IE900853L - cDNA coding for placental protein 9 (PP9), the isolation and¹use 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

67797 The invention relates to the isolation, of the cDNA which codes for placenta-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 5 has the following properties: a) a carbohydrate content of 5.57 ± 1.35%, including? hexoses 4.9 ± 1.0%; hexosEJiines 0.1 ± 0.1%; fucose 0.07 + 0.05%; neuraminic acid 0.5 ± 0.2%; b) a sedimentation coefficient s|0w 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 polyacrylaad.de gel containing sodium dodecyl sulfate (SDS) of 40,000 ± 4,000; e) an extinction coefficient (280 nun) of 14.6 ± 1.0; f) an elactrophoretic mobility in the region of the globulins; g) an isoelectric point in the region of pH 5.0-S.8.

The conventional isolation of PP9 described in the 20 abovesaantioned patent is very elaborate and thus the object was to isolate tha gene coding for PP9 in order to make the genetically engineered preparation of PP9 possible.

Surprisingly, when an expression gene bank was screened 25 with antibodies against a different placental protein PP5 (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, FP9-3 53, PP9-357, PPS-3S1 and PP9-3 62) . These clones contain the entire cDNA or parts 30 thereof. On sequencing of PP9-10 it was found that the cDNA appears to derive from, an incompletely processed hsterogeaaous nuclear RMA (HnEMA) because in the middle of the sequence - which is not quite complete however -coding for PP9 there is an intron about 500 bp in sis®, 5 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 cDMft. for PP5 by the above process.

The sequencing data yield the result that the processed 10 complete cDKA of PPS is 1394 base-pairs (bp) long and codes for a protein with 316 asaino acids (AA) (Tab „ 1) . The molecular weight of 35853d and the AA composition agree very wall with the data contained in the above-mentioned patent, (see Tab. 2).

Table 1 30 50 r^.^pa/-c\gccatggcaagccgtctcctgctcaacaacggcgccaagatgcccatcct gag * "* masrlllnngakmpil 70 90 110 GGGG*t*TGGGTACCTGGAAGTCCCCTCCAGGGCAGGTGACTGAGGCCGTGAAGGTGGCCAT glgtwksppgqvteavkvai 130 ISO 170 tgacgtcgggtaccgccacatcgactgtgcccatgtgtaccagaatgagaatgaggtggg d* vgyrhidcah v yqnenevg 190 210 230 ggtggccattcaggagaagctcagggagcaggtggtgaagcgtgaggagctcttcatcgt vaiqeklrsqvvkreelfiv 2S0 270 290 cagcaagctgtggtgcacgtaccatgagaagggcctggtgaaaggagcctgccagaagac. sklwctyhekglvkgacqkt 310 330 • 350 actcagcgacctgaagctggactacctggacctctaccttattcactggccgactggctt lsdlkldyldlylihwptgf 370 390 410 taagcctgggaaggaatttttcccattggatgagtcgggcaatgtggttcccagtgacac kpgkeffpldesgnvvpsdt 430 450 470 caacattctggacacgtgggcggccatggaagagctggtggatgaagggctggtgaaagc n i l dtwaameelvdeglvka 490 510 530 tatt g g c a t c t c caac tt caa c c at ct g caggt ggag at gat c ttaaacaaac c1gg ctt i g i s nfnhlqvemi l n k p g l 550 570 . • 590 gaagtataagcctgcagttaaccagattgagtgccacccatatctcactcaggagaagtt k y k pavnqi echpyl tqekl 610 630 650 aatccagtactgccagtccaaaggcatcgtggtgaccgcctacagccccctcggctctcc iqycqskgivvtaysplgsp 670 690 710 tgacaggccctgggccaagcccgaggacccttctctcctggaggatcccaggatcaaggc drpwakpedpslledprik-a 730 750 770 gatcgcagccaagcacaataaaactacagcccaggtcctgatccggttccccatgcagag iaakhnkttaqvlxrfpmqr 790 810 830 gaacttggtggtgatccccaagtctgtgacaccaga&cgcattgctgagaactttaaggt n l v v I p k s v t p eriaenfk v 850 870 890 C-T-ttgactttgaactgagcagccaggatatgac CAC cttactcagctacaacaggaactg f elssqdmttllsynrn w 910 930 9S0 gagggtctgtgccttgttgagctgtacctcccacaaggattaccccttccatgaagagtt kvcallsctsskdypfeeef 970 990 1010 ttgaagctgtggttgcctgctcgtccccaagtgacctatacctgtgtt-rCTTGCCTCATT 1030 1050 1070 tttttccttgcaaatgtagtatggcctgtgtcactcagcagtgggacagcaacctgtaga 1090 1110 1130 gtggccagcglagggcgtgtctagcttgatgttggatctcaagagccctgtcagtagagta 1150 1170 1190 gaagtctcttccagtttgctttgcccttctttctaccctgctggggaaagtacaacctga 1210 1230 12S0 ATAC C cttttctgac caaagagaag caaaatctac caggtcaaaatagtgccactaacgg 1270 1290 1310 ttgagttttgactgcttggaactggaatcctttcagcaagacttctctttgcctcaaata 1330 1350 1370 JWUUlGTGCTTTTGTGAAJUVAAJUUlAAAAAAAAAAAAAAAAAaAAAAAAAAAAAAAAAAAA 1390 a&aaaaaaaaaaaa rable 2 : Ami no acid composition of PP9 o acid Number CDMA PP9 (EP-B1-0 037 96 A = Ala t 19 6 .013 .69 B = » -a t» nSX 0 0.000 0.00 C = Cys 7 2.215 2 .29 D = Asp 4 .747 .30 E = Glu 23 7 .278 11.18 F » Pho 11 3.481 3.87 G = Gly 16 .063 .50 H = His 9 2.848 2 .58 1 ~ lie 18 .696 .28 K = Lys 7.911 8 .04 L Leu 34 .759 .55 M = M©t 6 1.899 1.4/ N = Asn 4.747 .30 P = Pro 2G 6 .329 6 .17 Q = Gin 13 4.114 11.18 H = Arg 11 3.481 3.49 S = Ssx 17 .380 .70 T = Thr 4 - 747 4 .29 V = Val 7.911 7 .12 W = Trp o 1.899 2 .36 Y = Tvr 11 3.481 3.95 Z = Glx 0 0.000 0 .00 A-s-G 11 .076 11.19 S*T 32 .127 9.99 D-KE 38 12 .025 — D+E+N+Q /» /» a a .886 _» H+K+R 45 14 .241 14.11 D+E-f-H+K+R 83 26 ^ i» .2oo .— 1-hL+M-rV 83 26 «"l f* <. 2o o 24.42 p+jy+v 28 8 .861 -18 — uOCeLJ = tota] OJ oi Asp Glu Asn Gin - s - The figure shows the position of lambda gtll-10 in relation to laxahda gtil-362 diagrassaatically. Two clones show a base exchange: PP9-3S1 at position 255 (C in place of G; AA Q in place 5 of E) asd PP9-3S2 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 ssgusn-10 ees« It was found that there are homologies to the DMA sequence of the aldose reductase of rats (Carper et al. (1987) FIBS Letters 220, 209-213) and to the genes of rho-crystal!in aad of aldehyde reductase. It is possible that these proteins belong to the same superfamily ae 15 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 PP9. It is furthermore possible, by the choice 20 of host,- to iafluence 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 pTacT7L (see the 25 examples) .

Knowing the asaino acid sequence of PP9, it is possible to prepare, by conventional or genetic engineering methodst, amino acid part.-sequences which are used as antigens for preparing polyclonal or monoclonal antibodies. Such, 3 0 antibodies can be employed not only for diagnostic purposes but also for the preparation of antibody colussns. PP9 e«axx 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 35 cDNA. or parts thereof, to isolate from a genomic bank the genomic clone which codas 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 5 enzyme and catalyses the reduction of aldose to the sugar alcohol. In the pathological scat® 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 10 in tha lens and to a thickening of the capillary membrane of the retina. Furthermore, increased aldose reductase levels are regarded as a causa of diabetic complications, such as of the nerves or of the kidney. For these reasons there is currently intensive work on developing, aldose 15 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 2 0 snakes 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 claixas and 25 explained further in the examples which follow.

The following abbreviations are used, where not explained in the text: = sodium ethylenediaminetetraacetate = sodium dodecyl sulfate = dithiothrextol = bovine serum albumin , = isopropyl thiogalactoside EDTA ' SDS DTT BSA 1PTG BacscapXes 1. Seree^dbag of an eaqpressic® cD5E&. bauaie froaa hxasasx pXaeeafca Tasiag1 aati-Fj?5 antibodies Mi eaqpression cDKA bank in phage lambda gtll from Genofit 5 GmbH, Heidelberg,, was plated out at a density of about 30,000 FPU per agar plats (13.5 em diameter) . For this, competent cells of the E.coli strain ylOSO (&TCC 37197) (R. A. Young and R.W. Davis, Science Vol. 222, 778-782/1083) were infected with the phages at 37°C for 30 r« sin and then, plated out in top agar on L broth plates. Tha 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 nM XPTG in water. The 15 plates with the filters were ©gain 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-HCl, pH 8.0, 150 mM NaCl, 0.05% Twees. 20 and 5% 2 0 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-PJ?5 rabbit antibodies in 15 ml of TBST per filter at room temperature for 1 h. (The solution of antibodies had previously 25 been diluted 1 s 2 Q 0 and saturated with non-recombinant, lambda gtll lyzed 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 mixi „ The filters were then incubated with the secondary anti-30 - rabbit antibody which was conjugated with alkaline phosphatase. (from Promega, TJSA - 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 sain. / 2s5 Finally, the eolor reaction was carried out to visualize the PBS-positive clones to which the primary and secondary antibodies were bound, by reaction of the alkaline phosphatase and a color reagent (ProtoBlot system from Frotogen) . For each color reaction, 99 p.I of MBT (nifcro blue tetragolium) substrate (50 mg/ml in 70% 5 dime thy 1 f oraamide) and 49.5 pi of 3CXP (5-bromo~4-chloro~ 3-indolyl phosphate) substrate (50 mg/ml in 70% dimethyl -formamide) were added to 15 ml of ftp buffer (100 mM tris-HC1 e pH 9.5, 100 mM NaCl, 5 mM MgCl2) for a nitrocellulose filter. The filters were swirled in the color 10 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 azid 5 mM EDTA) .

Positive signals ware assigned to the plaques on the 15 corresponding agar plate. The plaques 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 produce a single positive plaque. The result was the clones FPS-10, PP9-353, PP9-357, PP9-361 20 and PP9-362 with a positive reaction. 2 - EHS& sequence analysis Tha abovamsntioned phage clones were propagated, and the DMA of each was extracted, Tha particular EcoRI fragment was isolated and ligated into the EcoRI site of the 2 5 Bluescript Ml3 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 - Eaqpressxeaa of a PP3 fnisiaa protein. pTreSSC (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 si&e was ligated with the pTrc99C vector fragment described above. The resulting pXasmdd pTrc99C-PP9 is able to induce the synthesis of an approx- 36 kD protein in E. eoli cells. This protein can be iirjaunoprecipitated specifically with, tha aid of a monospecific rabbit anti-S PPS antiserum which has been raised by ixmmmination with PPS isolated from human placentae. PP9 expression in E. eoli calls was further demonstrated by Western blot analyses using the above serum. In this eseparimeat 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. eoli control extracts, and extracts which contained pTrcS9C»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 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, ia addition to the complete PP9 amino acid - sequence encoded by tha 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* un-30 translated region occurring in the PP9 cDNA. As a check, tha construction indicated hereinbefore was likewise carried out. with the eaqpressien vectors pTrc99A and pTrcSSB (Amasn et al. loe.cit.). These vectors differ from pTreSSC by merely 2 bp (pTrc99A) aad 1 bp (pTrc99B) , which bring about shifts in tha translation reading frame. As expected, neither pTrc99A-PP9 nor pTrc99B-PP9 was able to induce the synthesis of PP9 proteins reacting with anti-PP9 aatissra, 4. Sbsepressioia. of saafcare, laua.f'used PP9 proteiaa Tha 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 EcoRI fragment which is 1337. bp in 10 sissa (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 tha desired orientation (PP9 ATG initiation codon at the left-hand, 5" distal end) was obtained and propagated. The plasmid DNA was 15 digested completely with Hindi 11 and partially with Ncol. The Ncol-HindlU 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 2 0 pTrc9SJk-PP9M (■«MW stands for "saaturs") comprises 553 5 bp and, after IPTG induction, expresses the mature, unfused PP9 protein. The N~terminal amino acid sequence of tha 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 imaunoprecipitation,, it being possible to detect a protein about 3 6 kD in size. 3 0 It has r.o« been found that this mature PP9 protein is transported into the periplasm of E»eoli cells and displays its enzysaatic activity (aldose reductase) there.

In order further to increase the expression rate of aldose reductase in E„eoli, an improved expression vector was constructed (pTaeT7L), which uses the ribosome-binding site of gene 10 of the T7 phage. It is ko.o«a that this sequexiee immediately in front of a heterologous gene 5 is able to increase its eacpression rate due to more efficient rihosome binding (01 ins et al. (1988), Gene 73, 227-235) . pTaeT/L is essentially based 013. the known vector pK3L223 -3 (Brosius &. Holy (1984)„ Proc. Natl. Acad. Sci. XJS«, 81 s 6929-6933) „ but, in contrast to the latter, 10 has the abovementio&ed T7 sequence ismsxediately in front of the cloning linker. The abovementionad PP9-encoding He a J-Hind! II fragment which is 1415 bp long was ligated into the pTacT7L vector cut with the same restriction enzymes. The resulting plasmid pTa.cT7L~PPS likewise 15 mediates the expression of the imfusad PP9 protein,, but the. yield of PP9 is about 20 times that achieved by pTrc99A-PP9M.

- Aldose reductase, activity of tSae proteis. PPS after eaqpressioaa ixt 3-eoli Comparisons of BPS cDMA sequence homologies revealed a 94% homology (85% identity) in the computer analysis with the aldose reductase of rats (Carper et al. (1987) FEES Lett. 220,, 209-213) . Further homologies discovered were to rho-crystallin of the frog eye and to the aldehyde 25 redi.iet.ase of the rat lens. This finding leads to the suggestion that PP9 is another member of a relatively large protein £ easily and very probably is the human aldose reductase.

Periplassmie fractions were prepared from E.coli K12 30 W31101acIQ (pTae"f7L-PP9) by the method of Hsiung et al. (1985) (Bio/Teehaoiogy 4, 991-995) . These eactracts have an aldose reductase activity which is not present in corresponding control extracts. Aldose reductase activity was detected using know®, assay methods (for example 3JT Kawasaki et al., (1989) Biochim. Biophys. Acta 996.. 30-36) which are based on a decrease in the absorption at 340 ma due to the oxidation of the NADPH in the assay-mixture .

Claims (13)

- 14 - Patent Claims

1. A nucleotide sequence shown in Table 1, or a sequence derived therefrom on the basis of the degeneracy 5 of tha genetic code,, coding for placenta-specific protein PP9„

2. A gene structure containing a nucleic acid as claimed in claim 1.

3. A vector containing a nucleic acid as claimed in 10 claim 1.

4. Transformed cells containing a nucleic acid as claimed in claim 1.

5. A* process for the preparation of PP9, which comprises a nucleic acid aa claimed in claim 1 being 15 introduced into an expression system and expressed therein.

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

7. A nucleotide sequence according to claim 1, substantially 20 as hereinbefore described.

8. A gene structure according to claim 2, substantially as hereinbefore described and exemplified. 25 30

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

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

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

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. F.R. KELLY ( CO., AGENTS FOR THE APPLICANTS.