L-COOH. Fluorescence was detected using a Perkin-Elmer LS5 fluorescence photometer with excitation at 390 nm and emission at 475 nm.

Initial isolation of the enzyme is diagrammed in Figure 1. Throughout purification, protein peaks were detected by monitoring absorbance at 280 nm and peptidase activity. Eight hundred grams of rabbit liver was homogenized with a Teflon^® pestle in 2 liters of 20 mM Bis-tris buffer, pH 7.0, containing 0.25 M sucrose. The homogenate was centrifuged at 2000 x g for 10 minutes and the supernatant (approximately 1,500 mL) was further centrifuged at 10,000 x g for 15 minutes. The resulting supernatant (approximately 1,300 mL) was then centrifuged at 110,000 x g -for one hour. The precipitate was subsequently resuspended in the same buffer, and centrifuged at 110,000 x g for an additional hour. The wash step was repeated and the microsome fraction was recovered in the pellet. The endopeptidase activity was extracted with 1% Lubrol PX (Sigma Chemical Co. , St. Louis, MO), with stirring for 1 hour at 4°C. The extract was then centrifuged at 110,000 x g for 1 hour, and the supernatant was recovered. The activity from the microsomal extract was metal ion dependent, since it was totally inhibited by 1,10-phenanthroline. Therefore, the metal dependent activity was measured at each chromatography step.

The supernatant from the Lubrol extract of the microsome fraction (approximately 700 mL) was applied to a DEAE-Sepharose CL-6B column (Pharmacia) equilibrated with 20 mM Bis-tris, pH 7.0 containing 0.08 M NaCl, 0.1% Lubrol PX and 100 μm C0CI2 and the column was eluted with a linear gradient of 0.08 to 0.6 M NaCl. Active fractions were pooled and dialyzed against 20 mM Bis-trif?, pH 7.0 containing 0.03 M NaCl, 0.1% Lubrol PX, and 100 μM CoCl . The active fraction was then applied to reactive Green-19 Agarose (Sigma) and flow-through fractions were pooled.

The pooled fraction was then applied to a benzamidine-Agarose column (Sigma) and the active fractions were obtained with a step-wise elution of 0.02 M NaCl in 20 mM Bis-tris, 0.1% Lubrol PX, 100 μM CoCl₂, pH 7.0. The activity of the endopeptidase was not inhibited by benzamidine, but weakly binds to benzamidine- Agarose. Many tightly bound proteins can be efficiently removed by this chromatography step.

The active fractions were pooled and dialyzed against 20 mM Bis-tris, pH 7.0 containing 0.08 M NaCl, 0.1% Lubrol PX and 100 μm CoCl₂ and applied to a second DEAE-Sepharose CL-6B column. Proteins were eluted with a linear gradient of 0.08 to 0.5 M NaCl in the same buffer.

Enzyme fractions were pooled and applied to a hydroxylapatite column equilibrated with 45 mM sodium phosphate buffer, pH 7.0 containing 0.1 M NaCl, 0.1% Lubrol and 100 μm CoCl2* ^Tne column was eluted with a linear gradient of 45 mM to 150 mM sodium phosphate.

The active fractions were pooled and immediately applied to an organomercurial-agarose column (Bio-Rad Laboratories, Richmond, CA) equilibrated with 20 mM Bis- tris, pH 7.0 containing 0.5 M NaCl and 0.1% Lubrol PX. -The column was washed with the equilibration buffer and eluted with 1 mM J-mercaptoethanol in the same buffer. Enzyme-containing fractions (detected by activity assay) were pooled and concentrated using a Speed-Vac (Savant) . The concentrated pool was applied to a Sephacryl S-200 column (Pharmacia) equilibrated with 20 mM Bis-tris, pH 7.0 containing 0.5 M NaCl and 1 mM J-mercaptoethanol. Activity was eluted with an apparent Mr of 67,000 (equivalent to BSA) . The S-200 peak was analyzed by SDS-PAGE under reducing conditions. The enzyme fraction gave a major band with an apparent Mr of 65,000. Faint bands with Mr of 57,000 and 48,000 were also observed.

The purification procedure described above resulted in about 1400-fold purification of enzyme activity with a yield of 1% from the Lubrol extract of rabbit liver. Purification and yield after each step are presented in Table 1. The protein preparation was about 90% pure.

TABLE 1 PURIFICATION OF A MICROSOMAL ENDOPEPTIDASE

Lubrol Extract

DEAE - 1 C

Green 19- Agarose

Benzamidine-Agarose

DEAE - II

Hydroxylapatite

Organomercurial-Agarose

Sephacryl S-200

Protein was determined by BCA. Activity is expressed in units of n mol of peptide hydrolysis per min at 37 °C.

Example 2 Enzvme Characterization

The purified enzyme readily cleaved the processing sites of prothrombin (SEQ ID NO:l), factor IX (SEQ ID NO:2), protein Z (SEQ ID NO:2), protein C (SEQ ID NO:3), factor VII (SEQ ID NO:4) and factor X (SEQ ID NO:5) (see Figure 2) . Peptides were synthesized using an Applied BioSystems (Foster City, CA) solid phase synthesizer. Fifteen nmol of the peptide Ac-ARVRRANSFL (SEQ

ID NO:l) (derived from the prothrombin sequence) was mixed with 4 pmol of enzyme (enzyme:substrate ratio of 1:4,000). The mixture was incubated at 37°C in 0.1 M Na Borate, pH 8.7, containing 0.1 M NaCl, 0.1% Lubrol PX and 100 μM C0CI2. Aliquots were removed, and the reaction was stopped by the addition of TCA to 0.7%. Samples were injected into an Ultrasphere column (Beckman Instruments, Carlsbad, CA) , and peptides were eluted with a linear gradient of acetonitrile containing 0.1% TFA. The enzyme efficiently cleaved the peptide and after one hour more than 80% of total peptide was converted to Ac-ARVRR (SEQ ID NO:8) and NH₂-ANSFL (SEQ ID N0:9).

The peptidase activity was tested with prothrombin peptide at pH values ranging from 5.0 to 10.3. The maximal activity was obtained at pH 8.7, but the enzyme still retained about 50% of its activity at pH 10.3. However, the activity dropped sharply at acid pH, with only 25% at pH 6.0 and no activity at pH 5.0 (See Figure 3) . Various protease inhibitors were tested for their effects on the enzyme. Inhibitors were dissolved in H2O, methanol or ethanol and added as 10X concentrated solutions to 100 μL 20 mM Bis-tris, pH 7.0, containing 0.1 M NaCl, 0.1% Lubrol PX, 100 μM CoCl₂ and 0.3 μg purified protein. The mixtures were incubated for .10 minutes at room temperature, and the reaction was initiated by the addition of 15 nanomoles of the prothrombin-derived peptide (Ac-ARVRRANSFL) (SEQ ID NO:l). After 30 minutes incubation at 37°C, the reaction was stopped with TCA and assayed as described above. The serine protease inhibitors PMSF, DFP and TPCK had very little or no effect on enzyme activity. Even at 10 mM DFP there was no effect. Pepstatin had little effect. E-64 and leupeptin, inhibitors of cysteine proteases, had no effect, suggesting the presence of a critical free -SH group was not directly involved in catalysis. EDTA and 1, 10-phenanthroline strongly inhibited the enzyme activity, suggesting that the purified endopeptidase might be classified as a metallopeptidase. PCMS and Hg ion were also found to be strong inhibitors.

To further characterize the substrate specificity of the enzyme, peptides corresponding to the cleavage sites of factor IX (SEQ ID NO:2) and abnormal factor IX (SEQ ID NOS: 6, 7) were tested (see Figure 4) . The reaction was run at pH 8.7. In the abnormal peptides, arginine at the -4 position was replaced with glutamine or tryptophan. As shown in Figure 4, these peptides were cleaved by the enzyme, but reaction rates were decreased to 50% for the Trp(-4) peptide and 70% for the Gln(-4) peptide as compared to the normal factor IX peptide. These data suggest that the endopeptidase prefers Arg at the -4 position, but that this is not a strict requirement under the assay conditions used.

Example 3 Cloning of Rabbit Endopeptidase DNA The rabbit endopeptidase was purified essentially as described in Example 1, then digested with cyanogen bromide and proteases. The resultant fragments were separated by high performance liquid chromatography and sequenced using an automated sequenator (Applied Biosystems, Inc., Foster City, CA) . Families of degenerate primers were designed on the basis of the peptide sequence data and synthesized using conventional methods.

To obtain a clone, the primers were used to amplify sequences from a rabbit cDNA library. An amplified fragment was isolated and used to probe the same library. Two positive clones were isolated and sequenced. Analysis of the sequence data indicated that the clones overlapped. The sequences were aligned to give a cDNA of 2148 bp. A preliminary sequence analysis of this cDNA is shown in Figure 5 (SEQ ID NO. 12; see also SEQ ID NO. 13). The actual amino and carboxyl terminals of the protein have not yet been determined. The sequence shown in Figure 5 includes primer sequence at the 5' and 3' ends.

Example 4

Cloning of Human Endopeptidase DNA

Total mRNA from human HepG2 cells was obtained by the quanidinium isothiocyanate method of Chomozynski and Sacchi (Anal. Biochem. 16.2,:156-159, 1987). Poly(A) RNA was selected by chromatography on oligo(dT)-cellulose as disclosed by Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, 1989) . Twenty μg of poly(A) RNA was reverse transcribed into cDNA in a reaction of 100 μl containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, ' 3 mM MgCl₂, 1 mM dithiothreitol, 0.5 mM each of dGTP, dATP, dTTP and dCTP, 500 pmoles of oligo(dT)₁7 primer, and 2000 units of reverse transcriptase (Superscript, GIBCO BRL, Gaithersburg, MD) at 37°C for one hour. The reaction was terminated by phenol/chloroform extraction, and the cDNA was precipitated with three volumes of ethanol. The cDNA was centrifuged for ten minutes at 12,000 x g, and the precipitated cDNA was dissolved in 20 μl of water.

Oligonucleotides for polymerase chain reactions were synthesized by the phosphoramidite method in an Applied Biosystems 380B DNA synthesizer. The oligonucleotide primers, designated MP-5 and MP-6, have the sequences shown in Table 2. The underlined portion of MP-5 corresponds to nucleotides 309 to 332 of the partial rabbit sequence shown in .Figure 5 (SEQ ID NO. 12) . The underlined portion of MP-6 is complementary to nucleotides 1954 to 1979 of the same cDNA sequence (SEQ ID NO. 12) . The remaining sequence of each primer contains an Eco RI recognition sequence to facilitate subsequent cloning.

TABLE 2 MP-5 TTT GAA TTC TAA GAT GGG ATC TTT CAC CAG AGC (SEQ ID NO. 10) MP-6 TTT GAA TTC ACC TGG CAT ATT CAC TTG CAG CGT (SEQ ID NO. 11)

The cDNA synthesized from HepG2 poly(A) RNA was used in a polymerase chain reaction using the synthetic oligonucleotide primers MP-5 (SEQ ID NO. 10) and MP-6 (SEQ ID NO. 11) . The reaction was carried out in a total volume of 100 μl of 10 mM Tris-HCl, pH 9.0, 50 mM KC1, 1.5 mM MgCl₂, 0.01% gelatin, 0.1% Triton X-100, 0.2 mM of each of dGTP, dATP, dTTP and dCTP, 25 pmoles of each primer, 1 μl of HepG2 cDNA and 2.5 units of Ther us aguaticus (Taq) DNA polymerase (Promega Corp., Madison, -WI) . The samples were denatured at 94°C for one minute, annealed at 50°C for two minutes, and extended at 72°C for three minutes for 30 cycles in a Perkin-Elmer Cetus (Norwalk, CT) thermal cycler.

The product of the PCR reaction was analyzed on 0.8% agarose gels and showed a band of approximately 1.6 kb. The amplified DNA was extracted with phenol/chloroform and ethanol precipitated, then resuspended and digested with Eco RI to generate cohesive ends. The digested DNA was fractionated by electrophoresis in a 1% low melting point agarose gel, and the appropriate DNA band was excised. The DNA was electro-eluted from the gel and ethanol precipitated. The DNA was then cloned into the Eco RI site of the vector M13mpl9 and sequenced by standard dideoxy sequencing methods. From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Kawabata, Shunichiro Davie, Earl W.

(ii) TITLE OF INVENTION: MICROSOMAL ENDOPEPTIDASE

(iii) NUMBER OF SEQUENCES: 13

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Seed and Berry

(B) STREET: 6300 Columbia Center

(C) CITY: Seattle

(D) STATE: Washington

(E) COUNTRY: US

(F) ZIP: 98104

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/646,997

(B) FILING DATE: 28-JAN-1991

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Ma i, David J.

(B) REGISTRATION NUMBER: 31,392

(C) REFERENCE/DOCKET NUMBER: 990008.415PC

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (206)622-4900

(B) TELEFAX: (206)682-6031

(C) TELEX: 3723836

(2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(i i ) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note- "This amino acid is acetylated."

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

Ala Arg Val Arg Arg Ala Asn Ser Phe Leu 1 5 10

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: {A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note- "This amino acid is acetylated."

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:

Ala Arg-Pro Lys Arg Ala Asn Ser Phe Leu •1 5 10

(2) INFORMATION FOR SEQ ID N0:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note- "this amino acid is acetylated."

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

Arg He Arg Lys Arg Ala Asn Ser Phe Leu 1 5 10

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v)- FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note- "this amino acid is acetylated."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Ala Arg Arg Arg Arg Ala Asn Ser Phe Leu 1 5 10

^"(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note- "This amino acid is acetylated." ( ix) FEATURE:

(A) NAME/KEY: Modi fi ed-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note- "This amino acid is acetylated."

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:

Ala Arg Pro Thr Arg Ala Asn Ser Phe Leu 1 5 10

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note- "This amino acid is acetyl ted."

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:

Ala Gin Pro Lys Arg Ala Asn Ser Phe Leu 1 5 10

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(i i ) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-termi nal (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION : 1

(D) OTHER INFORMATION: /note- "This ^'amino acid is acetylated."

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

Ala Trp Pro Lys Arg Ala Asn Ser Phe Leu 1 5 10

(2) INFORMATION FOR SEQ ID N0:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE^": N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:

Ala Arg Val Arg Arg 1 5

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE^" DESCRIPTION: SEQ ID N0:9:

Ala Asn Ser Phe Leu 1 5 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: TTTGAATTCT AAGATGGGAT CTTTCACCAG AGC 33

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: TTTGAATTCA CCTGGCATAT TCACTTGCAG CGT 33

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2148 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 11..2075 (xi ) SEQUENCE DESCRIPTION : SEQ ID NO: 12 :

GAATTCCGGG CGCTGGCACN NCTGCACGCT GCTGTGGCCC GGGCTGTGGC GTCCCGGCCT 60

AGGCCGACCC GCGGGTGNCG GGGNGNGTGG GANTTGGNNG TNGGNCTNNN GGGAANCGNA 120

AAGNCAAGNA CGNNGNTTCT GNCGNTGNCG GNCTTGNGGN GCGCCCATNA TCGCCCGGTG 180

CTTTTCGGCT GTNCGAGGCC TCCACAGGGT TGGTGGTTCC AGGATTCTAT TCAAAATGAC 240

ATTAGGAAGA GAAGTGATGT CACCTCTTCA GGCAGTTTCT TCCTATACTG CAGCTGGCAG 300

GAATGTΠTA AGATGGGATC TΠ^"CACCAGA GCAAATCAAG ACAAGAACTG AGGAGCTCAT 360

TGCGCAGACC AAGCAGGTGT ATGATTCTGT TGGAATGCTG GATATCAAGG ACGTGACTTA 420

CGAGAACTGC TTGCAGGCGC TGGCAGATGT GGAAGTGAAG TACATAGTGG AAAGAACCAT 480

GCTAGACTTT CCTCAGCATG TTTCTACTGA CAGAGAAGTA CGGGCAGCAA GTACAGAAGC 540

GGACAAGAGG CTTTCTCGCT TTGATATTGA GATGAGCATG AGAGAAGATA TATTTCAGAG 600

AATCGTTCAC TTACAGGAAA CCTGTGATCT GGAGAAGATA AAGCCGGAAG CCAGACGATA 660

CTTGGAAAAG TCAGTTAAAA TGGGAAGAAG GNATGGGCTC CACCTTCCNN NAGAAGTACA 720

GAATGANATC AAATCAATGA AGAAAAGAAT GAGTGAGCTC TGTATTGACT πAACAAAAA 780

CCTCAATNAG GATGATACCN TCCNTGTGTT πCCAAGGCT GAGCTTGGTG CTCTTCCTGA 840

TGATTTCATT GACAGTTTAG AAAAGATGGA TGATGACAAG TATAAAATTA CCTTAAAATA 900

TCCACACTAT TTTCCTGTCA TGAAGAAATG TTGTATTCCT GAAACCAGAA GGAGAATGGA 960

AATGGCTΠΓT AATACAAGAT GCAAAGAGGN AAACACCGTC ATTCTGCAGC AGCTACTCCC 1020

ACTGCGGGCC CAAGTGGCCA AACTCCTGGG CTATAGCACA CATGCTGACT TCGTCCTCGA 1080

GATGAACACT GCAAAGAGCA CAAGCCGAGT AACAGCCTTT CTAGACGATT TAAGCCAGAA 1140

ATTAAAACCG CTGGGTGAGG CAGAACGAGA GTTTATCTTG AGTTTGAAGA AAAAGGAATG 1200

CGAAGAGAAA GGTTTTGAGT ATGACGGGAA AATCAATGCC TGGGACCTGC ATTACTACAT 1260

GACTCAGACA GAAGAGCTCA AGTACTCCAT CGACCAGGAG TTCATCAAGG AGTACTTNCC 1320

CCCNNTTNAG GGTGGTCANC CNAGGGGCCT GCTGAACATC TACCAGGAGT TGCTGGGACT 1380

TTNATTCGAG CAAGTGGCCG ACGCTCATGT TTGGAACCCG AGNGTCACAC TTTACACTGT 1440

GAAGGACAAG GCTACAGGAG AAGTGCTGGG GCAGTTCTAC CTGGACCTCT ATCCGNGGGA 1500

AGGAAAATAT AATCATGCAG NCTGCTTTGG TCTCCAGCCT GGCTGCCTGC TNCCCGATGG 1560

GAGCCGGATG CTGTCTGTGG CTGCCCTCGT GGTGAACTNN NCCCAGCCAG TGGCCGGCCG 1620 NCCCTCTCTG CTGAGGCGCG ATGAAGTGNG GNCTTACNCC CATGANTTTG GTCATGTCAT 1680

GCATCAGCπ TGTGCNCAGA CTGATTTTGC ACGATTTAGT GGAACAAATG TGGAAACTGA 1740

CTTTGTAGAG GTGCCTTCAC AAATGCTTGA GAACTGGGTG TGGGACATCG ACTCTCTCCG 1800

ANGATTNTNN AAACATTATA AAGATGGNNA CCCTATTGCA GATGATCTTC TTGAAAANCT 1860

TGTTGCTTCT AGACTGGTCA ACACAGGTCT TCTGACCCTT CGCCAGATCG TTTTGAGCAA 1920

AGTTGACCAG TCTCTGCATA CCAACTCATC CCTGGACGCT GCAAGTGAAT ATGCCAGGTA 1980

CTGCACCGAC ATTTTAGGTG TTGCCGCTAC NCCTGGAACA AACATGCCAG CTACTTTTGG 2040

GCATTTGGCA NNGGGATACG ATGGCCAGTA TTATGGATAT CTTTGGAGTG AAGNGNTTTC 2100

CATGGACATG TTNTACAGCT GCTTTAAAAA AAAAAANAAC CGGAATTC 2148 (2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 709 amtno acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Arg Trp His Xaa Cys Thr Leu Leu Trp Pro Gly Leu Trp Arg Pro Gly 1 _.5 10 15

Leu Gly Arg Pro Ala Gly Xaa Gly Xaa Xaa Gly Xaa Trp Xaa Xaa Xaa 20 25 30

Xaa Xaa Glu Xaa Xaa Xaa Gin Xaa Xaa Xaa Phe Xaa Xaa Xaa Arg Xaa

35 40 45

Xaa Xaa Ala Pro Xaa He Ala Arg Cys Phe Ser Ala Xaa Arg Gly Leu 50 55 60

His Arg Val Gly Gly Ser Arg He Leu Phe Lys Met Thr Leu Gly Arg 65 70 75 80

Glu Val Met Ser Pro Leu Gin Ala Val Ser Ser Tyr Thr Ala Ala Gly 85 90 95

Arg Asn Val Leu Arg Trp Asp Leu Ser Pro Glu Gin He Lys Thr Arg 100 105 110 Thr Glu Glu Leu He Ala Gin Thr Lys Gin Val Tyr Asp Ser Val Gly 115 120 125

Met Leu Asp He Lys Asp Val Thr Tyr Glu Asn Cys Leu Gin Ala Leu 130 135 140

Ala Asp Val Glu Val Lys Tyr He Val Glu Arg Thr Met Leu Asp Phe 145 150 155 160

Pro Gin His Val Ser Thr Asp Arg Glu Val Arg Ala Ala Ser Thr Glu 165 170 175

Ala Asp Lys Arg Leu Ser Arg Phe Asp He Glu Met Ser Met Arg Glu 180 185 190

Asp He Phe Gin Arg He Val His Leu Gin Glu Thr Cys Asp Leu Glu 195 200 205

Lys lie Lys Pro Glu Ala Arg Arg Tyr Leu Glu Lys Ser Val Lys Met 210 215 220

Gly Arg Arg Xaa Gly Leu His Leu Xaa Xaa Glu Val Gin Asn Xaa He 225 230 235 240

Lys Ser Met Lys Lys Arg Met Ser Glu Leu Cys He Asp Phe Asn Lys 245 250 . 255

Asn Leu Asn Xaa Asp Asp Thr Xaa Xaa Val Phe Ser Lys Ala Glu Leu 260 265 270

Gly Ala Leu Pro Asp^" Asp Phe He Asp Ser Leu Glu Lys Met Asp Asp 275 280 285

Asp Lys Tyr Lys He Thr Leu Lys Tyr Pro His Tyr Phe Pro Val Met 290 295 300

Lys Lys Cys Cys He Pro Glu Thr Arg Arg Arg Met Glu Met Ala Phe 305 310 315 320

Asn Thr Arg Cys Lys Glu Xaa Asn Thr Val He Leu Gin Gin Leu Leu 325 330 335

Pro Leu Arg Ala Gin Val Ala Lys Leu Leu Gly Tyr Ser Thr His Ala 340 345 350

Asp Phe Val Leu Glu Met Asn Thr Ala Lys Ser Thr Ser Arg- Val Thr 355 360 365

Ala Phe Leu Asp Asp Leu Ser Gin Lys Leu Lys Pro Leu Gly Glu Ala 370 375 380

Glu Arg Glu Phe He Leu Ser Leu Lys Lys Lys Glu Cys Glu Glu Lys 385 390 395 400 Gly Phe Glu Tyr Asp Gly Lys He Asn Ala Trp Asp Leu His Tyr Tyr 405 410 415

Met Thr Gin Thr Glu Glu Leu Lys Tyr Ser He Asp Gin Glu Phe He 420 425 430

Lys Glu Tyr Xaa Pro Xaa Xaa Xaa Gly Gly Xaa Xaa Arg Gly Leu Leu 435 440 445

Asn He Tyr Gin Glu Leu Leu Gly Leu Xaa Phe Glu Gin Val Ala Asp 450 455 460

Ala His Val Trp Asn Pro Xaa Val Thr Leu Tyr Thr Val Lys Asp Lys 465 470 475 480

Ala Thr Gly Glu Val Leu Gly Gin Phe Tyr Leu Asp Leu Tyr Pro Xaa 485 490 495 -

Glu Gly Lys Tyr Asn His Ala Xaa Cys Phe Gly Leu Gin Pro Gly Cys ^' 500 505 510

Leu Xaa Pro Asp Gly Ser Arg Met Leu Ser Val Ala Ala Leu Val Val 515 520 525

Asn Xaa Xaa Gin Pro Val Ala Gly Xaa Pro Ser Leu Leu Arg Arg Asp 530 535 540

Glu Val Xaa Xaa Tyr Xaa His Xaa Phe Gly His Val Met His Gin Leu 545 550 555 560

Cys Xaa Gin Thr Asp Phe Ala Arg Phe Ser Gly Thr Asn Val Glu Thr 565 570 575

Asp Phe Val Glu Val Pro Ser Gin Met Leu Glu Asn Trp Val Trp Asp 580 585 590

He Asp Ser Leu Arg Xaa Xaa Xaa Lys His Tyr Lys Asp Xaa Xaa Pro 595 600 605 lie-Ala Asp Asp Leu Leu Glu Xaa Leu Val Ala Ser Arg Leu Val Asn 610 615 620

Thr Gly Leu Leu Thr Leu Arg Gin He Val Leu Ser Lys Val Asp Gin 625 630 635 640

Ser Leu His Thr Asn Ser Ser Leu Asp Ala Ala Ser Glu Tyr Ala Arg 645 650 655

Tyr Cys Thr Asp He Leu Gly Val Ala Ala Xaa Pro Gly Thr Asn Met 660 665 670

Pro Ala Thr Phe Gly His Leu Ala Xaa Gly Tyr Asp Gly Gin Tyr Tyr 675 680 685 Gly Tyr Leu Trp Ser Glu Xaa Xaa Ser Met Asp Met Xaa Tyr Ser Cys 690 695 700

Phe Lys Lys Lys Xaa 705

Claims

ClaimsWe Claim:

1. An isolated microsomal endopeptidase characterized by: a) a molecular weight of 65,000 ± 3,000 daltons by SDS-PAGE under reducing conditions; b) cleaves proteins C-terminal to a basic amino acid residue; and c) retains at least 50% activity at pH 7.0- 10.0.

2. The endopeptidase of claim 1 wherein said endopeptidase is isolated from rabbit liver.

3. The endopeptidase of claim 1 wherein said endopeptidase is at least 90% pure.

4. The endopeptidase of claim 1 wherein said endopeptidase exhibits maximum activity at pH 8.7.

5. The endopeptidase of claim 1 wherein said endopeptidase exhibits essentially no activity at pH 5.0 or below.

6. The endopeptidase of claim 1 wherein said endopeptidase cleaves protein substrates having a basic amino acid residue at the -4 position.

7. The endopeptidase of claim 1 wherein said endopeptidase is inhibited by EDTA and 1,10-phenanthroline.

8. An isolated DNA molecule encoding the endopeptidase of any of claims 1-7.

9. An expression vector capable of directing the expression of a microsomal endopeptidase in a host cell, said expression vector comprising^' the following operably linked elements: a transcriptional promoter followed downstream by a DNA sequence encoding a microsomal endopeptidase characterized by: a) a molecular weight of 65,000 ± 3,000 daltons by SDS-PAGE under reducing conditions; b) cleaves proteins C-terminal to a basic amino acid residue; and c) retains at least 50% activity at pH 7.0- 10.0; and

- a transcriptional terminator.

10. A host cell transfected or transformed to express a microsomal endopeptidase characterized by: a) a molecular weight of 65,000 ± 3,000 daltons by SDS-PAGE under reducing conditions; b) cleaves proteins C-terminal to a basic amino acid residue; and c) retains at least 50% activity at pH 7.0- 10.0.

11. The cell of claim 10 wherein said cell is further transfected or transformed to express a protein which is a substrate for the endopeptidase.

12. The cell of claim 11 wherein the protein is selected from the group consisting^' of factor VII, factor IX, factor X, protein C, prothrombin, protein S, protein Z, bone gla protein, and t-PA.

13. The cell of claim 11 wherein said cell is a mammalian cell.

14. An isolated DNA molecule comprising from 14 to 2128 nucleotides of the nucleotide sequence shown in Figure 5, from nucleotide number 11 to nucleotide number 2138.