AU625082B2 - Cdna coding for placental protein 9 (pp9), the isolation and use thereof - Google Patents

Abstract

It has been possible to isolate the gene for placental protein 9 (PP9) from an expression cDNA gene bank by antibodies against placental protein 5. This makes possible the preparation of PP9 by genetic manipulation.

Description

COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class In Application Number: Lodged: Form it. Class Complete Specification Lodged: Accepted: Published: Priority Related Art BEHRINGWERKE AKTIENGESELLSCHAFT Name of Applicant 1-, Address of Applicant Actual Inventor: Address for Service D-3550 Marburg, Federal Republic of Germany.

ULRICH GRUNDMANN WATERMARK PATENT TRADEMARK ATTORNEYS.

LOCKED BAG NO. 5, HAWTHORN, VICTORIA 3122, AUSTRALIA Complete Specification for the invention entitled: cDNA CODING FOR PLACENTAL PROTEIN 9 (PP9), THE ISOLATION AND USE THEREOF u The following statement is a full description of this invention, including the best method of performing it known to la BEHRINGWERKE AKTIENGESELLSCHAFT HOE 89/B 007 Ma 749 Dr. Lp/rd I Description cDNA coding for placental protein 9 (PP9), the isolation and use thereof The invention relates to the isolation of the cDNA which codes for placental-specific protein 9 (PP9) and to the use thereof for the genetically engineered preparation of PP9. According to the description in EP-B1-0 037 963, PP9 has the following properties: a) a carbohydrate content of 5.57 1.35%, including: hexoses 4.9 hexosamines 0.1 fucose 0.07 0.05%; neuraminic acid 0.5 0.2%; b) a sedimentation coefficient s o.w of 3.2 0.2 S; c) a molecular weight determined in the ultracentrifuge of 35,100 3,800; d) a molecular weight determined in a polyacrylamide gel containing sodium dodecyl sulfate (SDS) of 40,000 j 20 4,000; 1 e) an extinction coefficient (280 nm) of 14.6 f) ai, electrophoretic mobility in the region of the p 1 globulins; g) an isoelectric point in the region of pH 5.0-6.8.

The conventional isolation of PP9 described in the abovementioned patent is very elaborate and thus the object was to isolate the gene coding for PP9 in order to make the genetically engineered preparation of PP9 possible.

2 Purprisingly, when an expression gene bank was screened with antibodies against a different placental protein (Bohn and Winkler, Arch. Gynak. 223, 179-186, 1977) which is not known to be similar to PP9, five individual clones were obtained (PP9-10, PP9-353, PP9-357, PP9-361 and PP9- 362). These clones contain the entire cDNA or parts thereof. On sequencing of PP9-10 it was found that the cDNA appears to derive from an incompletely processed heterogeneous nuclear RNA (HnRNA) because in the middle of the sequence which is not quite complete however coding for PP9 there is an intron about 600 bp in size, and no poly(A) sequence is present at the 3' end. The other four clones all lack an intron, but they do carry the poly(A) sequence. It was not possible to isolate the cDNA for PP5 by the above process.

The sequencing data yield the result that the processed complete cDNA of PP9 is 1394 base-pairs (bp) long and codes for a protein with 316 amino acids (AA) (Tab. 1).

The molecular weight of 35853d and the AA composition agree very well with the data contained in the abovementioned patent (see Tab. 2).

Ci I 202 0ai 0 o 90 0o 0 000r 0 0502 00 -3- Table 1 30

GAGCGCAGCAGCCATGGCAGCCGTCTCCTGCTCACACGGCGCCAGATGCCCATCCT

M A S R L L L N N G A K M L1 90 110

GGGGTTGGGTACCTGGAAGTCCCCTCCAGGGCAGGTGACTGAGGCCGTGAAGGTGGCCAT

G L G T W K Sp P G Q V T E A V K V A I 130 150 170 TGACGTCGGGTACCGC CACATCGACTGTGCC CATGTGTACCAGAATGAGAATGAGGTGGG D V G Y R H I D C A H V Y Q N E N E V G 190 210 230 GGTGGC CATTCAGGAGAAGCTCAGGGAGCAGGTGGTGA-GCGTGAGGAGCTCTTCATCGT V A I Q E K L R EQ V V K R E E LF7 IV 250 270 290

CAGCAGCTGTG-TGCACGTACCATGAGA.AGGGCCTGGTGAAAGGAGCCTGCCAGA.AGAC

S K L W C T Y H E K G L V K G A C Q K T 310 330 350

ACTCAGCGACCTGA.AGCTGGACTACCTGGACCTCTACCTTATTCACTGGCCGACTGGCTT

L S D L K L D Y L D L Y L I H W P T G F S*370 390 410 TAAGC CTGGGAAGGAATTTTTCCCATTGGATGAGTCGGGCAATGTGGTTCCCAGTGACAC K P G K E F F P L DE S G N V V p S D T 430 450 470

CAACATTCTGGACACGTGGGCGGCCATGGAAGAGCTGGTGGATGAAGGGCTGGTGAA.AGC

N I L D T W A A M E E L V D E G L V K A 490 510 530 TATTGGCATCTC CAACTTCAACCATCTCCAGGTGGAGATGATCTTAAACAAACCTGGCTT I G I S N F N H L Q V E M IL N K P G L 550 570 590 GAAGTATAAGCCTGCAGTTAACCAGATTGAGTGCCAC

CCATATCTCACTCAGGAGAAGTT

K Y K P A V N Q I E C H P Y L T Q E K L 610 630 650 AATC CAGTACTGC CAGTCCAAAGGCATCGTGGTGACCGCC-TACAGCCCCCTCGGCTCTCC I Q Y C Q S KG IV V T k Y S P L G S P 670 690 710

TGACAGGCCCTGGGCCAAGCCCGAGGACCCTTCTCTCCTGGAGGATCCCAGGATCAAGGC

D R P W A K P E D P S L L ED P R IK A 730 750 770 GATC GCAGC CAAGCACAATAAAACTACAGCCCAGGTCCTGATCCGGTTCCCCATGCAGAG I A A K H N K T T A Q V L I R F P M Q R 790 810 830 GAACTTGGTGGTGATCC CCAAGTCTGTGACACCAGAACGCATTGCTGAGAACTTTAAGGT -4- N L. V V IP K S V T PE R I A E N F K V 850 870 890 CTTTGACTTTGAACTGAGCAGC CAGGATATGAC CAC CTTACTCAGCTACAACAGGAACTG F D F E L S S Q D M T T L L S Y N R N W 910 930 950 GAGGGTC TG TGC CTTG TTGAGC TGTAC CTC CCACAAGGATTAC C CCTTC CATGAAGAGTT R V C A LLS C T S H K D Y P F H E E F 970 990 1010

TTGAAGCTGTGGTTGCCTGCTCGTCCCCAAGTGACCTATACCTGTGTTTCTTGCCTCATT

1030 .1050 1070 TTTTTC CTTGCAAATGTAGTATGGCCTGTGTCACTCAGCAGTGGGACAGCAACCTGTAGA 1090 1110 1130 G TGG CCAGC GAGGGC GTGTC TAGC TTGATGTTGGAT CT CAAGAGC CCTGT CAGTAGAG TA 1150 1170 1190

GAAGTCTCTTCCAGTTTGCTTTGCCCTTCTTTCTACCCTGCTGGGGAAAGTACAACCTGA

1210 1230 1250 ATAC CCTTTTCTGACCAAAGAGAAGCAAAATCTACCAGGTCAAAATAGTGCCACTAACGG 1270 1290 1310

TTGAGTTTTGACTGCTTGGAACTGGAATCCTTTCAGCAAGACTTCTCTTTGCCTCAAATA

1330 1350 1370

AAATCTTT

1390 -4 Table 2: Amino acid composition Amino acid Number of PP9 cDNA (EP-Bl-0 pp9 037 963) A Ala B Asx C Cys D Asp E Glu F Phe G Gly H His I Ile K Lys L Leu M Met N Asn P Pro Q Gin R Arg S Ser T Thr V Val W Trp Y Tyr Z Glx 6.013 0.000 2.215 4.747 7.278 3.481 5.063 2.848 5.696 7.911 10.759 1.899 4.747 6.329 4.114 3.481 5.380 4..747 7.911 1.899 3.481 0.000 11 .076 10. 127 12.025 20.886 14.241 26.266 26. 266 8.861 5.69 0.00 2.29 10.30 11.18 3.87 5.50 2.58 5.28 8.04 10.5.5 1.47 10.30 6.17 11.18 3.49 5.70 4.29 7.12 2.36 3.95 0.00 11.19 9.99 14.11 24.42 10.18 09& A+ G

S+T

D+E

D+E+N+Q

H+K+R

D+E+H+K+R

I+L+M+V

F+W+Y

total total of Asp of Glu Asn Gin

SI

-6 The figure shows the position of lambda gt11-10 in relation to lambda gt11-362 diagrammatically. Two clones show a base exchange: PP9-361 at position 255 (C in place of G; AA Q in place of E) and PP9-362 at position 925 (G in place of A; AA R in place of K).

Having the cDNA sequence of PP9 available in this way allowed comparisons with other known nucleic acid sequences. It was found that there are homologies to the DNA sequence of the aldose reductase of rats (Carper et al. (1987) FEBS Letters 200, 209- 213) and to the genes of rho-crystallin and of aldehyde reductase. It is possible that 1 0 these proteins belong to the same superfamily as aldehyde reductase; it is extremely probable that PP9 is identical with human aldose reductase.

It is possible according to the invention to use the coding cDNA to obtain DNA or RNA which hybridizes with the cDNA sequence of the invention. This RNA or DNA is obtained by conventional methods.

1 5 It is also possible according to the invention to use the coding cDNA, with the aid of suitable expression systems, to express PP9. It is furthermore possible, by the choice of host, to influence the PP9 modification form. Thus, there is no glycosylation in bacteria, while that in yeast cells differs from that in higher eukaryotic cells. PP9 is particularly advantageously expressed in E.coli with the expression vector pTrc99C or 2 0 pTacT7L (see the examples).

Another embodiment of the invention envisages the use of PP9 in conjunction with pharmaceutically acceptable carriers or excipients as a pharmaceutical.

Knowing the amino acid sequence of PP9, it is possible to prepare, by conventional or genetic engineering methods, amino acid part-sequences which are used as antigens for preparing polyclonal or monoclonal antibodies. Such antibodies can be employed not only for diagnostic purposes but also for the preparation of antibody columns. PP9 can thus be separated from solutions which contain it in addition to other proteins. It is also possible in a straightforward manner, with the aid of the cDNA or parts thereof, to isolate from a genomic bank the al&, -7 genomic clone which codes for PP9 and with whose aid not only is expression in eukaryotic cells facilitated but also further diagnostic information can be gained.

Aldose reductase (EC 1.1.1.21) is an NADPH-dependent enzyme and catalyzes the reduction of aldose to the sugar alcohol. In the pathological state of diabetes and of galactosemia, an increased aldose reductase activity in a number of tissues results in high levels of sorbitol and galactitol. This may lead to, for example, cataracts in the lens and to a thickening of the capillary membrane of the retina. Furthermore, increased aldose reductase levels are regarded as a cause of diabetic complications, such as of the nerves or of the kidney. For these reasons there is currently intensive work on developing aldose inhibitors. It has hitherto been necessary for this purpose to carry out an elaborate isolation of aldose reductase from animal tissues and organs, which moreover resulted in very low yields. The possibility of expression of aldose reductase in Escherichia coli (E.coli) now makes it possible to set up an assay system using bacterial extracts and thus supersedes the dependence on animal organs and tissues. The assay method is considerably simplified overall.

The invention is further defined in the patent claims and explained further in the examples which follow.

The following abbreviations are used, where not explained in the text: EDTA sodium ethylenediaminetetraacetate SDS sodium dodecyl sulfate DTT dithiothreitol BSA bovine serum albumin IPTG isopropyl thiogalactoside -8- Examples 1. Screening of an expression cDNA bank from human placenta using anti-PP5 antibodies An expression cDNA bank in phage lambda gtll from Genofit GmbH, Heidelberg, was plated out at a density of about 30,000 PFU per agar plate (13.5 cm diameter). For this, competent cells of the E.coli strain y1090 (ATCC 37197) Young and R.W. Davis, Science Vol. 222, 778- 782/1983) were infected with the phages at 37"C for min and then plated out in top agar on L broth plates.

The plates were incubated at 42 0 C 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 plates with the filters were again incubated at 37"C for 4 h. Before the filters were taken off again, the filter and plate were together marked with a needle dipped in carbon black. The filters were then incubated in TBST mM tris-HCl, pH 8.0, 150 mM NaC1, 0.05% Tween 20 and skim milk powder) at 4°C overnight. The filters were subsequently washed three times in TBST for 10 min at room temperature and then incubated with anti-PP5 rabbit antibodies in 15 ml of TBST per filter at room temperature for 1 h. (The solution of antibodies had previously been diluted 1:200 and saturated with non-recombinant lambda gtll lyzed E.coli cells on nitrocellulose filters for 1 After the incubation with the primary antibody, the filters were washed with TBST for 4 x 10 min. The filters were then incubated with the secondary antirabbit antibody which was conjugated with alkaline phosphatase (from Promega, USA marketed by Atlanta, Heidelberg) and previously diluted 1:5000 in TBST, with shaking for 1 h. The filters were then again washed with TBST for 4 x 10 min.

Finally, the color reaction was carried out to visualize the PP9-positive clones to which the primary and second- 9 ary antibodies were bound, by reaction of the alkaline phosphatase and a color reagent (ProtoBlot system from Protogen). For each color reaction, 99 pl of NBT (nitro blue tetrazolium) substrate (50 mg/ml in 70% dimethylformamide) and 49.5 ul of BCIP (5-bromo-4-chloro-3indolyl phosphate) substrate (50 mg/ml in 70% dimethylformamide) were added to 15 ml of AP buffer (100 mM tris- HC1, pH 9.5, 100 mM NaC1, 5 mM MgCl 2 for a nitrocellulose filter. The filters were swirled in the color solution in the dark for about 20 min to 1 h until positive plaques showed a sufficient blue coloration. The color reaction S was stopped by immersing the filters in a stop solution mM tris-HC1, pH 8.0 and 5 mM EDTA).

O o .o.o Positive signals were assigned to the plaques on the 0 0 e. 15 corresponding agar plate. The plaques were removed by 0 0 o ,o stabbing with a Pasteur pipette, resuspended in 1 ml of SM buffer (10 mM tris-HC1, pH 7.5, 10 mM MgC12) and singled out to produce a single positive plaque. The result was the clones PP9-10, PP9-353, PP9-357, PP9-361 0oo 20 and PP9-362 with a positive reaction.

ooo o 00 2. DNA sequence analysis The abovementioned phage clones were propagated, and the DNA of each was extracted. The particular EcoRI fragment was isolated and ligated into the EcoRI site of the Bluescript M13 vector (Stratagene, San Diego, CA, USA).

The sequence analysis was by the enzymatic dideoxy method of Sanger et al. (Proc.Natl.Acad.Sci.USA 74, (1977);5463- 5467). The sequence shows an open reading frame and codes for a protein with a maximum of 316 amino acids.

3. Expression of a PP11 fusion protein pTrc99C Amann et al., Gene 69, (1988), pp 301-315) was digested with EcoRI. The clone lambda gtll-361 was digested with EcoRI, and the EcoRI insert which is 1387 bp in size was ligated with the pTrc99C vector fragment 10 described above. The resulting plasmid pTrc99C-PP9 is able to induce the synthesis of an approx. 36 kD protein in E. coli cells. This protein can be immunoprecipitated specifically with the aid of a monospecific rabbit anti- PP9 antiserum which has been raised by immunization with PP9 isolated from human placentae. PP9 expression in E.

coli cells was further demonstrated by Western blot analyses using the above serum. In this experiment there is a reaction only with the extract from IPTG-induced cells containing the pTrc99C-PP9 plasmid, and a protein band of about 36 kD was again specifically visualized.

Plasmid-free E. coli control extracts, and extracts which contained pTrc99C-PP9 but had not been induced with IPTG, did not react with the abovementioned anti-PP9 antiserum.

The PP9 fusion protein produced by the plasmid construction produced hereinbefore had the following N-terminal amino acid sequence, defined by the nucleotide sequence thereafter: Vector/Linker 5'UT Region PP9 1 2 3 4 5 6 1 2 3 Met Gly Asn Ser Ala Ala Met Ala Ser CC ATG GGG AAT TCT GCA GCC ATG GCA AGC The PP9 fusion protein defined by this construction carries, in addition to the complete PP9 amino acid sequence encoded by the PP9 cDNA, six amino acids in front of the N terminus: four vector-encoded amino acids and two amino acids which are specified by the 5' untranslated region occurring in the PP9 cDNA. As a check, the construction indicated hereinbefore was likewise carried out with the expression vectors pTrc99A and pTrc99B (Amann et al. loc.cit.). These vectors differ from pTrc99C by merely 2 bp (pTrc99A) and 1 bp (pTrc99B), 11 which bring about shifts in the translation reading frame. As expected, neither pTrc99A-PP9 nor pTrc99B-PP9 was able to induce the synthesis of PP9 proteins reacting with anti-PP9 antisera.

4. Expression of mature, unfused PP9 proteins The PP9 cDNA has, besides the NcoI site (5'CCATGG3') at the initiation codon, another NcoI site in the structural gene. In order to achieve mature expression of the PP9 protein, first the EcoRI fragment which is 1387 bp in size (see above) was ligated into the vector pMa5-8 (Stanssens et al., 1989). A plasmid (pMa5-8-PP9) in which the EcoRI fragment was present in the desired orientation (PP9 ATG initiation codon at the left-hand, 5' distal 0 end) was obtained and propagated. The plasmid DNA was digested completely with HindIII and partially with NcoI.

The NcoI-HindIII fragment which is 1415 bp in size was isolated and ligated between the corresponding sites in the expression vector pTrc99A which had been cleaved with the same restriction enzymes. The resulting plasmid pTrc99A-PP9M stands for "mature") comprises 5535 bp and, after IPTG induction, expresses the mature, unfused PP9 protein. The N-terminal amino acid sequence of the PP9 protein is defined by the following nucleotide sequence: Met Ala Ser Arg Leu AGGAAACAGACC ATG GCA AGC CGT CTC The protein expressed in this way reacts with anti-PP9 antisera in Western blot and immunoprecipitation, it being possible to detect a protein about 36 kD in size.

It has now been found that this mature PP9 protein is transported into the periplasm of E.coli cells and displays its enzymatic activity (aldose reductase) there.

In order further to increase the expression rate of 12 aldose reductase in E.coli, an improved expression vector was constructed (pTacT7L), which uses the ribosome-binding site of gene 10 of the T7 phage. It is known that this sequence immediately in front of a heterologous gene is able to increase its expression rate due to more efficient ribosome binding (Olins et al. (1988), Gene 73, 227-235).

SpTacT7L is essentially based on the known vector pKK223-3 (Erosius Holy (1984), Proc. Natl. Acad. Sci. USA 81, 6929-6933), but, in contrast to the latter, has the abovementioned T7 sequence immediately in front of the cloning linker. The abovementioned PP9-encoding NcoI- HindIII fragment which is 1415 bp long was ligated into the pTacT7L vector cut with the same restriction enzymes.

The resulting plasmid pTacT7L-PP9 likewise mediates the expression of the unfused PP9 protein, but the yield of PP9 is about 20 times that achieved by pTrc99A-PP9M.

Aldose reductase activity of the protein PP9 after expression in E.coli Comparisons of PP9 cDNA sequence homologies revealed a 94% homology (85% identity) in the computer analysis with I the aldose reductase of rats (Carper et al. (1987) FEBS Lett. 220, 209-213). Further homologies discovered were to Rho-crystallin of the frog eye and to the aldehyde Sreductase of the rat lens. This finding leads to the i 25 suggestion that PP9 is another member of a relatively large protein family and very probably is the human aldose reductase.

Periplasmic fractions were prepared from E.coli K12 W31101acIQ (pTacT7L-PP9) by the method of Hsiung et al.

(1986) (Bio/Technology 4, 991-995). These extracts have an aldose reductase activity which is not present in corresponding control extracts. Aldose reductase activity was detected using known assay methods (for example Kawasaki et al., (1989) Biochim. Biophys. Acta 996, 30-36) which are based on a decrease in the absorption at 340 nm due to the oxidation of the NADPH in the assay mixture.

Claims (10)

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

2. A DNA sequence coding for placenta-specific protein PP9, containing the coding strand shown in Table 1.

3. A 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. o 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

7. PP9 whose preparation is genetically engineered by expression of the DNA sequence as claimed in claim 2 in E.coli, in yeast or animal cells.

8. A process for the preparation of PP9, which comprises a cDNA or RNA as claimed in claim 1, 2, 3 or 4 being introduced into an expression system and expressed therein. obtained by immunization with PP9 prepared by ic engineering, or parts thereof wit antigenic activity. A diagnostic aid w i contains a DNA or RNA as claimed in cla 1, 2, 3 or 4, in whole or in part.

11. A agnostic aid containing antibodies as claimed in claim 9. -14 9. Polyclonal or monoclonal antibodies specific for PP9 obtained by immunization with PP9 prepared by genetic engineering and encoded by the DNA sequence claimed in claim 1 or parts thereof with antigenic activity. A diagnostic aid which contains a DNA or RNA as claimed in claim 1, 2, 3 or 4, in whole or in part. 11. A diagnostic aid containing antibodies as claimed in claim 9.

12. A diagnostic method, which comprises contacting body fluids, tissue or nucleic acids isolated therefrom with a diagnostic aid as claimed in claim 10 or 11. o 0 0 S 13. PP9 encoded by the DNA sequence claimed in claim 1 in adjunct with 0: pharmaceutically acceptable carriers and excipients as a pharmaceutical. O o

14. A method for detecting or screening aldose reductase inhibitors, which comprises screening a sample potentially including aldose reductase with PP9 as claimed in claim 9. .0 DATED this 13th day of March 1992. 0 0 0 00 0 BEHRINGWERKE AKTIENGESELLSCHAFT O 4 WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 0" 290 BURWOOD ROAD S. HAWTHORN VICTORIA 3122 a0 AUSTRALIA 111 111 e DBM/KJS/CH (DOC.10) AU5110390.WPC a e I I Sac I Hpa I HaeII~~ aa m~Q B H HI I A 59 N C I n tro n xgtll I Xgt 11 362 0500 1000o bp