IE67797B1 - 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

The invention relates to the isolation of the cDNA which codes for placenta-specific protein 9 (PPS) and to the use thereof for the genetically engineered, preparation of PPS. According to the description in ΞΡ-Β1-0 037 963, PP9 has the following properties: a) a carbohydrate content of 5.57 + 1.35%, including: hexoses 4.9 + 1.0%; hexosamines 0.1 ± 0.1%; fucose 0.07 ± 0.05%; neuraminic acid 0.5 ± 0.2%; b) a sedimentation coefficient s°o.w 3-2 χ 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 ± 4,000; e) an extinction coefficient (280 nm) of 14. S ± 1.0; f) an electrophoretic mobility in the region of the βχglobulins; g) an isoelectric point in the region of pH 5.0-S.8.

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

Surprisingly, when an expression gene bank was screened with antibodies against a different placental protein PPS (Bohn and Winkler, Arch. Gynak- 223, 179-186, 1977) which is not known, to be similar to PPS, five individual clones were obtained (PP9-10, PP9-353, PP9-357, PPS-361 and PPS-362). These clones contain the entire cDNA or parts thereof. On sequencing of PPS-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 10 complete cDNA of PPS 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).

Table 1 3U riGCAGCCATGGCAAGCCGTCTCCTGCTCAACAACGGCGCCAAGATGCCCATCCT GA^^- - MASrlllnngakm?il 90 110 gggg't’TGggtacctggaagtcccctccagggcaggtgactgaggccgtgaaggtggccat GLGTWKSPPGQVTEAVKVAI 130 iso 170 TGACGTCGGGTACCGCCACATCGACTGTGCCCATGTGTACCAGAATGAGAATGAGGTGGG ο V G Y R Η I D C A Η V YQNENEVG 190 210 230 GG’T’GGCCATTCAGGAGAAGCTCAGGGAGCAGGTGGTGAAGCGTGAGGAGCTCTTCATCGT VAIQEKLRSQVVKREELFIV 2S0 270 290 CAGCAAGCTGTGGTGCACGTACCATGAGAAGGGCCTGGTGAAAGGAGCCTGCCAGAAGAC SKLWCTYHEKGLVKGACQKT 310 330 · 350 ACTCAGCGACCTGAAGCTGGACTACCTGGACCTCTACCTTATTCACrGGCCGACTGGCTT lsdlkldyldlylxewptg? 370 390 410 TAAGCCTGGGAAGGAATTTTTCCCATTGGATGAGTCGGGCAATGTGGTTCCCAGTGACAC KPGKEFFPLDESGNVVPSDT 430 450 470 CAACATTCTGGACACGTGGGCGGCCATGGAAGAGCTGGTGGATGAAGGGCTGGTGAAAGC Ν I LDTWAAKEELVDEGLVKA 490 510 530 TATTGGCATCTCCAACTTCAACCATCTGCAGGTGGAGATGATCTTAAACAAACCTGGCTT I G I S N FNHLQVEM I L Ν K P G L 530 570 . ‘ '590 GAAGTATAAGCCTGCAGTTAAGCAGATTGAGTGCCACCCATATCTCACTCAGGAGAAGTT K Y K P A v N Q I E C η P Y L T Q Ε K L 610 630 650 AATCCAGTACTGCCAGTCCAAAGGCATCGTGGTGACCGCCTACAGCCCCCTCGGCTCTCC IQYCQSKGIVVTAYSPLGSP 670 690 710 TGACAGGCCCTGGGCCAAGCCCGAGGACCCTTCTCTCCTGGAGGATCCCAGGATCAAGGC drpwakpedpslledprik a 730 750 770 gatcgcagccaagcacaataaaactacagcccaggtcctgatccggttccccatgcagag iaakhnkttaqvlirfpmqr 790 810 830 gaacttggtggtgatccccaagtctgtgacaccagaacgcattgctgagaactttaaggt w L V V I Ρ K S V Τ P ERIAENFK V 850 870 890 C-tt^GACTTTGAACTGAGCAGC caggatatgac cac cttactcagctacaacaggaactg f*d f elssqdmttllsynrn w 910 930 9S0 GAGGGTCTGTGCC TTGTTGAGCTGTAC CTC C CACAAGGATTAC C C CTTCCATGAAGAGTT RV CALLSCTSSKDYPFEEEF 970 990 1010 TTGAAGCTGTGGTTGCCTGCTCGTCCCCAAGTGACCTATACCTGTGTTTCTTGCCTCATT 1030 1050 1070 tttttccttgcaaatgtagtatggcctgtgtcactcagcagtgggacagcaacctgtaga •1090 1110 1X30 gtggccagcgagggcgtgtctagcttgatgttggatctcaagagccctgtcagtagagta 1150 1170 1190 gaagtctcttccagtttgctttgcccttctttctaccctgctggggaaagtacaacctga 1210 1230 1250 ATAC C CTTTT CTGAC CAKAGAGAAG CAAAATCTAC CAGGT CAAAATAGTGC CACTAACGG 1270 1290 1310 TTGAGTTTTGACTGCTTGGAACTGGAATCCTTTCAGCAAGACTTCTCTTTGCCTCAAATA 1330 1350 1370 AAAAGTGCTTTTGTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 1390 AAAAAAAAAAAAAA Table 2 s Amino acid composition, of PP9 Amino acid Number cDNA (EP-B1-0 PP9 037 96 A = Ala t 19 6.013 5.69 B = Asx 0 0.000 0.00 C Cys 7 2.215 2.29 D ~ Asp 15 4.747 10.30 E = Glu 23 7.278 11.18 F - Phe 1 1 ·=» -»» 3.481 3.87 G = Gly 16 5.063 5.50 H = His 9 2.848 2.58 1 ~ lie 18 5.696 5.28 K = Lys 25 7.911 8.04 L - Leu 34 10.759 10.55 M = Met 6 1.899 1.47 N = Asn 15 4.747 10.30 P = Pro 2C 6.329 6.17 Q = Gin 13 4.114 11.18 R - Arg 11 3.481 3.49 S = Sex 17 5.380 5.70 T = Thr 15 4.747 Λ 0 0 'S β om «X V = Val 25 7.911 7 i 9 • β <Λ> «0 W — Trp t* o 1.899 2.36 γ = Tvr *** «=> 11 3.481 3.95 Z = Glx 0 0.000 0.00 A—G 35 11.076 11.19 S4-T 32 10.127 9.99 DtS 38 12.025 —— D+E+N+Q / m α o 20.886 — of Ώ Aa » A « Λ 45 *1 Λ *> Al *3 X "3 ® X "I X 14.11 d+e+h+k+r 83 2 β ., 2 ο o .— I+L+M+V 83 26.266 24.42 F+w+r 28 8.861 10.18 * = total of Asp ™ Asa ** = total of Glu + Gin The figure shows the position of lambda gtll-10 in relation to lambda gtll-3S2 diagrammatically. Two clones show a base exchange: PFS-3SX at position 255 (C in place of G; AA Q in place of S) and PP9-3S2 at position 925 (G in place of A; AA 2. 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) PKBS Letters 220, 209-213) and to the genes of rho-crystallin and of aldehyde reductase. It is possible that 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, with the aid of suitable expression systems, to express ΡΡ9» 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.eoli with the expression vector pTrc99C or pTacT7L (see the examples).

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 frost 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 genomic clone which, codes for PP9 and with whose aid not only is expression in eukaryotic cells facilitated but also further diagnostic infonoation can be gained.

Aldose reductase (SC 1.1.1.21) is an NADPEE-dependent enzyme and catalyses 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 ethylenediaxninetetraacetate SDS = sodium dodecyl sulfate DTT ~ dithiothreitol BSA = bovine serum albumin .

XPTG « isopropyl thiogalaetoside BxaapleS 1. Screening of an expression cDMA bank f res human, placenta wsing as.fci-PP5 antibodies An expression cDNA bank in phage lambda gtll from Genafit 5 GmbH, Heidelberg, was plated out at a density of about ,000 PFU per agar plate (13.5 cm diameter). Fox this, competent cells of the E.coli strain ylOSO (ATCC 37197) (R.A. Young and R.W» Davis, Science Vol. 222, 778782/1983) were infected with the phages at 37°C for 30 fu min and then plated out in top agar on L broth plates» The plates were incubated at 42°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 (10 mM tris-BCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20 and 5% skim milk powder) at 4°C overnight. The filters were subsequently washed three times in TBST for 10 min at room teaperature and then incubated with anti-P?5 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 lysed E.coli cells on nitrocellulose filters for 1 h) . 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 anti30 - rabbit 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.

J Finally, th® color reaction was carried out to visualize the PPS-positive clones to which the primary and — 9 — secondary antibodies were bound, by reaction of the alkaline phosphatase and a color reagent (ProtoBlot system from Protogen) . For each color reaction, 99 μ.1 of N3T (nitro blue tetrasolium) substrate (50 mg/ml in 70% dimethylformamide) and 49.5 μΐ of BCIP (5-bromo-4-chloro3-indolyl phosphate) substrate (50 mg/al in 70% dimethylformamide) were added to 15 ml of AP buffer (100 oM trisHCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) 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 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 plagues 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 fo produce a single positive plague. The result was the clones PP9-10, PP9-353, PP9-357, PPS-361 and PP9-3S2 with a positive reaction. 2. EJB£A 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);54635467) . The sequence shows an open reading frame and codes for a protein with a maximum of 316 amino acids». 3. Bhqjressxon of a FP9 fusion protein. pTre99C (E. 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 described above. The resulting plasmid pTre99C-PP9 is able to induce the synthesis of an approx. 36 kD protein ia S. coli cells. This protein can be immunopreeipitated specifically with the aid of a monospecific rabbit anti5 PP9 antiserum which has been raised by immunization with PPS 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 pTrcSSC-PPS 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 / 'UT Region / PP9 123456123 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 5s un30 translated region occurring in the PP9 cDNA. As a check, th© construction indicated hereinbefore was likewise carried out with the expression vectors pTrc99A and pTrcSSB (Ara&sn et al. loc.cit.). These vectors differ from pTrc99C by merely 2 bp (pTrc99A) and 1 bp (pTre99B) , which bring about shifts in the translation reading frame. As expected, neither pTrc99A-PP9 nor pTrc9S3-PPS was able to induce the synthesis of PP9 proteins reacting with anti-PP9 antisera. 4. Expression. of aafcura, TOfosed Ps’9 proteins The PP9 cDNA has, besides the Ncol site (5'CCATGG3') at the initiation codon, another Ncol site in the structural gene. In order to achieve mature expression of the PP9 protein, first the EcoRl fragment which is 1387. bp in size (see above) was ligated into the vector pMa5-8 (Stanssens et al., 1989). A plasmid (pMaS-S-BPS) in which the EcoRl fragment was present in the desired orientation (PP9 ATG initiation codon at the left-hand, 5' distal end) was obtained and propagated. The plasmid DNA was digested completely with HindHI and partially with Ncol. The Ncol-Hindi!Ϊ fragment which is 14.15 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 (-'’Μ8 stands for "mature") comprises 5535 bp and, after IPTG induction, expresses the mature, unfused PP9 protein. The 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-PPS antisera in a 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 aldose reductase ia E.coli, aa improved expression vector was constructed (pTacT7L), which uses the ribosoaeblading site of gene 10 of the T7 phage. It is known that this sequence immediately ia 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) . pTacT7L is essentially based oa the known vector pKX223-3 (Brosius & 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 Ncol-Hiadlll fragment which is 1415 bp long was ligated into the pTacT7I« 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 rednetase. activity of the protein PP9 after expression in Ξ-coli Comparisons of PP9 cDNA sequence homologies revealed a 94% homology (85% identity) in the computer analysis with the aldose reductase of rats (Carper et al. (1987) FEBS Lett. 220, 209-213). Further homologies discovered were to rho-crystal!in of the frog eye and to the aldehyde reductase of the rat leas. This finding leads to the suggestion that PP9 is another- member of a relatively large protein family and very probably is the human aldose reductase.

Periplasmic fractions were prepared froxa E.coli KI2 W31101acIQ (pTacT7L-PP9) by the method of Hsiung et al. (1986) (Bio/Techaology 4, 991-995)„ These extracts have sn aldose reductase activity which is not present in corresponding control extracts. Aldose reductase activity was detected using known assay methods (for example 3fT Kawasaki et al., (1989) Biochia. Biophys. Acta 996, 30-36) which are based on a decrease in the absorption at 340 nra due to ths oxidation of the NADPH in the assay mixture.

Claims (9)

1. Patent Claim®

1. A nucleotide sequence shown in Table 1, or a sequence derived therefrom on the basis of the degeneracy of the genetic code, coding for placenta-specific protein PPS. 2. A gene structure containing a nucleic acid as claimed ia claim 1. 3. A vector containing a nucleic acid as claimed in claim 1. 4. Transformed cells containing a nucleic acid as claimed ia claim 1.

2. 5. .¾ process for the preparation of PPS, which comprises a nucleic acid as claimed in claim 1 being introduced into an expression system and expressed therein.

3. 6» A diagnostic aid which contains a nucleic acid as claimed in claim 1 ...

4. 7. A nucleotide sequence according to claim 1, substantially as hereinbefore described.

5. 8. A gene structure according to claim 2, substantially as hereinbefore described and exemplified.

6. 9. A vector according to claim 3, substantially as hereinbefore described and exemplified.

7. 10. A transformed cell according to claim 4, substantially as hereinbefore described and exemplified.

8. 11. A process according to claim 5 for the preparation of PP9 , substantially as hereinbefore described and exemplified.

9. 12. PP9 whenever prepared by a process claimed in claim 5 or 11 . '13. A diagnostic aid according to claim 6, substantially as hereinbefore described.