FR2649720A1 - Recombinant gene which encodes a protein such as urate oxidase - Google Patents

Abstract

The invention relates to a recombinant gene containing a DNA sequence which hybridises with a probe of formula: T C D1 A T D2 T C D3 A A D4 T A D5 T G in which the symbols D1, D2, D3, D4 and D5 represent A, G or T, T or C, G or A, G or A and G or A respectively, as well as with a probe of formula: A E1 E2 A A E3 C C C C A E4 A A E5 T G in which the symbols E1, E2, E3, E4 and E5 represent G or A, G or A, A, G, C, or T, G or A and T or C and which encodes a protein with an apparent molecular weight of about 33kDa, which catalyses the degradation of a metabolite.

Description

Recombinant gene encoding a protein such as urate oxidase.

 The invention relates to a new recombinant gene coding for a protein such as urate oxidase which catalyzes the degradation of a metabolite, an expression vector carrying this gene, prokaryotic microorganisms transformed with this expression vector, and that a new recombinant protein obtained using this gene and a drug containing the latter.

 Urate oxidase (EC 1.7.3.3.), Also known as uricase, is an enzyme in the purine degradation pathway. This enzyme does not exist in primates (such as humans), birds, some reptiles or most insects. Some dogs (like daLmatien) are also lacking.

acide urique + 2 . In humans, purine bases, adenine and guanine, are converted to xanthine. Xanthine is oxidized by Xanthine oxidase to form uric acid according to the xanthine + H2O + O2 reaction

uric acid + 2.

The radical 0. superoxide dismutase substrate is converted by the latter into hydrogen peroxide
Uric acid, a metabolite present in the blood, is normally found essentially as a soluble monosodium salt. However, it can happen that, in some people,
Uric acid precipitates and forms stones. Hyperuricemia, an increase in the amount of uric acid circulating in the blood, causes the deposition of uric acid in the cartilaginous tissues, which causes gout. Hyperuricemia can also have consequences on the kidneys: an excess of uric acid in the urine.

and in your kidneys can lead to uric acid nephrolithiasis, that is to say the accumulation of kidney stones that are very painful and can damage the kidney. These calculations are composed of uric acid possibly associated with phosphate salts and oxalates.

The overproduction of uric acid can be caused by various congenital metabolic defects, Lesch-Nyhan syndrome, excessive intakes of purine or proteins, treatments with uricosuric drugs, etc. (Gutman, AB and YU, T.F. (1968) Am. J.

Med. 45-756-779).

Urate oxidase, an enzyme that catalyzes the degradation of
Uric acid in allantoin (a much more soluble compound than uric acid, which does not crystallize at the concentrations reached in biological fluids), is therefore of therapeutic interest. Used in injection, it has a large number of advantages in the treatment of hyperuricemia and nephrolithiasis: speed of the hypo-uricemic effect (decrease of
The order of 50% of the hyperuricemia in less than 24 hours), better protection of the kidney against lithiases compared to other drugs such as allopurinol (xanthine oxidase inhibitor), etc.

 The urate oxidase presently used as a medicament is obtained by a process comprising the cultivation of an Aspergillus flavus mycelium and the isolation of urate oxidase by extraction of the culture medium as well as several steps of purification of this protein. This method, which allows obtaining high purity urate oxidase, however, has disadvantages. Indeed, the physiology and especially the genetics of A. flavus are not readily workable (WOLOSHUK et al., (1989) Applied approx.

Microbiol. flight. 55, p. 86-90). It is therefore not possible to obtain strains that produce this enzyme in large quantities.

On the other hand, A. flavus is likely to produce aflatoxins, which are sometimes difficult to separate. It should therefore be checked that the purified product is free of these toxins.

 There is therefore a need for tools and techniques of genetic engineering to overcome these disadvantages.

 The Applicant has constructed a cDNA library from the messenger RNAs of an A strain. flavus grown under urate oxidase production conditions, from which it was isolated, using two probes constructed on the basis of the partial sequence of A. urate oxidase. flavus determined experimentally and then sequenced a cDNA encoding urate oxidase. She then constructed an expression vector comprising this cDNA, transformed a strain of E. coli. coli K12 with the latter, cultured it and verified that the cell lysate contained a recombinant protein of the expected molecular weight, which has a urate oxidase activity (ability to degrade uric acid).

The invention therefore relates to a recombinant gene characterized in that it comprises a DNA sequence which hybridises with a probe of formula
TC D1 AT D2 TC D3 AA D4 AT D5 TG in which the symbols D1, D2, D3, D4 and D5 respectively represent (A, G or T), (T or C), (G or A) (G or A ) and (G or
A) as well as with a probe of formula
A E1 E2 AA E3 CCCCA E4 Y E E5 TG in which the symbols E1, E2, E3, E4 and E5 respectively represent (G or A), (G or A), (A, G, C or T), (G or A) and (T or C) and encodes a protein of apparent molecular weight of about 33 kDa, which catalyzes the degradation of a metabolite.

 Preferably this metabolite is uric acid.

A particularly preferred recombinant gene is that encoding the primary sequence protein hereinafter
MetSerAlaValLysAlaALaArgTyrGly LysAspAsnValArgValTyrLysValHis
LysAspGluLysThrGlyValGlnThrVal TyrGluMetThrValCysValLeuLeuGlu
GlyGluIleGluThrSerTyrThrLysAla AspAsnSerValIleValAlaThrAspSer
IleLysAsnThrIleTyrIleThrAlaLys GlnAsnProValThrProProGluLeuPce
GlySerIlELeuGlyThrHisPheIleGlu LysTyrAsnHisIleHisAlaAlaHisVal AsnfleValCysHisArgTrpThrArgMet AspIleAspGlyLysProHisProHisSer
PheIleArgAspSerGluGluLysArgAsn ValGlnValAspValValGluGlyLysGly
IleAspIleLysSerSerLeuSerGlyLeu ThrValLeuLysSerThrAsnSerGlnPhe
TrpGlyPheLeuArgAspGluTyrThrThr LeuLysGluThrTrpAspArgIleLeuSer ThrAspValAspAlaThrTrpGlnTrpLys AsnPheSerGlyLeuGlnGluValArgSer
HisValProLysPheAspAlaThrTrpAla ThrAlaArgGluValThrLeuLysThrphe AlaGluAspAsnSerAlaSerValGlnAla ThrMetTyrLysMetAlaGluGlnIleLeu AlaArg6lnGlnLeuIleGluThrValGlu TyrSerLeuProAsnLysHisTyrPheGlu
IleAspLeuSerX1HisLysGlyLeuGln AsnThrGlyLysAsnAlaGluValPheAla ProGlnSerAspProAsnGlyLeuIleLys CysThrValGlyArgSerSerLeuLysSer
LysLeu in which X1 represents Trp or Gly.

 Preferably X1 represents Trp.

Because of the degeneracy of the genetic code, there are a large number of DNA sequences encoding a protein whose sequence corresponds to the formula given above. Among these, a preferred sequence is the following:
ATGTCTGCGG TAAAAGCAGC GCGCTACGGC AAGGACAATG TTCGCGTCTA
CAAGGTTCAC AAGGACGAGA AGACCGGTGT CCAGACGGTG TACGAGATGA
CCGTCTGTGT GCTTCTGGAG GGTGAGATTG AGACCTCTTA CACCAAGGCC
GACAACAGCG TCATTGTCGC AACCGACTCC ATTAAGAACA CCATTTACAT
CACCGCCAAG CAGAACCCCG TTACTCCTCC CGAGCTGTTC GGCTCCATCC
TGGGCACACA CTTCATTGAG AAGTACAACC ACATCCATGC CGCTCACGTC
AACATTGTCT GCCACCGCTG GACCCGGATG GACATTGACG GCAAGCCACA
CCCTCACTCC TTCATCCGCG ACAGCGAGGA GAAGCGGAAT GTGCAGGTGG
ACGTGGTCGA GGGCAAGGGC ATCGATATCA AGTCGTCTCT GTCCGGCCTG
ACCGTGCTGA AGAGCACCAA CTCGCAGTTC TGGGGCTTCC TGCGTGACGA
GTACACCACA CTTAAGGAGA CCTGGGACCG TATCCTGAGC ACCGACGTCG
ATGCCACTTG GCAGTGGAAG AATTTCAGTG GACTCCAGGA GGTCCGCTCG
CACGTGCCTA AGTTCGATGC TACCTGGGCC ACTGCTCGCG AGGTCACTCT
GAAGACTTTT GCTGAAGATA ACAGTGCCAG CGTGCAGGCC ACTATGTACA
AGATGGCAGA GCAAATCCTG GCGCGCCAGC AGCTGATCGA GACTGTCGAG
TACTCGTTGC CTAACAAGCA CTATTTCGAA ATCGACCTGA GCTGGCACAA
GGGCCTCCAA AACACCGGCA AGAACGCCGA GGTCTTCGCT CCTCAGTCGG
ACCCCAACGG TCTGATCAAG TGTACCGTCG GCCGGTCCTC TCTGAAGTCT
AAATTG.

 The invention also relates to an expression vector which carries, with the means necessary for its expression, the recombinant gene defined above.

 For expression in prokaryotic microorganisms, in particular in Escherichia coli, the coding sequence must be inserted into an expression vector comprising in particular an effective promoter, followed by a ribosome binding site upstream of the gene to be expressed, as well as an efficient transcription stop sequence downstream of the gene to be expressed. This plasmid must also include an origin of replication and a selection marker. All these sequences must be chosen according to the host cell.

For expression in eukaryotic cells such as animal cells, especially in CHO Chinese hamster ovary cells, the coding sequence is inserted into a plasmid (e.g. derived from pBR 322) having two expression units. A first unit in which the gene of interest is inserted in front of an effective promoter (for example the SV40 early promoter.) The sequence around the initiation ATG is preferably chosen according to the consensus sequence described by
KOZAK (M. KOZAK (1978), 15, 1109-1123). An intronic sequence, for example an intron of mouse α-globin, can be inserted upstream of the gene of interest as well as an sequence comprising a polyadenylation site, for example a Sv40 polyadenylation sequence, downstream of the gene of interest. The second expression unit comprises a selection marker (for example a DNA sequence) coding for dihydrofolate reductase (abbreviated enzyme DHFR). The plasmid is transfected into eukaryotic cells, for example CHO DHFR cells (unable to express DHFR). A line is selected for its resistance to methotrexate and expresses at a sufficient level the gene of interest.

 For expression in eukaryotic cells such as yeast, for example Saccharomyces cerevisiae, the coding sequence should be inserted between, on the one hand, sequences recognized as an effective promoter, on the other hand a transcription terminator. The promoter-sequence sequence-terminator, called expression cassette, is cloned either in a multicopy vector for yeast, or integrated in multicopy in the genome of the yeast. The interest of yeast is to present a cellular machinery close to that of the natural host, A. flavus.

The invention also relates to a novel recombinant protein sequence as follows:
SerAlaValLYsAlaAlaArgTyrGly LysAspAsnValArgValTyrLysValHis LysAspGluLysThrGLyVaLGLnThrVal TyrGluMetThrValCysVaLLeuLeuGLu
GlyGluIleGluThrSerTyrThrLysAla AspAsnSerValIleValAlaThrAspSer
IleLysAsnThrIleTyrIleThrAlaLys GlnAsnProValThrProProGluLeuPhe GlySerIleLeuGLyThrHisPheILeGlu LysTyrAsnHisIleHisAlaAlaHisVaL
AsnIleValCysHisArgTrpThrArgMet AspIleAspGlyLysProHisProHisSer
PheIleArgAspSerGlyGluLysArgAsn ValGlnValAspValValGluGlyLysGly
IleAspIleLysSerSerLeuSerGlyLeu ThrValLeuLysSerThrAsnSerglnPhe
TrpGlyPheLeuArgAspGluTyrThrThr LeuLysGluThrTrpAspArgîleLeuSer ThrAspValAspAlaThrTrpClnTrpLys AsnPheSerGlyLeuGlnGluValArgSer HisValProLysPheAspAlaThrTrpAla ThrAlaArgGluvalThrLeuLysThrPhe
AlaGluAspAsnSerAlaSerValGlnAla ThrMetTyrLysMetAlaGluGlnIleLeu
AlaArgGlnGlnLeuIleGluThrValGlu TyrSerLeuProAsnLysHisTyrPheGlu
IleAspLeuSerTrpHisLysGlyLeuGln AsnThrGlyLysAsnAlaGluValPheAla ProGlnSerAspProAsnGlyLeuIleLys CysThrValGlyArgSerSerLeulysSer.

LysLeu possibly preceded by a methionine.

 Indeed, the protein encoded by the cDNA sequences given above can be processed by methionyl aminopeptidase, which cleaves it from its amino-terminal methionine residue.

 The invention also relates to the medicament containing the protein defined above.

The invention will be better understood using the following examples:
much of the following set of techniques, well known to those skilled in the art, is set forth in detail in
Maniatis et al: "Molecular cloning: a laboratory manual" published in 1984 by Cold Spring Harbor Press at
New York.

EXAMPLE 1 Isolation of messenger RNAs of Aspergillus flavus
The strain of A. urate oxidase producing flavus was cultured under conditions of urate oxidase production, that is to say in a medium containing uric acid of the following composition: glucose 15 g / l, MgSO 4, 7H 2 O 1 g / l , KH2PO4 0.75 g / l, CaCO3 1.2 g / l, uric acid 1.2 g / l, KOH 0.5 g / l, soybean oil 0.66 ml / l, FeSO4.7H20 10 mg / l 1, CuSO 4, 5H 2 O 1 mg / l, ZnSO 4, 7H 2 O 3 mg / l, MnSO 4, H 2 O 1 mg / l.

The medium is adjusted to pH 7 with 1M H2SO4 and is sterilized at 120 ° C for 80 min.

 In a 5-liter Erlenmeyer flask, 1.5 l medium is seeded with about 1 to 3,107 spores.

 The culture is incubated for about 40 hours at 30 ° C. with stirring (120 rpm). The mycelium is recovered by filtration on gauze, washed with water and frozen in liquid nitrogen.

 15 g of mycelium (wet weight) are thawed and resuspended in 45 ml of lysis buffer and then taken up in the same volume of beads (0.45 m in diameter). The lysis buffer consisted of 4 M guanidine thiocyanate, 10 mM Tris-HCl pH 7.6, 10 mM EDTA, 50 ml / l ss-mercaptoethanol. The mycelial suspension is ground in a Zellmühler mill (vibrogene) for 5 min.

 The ground material is recovered and the logs are decanted. The supernatant is removed (approximately 45 ml) and brought back to the final 3 M lithium chloride and stored at OOC.

 After two days, it is centrifuged for 60 minutes at 10,000 rpm. The supernatant is discarded and the pellet is taken up in 40 ml of 3M LiCl and recentrifuged at 10,000 rpm for 1 hour 30 minutes.

Proétinase K (SIGMA) was added with 40 g / ml of SDS (0.1% w / v) and 20 mM EDTA. Incubated at 370C for 3 h. The precipitate is precipitated with 2 volumes of ethanol, followed by washing with 70% ethanol. The pellet is taken up in 0.5 ml of TE buffer (tris
10 mM EDTA, 1 mM pH 7.5), extracted twice with chloroform, precipitated with ethanol. RNAs are stored at -800C in alcohol.

EXAMPLE 2 Purification of the poly A + fraction of RNAs
About 1 mg of RNA is precipitated for 20 minutes at 40 ° C. (15,000 rpm), then washed with 70% ethanol and then dried.

The pellet is taken up in 1 ml of TE buffer and resuspended by vortexing. The oligo dT-cellulose type 3 (marketed by
Collaborative Research Inc., Biomedicals Product Division) is prepared according to the manufacturer's recommendations. The RNA is deposited on oligo dT, gently stirred to resuspend your beads, then heated for 1 min at 650C.

The suspension is adjusted to 0.5 M NaCl and then stirred gently for 10 min. The suspension is then centrifuged for 1 min at 1000 rpm, the supernatant is removed, the pellet is washed twice with 1 ml of TE buffer containing 0.5 M NaCl. Supernatants are eliminated. The elution of the polyadenylated fraction of the RNAs (consisting of the messenger RNAs) is obtained by suspending the beads in 1 ml of TE buffer and then heating this suspension at 600C for 1 min, followed by stirring for 10 min on a tilting plate. . It is then centrifuged for 1 minute at 1000 rpm, which makes it possible to recover, on the one hand, the supernatant containing
Free mRNA in solution and secondly the pellet of celluLose beads. All of the above operations (from elution) are repeated. The supernatants thus obtained are combined, the excess beads are removed by centrifugation and the supernatant is precipitated with ethanol containing NaCl according to the usual techniques. (Maniatis: op.cit.

EXAMPLE 3 Constitution of the cDNA library
From isolated messenger RNAs as described in
The previous example, a cDNA library was made in the vector pTZ19R (marketed by PHARMACIA). This vector is a plasmid comprising a polylinker containing unique restriction sites.

The cloning technique used is that described by
Caput et al., (Primer-adapter technique): (Caput et al, Proc Natl Acad Sci (USA) (1986) 83, 16701674)).

It consists on the one hand in digesting the vector with PstI, adding a polyhydric tail on the protruding 3 'end and then digesting the plasmids thus obtained with BamHI. The fragment corresponding to the vector is purified on a column of Sepharose CL4B (Pharmacia). It therefore comprises a polydC tail at one end, the other end being cohesive, of the BamHI type. On the other hand,
Messenger RNAs are reverse transcribed from a primer whose sequence is as follows: ## STR2 ##

Thus, the cDNAs have at their 5 'end the sequence
GATCC complementary to the BamHI cohesive end.

The RNA-DNA hybrids obtained by the action of the reverse transcriptase are subjected to an alkaline hydrolysis which makes it possible to get rid of the RNA. The single-stranded cDNAs are then purified by 2 cycles on a Sepharose CL4B column and subjected to terminal transferase treatment so as to add 3 'polydGs. The cDNAs are inserted in single-stranded form into the vector prepared as described above. A second "adapting" oligonucleotide complementary to the primer is necessary to generate an "open" BamHI site at
The 5 'end of the cDNAs. After hybridization of the vector, the cDNA and the "adapter:", the recombinant molecules are circularized by
The action of T4 phage ligase. The single-stranded regions are then repaired by phage T4 DNA polymerase.

The plasmid pool thus obtained serves to transform strain MC 1061 for ampicillin resistance (Casabadan Chou and Cohen J.

Bact. (1980) 143 pages 9971-980).

EXAMPLE 4 Determination of the partial sequence of urate
oxidase
A urate oxidase preparation of A. flavus (SIGMA) was repurified by chromatography on a column Red-agarose 120 (SIGMA), followed by filtration on an acrylamide-agarose gel
Ultrogel Aca 44 (IBF).

 In order to obtain information on the amino acid sequence of the purified urate oxidase, allowing synthesis of the probes necessary for cDNA cloning, direct aminoterminal sequencing of the protein was attempted. The latter did not succeed, probably because of an amino-terminal blockade of the protein.

 The following strategy was therefore developed for obtaining the partial sequence of urate oxidase - cleavage of the protein by proteolytic enzymes (using the trypsin and V8 protease enzymes of Staphylococcus aureus) - separation of the polypeptides obtained by reverse phase HPLC - sequencing of the purified peptides.

1) Hydrolysis of urate oxidase by trypsin, purification and sequencing of peptides
9 mg / ml urea oxidase in 100 mM ammonium carbonate buffer pH 8.9 was trypsin-donated (Worthington,
TPCK) with a urate oxidase / trypsin ratio of 30/1 by weight at 300C for 24 h.After tryptic hydrolysis, 60 μg of digested urate oxidase was directly injected onto a Brownlee G18 grafted silica reverse phase HPLC column. (column 10 x 0.2 cm), equilibrated with acetonitrile 1 X (v / v), trifluoroacetic acid 0.1 X (v / v) in water. The peptides were then eluted by a linear gradient of acetonitrile in a solution of trifluoroacetic acid (0.1 X v / v) in water ranging from 1 X to 60 X of acetonitrile in 60 min, with a flow rate of 150 μl / min. The peptides at the outlet of the column were detected by measuring the optical density at 218 nm.

 The elution profile is shown in Figure 1, in which the numbers following the letter T (Trypsin) correspond to the identified peaks.

 Each peak was collected and stored at -200C until analysis of a protein sequencer (Applied Biosystems Model 470A), equipped with a chromatograph (Apptied Biosystems model 430A) that analyzed continuously The phenylthiohydantic derivatives formed, after each cycle of degradation.

Table (1) below shows the peptide sequences of the 9 identified peaks.

2) Hydrolysis of urate oxidase by V8 protease, purification and sequencing of peptides.

 Urate oxidase, at a concentration of 2 mg / ml in a 100 mM ammonium acetate buffer pH 6.8, was digested with Staphylococcus aureus protease V8 (Boehringer-Mannheim) with a urate oxidase / protease V8 ratio. from 60/1 to 300C for 72 hours.

160 g of digested urate oxidase were then injected onto a reversed phase HPLC column of G18 Brown grafted silica (10 x 0.2 cm column); particles (7 x 0.03 μm) equilibrated with 1% acetonitrile, 0.1% trifluoroacetic acid (v / v) in water. The peptides were then eluted by a linear gradient of acetonitrile in a solution of trifluoroacetic acid in water (0.1 X (v / v)) ranging from 1 X to 60 X acetonitrile in 60 min, with a flow rate of 150 μl / mI. The peptides at the outlet of the column were detected by measuring the optical density at 218 nm.

 The elution profile is shown in FIG. 2, in which the numbers following the letter V (protease V8) correspond to the identified peaks.

 Each peak was collected and stored at -20 ° C until analysis on the protein sequencer already mentioned.

 Table (1) below shows the peptide sequences of the 5 identified peaks.

TABLE (1)

<SEP> Sequencing <SEP> of <SEP> products <SEP> obtained <SEP> by <SEP> hydrolysis
<tb><SEP> T <SEP> 17 <SEP> Asn <SEP> - <SEP> Val <SEP> - <SEP> Gln <SEP> - <SEP> Val <SEP> - <SEP> Asp <SEP> - <SEP> Val <SEP> - <SEP> Val <SEP> - <SEP> Glu <SEP> - <SEP> Gly <SEP> - <SEP> Lys
<tb><SEP> T <SEP> 20 <SEP> Asn <SEP> - <SEP> Phe <SEP> - <SEA> Ser <SEP> - <SEA> Gly <SEP> - <SEA> Leu <SEP> - <SEP> Gln <SEP> - <SEP> Glu <SEP> - <SEP> Val
<tb> The <SEP> trypsin <SEP> T <SEP> 23 <SEP> Phe <SEP> - <SEP> Asp <SEP> - <SEP> Ala <SEP> - <SEP> Thr <SEP> - <SEP > Trp <SEP> - <SEP> Ala
<tb><SEP> T <SEP> 27 <SEP> His <SEP> - <SEP> Tyr <SEP> - <SEP> Phe <SEP> - <SEP> Glu <SEP> - <SEP> Ile <SEP><SEP> Asp <SEP> - <SEP> Leu <SEP> - <SE> Ser
<tb><SEP> T <SEP> 28 <SEP> Ile <SEP> - <SEP> Leu <SEP> - <SEA> Ser <SEP> - <SEP> Thr <SEA> - <SEA> Asp <SEP> - <SEP> Val <SEP> - <SEP> Asp <SEP> - <SEP> Ala <SEP> - <SEP> Thr <SEP> - <SEP> Trp <SEP> - <SEP> Gln <SEP> - <SEP> Trp <SEP> - <SEP> Lily
<tb><SEP> T <SEP> 29 <SEP> His <SEP> - <SEP> Tyr <SEP> - <SE> Phe <SEP> - <SEP> Glu <SEP> - <SEP> Ile <SEP> - <SEP> Asp <SEP> - <SEP> Leu <SEP> - <SE> Ser <SEP> - <SEP> Trp <SEP> - <SEP> His <SEP> - <SEP> Lys
<tb><SEP> T <SEP> 31 <SEP> Ser <SEP> - <SEP> Thr <SEP> - <SEP> Asn <SEP> - <SEA> Ser <SEP> - <SEP> Gln <SEP> - <SEP> Phe <SEP> - <SEP> Trp <SEP> - <SEP> Gly <SEP> - <SEP> Phe <SEP> - <SEP> Leu <SEP> - <SEP> Arg
<tb><SEP> T <SEP> 32 <SEP> Gln <SEP> - <SEP> Asn <SEP> - <SEP> Pro <SEP> - <SEP> Val <SEP> - <SEP> Thr <SEP> - <SEP> Pro <SEP> - <SEP> Pro <SEP> - <SEP> Glu <SEP> - <SE> Leu <SEP> - <SEP> Phe <SEP> - <SEP> Gly <SEP> - <SEP> Ser <SEP> - <SEP> Ile <SEP>
<SEP> Leu <SEP> - <SEP> Gly <SEP> - <SEP> Thr
<tb><SEP> T <SEP> 33 <SEP> Gln <SEP> - <SEP> Asn <SEP> - <SEP> Pro <SEP> - <SEP> Val <SEP> - <SEP> Thr <SEP> - <SEP> Pro <SEP> - <SEP> Pro <SEP> - <SEP> Glu <SEP> - <SE> Leu <SEP> - <SEP> Phe <SEP> - <SEP> Gly <SEP> - <SEP> Ser <SEP> - <SEP> Ile <SEP>
<SEP> Leu <SEP> - <SEP> Thr
<tb><SEP> V <SEP> 1 <SEP> Tyr <SEP> - <SE> Leu <SEP> - <SEP><SEP> - <SEP> Asn <SEP> - <SEP><SEP> Lys - <SEP> His <SEP> - <SEP> Tyr <SEP> - <SEP> Phe <SEP> - <SEP> Glu <SEP> - <SEP> Ile <SEP> - <SEP> Asp <SEP> - <SEP> Leu <SEP>
<SEP> Ser <SEP> - <SEP> Trp <SEP> - <SEP> His <SEP> - <SEP> Lys
<tb> A <SEP> The help <SEP> of <SEP> V <SEP> 2 <SEP> Val <SEP> - <SEP> Thr <SEP> - <SEP> Leu <SEP> - <SEP> Lys <SEP> - <SEP> Thr <SEP> - <SEP> Phe <SEP> - <SEP> Ala <SEP> - <SEP> Glu <SEP> - <SEA> Asp <SEP> - <SEP> Asn <SEP > - <SEP> Ser <SEP> - <SEP> Ala <SEP> - <SEA> Ser <SEP>
<SEP> Val <SEP> - <SEP> Gln <SEP> - <SEP> Ala
<tb> the <SEP> protease
<tb><SEP> V8 <SEP> V <SEP> 3 <SEP> Thr <SEP> - SEP> Ser <SEP> - <SEP> Tyr <SEP> - <SEP> Thr <SEP> - <SEP> Lys <SEP> - <SEP> Ala <SEP> - <SEP> Asp <SEP> - <SEP> Asn <SEP> - <SEA> Ser <SEP> - <SEP> Val <SEP> - <SEP> Ile <SEP> - <SEP> Val <SEP> - <SEP> Asp <SEP>
<SEP> Thr <SEP> - <SEP> Asp <SEP> - <SEP> Ser <SEP> - <SEP> Ile <SEP> - <SEP> Lys <SEP> - <SEP> Asn <SEP> - <SEP > Thr <SEP> - <SEP> Ile <SEP> - <SEP> Tyr <SEP> - <SEP> Ile <SEP> - <SEP> Thr
<tb><SEP> V <SEP> 5 <SEP> Gly <SEP> - <SEP> Lys <SEP> - <SEP> Gly <SEP> - <SEP> Ile <SEP> - <SEP> Asp <SEP> - <SEP> Ile <SEP> - <SEP> Lys <SEP> - <SEA> Ser <SEP> - <SEA> Ser <SEA> - <SEA> Leu <SEP> - <SEA> Ser <SEA> - <SEP> Gly <SEP> - <SEP> Leu <SEP>
<SEP> Thr <SEP> - <SEP> Val <SEP> - <SEP> Leu <SEP> - <SEP> Lys <SEP> - <SEA> Ser <SEP> - <SEP> Thr <SEP> - <SEP > Asn <SEP> - <SEP> Ser <SEP> - <SEP> Gln <SEP> - <SEP> Phe <SEP> - <SEP> Trp <SEP> - <SEP> Gly <SEP> - <SEP> Phe <September>
<SEP> Leu <SEP> - <SEP> Arg
<tb><SEP> V <SEP> 6 <SEP> Gly <SEP> - <SEP> Lys <SEP> - <SEP> Gly <SEP> - <SEP> Ile <SEP> - <SEP> Asp <SEP> - <SEP> Ile <SEP> - <SEP> Lys <SEP> - <SEA> Ser <SEP> - <SEA> Ser <SEA> - <SEA> Leu <SEP> - <SEA> Ser <SEA> - <SEP> Gly <SEP> - <SEP> Leu <SEP>
<SEP> Thr <SEP> - <SEP> Val <SEP> - <SEP> Leu <SEP> - <SEP> Lys
<Tb>
EXAMPLE 5 Screening of Bacteria
1) Preparation of labeled probes
Two probe pools deduced from amino acid sequences of the protein were synthesized using a DNA synthesizer
Biosearch 4600. The first pool corresponds to the residue sequence
His-Tyr-Phe-Glu-Ile-Asp (part of the sequence of T 27), ie from 5 'to 3'
ATGGG
TCGAT TC AA TA TG
TCAAA
This pool consists of 24 x 3 = 48 different oligonucleotides representing all possible combinations.

The second pool corresponds to the sequence of amino acid residues
Gln-Phe-Trp-Gly-Phe-Leu (part of the V5 sequence), from 5 'to 3'
GG AGT
AAGCCCCA 'AA TG
AA CAC
T
This pool consists of 24 x 4 = 64 combinations.

Probes are terminally labeled deoxynucleotide transferase (TdT) (commercially available from IBI, Inc.).

 The reaction is carried out on 100 ng of a mixture of oligonucleotides in solution (100 mg / ml) in "Cobalt" reaction buffer (supplied 10 times concentrated by IBI inc .: 1.4 M potassium cocodylate). pH 7.2, 300 mM dithiothreitol, 1 l deoxynucleotidyl-terminal-transferase enzyme (IBI inc) and 50 μCi deoxycytidyttriphosphate dCTP labeled with P32.

The reaction is carried out at 37 ° C. for 10 minutes and then is stopped by adding 1 μl of 0.5 M EDTA.

Extracted with phenol and dialyzed on a polyacrylamide column
Biogel P 10 (Biorad: 150-1050).

2) Hybridization and colony detection containing the urate oxidase cDNA:
About 40,000 colonies are screened by the in situ hybridization technique developed by Grunstein and Hogness (1975,
Proc. Natl. Acad. Sci. (USA), 72,396). About 6,000 bacteria are plated on Petri dishes to obtain isolated colonies. After incubation for 24 h at 370 ° C., each dish is replicated on 2 filters, each filter being intended to be treated with one of the 2 probe pools, so that all the colonies obtained are tested with the 2 probe pools in parallel.

The filters are hybridized with one of the 2 probe pools in a buffer containing 6 x SSC, 10 x Denhardt's and 100 g / ml of sonicated and denatured salmon sperm DNA (SIGMA). The hybridization temperature is 420.degree. C. and the duration is 16 hours. The 6 x SSC solution is obtained by diluting a 20 x SSC solution. The preparation of the 20 x SSC buffer is described in Maniatis, Fritsch and
Sambrook (op.cit.). In summary, this buffer contains 175.3 g / l of NaCl; 88.2 g / l of sodium citrate and is adjusted to pH 7 by a few drops of 10N NaOH. The 10 x Denhardt's solution contains 1 g of Ficoll, 1 g of polyvinylpyrrolidone, 1 g of human serum albumin per 500 ml of final volume.

 After washing in the 6 × SSC solution at 42 ° C. (3 h with 5 bath changes), the filters are wiped with Joseph paper and subjected to autoradiography. The filters are revealed after 16 hours. it appeared that about 0.5% of the colonies hybridized with the 2 pools of probes.

 5 of these colonies were taken and purified.

Plasmid DNA was prepared from each of these colonies, and this DNA was analyzed by digestion with either BamHI or HindIII, or both BamHI and HindIII.

 After analysis on agarose gel, it was found that the plasmids obtained are linearised with BamHI and with HindIII. The double digestions make it possible to release a fragment corresponding to the entirety of the cloned cDNA. The size of this fragment is about 1.2 Kb in 3 cases, about 0.9 Kb in the other 2 cases. For the determination below, one of the 0.9 Kb fragments and one of the 1.2 Kb fragments which were recloned were selected (see Example 6 below).

EXAMPLE 6 Determination of the sequence of the urate cDNA
oxidase
One of the fragments of 0.9 Kb on the one hand (clone 9A) and one fragment of 1.2 Kb (clone 9C) was recloned on the other hand in the DNA of the single-stranded phage replicative form. M13. The DNA of the M13 clones containing, on the one hand, the 0.9 Kb fragment and, on the other hand, the 1.2 Kb fragment was subjected to exonuclease digestion so as to generate a series of overlapping M13 clones (procedure Cyclone I Biosystem (I8I), which were sequenced by the dideoxyribonucleotide method (Sanger et al.
PNAS-USA-1977, 14, 5463-5467).

 The nucleotide sequence of clone 9C is shown in FIG. 3, which also indicates with an arrow + the beginning of clone 9A and by a nucleotide symbol provided with an asterisk * the sequence nucleotides of clone 9A that are not identical to those of clone 9C ( when we match the two sequences).

We observe that
the nucleotide sequence of the longest fragment (clone 9C) overlaps that of the shortest fragment (clone 9A), with two differences (v FIG. 3). One of the differences is silent, the other is a change from a tryptophan residue to a glycine residue. These differences may be due either to differences in the isolated messenger RNAs (see Example 2 above), or to the reverse transcriptase errors used in the constitution of the cDNA library (see Example 3 above).

 In the case of the longest fragment, an ATG codon (at position 109 in FIG. 3) opens an open phase corresponding to a polypeptide of 302 amino acids with a molecular mass of about 34240 Da, the sequence of which corresponds to the partial sequence. urate oxidase of A. purified flavus (see Example 4).

 FIG. 4 shows the open DNA sequence by the ATG codon and the encoded polypeptide, as well as by arrows opposite the polypeptide encoded with the sequenced peptides (Cf.

Example 4) obtained by hydrolysis of A urate oxidase. flavus using trypsin and protease Ve.

 It is found that the polypeptide sequence ends with the Ser-Lys-Leu triplet, typical of peroxisomal localization enzymes (Gould S.J. et al., J. Cell, Biology 108 (1989) 1657-1664).

EXAMPLE 7 Construction of an expression vector of cDNA
urate oxidase
Plasmid p466, an expression vector in
E. coli. The latter comprises a fragment of pBR327 including the origin of replication and the ampicillin resistance gene, it also comprises a synthetic promoter of E. coli (R.

RODRIGUEZ and M. CHAMBERLIN "Promoters-Structure ans function (1982)
Preager), a Shine-Dalgarno sequence, followed by a polylinker presenting the unique Ndel and KpnI sites, a transcription terminator (derived from phage fd), and the lac i gene.

 This plasmid was constructed from Enns E. coli hGH expression plasmid (p462) by substitution of a hGH gene-bearing fragment with urate oxidase cDNA.

 The construction of plasmid p466 will now be described in more detail in the following discussion in which reference will be made to FIGS. 5, 6, 7, 8, 9.

= Segment d'ADN issu du plasmide pBR322 = Localisation de l'origine de réplication (ORI) = Segment d'ADN contenant la séquence codant pour un
précurseur naturel de l'hGH

= Segment d'ADN du phage fd contenant un terminateur
de transcription = Segment d'ADN contenant un promoteur-opérateur
hybride tryptophane lactose UV5 = Segment d'ADN codant pour la !-lactamase (ApR
résistance à l'ampicilline).Figure 5 shows a restriction map of plasmid p163.1. The different restriction segments are marked arbitrarily according to the legend below

DNA segment from plasmid pBR322 = Localization of the origin of replication (ORI) = DNA segment containing the coding sequence for a
natural precursor of hGH

Phage fd DNA segment containing a terminator
of transcription = DNA segment containing a promoter-operator
tryptophan hybrid lactose UV5 = DNA segment encoding β-lactamase (ApR
ampicillin resistance).

FIG. 6 represents the restriction map of a plasmid p160 whose PvuI-XhoI-BamHI (1) and PvuI-ORI-BamHI (2) fragments respectively originate from plasmids p163,1 and pBR327 and whose small BamHI fragment (2 BamHI (1) is the fragment 3 described below.

= Séquence PvuI-BamHI issue du plasmide pBR327 = Séquence PvuI-XhoI issue du plasmide p163,1 = Séquence XhoI-HincII issue du plasmide p163,1

Figure 7 shows the restriction map of plasmid p373,2. The different restriction segments are marked arbitrarily according to the legend below

PvuI-BamHI sequence derived from plasmid pBR327 = PvuI-XhoI sequence derived from plasmid p163.1 = XhoI-HincII sequence derived from plasmid p163.1

= Fragment 3 décrit ci-après = Segment d'ADN du phage fd contenant un terminateur
de transcription
La' figure 8 représente une carte de restriction du plasmide p462, le fragment synthétique BglII-HindIII défini ci apres étant représenté par

<tb><SEP> ClaI <SEP> NdeI <SEP> Pstl
<tb> (Hindi) <SEP> It <SEP> I
<Tb>
Fragment 4 described below

Fragment 3 described below = DNA segment of phage fd containing a terminator
of transcription
FIG. 8 represents a restriction map of the plasmid p462, the synthetic fragment BglII-HindIII defined below being represented by

1) Construction du plasmide p373,2
La stratégie mise en oeuvre fait appel à des fragments obtenus à partir de plasmides préexistants accessibles au public et à des fragments préparés par voie de synthèse selon les techniques maintenant couramment utilisées. Les techniques de clonage employées sont celles décrites par T. MANIATIS EF, FRITSCH et J.FIG. 9 represents a restriction map of plasmid p466, the NdeI-KpnI fragment comprising the gene coding for urate oxidase being represented by

1) Construction of plasmid p373,2
The strategy used uses fragments obtained from publicly available pre-existing plasmids and fragments prepared by synthesis according to the techniques now commonly used. The cloning techniques employed are those described by T. MANIATIS EF, FRITSCH and J.

SAMBROOK, Cold Spring Harbor Laboratory (1982). The synthesis of the oligonucleotides is carried out using a DNA synthesizer
Biosearch 4,600.

Plasmid p163.1 (FIG. 5), described in patent application EP-A-0245138 and deposited at the CNCM under the reference I-530 on February 17, 1986, was subjected to digestion by enzymes.
PvuI and BamHI. This plasmid contains the gene coding for hGH. The PvuI-BamHI fragment - hereinafter fragment 1 - containing the site of action of the restriction enzyme XhoI, shown in FIG. 5, was purified.

Similarly, plasmid pBR327, well known to those skilled in the art (see Soberon, X et al., Gene, 9 (1980) 287-305), was digested with PuvI and BamHI enzymes. . The PvuI-BamHI fragment
- hereinafter fragment 2 -, containing the origin of replication, has been
purified.

Then we prepared fragment 3, which is a fragment
synthetic BamHI (1) -BamHI (2) containing the gene lac i and its promo
whose sequence is the following one on which the two ext
mites of the strand are identified by the numbers 1 and 2 to specify
the orientation of the fragment in the plasmids described in FIGS.
and 7.

FRAGMENT 3
BamHI (1) 5 'GATCC GCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT
GAGCTAACTT ACATTAATTG CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG
GAAACCTGTC GTGCCAGCTG CATTAATGAA TCGGCCAACG CGCGGGGAGA
GGCGGTTTGC GTATTGGGCG CCAGGGTGGT TTTTCTTTTC ACCAGTGAGA
CGGGCAACAG CTGATTGCCC TTCACCGCCT GGCCCTGAGA GAGTTGCAGC
AAGCGGTCCA CGCTGGTTTG CCCCACCACC CGAAAATCCT GTTTGATGGT
GGTTAACGGC GGGATATAAC ATGAGCTGTC TTCGGTATCG TCGTATCCCA
CTACCGAGAT ATCCGCACCA ACGCGCAGCC CGGACTCGGT AATGGCGCGC
ATTGCGCCCA GCGCCATCTG ATCGTTGGCA ACCAGCATCG CAGTGGGAAC
GATGCCCTCA TTCAGCATTT GCATGGTTTG TTGAAAACCG GACATGGCAC
TCCAGTCGCC TTCCCGTTCC GCTATCGGCT GAATTTGATT GCGAGTGAGA
TATTTATGCC AGCCAGCCAG ACGCAGACGC GCCGAGACAG AACTTAATGG
GCCCGCTAAC AGCGCGATTT GCTGGTGACC CAATGCGACC AGATGCTCCA
CGCCCAGTCG CGTACCGTCT TCATGGGAGA AAATAATACT GTTGATGGGT
GTCTGGTCAG AGACATCAAG AAATAACGCC GGAACATTAG TGCAGGCAGC
TTCCACAGCA ATGGCATCCT GGTCATCCAG CGGATAGTTA ATGATCAGCC
CACTGACGCG TTGCGCGAGA AGATTGTGCA CCGCCGCTTT ACAGGCTTCG
ACGCCGCTTC GTTCTACCAT CGACACCACC ACGCTGGCAC CCAGTTGATC
GGCGCGAGAT TTAATCGCCG CGACAATTTG CGACGGCGCG TGCAGGGCCA
GACTGGAGGT GGCAACGCCA ATCAGCAACG ACTGTTTGCC CGCCAGTTGT
TGTGCCACGC GGTTGGGAAT GTAATTCAGC TCCGCCATCG CCGCTTCCAC
TTTTTCCCGC GTTTTCGCAG AACGTGGCT GGCCTGGTTC ACCACGCGGG
AAACGGTCTG ATAACAGACA CCGGCATACT CTGCGACATC GTATAACGTT
ACTGGTTTCA CATTCACCAC CCTGAATTGA CTCTCTTCCG GGCGCTATCA
TGCCATACCG CGAAAGGTTT TGCGCCATTC GATGGTGTCCG 3 'BamHI (2)
Fragments 1, 2 and 3 were then ligated to obtain the plasmid p160 shown in FIG.

This plasmid was subjected to partial digestion with the restriction enzymes HincII and PstI. The big fragment
HincII-PstI, containing the origin of replication and shown in Figure 6, was then ligated to fragment 4, shown hereinafter, which is a synthetic DNA fragment carrying a coding sequence for the first 44 amino acids of a precursor of the hGH and upstream of this sequence of regulation signals.

FRAGMENT 4

-1 1
GAC AAC GCT ATG CTC CGC GCC CAT CGT CTG CAC CAG CTG GCC TTT GAC ACC TAC
CTG TTG CGA TAC GAG GCG CGG GTA GCA GAC GTG GTC GAC CGG AAA CTG TGG ATC
DNAMLRAHRLHQLAFLTY
Pst
GAAG GA GA GA GA GA TAT ATC CCA GA GA GAAG AAG TAT TCA TTC CTG CA
GTC CTC AAA CTT CTT CGG ATA TAG GGT TTC CTT GTC TTC ATA AGT AAG G
QEFEEAYIPKEQKYSF
44
In this fragment, the amino acids are designated by letters according to the following code:
A = Alanine M = Methionine
C = Cysteine N = Asparagine
D = Aspartic acid P = Proline
E = Glutamic acid Q = Glutamine
F = Phenylalanine R = Arginine
G = Glycine S = Serine
H = Histidine T = Threonine
I = Isoleucine V = Valine
K = Lysine W = Tryptophan
L = Leucine Y = Tyrosine
The sequences -35 (TTGCTT) and -10 (TATAAT) of the promoter sequence are successively underlined in this fragment, and the sequence of Shine and Dalgarno well known to those skilled in the art.

 The plasmid p380.1 was thus obtained.

Plasmid p380.1 was then digested with Clal and NdeI restriction enzymes so as to remove the small ClaI-NdeI fragment of fragment 4 above and to replace it with the ClaI-NdeI fragment below.
CLAI 5 'CGATAGCGTATAATGTGTGGAATTGTGAGCGGATAACA
TATCGCATATTACACACCTTAACACTCGCCTATTGT
Nde
ATTTCACACAGTTTTTCGCGAAGAAGGAGATATACA
TAAAGTGTGTCAAAAAGCGCTTCTTCCTCTATATGTAT 5 '
The resulting plasmid is plasmid p373,2 (Figure 7).

2) Construction of plasmid p466:
Plasmid p373.2 was double digested with BglII and HindIII enzymes. The large fragment resulting from this digestion was purified and ligated with a synthetic DNA fragment, whose sequence given below is intended to reconstitute the end of the hGH gene followed 3 'KpnI cloning sites and
SnaBI.

B
g
l
I
I
GATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGAT
---- ± -------- ± -------- ± -------- ± -------- + A TTCGTCTGGATGTCGTTCAAGCTGTGTTTGAGTGTGTTGCTA
GACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTC
--------- ± -------- ± -------- ± -------- ± -------- ± ---- ---- +
CTGCGTGATGAGTTCTTGATGCCCGACGAGATGACGAAGTCCTTCCTGTACCTGTTCCAG
F
S
P
I
GAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCTAGTAA
--------- ± -------- ± -------- ± -------- ± -------- ± ---- ---- + CTCTGTAAGGACGCGTAGCACGTCACGGCGAGACACCTCCCGTCGACACCGAAGATCATT
H
i
S n
K nd
pai
n BI
III
GGTACCCTGCCCTACGTACCA --------- ± -------- ± ---
CCATGGGACGGGATGCATGGTTCGA
This fragment includes the BglII and
HindIII. The new plasmid thus formed p462 (see Fig. 8) thus comprises a KpnI site and an NdeI site which will be used to clone the fragment bearing the urate oxidase cDNA into the expression vector.

 The hybrid plasmid derived from pTZ19R carrying the cDNA of about 1.2 Kb (clone 9C), urate oxidase (see Example 3) comprises a unique KpnI site. This site is located a few base pairs downstream of the cloning site of the cDNA. On the other hand, the urate oxidase cDNA contains an AccI site located near the 5 'end.

The AccI-KpnI fragment comprising the major part of this cDNA was therefore isolated and purified. On the other hand, two complementary oligonucleotides were synthesized, the sequence of which is given below: ## EQU2 ##
ACAGACGCCATTTTCGTCGCGCGATGCCGTTCCTGTTACAAGCGCAGA-5 'is intended to reconstitute the 5' end of the cDNA. This synthetic fragment thus obtained has one NdeI end and another AccII. The fragment and the synthetic sequence were ligated with the expression vector cut with KpnI and NdeI. This three-fragment ligation makes it possible to obtain the expression vector named p466, urate oxidase for E. coli (cf. FIG. 9). This plasmid was subjected to a series of enzymatic hydrolyses by restriction enzymes. , which made it possible to check the presence of the expected restriction sites, in particular those carried by the gene coding for urate oxidase.

The plasmid p466 therefore contains, by construction, a gene coding for the urate oxidase of sequence hereinafter:
ATGTCTGCGG TAAAAGCAGC GCGCTACGGC AAGGACAATG TTCGCGTCTA
CAAGGTTCAC AAGGACGAGA AGACCGGTGT CCAGACGGTG TACGAGATGA
CCGTCTGTGT GCTTCTGGAG GGTGAGATTG AGACCTCTTA CACCAAGGCC
GACAACAGCG TCATTGTCGC AACCGACTCC ATTAAGAACA CCATTTACAT
CACCGCCAAG CAGAACCCCG TTACTCCTCC CGAGCTGTTC GGCTCCATCC
TGGGCACACA CTTCATTGAG AAGTACAACC ACATCCATGC CGCTCACGTC
AACATTGTCT GCCACCGCTG GACCCGGATG GACATTGACG GCAAGCCACA
CCCTCACTCC TTCATCCGCG ACAGCGAGGA GAAGCGGAAT GTGCAGGTGG AÇGTGGTCGA GGGCAAGGGC ATCGATATCA AGTCGTCTCT GTCCGGCCTG
ACCGTGCTGA AGAGCACCAA CTCGCAGTTC TGGGGCTTCC TGCGTGACGA
GTACACCACA CTTAAGGAGA CCTGGGACCG TATCCTGAGC ACCGACGTCG
ATGCCACTTG GCAGTGGAAG AATTTCAGTG GACTCCAGGA GGTCCGCTCG
CACGTGCCTA AGTTCGATGC TACCTGGGCC ACTGCTCGCG AGGTCACTCT
GAAGACTTTT GCTGAAGATA ACAGTGCCAG CGTGCAGGCC ACTATGTACA
AGATGGCAGA GCAAATCCTG GCGCGCCAGC AGCTGATCGA GACTGTCGAG
TACTCGTTGC CTAACAAGCA CTATTTCGAA ATCGACCTGA GCTGGCACAA
GGGCCTCCAA AACACCGGCA AGAACGCCGA GGTCTTCGCT CCTCAGTCGG
ACCCCAACGG TCTGATCAAG TGTACCGTCG GCCGGTCCTC TCTGAAGTCT
AAATTG.

(Nucleotides different from nucleotides of cDNA isolated from A.

flavus are underlined in the sequence above. These differences were introduced on the Acci-Kpni synthetic fragment so as to have downstream of the ATG a nucleotide sequence more in line with those usually encountered in a prokaryotic gene).

EXAMPLE 8 Expression of the urate oxidase cDNA
E. coli strain K12 RR1 (Bethesda research Lab Inc.) was transformed for ampicillin resistance with plasmid p466 and with a negative control plasmid pBR322.

Colonies resistant to ampicillin were obtained in both cases.

1 colony of each type was cultured in medium (LB + ampicillin 100 μg / ml). After stirring for one night at 370 ° C., the two cultures were diluted 100 times in medium (LB + ampicillin 100 g / ml). After 1 h of culture is added IPTG (Isopropyl BDThiogalactoside) 1 mM for 3 h.

Immunodetection of urate oxidase by Western blot 1) Procedure:
From the culture medium obtained after 3 hours of induction with IPTG, an aliquot fraction corresponding to 0.2 ml of OD = 1 is taken. The latter is centrifuged and the supernatant is removed. The pellet is then subjected to a Western blot, a technique well known to those skilled in the art, which comprises the following steps: - Solubilization of the pellet by boiling for 10 min in a buffer called loading buffer consisting of Tris HCl 0.125 M pH 6.8 x 4 X SDS, 0.002 X bromophenol blue, 20 X glycerol, p-mercaptoethanol Z (according to the protocol described by LAEMMLI (UK)

LAEMMLI, Nature, 227 (1970), 680-685)), electrophoretic separation of the different proteins contained in the solubilisate according to the protocol described by LAEMMLI (U.K.

 LAEMMLI, Nature, 227 (1970), 680-685), - transfer of said proteins contained in the gel to a nitrocellulose filter (according to the technique of H. TOWBIN et al., Proc.

Native. Acad. Sci. USA 76 (1979) 4350-4354),
Immunodetection, performed using the technique of BURNETTE (WW

BURNETTE Ana. Biochem. 112 (1981) 195-203), it involves successively
Rinse the nitrocellulose filter for 10 min with a
buffer A (10 mM Tris-HCl, 170 mM NaCl, 1 mM KI).

The contacting of the nitrocellulose filter for 30 minutes
at 370C with buffer B (buffer A plus serum
bovine albumin at a rate of 3 g per 100 ml).

. The contacting of the nitrocellulose filter for 1 hour at
370C with antiserum (polyclonal antibodies recognize
the urate oxidase of A. flavus).

 Rinsing the nitrocellulose filter with buffer B.

The contacting of the nitrocellulose filter for 1 hour at
37 C with a solution of protein G arched with iodine 125 to
0.1 microcurie / ml.

 . Rinsing the filter with buffer A.

 . The drying of the filter between two absorbent sheets.

 . The contacting of a film. ray.

 . The revelation of the film.

2) Results:
It is found that the strain transformed with the plasmid p466 overproduces a protein of apparent molecular weight of about 33 KDa which is recognized by the antibodies directed against the urate oxidase A. flavus and which is absent in the control strain.

EXAMPLE 9 Assay of the urate oxidase activity
From the culture medium obtained after 3 h of IPTG induction under the culture conditions described in the preceding example, an aliquot fraction corresponding to the equivalent of 0.5 ml at OD = 1 is taken. The latter is centrifuged. and we eliminate the supernatant. The pellets are taken up in 1 ml of 0.05M TEA buffer (Triethanolamine), pH 8.9. The cell suspension is sonicated 2 times for 30 seconds in the ice with ultrasonic sounder W10 (set at power 8 and intensity 4). The extracts are centrifuged at 10,000 g for 10 min, supernatants are used for the assay.

 The above operations are carried out for four colonies randomly selected from E. coli K12 transformed with the plasmid p466 (colonies A1, B1, C1 and D1) and a colony transformed with the plasmid pBR322).

1) Principle
The transformation of uric acid to allantoin is followed by the decrease in absorbance at 292 nm. The reaction is as follows:

<tb>S;<SEP> 4 <SEP> Urate <SEP> Oxidase <SEP> Yo
<tb> H2D <SEP> + <SEP> 02 <SEP> H20 <SEP> + <SEP> C 2 <SEP> -
Uric acid Allantoine
(absorbing at 292 nm) 2) Reagents
a) 0.05 M TEA buffer pH 8.9 / EDTA - dissolve 7.5 g of TEA (reagent for analysis-Prolabo ref.

287.46.266) in 400 ml of distilled water, dissolve 0.372 g of Complexon III (Merck-ref 8418) in 50 ml of distilled water, combine the two solutions and make up to 500 ml (solution 1), adjust the pH of this solution to 8.9 with 2N HCl, make up to 1000 ml with distilled water (solution 2).

 b) Uric acid Stock solution - dissolve 100 mg of uric acid (Carbiochem No. 6671) in 50 ml of solution 1, - adjust with 0.2N HCl to pH 8.9, - make up to 100 ml with distilled water.

 The resulting solution can be stored for one week at 40C.

 c) Uric acid Solution Substrate - withdraw 1.5 ml uric acid stock solution (carbiochem ref.

6671) and dilute to 100 ml with TEA buffer / reagent for analysis
Prolabo ref. 287.46.266).

 This solution should be used during the day.

3) Operating mode
The following volumes are introduced into the quartz vat of a spectrophotometer set at 292 nm and thermostated at 300 ° C.: - 600 l of uric acid substrate solution (preheated to 30 ° C.), - 100 l of the above supernatants to which added 200 l of TEA pH 8.9 (preheated to 3O0C).

 The optical density change is mixed and read every 30 seconds for 5 minutes. LE is deduced, variation of the optical density per minute.

4) Results
Activity A enzymatic urate oxidase expressed in U / ml
DO 1 is calculated from the measure of #E using the formula
A = #E x Vr xd
#I x VPE in which the symbols Vr, d, EI and VpE respectively represent the reaction volume (0.9 ml), the dilution ratio (2), the extinction coefficient of uric acid at 292 nm (12 , 5) and the volume of the test sample (0.1 ml).

The results obtained are collated in Table (II) below:
TABLE (II)

<tb><SEP> Strain <SEP> of E. <SEP> coLi <SEP> K12 <SEP> Activity <SEP> urate <SEP> oxidase
<tb><SEP> transformed <SEP> with <SEP> (U / ml <SEP> DO <SEP> 1)
<tb> pBR322 <SEP><<SEP> 0.001
<tb><SEP> colony <SEP> A1 <SEP> 0.086
<tb><SEP> colony <SEP> B1 <SEP> 0.119
<tb> p466
<tb><SEP> colony <SEP> C1 <SEP> 0.135
<tb><SEP> colony <SEP> D1 <SEP> 0.118
<Tb>
It is clear from the table above that the cells of E. coli transformed with plasmid p466 are capable in the presence of IPTG to produce urate oxidase activity.

Claims (9)

1. Recombinant gene characterized in that it comprises a DNA sequence which hybridizes with a probe of formula:  TC D1 TO T D2 TC D3 AA D4 TA D5 TG in which symbols D1, D2, D3, D4 and D5 respectively represent (A, G or T), (T or C), (G or A), CG or A ) and (G or A) as well as with a probe of formula:  A E1 E2 AA E3 CCCCA E4 Y E E5 TG in which the symbols E1, E2, E3, E4 and E5 respectively represent (G or A), (G or A), (A, G, C or T), (G or A) and (T or C) and encodes a protein of apparent molecular weight of about 33 kDa, which catalyzes the degradation of a metabolite. 2. Recombinant gene according to claim 1, characterized in that the metabolite is uric acid. 3. recombinant gene according to one of claims 1 or 2, characterized in that the protein has the following sequence MetSerAlaValLysAlaAlaArgTyrGly LysAspAsnValArgValTyrLysValHis LysAspGluLysThrGlyValGlnThrVal TyrGluMetThrValCysValLeuLeuGlu GlyGluIleGluThrSerTyrThrLysALa AspAsnSerValIleValAlaThrAspSer IleLysAsnThrIleTyrIleThrAlaLys GlnAsnProValThrProProGluLeuPhe GlySerIleLeuGlyThrHisPheIleGly LysTyrAsnHisIleHisAlaAlaHisVal AsnIleVatCysHisArgTrpThrArgMet AsplIeAspGlyLysProHisProHisSer PheIleArgAspSerGluGluLysArgAsn ValGlnValAspValValGluGlyLysGly IleAspIleLysSerSerLeuSerGlyLeu ThrValLeuLysSerThrAsnSerGlnPhe TrpGlyPheLeuArgAspGluTyrThrThr LeuLysGluThrTrpAspArgIleLeuSer ThrAspValAspAlaThrTrpGlnTrpLys AsnPheSerGlyLeuGlnGluValArgSer HisValProLysPheAspAlaThrTrpAla ThrAlaArgGluValThrLeuLysThrphe AlaGluAspAsnSerAlaSerValGlnAla ThrMetTyrLysMetAlaGluGlnlleLeu AlaArgGlnGlnLeuIleGluThrValGlu TyrSerLeuProAsnLysHisTyrPheGlu IleAspLeuSerXiHisLysGlyLeuGln AsnThrGlyLysAsnAlaGluValPheAla ProGlnSerAspProAsnGlyLeuIleLys CysThrValGlyArgSerSerLeuLysSer LysLeu in which X1 represents Trp or Gly. 4. Recombinant gene according to claim 3, characterized in that X1 represents Trp. 5. Recombinant gene according to any one of claims 1 to 4, characterized in that said DNA sequence contains the following sequence: ATGTCTGCGG TAAAAGCAGC GCGCTACGGC AAGGACAATG TTCGCGTCTA CAAGGTTCAC .AAGGACGAGA AGACCGGTGT CCAGACGGTG TACGAGATGA CCGTCTGTGT GCTTCTGGAG GGTGAGATTG AGACCTCTTA CACCAAGGCC GACAACAGCG TCATTGTCGC AACCGACTCC ATTAAGAACA CCATTTACAT CACCGCCAAG CAGAACCCCG TTACTCCTCC CGAGCTGTTC GGCTCCATCC TGGGCACACA CTTCATTGAG AAGTACAACC ACATCCATGC CGCTCACGTC AACATTGTCT GCCACCGCTG GACCCGGATG GACATTGACG GCAAGCCACA CCCTCACTCC TTCATCCGCG ACAGCGAGGA GAAGCGGAAT GTGCAGGTGG ACGTGGTCGA GGGCAAGGGC ATCGATATCA AGTCGTCTCT GTCCGGCCTG ACCGTGCTGA AGAGCACCAA CTCGCAGTTC TGGGGCTTCC TGCGTGACGA GTACACCACA CTTAAGGAGA CCTGGGACCG TATCCTGAGC ACCGACGTCG ATGCCACTTG GCAGTGGAAG AATTTCAGTG GACTCCAGGA GGTCCGCTCG CACGTGCCTA AGTTCGATGC TACCTGGGCC ACTGCTCGCG AGGTCACTCT GAAGACTTTT GCTGAAGATA ACAGTGCCAG CGTGCAGGCC ACTATGTACA AGATGGCAGA GCAAATCCTG GCGCGCCAGC AGCTGATCGA GACTGTCGAG TACTCGTTGC CTAACAAGCA CTATTTCGAA ATCGACCTGA GCTGGCACAA GGGCCTCCAA AACACCGGCA AGAACGCCGA GGTCTTCGCT CCTCAGTCGG ACCCCMCGG TCTGATCAAG TGTACCGTCG GCCGGTCCTC TCTGAATCTCT AMTTG. 6. Expression vector, characterized in that it carries, with the means necessary for its expression, a recombinant gene according to any one of claims 1 to 5. 7. Prokaryotic microorganisms, characterized in that they are transformed by an expression vector according to claim 6. 8. Recombinant protein characterized in that it has the following sequence  SerAlaValLysAlaAlaArgTyrGly LysAspAsnValArgValTyrLysValHis LysAspGluLysThrGlyValGlnThrVal TyrGluMetThrValCysValLeuLeuGlu GlyGlulleGluThrSerTyrThrLysAla AspAsnSerValIleValAlaThrAspSer IleLysAsnThrIleTyrIleThrAlaLys GlnAsnProValThrProProGluLeuPle GlySerIleLeuGlyThrHisPheIleGlu LysTyrAsnHisIleHisAlaAlaHisVal AsnlleValCysHisArgTrpThrArgMet AsplleAspGlyLysProHisProHisSer PheIleArgAspSerGluGluLysArgAsn ValGlnValAspValValGluGlyLysGly IleAsplleLysSerSerLeuSerGlyLeu ThrValLeuLysSerThrAsnSerGlnPhe TrpGlyPheLeuArgAspGluTyrThrThr LeuLysGluThrTrpAspArgIleLeuSer ThrAspValAspAlaThrTrpGlnTrpLys AsnPheSerGlyLeuGlnGluValArgSer HisValProLysPheAspAlaThrTrpAla ThrAlaArgGluValThrLeuLysThrPhe AlaGluAspAsnSerAlaSerValGlnAla ThrMetTyrLysMetAlaGluGlnIleLeu AlaArgGlnGlnLeulleGluThrValGlu TyrSerLeuProAsnLysHisTyrPheGlu IleAspLeuSerTrpHisLysGlyLeuGln AsnThrGlyLysAsnAlaGluValPheAla ProGlnSerAspProAsnGlyLeuIleLys CysThrValGlyArgSerSerLeuLysSer LysLeu possibly preceded by a methionine. 9. Medicinal product characterized in that it contains the recombinant protein according to claim 8.