WO1995016043A1

WO1995016043A1 - Cloning of endothelin converting enzyme

Info

Publication number: WO1995016043A1
Application number: PCT/GB1994/002673
Authority: WO
Inventors: William Mark Abbott
Original assignee: Zeneca Limited
Priority date: 1993-12-09
Filing date: 1994-12-07
Publication date: 1995-06-15
Also published as: AU1246395A; GB9325221D0

Abstract

Recombinant mammalian endothelin converting enzymes, their preparation and use.

Description

CLONING OF ENDOTHELIN CONVERTING ENZYME.

Endothelin-l (ET-1) is a 21 amino acid peptide derived from vascular endothelial cells. It has potent vasoconstricting properties on coronary arteries in vitro and produces a long lasting hypertensive or pressor effect in vivo. ET-1 is generated from a precursor polypeptide of 212 residues by the action of several peptidase enzymes. The final step is the cleavage of 38 or a 39 residue peptide termed big endothelin-l (bigET-1) at the Trp21-Val22 bond by a specific endopeptidase known as endothelin converting enzyme (ECE) . The final product ET-1 is >100-fold more potent than bigET-1 such that ECE is essential to generate the full biological effect.

Considerable effort has been expended on the characterisation of ECE. Recent studies have suggested that ECE is likely to be a membrane bound neutral metallopeptidase sensitive to the inhibitor phosphoramidon. In addition several groups have reported a partial purification of ECE (Ohnaka et al,

Biochem.Biophys.Res.Commun, 1992, 185. 611-616; Takahashi et al, 1993, 268, 21394-21398; Parker-Botehlo et al- W092/13944) . However all efforts to characterise ECE definitively and clone the gene appear to have been unsuccessful.

According to one aspect of the present invention we now provide DNA encoding a human endothelin converting enzyme (ECE) .

The DNA of the invention conveniently corresponds to the insert sequence of a cDNA clone identifiable using a DNA or RNA probe as set out in Table 1 or Table 2 to screen a human cDNA library. The cDNA library is preferably derived from materials rich in endothelial cells. Convenient cDNA libraries are derived from lung and placental material. A particular cDNA library is a library constructed using the ECV304 cell line (ATCC acccession no. CRL 1998) .

The DNA or RNA probe is labelled, for example radiolabelled by standard techniques (Sambrook et al, Molecular Cloning; A 32 Laboratory Manual, 2nd Edition, Cold Spring Harbor Press) with P, and used to screen, for example in duplicate, a cDNA library.

The cDNA insert sequences of the clones are determined by standard DNA sequencing techniques.

In a further aspect the DNA of the invention corresponds to the insert sequence of a cDNA clone identifiable by repeat screening of a cDNA library. The library is conveniently screened twice, up to three, four or five times. If required the library may be screened up to ten times. In a preferred aspect positively hybridising single clones are purified to homogeneity, said screening being initiated using a DNA or RNA probe as set out in Table 1 or Table 2.

In a further preferred aspect the DNA of the invention comprises the complete protein coding sequence of a human ECE. Such DNA is typically comprised in a cDNA of at least 2.5 or 5 kilobases such as 5-15 kilobases or 5-10 kilobases.

DNA of the present invention may be used to prepare ECE by recombinant techniques. Methods for DNA expression will be apparent to the molecular biologist of ordinary skill and include those set out in the Maniatis cloning manual (Molecular Cloning: A Laboratory Manual - 2nd Edition (1989) - J. Sambrook, E.F. Fritsch & Maniatis), and in Current Protocols in Molecular Biology - 1987 - P. Sharp (Ed.) . Particular expression systems include the mouse erythroleukaemia (mel) cell expression system claimed in WO-89/01517 (Grosveld) and, more particularly, as claimed in Wθ-92-11380 (Hollis et al) . An alternative expression system is the Baculovirus Expression System (Clontech) - A. Prokop et al, Recombinant DNA Technology & Applications, 1991, 97-152.

Therefore according to a further aspect of the invention we provide recombinant human ECE and convenient fragments thereof. Convenient fragments include those of up to 50, 100, 500, and 1000 amino acid residues extending from the N and C terminii respectively. The recombinant human ECE of the present invention is used to identify agents that modulate the action of ECE, and thereby produce a desirable therapeutic effect. In general this would arise by inhibition of the action of the endothelin converting enzyme. Such agents may provide anti-hypertensive effects. They may also be used in the treatment and therapy of other vascular diseases including stroke, reno-vascular disease, and pulmonary hypertension.

Therefore in a further aspect of the present invention we provide a method for the identification of an agent which modulates the action of recombinant ECE which method comprises contacting a test compound with recombinant ECE in a test system comprising an ECE substrate for ECE and identifying any modulation of the action of recombinant ECE due to presence of the test compound.

In a still further aspect of the invention we provide a method for the identification of an agent which modulates the action of recombinant ECE, which method comprises contacting a test compound with a cell expressing recombinant ECE and identifying any change in ECE expression over that found in the absence of the test compound. This method may involve the use of any convenient in vitro assay. In general, a cell expressing the ECE gene is contacted with an agent capable of transcriptionally modulating expression of the gene, thereby affecting the level of protein encoded by the gene which is expressed by the cell. Convenient procedures are described in WO-91/01379 (Oncogene) . Any modulation such as an increase or decrease in transcription may be monitored by blotting techniques using probes derived from the cDNA encoding ECE, or by PCR using primer sequences derived from the cDNA. Protein expression levels can be conveniently monitored by the use of antibodies and enzyme activity measured as described above.

The cDNA of the invention encoding ECE, or any convenient fragment thereof, may be used to provide DNA probes. The molecular biologist of ordinary skill will be aware that these DNA probes (or RNA probes derived therefrom) may be used in a number of procedures. These include the identification and cloning of homologous mammalian and non-mammalian cDNAs, for example for isoenzymes that cleave bigET-1, bigET-2 or big ET-3. Such homologous cDNAs represent further independent aspects of the invention. They may be cloned into vectors (such as those commercially available) in order to produce useful fusion proteins or cloned into expression vectors to construct high level expressing cell lines. They may also be used in gene cloning studies in order to analyse a number of regulatory elements including promoters, enhancers and introns. In addition they may be used to investigate gene expression in vivo. They may also be used in the preparation of transgenic animals, such as mice or rats expressing ECE.

According to a further aspect of the invention we provide recombinant mammalian ECE and convenient fragments thereof.

As outlined earlier above the recombinant ECE of the invention, and convenient fragments thereof, may be used to identify agents which inhibit the enzyme. It may also be used in molecular modelling and X-ray crystallography studies. In addition it can be used to map binding site(s) .

The recombinant ECE of the invention, and convenient fragments thereof, may also may be used to raise antibodies. Such antibodies have a number of uses which will be evident to the molecular biologist of ordinary skill. These include (i) monitoring levels of expression in native cells and clones which express recombinant ECE, (ii) the development of assays, (iii) the precipitation of ECE and other proteins which associate with ECE leading to identification of these proteins and, (iv) investigation of localisation.

Furthermore recombinant ECE may be used to screen compound libraries such as peptide libraries, including synthetic peptide libraries and peptide phage libraries, to identify inhibitors of the enzyme. TABLE 1

N-terminal amino acid sequence of ECE with the corresponding DNA and RNA sequences

residue 1 2 3 4 5 6 7 8 9

amino X G Q R E W A A R acid

DNA GGN CAA CGN GAA TGG GCN GCN CGN

G A G A

RNA GGM CAA CGM GAA UGG GCM GCM CGM

G A G A

Residue 10 11 12 13 14 15 16 17 18

amino T Q V E K/E R L V V acid

DNA ACN CAA GTN GAA AAA CGN TTN GTN GTN

G G G G A C

RNA ACM CAA GUM GAA AAA CGM UUM GUM GUM

G G G G A C

Legend

1) The internationally accepted single letter codes for amino acids are used (cf. "Molecular Biology of the Cell", Alberts et al.) . X=unknown

Residue 14 appeared as either K or E

2) N= A,C,G or T M= A,C,G or U Table 2

ECE amino acid number & identity

10 11 12 13 14 15 16 17 18

W A V K/E V V

Probes

5'

a) GGC CAG CGG GAG TGG GCT GCC CGG ACC CAG GTG GAG RAG CGG CTG GTG GT

b) GGI CAI IGI GAI TGG GCI GCI IGI ACI CAI GTI GAI IAI IGI ITI GTI GT

c) GGI CAR MGI GAR TGG GCI GCI MGI ACI CAR GTI GAI RAG MGI CTI GTI GT

R = A or G M = A or C S = C or G W = A or T I = Inosine Table 3

ECE amino acid number and identity

10 11 12 13 14 15 16 17 18

X R W V E K/E V V

Primers

Gl) 3' TGN GTY CAN CTY YTY KGN RAN CAN CA 5'

G2) 3' ACC CGN CGN KCN TGN GTY CAN CTY YT 5'

II) 3' TGI GTY CAI CTY YTY KGI RAI CAI CA 5'

12) 3' ACC CGI CGI KCI TGI GTY CAI CTY YT 5'

R = G or A Y = C or T K = G or T I = Inosine Table 4

Full length sequence of human endothelin converting enzyme.

CGCCCCCCCG GTGTCCGCCC TGCTGTCGGC GCTGGGGATG TCGACGTACA

51 AGCGGGCCAC GCTGGACGAG GAGGACCTGG TGGACTCGCT CTCCGAGGGC

101 GACGCATACC CCAACGGCCT GCAGGTGAAC TTCCACAGCC CCCGGAGTGG

151 CCAGAGGTGC TGGGCTGCAC GGACCCAGGT GGAGAAGCGG CTGGTGGTGT

201 TGGTGGTACT TCTGGCGGCA GGACTGGTGG CCTGCTTGGC AGCACTGGGC

251 ATCCAGTACC AGACAAGATC CCCCTCTGTG TGCCTGAGCG AAGCTTGTGT

301 CTCAGTGACC AGCTCCATCT TGAGCTCCAT GGACCCCACA GTGGACCCCT

351 GCCATGACTT CTTCAGCTAC GCCTGTGGGG GCTGGATCAA GGCCAACCCA

401 GTCCCTGATG GCCACTCACG CTGGGGGACC TTCAGCAACC TCTGGGAACA

451 CAACCAAGCA ATCATCAAGC ACCTCCTCGA AAACTCCACG GCCAGCGTGA

501 GCGAGGCAGA GAGAAAGGCG CAAGTATACT ACCGTGCGTG CATGAACGAG

551 ACCAGGATCG AGGAGCTCAG GGCCAAACCT CTAATGGAGT TGATTGAGAG

601 GCTCGGGGGC TGGAACATCA CAGGTCCCTG GGCCAAGGAC AACTTCCAGG

651 ACACCCTGCA GGTGGTCACC GCCCACTACC GCACCTCACC CTTCTTCTCT

701 GTCTATGTCA GTGCCGATTC CAAGAACTCC AACAGCAACG TGATCCAGGT

751 GGACCAGTCT GGCCTGGGCT TGCCCTCGAG AGACTATTAC CTGAACAAAA 801 CTGAAAACGA GAAGGTGCTG ACCGGATATC TGAACTACAT GGTCCAGCTG

851 GGGAAGCTGC TGGGCGGCGG GGACGAGGAG GCCATCCGGC CCCAGATGCA

901 GCAGATCTTG GACTTTGAGA CGGCACTGGC CAACATCACC ATCCCACAGG

951 AGAAGCGCCG TGATGAGGAG CTCATCTACC ACAAAGTGAC GGCAGCCGAG

1001 CTGCAGACCT TGGCACCCGC CATCAACTGG TTGCCTTTTC TCAACACCAT

1051 CTTCTACCCC GTGGAGATCA ATGAATCCGA GCCTATTGTG GTCTATGACA

1101 AGGAATACCT TGAGCAGATC TCCACTCTCA TCAACACCAC CGACAGATGC

1151 CTGCTCAACA ACTACATGAT CTGGAACCTG GTGCGGAAAA CAAGCTCCTT

1201 CCTTGACCAG CGCTTTCAGG ACGCCGATGA GAAGTTCATG GAAGTCATGT

1251 ACGGGACCAA GAAGACCTGT CTTCCTCGCT GGAAGTTTTG CGTGAGTGAC

1301 ACAGAAAACA ACCTGGGCTT TGCGTTGGGC CCCATGTTTG TCAAAGCAAC

1351 CTTCGCCGAG GACAGCAAGA GCATAGCCAC CGAGATCATC CTGGAGATTA

1401 AGAAGGCATT TGAGGAAAGC CTGAGCACCC TGAAGTGGAT GGATGAGGAA

1451 ACCCGAAAAT CAGCCAAGGA AAAGGCCGAT GCCATCTACA ACATGATAGG

1501 ATACCCCAAC TTCATCATGG ATCCCAAGGA GCTGGACAAA GTGTTTAATG

1551 ACTACACTGC AGTTCCAGAC CTCTACTTTG AAAATGCCAT GCGGTTTTTC

1601 AACTTCTCAT GGAGGGTCAC TGCCGATCAG CTCAGGAAAG CCCCCAACAG

1651 AGATCAGTGG AGCATGACCC CGCCCATGGT GAACGCCTAC TACTCGCCCA 1701 CCAAGAATGA GATTGTGTTT CCGGCCGGGA TCCTGCAGGC ACCATTCTAC

1751 ACACGCTCCT CACCCAAGGC CTTAAACTTT GGTGGCATAG GTGTCGTCGT

1801 GGGCCATGAG CTGACTCATG CTTTTGATGA TCAAGGACGG GAGTATGACA

1851 AGGACGGGAA CCTCCGGCCA TGGTGGAAGA ACTCATCCGT GGAGGCCTTC

1901 AAGCGTCAGA CCGAGTGCAT GGTAGAGCAG TACAGCAACT ACAGCGTGAA

1951 CGGGGAGCCG GTGAACGGGC GGCACACCCT GGGGGAGAAC ATCGCCGACA

2001 ACGGGGGTCT CAAGGCGGCC TATCGGGCTT ACCAGAACTG GGTGAAGAAG

2051 AACGGGGCTG AGCACTCGCT CCCCACCCTG GGCCTCACCA ATAACCAGCT

2101 CTTCTTCCTG GGCTTTGCAC AGGTCTGGTG CTCCGTCCGC ACACCTGAGA

2151 GCTCCCACGA AGGCCTCATC ACCGATCCCC ACAGCCCCTC TCGCTTCCGG

2201 GTCATCGGCT CCCTCTCCAA TTCCAAGGAG TTCTCAGAAC ACTTCCGCTG

2251 CCCACCTGGC TCACCCATGA ACCCGCCTCA CAAGTGCGAA GTCTGGTAAG

2301 GACGAAGCGG AGAGAGCCAA GACGGAGGAG GGGAAGGGGC TGAGGACGAG

2351 ACCCCCATCC AGCCTCCAGG GCATTGCTCA GCCCGCTTGG CCACCCGGGG

2401 CCCTGCTTCC TCACACTGGC GGGTTTTCAG CCGGAACCGA GCCCATGGTG

2451 TTGGCTCTCA ACGTGACCCG CAGTCTGATC CCCTGTGAAG AGCCGGACAT

2501 CCCAGGCACA CGTGTGCGCC ACCTTCAGCA GGCATTCGGG TGCTGGGCTG

2551 GTGGCTCATC AGGCCTGGGC CCCACACTGA CAAGCGCCAG ATACGCCACA 2601 AATACCACTG TGTCAAATGC TTTCAAGATA TATTTTTGGG GAAACTATTT

2651 TTTAAACACT GTGGAATACA CTGGAAATCT TCAGGGAAAA ACACATTTAA

2701 ACACTTTTTT TTTTAAGCCC

Table 5

Amino acid sequence of human endothelin converting enzyme

MSTYKRATIDEEDLVOSLSEGDAYPNGLQVNFHSPRSGQRCWAA

RTQVΕKRLV _,VVI_LAAGLVACLAALGIQYQTRSPSVCLSEACVSVTSSILSSMDPTV

DPCHDFFSYACGGWIKANPVPDGHSRWGTFSNLWEHNQAIIKHLLENSTASVSEAERK

AQVYYRACMNETRIEELRAKPLMELIERLGGWNITGPWAKDNFQDTLQWTAHYRTSP

FFS YVSADSKNSNSNVIQVDQSGLGLPSRDYYLNKTENEKVLTGYI-NYMVQLGKLLG

GGDEEAIRPQMQQILDFETAIANITIPQEKRRDEELIYHKVTAAELQTLAPAINWLPF

__NTIFYPVΕ:iNESEPIvVYDKEYLEQISTLINTTDRCLLNNYMIWNLVRKTSSFLDQR

FQDADEKFMEV-^GTKKTCLPRWKFCVSDTENNLGFALGPMFVKATFAEDSKSIATEI

ILEIKKAFEΞSLSTLKWMDEETRKSAKEKADAIYNMIGYPNFIMDPKELDKVFNDYTA

VPDLYFENAMRFFNFSWRVTADQLRKAPNRDQWSMTPPMVNAYYSPTKNEIVFPAGIL

QAPFYTRSSPKALNFGGIGVWGHELTHAFDDQGREYDKDGNLRPWWKNSSVEAFKRQ

TECMVΕQYSNYSVNGEPVNGflHTLGENIADNGGLKAAYRAYQNWVKKNGAEHSLPTLG

LTNNQLFFLGFAQVWCSVRTPESSHEGLITDPHSPSRFRVIGSLSNSKEFSEHFRCPP

GSPMNPPHKCEVW The invention will now be illustrated but not limited by reference to the following Figure and Examples wherein Figure 1 shows:

__. a) SDS-PAGE analysis of fractions from the Superdex 200 column. The fractions from left to right are; molecular weight markers, fractions 8-16. The band at ~220kDa is arrowed. The molecular weight markers are, from bottom to top, 45kDa, 65kDa, 97kDa, 116kDa and 205kDa.

b) Enzyme activity of fractions 8-14.

The band at -220 kDa is most prominent in fractions 11 and 12 which is where the peak of ECE activity occurs.

Example 1

Identification of N-terminal amino acid sequence of ECE

Partial Purification of ECE

ECE was partially purified from the human endothelial cell line ECV304 (ATCC accession no. CRL 1998) by the following route. Approximately 2x10 cells were homogenised in 50mM sodium phosphate buffer pH 7.8 (buffer A) using a glass teflon homogeniser and the membranes pelleted by centrifugation at 100,000g for 1 hour. The pellet was resuspended in 400ml buffer A containing 0.75% (3- [ (3-Cholamidopropyl)dimethylammonio] -1-propane sulphonate (CHAPS) to solubilise the membrane proteins. After gentle stirring for 2 hours a clear supernatant was then obtained by centrifugation at 100,000g for 1 hour.

The solubilised membranes were applied to a 50ml Q-Sepharose Fast Flow (Pharmacia) column equilibrated in buffer A plus 0.75% CHAPS (buffer B) . The column was washed with buffer B and eluted by a 500ml linear gradient from 0-0.5M NaCl in buffer B. The active fractions were pooled and concentrated to approximately 50ml in an Amicon concentrator with a YM30 membrane. This material was then applied to a 15ml RCA-agarose (Sigma) column equilibrated in 0.2M Tris pH7.2, 0.75% CHAPS (buffer C) . After washing in buffer C the column was eluted using 60ml of 0.1M galactose in buffer C. The eluted material was concentrated using an Amicon concentrator with a YM30 membrane to 5ml, dialysed into 20mM Tris pH7.2, 0.75% CHAPS (buffer D) and applied to a heparin-Sepharose (Pharmacia) column equilibrated in buffer D. After washing the bound protein was eluted in 7.5ml buffer D containing 1M NaCl. This material was concentrated in Centriprep-10 and Centricon-10 (Amicon) devices to 0.2ml and subjected to gel filtration on a Superdex 200 column (Pharmacia) , equilibrated and developed in buffer C. The active fractions were pooled for further analysis.

Characterisation of purified ECE

ECE activity was measured by first dialysing samples against 50 volumes of 0.2M Tris pH7.2 and then incubating 67ul in a final volume of lOOul containing lOuM bigET-1, lmM p-Chloromercuriphenylsulphonic acid (PCMS) , 0.2mM phenylmethylsulphonylfluoride (PMSF) and 2uM pepstatin. After 6 hours the reaction was terminated by the addition of 160ul 5% TFA, insoluble material removed by centrifugation and 200ul loaded onto a Rainin C18 reverse phase column (Anachem) . Chromatography was performed by a 5min isocratic elution with H2O/0.1%TFA followed by a lOmin linear gradient from 0-35% acetonitrile/0.1%TFA (solventB) , a 15min linear gradient from 35-45% solvent B and lOmin gradient from 45-100% solvent B. The absorbance at 230nm was monitored throughout the chromatography procedure and the position of elution of standard samples of bigET-1, ET-1 and the C-terminal fragment (CTF) determined. ECE activity was thereby determined by the size of the ET-1 and CTF peaks produced during the incubation.

The ECE activity of the purified material was inhibited by phosphoramidon with an IC50 of ~2uM. Thiorphan and Captopril showed no inhibition at lOOuM. The activity was completely abolished by lOuM EDTA, an effect which was reversed by the subsequent addition of 20uM ZnCl,, . 2

The moleeular weight of the activity on the Superdex 200 gel filtration column was estimated to be ~300kDa by comparison to the position of migration of known molecular weight markers (Pharmacia) .

SDS-PAGE analysis of purified samples was performed on 8-16% gradient gels according to the methods disclosed by Laemmli (Nature, 1970, 227. 680-685) .

Identification of ECE

The material purified through the above 4-step procedure was -200-500 fold enriched over the initial solubilised preparation but was not homogeneous. In order to identify ECE from the several species that were present, samples from the final two chromatographic steps were carefully analysed both for activity and by SDS-PAGE using silver staining to visualise the proteins. Only one band could be identified that consistently co-migrated with activity. This band had an apparent mwt of -220kDa on SDS-PAGE (Figure la) in the presence of a reducing agent (1% 2-mercaptoethanol) .

N-terminal sequence analysis

To obtain N-terminal sequence information the active fractions from the Superdex 200 column were pooled and prepared for SDS-PAGE by the method of Wessel and Flugge (Anal.Biochem. , 1984, 138, 141-143. All the material from one purification was loaded into one track of a 6% SDS-PAGE gel. After electrophoresis the gel was blotted onto a Problot membrane (Applied Biosystems) stained with coomassie blue and a band at ~220kDa was cut out and subjected to automated sequence analysis (Applied Biosystems 475 sequencer) to generate the sequence shown in Table 1. Example 2

Cloning of cDNA encoding ECE

The amino acid sequence of ECE as shown in Table 1 is used to isolate cDNA encoding the complete enzyme.

Method 1

The amino acid sequence is translated into nucleic acid sequence, oligonucleotides can then be synthesised, which can be radiolabelled and used as probes to screen suitable cDNA libraries.

(a) A probe is synthesised so that positions of ambiguity in the nucleic acid sequence are assigned to be one of the 4 standard bases by reference to a codon usage table for human genes (Wada et al. Nucleic Acids Research, 2J3, S2111-2118) and the described methods for its application (Lathe, J. Molecular Biology, 183, 1-12) . A convenient probe is illustrated in Table 2a. Shorter versions thereof may also be used. These probes are used to hybridise selectively to the target, ECE, cDNA (cf. Ulrich et al. , Nature, 309, 418-425; &, ibid, 313, 756-761; &, ibid, 316, 701-705; &, Jaye et al . , Nucleic Acids Research, 13., 2325-2335) .

(b) A probe is synthesised wherein positions of ambiguity are represented by the base, inosine (cf. Ohtsuka et al, J. Biological Chemistry, 260, 2605-2608; Takahashi et al, Proc.Natl .Acad.Sci.USA,

82 , 1931-1935. A convevient probe is shown in Table 2b.

(c) Probes based on a combination of (a) and (b) are also used. The number of inosines in the probe are reduced by incorporating either a predicted base as in (a) or a mixture of potential natural bases at positions of minimal uncertainty whilst retaining an inosine at positions of maximal uncertainty. A convenient probe is shown in Table 2c

A cDNA library is constructed as follows. PolyA+ RNA is prepared from ECV304 cells by standard techniques (ibid) and cDNA synthesised using random hexamer primers, under standard conditions, as specified by the manafacturers (Gibco-BRL, cDNA synthesis kit) . cDNAs is cloned and propogated in a suitable bacteriophage vector, eg.

Lambda Zap, under conditions described by the manafacturer (Stratagene) . Alternatively, a commercially available cDNA library (Clontech or Stratagene) constructed from tissues or cells that are expected to express ECE, for example endothelial cells, is screened by the same procedures.

The above probes are radiolabelled by standard techniques

(Sambrook et al, Molecular Cloning; A Laboratory Manual, 2nd Edition,

32 Cold Spring Harbor Press) with P, and used to screen, in duplicate, a cDNA library transferred to nylon filters as described (ibid) .

Convenient cDNA library screeninng conditions are: 50ng radiolabelled oligonucleotide probe in 5x saturated sodium phosphate/sodium chloride/EDTA buffer - pH7 (SSPE) , lOOug/ml of sonicated and denatured salmon sperm DNA, 0.1% SDS, 0.25% non-fat dried milk (Marvel) at

40-60 C for 16 hours. Filters are then be washed at room temperature for 30 minutes in 5x SSPE, 0.1% SDS followed by 30 minutes in the same solution at 40-60 C (references ibid) .

Positive clones are isolated and rescreened as described above until positively hybridisisng single clones have been purified to homogeneity. The cDNA insert sequences of these clones are then determined by standard DNA sequencing techniques (ibid & Stratagene) and the sequence translated into all 6 possible amino acid sequences. Comparison of these translated sequences to the known sequence of ECE as set out in Table 1 reveals which of these cDNA clones encodes this region of ECE. An isolated clone that encodes the amino terminal sequence of ECE, but not the full length protein coding sequence, is then used as a specific probe to re-screen the cDNA libraries under conditions described above or variations thereof (ibid) . This process is repeated until the entire protein coding sequence of ECE is isolated.

The probes are radiolabelled by standard techniques (Sambrook et al, Molecular Cloning; A Laboratory Manual, 2nd Edition,

32 Cold Spring Harbor Press) with P, and used to screen, in duplicate, cDNA libraries (see below) transferred to nylon filters as described

(ibid) . Conditions for screening the cDNA library will be: 50ng radiolabelled oligonucleotide probe in 5x SSPE, lOOug/ml of sonicated and denatured salmon sperm DNA, 0.1% SDS, 0.25% non-fat dried milk at o 40-60 C for 16 hours. Filters will then be washed at room temperature for 30 minutes in 5x SSPE, 0.1% SDS followed by 30 minutes in the same solution at 40-60 C (references ibid) .

Method 2

The amino acid sequence of ECE as shown in Table 1 is used to design polymerase chain reaction (PCR) primers. These are used on suitable cDNA to yield a fragment of ECE cDNA. This fragment is then used as a specific nucleic acid probe for cDNA library screening.

The amino acid sequence of ECE as shown in Table 1, is used to design and synthesise mixtures of degenerate oligonucleotides. Examples of convenient primers Gl and G2 are shown in Table 3.

In addition, oligonucleotides containing inosine residues at positions of maximum uncertainty are designed and synthesised. Examples of convenient primers II & 12 are also shown in Table 3.

Single stranded cDNA is synthesised from ECV304 cell polyA+ RNA using a commercially available kit under conditions described by the manafacturer (Pre-amp kit, Gibco-BRL) . The cDNA is then subjected to Rapid Amplification of cDNA Ends (RACE) PCR following the modifications of Edwards et al, Nucleic Acids Research, 23 , Sill-Sill , and Troutt et al, Proc. Natl. Acad. Sci., ______ 9823-9825; under conditions as described by the commercial supplier of the kit (5' Amplifinder RACE kit, Clontech) . In the first round of PCR oligonucleotide mixes Gl or II are utilised as the 3' primer, along with the manufacturer's recommended 5' primer, under conditions specified by the manufacturer of the Taq polymerase (Perkin-Elmer) . Amplification is optimised by varying cycle times and temperatures, as experimentally determined. The amplification products are then subjected to a second round of nested PCR using G2 or 12 as*the 3' primer and the manafacturer's recommended 5' nested primer. PCR products are then cloned directly into the TA cloning vector under conditions described in the TA cloning kit (In Vitrogen) . The resulting recombinants are then subjected to DNA sequence analysis and those found to contain the amino acid sequence presented in Table 1 are partial cDNA clones of ECE. These sequences are then used as specific radiolabelled probes to screen cDNA libraries as set out in Method 1 above (cf. Sambrook et al, ibid) .

Similarly cDNA library DNA, (Method 1) is used as the template for PCR in conjunction with nested 5' primers designed from the vector DNA that flanks the cDNA cloning site.

Method 3

The amino acid sequence of ECE as shown in Table 1 is used to produce ECE peptides that can be used as immunogens to raise, in animals, an antiserum. This antiserum is used to screen a suitable cDNA expression library.

Peptides based on the ECE amino acid sequence presented in Table 1 are synthesised. These are then coupled to immunogenic carrier proteins such as BSA or keyhole limpet haemocyanin using standard chemical techniques as described for example by Van Regenmortel et al, Synthetic Polypeptides as Antigens, Elsevier Press) . Animal immunisations, antiserum analysis and purification then follow using standard techiques as described therein. The antiserum is then used to screen a cDNA library constructed in a bacteriophage expression vector such as lambda Zap or gtll. The conditions used are as descibed by the manafacturer of an antibody based screening kit (Clontech, Click kit) . The cDNA library is as described in Method 1 above. The full length cDNA sequence of human endothelin converting enzyme is set out in Table 4. This sequence was disclosed to the EMBL database by Burkhard Kroeger of the Department of Pharmaceutical Research, BASF on 21st July 1994.

Example 3

Expression of ECE cDNA in heterologous cells

The cDNA encoding the protein coding sequence of ECE, isolated as disclosed in Method 2 above, is expressed in a heterologous cell system.

The cDNA is expressed in the baculovirus vector and insect cell system using standard methodologies (Summers et al, A Manual of Methods for Baculovirus Vectors & Insect Cell Culture, MAN2.TEX) and commercially available vectors and cells (In Vitrogen) . We have recently exemplified this approach to recombinant protein production (Aharony et al, Molec.Pharmacol. , 44,, 356-363).

Alternatively the cDNA is expressed in mammalian cells, initially MEL cells (Friend et al, Proc Natl Acad Sci USA, 68. 377-382) under the control of the beta-globin gene, locus control region, (Grosveld et al., Cell, 5_6, 975-985). We have recently exemplified this system for recombinant protein production (Needham et al., Nuc. Acids Res.,20., 997-1003). In addition, the cDNA may be expressed in yeasts, for example, Saccharomyces cerevisiae and Pischia pastoris, using commercially available vectors and strains (In Vitrogen) , as well as in E.Coli using standard expression systems and commercially available vectors and strains.

The complete amino acid sequence of human endothelin converting enzyme is set out in Table 5. This sequence was disclosed to the EMBL database by Burkhard Kroeger of the Department of Pharmaceutical Research, BASF on 21st July 1994.

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(1) GENERAL INFORMATION

(i) APPLICANT: ZENECA Limited

(ii) TITLE OF INVENTION: NUCLEIC ACID

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(A) MEDIUM TYPE: DISKETTE, 3.5 INCH, 1.44 mB Storage

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: PC-DOS 3.20

(D) SOFTWARE: ASCII from WPS-PLUS

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER

(B) FILING DATE:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NO: 9325221.1

(B) FILING DATE: 09-Dec-93 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

XGQREWAARTQVEXRLW

(2) INFORMATION FOR SEQ ID N0:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

GGNCAACGNG AATGGGCNGC NCGNACNCAA GTNGAAAAAC GNTTNGTNGT N 51 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GGMCAACGMG AAUGGGCMGC MCGMACMCAA GUMGAAAAAC GMUUMGUMGU M 51

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

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

GGCCAGCGGG AGTGGGCTGC CCGGACCCAG GTGGAGRAGC GGCTGGTGGT 50 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GGNCANNGNG ANTGGGCNGC NNGNACNCAN GTNGANNANN GNNTNGTNGT 50

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GGNCARMGNG ARTGGGCNGC NMGNACNCAR GTNGANRAGM GNCTNGTNGT 50 ( 2 ) INFORMATION FOR SEQ ID NO : 7 :

(i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 26 base pairs

(B) TYPE : nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

ACNACNARNG KYTYYTCNAC YTGNGT 26

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

TYYTCNCAYT GNGTNCKNGC NGCCCA (2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

ACNACNARNG KYTYYTCNAC YTGNG 25

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

TYYTCNACYT GNGTNCKNGC NGCCCA 26 (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2720 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11 :

CGCCCCCCCG GTGTCCGCCC TGCTGTCGGC GCTGGGGATG TCGACGTACA AGCGGGCCAC GCTGGACGAG GAGGACCTGG TGGACTCGCT CTCCGAGGGC GACGCATACC CCAACGGCCT GCAGGTGAAC TTCCACAGCC CCCGGAGTGG CCAGAGGTGC TGGGCTGCAC GGACCCAGGT GGAGAAGCGG CTGGTGGTGT TGGTGGTACT TCTGGCGGCA GGACTGGTGG CCTGCTTGGC AGCACTGGGC ATCCAGTACC AGACAAGATC CCCCTCTGTG TGCCTGAGCG AAGCTTGTGT CTCAGTGACC AGCTCCATCT TGAGCTCCAT GGACCCCACA GTGGACCCCT GCCATGACTT CTTCAGCTAC GCCTGTGGGG GCTGGATCAA GGCCAACCCA GTCCCTGATG GCCACTCACG CTGGGGGACC TTCAGCAACC TCTGGGAACA CAACCAAGCA ATCATCAAGC ACCTCCTCGA AAACTCCACG GCCAGCGTGA GCGAGGCAGA GAGAAAGGCG CAAGTATACT ACCGTGCGTG CATGAACGAG ACCAGGATCG AGGAGCTCAG GGCCAAACCT CTAATGGAGT TGATTGAGAG GCTCGGGGGC TGGAACATCA CAGGTCCCTG GGCCAAGGAC AACTTCCAGG ACACCCTGCA GGTGGTCACC GCCCACTACC GCACCTCACC CTTCTTCTCT GTCTATGTCA GTGCCGATTC CAAGAACTCC AACAGCAACG TGATCCAGGT GGACCAGTCT GGCCTGGGCT TGCCCTCGAG AGACTATTAC CTGAACAAAA CTGAAAACGA GAAGGTGCTG ACCGGATATC TGAACTACAT GGTCCAGCTG GGGAAGCTGC TGGGCGGCGG GGACGAGGAG GCCATCCGGC CCCAGATGCA GCAGATCTTG GACTTTGAGA CGGCACTGGC CAACATCACC ATCCCACAGG AGAAGCGCCG TGATGAGGAG CTCATCTACC ACAAAGTGAC GGCAGCCGAG CTGCAGACCT TGGCACCCGC CATCAACTGG TTGCCTTTTC TCAACACCAT CTTCTACCCC GTGGAGATCA ATGAATCCGA GCCTATTGTG GTCTATGACA AGGAATACCT TGAGCAGATC TCCACTCTCA TCAACACCAC CGACAGATGC CTGCTCAACA ACTACATGAT CTGGAACCTG GTGCGGAAAA CAAGCTCCTT CCTTGACCAG CGCTTTCAGG ACGCCGATGA GAAGTTCATG GAAGTCATGT ACGGGACCAA GAAGACCTGT CTTCCTCGCT GGAAGTTTTG CGTGAGTGAC ACAGAAAACA ACCTGGGCTT TGCGTTGGGC CCCATGTTTG TCAAAGCAAC CTTCGCCGAG GACAGCAAGA GCATAGCCAC CGAGATCATC CTGGAGATTA AGAAGGCATT TGAGGAAAGC CTGAGCACCC TGAAGTGGAT GGATGAGGAA ACCCGAAAAT CAGCCAAGGA AAAGGCCGAT GCCATCTACA ACATGATAGG ATACCCCAAC TTCATCATGG ATCCCAAGGA GCTGGACAAA GTGTTTAATG ACTACACTGC AGTTCCAGAC CTCTACTTTG AAAATGCCAT GCGGTTTTTC AACTTCTCAT GGAGGGTCAC TGCCGATCAG CTCAGGAAAG CCCCCAACAG AGATCAGTGG AGCATGACCC CGCCCATGGT GAACGCCTAC TACTCGCCCA CCAAGAATGA GATTGTGTTT CCGGCCGGGA TCCTGCAGGC ACCATTCTAC ACACGCTCCT CACCCAAGGC CTTAAACTTT GGTGGCATAG GTGTCGTCGT GGGCCATGAG CTGACTCATG CTTTTGATGA TCAAGGACGG GAGTATGACA AGGACGGGAA CCTCCGGCCA TGGTGGAAGA ACTCATCCGT GGAGGCCTTC AAGCGTCAGA CCGAGTGCAT GGTAGAGCAG TACAGCAACT ACAGCGTGAA CGGGGAGCCG GTGAACGGGC GGCACACCCT GGGGGAGAAC ATCGCCGACA ACGGGGGTCT CAAGGCGGCC TATCGGGCTT ACCAGAACTG GGTGAAGAAG AACGGGGCTG AGCACTCGCT CCCCACCCTG GGCCTCACCA ATAACCAGCT CTTCTTCCTG GGCTTTGCAC AGGTCTGGTG CTCCGTCCGC ACACCTGAGA GCTCCCACGA AGGCCTCATC ACCGATCCCC ACAGCCCCTC TCGCTTCCGG GTCATCGGCT CCCTCTCCAA TTCCAAGGAG TTCTCAGAAC ACTTCCGCTG CCCACCTGGC TCACCCATGA ACCCGCCTCA CAAGTGCGAA GTCTGGTAAG GACGAAGCGG AGAGAGCCAA GACGGAGGAG GGGAAGGGGC TGAGGACGAG ACCCCCATCC AGCCTCCAGG GCATTGCTCA GCCCGCTTGG CCACCCGGGG CCCTGCTTCC TCACACTGGC GGGTTTTCAG CCGGAACCGA GCCCATGGTG TTGGCTCTCA ACGTGACCCG CAGTCTGATC CCCTGTGAAG AGCCGGACAT CCCAGGCACA CGTGTGCGCC ACCTTCAGCA GGCATTCGGG TGCTGGGCTG GTGGCTCATC AGGCCTGGGC CCCACACTGA CAAGCGCCAG ATACGCCACA AATACCACTG TGTCAAATGC TTTCAAGATA TATTTTTGGG GAAACTATTT TTTAAACACT GTGGAATACA CTGGAAATCT TCAGGGAAAA ACACATTTAA ACACTTTTTT TTTTAAGCCC

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 753 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:12:

MSTYKRATI-DEEDLVDSLSEGDAYPNGLQVNFHSPRSGQRCWAARTQVEKRLVVLVVLIIAAGLVACLAAL GIQYQTRSPSVCLSEACVSVTSSILSSMDPTVDPCHDFFSYACGGWIKANPVPDGHSRWGTFSNLWEHNQ AIIKHLLENSTASVSEAERKAQVYYRACMNETRIEELRAKPLMELIERLGGWNITGPWAKDNFQDTLQW TAHYRTSPFFSVYVSADSKNSNSNVIQVDQSGLGLPSRDYYI_NKTENEIΘAITGY___NYMVQLGKLLGGGDE EAIRPQMQQILDFETAIIANITIPQEKRRDEELIYHKVTAAELQTIIAPAINWLPFLNTIFYPVEINESEPI VVYDKEYLEQISTLIΪΠ'TDRCLLNNYMIWNLVRKTSSFLDQRFQDADEKFMEVMYGTKKTCLPRWKFCVS DTENNLGFALGPMFVKATFAEDSKSIATEIILEIKKAFEESLSTLKWMDEETRKSAKEKADAIYNMIGYP NFIMDPKEIJDKVFNDYTAVPDLYFENAMRFFNFSWRVTADQLRKAPNRDQWSMTPPMVNAYYSPTKNEIV FPAGILQAPFYTRSSPKALNFGGIGVVVGHELTHAFDDQGREYDKDGNLRPWWKNSSVEAFKRQTECMVE QYSNYSVNGEPVNGRHTLGENIADNGGLKAAYRAYQNWVKKNGAEHSLPTLGLTNNQLFFLGFAQVWCSV RTPESSHEGLITDPHSPSRFRVIGSLSNSKEFSEHFRCPPGSPMNPPHKCEVW

Claims

Claims :

Recombinant mammalian endothelin converting enzyme.

2. Recombinant human endothelin converting enzyme.

3. cDNA encoding a mammalian endothelin converting enzyme.

4. cDNA encoding a human endothelin converting enzyme.

5. A cDNA as claimed in claim 3 or claim 4 identifiable using a probe comprising nucleic acid as set out in Table 1 or Table 2 herein.

6. A cDNA as claimed in claim 3 or claim 4 identifiable using PCR amplification primers as set out in Table 3 herein.

7. A cDNA clone comprising as an insert sequence a cDNA as claimed in claim 4 or claim 5.

8. A method for the identification of an agent which modulates the action of a recombinant endothelin converting enzyme, which method comprises contacting a test compound with a recombinant endothelin converting enzyme in a test system comprising a substrate for the enzyme and identifying any modulation of the action of the enzyme due to the presence of the test compound.

9. A method for the identification of an agent which modulates the action of a recombinant endothelin converting enzyme, which method comprises contacting a test compound with a cell expressing a recombinant endothelin converting enzyme and identifying any change in enzyme expression over that found in the absence of the test compound.

10. Antibodies raised to a recombinant endothelin converting enzyme as claimed in claim 1 or claim 2.

11. The use of a recombinant endothelin converting enzyme as claimed in claim 1 or claim 2 in the screening of a compound library to identify compounds which modulate the activity of the enzyme.