CA2011877A1 - Cdna coding for placental protein 9 (pp9), the isolation and use thereof - Google Patents
Cdna coding for placental protein 9 (pp9), the isolation and use thereofInfo
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- CA2011877A1 CA2011877A1 CA002011877A CA2011877A CA2011877A1 CA 2011877 A1 CA2011877 A1 CA 2011877A1 CA 002011877 A CA002011877 A CA 002011877A CA 2011877 A CA2011877 A CA 2011877A CA 2011877 A1 CA2011877 A1 CA 2011877A1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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Abstract
Abstract of the disclosure cDNA coding for placental protein 9 (PP9), the isolation and use thereof 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 the genetically engineered preparation of PP9 possible.
Description
20~1~77 B~RI~G~ERRE ARTI~MOE SELLSCHAPT HOE 89/B 007 - Na 749 Dr. Lp/rd Description cDN~ coding for placental protein 9 (PP9), the isolation and u~e thereof The invention relatas to the i~olation o 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-Bl-0 037 963, PP9 has the following properties:
a) a carbohydrate content of 5.57 ~ 1035~, including:
hexo~e~ 4.9 ~ 1.0~; hexosamine~ 0.1 ~ 0.1%; fucose 0.07 i 0.05%; neuraminic acid 0.5 ~ 0O~%;
b) a sedimentation coefficient S20,W of 3.2 i 0.2 S;
c) a molecular weight determined in the ul~racentrifuge of 35,1~0 i 3,800;
d) a molecular weight determined in a polyacrylamide gel containing sodium dodecyl ~u~fa~e (SDS) of 40,000 i 4,000;
e) an extinction coefficient El~m (280 nm) of 14.6 + 1.0;
f3 an electrophoretic mobility in the region of the ~'-globulins;
g) an i~oelectric point in the region of p~ 5.0-6.8.
The conventional i~olation of PP9 de~cribed in the abovementioned patent is very elaborate and thus the object was to i601ate the gene coding for PP9 in order to make the genetically engineered preparation of PP9 possible.
a) a carbohydrate content of 5.57 ~ 1035~, including:
hexo~e~ 4.9 ~ 1.0~; hexosamine~ 0.1 ~ 0.1%; fucose 0.07 i 0.05%; neuraminic acid 0.5 ~ 0O~%;
b) a sedimentation coefficient S20,W of 3.2 i 0.2 S;
c) a molecular weight determined in the ul~racentrifuge of 35,1~0 i 3,800;
d) a molecular weight determined in a polyacrylamide gel containing sodium dodecyl ~u~fa~e (SDS) of 40,000 i 4,000;
e) an extinction coefficient El~m (280 nm) of 14.6 + 1.0;
f3 an electrophoretic mobility in the region of the ~'-globulins;
g) an i~oelectric point in the region of p~ 5.0-6.8.
The conventional i~olation of PP9 de~cribed in the abovementioned patent is very elaborate and thus the object was to i601ate the gene coding for PP9 in order to make the genetically engineered preparation of PP9 possible.
2~7 ~77 Surprisingly, when an expression gene bank was screened with antibodies against a different placental protein PP5 (Bohn and Winklerl Arch~ Gynak. 223, 179-186, 1977) which i8 not known to be similar to PP9, five individual clonPs were obtained (PP9-10, PP9-353, PP9-357, ~P9-361 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 tha sequencé - which i8 not quite complete however -coding for PP9 there is an intron about 600 bp in size, and no poly(A) sequence is pre~ent 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 PPS by the above process.
The sequencing data yield the result that the processed complete cDNA of PP9 is 1394 ba~e-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 above-mentioned patent ~see Tab. 2).
_ 3 ~ 7~
Table l GAGcGc~AGc;~GccA~GGcA~GccGTcTccTGcTcAAc~AcGGcGccAAGATGcccATccT
M A S R L L L N N G A K M P I L
GGGGl~TGGGTAccTGGAAGTccccTccl~GGGcAGGTGAcTGAGGccGTGAAGGTGGccAT
G L G T W K S P P G Q V T E A V R V A
TGAcGTcGGGTAccGccAcATcGAcTGTGcccATGTGTAccAGAATGAGAATGAGGTGGG
D v G Y R H I D C A H V Y Q N E N E V G
GGTGGccATTcAGGAGAAGcTcAGGGAGcAGGTGGTGAAGcGTGAGGAGcTcTTcATcGT
v A I Q E K L R E Q V V K R E E L F I V
CAGcAAGcTGTGGTGc~cGTAccATGAGAAGG&ccTGGTGAAAGGAGccTGccAGAAGAc S K L W C T Y H E ~ G L V R G A C Q R T
ACTCAGCGACCTGAAGCTGGACTACCTGGACCTCTACCTTATTCACTGGCCGACTGGCTT
L S D L ~ L D Y L D L Y L I H W P T G F
TAAGCCTGGGAAGGAATTTTTCCCATTGGATGAGTCGGGCAATGTGGTTCCCAGTGACAC
K P G K E F F P L D E S G N V V P S D T
C~ACATTCTGGACACGTGGGCGGCCATGGAAGAGCTGGTGGATGAAGGGCTGGTGAAAGC
N I L D T W A A M E E L V D E G L V R A
TATTGGCATCTCCAACTTC~ACCATCTCCAGGTGGAGATGATCTTAAAC~AACC~GGCTT
I G I S N F N H L Q V E M I L N ~ P G L
550 ~570 ~ 590 G~AGTAT~AGCCTGCAGTTAACCAGATTGAGTGCCACCC~TATCTC~CTCAGGAGAAGTT
R Y ~ P A V N Q I E C ~ P Y L T Q E ~ L
610 630 6~0 AATCCAGTAGTGCCAGTCCAAAGGCATCGTGGTGAGCGCCTA~GCCCCCTCGGCTCTCC
I Q Y C Q S X G I V V T A Y S P L G S P
TGACAGGCCCTGGGCCAAGCCCGAGGACCCTTCTCTCCTGGAGGATCC~G~ATCAAGGC
D R P W A R P E D P S L L E D P R I ~ A
GA~CGCAGCCl~AGCACAATAA ~CTACAGCCC~GGTCCTGATCeGGTTCCCC~TGCAGAG
I A A R H N ~ T T A Q V L I R F P M Q R
790 ~1~ 830 G~ACTTGGTGGTGATCCCC~AGTCTGTGACAC~AGAAC&C~TTGCTG~GA~C~TTAAGGT
20~877 N ~ ~ V I P R S V T P E R I A E N F R V
~50 870 890 CTTTGAcTTTGAAclrGAGcAGccAGGATATGAccAccTTAcTcAGcTAcAAcAGGAAcTG
F D F E L S S Q D M T T L L S Y N R N
GAGGGTCTGTGCCTT(;TTGAGCTGTACCTCCCACAAGGATTACCCCTTCCATGAAG~G'rT
R v C A L L S C T S ~ X D Y Y F H E E F
TTG~AGCTGTGGTTGCCTGCTCGTCCCCAAGTGACCTATACCTGTGTTTCTTGCCTCATT
TTTTTCCTTGC~AATGTAGTATGGCCTGTGTCACTCAGCAGTGGGACAGC~ACCTGTAGA
1090 lllO 1130 GTGGCCAGCG~GGGCGTGTCTAGCTTGATGTTGGATCTC~AGAGCCCTGTCAGTAGAGTA
GAAGTCTCTTCCAGTTTGCTTTGCCCTTCTTTCTACCCTGCTGGGGAAAGTACAACCTGA
121~ 1230 1250 ATACCCTTTTCTGACCAAAGAGAAGCAAAATCT~CCAGGTCAAAATAGTGCCACTAACGG
TTGAGTTTTGACTGCTTGGAACTGGAATCCTTTCAGCAAGACTTCTCTTTGCCTCAAATA
AAAAGTGCTTTTGTG
A~A~
20~ ~ 877 Table 2 ~ Amino acid composition of PP9 Amino acidNumber cDNA PP9 (EP - B1 - 0 037 963) A a Ala 19 6.013 5.69 ~ = 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 11 3.481 3.87 G = Gly 16 5.063 5.50 H = His 9 2.848 2.58 I - Ile 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 2G 6.329 6.17 Q = Gln 13 4.114 11.18 R = Arg 11 3.481 3.49 S = Ser 17 5.380 5.70 T = Thr 15 4.747 4.29 V = Val 25 7.911 7.12 W = Trp 6 1.899 2.36 Y = Tyr 11 3.481 3.95 Z = ~lx O O . 0~0 0 . 00 A~G 35 11.076 11.19 S+T 32 10.127 9.99 D+E 38 12.025 - -DfE+N+Q 66 20.886 --H+K~R 45 I4.241 14.11 D+E+H+K+R83 26.266 --I+L+M+V 83 26.266 24.42 F+W+Y 28 8.861 10.18 * = total of Asp + A3n ** = total of Glu + Gln - 6 - ~ 7 The invention iB further de~cribed with reference to the drawlng in which Figure 1 shows the position of lambda gtll-10 in relation to lambda gtll-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 po~ltion 925 (~ 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 sequen-ces. It was found that there are homologies to the DNAsequence of the aldose reductase of rats (Carper et al.
(1987) FEBS 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 i8 extremely probable that PP9 i5 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 i8 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 pTac~7L (see the examples).
Knowing the amino acid sequence of PP9, it i8 possible to prepare, by conventional or genetic engineering methods, amino acid part-sequences which are used as antigens for preparing polyclonal or monoclonal antibodie 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 2~877 genomic clone which codes for PP9 and with who~e aid not only is expression in eukaryotic cells facilitated but also further diagnostic information can be gained.
Aldose reductase (EC 1.1~1. 2 1 ) i8 an NADPH-dependant enzyme and cataly~es the reduction of aldose to the ~u~ar alcohol. In the pathological state of diabetes and of galactosemia, an increased aldo~e reducta~e activity in a number of ti~sues results in high level~ of sorbitol and galactitol. This may lead to, for example, cataracts in the 18n8 and to a thickening of the capillary membrane of the retina. Furthermore, increa~ed aldo~e reductase levels axe regarded as a cause of diabetic complications, such as of the nerves or of the ki~ney. For these reasons there is currently inten~ive work on developing aldose inhibitors. It has hitherto been nece3sary for this purpose to carry out an elaborate isolation of aldose reductase from animal tis~ues and organs, which moreover resulted in very low yields. The possibility of e~pres-sion of aldose reductase in Escherichia coli ~E.coli) now makes it possible to set up an assay system using bac-terial extracts and thus ~upersedes the dependence on animal organs and tissues. The assay method i5 consider-ably simplified overall.
The invention iB further defined in the patent claLms and ~5 explained further in the Pxamples which follow.
The following abbreviations are used, whexe not explained in the text:
EDTA = sodium ethylenediaminetetraacetate SDS = sodium dodecyl sulfate DTT = dithiothreitol BSA = bovine ~erum albumin IPTG = isopropyl thiogalactoside - 8 - 2 ~ ~ 3 ~77 _ample3 1. Screening of an expression cD~a bank from human placenta u~ing anti~PP5 ~ntibodies An expreseion cDNA bank in phage lambda gtll from Genofit GmbH, Heidelberg, was plated out at a den~ity of about 30,000 PFU per agar plate tl3.5 cm diameter~. For this, competent cells of the E.coli strain ylO90 (ATCC 37197) (R.A. Young and R.W. Davi~, Science Vol. ~22, 778-782/1983) were infected with the phages at 37~C for 30 min and then plated out in top agar on L broth plate~.
The plate~ 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 filter~ had previously been saturated with 10 mM IPTG in water. The plates with the filters were again incubated at 37DC 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% Tween 20 and 5%
skim milk powder) at 4C 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 tempera-ture for 1 h. (The ~olution of antibodies had previously been diluted 1:200 and saturated with non-recombinant lambda gtll lyzed E.coli cell~ on nitrocellulose fil~ers for 1 h). After the incubation with the primary antibody, the filters were washed with TBST for 4 x 10 ~in. The filters were then incubated with the ~econdary anti-rabbit antibody which was conjug tPd with alkalinephosphatase (from Promega, USA - marketed by Atlanta, Heidelberg) and previou~ly diluted 1:5000 in T~ST, 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 prLmary and second-2~8~7 ary antibodies were bound, by reaction of the alkaline phosphatase and a color reagent (ProtoBlot system from Protogen). For aach color reaction, 99 ~1 of NBT (nitro blue tetrazolium) substrate (50 mg/ml in 70% dimethyl-formamide) and 49.5 ~1 of BCIP (5-bromo-4-chloro-3-indolyl phosphate) substrate (50 mg/ml in 70% dimethyl-formamide) were added to 15 ml of AP buffer (100 mM tris-HCl, 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 ~ufficient blue coloration. The color reaction was stopped by immersiny the filters in a stop solution (20 mM tris-HCl, pH 8.0 and 5 mM ~DTA).
Positive signals were assigned to the plaques on the 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 ~ingle positive plaque. The result was the clones PP9-10, PP9-353, PP9-357, PP9-361 and PP9-362 with a positive reaction.
2. DNa ~equence analy8i8 The abovementioned phage clones were propagated, and the ~NA of each was extracted. The particular EcoRI fragment was isolated and ligated into the EcoRI site of the Bluescript M13 vector (Stratagene, San Diego, C~, USA~.
The sequence analysis was by the en2ymatic dideoxy method of Sang~r et al. (Proc.Natl.Acad.Sci.U5A 74, (1977)g5463-5467~. The sequence shows an open reading frame and codes for a protein with ~ maximum of 316 amino acids.
The sequencing data yield the result that the processed complete cDNA of PP9 is 1394 ba~e-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 above-mentioned patent ~see Tab. 2).
_ 3 ~ 7~
Table l GAGcGc~AGc;~GccA~GGcA~GccGTcTccTGcTcAAc~AcGGcGccAAGATGcccATccT
M A S R L L L N N G A K M P I L
GGGGl~TGGGTAccTGGAAGTccccTccl~GGGcAGGTGAcTGAGGccGTGAAGGTGGccAT
G L G T W K S P P G Q V T E A V R V A
TGAcGTcGGGTAccGccAcATcGAcTGTGcccATGTGTAccAGAATGAGAATGAGGTGGG
D v G Y R H I D C A H V Y Q N E N E V G
GGTGGccATTcAGGAGAAGcTcAGGGAGcAGGTGGTGAAGcGTGAGGAGcTcTTcATcGT
v A I Q E K L R E Q V V K R E E L F I V
CAGcAAGcTGTGGTGc~cGTAccATGAGAAGG&ccTGGTGAAAGGAGccTGccAGAAGAc S K L W C T Y H E ~ G L V R G A C Q R T
ACTCAGCGACCTGAAGCTGGACTACCTGGACCTCTACCTTATTCACTGGCCGACTGGCTT
L S D L ~ L D Y L D L Y L I H W P T G F
TAAGCCTGGGAAGGAATTTTTCCCATTGGATGAGTCGGGCAATGTGGTTCCCAGTGACAC
K P G K E F F P L D E S G N V V P S D T
C~ACATTCTGGACACGTGGGCGGCCATGGAAGAGCTGGTGGATGAAGGGCTGGTGAAAGC
N I L D T W A A M E E L V D E G L V R A
TATTGGCATCTCCAACTTC~ACCATCTCCAGGTGGAGATGATCTTAAAC~AACC~GGCTT
I G I S N F N H L Q V E M I L N ~ P G L
550 ~570 ~ 590 G~AGTAT~AGCCTGCAGTTAACCAGATTGAGTGCCACCC~TATCTC~CTCAGGAGAAGTT
R Y ~ P A V N Q I E C ~ P Y L T Q E ~ L
610 630 6~0 AATCCAGTAGTGCCAGTCCAAAGGCATCGTGGTGAGCGCCTA~GCCCCCTCGGCTCTCC
I Q Y C Q S X G I V V T A Y S P L G S P
TGACAGGCCCTGGGCCAAGCCCGAGGACCCTTCTCTCCTGGAGGATCC~G~ATCAAGGC
D R P W A R P E D P S L L E D P R I ~ A
GA~CGCAGCCl~AGCACAATAA ~CTACAGCCC~GGTCCTGATCeGGTTCCCC~TGCAGAG
I A A R H N ~ T T A Q V L I R F P M Q R
790 ~1~ 830 G~ACTTGGTGGTGATCCCC~AGTCTGTGACAC~AGAAC&C~TTGCTG~GA~C~TTAAGGT
20~877 N ~ ~ V I P R S V T P E R I A E N F R V
~50 870 890 CTTTGAcTTTGAAclrGAGcAGccAGGATATGAccAccTTAcTcAGcTAcAAcAGGAAcTG
F D F E L S S Q D M T T L L S Y N R N
GAGGGTCTGTGCCTT(;TTGAGCTGTACCTCCCACAAGGATTACCCCTTCCATGAAG~G'rT
R v C A L L S C T S ~ X D Y Y F H E E F
TTG~AGCTGTGGTTGCCTGCTCGTCCCCAAGTGACCTATACCTGTGTTTCTTGCCTCATT
TTTTTCCTTGC~AATGTAGTATGGCCTGTGTCACTCAGCAGTGGGACAGC~ACCTGTAGA
1090 lllO 1130 GTGGCCAGCG~GGGCGTGTCTAGCTTGATGTTGGATCTC~AGAGCCCTGTCAGTAGAGTA
GAAGTCTCTTCCAGTTTGCTTTGCCCTTCTTTCTACCCTGCTGGGGAAAGTACAACCTGA
121~ 1230 1250 ATACCCTTTTCTGACCAAAGAGAAGCAAAATCT~CCAGGTCAAAATAGTGCCACTAACGG
TTGAGTTTTGACTGCTTGGAACTGGAATCCTTTCAGCAAGACTTCTCTTTGCCTCAAATA
AAAAGTGCTTTTGTG
A~A~
20~ ~ 877 Table 2 ~ Amino acid composition of PP9 Amino acidNumber cDNA PP9 (EP - B1 - 0 037 963) A a Ala 19 6.013 5.69 ~ = 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 11 3.481 3.87 G = Gly 16 5.063 5.50 H = His 9 2.848 2.58 I - Ile 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 2G 6.329 6.17 Q = Gln 13 4.114 11.18 R = Arg 11 3.481 3.49 S = Ser 17 5.380 5.70 T = Thr 15 4.747 4.29 V = Val 25 7.911 7.12 W = Trp 6 1.899 2.36 Y = Tyr 11 3.481 3.95 Z = ~lx O O . 0~0 0 . 00 A~G 35 11.076 11.19 S+T 32 10.127 9.99 D+E 38 12.025 - -DfE+N+Q 66 20.886 --H+K~R 45 I4.241 14.11 D+E+H+K+R83 26.266 --I+L+M+V 83 26.266 24.42 F+W+Y 28 8.861 10.18 * = total of Asp + A3n ** = total of Glu + Gln - 6 - ~ 7 The invention iB further de~cribed with reference to the drawlng in which Figure 1 shows the position of lambda gtll-10 in relation to lambda gtll-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 po~ltion 925 (~ 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 sequen-ces. It was found that there are homologies to the DNAsequence of the aldose reductase of rats (Carper et al.
(1987) FEBS 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 i8 extremely probable that PP9 i5 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 i8 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 pTac~7L (see the examples).
Knowing the amino acid sequence of PP9, it i8 possible to prepare, by conventional or genetic engineering methods, amino acid part-sequences which are used as antigens for preparing polyclonal or monoclonal antibodie 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 2~877 genomic clone which codes for PP9 and with who~e aid not only is expression in eukaryotic cells facilitated but also further diagnostic information can be gained.
Aldose reductase (EC 1.1~1. 2 1 ) i8 an NADPH-dependant enzyme and cataly~es the reduction of aldose to the ~u~ar alcohol. In the pathological state of diabetes and of galactosemia, an increased aldo~e reducta~e activity in a number of ti~sues results in high level~ of sorbitol and galactitol. This may lead to, for example, cataracts in the 18n8 and to a thickening of the capillary membrane of the retina. Furthermore, increa~ed aldo~e reductase levels axe regarded as a cause of diabetic complications, such as of the nerves or of the ki~ney. For these reasons there is currently inten~ive work on developing aldose inhibitors. It has hitherto been nece3sary for this purpose to carry out an elaborate isolation of aldose reductase from animal tis~ues and organs, which moreover resulted in very low yields. The possibility of e~pres-sion of aldose reductase in Escherichia coli ~E.coli) now makes it possible to set up an assay system using bac-terial extracts and thus ~upersedes the dependence on animal organs and tissues. The assay method i5 consider-ably simplified overall.
The invention iB further defined in the patent claLms and ~5 explained further in the Pxamples which follow.
The following abbreviations are used, whexe not explained in the text:
EDTA = sodium ethylenediaminetetraacetate SDS = sodium dodecyl sulfate DTT = dithiothreitol BSA = bovine ~erum albumin IPTG = isopropyl thiogalactoside - 8 - 2 ~ ~ 3 ~77 _ample3 1. Screening of an expression cD~a bank from human placenta u~ing anti~PP5 ~ntibodies An expreseion cDNA bank in phage lambda gtll from Genofit GmbH, Heidelberg, was plated out at a den~ity of about 30,000 PFU per agar plate tl3.5 cm diameter~. For this, competent cells of the E.coli strain ylO90 (ATCC 37197) (R.A. Young and R.W. Davi~, Science Vol. ~22, 778-782/1983) were infected with the phages at 37~C for 30 min and then plated out in top agar on L broth plate~.
The plate~ 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 filter~ had previously been saturated with 10 mM IPTG in water. The plates with the filters were again incubated at 37DC 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% Tween 20 and 5%
skim milk powder) at 4C 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 tempera-ture for 1 h. (The ~olution of antibodies had previously been diluted 1:200 and saturated with non-recombinant lambda gtll lyzed E.coli cell~ on nitrocellulose fil~ers for 1 h). After the incubation with the primary antibody, the filters were washed with TBST for 4 x 10 ~in. The filters were then incubated with the ~econdary anti-rabbit antibody which was conjug tPd with alkalinephosphatase (from Promega, USA - marketed by Atlanta, Heidelberg) and previou~ly diluted 1:5000 in T~ST, 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 prLmary and second-2~8~7 ary antibodies were bound, by reaction of the alkaline phosphatase and a color reagent (ProtoBlot system from Protogen). For aach color reaction, 99 ~1 of NBT (nitro blue tetrazolium) substrate (50 mg/ml in 70% dimethyl-formamide) and 49.5 ~1 of BCIP (5-bromo-4-chloro-3-indolyl phosphate) substrate (50 mg/ml in 70% dimethyl-formamide) were added to 15 ml of AP buffer (100 mM tris-HCl, 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 ~ufficient blue coloration. The color reaction was stopped by immersiny the filters in a stop solution (20 mM tris-HCl, pH 8.0 and 5 mM ~DTA).
Positive signals were assigned to the plaques on the 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 ~ingle positive plaque. The result was the clones PP9-10, PP9-353, PP9-357, PP9-361 and PP9-362 with a positive reaction.
2. DNa ~equence analy8i8 The abovementioned phage clones were propagated, and the ~NA of each was extracted. The particular EcoRI fragment was isolated and ligated into the EcoRI site of the Bluescript M13 vector (Stratagene, San Diego, C~, USA~.
The sequence analysis was by the en2ymatic dideoxy method of Sang~r et al. (Proc.Natl.Acad.Sci.U5A 74, (1977)g5463-5467~. The sequence shows an open reading frame and codes for a protein with ~ maximum of 316 amino acids.
3. ~spre~sio~ of a PPll fusion protein pTrc99C (E. Amann et al., Gene 69, (1988), pp 301-315) was digested with EcoRI~ The clone lambda gtll-361 was digested with ~coRI, and the EcoRI insert which is 1387 bp in size was ligated with the pTrc99C vec$or fragment 2~ ,77 described above. The re~ulting plasmid pTrc99C-PP9 i~
able to induce the synthesi~ of an approx. 36 kD protein in E. coli cells. This protein can be immunoprecipitated ~pecifically with the aid of a monospecific rabbit anti-PP9 anti3erum which ha~ been raised by immunization withPP9 isolated from human placentae. PP9 expression in E.
coli cells wa~ further demon~trated by Western blot analyses using the above serum. In thi~ experiment there is a reaction only with the extract from IPTG-induced cells containing the pTrc99C-PP9 pla~mid, and a protein band of about 36 kD was again specifically vi~ualized.
Plasmid-free E. coli control extracts, and ~xtracts 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 construc-tion produced hereinbefore had the following N-terminal amino acid sequence, defined by the nucleotide sequence thereafter:
Vector/Linker /
5'UT Region Met Gly Asn Ser Ala Ala Met Ala Ser . . . CC ATG GGG AAT TCT GCA ( :CC ATG GCA AGC
The PP9 fusion protein defined by this con~truction carries, in addition to the complete PP9 amino acid sequence encoded by the PP9 cDNA, ~ix amino acid~ in front of the N terminus: four vector-encoded ~mino acids and two amino acid~ which are specified by the 5' un-translated region occurring in the PP9 cDNA. As a check,the construction indicated hereinbefore was likewise carried out with the expression vectors pTrc99A and pTrc99B (~mann et al. loc.cit.). These vectors differ from pTrc99C by merely 2 bp (pTrc99A) and 1 bp (pTxc99B), which bring about shift~ in the translation reading frame. As expec~ed, neither pTrc99A-PP9 nor pTrc99B-PP9 was able to induce the synthesi~ of PP9 proteins reacting with anti-PP9 antisera.
able to induce the synthesi~ of an approx. 36 kD protein in E. coli cells. This protein can be immunoprecipitated ~pecifically with the aid of a monospecific rabbit anti-PP9 anti3erum which ha~ been raised by immunization withPP9 isolated from human placentae. PP9 expression in E.
coli cells wa~ further demon~trated by Western blot analyses using the above serum. In thi~ experiment there is a reaction only with the extract from IPTG-induced cells containing the pTrc99C-PP9 pla~mid, and a protein band of about 36 kD was again specifically vi~ualized.
Plasmid-free E. coli control extracts, and ~xtracts 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 construc-tion produced hereinbefore had the following N-terminal amino acid sequence, defined by the nucleotide sequence thereafter:
Vector/Linker /
5'UT Region Met Gly Asn Ser Ala Ala Met Ala Ser . . . CC ATG GGG AAT TCT GCA ( :CC ATG GCA AGC
The PP9 fusion protein defined by this con~truction carries, in addition to the complete PP9 amino acid sequence encoded by the PP9 cDNA, ~ix amino acid~ in front of the N terminus: four vector-encoded ~mino acids and two amino acid~ which are specified by the 5' un-translated region occurring in the PP9 cDNA. As a check,the construction indicated hereinbefore was likewise carried out with the expression vectors pTrc99A and pTrc99B (~mann et al. loc.cit.). These vectors differ from pTrc99C by merely 2 bp (pTrc99A) and 1 bp (pTxc99B), which bring about shift~ in the translation reading frame. As expec~ed, neither pTrc99A-PP9 nor pTrc99B-PP9 was able to induce the synthesi~ of PP9 proteins reacting with anti-PP9 antisera.
4. ~xpression of mature, unfused PP9 proteins The PP9 cDNA has, besidss the NcoI site (5'CCA'rGG3') at the initiation codon, another NcoI site in the structural gene. In order to achiove matuxe expression of the PP9 protein, first the EcoRI fragment which is 1387 bp in size (see above) waæ ligated into the vector pMa5-8 (Stanssens et al., 1989). A plasmid (pMa5-8-PP9) in which the EcoRI frasment was present in the desired orientation (PP9 ATG initiation codon at the left-hand, 5' di~tal end) was obtained and propagated. The plasmid DNA was lS 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 ~"M~' s ands for "mature"~ comprises 5535 bp and, after IPTG induction, expresses the mature, unfused PP9 protein. The N-terminal amino acid sequenca of the PP9 protein is defined by the following nucleotide sequence:
Met Ala Ser Arg Leu ... AGG~CAGACC A.TG GCA AGC CGT CTC ...
The protein expressed in this way reacts with anti-PP9 antisera in W~stern blot and immunoprecipitation, it being possible to detect a protein about 36 kD in si~e.
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 - 2~ 77 aldose reductase in E.coli, an improved expression ~ector was cons~ructed (pTacT7L), which useA 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 ribosomQ binding (Olin~ et al. tl988), Gene 73, 227-235).
pTacT7L is essentially based on the known vector pKX223-3 (Brosius ~ Holy (1984), Proc. Natl. Acad. Sci. USA 81, 6929-6333), but, in contrast to the latter, has the abovementioned T7 ~equence 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 ~ame restriction enzymes.
The xesulting plasmid pTacT7L-PP9 liXewise mediates the expre~sion of the unfused PP9 protein, but the yield of PP9 is about 20 times that achieved by pTrc99A-PP9M.
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 ~"M~' s ands for "mature"~ comprises 5535 bp and, after IPTG induction, expresses the mature, unfused PP9 protein. The N-terminal amino acid sequenca of the PP9 protein is defined by the following nucleotide sequence:
Met Ala Ser Arg Leu ... AGG~CAGACC A.TG GCA AGC CGT CTC ...
The protein expressed in this way reacts with anti-PP9 antisera in W~stern blot and immunoprecipitation, it being possible to detect a protein about 36 kD in si~e.
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 - 2~ 77 aldose reductase in E.coli, an improved expression ~ector was cons~ructed (pTacT7L), which useA 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 ribosomQ binding (Olin~ et al. tl988), Gene 73, 227-235).
pTacT7L is essentially based on the known vector pKX223-3 (Brosius ~ Holy (1984), Proc. Natl. Acad. Sci. USA 81, 6929-6333), but, in contrast to the latter, has the abovementioned T7 ~equence 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 ~ame restriction enzymes.
The xesulting plasmid pTacT7L-PP9 liXewise mediates the expre~sion of the unfused PP9 protein, but the yield of PP9 is about 20 times that achieved by pTrc99A-PP9M.
5. Aldose reductase activity of the protein PP9 after expression in ~.coli Comparisons of PP9 cDNA sequence homologies revealed a 94~ homology (85% identity) in the computer analysis with the aldose reducta~e of rats tCarper et al. ~1987) FEBS
Lett. 220, 20g-213). Further homologies discovered were to Rho-crystallin of the frog eye and to the aldehyde reductase of the rat lens. This finding lead6 to the suggestion that PP9 is another member of a relatively large protein family and very probably is the human aldose reducta~e.
Periplasmic fractions were prepared from E.coli R12 W31101acIQ (pTacT7L-PP9) by the method of ~iung et al.
(1986) ~Bio/Technolosy 4, 991-995). These extracts have an aldosereductas2 activitywhich is not present incorres-ponding control extracts. Aldose reductase activity was detected u~ing 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 as~ay mixture.
Lett. 220, 20g-213). Further homologies discovered were to Rho-crystallin of the frog eye and to the aldehyde reductase of the rat lens. This finding lead6 to the suggestion that PP9 is another member of a relatively large protein family and very probably is the human aldose reducta~e.
Periplasmic fractions were prepared from E.coli R12 W31101acIQ (pTacT7L-PP9) by the method of ~iung et al.
(1986) ~Bio/Technolosy 4, 991-995). These extracts have an aldosereductas2 activitywhich is not present incorres-ponding control extracts. Aldose reductase activity was detected u~ing 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 as~ay mixture.
Claims (17)
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.
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. 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.
9. Polyclonal or monoclonal antibodies specific for PP9, obtained by immunization with PP9 prepared by genetic engineering, or parts thereof with antigenic activity.
10. 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.
13. PP9 as pharmaceutical.
14. A pharmaceutical which contains PP9.
15. A method for detecting or screening aldose reductase inhibitors, which comprises employing PP9 as claimed in claim 7.
16. The use of PP9 as claimed in claim 7 for screening or detecting aldose reductase inhibitors.
17. The DNA sequence as claimed in claim 1, and substantially as described herein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3907744A DE3907744A1 (en) | 1989-03-10 | 1989-03-10 | FOR PLAZENTAPROTEIN 9 (PP9) ENCODING CDNA, ITS INSULATION AND USE |
| DEP3907744.6 | 1989-03-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2011877A1 true CA2011877A1 (en) | 1990-09-10 |
Family
ID=6375984
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002011877A Abandoned CA2011877A1 (en) | 1989-03-10 | 1990-03-09 | Cdna coding for placental protein 9 (pp9), the isolation and use thereof |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0386733B1 (en) |
| JP (1) | JPH02295486A (en) |
| KR (1) | KR900014595A (en) |
| AT (1) | ATE124722T1 (en) |
| AU (1) | AU625082B2 (en) |
| CA (1) | CA2011877A1 (en) |
| DE (2) | DE3907744A1 (en) |
| DK (1) | DK0386733T3 (en) |
| ES (1) | ES2076238T3 (en) |
| IE (1) | IE67797B1 (en) |
| PT (1) | PT93381B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2680229B2 (en) * | 1992-02-12 | 1997-11-19 | 三菱瓦斯化学 株式会社 | Method for measuring human aldose reductase |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3013724A1 (en) * | 1980-04-10 | 1981-10-15 | Behringwerke Ag, 3550 Marburg | NEW PROTEIN PP (DOWN ARROW) 9 (DOWN ARROW), METHOD FOR ITS ENRICHMENT AND PRODUCTION AND ITS USE |
-
1989
- 1989-03-10 DE DE3907744A patent/DE3907744A1/en not_active Withdrawn
-
1990
- 1990-03-07 AT AT90104352T patent/ATE124722T1/en not_active IP Right Cessation
- 1990-03-07 ES ES90104352T patent/ES2076238T3/en not_active Expired - Lifetime
- 1990-03-07 DE DE59009360T patent/DE59009360D1/en not_active Expired - Fee Related
- 1990-03-07 EP EP90104352A patent/EP0386733B1/en not_active Expired - Lifetime
- 1990-03-07 DK DK90104352.1T patent/DK0386733T3/en active
- 1990-03-08 AU AU51103/90A patent/AU625082B2/en not_active Ceased
- 1990-03-09 PT PT93381A patent/PT93381B/en not_active IP Right Cessation
- 1990-03-09 CA CA002011877A patent/CA2011877A1/en not_active Abandoned
- 1990-03-09 JP JP2059800A patent/JPH02295486A/en active Pending
- 1990-03-09 IE IE85390A patent/IE67797B1/en not_active IP Right Cessation
- 1990-03-09 KR KR1019900003102A patent/KR900014595A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0386733A1 (en) | 1990-09-12 |
| KR900014595A (en) | 1990-10-24 |
| DE59009360D1 (en) | 1995-08-10 |
| ES2076238T3 (en) | 1995-11-01 |
| DE3907744A1 (en) | 1990-09-20 |
| AU625082B2 (en) | 1992-07-02 |
| PT93381A (en) | 1990-11-07 |
| IE900853L (en) | 1990-09-10 |
| ATE124722T1 (en) | 1995-07-15 |
| PT93381B (en) | 1996-02-29 |
| JPH02295486A (en) | 1990-12-06 |
| IE67797B1 (en) | 1996-05-01 |
| AU5110390A (en) | 1990-09-13 |
| DK0386733T3 (en) | 1995-11-20 |
| EP0386733B1 (en) | 1995-07-05 |
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