CA2006535A1 - Cdna coding for placenta protein 11 (pp11), the isolation and use thereof - Google Patents

Abstract

BEHRINGWERKE AKTIENGESELLSCHAFT 88/B 045 - Ma 738 Dr. Lp/rd Abstract of the disclosure cDNA coding for placenta protein 11 (PP11), the isolation and use thereof The invention relates to the isolation of the gene which codes for placenta-specific protein 11 (PP11) and to the use thereof for the preparation of PP11 by genetic manipulation.

Description

BEHRING~ERKE AKTIENGESELLSCHAET 88~B 045 - Ma 738 Dr. Lp/rd cDNA coding for placenta protein ll (PP11), the isolation and use thereof The invention relates to the isolation of the gene which codes for placenta-specific protein 11 (PP11) and to the use thereof for the preparation of PPll by genetic manipulation.

PPll has the following properties according to the description in EP-B10,029,191:

a) a carbohydrate content of 3.9 + 0.9%, composed of 2.6 i 0.5% hexoses, 1.0 i 0.3% hexosamines, 0.05 i 0.03~ fucose and 0.26 i 0.07% neuraminic acid;
b) a sedimentation coefficient S of 3.5 i 0.2 S;
5 c) a molecular weight, determined in an ultracentri-fuge, of 44,300 + 6,000;

d) a molecular weight, determined in a sodium dodecyl sulfate (SDS)-containing polyacrylamide gel, of 62,000 i 3,000;

e) an extinction coefficient E (280nm) of 13.4 i 1.0 and f) an electrophoretic mobility in the region of the alpha1 globulins as well as the amino acid composition depicted in Table 1.

Z~06S3~i Tab. l Amino acid composition of PP11 (residues per 100 residues in mol-~) CV%
(Coefficient of variation) Lysine 6.26 7.00 Histidine 3.34 1.72 Arginine 3.31 5.60 Aspartic acid 10.75 2.43 Threonine 3.31 10.49 Serine 9.63 1.84 Glutamic acid 13.81 2.09 Proline 4.10 4.68 Glycine 6.23 6.26 Alanine 6.30 1.82 Cystine 1/2 3.37 4.53 Valine 4.53 5.40 : Methionine 1.00 26.22 Isoleucine 3.60 2.24 Leucine 6.74 1.05 Tyrosine 5.90 3.02 i Phenylalanine 6.06 2.52 Tryptophan 1.66 20.87 Since the conventional i~olation of PPll described in the abovementioned patent is extremely laborious, the ob~ect therefore was to isolate the gene coding for PP11 in order to make it possible to prepare PP11 by genetic manipulation.

Initially, polyclonal antibodies were raised with PP11 purified conventionally as described above and were used to search for positive clones in a commercially available : cDNA expression bank of human placenta (Genofit GmbH, Heidelberg; HL 1008 Human Placenta gtll Placental tissue, 34 weeks old). One clone out of 1.2 million recombinant clones reacts with the antiserum. It was called PPl1-93 - 2~0~i~35 and contained an insert of about 1,800 base-pairs ~bp).
This insert had an open reading frame of about 860 bp but had no start methionine. Subsequently the insert of PP11-93 was labelled and used as probe in a placenta cDNA
bank in lambda gtlO prepared by u5. Two further clones which overlap with PP11-93 were found (PP11-166 and PP11-169), and their sequence was determined. Although clone PP11-169 is shorter than clone PP11-93, it overlaps the latter at the 5' end by 270 bp and has a methionine, which must be the start methionine. Using a 5'-terminal 46mer oligonucleotide of the clone PP11-169 (5~
CTCCAACTGGGCACCATGAGGGCCTGCATCTCCCTGGTATTGGCCG), finally the entire 5'-untranslated sequence was found in the clone PP11-318, which shows that there are now only stop codons in front the abovementioned presumptive start methionine and no further methionine appears. Thus, the complete cDNA of PPll is, including the poly(A) sequence, 2399 bp long and has an open reading frame for 369 amino acids, which corresponds to a protein of 42,120 D. 154 bp at the 5' end are untranslated, 1138 bp are untranslated at the 3' end, and a 1107 bp coding sequence iB enC108ed.
The complete cDNA sequence is listed in Table 2, and the figure shows the position of the individual clones within the cDNA as well as the coding region. "N" therein represents the N terminus, "C" represents the C terminus of the coding region and "A" represents the poly(A) sequence.

It is possible according to the invention for the coding cDNA to be used, by means of suitable expression systems, for expressing PPll. It is furthermore possible, by the choice of the host, to influence the type of modification of PPll. Thus, no glycosylation takes place in bacteria, and that takinq place in yeast cells differs from that in higher eukaryotic cells. The expression of PPll takes place particularly advantageously in E.coli with the expression vector pTrc 99A or pTrc 99C (see Example 11).
It has furthermore been found that PPll, preferably prepared as above by genetic manipulation in E.coli or i1653S

yeast, can be employed for the treatment of coagulation disorders, disorders of the complement system or in connection with disorders of fibrinolysis, because it has an amidolytic activity.

It is possible, knowing the amino acid sequence of PPll, to prepare, by conventional methods of genetic manipu-lation, amino acid partial sequences which act as anti-gens for the preparation of polyclonal or monoclonal antibodies. Antibodies of these types can be employed not only for diagnostic purposes but also for the preparation of antibody columns. PP11 can thus be removed from solutions which contain it in addition to other proteins.
It i8 also possible with the aid of the cDNA or parts thereof to isolate in a straightforward manner from a genomic bank the genomic clone coding for PP11, with whose aid not only is expression in eukaryotic cells facilitated but also further diagnostic conclusions can be drawn.

The invention is furthermore defined in the patent claims and detailed in the examples which follow.

Where not explained in the text, the following abbrevi-ations are used:

EDTA = Sodium ethylenediaminetetraacetate SDS = Sodium dodecyl sulfate DTT = Dithiothreitol BSA = Bovine serum albumin Examples:
1. Isolation of RNA from human placenta RNA was obtained from mature human placenta (method of Chirgwin et al., Biochemistry 18 (1079) 5294-5299)).
About 10 g of placenta tissue were ground in a mortar under liquid nitrogen, suspended in 80 ml of 4 M guani-dinium thiocyanate containing 0.1 M mercaptoethanol and ZC~ 535 treated in a homogenizer (Ultraturrax) at 20,000 rpm for 90 sec. The lysate was centrifuged at 7,000 rpm for 15 min (Sorvall GSA rotor) and the supernatant was precipi-tated with 2 ml of 1 M acetic acid and 60 ml of ethanol abs. at -20C overnight. After sedimentation at 6,000 rpm and -10C for 10 min, the nucleic acids were completely dissolved in 40 ml of 7.5 M guanidinium hydrochloride (pH
7.0) and precipitated with a mixture of 1 ml of 1 M
acetic acid and 20 ml of ethanol abs. To remove the DNA, the precipitation was repeated once more with each of the volumes halved. The RNA was dissolved in 12 ml of H2O, precipitated with a mixture of 1.2 ml of 4 M potassium acetate and 24 ml of ethanol abs., sedimented and finally taken up again in 10 ml of H2O (1 ml per g of tissue).

2. Obtaining poly(A~-containing placenta mRNA

The placenta RNA was fractionated by oligo(dT)-cellulose chromatography (Aviv and ~eder, Proc. Natl. Acad. Sci.
~SA 69 (1973) 1408-1412) in 2 ml Pasteur pipettes in LiCl in order to obtain poly(A)-containing mRNA. About 5 mg of placenta RNA in buffer 1 (500 mN LiCl, 20 mM Tris (pH
7.S), 1 mM EDTA, 0.1% SDS) were loaded onto the column.
Whereas the poly(A)+-RNA was bound to oligo(dT)-cellulose, it was possible to elute the poly(A) -RNA again. After a washing step with buffer 2 (100 mN LiCl, 29 mM Tris (pH
7.5), 1 mM EDTA, 0.1% SDS), the poly(A)t-RNA (placenta mRNA) was eluted from the column with buffer 3 (5 mM Tris (pH 7.5), 1 mm EDTA, 0.05% SDS).

For further purification, the poly(A)+-RNA was ad~usted to buffer 1 and again chromatographed on oligo(dT)-cellulose. The yield of placenta poly(A)+-RNA after this second purification step was about 4% of the RNA
employed.

3. Synthesis of cDNA from human placenta (placenta cDNA) and double-stranded cDNA (dsDNA) .

2~G535 Poly(A)-containing placenta mRNA was checked for in tegrity before the cDNA synthesis in a 1.5% agarose gel.

Then 4 ~g of placenta mRNA were dissolved in 65.5 ~l of H20, denatured at 70C for 10 min and cooled on ice.

The cDNA was synthesized in a 100 ~l mixture after addition of 20 ~l of RTl buffer (250 mM Tris (pH 8.2) at 42C, 250mM KCl, 30mM MgCl2), 2.5 ~l of 20 mM dNTP (i.e.
all four deoxynucleoside triphosphates), 1 ~l of oli-gotdT) of 1 ~g/ml, 1 ~l of 1 M DTT, 2 ~l of RNAsin and 8 ~l of reverse transcriptase (24 U/~l) at 42C for 90 min. Double-stranded cDNA (dsDNA) was synthesized by the method of Gubler and Hoffmann (Gene 25 (1983) 263-269).
The synthesis was carried out immediately after the cDNA
synthesis by adding 305.5 ~l of ~2~ 80 ~l of RT2 buffer (100 mM Tris (pH 7.5), 25 mM MgCl2, 500 mM KCl, 50 mM DTT, 250 ~g/ml BSA), 2 ~l of RNase H (2 U/~l), 2.5 ~l of E.coli DNA ligase (5 U/~l), 5 ~1 of 15 mN ~-NAD, and 5 ~1 of DNA polymera~e I (5 U/~l) and incubation at 15C for 5 h. The reaction was stopped by heat inactivation (70C, 30 min).

55 ~1 of 250 ~M dNTP, 55 ~l of 10 mM Tris (pH 7.5), 10 mM
MgCl2, 10 ~g/ml BSA, 3 ~l of T4 DNA polymerase I (1 U/~l), 2 ~l of RNase H (2 U/~l) and 2 ~l of RNa~e A (2 ~g/ml) were added to the reaction mixture, which was then incubated at 37C for a further 30 min in order to ensure that the synthesis of the second DNA strand was complete (repair reaction).

4. Ligation of EcoRI linkers to the dsDNA and opening of the linkers To set up a placenta cDNA bank, the dsDNA wa~ provided with EcoRI ends in order to be able to ligate it into the EcoRI cleavage 6ite of the phage vector lambda gtlO
(T. Maniatis et al. (1982), Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor). For this purpose, the dsDNA was a) treated with EcoRI methylase in order to protect internal EcoRI cleavage sites in the dsDNA, b) provided with EcoRI linkers which c) were then opened with EcoRI.

Re a):

The methylase reaction of the dsDNA was carried out immediately following the repair reaction after addition of 25 ~1 of 500 mN EDTA (pH 8.0), 60 ~1 of methylase buffer (100 mN NaOAc (pH 5.2), 2 mg of S-adenosyl-L-methionine) and 2 ~1 of EcoRI methylase (20 U/~l) by incubation at 37C for 30 min. The reaction mixture was extracted with phenol, and the dsDNA was precipitated with 60 ~1 of 4M NaOAc and 1300 ~1 of ethanol. The dsDNA
was washed twice with 70~ ethanol, extracted by shaking once with ether and dried.

Re b):

The EcoRI-methylated dsDNA was dissolved in 88 ~1 of H20 and, after addition of 10 ~1 of ligase buffer (500 mM
Tris (pH 7.4), 100 mM MgCl2, 100 mM DTT, 10 mM spermidine, 10mM ATP, 1 mg/ml BSA), 1 ~1 of T4 DNA ligase (10 U/~l), ligated with 1 ~1 of EcoRI linkers (0.5 ~g/~l) (pGGAATTCC
and pAGAATTCT) at 15C overnight.

Re c):

The volume of the ligase mixture was adjusted to 120 ~1 with 6 ~1 of H2O, 12 ~1 of 10 x EcoRI buffer and 2 ~1 of EcoRI (120 U/~l). The EcoRI digestion was carried out at 37C for 2 h.

5. Removal of unbound linkers on potassium acetate , . .- - .

~ ~0~)~s3s gradients and selection of the dsDNA for size To remove all unbound EcoRI linkers from the dsDN~, the EcoRI reaction mixture was loaded in toto onto a potas-sium acetate gradient (5-20% XAc, l mM EDTA, l ~l/ml ethidium bromide) and centrifuged at 50,000 rpm and 20C
for 3 h (Beckman SW 65 rotor).

The gradient was fractionated from below in such a way that the volume of the first five fractions was 500 ~il and that of the remainder was 100 ~1. The fractions were precipitated with 0.01 volume of acrylamide (2 mg/ml) and 2.5 volumes of ethanol, washed once with 70~ strength ethanol, dried and taken up in 5 ~il of H20 in each case.

To determine the ~ize of the dsDNA, 1 ~l of each fraction was analyzed in a 1.5% agarose gel. In addition, the quantity of dsDNA was determined using l ~il of each fraction.

Fractions with dsDNA over 1,000 bp were combined and the sample was concentrated until the final concentration was 27 ~g/ml.

6. Incorporation of the dsDNA into the phage vector lambda gtlO and in vitro packaging reaction The dsDNA was incorporated into the EcoRI cleavage site of the phage vector lambda gtlO (Vector Cloning Systems, San Diego, CA) in a 4 ~il ligase mixture: 2 ~il of dsDNA, 1 ~l of lambda gtlO x EcoRI (1 ~g/ml), 0.4 ~l of ligase buffer, 0.5 ~il of H~O, 0.1 ~il of T4 DNA ligase. The mixture was incubated at 15C for 4 h.

To establish the placenta cDNA bank in the phage vector lambda gtlO, an in vitro packaging reaction of the ligase mixture was subsequently carried out with the lambdalysogenic cell extracts E.coli NS 428 and NS 433 at room temperature for 2 h (Vector Cloning Systems, San , , 2~S35 g Diego, CA; Enquist and Sternberg, Methods in Enzymology 68, (1979), 281-298). The reaction was stopped with 500 ~1 of suspending medium (SM: 0.1 M NaCl, 8 mM MgSO4, 50 mM Tris (pH 7.5), 0.01~ gelatin) and 2 drops of chloroform.

7. Determination of the titer and analysis of the placenta cDNA bank The number of plaque-forming units (PFU) of the placenta cDNA bank was determined using competent cells of the E.coli K 12 strain C600 HFL: it was 1 x 106 PFU. About 80~ of all the phages contained DNA inserts which were larger than 1,000 base-pairs.

8. Screening of an expression cDNA bank from human placenta with anti-PP11 antibodies An expression cDNA bank in phage lambda gtll from Genofit (loc. cit.) was plated out at a density of about 30,000 PPU per agar plate (13.5 cm diameter). For this, competent cells of the E.coli strain ylO90 (ATCC 37197) (R.A. Young and R.W. Davis, Science Vol. 222, 778-782/1983) were infected with the phages at 37-C for 30 min and then plated out in the top agar on L broth plates. The plates were incubated at 42C for 4 h and then each was covered with a dry nitrocellulose filter (Schleicher and Schuell, BA 85, Ref. No. 401124). The filters had previously been saturated with 10 mM IPTG in water. The plate with the filter~ was sub~ected to renewed incubation at 37C for 4 h. Before the filter~
were removed again, the filters and plate were marked simultaneously with a needle dipped in carbon black. The filters were then incubated in TBST (10 mM Tris-HCl, pH
8.0, 150 mN NaCl, 0.05% Tween 20 and 5% skim milk powder) at 4C overnight. The filters were subsequently washed three more times for 10 min in TBST at room temperature and then incubated with rabbit anti-Pill antibodies in 15 ml of TBST per filter at room temperature for 1 h.

, .

(The solution of the antibodies had previously been diluted 1:200 and saturated with non-recombinant lambda gtll lysed E.coli cells on nitrocellulose filters for 1 h). After the filters had been incubated with the prLmary antibody they were washed 4 x 10 min with T~ST. The filters were then incubated, shaking for 1 h, with the ~econdary anti-rabbit antibody which was con~ugated to alkaline phosphatase (from Promega, USA - marketed by Atlanta, Heidelberg) and had previously been diluted 1:5,000 in TBST. The filters were then washed again 4 x 10 min with TBST. Finally, the color reaction was carried out in order to visualize the PPll-positive clones to which both primary and secondary antibodies were bound, by reaction of the alkaline phosphatase and a color reagent (ProtoBlot system from Protogen). For each color reaction, 15 ml of AP buffer (100 mN Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) were mixed with 99 ~1 of NBT
(nitro blue tetrazolium) substrate (50 mg/ml in 70%
dimethylformamide) and 49.5 ~1 of BCIP (5-bromo-4-chloro-3-indolyl phosphate) substrate (50 mg/ml in 70~ dimethyl-formamide) for one nitrocellulose filter. The filters were agitated in the stain solution in the dark for about 20 min to 1 h until positive plaques showed an adequate blue stain. The color reaction was stopped by immersing the filters in a stop solution (20 mM Tris-HCl, pH 8.0 and 5 mM EDTA).

Positive signals were assigned to the plaques on the corresponding agar plate. The plaque~ were removed by stabbing with a Pasteur pipette, resuspended in 1 ml of SM buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2) and singled out to obtain a single positive plaque. From the expression gene bank used here there wa~ found a clone (PP11-93) which, however, did not contain the complete gene but, with the total length of the insert being about 1800 bp, had an open reading frame - without start methionine - of 860 bp.

9 a) Preparation of a PPll-specific probe 2~535 ; The cDNA was isolated by known methods from the PPll-g3 clone and labelled with 32p as follows.

10 ng of DNA were labelled using the Multiprime DNA
labelling system (from Amersham, Braunschweig) with Klenow polymerase in the presence of alpha-32P-dCTP
(35 ~Ci/25 ~1 of reaction mixture were employed). The DNA
probe had a specific activity of 330 MBq/pmol.

9 b) Screening of the placenta cDNA bank, prepared by us as in 1. to 7., with PP11-specific probes For this, 3 x 104 PFU were plated out with cells of the E.coli K 12 strain C 600 HFL in soft agar on 13.5 cm Petri dishes and incubated at 37DC for 6 h. Lysis was not yet complete at this time. The plates were incubated in a refrigerator overnight, and the pha~es were transferred onto nitrocellulose filters ( Schleicher & SchUll, BA 85, Ref. No. 401124) (duplicates). Nitrocellulose filters and Petri dishes were marked with an in~ection needle in order to allow subsequent assignment. The Petri dishes were stored in a cold room while the nitrocellulose filters were being processed. The DNA present on the nitrocellulose filters was denatured by placing the filters for 5 min on filter paper (Whatman M3) impreg-nated with 1.5 M NaCl, 0.5 M NaOH. The filters were subsequently renatured in the same way with 1.5 M NaCl, 0.5 M Tris (pH 8.0) and washed with 2 x SSPE (0.36 M
NaCl, 16 mM NaOH, 20 mM NaH2PO4, 2 mM EDTA). The filters were then dried in vacuo at 80 C for 2 h. The filters were prehybridized at 65C for 4 h (prehybridization solution: 0.6 M NaCl, 0.06 M Tris (pH 8.3), 6 mM EDTA, 0.2% non-ionic synthetic sucrose polymer, (Ficoll), 0.2%
polyvinylpyrrolidone 40, 0.2% BSA, 0.1% SDS, 50 ~g/ml denatured herring sperm DNA). The filter~ were finally incubated overnight with addition of 100,000 - 200,000 Bq of the labelled oligonucleotide/ml of hybridization solution (as prehybridization solution but without .. - .
-, 2~ ;35 herring sperm DNA) in beakers or in sealed polyethylenefilms, shaking gently. The hybridization temperature was 65C. The nitrocellulose filters were washed with 6 x SSC, 0.05 M sodium pyrophosphate at room temperature for one hour and at the particular hybridization temperature for a further hour. The filters were dried and auto-radiographed overnight. X-ray film signals which occurred on both duplicates were assigned to the Petri dish, and the region (about 50 plaques) was removed by stabbing with the wide end of a Pasteur pipette, and the phages were resuspended in 1 ml of SM buffer. Positive phages were singled out in three cycles until a single clone was obtained.

In total, 1 x 106 PFU of the placenta cDNA bank were examined. 2 further signals were identified on duplicate filters (clones PP11-166 and PP11-169). Finally, with the aid of the 46mer from PP11-169, which is mentioned above (p.3), the clone PP11-318 which covers the entire 5' end of the PP11 cDNA was found. These 4 clones together carry a PP11-encoding sequence as depicted in Tab. 2 and the figure.

10. DNA sequence analysis The phage clones (clones PP11-93, PP11-166, PP11-169 and PP11-318) were multiplied and the DNA of each was ex-tracted. The relevant EcoRI fragment was isolated andligated into the EcoRI site of the Bluescript M13 vector (Stratagene, San Diego, CA, USA) for restriction analyses and sequence analyses by the enzymatic dideoxy method of Sanger. The sequence has an open reading frame and codes for a protein with a maximum of 369 amino acids. Thus, there are 154 bp not translated at the 5' end, and these are followed by 1107 bp of coding sequence and finally 1138 bp of untranslated sequence at the 3' end.

11. Expression of PPll in bacterial cells _ 13 -a) Expression of ~ PPll fu~ion protein pTrc99C (E. Amann et al., Gene 69 tl988) 301-315) was completely digested with EcoRI and XbaI, and the fragment 4149 base-pairs in size was isolated by gel electro-phoresis. The PP11 clone lambda gtll-169 was digested with EcoRI, and the EcoRI insert which is 1785 base-pairs in size, including the linker portion, was likewise \ isolated. The PPll cDNA of the clone lambda gtll-169 starts at position 140 in Figure ~. The resulting fragment was then cut with XbaI, and the EcoRI-XbaI
fragment which is 1339 base-pairs in ~ize was isolated and ligated to the pTrc99C vector fragment described above. The 5488bp plasmid pTrc99C-PPll obtained in this way is able to induce the synthesis of an approximately 42 kD protein in Escherichia coli cells. This protein can be specifically immunoprecipitated with aid of a mono-specific rabbit anti-PPll antiserum which has been raised by immunization with PPll isolated from human placenta.
PPll expression in Escherichia coli cells is additionally detected by Western blot analyses using the above serum.
In this experiment, only the extract from IPTG-induced cells containing pTrc99C-PPll plasmid reacted, once again a protein band of about 42 kD being specifically visual-ized. Escherichia coli control extracts without plasmid, and extracts containing pTrc99C-PPll but not induced with IPTG, did not react with the abovementioned anti-PPll antiserum. The PPll fusion protein described by the pla~mid construction generated hereinbefore has the following N-terminal amino acid sequence, defined by the following nucleotide seguence:
Vector/linker / 5' UT region / PPll Met Gly Asn Ser Leu Gln Leu Gly Thr Met Arg Ala ..CC ATG GGG AAT TCT CTC CAA CTG GGC ACC ATG AGC GCC...

The PPll fusion protein defined by this construction carries, in addition to the complete PPlI amino acid sequence encoded by the PPll cDNA, nine amino acids in .
` ' ' '' ' . , ZC~ 53~

front of the N-terminus: four vector-encoded amino acids plus five amino acids specified by the 5' untranslated region occurring in the PP11 cDNA. As a check, the construction indicated hereinbefore was likewise carried out with the expression vectors pTrc99A and pTrc99B
(German Patent Application P 38 19 463.5 and Amann et al.
(1988) Gene 69, ~01-315). These vectors differ from pTrc99C only by 2 base-pairs (pTrc99A) and 1 base-pair (pTrc99B), which cause shifts in the translation reading frame. As expected, neither pTrc99A-PPll nor pTrc99B-PP11 was able to induce the synthesis of PPll proteins react-ing with anti-PPll antisera.

It was found, surprisingly, that the PPll protein induced by plasmid pTrc99C-PP11 is processed in E~cherichia coli.
The signal sequence which in nature precedes the mature PPll protein (also called leader sequence) is recognized by E.coli cells and cleaved by the signal peptidase. It is possible to conclude this from the finding that the PP11 protein detected in the periplasmic fraction after fractionation of cell extracts is smaller than in the membrane-associated fraction, with the observed molecular weight difference corresponding to the expected dif-ference after elimination of the signal sequence.

b) Expression of the mature unfused protein:

To express the mature PP11 protein lacking the naturally occurring signal sequence, the EcoRI-XbaI fragment which is 1339 base-pairs long and is described hereinbqfore was ligated to the mutagenesis vector pMa5-8 which had been digested with the same restriction enzymes. The muta-genesis vector pMa5-8 carries a cloning polylinker region and the origin of replication of the single-stranded bacteriophage F1, in addition to a bacterial origin of replication and an antibiotic-resistance marker. The resulting plasmid pMa5-8-PP11 was used to prepare a single strand. The single strand of the cloned PP11 cDNA
obtained in this way was then isolated by known methods 6~35 _ 15 -and subjected to the published gapped duplex mutagenesis protocol (Kramer et al. (1984) Nucl. Acids. Res. 12, 9441-9456), using the following oligodeoxynucleotide, 5~ TTTGTGGTCCTCCATGGAGGCCAGGCCACA 3' A clone which had the desired NcoI mutation (creation of a new NcoI site, which was not present in the originally isolated cDNA, immediately in front of the coding se-quence for the mature PPll protein) was identified by appropriate restriction analysis and called pMa5-8-PPll-NcoI. Following partial NcoI and complete XbaI digestion,the NcoI-XbaI fragment which is 1268 bp in size was isolated from this plasmid and ligated into the vector pTrc99A which had been cut in the same way. The resulting plasmid contains 5412 bp and, after induction of the trc promoter with IPTG, expresses the mature unfused PPll protein with a molecular weight of about 42 kD, which in turn specifically reacts with anti-PP11 antisera, as already described hereinbefore.

12. Amidolytic activity of PP11 PP11 was shown to have protease activity in the following colorimetric assay mixture. Periplasmic or cytoplasmic fractions of E.coli strain RB791 (R. Brent and M. Ptashne (1981) Proc.Natl.Acad.Sci ~SA 78, 4204-4208) transformed with pTrc99C or pTrc99C-PP11 were measured in an optical assay using the chromogenic substrate Chromozym0 TH
(Boehringer Mannheim GmbH). 400 ~1 of triethanolamine buffer and 100 ~1 of substrate were mixed with 300 ~1 of each E.coli extract, these extracts having been diluted to a protein concentration of 16 mg/ml. The reaction cuvette was incubated at 37C, and the change in absorp-tion per minute at 405 nm was recorded. In some experi-ments the E.coli extract and control extract were pre-incubated with a rabbit anti-PP11-antiserum, and the amidolytic activity of the antiserum was subtracted in the calculation of the amidolytic activities of the extracts. At the same total protein concentration, the PP11-containing E.coli extract brought about a larger .

~0~6535 increase in absorption (~A = 0.04 per minute) than the control (~A = 0.024 per minute), corresponding to 32 mU/ml and 19.2 mU/ml respectively. The amidolytic activity of PP11 was almost completely inhibited by pre-incubation with anti-PP11 antiserum. Furthermore, the cytoplasmic unprocessed PP11 with the molecular weight of 45 kD did not have this amidolytic activity. Since the amidolytic activity is inhibited by diisopropyl fluoro-phosphate, PPll is probably a serine proiease.

-` 2~535 CTTCCTGAAAGGATCTGGAGACACCAGCTCCACAAGTCCTGGTGTCTTTAAAAGGATCAG

CTTGAGGAATAAGGCTCGTCTGAGAGCTGTGACATTCATCTGACTCTAGTGAAAGTCCAA

M R A C I S L V L

GGCCGTGCTGTGTGGCCTGGCCTGGGCTGAGGACCACAAAGAGTCAGAGCCATTGCCACA
A V L C G L A W A E D H R E S E P L P Q

GCTGGAGGAAGAGACAGAAGAGGCCCTCGCCAGCAACTTGTACTCGGCACCCACCTCCTG
L E E E T E E A L A S N L Y S A P T S C

CCAGGGCCGCTGCTACGAAGCCTTTGACAAGCACCACCAATGTCACTGCAATGCCCGCTG
Q G R C Y E A F D K H H Q C H C N A R C

CCAAGAGTTTGGGAACTGCTGCAAGGATTTTGAGAGCCTGTGTAGTGACCACGAGGTCTC
Q E F G N C C K D F E S L C S D H E V S

CCACAGCAGTGATGCCATAACAAAAGAGGAGATTCAGAGCATCTCTGAGAAGATCTACAG
H S S D A I T K E E I Q S I S E K I Y R

GGCAGACACCAACAAAGCCCAGAAGGAAGACATCGTTCTCAATAGCCAAAACTGCATCTC
A D T N K A Q K E D I V L N S Q N C I S

CCCGTCAGAGACCAGAAACCAAGTGGATCGCTGCCCAAAGCCACTCTTCACTTATGTCAA
P S E T R N Q V D R C P R P L F T Y V N

TGAGAAGCTGTTCTCCAAGCCCACCTATGCAGCCTTCATCAACCTCCTCAACAACTACCA
E K L F S R P T Y A A F I N L L N N Y Q

GCGGGCAACAGGCCATGGGGAGCACTTCAGTGCCCAGGAGCTGGCCGAGCAGGACGCCTT
; R A T G H G E H F S A Q E L A E Q D A F

CCTCAGAGAGATCATGAAGACAGCAGTCATGAAGGAGCTCTACAGCTTCCTCCATCACCA
L R E I M K T A V M R E L Y S F L H H Q

GAATCGCTATGGCTCAGAGCAAGAGTTTGTCGATGACTTGAAGAACATGTGGTTTGGGCT
N R Y G S E Q E F V D D L K N M W F G L

Tab. 2 .
-2C~)6535 CTATTCGAGAGGCAATGAAGAGGGGGACTCGAGTGGCTTTGAACATGTCTTCTCAGGTGA
Y S R G N E E G D S S G F E H V F S G E

GGTAAAAAAAGGCAAGGTTACTGGCTTCCATAACTGGATCCGCTTCTACCTGGAGGAGAA
V K K G K V T G F H N W I R F Y L E E K

GGAGGGTCTGGTTGACTATTACAGTCACATCTACGATGGGCCTTGGGATTCTTACCCCGA
E G L V D Y Y S H I Y D G P W D S Y P D

TGTGCTGGCAATGCAGTTCAACTGGGACGGCTACTATAAGGAAGTGGGCTCTGCTTTCAT
V L A M Q F N W D G Y Y K E V G S A F

CGGCAGCAGCCCTGAGTTTGAGTTTGCACTCTACTCCCTGTGCTTCATCGCCAGGCCAGG
G S S P E F E F A L Y S L C F I A R P G

CAAAGTGTGCCAGTTAAGCCTGGGAGGATATCCCTTAGCTGTCCGGACATATACCTGGGA
K V C Q L S L G G Y P L A V R T Y T W D

CAAGTCCACCTATGGGAATGGCAAGAAGTACATCGCCACAGCCTACATAGTGTCTTCCAC
K S T Y G N G K K Y I A T A Y I V S S T

CTAATAGAACTTCGAGCCAGAAAGGGGCATGAGGGCTCTTGCGAGACTGAAGTGCTATCT

TCTCTGGACTAGAGAGAAGAGGGAGAGGACTGGAAGGGATCACCAAATCTCAAAGCAATG

AGAAGCATTCCTAAATCCCAAAGTGCCCACATGGGAAAGAGATAAAATGTACAAATTAGA

AAAATGTGGATAAACAGTCAAACCTTTATCCTCTAGAATTTTGGCAATGTTGACTAAGAA

ACAGAGTCCAAGCAGAGAAGGTAGGAACCCTCCATAGCTCTCTGCCCTGATGTGTGGGGG

AACTAGGAAGAAGTCCTTTGACCTCACCAGGCCTCATGCTTCCCTTTAATGTAAAGGGAA

GGGGTTTGCCCACTTTCCTCTTTTTGGGGTTGGTGAGAGGGCAAACCCTGATATTTTTAC

TGTGAAGGTGTTTTCAGTTGTTCTTAGGAAGAACAGCTGATAGAAATTCAAGATTACTAT

AATGGCTGTTATTATACACAGCTCTGTAAACTACCACTCAGCCCTGTGTTGGGGTCCTCA

Tab. 2 continued ~)6S:~S

AAGAAGTAAGGCCACAGTAATCAAGCAAGGGCCTTTGGTTTTTTCCAGAGTTAGATCCTC

TCAGAACAGAGTCTGGGAGAACTCCAATGCTGAATGGAGAAGGGTAATAGGTTGGTTGCA

GTGAATGGGCTGGGGGTGGGGTGGCCTTCTCCAGGCCTGAGTGTTTTTGTGTCCAGCTCA

GTATCTGCAACAAGAAGTTTCCCACTTGTGGATGTTTAGTGCAGCCACAGACTTGTATTT

TGATCCCCAATTTTTTTTGAAAGAGTTCTCCTCATAGGAGGATGATTCAGCATCAGAAGA

AGAAGGAACCCATAGCTTGGTGTCATTAACATAATTATTTTAAGCCTTATCCAGCAGCCA

TAATTTGAATAACTCTACGAGACCAGAGAGACTGTAGTTCCCTATTTTAACCTCAATTAT

GCATTTGTCCCCAACCCCACTGAGAACTAAATGCTGTACCACAGAGCCGGGTGTGAACTA

TGGTTTAGAAGGTTcAAGTTTccAATTAAAGTcATTGAAGAb8-~uu4~uu~u~AAAA~A

Tab. 2 continued

Claims (18)

1. A DNA sequence coding for the amino acid sequence shown in Table 2, the alleles and variants thereof.

2. A DNA sequence coding for placenta-specific protein PP11, containing the coding strand shown in Table 2.

3. DNA or RNA which hybridizes with the DNA as claimed in claim 1 under stringent conditions.

4. A gene structure containing a DNA or RNA as claimed in claim 1, 2 or 3.

5. A vector containing a DNA or RNA as claimed in one or more of the preceding claims.

6. Transformed cells containing DNA or RNA as claimed in claim 1, 2, 3, 4 or 5.

7. PP11 obtainable by genetic manipulation, which has the amino acid sequence shown in Table 2.

8. PP11 which is prepared by genetic manipulation in E.coli by expression of the DNA sequence as claimed in claim 2.

9. PP11 which is prepared by genetic manipulation in yeast by expression of the DNA sequence as claimed in claim 2.

10. A process for the preparation of PP11, which comprises a cDNA or RNA as claimed in claim 1, 2, 3 or 4 being introduced into an expression system and expressed therein.

11. Polyclonal or monoclonal antibodies specific for PP11, obtained from PP11 prepared by genetic manipulation, or parts thereof having antigenic activity.

12. A diagnostic aid which contains a DNA or RNA as claimed in claim 1, 2, 3 or 4, in whole or in part.

13. A diagnostic aid which contains a DNA or RNA, in whole or in part, which is complementary to the DNA as claimed in claim 1.

14. A diagnostic aid containing antibodies as claimed in claim 11.

15. A diagnostic method which comprises contacting body fluids, tissue, or nucleic acids isolated therefrom with a diagnostic aid as claimed in claim 12, 13 or 14.

16. PP11 for use as a pharmaceutical.

17. A pharmaceutical which contains PP11 prepared as claimed in claim 7.

18. The DNA sequence as claimed in claim 1, and substantially as described herein.