AU616248B2 - Protein pp 15 prepared by genetic manipulation - Google Patents

Abstract

The cDNA coding for PP15 is described. This cDNA can be used to prepare PP15 in pro- or eukaryotic cells. <IMAGE>

Description

Nlu~U~ .11,1 mi ii.i...ii,.,ii...,.ii.i,.,,ii^unBC3gg^wjwA 616248 Form COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class Application Number: Lodged: Int. Class Cbmplete Specification Lodged: Accepted: SPublished: Priority C C o Related Art: N'me of Applicant: Address of Applicant: Actual Inventor: Address for Service BEHRINGWERKE AKTIENGESELLSCHAFT D-3550 Marburg, Federal Republic of Germany ULRICH GRUNDMANN, KARL-JOSEF ABEL and EUGEN AMANN EDWD. WATERS SONS, QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled: PROTEIN PP PREPARED BY GENETIC MANIPULATION The following statement is a full description of this invention, including the best method of performing it known to Us -1a- Behringwerke Aktiengesellschaft HOE 88/B 007 Ma 671 Dr. LP/AW Description Protein PP 15 prepared by genetic manipulation The protein PP 15, which has an immunosuppressant action, is described in DE-A 29 52 792 (US-A 4,348,316) with the foLLowing parameters: n 0 0 S 0O 10 0 0 o 0 4 a 0 0 0 a) a carbohydrate content of 3.35 composed of 2.8 0.5% hexoses, 0.3 0.2% hexosamines, 0.05 0.05% fucose and 0.20 0.15% neuraminic acid; b) a sedimentation coefficient S20.w 0 of 2.9 0.2 S; c) a molecular weight determined in the ultracentrifuge of 30,700 3,200 (dimer); d) an extinction coefficient E 1% cm (280 nm) of 14.2 1.0, and e) an electrophoretic mobility in the region of that of aLbumin, as well as f) an isoelectric point of 4.4 0.1; g) the amino acid composition Amino acid Lysine His t i dine Argin i ne Aspartic acid Threonine Ser in e Glutamic acid Pro ine Glycine ALanine Cystine 1/2 Residues per 100 residues (mol-%) 4.74 3.81 1.62 13.39 3.85 6.38 13.43 4.35 6.87 6.51 2.48 Coefficient of variation 3.30 5.43 3.43 5.08 5.35 2.81 5.32 14.25 2.13 8.26 4.55 11

II

2 Amino acid Residues per Coefficient of 100 residues variation (mol-%) Valine 2.29 15.67 Methionine 2.87 10.86 Isoleucine 8.39 8.18 Leucine 8.18 6.72 Tyrosine 2.09 8.49 10 PhenylaLanine 6.27 2.27 o Tryptophan 2.51 6.81 °nn Determination of the moLecular weight by SDS polyacryL- 0o 15 amide geL electrophoresis yielded a molecuLar weight of about 15,000 d (monomer).

00 0 oo Because of the therapeutic interest aroused by the immunosuppressant properties, and of the diagnostic .00 20 interest, a preparation of this protein by genetic manipulation is extremely desirable. Consequently, the invention relates to a process for the preparation of 0o PP15 by genetic manipulation, to the mRNA necessary for this, to the cDNA obtained therefrom, to DNA structures 25 and vectors containing this DNA in whole or in part, to cells transformed with such DNA, to the polypeptide expressed by these cells, and to the use thereof as pharmaceuticals. The invention further relates to the amino acid sequence and to part-sequences of the amino acid sequence of PP15, to specific antibodies obtained therewith, to diagnostic aids and antibody columns prepared from these antibodies, and to the polypeptide obtained using such columns. A further embodiment of the invention relates to diagnostic aids which contain, in whole or in part, RNA or DNA encoding PP15, or complementary thereto, and to diagnostic methods with which body fluids and tissue are examined using such diagnostic aids.

Further aspects of the invention are explained in detail hereinafter and defined in the patent claims.

i -3 Initially, an attempt was made, using specific antibodies against PP15, to detect in a commercialLy available cDNA expression bank composed of mRNA from mature human placenta- (f-rom Genofit, Heidelberg) clones which express PP15. It was known that PP15 has an immunosuppressant action, and consequently it was possible to prepare specific antibodies only unsatisfactorily, if at all, which is why specific antibodies against peptide fragments were prepared.

S I For this reason, the protein PP15 was broken down by 0 e cleavage with cyanogen bromide, trypsin or proteinase V8 o into specific fragments which were subsequently sequenced.

o The following fragments were obtained: 0 15 MVV G Q LKADEDP IMGF HQ M F F R L A L H N F G o V S V Y A E A A E R .o L S S L P F Q K I Q (H) DN D RTQ LG AI YID A S- LT- E 20 So L L K N I N D A WT Peptides A, B and C were synthesized by generally known 0" methods, and specific antibodies were raised in rabbits °o by customary processes. It was not possible to locate positive clones in the abovementioned cDNA expression bank, which contained 1 x 106 recombinant lambda gtll clones. Thus, antibodies against peptide A and peptide B precipitated PP15 in control experiments, whereas antibodies against peptide C did not react. Moreover, as will be seen later, peptide C is not present in the protein sequence of PP15 subsequently derived from the cDNA sequence, so that it ought probably to be assigned to concomitant proteins of Subsequently, statistical data by R. Lathe Mol. Biol.

(1985) 183, 1-12) were used to select from the oligonucleotides coding for PP15 oligopeptide A the PP15 oligonucleotide 103 GCCAGCTGAA GGCTGATGAG GACCCC, I -4and correspondingly from the oligopeptide E the oligonucleotide 140 ACCGGACCCA GCTGGGCGCC ATCTACATTG

ATGC

and from the oligopeptide F a 64-fold degenerate oligonucleotide 139 G C C C C

A

These oligonucleotide probes were used to screen a cDNA bank prepared from mRNA from mature human placenta. The mRNA was initially isolated from the placenta and then °o15 used to prepare the cDNA. The Latter was provided with EcoRI ends and ligated into the EcoRI cleavage site of S" the phage vector lambda gt10. 2 clones (PP15-24 and 28) which contain the complete cDNA of PP15 were detected. DNA sequencing was carried out by methods known per se; the complete sequence of PP15 cDNA (coding strand) is shown in Tab. 1. This cDNA is 894 base-pairs long, has a 99 bp untranslated sequence at the end, has an open reading frame of 381 bp, and leaves 414 bp, including eight bases of poly(A), untranslated '2 .25 at the 3' end.

The positions of the nucleotide probes are indicated by underlining in Table 1, and the amino acid sequence is additionally inserted.

S 0 4 It is possible according to the invention for the coding cDNA to be used, with the aid of suitable expression systems, to express PP15. Furthermore, the type of modification of PP15 can be influenced by the choice of the host. Thus, no glycosylation takes place inbacteria, while that taking place in yeast cells differs from that in higher eukaryotic cells. t Knowing the amino acid sequence of PP15, it is possible 5 to prepare, by conventional or genetic manipulation methods, amino acid part-sequences which can be used as antigens for the preparation of polycLonaL or monoclonal antibodies. Such antibodies can be used not only for diagnostic purposes but also for the preparation of antibody columns with which it is possible to separate from solutions which contain it together with other proteins.

10 It is also possible using the cDNA, or parts thereof, to isolate in a straightforward manner from a genomic bank Sthe genomic clone which codes for PP15 and which not only .ooo facilitates the expression in eukaryotic cells but also "OD allows further diagnostic conclusions to be drawn.

ci O0 00 S The invention is further defined in the patent claims and is explained in detail in the Examples which follow.

oo o 00 The following abbreviations are used, apart from those 20 explained in the text: o 00 EDTA sodium ethyLenediaminetetraacetate SDS sodium dodecyl sulfate 00 DTT dithiothreitol o25 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 (1979) 5294-5299).

About 10 g of placental tissue were ground in liquid nitrogen in a mortar, suspended in 80 ml of 4 M guanidinium thiocyanate containing 0.1 M mercaptoethanol, and treated in a homogenizer (Ultraturrax) at 20,000 rpm for sec. The lysate was centrifuged (Sorvall GSA rotor) at 7,000 rpm for 15 min, and the supernatant was precipitated with 2 ml of 1 M acetic acid and 60 ml of abs.

ethanol at -20 0 C overnight. The nucleic acids were 1 I It 6 sedimented at 6,000 rpm and -100C for 10 min and then 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 abs. ethanoL. To remove the DNA, the precipitation was repeated once more with each of the voLumes being halved. The RNA was dissolved in 12 mL of H 2 0, precipitated with a mixture of 1.2 ml of 4 M potassi'jm acetate and 24 ml of abs. ethanol sedimented and, finally, again taken up in 10 mL of H 2 0 (1 ml per g of tissue).

2. Obtaining poly(A)-containing placental mRNA 000 44o o To obtain poly(A)-containing mRNA, the placental RNA was S 15 fractionated by oligo(dT)-cellulose chromatography (Aviv Sand Leder, Proc. Natl. Acad. Sci. USA 69 (1973) 1408- 1412) in 2 ml Pasteur pipettes in LiCL. About 5 mg of °O placental RNA in buffer 1 (500 mM LiCL, 20 mM Tris (pH 0o0 1 mM EDTA, 0.1% SDS) were applied to 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 mM LiCL, 29 mM Tris (PH 1 mM EDTA, 0.1% SDS), the poly(A) 1 RNA (placental mRNA) was eLuted from the column with buffer 3 25 (5 mM Tris (pH 1 mM EDTA, 0.05% SDS).

For further purification, the poLy(A) RNA was adjusted to buffer 1 and again chromatographed on oligo(dT)cellulose. The yield of placental poly(A) RNA after this second purification step was about 4% of the RNA used.

3. Synthesis of cDNA from human placenta (placental cDNA) and double-stranded cDNA (dsDNA) The integrity of the poLy(A)-containing placental mRNA was checked in a 1.5% agarose gel before the cDNA synthesis.

Then 4 pg of placental mRNA were dissolved in 65.5 Iu of H 2 0, denatured at 700C for 10 min and cooled in ice.

i, -4 7 The cDNA was synthesized in a 100 pl mixture after addition of 20 p1 of RT 1 buffer (250 mM Tris (pH 8.2) at 420C, 250 mM KCI, 30 mM MgCL 2 2.5 pL of 20 mM dNTP all four deoxynucLeoside triphosphates), 1 pu of oLigo(dT) of 1 pg/ml, 1 pL of 1 M DTT, 2 ul of RNAsin (Boehringer Mannheim) and 8 pl of reverse transcriptase (24 U/pL Boehringer Mannheim) at 42 0 C for 90 min. DoubLe-stranded cDNA (dsDNA) was synthesized by the method of GubLer and Hoffmann (Gene (1983) 263-269). The synthesis was carried out immedi- 10 ately after the cDNA synthesis by addition of 305.5 uL of

H

2 0, 80 pL of RT 2 buffer (100 mM Tris (pH 25 mM MgCL2, 500 mM KCI, 50 mM DTT, 250 pg/ml BSA), 2 pl of RNase ,066~Q So° H (2 U/pl), 2.5 pl of E. coli DNA Ligase (5 U/pl), 5 pl of 6 9o 0 0 15 mM B-NAD, and 5 pl of DNA poLymerase I (5 U/pl) and incubation at 150C for 5 h. The reaction was stopped by heat inactivation (700C, 30 min).

6 After addition of 55 pl of 250 pM dNTP, 55 pL of 10 mM Tris (pH 10 mM MgCL 2 10 pg/mL BSA, 3 p1 of T4 DNA polymerase I (1 U/pI), 2 L1 of RNase H (2 U/pL) and 2 pL of RNase A (2 pg/ml) to the reaction mixture it was 64 incubated at 370C for a further 13 min in order to en- Ssure that the synthesis on 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 placental cDNA bank, the dsDNA was provided with EcoRI ends in order to be able to Ligate it into the EcoRI cleavage site of the phage vector Xgt10 (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 of the dsDNA, and b) provided with EcoRI linkers which 8 c) were then opened with EcoRI.

i Re a): The methylase reaction of dsDNA was carried out directly foLLowing the repair reaction after addition of 25 pl of 500 mM EDTA (pH 60 pL of methyLase buffer (100 mM NaOAc (pH 2 mg of S-adenosyl-L-methionine) and 2 pL of EcoRI methylase (20 U/pL) by incubation at 37°C for 30 min.

i 10 The reaction mixture was extracted with phenoL, and the dsDNA was precipitated with 60 pL of 4 M NaOAc and i: 130C pL of ethanoL. The dsDNA was washed twice with ethanoL, extracted by shaking once with ether, and dried.

0 15 Re b): o The EcoRI-methyLated dsDNA was dissoLved in 88 pL of H 2 0 and, after addition of 10 pL of Ligase buffer (500 mM 000 Tris (pH 100 mM MgCL 2 100 mM DTT, 100 mM spermidine, 10 mM ATP, 1 mg/mL BSA) and 1 pL of T4 DNA Ligase (10 U/pL), S 20 was Ligated with 1 pL of EcoRI Linkers (0.5 pg/pL) (pGG-AATTCC and pAGAATTCT) at 15 0 C overnight.

Re c): The voLume of the Ligase mixture was made up to 120 pL 25 with 6 pL of H 2 0, 12 yL of 10 x EcoRI buffer and 2 pl of EcoRI (120 U/pL). The EcoRI digestion was carried out at 370C for 2 h.

RemovaL of unbound Linkers on a potassium acetate gradient, and seLection of the dsDNA for size ALL unbound EcoRI Linkers were removed from the dsDNA by appLying the EcoRI reaction mixture in toto to a potassium acetate gradient (5-20% KOAc, 1 mM EDTA, 1 pl/mL ethidium bromide) and centrifuging (Beckman SW 65 rotor) at 50,000 rpm and 200C for 3 h.

The gradient was fractionated from beLow in such a way that the first five fractions measured 500 pL, and aLL the remainders measured 100 pL. The fractions were preiF i S- 9cipitated with 0.01 volume of acryLamide (2 mg/ml) and volumes of ethanol, washed once with 70% strength ethanol and dried, and each was taken up in 5 pL of H 2 0.

To determine the size of the dsDNA, 1 pl of each fraction was analyzed in a 1.5% agarose gel. In addition, the *t quantity of dsDNA was determined using 1 I L OT each fraction.

Fractions containing dsDNA above 500 bp were combined, and the sample was concentrated until the final concen- S 0 tration was 27 pg/ml.

0oes 6. Insertion of the dsDNA into the phage vector and in vitro packaging reaction 0 0 The dsDNA was inserted into the EcoRI cleavage site of the phage vector Xgt10 (Vector Cloning Systems, San Diego, CA) in a 4 pl ligase mixture: 2 pl of dsDNA, 20 1 pL of Xgt10 x EcoRI (1 pg/ml), 0.4 pL of ligase buffer, S 0.5 pl of H 2 0, 0.1 pI of T4 DNA ligase. The mixture was incubated at 150C for 4 h.

To establish the placental cDNA bank in the phage vector 25 Xgt10, the ligase mixture was subsequently subjected to an in vitro packaging reaction with the X-lysogenic cell extracts E. coli NS 428 and NS 433 at room temperature for 2 h (Vector Cloning Systems, San Diego, CA; Enquist and Sternberg, Methods in Enzymology 68, (1979), 281-298).

The reaction was stopped with 500 pl of suspending medium (SM: 0.1 M NaCL, 8 mM MgS0 4 50 mM Tris (pH 0.01% gelatin) and 2 drops of chloroform.

7. Titer determination and analysis of the placental cDNA bank The number of plaque-forming units (PFU) of the placental cDNA bank were determined using competent cells of E. coli K 12 strain C600 HFL: it was 1 x 106 PFU.

8. Oligonucleotide probes for screening the placental cDNA bank OligonucLeotide probes (PP15 oligonucleotide 103 and 140) and a pool of oLigonucleotides (PP15 oLigonucleotide pool 139) were synthesized for the analysis of the placental cDNA bank. Their sequences were derived from the amino acid sequence of three cyanogen bromide fragments of The manner of construction and the use of the probes essentially followed the rules of R. Lathe, Inc. cit.

0 0 0 The oligonucleotide sequences were labeled at the 5' end 000 32 90o0 using T4 polynucleotide kinase in the presence of P) S 15 ATP (using 60 pCi/40 pL of reaction mixture). The probes 8 6 had a specific activity of 1 x 10 Bq/pl or 1.5 x 10 Bq/ pmol.

0oo 9. Screening of the placental cDNA with 0 20 oligonucleotides 1 x 106 PFU of the placental cDNA bank were examined with the PP15 oligonucleotide probes 103, 140 and 139 too, gether. For this purpose, 3 x 10 PFU were plated out with o 25 cells of the E. coli K 12 strain C 600 HFL in soft agar on 13.5 cm Petri dishes and incubated at 370C for 6 h.

Lysis was still incomplete at this time. The plates were incubated in a refrigerator overnight, and the phages were transferred to nitrocellulose filters (Schleicher SchulL, BA 85, Ref. No. 401124) (duplicates). The nitrocellulose filters and Petri dishes were marked with an injection needle to allow later assignment of positive plaques.

SDuring the processing of the nitrocellulose filters, the Petri dishes were stored in a cold room. The DNA on the nitrocellulose filters was denatured by placing the filters on filter paper (Whatman M3) impregnated with M NaCL, 0.5 M NaOH for 5 min. The filters were then renatured in the same way using 1.5 M NaCI, 0.5 M Tris (pH and washed with 2 x SSPE (0.36 M NaCL, 16 mM NaOH, *1 L! I:

C

,J.B

~I

f 044000 S o 0 0 0 0 00 0 S0 440 0 0 bYBO O o 0 0 V 00 Q 00 4 00 C0 0 11 20 mM NaH 2 P04, 2 mM EDTA). The filters were then dried in vacuo at 800C for 2 h. The filters were washed in 3 x SSC, 0.1% SDS (20 x SSC 3 M NaCL, 0.3 M Na citrate) at 65 0

C

for 4 h and prehybridized at 65 0 C for 4 h (prehybridization solution: 0.6 M NaCl, 0.06 M Tris (pH 6 mM EDTA, 0.2% non-ionic synthetic sucrose polymer (RFicoll), 0.2% polyvinylpyrrolidone 40, 0.2% BSA, 0.1% SDS, 50 pg/m lenatured herring sperm DNA). The filters were incubated overnight with the addition of 100,000-200,000 Bq of the labeled oligonucleotide per ml of hybridization solution (as prehybridization solution but without herring sperm DNA) in beakers or in sealed polyethylene films, shaking gently. The hybridization temperature was 460C for oligonucleotide probe 139 and 52 0 C for the other probes. The 15 nitro-cellulose filters were washed with 6 x SSC, 0.05 M sodium pyrophosphate at room temperature for one hour and at the relevant hybridization temperature for a further hour. The filters were dried and autoradiographed overnight. Signals which appeared on both duplicates of the X-ray film were assigned to the Petri dishes, and the region (about 50 plaques) was punched out with the wide end of a Pasteur pipette, and the phages were resuspended in 1 ml of SM buffer. Positive phages were singled out over three cycles until a single clone was obtained.

0 0 0 0 0"000 Three samples each of 1 x 10 6 PFU of the placental cDNA bank were examined. Not until the third screening were 2 signals identified on duplicate filters. The two clones PP15-24 and PP15-28 contain the romplete cDNA of Tab. 2 compares the oligonucleotide sequences 103, 139 and 140 with the PP15 sequence found.

Table 2 sequence vs. PP15 oligonucleotide 103 S 1-- '1 s ~401 AC if! 111 S~lil s351 GC 3 G( 1 1 j "K 4 15 444 151 TA 12 rCACCGCGCAGGACCATCAGCCCACTCCAGATAGCTGCATCATCAGCAT 350 AT 2 ;TTGTGGGCCAGCTTAAGGCGGATGAAGACCCCATCATGGGGTTCCACC 400 )TGGTGGGCCAGCTGAAGGCTGATGAGGACCC 36 jequenz vs. PP15-Oligonukleotid 139 ;ATGTTCCTATTAAAGAACATCAACGATGCTTGGGTTTGCACCAATGAC 1450 AAGAACATCAACGATGCCTGGAC.............23 equenz vs. PP15-Oligonukleotid 140 4 48 4 1..

CTACCAGTTATTTGATAATGATAGAACCCAACTAGGCGCAATrTACAT 200 TTTGACAATGACCGGACCCAGCTGGGCGCCATCTACAT 38 201 TGACGCGTCATGCCTTACGTGGGAAGGACAACAGTTCCAGGGGAAAGCTG 250 39 TGATGC 44 .444 4 4 4~ 4 4 4 4 46 444 4 44 4, 30 10. DNA sequence analysis The phage clones PP15-24 and PP15-28 were propagated, and the DNA of each of them was extracted. In each case the EcoRI fragment was isolated and Ligated into the EcoRI site of the BLuescript M13 vector (Stratagene, San Diego, CA, USA) for restriction analyses and sequence analyses using the enzymatic dideoxy method of Sanger.

The sequence shows an open reading frame and codes for a protein having a maximum of 127 amino acids. PP15 has a calculated molecular weight of 14478 d (including methionine), which agrees well with the figure, mentioned in the introduction, from the Patent DE-A 2,952,792.

It 13 TabLe 1 30

GGAAGGGACAGTCGGCCGCAGACCGCGCTGGGTTGCCGCTGCCGCTGCCGCCATCGTGCC

90 110

AGCCCCTCGGGTCTCCGTGAGGCCGGGTGACGCTCCAGAATGGGAGACAAGCCAATTTGG

M G D K P1IW hi130 150 170 GAGCAGATTGGATCCAGCTTCATTCAACATTACTACCAGTTATTTGATAATGATAGAAC

C

E Q I G S SF1I Q H Y Y Q L F D N D R T 190 210 230 CAAC TAG G CGCAATTTACATTGAC GC GTCATG CC TTAC GTGGGAAGGACAACAGTTC CAG QL G A I Y I D A S C L T W E G Q Q F Q 250 270 290 GGGAAAGCTGC CATTG TGGAGAAGTTGTC TAG C CTTC CGTTC CAGAAAATTCAG CA CAGC 0 0 G K A A I V E K L S S L P F Q K I Q H-iS 310 330 350 ATCACCGCGCAGGAC CATCAGCCCACTCCAGATAGCTGCATCA:TCAGCATGGTTGTGGGC 0 v I T A Q D H Q P T P D S C I I S M V V G 370 390 410 CAG CTTAAGGCGGATGAAGAC CC CAT CATGGGGTTC CAC CAGATG TTC CTATTAAAGAAC Q L K A D E D P INMG F H Q M F L L F N 430 450 470

ATCAACGATGCTTGGGTTTGCACCAATGACATGTTCAGGCTCGCCCTGCACAACTTTGGC

I N D A W V C T N D M F R L A L H N F G 490 510 530

TGACCTCCTCTCAGCTAGGCACTCACGCTGTTTCCTCCTCCCTCCTCTTCCCAATACTAT

550 570 590 TCCCACTCCTCCAGATGrTCCAAATATCATGCACAA.ATGAGCAGGGCCGCGGTGGGAGTG 610 630 650

GGCGCAGTGCGCTGCTGCCACTGAGGTGTTGTGCATGATGTTTGGATGCTAGACTAGTTG

670 690 710 CATCTGACGGGAGAAGTTTGTGTTGTACCAGCGCATGCCTTGGAAAGAC ;TAAGTAATGC 730 750 770

AAAAGGTTGTCCTTTTTTTTTTTTTTTTTTTTTTAATCTACTGACAAGTTGCTCTAGTAA

14790 810 830 CCCAAAGAAGTGAAGGAGAA.AGCAGCTGCCTCACCGC CCAGACATTGATTTGTTCAGATG 850 870 890

TTTCAATGCCTCATGATACAATAAAACCACAAAAATTTTCTTAACAAAAAA

-14 11. Expression of the immunosuppressive protein The vector pTrc99A Amann et al. (1988) Gene 69, 301- 315) was used to express the non-fused mature PP15 protein in E. coli. The DNA sequence of the PP15 cDNA at the initiation codon is as follows: Met Gly Asp CGCTCCAGA ATG GGA GAC 3' Since there is no NcoI site at the ATG, it is impossible for this DNA to be cloned directly into the pTrc99A ex- 't pression vector. However, an NcoI site can be achieved by mutagenesis, by two base-exchanges in the PP15 sequence: GAATGG 3' to 5' CCATGG The second amino acid (GLy) is unaffected by this manipulation, because the second codon of the PP15 structural sequence starts with a For the mutagenesis, an EcoRI fragment 902 base-pairs in size was isolated from the PP15 cDNA clone PP15-28 and oLa igated into the mutagenesis vector pMa5-8 (Fig. 1) which o, had likewise been cut with EcoRI and had been dephosphory- Lated. The resulting plasmid pMa5-8-PP15 (with the correct orientation of the PP15 EcoRI insert in relation to F1-orik was then subjected to the gapped duplex mutagenesis protocol (Kramer et al. (1984) Nucl. Acids. Res. 12, 9441-9456), using the following oligodeoxynucleotide: GGCTTGTCTCCCATGGTGGAGCGTCAC 3' One clone which had the desired mutation was identified by restriction analysis and was called pMc5-8-PP15-NcoI.

The NcoI-EcoRI fragment 798 base-pairs in size was isolated from this plasmid and Ligated into the correspondingly cut pTrc99A vector. The resulting plasmid pTrc-99Aembraces 4918 base-pairs and, after inJuction of the trc promoter, expresses the no -fused PP15 protein about kD in size.

F Key to Fig. 1: Map of the pLasmids pMac5-8 (~pMa5-8 and pMc5-8).

Fi-ORI: origin of repLication of the phage fl; ORI: origin of repLication of the CoLEl type; CAT: region coding for chioramphenicoL acetyltransferase; AMP: region coding for -Lactamase.

pMa5-8 has an amber mutation in CAT (A at position 3409) and pMc5-8 has an amber mutation in AMP (C at positioni 2238) a 02

__A

Claims (10)

1. A substantially purified DNA sequence coding for the amino acid sequence shown in Table 1, or functional parts thereof.

2. A substantially purified DNA or RNA which hybridized with the DNA as claimed in claim 1 under stringent conditions, or parts thereof.

3. A substantially purified DNA sequence as claimed in claim 1, coding for protein and containing the coding strand sown in Table 1, or functional parts thereof.

4. Substantially purified gene structures or vectors containing a DNA as claimed in claim 1, 2 or 3. A transformed cell containing DNA as claimed in claim 1, 2, 3 or 4.

6. PP15 obtained by genetic manipulation and having the amino acid sequence shown in Table 1.

7. PP15 obtained by genetic manipulation, which comprises expressing the DNA 4 sequence as claimed in claim 1 in Escherichia coli.

8. PP15 obtained by genetic manipulation, which comprises expressing the DNA *sequence as claimed in claim 1 in yeast. e ae S 9. A process for the preparation of PP15, which comprises insertion of a cDNA as claimed in claim 1 or 3 into an expression system, and bringing about expression therein. Polyclonal or monoclonal antitodies specific for PP15 and obtained from prepared by genetic manipulation, or from parts thereof having antigenic activity, as claimed in claim 6, 7 or 8.

11. A diagnostic aid which contains a DNA as claimed in claim 1, 2, 3 or 4 or functional parts thereof. S17 1 2. A diagnostic aid containing antibodies as claimed in claim

13. A diagnostic aid containing PP15, or functional parts thereof, as claimed in claim 6.

14. A diagnostic method which comprises contacting body fluids, tissue, or nucleic acids isolated therefrom, with a diagnostic aid as claimed in claim 11, 12 or 13. Pharmaceutical which contains PP15 as claimed in claim 6. Dated this 19th day of July 1991. SBEHRINGWERKEAKTIENGESELLSCHAFT I WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM S 290 BURWOOD ROAD S HAWTHORN VICTORIA 3122 AUSTRAUA DBM:JMW:PL 6 e e 4 0 0 a o o o a a a so oe J o a s o s',