AU636637B2 - Urate oxidase activity protein, recombinant gene coding therefor, expression vector, micro-organisms and transformed cells - Google Patents

Abstract

The invention concerns a new urate oxidase activity protein which has the sequence (I), possibly preceeded by a methionine, or in that it may present a degree of substantial homology with this sequence. The invention is also aimed at medicines containing this protein, as well as the genetic engineering implements to obtain it.

Description

636637 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION NAME ADDRESS OF APPLICANT: Sanofi avenue George V Paris 75008 France NAME(S) OF INVENTOR(S): Daniel CAPUT Pascual FERRARA Jean-Claude GUILLEMOT Mourad KAGHAD Richard LEGOUX Gerard LOISON Elisabeth LARBRE Johannes LUPKER Pascal LEPLATOIS Marc SALOME Patrick LAURENT ADDRESS FOR SERVICE: DAVIES COLLISON Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: Urate oxidase activity protein, recombinant gene coding therefor, expression vector, micro-organisms and transformed cells The following statement is a full description of this invention, including the best method of performing it known to me/us:la The invention relates to a novel protein possessing urate oxidase activity the invention also concerns the drugs containing this protein as well as the genetic engineering tools for producing that protein and notably the recombinant gene coding for that protein, the expression vector carrying that gene and the eukaryotic cells or the prokaryotic microorganisms transformed by this expression vector.

Urate oxidase (EC which is also called uricase, is an enzyme of the purine degradation pathway.

This enzyme does not exist in primates (such as man), birds, a few reptiles or most insects. It is also nonexistent in some dogs (such as the dalmatian).

In man, the purine bases adenine and guanine are converted to xanthine. The xanthine is oxidized by xanthine oxidase to form uric acid according to the following reaction: xanthine H20 02 uric acid 02- The 02- radical, which is the substrate for superoxide disnutase, is converted by the latter to hydrogen peroxide.

Uric acid, a metabolite present in blood, is normally found essentially in the form of the soluble monosodium salt. However, in certain people, it may happen that the uric acid precipitates and forms calculi.

Hyperuricemia, which is an increase in the amount of uric acid circulating in the blood, causes uric acid to deposit in the cartilaginous tissues, leading to gout.

Hyperuricemia can also have consequences on the kidneys: an excess of uric acid in the urine and in the kidneys can result in uric acid nephrolithiasis, i.e. the 2 accumulation of renal calculi, which are very painful and can damage the kidney. These calculi are composed of uric acid possibly associated with phosphate and oxalate salts. Overproduction of uric acid can have a variety of origins congenital metabolic defects, Lesch-Nyhan syndrome, excess ingestion of purine or proteins, treatments with uricosuric drugs,treatments of the hemopathies, particularly the cancerous hemopathies by cytolytic agents (chemotherapy) or by radiotherapy. (Gutman, A.B. and YU, T.F.

(1968) Am. J. Med. 45 756-779).

Urate oxidase, the enzyme which catalyzes the degradation of uric acid to allantoin (a compound which is much more soluble than uric acid and does not crystallize at the concentrations reached in biological fluids), therefore has therapeutic value. Used in injections, it has a large number of advantages in the treatment of hyperuricemia and nephrolithiasis: speed of the hypouricemic effect (reduction of hyperuricemia of the order of 50% in less than 24 better protection of the kidney against lithiasis compared with other drugs such as-allopurinol (a xanthine oxidase inhibitor), etc.

At the present time, this enzyme is mainly used as adjuvant for the cvtolytic agents in chemotherapy.

The urate oxidase currently used as a drug is obtained by a method comprising the culture of a mycelium of Aspergillus flavus and isolation of the urate oxidase from the culture medium by extraction, together with several steps for purifying this protein. This method, which makes it possible to obtain urate oxidase of high purity, nevertheless has disadvantages. In fact, the physiology and especially the genetics of A. flavus are not easy to work with (WOLOSHUK et al. (1989) Applied environ. microbiol., vol. 55, p. 86-90). It is therefore impossible to obtain strains which produce this enzyme in substantial amounts. Furthermore, A. flavus is liable to produce aflatoxins, which are sometimes difficult to separate off. The purified product should consequently be checked to ensure that it is free from these toxins.

-ON

3 There is therefore a need for a purer urate oxidase of A. flavus as well as for genetic engineering tools and techniques whereby these disadvantages can be overcome.

The Applicant purified the urate oxidase extracted from A. flavus, named thereafter the urate oxidase extract, up to a purity degree higher than that already known for this protein the Applicant also determined the partial sequence of that protein and built two pools of labelled probes able to hybridize with the nucleotides coding for two portions of that protein.

It then constructed an expression vector comprising this cDNA, transformed a strain of E. coli K12 with the latter, cultivated said strain and verified that the lyzate of the cells contained a recombinant protein of the expected molecular weight, which possesses urate oxidase activity (capacity to degrade uric acid in allantoine).

The Applicant also constructed several vectors for expression in eukaryotic cells, comprising a recombinant gene coding for urate oxidase whose sequence contains variations, relative to the isolated cDNA, introduced for the purpose of inserting codons which are customary in eukaryotic cells, transformed different eukaryotic cells with the aid of these vectors, cultivated said cells in a small volume as well as in a larger volume (fermenter), and found that the lyzates of the cells contained a substantial proportion of a recombinant protein of the expected molecular weight, possessing urate oxidase activity. It purifiea this reconbinant protein and partially charactcrized it, comnaratively towards the urate oxidase extract.

~L 4- Therefore, the present invention relates to a novel protein possessing a specific urate oxidase activity of at least 6 U/mng, which has the following sequence: Sen Ala Vat Lys ALa A~a Ang Tyr GLy Lys Asp Asn Vat Ang Vat Tyr Lys Vat H is Lys Asp GI. Lys Th r Gty VaL G~n Thr Vat Tyr Gbu Met Thr VaL Cys Vat Leu Leu Gbu Gly GU TL~e GLu Thr Sen Tyr Thr Lys ALa Asp Asn Sen Vat ILe Vat ALa Thr Aso Sen 11,e Lys Asn Th r TL~e Tyr I~e Thr ALa Lys G~n Amn Pro VaL Thr Pro Pro Gbu Leu Phe GLy Ser !Le Leo GLy Thn His Phe TLe GL,, Lys Tyr Am 'His Ito His Ala Ala His Vat Asn Ie Vat Cys His A rc Tro Thr Ang M e Asp 11te Asp GLy Lys Pro H is Pro Hi S Sen Phe I't, Ang A::p Ser GIb GL12 Lys Arg Asn VaL G~n Vat Aso Vat Vat Gtu SlYl Lys -ty 'et Asp I Ic Lys Sen Sen Leu Sen GLy Leo Thr Vat Leo LYS ,en TIr A s Ser Phe Trp 2.y Lco Ayr Asp rwTn Thn Leo Lys Sb~ Thrn Tro Aso Ang Leu Sen Thn Asp VaL Asp Ala Thr Tro GLn Tro Lys Asn Phc Sen Sty t o Sbu V Arg SenF H 41S Vat Pro Lys Phe AsD Ala Tnr 7 r. Ala Thn A 1 Ang Gb T hr Leo Ly S Thr Phe Ala G L A s o Aso7 Senr Al3 sen V at L Gn A La Thn Me t Ty r Lys M et Ala Gbo G 'Ln -"e Leo A La A rc at n '-eo Le 0o .n,1 V at ut Sen L eo Pro Asn Lys HsTyr Phe Gb Le Asp Le Sen TrL HsL y Leo Gtn Asn Thr Gly Lys Asn Ala Q .u VaL Phe At a Pro Gk_- Sen Aso Pro Asr Sty Leo Ie Lys 'y s -hr 'dat G ty Ang S en r7enr ~eu Lys Sen Lys Leo op~oai-Ypreceded by a methionine or wlci present A. substantial degree of homology with that sequence.

Preferably the specific orate oxidase activity of the nvention protein is of about 30 U/mg.

A preferred protein of that type is the protein, which, by analysis on a bidimensional gel, presents a spot of molecular mass of about 33.5 kDa and an isoelectric point around representing at least 90 of the protein mass.

Preferably the purity degree of the invention protein, determined by liquid chromatography on aC8 grafted silica column, is higher than 80 An interesting protein of that type is the protein having an isoelectric point of 8.0. Preferably the amino-terminal serine of that protein carries a blocking group, having preferably a mass around 43 units of atomic mass, such as for example the acetyl group.

5 The present invention also relates to the drug which contains the inventicn protein in combination with a pharmaceutically acceptable carrier. The invention protein may advantageously replace, in its different uses, the urate oxidase extract possessing a specific urate oxidase activity of about 8 U/mg, which is sold in the injectable form under the trade mark "Uricozyme" (Vidal 1990).

The invention also relates to a recombinant gene which comprises a DNA sequence coding for the protein hnving the following sequence Met Ser ALa Val Lys Ala Ala Arg Tyr Gly Lys Asp Asn VaL Arg VaL Tyr Lys Vat His Lys Asp GLu Lys Thr Gly VaL GLn Thr VIL Tyr Glu Met Thr VaL Cys Vat Leu Leu GLu Gly Glu ILe GLu Thr Ser Tyr Thr Lys ALa Asp Asn Ser VaL ILe Vat ALa Thr Asp Ser Ile Lys Asn Thr ILe Tyr ILe Thr ALa Lys GLn Asn Pro VaL Thr Pro Pro GLu Leu Phe GLy Ser ILe Leu Gly Thr His Phe ILe GLu Lys Tyr Asn His ILe His ALa ALa His Vat Asn ILe VaL Cys His Arg Trp Thr Arg Met Asp ILe Asp GLy Lys Pro His Pro His Ser Phe ILe Arg Asp Ser GLu Glu Lys Arg Asn VaL GLn Val Asp Vat Vat Glu Gly Lys GLy ILe Asp ILe Lys Ser Ser Leu Ser GLy Leu Thr Val Leu Lys Ser Thr Asn Ser GLn Phe Trp GLy Phe Leu Arg Asp GLu Tyr Thr Thr Leu Lys GLu Thr Trp Asp Arg ILe Leu Ser Thr Asp VaL Asp Ala Thr Trp GLn Trp Lys Asn Phe Ser Gly Leu GLn Glu Vat Arg Ser His Vat Pro Lys 2 Phe Asp ALa Thr Trp ALa Thr Ala Arg GLu Vat Thr Leu Lys Thr Phe ALa GLu Asp Asn Ser ALa Ser Vat GLn ALa Thr Met Tyr Lys Met ALa GLu GLn ILe Leu ALa Arg GLn GLn Leu ILe GLu Thr Vat GLu Tyr Ser Leu Pro Asn Lys His Tyr Phe Glu ILe Asp Leu Ser Trp His Lys GLy Leu GLn Asn Thr GLy Lys Asn Ala Glu Val Phe ALa Pro GLn Ser Asp Pro Asn Gly Leu ILe Lys Cys Thr Vat GLy Arg Ser Ser Leu Lys Ser Lys Leu Because of the degeneracy of the genetic code, there are a large number of DNA sequences coding for a protein whose sequence corresponds to the formula given above. One preferred DNA sequence, particularly appropriate for an expression in the prokaryotic microorganisms, is as follows

ATGTCTGCGG

CAAGGTTCAC

CCGTCTGTGT

GACAACAGCG

CACCGCCAAG

TGGGCACACA

AACATTGTCT

CCCTCACTCC

AC'UTGGTCGA

ACCGTGCTGA

GTAC ACCACA

ATGCCACTTG

CACGTGCCTA

GAAGACTTTT

AGATGGCAGA

TACTCGTTGC

GGGCCTCCAA

ACCCCAACGG

AAATTG.

TAAAAGCAGC

AAGGACGAGA

GCTTCTGGAG

TCATTGTCGC

CAGAACCCCG

CTTCATTGAG

GCCACCGCTG

TTCATCCGCG

GGGCAAGGGC

AGAGCACCAA

CTTAAG GAGA

CCAGTGGAAG

AGTTCGATGC

GCTGAAGATA

GCAAATCCTG

CTAACAAGCA

AACACCGGCA

TCTGATCAAG

GCGCTACGGC

AGACCGGTGT

GGTGAGATTG

AACCGACTCC

TTACTCCTCC

AAGTACAACC

GACCCGGATG

ACAGCGAGGA

ATCGATATCA

CTCGCAGTTC

CCTGGGACCG

AATTTCAGTG

TACCTGGGCC

ACAGTGCCAG

GCGCGCCAGC

CTATTTCGAA

AGAACGCCGA

7GTACCGTCG

AAGGACAATG

CCAGACGGTG

AGACCTCTTA

ATTAAGAACA

CGAGCTGTTC

ACATCCATGC

GACATTGACG

GAAGCGGAAT

AGTCGTCTCT

TGGGGCTTCC

TATCCTGAGC

GACTCCAGGA

ACTGCTCGCG

CGTGCAGGCC

AGCTGATCGA

ATCGACCTGA

GGTCTTCGCT

GCCGGTCCTC

TTCGCGTCTA

TACGAGATGA

CACCAAGGCC

CCATTTACAT

GGCTCCATCC

CGCTCACGTC

GCAAGCCACA

GTGCAGGTGG

GTCCGGCCTG

TGCGTGACGA

ACCGACGTCG

GGTCCGCTCG

AGGTCACTCT

ACTATGTACA

GACTGTCGAG

GCTGGCACAA

CCTCAGTCGG

TCTGAAGTCT

Another preferred DNA sequence, which is particularly suitable for expression in eukaryotic cells, such as yeast, is as follows 7 ATGTCTGCT6 CA AGGT T CA C CCGTCTIG-iGT GA CA AC AG CG CA CC GCC AAG

TGGGCACACA

AACATTG-CT

CCC TC AC'TCC A C GT G G TCGA A iC CATGCCT GA ATA C ACC AC A A'T3C C CACTT G C ACG G ACC TA GPAGAC 'TTT A G A TA ,G C, C A TACTCG TTGC G] C, C C T C fC A A A C CCC A T C G C

AAATTG.

T TAAIG GCT AC A AG GA CGAAGA ACT TC TAGAG T CATTAT C GC

CAGAACCCCG

CTT CAT TAAG

GCCACCGCTG

TTCATCCGCG

G GC AAG G GC A GAGC AC CA A CT T AAGGAG A

GCAGTGAAAG

A GTT7C'A T C G C TA A A A ATA AC A;AAT CC TA C TA A..AtCC A A ACAC C GCA T CTA AT C, A T AG ATA CAC AGA C C GArT GGTGAG A TTA

AACCAACTCC

TTACTCCTCC

A AG TA CAAC C

AACCCAGATA

ACAACGAAG GA A TC GA TATC A

CTCGCAGTTC

CC TAGGAC CA ,AAT T TCAG TA TAC C TAGAC C A C A ATGAC A GAC A CAGC C AG C

CTATTTCGAA

AG AfACAC CCA TGTACCGTrG A AGAA CAAC A C CAG ACGGTA A GA CCTCT TA AT TAAG AA CA

CGAACTGTTC

ACATC CATAC

AACATTAACA

A AAAGCA AAT AG TC TC T

TAAGG'TTCC

TATCCTGAC

AACTCCAGA

ACTGCTCACA

CATACAAGCC

AACTAATCGA

ATCAACCTGA

GGTCTTCACT

AC C A T V CT C TTAAAGTC TA

TACAAGATA

CACCAAGACC

C CAT TTAC AT

GACTCCATCC

CACTCACATC

ACAAACCACA

A TACAGGTG A TC CGGCC TA TA CGATGA CGAA AC CAACG TC A GGTC CCC TCA

AAATCACTCT

AC TA TATACA

AACTATCAAG

GCTAACACAA

CC TCAGTCGG T C TAAA T CT Another preferred DNA sequence. which is notably suiLable for expression in animal rells, is as follows 8-

CAATGTCCGC

CGGTOTACSA

SCT TACA CC A S 'A CA CCAT T TrTTCCGCTC

CAT.GCCGCTC

T GA S G CA AG

GGA:ATSISCA

TCTCTG FCCUS GW7'CFGCCT T G I' C A C C CAGGACI IC C TCFSC SAc-C TC G C C T AT ~TCGA G CISAC CT G G T CTCCTCA 7CCTCT,

-ATGTC

GTCTAGAAGG

G GG C G TC

AGGCGGACAA

TA CAT C ACC G C AT CCTG6GC

AGGTCAACAT

CCACACCCTFC

GGTGGACGIG

G CC TGA CCG T GACG S AC A IITCGATGCCu S C TCCCA CG T A C C T 'I A 'STA A A G A T Sj T C GA STAC T C

CACACISGCC

GST G 4S', C C ASC T A A A T

CGCAGTAAAA

TTCA CAAGGA TGSTG T GCT TC

GAGCGTCATT

C C AAGC AGA A ACAC AC TTC A

TGTC!GCCAC

A CCTTC A G TC GAGSC A GC 1 GA AGAG C C CA CA CT FA A ACT TGGC A'T

GCCTAAGITC

CTTT TG' SGC,^SAG CAAA

GTTIGCCTAAC

TG GAAAACAC A A C G GT CTG

GCAGCCCGCT

C GAS AAGAC C

TGGAGGGTGA

GTCGCAACCG

CC CCGTTACT IrT GAG AAG TA

CGCTGGACCC

CCGCGACAGC

AGGG5CA TCGA ACC AA C TC C GGjAGAGCTGG GGAAG A ATTT

GATGCTACCT

A GATA A CAG T I CCTGG SCSC G A AGC A CTAT T

CGGCAAGAAC

F C AAGT STA C

GGTGTCCAGA

GAT TGAG AC C

AGTCCATTAA

CCTGCCGAGC

CAACGAGATC

G GA TGG ACAT GA GGAG AA C TAT CAA ST CS A ST TCTGSC C SAC GGTATCC C AG TSSAC IC

GGGCCACTGC

GCCAGCGTC

CCAGCASCTIS

TCGAAATCGA

GCCGAGGTC

T

C GTCGG C yr-eceded by a non-translated 5' sequence favoring expression in animal cells. A preferred non-translated seqiience of this type is the ono comprising the sequence AC_"CTT7_O(CDGCCACT, located immediately upstream from the sequence described above.

I t wil1l. be not ic ed tha t Ltc;e prot oi n coded fo r by thle cP)N. sequ,.ences given above can undergo processi*ng by metnionyl a-'inope[,tidlase, which cleaves it from its amino-termina.

methl'onine residue.

3 The invention further relates to an expression vector carrying the above-defined recombinant gene with the means necessary for its expression.

For expression in prokaryotic microorganisms, in particular in Esoheriohia coli, the coding sequence must be inserted into an expression vector containing especially an effective promoter, followed by a ribosome binding site upstream from the gene to be expressed, and also an effective transcription stop sequence downstream 22- 9 from the gene to be expressed. This plasmid must also contain an origin of replication and a selection marker.

All these sequences must be chosen as a function of the host cell.

For expression in eukaryotic cells, the expression vector according to the invention carries the abovedefined recombinant gene with the means necessary for its expression, for its replication in eukaryotic cells and for selection of the transformed cells. Preferably, this vector carries a selection marker, chosen for example to complement a mutation of the recipient eukaryotic cells, which makes it possible to select those cells which have integrated a large number of copies of the recombinant gene either into their genome or into a multicopy vector.

For expression in animal cells, especially in the cells of Chinese hamster ovaries, CHO, the coding sequence is inserted into a pla-mid (for example derived from pBR322) containing two expression units, a first unit, into which the recombinant gene is inserted, before an effective promoter (for e.ample thL early promoter). The sequence around the initiation ATG is preferably chosen as a function of the consensus sequence described by KOZAK KOZAK (1978) Cell, 1109-1123). An intron sequence, for example the intron of mouse a-globin, can be inserted upstream from the recombinant gene, and a sequence containing a polyadenylation site, for example an SV40 polyadenylation sequence, can be inserted downstream from the recombinant gene. The second expression unit contains a selection marker (for example a DNA sequence) coding for dihydrofolate reductase (an enzyme abbreviated hereafter to DHFR). The plasmid is transfected in animal cells, for example DHFR- CHO cells (incapable of expressing DHFR).

A line is selected for its methotrexate resistance: it has integrated a large number of copies of the recom- 0 binant gene into its genome and expresses said recombinant gene at a sufficient level.

For expression in eukaryotic cells such as yeast, for example Saccharomyces cerevisia, the coding sequence should be inserted between, on the one hand, sequences recognized as an effective promoter and, on the other hand, a transcription terminator. The array promoter/ coding sequence/terminator, which is called an expression cassette, is either cloned in a plasmid vector (singlecopy or multicopy) for the yeast, or integrated as a multicopy into the genome of the yeast.

The invention further relates to the eukaryotic cells transformed by the above expression vector. Of value among these eukaryotic cells are strains of the species Saccharomyces cerevisiae, in particular those which contain a mutation on one of the genes responsible for the synthesis of leucine or uracil, for example the LEU2 gene or the URA3 gene.

The invention further relates to -he animal cells containing this recombinant gene with the means necessary for its expression. Said recombinant gene may, for example, have been introduced into the cells by transfection by the above expression vector, by infection with a virus or a retrovirus carrying said expression vector, or by microinjection.

The invention further relates to the process for producing a recombinant urate oxidase which comprises the steps of 1) cultivating transformed cells as hereinabove defined 2) producing the lysis of that cells 3) isolating and purifying the urate oxidase contained in the obtained lysate.

l I- The invention will be understood more clearly with the aid of the Examples below.

Many of the following techniques, which are well known to those skilled in the art, are described in detail in the work by Maniatis et al.: "Molecular cloning: a laboratory manual" published in 1984 by Cold Spring Harbor Press in New York.

E AMELELZ Islation of t'he messenger A'frm Asnergillus flavu The strain of A. flaNu which produces urate 12 oxidase was cultivated under conditions appropriate for the production of urate oxidase, i.e. in a medium containing uric acid and having the following composition: glucose 15 g/l, MgSO4.7H20 1 g/l, KH2PO4 0.75 g/l, CaCOs 1.2 g/1, uric acid 1.2 g/l, KOH 0.5 g/1, soy bean oil 0.66 ml/1, FeSO4.7H20 10 mg/l, CuSO4.5H20 1 mg/l, ZnSO4.7H20 3 mg/l, MnSO4.H20 1 mg/l. The medium is adjusted to pH 7 with H2S04 1 M and sterilized at 120°C for 80 min.

In a 5 1 Erlenmeyer flask, 1.5 1 of medium are inoculated with about 1 to 3.107 spores.

The culture is incubated for about 40 h at 30 0

C,

with agitation (120 rpm). The mycelium is recovered by filtration on gauze, washed with water and frozen in liquid nitrogen.

g of mycelium (wet weight) are thawed, resuspended in 45 ml of lysis buffer and then taken up in the same volume of beads (0.45 um in diameter). The lysis buffer consists of guanidine thiocyanate 4 M, Tris-HC1 10 mM pH 7.6, EDTA 10 mM, 3-mercaptoethanol 50 ml/l. The mycelian suspension is ground in a Zellmiihler mill (vibrogenic) for 5 min.

The ground material is recovered and the beads are decanted. The supernatant is removed (about 45 ml), brought back to a final concentration of 3 M in respect of lithium chloride and stored at OC.

After two days, it is centrifuged for 60 min at 10,000 rpm. The supernatant is discarded and the residue is taken up in 40 ml of LiCI 3 M and centrifuged again at 10,000 rpm for 1 h 30 min.

The following are added: proteinase K (SIGMA) ug/ml, SDS w/v) and EDTA 20 mM. The mixture is incubated at 37 0 C for 3 h. Precipitation with 2 volumes of ethanol is followed by washing with 70% ethanol. The residue is taken up in 0.5 ml of TE buffer (Tris-HCl 13 mM, EDTA 1 mM pH the mixture is extracted twice with chloroform and precipitation is carried out with ethanol. The RNA's are stored at -80°C in alcohol.

EXAMPLE 2: Purification of the poly A- fraction of the RNA's About 1 mg of RNA is precipitated for 20 min at 4°C (15,000 rpm) and then washed with 70% ethanol and dried. The residue is taken up in 1 ml of TE buffer and resuspended by agitation in a Vortex. 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 the oligo dT, agitated gently to resuspend the beads and then heated for 1 min at The suspension is a justed to 0.5 M NaCI and then agitated gently for 10 min. It is then centrifuged for 1 min at 1000 rpm, the supernatant is removed and the residue is washed twice with 1 ml of TE buffer containing M NaC1. The supernatants are removed. The polyadenylated fraction of the RNA's (consisting of the messenger RNA's) is eluted by suspending the beads in 1 ml of TE buffer, then heating this suspension at 60°C for 1 min and subsequently agitating it for 10 min on a tilting plate. It is then centrifuged for 1 min at 1000 rpm, which makes it possible to recover on the one hand the supernatant containing free mRNA's in solution, and on the other hand the residue of cellulose beads. The above series of operations (starting from elution) is repeated. The supernatants obtained in this way are pooled, the excess beads are removed by centrifugation and the supernatant is precipitated with ethanol containing NaC1 in accordance with the usual techniques (Maniatis: op. cit.).

EXAMPLE 3: Building of the cDNA library The messenger RNA's isolated as described in the 14 previous Example were used to build a cDNA library in vector pTZ19R (marketed by PHARMACIA). This vector is a plasmid comprising a polylinker containing unique restriction sites.

The cloning technique used is the one described by Caput et al. (primer-adapter technique: Caput et al., Proc. Natl. Acad. Sci. (1986) 83, 1670-1674).

It consists firstly in digesting the vector with Pstl, adding a polydC tail to the protuberant 3' end and then digesting the resulting plasmids 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 a sticky end of the BamHI type. Secondly, the messenger RNA's are subjected to reverse transcription starting from a primer having the sequence 5'<GATCCGGGCCCT( 1 Thus the cDNA's have at their 5' end the sequence GATCC complementary to the BamHI sticky end. The RNA-DNA hybrids obtained by the action of reverse transcriptase are subjected to alkaline hydrolysis, enabling the RNA to be removed. The single-stranded cDNA's are then purified by 2 cycles on a column of Sepharose CL4B and subjected to a treatment with terminal transferase so as to add polydG's at the 3' end. The cDNA's are inserted in single-stranded form into the vector prepared as described above. A second oligonucleotide, the adapter, complementary to the primer, is necessary in order to generate an "open" BamHI site at the 5' end of the cDNA's. After hybridization of the vector, the cDNA and the adapter, the recombinant molecules are circularized by the action of the ligase of phage T4. The singlestranded regions are then repaired by means of the DNA polymerase of phage T4. The plasmid pool obtained in this way is used to transform the MC1061 strain for ampicill:Ln resistance (Casabadan, Chou and Cohen, J.

Bact. (1980) 143, pages 971-980.

EAMPLE_: Purification of ute oxidaseextracted from A. flavus and characterization thereof 1) Purification of urate oxidase extracted from A. flavua A preparation of urate oxidase extracted from Aflavus (Uricozyme Laboratoires Clin Midy), having a specific urate oxidase activity of 8 U/ml (the specific urate oxidase activity is the ratio of the urate oxidase activity measured by the test described in Example 9 to the weight of total proteins measured by the Bradford method: Anal. Biochem-, 72, 248-254), was repurified by chromatography on a column of Red-agarose 120 grafted agarose (SIGMA), concentration by ultrafiltration and filtration on Ultrogel Aca 44 (IBF), a polyacrylamideagarose gel, according to the following protocol: Step 1: Affinity chromatography on grafted agarose Temperature: 4°C Column: PHARMACIA K50/30 diameter 50 mm length 33 cm Resin: Red 120 Agarose (3.000 CL/R-0503 SIGMA) (volume of gel 410 ml height of gel 20 cm) Equilibration buffer: glycine/NaOH 20 mM pH 8.3 Elution buffer: glycine/NaOH 20 mM, NaCl 2 M pH 8.3 Conditioning flow rate: 250 ml-h- 1 Operating flow rate: 160 ml-h- 1 Elution flow rate: 60 ml.h- 1 1) Deposit the solution of Uricozyme on the top of the column with the aid of a constant-flow pump.

"16 2) After adsorption, wash the column with twice its volume of equilibration buffer 3) Elute with an ionic strength gradient having the following composition: glycine, NaOH, 20 mM pH 8.3/glycine, NaOH, 20 mM NaCl 2 M pH 8.3 The total volume of the gradient is equal to 10 times the volume of the column, divided up equally between the two constituents.

Chromatographic recording is carried out at A 280 nm; the urate oxidase pool is collected after combination of the fractions which have a specific urate oxidase activity greater than or equal to 16 U/mg.

St p 2: Concentration of the urate oxidase pool by ultrafiltration with the aid of a Biopass system comprising a 10 kDa ultrafiltration membrane Temperature: Column: Resin: 4 C PHARMACIA K 50/100 diameter 50 mm length 300 cm polyacrylamide-agarose with amine and hydroxyl groups: Ultrogel ACA 44 (IBF) volume of gel 1.6 1 height of gel 80 cm >uffer: glycine/NaOH 20 mM pH 8.3 Equilibration b Conditioning flow rate: Operating flow rate: 40 ml.h- 1 24 ml.h-1 1) Deposit the concentrated urate oxidase pool on the top of the column with the aid of a constant-flow pump- 2) After the sample has been deposited, continue to 1 -17supply the column with the buffer glycine/NaOH 20 mM pH 8.3 3) After chromatography, wash with NaCl 2 M until the UV absorbance value (A 280 nm) 0.05.

Store under NaCl 2 M at 4°C.

Chromatographic recording is carried out at 280 nm; the urate oxidase pool is collected after combination of the fractions which conjointly have: a specific urate oxidase activity greater than or equal to 20 U/mg; and only bands electrophoresi3 under denaturing conditions (presence of SDS) and with silver nitrate developing (Biorad staining kit), name i y.

major band of 33-34 kDa a minor Land of 70-71 kDa.

2) Chariicterization of purified urate oxidase extracted from A. flaus a) Partial sequencing Direct amino-terminal sequencing of the protein wais attempted in order to obtain information on the amino acid sequence of the purified urate oxidase extract, making it possible to synthesize the probes necessary for cloning the cDNA. This sequencing was not successful because of amino-terminal blocking of the protein (cf. f) below)- The following strategy was therefore developed to obtain the partial sequence of urate oxidase: cleavage of the protein with proteolytic enzymes (using the enzymes trypsin and protease V8 of 9taph.VQlQccu separation of the resulting polypeptides by reversed L I 18 phase HPLC sequencing of the purified peptides.

a) Hydrolvyis of the urate oxidase with trypsin. Durificatin and seuencin of the peptides The urate oxidase, at a concentration of 9 mg/ml in an ammonium carbonate buffer 100 mM pH 8.9, was digested with trypsin (Worthington, TPCK), in a ratio urate oxidase/trypsin of 30/1 by weight, at 30*C for 24 h. After tryptic hydrolysis, 60 pg of digested urate oxidase were directly injected on to a reversed phase HPLC column of Brownlee G18 grafted silica (column: 10 x 0.2 cm) equilibrated with acetonitrile 1% and trifluoroacetic acid 0.1% in water_ The peptides were then eluted by a linear gradient of acetonitrile in a solution of trifluoroacetic acid v/v) in water, varying from 1% to 60% of acetonitrile in 60 min, at a rate of 150 pl/min_ The peptides leaving the column were detected by measurement of 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 peaks identified.

Each peak was collected and stored at -20°C until analyzed on a protein sequencer (model 470 A from Applied Biosystems) equipped with a chromatograph (model 430 A from Applied Biosystems), which continuously analyzes the phenylthiohydantoic derivatives formed, after each degradation cycle. Table I below shows the peptide sequences of the 9 peaks identified.

0) Hydrolyis of the urate oxidase with protease V8, nurification and seencing of the pentides The urate oxidase, at a concentration of 2 mg/ml 19 in an ammonium acetate buffer 100 mM pH 6.8, was digested with the protease V8 of Staphyloocus aureus (Boehringer-Mannheim), in a ratio urate oxidase/protease V8 of 60/1, at 30°C for 72 h. 160 pg of digested urate oxidase were then injected on to a reversed phase HPLC column of Brownlee G18 grafted silica (column: 10 x 0.2 cm; particles: 7 x 0.03 jim), equilibrated with acetonitrile 1% and trifluoroacetic acid 0.1% in water.

The peptides were then eluted by a linear gradient of acetonitrile in a solution of trifluoroacetic acid in water varying from 1% to 60% of acetonitrile in 60 min, at a rate of 150 pl/min. The peptides leaving the column were detected by measurement of the optical denoiit. at 218 nmi The elution profile is shown in Figure 2, in which the numbers following the letter V (protease V8) correspond to the peaks identified.

Each peak was collected and stored at -20°C until analyzed on the protein sequencer already mentioned- Table I below shows the peptide sequences of the peaks identified.

7;l4 0<

TABLEAI

Sequencing of the products obtained by hydrolysis T 17 Asn -Vai- Gin-Vai-Asp -Vali-Val- Glu-Oly-Lys T 20 An Pe-Ser Giy-Leu -Gln Glu-Val T 23 Phe -Asp- Aa -Thr -Trp- Ala T 27 Hi -Tyr he Giu- Ile -Asp -Leu -Ser With the aid of trypsin T 28 Ile -Lei1- Ser -Thr- Asp -Val- Asp -Ala Thr-Trp-GIn Trp -Lys T 29 Hi -Tyr -Phe- Glu- Ile- Asp -Leu -Ser-Trp -His-Lys T 31 Ser Tbr-Asn -Ser -Gln -Phe -Tr-p- Gy-Phe -Leu-Arg T 32 Gln An-Pro -Va-Thr -Pro -Pro GuLeu -Phe-Gy -Ser -Ile Len. Gly Thr T 33 GLn Asn -Pro-Va -Thr Pro -Pro GluLeu -Phe Gy -Ser -Ile I Leu Giy-Thr V 1 Tyr Ser Ser Trp ILen His Pro [Lys Asn Lys His Tyr Phe Giu Ile Asp Len With the aid of protease V8 V 2 Vai-Thr -Leu -Ly Thr -Phe -Ala Glu -Asp -Asn -Ser Ala-Ser Val Gln Ala V 3 Thr -Ser -Tyr -Thr -Ly Ala -Asp-Asn -Ser Ile a As Tbr-Asp- Ser Ile Lys Asn Thr -Ile Tyr Ile Thr- V 5 Gy -Lys- Gy- Ie -Ap-:e -Lys-Scr- Ser-[eu-Ser-Giy-[Eu- Thr Val [eu Lys Ser Thr Asn Ser Gin Phe Trp Gly Phe Leu Arg V 6 Gly -Lys -Gly Ile Asp Tle Lys Ser Ser [eu Ser (fly [eu Thr Val IAEu Lys 21'b) Specific activity The purified urate oxidase extract has a specific activity of about 30 U/mg.

c) Electrophoresis under denaturing conditions Electrophoresis of the purified urate oxidase extract on polyacrylamide gel in the presence of SDS (sodium dodecylsulfate), followed by silver developing, reveals a high intensity band of about 33-34 kDa and a very low intensity band of about 70-71 kDa.

d) Determination of the isoelectric point UF-e; of ready-to-use gels, namely LKB Ampholines gel plates from Pharmacia with pH ranges of (3.5 9-5) and (5 8).

Deposition of 10 pl of LKB standard proteins (range of isoelectric points of the standard proteins: 3-5 and 4 pg and 8 pg of purified urate oxidase (on two different lanes).

Run 1 h 30 min, 12 V, 6*C.

Then staining with Coomassie blue in ethanol, 8% acetic acid) to stain the proteins, followed by decolorization with a solution containing 25% of ethanol and 8% of acetic acid (to eliminate the background).

Results: Observation of two close bands (doublet), of 22isoelectric points 8.1 and 7.9, on each of the two lanes e) Two-dimensional gel analysis Two-dimensional gel analysis makes it possible to separate the proteins in a first stage according to their isoelectric points and in a second stage according to their molecular weights.

Protocol Sample: solution of purified urate oxidase extract in a glycine buffer 20 mM! pH 8.3 Preparation of the sample Two samples of 5 pg and 10 pg of urate oxidase.

Drying by vacuum centrifugation and taking-up in 5 il of a lysis buffer having the following composition: urea 2.5 M, 3-(3-cholamidopropyl)dimethylammoniopropane-1sulfonate, CHAPS (Sigma), 2% Ampholines amphoterics (LKB) of pH ranges 5 8 and 3.5 9.5, and 0-mercaptoethanol Isoelectrofocusina gel Preparation of a solution containing urea 9.5 M, CHAPS LKB Ampholines (pH (3.5 9.5) pH (5 8) acrylamide/bisacrylamide 3.5% final concentration, Filtration and degassing of the solution, followed by addition of 0.075% of tetramethylethylenediamine, Temed (Pharmacia), and 0.015% of ammonium persulfate.

23 Introduction of the solution into tubes (16 x 0.12 cm) polymerization overnight at Cathodic solution: NaOH 0.1 M, degassed.

Anodic solution: HaPO4 25 mM.

Prerun 45 min at 4 mA (voltage 300 V i 1000 V).

Deposition of the samples at the cathode.

Run 19 h at 1000 V and at Demolding of the gels and equilibration for 10 min at in a buffer (Tris 0.375 M pH 8-8; SDS dithiothreitol, DTT, 50 mM).

PAGE/SDS denaturjing-gel Preparation of a solution containing acrylamide/bisacrylamide 15% final concentration, Tris- HC1 (pH 8.8) 0.375 M, Filtration and degassing of the solution, followed by addition of SDS ammonium persulfate 0.05% and Temed 0.05%.

Polymerization overnight at 4°C (gel 16 x 20 x 0.15 cm).

After equilibration, deposition of the isoelectrofocusing gel on the surface of the PAGE/SDS gel, followed by sealing with agarose.

Electrophoresis buffer: (Tris-HCl 25 mM pH 8.3, glycine 0.192 M, SDS 24 Run 100 mA 6 h at 6*C.

Fixing of the gel in 50% methanol, 10% acetic acid, followed by silver nitrate staining (method of Blum, Electrophoresis 1987, 8, p. 93-99).

Scanning of the gel on a Visage 2000 image analyzer from Kodak for determination of the optical density and surface area of each spot and hence for calculation of the quantitative ratio between the spots.

Determination of the molecular weight of the protein by preparation of a two-dimensional gel in the presence of Amersham standard proteins.

Result Two spots with a molecular weight of the order of 33.5 kDa are observed, one being the majority spot with an isoelectric point of the order of 8.0, intensity 5.2 (representing about 93% of the weight of proteins), and the other being the minority spot with an isoelectric point of the order of 7_4, intensity 0.41 (representing about 7% of the weight of proteins).

f) Determination of the amino-terminal sequence and the mass of the blocking amino-terminal group a) Demonstration of the blocked character of the amino-terminal sequence The amino-terminal sequence was analyzed with the aid of an Applied Biosystem model 470A sequencer coupled with an Applied Biosystem model 120A analyzer of phenylthiohydantoic derivatives. The purified urate oxidase (200 pmol, checked by amino acid analysis) was deposited on the rFquencer in the presence of 20 pmol of 3-lactoglobulin, a standard protein.

No amino-terminal sequence corresponding to a urate oxidase sequence was detected (by contrast, the amino-terminal sequence of the standard protein was detected, showing that the sequenrer was working).

A._flavus urate oxidase therefore has the aminoterminal end blocked.

0) Determination of the sequence of an aminoterminal peptide of 32 amino acids and the mass of the blocking amino-terminal group Mthod: Di.eatio with yanogebxromids The purified urate oxidase extract is subjected to gel filtration on Sephadex G25 (PD10 Pharmacia), a gel obtained by crosslinking dextran with epichlorohydrin, equilibrated with a solution containing 7% of formic acid, making it possible to remove the salts and change the buffer. The formic acid concentration is increased to 70% by vacuum centrifugation- Cyanogen bromide is then added to a final concentration of 0.2 M and the reaction is allowed to proceed for 20 h under argon, in the absence of light and at room temperature.

Separation by ion exchange hroma of the peptides derived from diestion of the protein with canogen brmide The peptides were separated on an ion exchange column based on mono S hydrophilic resin (Pharmacia).

Buffer A: ammonium acetate 10 mM pH 6.2 26 Buffer B: ammonium acetate 1 M pH 6.2 Flow rate: 0.6 ml/min, peak detection by measurement of the optical density at 278 nm Gradient: from 0% of B to 100% of B in 30 min collection of 1 ml fractions The fractions derived from the ion were analyzed by PAGE/SDS gel according to described by Schagger and Von Jagow (1987) 166 p. 368-379.

exchange step the method Anal_ Biochem.

Purification of the amino-terminal peptide by reversed phase HPLC and analysis thereof by mass aectrometry The peptide derived from the ion exchange step, having a molecular weight of about 4000 Da (on PAGE/SDS gel), was purified on a Beckman Altex C18 column (250 x 2.1 mm), which is a reversed phase HPLC column based on C18 grafted silica.

Flow rate: Buffer A: Buffer B: Gradient: 0.3 ml/min, peak detection by measurement of the optical density at 218 nm H20/0.1% TFA (trifluoroacetic acid) acetonitrile/0.1% TFA from 1 to 50% of B in 60 min.

The peptide collected after a first reversed phase HPLC step was repurified on the same reversed phase HPLC column, but with a different gradient Gradient: from 1 to 50% of B in 10 min.

The peak collected was subjected to analysis by fast atom bombardment mass spectrometry (FAB/MS) with a glycerol thioglycerol matrix.

27 Digestion of the amino-terminal peptide with chvmo- _trNEJ _an and amino acidan.alv.a f thE chvmotryptic peJptide separated by reversed phase HPLC To establish the sequence of the peptide purified by reversed phase HPLC, said peptide was digested with chymotrypsin. The chymotryptic peptides were separated by reversed phase HPLC on a Beckman Altex C18 column (250 x 2.1 mm).

Flow rate: 0.3 ml/min, peak detection by measurement of the optical density at 218 nm Buffer A: H20/0.11% TFA Buffer B: acetonitrile/0_08% TFA Gradient: from 1% of B to 50% of B in 60 min collection of the peaks.

The chymotryptic peptides were identified by amino acid analysis on an Applied Biosystem analyzer (model 420-130A).

Resulta The results presented below, which were established after determination uf the sequence of the cDNA of A. flayvu urate oxidase and the deduced amino acid sequence (cf. Example can only be understood in the light of the following: Analysis of the amino-terminal peptide by mass spectrometry A difference of about 42 atomic mass units is observed between the two molecular weights determined by mass spectrometry, 3684 and 3666, and the theoretical 28 molecular weights determined from the following sequence (amino acid sequence deduced from the cDNA of A. flavus urate oxidase with cleavage of the amino-terminal methionine group and peptide cleavage with cyanogen bromide after the first methionine residue): SerAlaValLysAlaAlaArgTyrGly LysAspAsnValArgValTyrLysValHis LysAspGluLysThrGlyValGlnThrVal TyrGlu (1) with a carboxy-terminal methionine residue modified by reaction with cyanogen bromide to give either homoserine, 3642, or homoserine lactone, 3624.

There is therefore a blocking group on the aminoterminal serine which accounts for an additional mass of about 42 atomic mass units, probably corresponding to acetylation of said amino-terminal serine (mass of CHsCO mass of H 42 atomic mass units).

Amino acid analysis of the chymotryptic peptides This analysis made it possible to show unambiguously that the sequence of the amino-terminal peptide obtained by digestion with cyanogen bromide comprises the sequence described above.

The complete amino acid sequence of urate oxidase is shown hereinafter.

29 Sen Ala Val Lys ALa ALa Ang Tyr GLy Lys Asp Asn VaL Ang Val Tyr Lys Val H is Lys Asp GLu Lys Thn GLy VaL G~n Thn VaL Tyn G Lu Met Thn VaL Cys \JaL Leu Leu Glu GLy Glu Ie GLu Thr Sen Tyn Thn Lys Ala Asp Asn Sen VaL Ie Val Ala Thr Asp Sen Ie Lys Asn Thn I~e Tyn Ie Thn Ala Lys G~n Asn Pno Val Thn Pno Pno Glu Leu Phe Gly Sen TL~e Leu GLy Thn His Phe Ie Glu Lys Tyn Asn His Ie His Ala Ala His Val Asn Ie Val Cys His Ang Tnp Thn Ang Met Asp Ie Asp GLy Lys Pno His Pno His Sen Phe Ie Any Asp Sen GLu G,.u Lys Any Asn VaL G~n VaL Asp VaL VaL G~u Gly Lys Gly I Le Asp I Le Lys Sen Sen Leu Sen GLy Leu Thn VaL Leu Lys Sen Thn Asn Sen G~n Phe Tnp Gly Phe Leu Any Asp GLu Tyn Thn Thn Leu Lys G~u Thn Tnp Asp Any Ie Leu Sen Thr Asp VaL Asp Ala Thn Tnp G~n Tnp Lys Asn Phe Sen Gly Leu G~n GLu Val Arg Sen His VaL Pno Lys Phe Asp Ala Thn 1np Ala Thn Ala Any G~u VaL Thn Leu Lys Thn Phe Ala G~u Asp Asn Sen Ala Sen VaL G~n Ala Thn Met Tyr Lys Met Ala GLu G~n I~e Leu Ala Any G~n G~n Leu I e G~u Thn VaL G~u Tyn Sen Leu Pro Asn Lys His Tyn Phe G~u Ie Asp Leu Sen Tnp His Lys GLy Leu G~n Asn Thn GLy Lys Asn Ala G~u VaL Phe ALa Pno G~n Sen Asp Pno Asn Gly Leu Ie Lys Cys Thn VaL Gly Any Sen Sen Leu Lys Sen Lys Leu 30 EXAMPLE 5: Screening of the bacteria 1) Preparation of the labeled probes Two pools of probes deduced from amino acid sequences of the protein were synthesized with the aid of a Biosearch 4600 DNA synthesizer. The first pool corresponds to the sequence of residues His-Tyr-Phe-Glu-Ile-Asp (part of the sequence of T 27), i.e. from 5' to 3': A T G G G TCGAT TC AA TA TG T C A A A This pool in fact consists of 24 x 3 48 different oligonucleotides, representing all the possible combinations. The second pool corresponds to the sequence of amino acid residues Gln-Phe-Trp-Gly-Phe-Leu (part of the sequence of V i.e. from 5' to 3': GG A G T A AAGCCCCA AA TS AA C A C

T

This pool consists of 24 x 4 64 combinations. The probes are labeled with terminal deoxynucleotide transferase (TdT) (marketed by 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 as a 10-fold concentrate by IBI Inc.): 1.4 M potassium cacodylate pH 7.2, 300 mM dithiothreitol, 1 pl of the enzyme terminal deoxynucleotide transferase (IBI Inc.) and 50 pCi of deoxycytidyl triphosphate, dCTP, labeled with P32. The reaction is carried out at 37C for 10 min and is then stopped by the addition of 1 pl of EDTA 0.5 M. A phenol I- 31extraction is carried out and the extract is dialyzed on a column of Biogel P10 polyacrylamide (Biorad: 150-1050).

2) Hybridization and detection of the colonies containing urate oxidase cDNA About 40,000 colonies are screened by the in sit hybridization technique developed by Grunstein and Hogness (1975, Proc. Natl. Acad. Sci. 12, 3961). About 6000 bacteria are plated out in Petri dishes to give isolated colonies. After incubation for 24 h at 37°C, each dish is replicated on 2 filters, each filter being intended to be treated with one of the 2 pools of probes, so that all the colonies obtained are tested with the 2 pools of probes in parallel.

The filters are hybridized with one of the 2 pools of probes in a buffer containing 6 x SSC, 10 x Denhardt's solution and 100 pg/ml of sonicated and denatured salmon sperm DNA (SIGMA). The hybridization is carried out at a temperature of 42°C for 16 h. The 6 x SSC solution is obtained by diluting a 20 x SSC solution.

The preparation of the 20 x SSC buffer is described by Maniatis, Fritsch and Sambrook (op. cit.). In summary, this buffer contains 175.3 g/1 of NaCl and 88.2 g/l of sodium citrate and is adjusted to pH 7 with a few drops cf NaOH 10 N. The 10 x Denhardt's solution contains 1 g of Ficoll, 1 g of polyvinylpyrrolidone and 1 g of human serum albumin per 500 ml of final volume.

After washing in the 6 x SSC solution at 42°C (3 h with 5 changes of bath), the filters are wiped with Joseph paper and subjected to autoradiography. The filters are developed after 16 h. A fraction of about of the colonies was found to have hybridized with the 2 pools of probes.

colonies from this fraction were taken up and purified. The plasmid DNA was prepared from each of these colonies and this DNA was analyzed by digestion t i 32with either BamHI, or HindIII, or both BamHI and HindIII.

After analysis on agarose gel, the 5 plasmids obtained were found to have been linearized by BamHI and by HindIII. The double digestions make it possible to release a fragment corresponding to the whole of the cloned cDNA. TLe size of this fragment is about 1.2 kb in 3 cases and about 0.9 kb in the other 2 cases. For the following determination, one of the 0.9 kb fragments and one of the 1.2 kb fragments were selected and recloned (see Example 6 below).

E~ZXAELE Determination of the seaQjuce of urate oxidase On the one hand one of the 0.9 kb fragments (clone 9A) and on the other hand one of the 1.2 kb fragments (clone 9C) were recloned in the DNA of the replicative form of single-stranded phage M13. The DNA of the M13 clones, containing the 0.9 kb fragment, on the one hand and the 1.2 kb fragment on the other, was digested with exonuclease so as to generate a series of overlapping M13 clones (procedure: "Cyclone I Biosystem" of IBI). Said clones were sequenced by the dideoxyribonucleotide method (Sanger et al., PNAS-U.S.A. 1977, 14, 5463-5467).

The nucleotide sequence of clone 9C is shown in Figure 3, which also indicates, with an arrow, the start of clone 9A and, with a nucleotide symbol followed by an asterisk the sequenced nucleotides of clone 9A which are not identical to those of clone 9C (when matching the two sequences and the AccI and BamHI restriction sites used in the subsequent constructions (cf. Example It is found that the nucleotide sequence of the longer fragment (clone 9C) overlaps that of the shorter fragment (clone 9A) but for two differences (see Figure One of the differences is quiescent and the other corresponds to a 33 change from a tryptophan residue to a glycine residue. These differences may be due either to differences in the messenger RNA's isolated (cf.

Example 2 above) or to errors in the reverse transcriptase used when building the cDNA library (cf. Example 3 above). The sequencing of the genomic DNA of A. flavus-urate oxidase has made it possible to overcome this ambiguity: it is a tryptophan residue (hence probably an error of the reverse transcriptase.

In the case of the longer fragment, an ATG codon (in position 109 in Figure 3) opens an open reading frame corresponding to a polypeptide of 302 amino acids, with a molecular weight of about 34,240 Da, whose sequence corresponds to the partial sequence of purified A. flaus urate oxidase (cf. Example 4).

Figure 4 shows the DNA sequence opened by the ATG codon and the polypeptide coded for, and, with arrows opposite the polypeptide coded for, the sequenced peptides (cf. Example 4) obtained by hydrolysis of A. flavua urate oxidase with trypsin and protease V8.

It is found that the sequence of the polypeptide terminates in the triplet Ser-Lys-Leu, which is typical of peroxisomal location enzymes (Gould S.J. et al., J.

Cell Biology 108 (1989) 1657-1664).

EXAMPLE7: Construction of an expression vector for urate oxidase cDNA Plasmid p466, a vector for expression in E. coli, was prepared. It comprises a fragment of pBR327 including the origin of replication and the ampicillin resistance gene; it also comprises a synthetic promoter of E. coli RODRIGUEZ and M. CHAMBERLIN, "Promoters Structure and function (1982), Preager), a Shine-Dalgarno sequence followed by a polylinker containing the unique NdeI and KpnI sites, a transcription terminator (derived from phage fd) and the lac i gene.

This plasmid was constructed from an expression plasmid for hGH in E. coli (p462) by replacing a fragment carrying the hGH gene with urate oxidase cDNA.

The construction of plasmid p466 will now be 34 described in greater detail in the following account, which will refer to Figures 5, 6, 7, 8 and 9.

Figure 5 shows a restriction map of plasmid p163,1. The different restriction segments are labeled arbitrarily according to the following legend: DNA segment derived from plasmid pBR322 Location of the origin of replication

(ORI)

DNA segment containing the sequence coding for a natural precursor of hGH DNA segment of phage fd containing a transcription terminator DNA segment containing a tryptophanlactose UV5 hybrid promoter-operator 77 DNA segment coding for 1-lactamase (ApR: ampicillin resistance) Figure 6 shows the restriction map of plasmid whose PvuI-XhoI--BamHI(1) and PvuI-ORI-BamHI(2) fragments originate respectively from plasmids p163,1 and pBR327 and whose small BamHI(2)-BamHI(1) fragment is fragment 3 described below.

Figure 7 shows the restriction map of plasmid 35 p3 7 3,2. The different restriction segments are labeled arbitrarily according to the following legend: PvuI-BamHI sequence derived from plasmid pBR327 PvuI-XhoI sequence derived from plasmid pl 63 1 XhoI-HincII sequence derived from plasmid p16 3 ,1 ClalI Ndel PstI (HincII) "f Cj (It Fragment 4 described below

X>XXX

Fragment 3 described below DNA segment of phage fd containing a transcription terminator Figure 8 shows a restriction map of plasmid p462, the synthetic BglII-HindIII fragment defined below being represented by: Figure 9 shows a restriction map of plasmid p 4 6 6 the NdeI-KpnI fragment, comprising the gene coding for urate oxidase, being represented by:A A A A A A 36 1) Construction of plasmid p373,2 The strategy employed uses fragments obtained from pre-existing plasmids available to the public, and fragments prepared synthetically by the techniques now in common use. The cloning techniques employed are those described by T. MANIATIS, E.F. FRITSCH and J. SAMBROOK, Cold Spring Harbor Laboratory (1982). The oligonucleotides are synthesized with the aid of a Biosearch 4600 DNA synthesizer.

Plasmid p163,1 (Figure described in European patent application A-0245138 and deposited in the CNCM under the reference 1-530 on 17 February 1986, was digested with the enzymes PvuI and BamHI. This plasmid contains the gene coding for hGH. The PvuI-BamHI fragment hereafter called fragment 1 containing the site of action of the restriction enzyme XhoI, shown in Figure was purified.

Likewise, plasmid pBR327, which is well known to those skilled in the art SOBERON, X. et al., Gene, 9 (1980) 287-305), was digested with the enzymes PvuI and BamHI. The PvuI-BamHI fragment hereafter called fragment 2 containing the origin of replication, was purified.

Fragment 3 was then prepared; this is a synthetic BamHI(1)-BamHI(2) fragment containing the lac i gene and its promoter and it has the following sequence, in which the two ends of the strand are identified by the numbers 1 and 2 in order to specify the orientation of the fragment in the plasmids described in Figures 6 and 7: 37 FAG-MFNT3- DarnHI (1)

GATCC

GAGCTAACTT

GAAACCTGTC

GGCGGTTTGC

CGGGCAACAG

AAGCGGTCCA

GGTTAACGGC

C TACCG AG AT

ATTGCGCCCA

GATGCCCTCA

TCCAGTCGCC

TATTTATGCC

GC C CGCTAAC

CGCGAGTCG

GTCTGGTCAG

TTCCACAGCA

CACTGACGCG

ACGCCGCTTC

GGCGCGAGAT

GACTGGAGGT

TGTGCCACGG

TTTTTCGCGC

AAACGGTCTG

ACTG G 7T T CA

TGCCATACCG

G C GGA AGCAT

ACATTAATTG

GTGGCCAG CTG

GTATTGGGCG

C TGAT TG CC C CG7:TGGTTTG GGGATA TAA C ATCCG C AGCA

GCGCGATCTG

TTGAGCATTT

TTC C CGTTC C AGC CAGC CAG

AGCGCGATTT

GGTACGGTCT

AGAGATCAAG

ATGGCATGCT

T TGC CGC GAG A GTTG TAGCCAT

TTAATCGCG

GGAACGA

GGTTGGGAAT

GTTTTG AG A TA ACAG AC A CATTGAG GAG

CGAAAGGTTT

AAAGTGTAAA

CGTTGCGCTC

GATTAATGAA

GCAGGGTGGT

TTCAC CGC CT CCC GAGCACC A TG ASCTGT C AC GC GCAGC C

ATGGTTGGCA

GCATGGTTTG

GCTATGGGCT

ACGCAGACGC

GCTGGTGACC

TCATGGGAGA

AAA TAAC GC C GGTGATG GAG

AGATTGTGCA

CGA AG GAGCC C GA CAA T TTG

ATGAGCAACG

C TAATTC A GC

AAACGTGGGT

CCGGGCATACT

CCTGAATTGA

TGCGCCATTC

GCCTGGGGTG

AC TGC C CGCT

TCGGCCAACG

TTTTICTTTTC

GGCCGTGAGA

CGAAAATCCT

T TCGG TAT C

CGGACTGGGT

AC CAG CATCG

TTGAAAACCG

GAATTTGATT

GCCGAGACAG

CAATGCGAC C

AAATAATACT

GGA AGAT TAG CGCGAT ACTTA

CCGCCGCTTT

ACGTGG GAG C GA CGGC GCG

AGTGTTTGGC

TCCGGATCG

GGGGTGGTTG

C TG CGAG ATC

GTGTGTTGCG

GATGGTGTGC

CC TAATCAGT

TTCCACTCGG

CGCGGGAGA

AC CAGTCA GA GAG TTGCAGC

GTTTCATGGT

TCGTATCCCA

AATGGCCC

CAGTGGGAAC

GAG AT CCCA C

GCGAGTGAGA

ACTTAATGG

ACATGCTCCA

GTTGATGGGT

TGCAGGCAGC

A T ATC AGC C AC AGG CITTC G

CGAGTTGATG

TC CAGG CCCA

CGGCAGTTGT

CGCTTCGAG

AGGACGGGG

GT AT AA C GTT GGCGC TAT CA BamHI (2) Fragments 1, 2 and 3 were then ligated to give ?.lasmid p160, shown in Figure 6.

This plasrnid was partially digested with the restriction enzymes HincII and PstI. The large HinclI- 38 PstI fragment, containing the origin of replication and shown in Figure 6, was then ligated with fragment 4, shown below, which is a synthetic DNA fragment carrying a sequence coding for the first 44 amino acids of a natural precursor of hGH and, upstream from this sequence, regulatory signals.

39 C L aI 51 TCGAGCTGACTGACCTGTTGjCTTATATTACATCGA 0 5-

AGCTCGACTGACTGGACAACGAATATAATGTAGCT

A

Ndel TAGCGTATAATGTGTGGAATTGTGAGCGATAACAA1TC" CACAGTFTAAC TTTAAGAAGGJAGATATACAT ATCGATATTACACACC TTAACACTCGCCTATTGTTAAAGTGTGTCAATTGAAATTCTTC CTCTATATGTA ATG GCT ACC GGA TCC CGG ACT AGT CTG CTC CTG GCT TTT (JGC CTG CTC TGC CTG TAC CGA TGG CCT AGG GCC TGA TCA GAC GAG GAC CGA AAA CCG GAC GAC ACG GAC

A

M A T G S R T S L L L A F G L L C L -26 XbaI CCC TGG CTT CAA GAG GGC AGT GCC TTC CCA ACC ATT CCC TTA TCT AGA CTiT TTT GGG ACC GAA GTT CTC CCG TCA CGG MAG GGT TGG TMA GGG MAT AGA TCT GM A P W L Q E G S A F P T I P L S R L F -1 1 GAC AAC GCT ATG CTC CGC GCC CAT CGT CTG CAC CAG CTG GCC TTTi GAC ACC TAC CTG TTG CGA TAC GAG GCG CGG GTA GCA GAC GTG GTC GAC CCL3 fM CTG TGG ATC D NA M L R A H R L HQ L A F L T Y Pst I CAG GAG TTT GAA GMA GCC TAT ATC CCA AAG GAA CAG AAG TAT TCA TTC CTG CA 3 0- GTC CTC AMA C7T CTT CGG ATA TAG GGT FTC CTT GTC TTC ATA AGT MAG G G E F EE A Y IP KEGQ K Y SF 40 In this fragment, the amino acids are designated by letters according to the following code: A Alanine C Cysteine D Aspartic acid E Glutamic acid F Phenylalanine G Glycine H Histidine I Isoleucine K Lysine L Leucine M Methionine N Asparagine P Proline Q Glutamine R Arginine S Serine T Threonine V Valine W Tryptophan Y Tyrosine The sequences -35 (TTGCTT) and -10 (TATAAT) of the promoter sequence, and the Shine-Dalgarno sequence well known to those skilled in the art, are successively underlined in this fragment.

Plasmid p380,1 was obtained in this way.

Plasmid p380,1 was then digested with the restriction enzymes Clal and NdeI so as to remove therefrom the small ClaI-NdeI fragment of fragment 4 above and to replace it with the ClaI-NdeI fragment below: Clal

CGATAGCGTATAATGTGTGGAATTGTGAGCGGATAACA

TATCGCATATTACACACCTTAACACTCGCCTATTGT

Ndel

ATTTCACACAGTTTTTCGCGAAGAAGGAGATATACA

TAAAGTGTGTCAAAAGCGCTTCTTCCTCTATATGTAT

The resulting plasmid is plasmid p373,2 (Figure 41 2) Construction of plasmid p466 Plasmid p373,2 was subjected to a double digestion with the enzymes BglII and HindIII. The large fragment derived 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 at the 3' end by the KpnI and SnaBI cloning sites.

42

B

g GATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACI7 '.CAACGAT

AAGTTCGTCTGGATGTCGTTCAAGCTGTGTTTGAGTGTGTTGCTA

GACGCACTACTCAAGAACTACGGGCTGC'TCTACTGCTITCAGUGAAGGACATGGACAAGGTC

CT-2TGATGAGTTCTTGATGCCCGACGAGATGACGAAGTCCTTCCTGTACCTGTTCCAG

F

S

p

I

GAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGG AGGGCAGCTGTGGCTTCTAGTAA CTCTGTAAGGAC GCGTAGCAC GTCAC GGC GAGAC AC C TCC CG C GACACC GAAGATC ATT

H

S n K n d p a n I GG TAC C CTGCC CTAC GTAC CA

CCATGGGACGGGATGCATGGTTCGA

This fragment comprises the BglII and HindlII sticky ends. The novel plasmid formed in this way, p462 (cf. Figure thus comprises a KpnI site and an NdeI 43 site, which will be used for cloning the fragment containing urate oxidase cDNA in the expression vector.

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

The AccI-KpnI fragment, comprising the greater part of this cDNA, was therefore isolated and purified.

Two complementary oligonucleotides were also synthesized, whose sequence, given below: is intended to reconstitute the 5' end of the cDNA. This synthetic fragment obtained in this way has an NdeI end and another AccII end. The fragment and the synthetic sequence were ligated with the expression vector cut by KpnI and by NdeI. This three-fragment ligation makes it possible to obtain the expression vector, called p466, for E _cli urate oxidase (cf. Figure This plasmid was subjected to a series of enzymatic hydrolyses with restriction enzymes, which made it possible to verify the presence of the expected restriction sites, in particular those carried by the gene coding for urate oxidase.

Plasmid p 4 6 6 therefore contains, by construction, a gene coding for urate oxidase, having the following sequence: 44

ATGTCTGCGG

CAAGG TICAC

CCGTCTGTGT

GACAACAGCG

CACCGCCAAG

TGGGCACACA

AACATTGTCT

CCCTCACTCC

ACGTGGTCGA

ACCGTGCTGA

GTACCCACA

ATGCCACTTG

CAC'T)CCTA

GAAGACTTTT

AGATGGCAGA

TACTCGTTGC

GGCC:TCCAA

ACCCCAACSG

AAATTG.

TAAAAGCAGC

AAGGACGAGA

GCTTCTGGAG

TCATTGTCGC

CAGA ACCCCG

CTTCATTGAG

GCCACCGCTG

TTCATCCGCG

GGGCAAGGGC

AGAGCACCAA

CTTAAGGAGA

GCAGTGGAAG

AGTTCGATGC

GCTGAAGATA

GCAAATCCTC

CTAACAAGCA

AACACCGGCA

TCTGAYCAAG

GCGCTACGGC

AGACCGGTGT

GGTGAGATTG

AACCGACTCC

TTACTCCTCC

AAGTACAACC

GACCCGGATG

ACAGCGAGGA

ATCGATATCA

CTCGCAGTTC

CCTGGGACCG

AATTTCAGTC

TACCTGGOCC

ACAG 7CCAG

GCGCGCCAGC

CTATTTCGAA

AGAACGCCGA

TGTACCGTCG

AAGGACAATG

CCAGACGGTG

AGACCTCTTA

ATTAAGAACA

CGAGC TGTC

ACATCCATGC

GACATTGACG

GAAGCGGAAT

AGTCGTCTCT

TGGGGCTTCC

TATCCTGAGC

GACTCCAGCA

AC TGCT CCCG

CGTGCAGGCC

AGCTGATCGA

IGAC7SA G3TCTTC3C' CCCGTC CIC

TTCGCGTCTA

TACGAGATGA

CACCAAGGCC

CCATTTACAT

GGCTCCATCC

CGCTCACGTC

GCAAGCCACA

GTGCAGGTGG

GTCCGGCCTG

TGCGTGACGA

ACCGACGTCG

CCTCCCCTCG

AGC 7CACTCT

ACTATGTACA

GACT CGAG

GCTGGCACAA

CC

T

CACTCGG

TCTCAAGTCT

(The nucleotides which are different from the nucleotides of the cDNA isolated from A. flavus are underlined in the above sequence. These differences were introduced into the synthetic AccT-KpnI fragment so as to have, downstream from the ATG, a nucleotide sequence corresponding more closely to those normally encountered in a prokaryotic gene.) EXAMPLE 8: Exoressin of urate oxidase cDNA The E. coli K12 RRl strain (Bethesda Research Lab. Inc.) was transformed for ampicillin resistance with plasmid p466 and with a negative control plasmid, pBR322.

Ampicillin-resistant colonies were obtained in both cases. 1 colony of each type was cultured in a medium (LB ampicillin 100 pg/ml). After one night at 37'C, with agitation, the two cultures were diluted 100-fold in 45 the medium (LB ampicillin 100 ug/ml). After culture for 1 h, IPTG (isopropyl--D-thiogalactoside) 1 mM is added for 3 h.

Immunodetection of the urate oxidase by Western blot 1) Procedure An aliquot corresponding to 0.2 ml at OD 1 is taken from the culture medium obtained after induction with IPTG for 3 h. This aliquot is centrifuged and the supernatant is removed. The residue 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 residuu by boiling for 10 min in a buffer, called a loading buffer, consisting of Tris-HCl 0.125 M pH 6.8, SDS bromophenol blue 0.002%, glycerol 20%, 1-mercaptoethanol 10% (according to the protocol described by LAEMMLI LAEMMLI, Nature, 227 (1970) 680-685)); electrophoretic separation of the different proteins contained in the solubilizate, according to the protocol described by LAEMMLI LAEMMLI, Nature, 227 (1970) 680-685); and transfer of said proteins contained in the gel on to a nitrocellulose filter (according to the technique of H.

TOWBIN et al., Proc. Natl. Acad. Sci. USA 7S (1979) 4350- 4354).

Immunodetection, performed according to the technique of BURNETTE BURNETTE, Ana. Biochem. 112 (1981) 195- 203), involves the following successive operations: rinsing the nitrocellulose filter for 10 min with a buffer A (Tris-HCl 10 mM, NaCI 170 mM, KC1 1 mM); bringing the nitrocellulose filter into contact with a buffer B (buffer A with bovine serum albumin added at a rate of 3 g per 100 ml) for 30 min at 370C; *bringing the nitrocellulose filter into contact with an immune serum (polyclonal antibodies recognizing A.

46 flavus urate oxidase) for 1 h at 37*C; rinsing the nitrocellulose filter with buffer B; bringing the nitrocellulose filter into contact with a solution of protein G, labeled with iodine 125 at a rate of 0.1 microcurie/ml, for 1 h at 37 0

C;

Srinsing the filter with buffer A; drying the filter between two absorbent sheets; bringing the filter into contact with an X-ray film; and developing the film.

2) Results It is found that the strain transformed by plasmid p46 6 overproduces a protein with an apparent molecular weight of about 33 kDa, which is recognized by antibodies directed against A. flavus urate oxidase and which is absent from the control strain.

EXAMPLE 9: ssav of the urate oxi dae _activiy An aliquot corresponding to the equivalent of ml at OD 1 is taken from the culture medium obtained after induction with IPTG for 3 h under the culture conditions described in the previous Example. This aliquot is centrifuged and the supernatant is removed. The residues are taken up in 1 ml of TEA (triethanolamine) buffer 0.05 M pH 8.9. The cell suspension is sonicated twice for 30 s in ice with a W10 ultrasonic sonicator (set to strength 8 and intensity The extracts are centrifuged at 10,000 g for 10 min and the supernatants are used for the assay.

The above operations are carried out for four colonies taken at random from E. coli K12 transformed by plasmid p466 (colonies Ai, Bi, Ci and Di) and one colony transformed by plasmid pBR322.

1) Principle The conversion of uric acid to allantoin is followed by the decrease in absorbance at 292 nm. The 47 reaction is as follows: 0 o Urate oxidase *IN .1

H

2 XC0f 0O 0 N H H

:L

02 H20 CO2 Uric acid Allantoin (absorbs at 292 nm) 2) Reagents a) TEA 0.05 M pH 8.9/EDTA buffer 7.5 g of TEA (reagent for analysis Prolabo ref.

287.46.266) are dissolved in 400 ml of distilled water; 0.372 g of Complexon III (Merck ref. 8418) is dissolved in 50 ml of distilled water; the two solutions are combined and made up to 500 ml (solution 1); the pH of this solution is adjusted to 8.9 with HC1 0.2 N; and the volume is made up to 1000 ml with distilled water (solution 2).

b) Uric acid stock solution 100 mg of uric acid (Carbiochem ref. 6671) are dissolved in 50 ml of solution 1; the pH is adjusted to 8.9 with HC1 0.2 N; and the volume is made up to 100 ml with distilled water.

The solution obtained can be stored for one week at 4 0

C.

c) Uric acid substrate solution 1.5 ml of uric acid stock solution (Carbiochem ref.

6671) are taken and diluted to 100 ml with TEA buffer (reagent for analysis Prolabo ref. 287.46.266).

This solution must be used the same day.

48 3) Procedure The following volumes are introduced into the quartz cell of a spectrophotometer set to 292 nm and thermostated at 600 il of uric acid substrate solution (preheated to and 100 pl of the above supernatants to which 200 pl of TEA pH 8.9 have been added (preheated to 300C).

After mixing, the change in optical density is read off every 30 s for 5 min. AE, the variation in optical density per minute, is deduced from these readings.

4) Results The urate oxidase enzymatic activity A, expressed in U/ml OD 1, is calculated from the AE measurement with the aid of the formula £I x VpE in which the evnbols Vr, d, El and VPE respectively represent the reaction volume (0.9 ml), the dilution factor 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: 49 TABLE II E. coli K12 strain Urate oxidase activity transformed by (U/ml OD 1) pBR322 0.001 colony Al 0.086 colony Bi 0.119 p466 colony C 1 0.135 colony Di 0.118 The above Table clearly shows that the E. coli cells transformed by plasmid p468 are capable of pro- 1 ducing urate oxidase activity in the presence of IPTG.

EXAMELEGU Construction of three cres n vectors for urate oxidasa cDNAa veat- niasmids PEMR469, DEMR473 and rlEMR R The strategy employed use-' fragments obtained from pre-existing plasmids available to the public, and fragments prepared synthetically by the techniques now in common use. The cloning techniques employed are those described by T. MANIATIS, E.F. FRITSCH and J. SAMBROOK in "Molecular Cloning, a laboratory manual" (Cold Spring 2 Harbor Laboratory, 1984). The oligonucleotides are synthesized with the aid of a Biosearch 4600 DNA synthesizer.

The following description will be understood more clearly with reference to Figures 10, 11 and 12, which respectively show restriction maps of plasmids pEMR414, pEMR469 and pEMR473. The symbols used in these Figures will be specified in the description below. In the case where a site has been blunted by Klenow polymerase, it carries the index where the sites have been eliminated by ligation, they are indicated in brackets.

50 1) Construction of plasmid pEMR469 This plasmid was constructed from the shuttle vector E. coli-yeast pEMR414, constructed by successive ligations of the following components: the PstI-HindIII° fragment symbolized by in Figure 10 of plasmid pJDB207 (BEGGS, 1978: Gene cloning in yeast p. 175-203 in: Genetic Engineering, vol. 2 WILLIAMSON Academic Press London UK) comprising the upstream part of the ampicillin resistance gene AmpR of pBR322 (Sutcliffe, 1979, Cold Spring Symp.

Quart. Biol. 43, 779) and an endogenous 2p fragment, B form, carrying the LEU2 gene of S. cerevisiae partially modified by the deletion of its promoter (called LEU2d), the locus STB (REP3) and the origin of replication of the 2p fragment (HARTLEY and DONELSON, 1980, Nature, 286, 860-865). The HindIII end of this fragment has been blunted by the action of Klenow polymerase. It is denoted by HindIII° in Figure the HindIII-Smal fragment represented by r7 in Figure 10 of chromosome V of yeast containing the URA3 gene with its promoter (ROSE et al., 1984, Gene, 29, p. 113-124). This HindIII-Smal fragment originates from plasmid pFL1 (CHEVALLIER et al., 1980, Gene 11, 11-19). The HindIII end of this plasmid has been blunted by the action of Klenow polymerase.

an SamI-BamHI fragment symbolized by in Figure 10 containing a synthetic version of the promo' .r of the ADH2 gene which differs from the natural version described by RUSSEL and SMITH (RUSSEL et al.

(1983) J. Biol. Chem. 258, 2674-2682) only by a few base pairs intended for introducing restriction sites. (The natural sequence could be used with only slightly different results.) The sequence of this fragment is given below: 51 S M m I a U 1 1 "GGCCT7C CGCGACP QG TCCAC TAT GTTGGAG CCCTGCGCPGA0GGCGGCCT TGTGGCCCGTAGAGGT TGPTAT TCAACCTC AATAAGAG T TGAT T A GA~AAAA A ArA G AGG 7TRTTTAATTACCCTCTTT I TTTTTTTTTTTCCGTC-TCCTCT

S

P

CAGT TCCGAAAGGOO CT AT GAGlTuA I GAUG~i~ARP TRTCACTTCTTT CTGAPTGAACRCA~~AGAAnTA C a I m a H 1 a rn

H

GACTCTAGAGV

C7GAG ATCTCCTAG 52 the BgIII-HindIII fragment symbolized by s in Figure 10 carrying the 3' end of the yeast PGK gene. This fragment originates from complete digestion with BgIII of the HindIII fragment of the yeast chromosomal DNA, carrying the PGK gene described by HITZEMAN et al. (1982, Nucleic Acids Res., 10, 7791- 7808), which has only one BgIII site. This digestion makes it possible to obtain two HindIII-BgIII fragments of which the smaller, of about 0.4 kb, which carries the 3' end of the yeast PGK gene, is retained. The sequence of the latter fragment is described by HITZEMANN et al.

(op. cit.). The BgIII site is cloned in the BamHI site of the previous fragment (the BamHI and BgIII sites therefore disappearing), and the HindIII site, blunted by the action of Klenow polymerase, is cloned in the PvuII site of the PvuII-PstI fragment of pBR322, described below.

the PvuII-PstI fragment symbolized by xxx in Figure 10 of pBR322, containing the origin of replication and the downstream part of the ampicillin resistance gtne AmpR.

Plasmid pEMR414 formed in this way therefore contains the following components: an origin of replication and an ampicillin resistance gene AmpR permitting the replication and selection of the plasmid in E. coli cells. These components permit transformation in E. coli cells.

an origin of replication for the yeast (ARS), the locus STB and the LEU2 gene of S. cerevisiae without promoter and the URA3 gene of S. cerevisiae with its promoter. These components permit the replication and selection of the plasmid in S. cerevisiae cells and a sufficient partition efficacy in cells containing the endogenous 2p plasmid.

Plasmid pEMR414 was completely digested with the 53 restriction enzymes NheI and Clal. The small NheI-ClaI fragment containing the URA3 gene, hereafter called fragment A, was purified.

Plasmid pEMR414 was completely digested with the enzymes NheI and BamHI. The large NheI-BamHI fragment containing especially the LEU2d gene and the origin of replication of plasmid pBR322, hereafter called fragment B, was purified.

The synthetic ClaI-AccI fragment, containing the start of a gene coding for the protein deduced from the urate oxidase cDNA sequence (clone 9C), was also prepared. This fragment contains modifications, relative to clone 9C, introduced for the purpose of inserting codons which are customary in yeast SHARP et al., 1986, Nucl. Ac. Res., vol. 14, 13, pp. 5125-5143) without changing the amino acids coded for. The sequence of this fragment, hereafter called fragment C, is as follows (the underlined nucleotides are those modified relative to clone 9C): C A L c a c I I

CGATATACACAATGTCTGCTGTTAAGGCTGCTAGATACGGTAAGGACAACGTTAGAGT

TATATGTGTTACAGACGACAATTCCGACGATCTATGCCATTCCTGTTGCAATCTCAGA

The plasmid of clone 9C (cf. Figure 3) was digested with the enzymes AccI and Bamlil. The AccI-BamHI fragment, which contains the end of urate oxidase cDNA, hereafter called fragment D, was purified. This fragment has the following sequence: 54 Acc I

CTAC.ACCTTCACAACACGAGAAC

CrT TC CAAGT CT TCC TGC TC TTC

ACCCCTCTCCACACCCTCTACGAGATGACC

TCCCCACACCTCTCCACATCCTCTACTCC

ACCTCTTACACCAAGGCCCACAkACACCGTC

TCCACAATCTGCTTCCCCCTCTTCTCCCAG

ATT TACATOAC CGCCCAAGCACAACC C CGT T TAAATCTACTCGCCCTTCCTCTTGCGC A CGCACACACT7CATTCACA.ACTACAACCAC CCCTCTGTCACTAACT2-TTCAn rGTTCCTG

CACCCCTCCACCCCATCCACATTGACCGC

CTGCCACCTGGCCCTACCTCTAACTCCC

A.C C A AGXGAGCCG, TC CCACG GGGAC TCGCCCTCTTCCCCTTACACC7CCACCTC

TCCTCTCTCTCCCCCCTGACCCTCCTCAC

AC CAAAC C C CCAG~C 7CCCACGCACTT72 CC7CACCAG7ACACCACACT TAACCACACC CCACTGCC7 CATC7CTTTGATTCC TCTCC CCCACTTCCCACTCCAACA.ATTTCAC7CCA CCCTCAACCC7CACCTTCT TAAACTCACCT T TCCATCCTACCTCCCCCACTCC CCA

AACCTACCATCCACCCCCTCACCACCCTC

ACTCC CAGC CTGCCAC CCCC TAT C TACAAC TC z.CCC7CCCACCTCCCGTCATACATC7TC

CTC.ATCCACACTCTCCACTACTCCTTCCCT

CACTACCTCTCACACCTCATGACCAACCCA

TOC CACAACC CC CTCC.AAAACAC CGC CAAC AC CC TGTTC CC CCAGCTTT TC TCCCCTTC CCCA.ACCCTC TCLT CAAC TCTAC CC TCC

CCCTTCCCAC.ACTACTTCACATCCCACCC

AACATGCAT TC TCACCTT CCCCACT TTCCA.A TTGTACTPAAATC7ACCCCTCAX ACCTT

CTCTCTCTCCTTCTCCACCCTCAGATTCAC

CACACACACC;AACACCTCCCACTCTAAC7,C AT TCTCC CACCCAC TCCAT 7AACAACACC

TAACAGCCCTTCCTCACCTAATTCTTCTCCG

ACTCCTCCCC-ACCTCTTCCCCTCCA7TCC7C

TCACCACCCCTCCACA.ACCCACCTACCAC

A TCCTGC C CCCT CA'CGCTCAAPC AT TCT C C TAGCCTACGCCCCTCCAC T TCT;7AC;,ACC AA:,CCCCACCC 7CACTCCT TCATCCC'CCAC, T' CCCTCTCCCGACTGACCAAZ3TACCCC"C:CG C 7CT CCCCG GC CAAIGGC C T C CATAT CAAC7 CAC CAC CCCC CTTCC CGCTACTATAC -T 7C

AGCACCAA,'CTCC;CACTTCTCCCCGCTTCCTCG

7TCCG77CA2,CCTCAAC;kACCCCCXACCAC 7GGGCACCC7TTCCAGC:ACCCACCTCCAT ACCCTCCCATACCGAC7CCTCCCTCACCTA C TC CAG CAGC TC CCCG CACC TC C 7AA CACCGTCC TCCAGCCACCTCCACCCAT TC CTCACTC TG CACA CTfTTCTCAACATIA-AC CACTCACACT'TCTCIAAACCACTTCTiATTC ATCC CA CACCAAAAT CC TGCCCCC CCACC AC T AC CC TCTC CTT TACCACCGCC CCTCC TC A-ACAACCAC TAT T TCCAAATCGACCT.AC

TTCTTCGTCAT-AACTTTACCTCCACTCC

AACCCGAC CTCTTC CC TCCT CACT CCCAC

TTCCCTCC.AGAACACCACTCACCCTC

CCCTCCTCTCTATCTATTCTAA.CC

GCCACCACACACTTCACATTTAA3CATTTCC GCCAAAkCTCTATATACGTCTCCCATACCCTA

CCCTTTCACATATATCACACCCTATCCCAT

TACCATTCATTCACTTCTTTTTTACTTCCA

ATCCTAACTAACTCA.ACAAAAAA TCAACCT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT AAAAAAAAAAAkAAAAAAAAAAG C C CCG TTTTTTTTTTTTTTTTTTTTTCCCCCCCCT AC eornH' 55 Fragments A, B, C and D were ligated to give plasmid pEMR469 shown in Figure 11, in which the symbols have the same meanings as in Figure 10, the novel ClaI- AccI and AccI-BamHI fragments being symbolized by 2) Construction of hlasmid DEMR473 Plasmid pEMR469 was completely digested with the enzymes Miul and SphI. The large MluI-SphI fragment, containing the urate oxidase gene, was then ligated with the synthetic fragment, whose sequence is given below, corresponding to a part (200 bp) of the sequence upstream from the TATA component of promoter GAL7 of S.

cerevisiae, said part comprising the upstream activation sequences (UAS).

U

POP TAT GAAGCCTCGTaCPACTCGCTTCCGGTAAT CTATATAAAAGACAGTA TTTCCTTAACCCAAAAATAAGOAGAGG GOTCCAAAAGCGCTCGGACAACTGTTGACCGT

AAAGGAATTGGGTTTATTCCCTCTCCCAGGTTTTTCGCGAGCCTGTTGACAACTGGCA

GATCCGAAGGACTGCTTATACAGTGTTCACAAATAGCCAAGCTGAAAATAATGTGTAGC

CTAGGCTTCCTGACCGAT A TGTCCAAGTGTT TTATCGGTTCGCTTTTAT TACACTCG

P

CTTTAGCTATOTTCAGTTAGTTTGGCATG-

GAAATCGATACAAGTCAATCAAACC,

Plasmid pEMR473 obtained in this way is shown in Figure 12, in which the symbols have the same meanings as in Figure 11, the novel MluI-Sphl fragment introduced being symbolized by 1 S1 S56 3) Construction of plasmid PEMR515 Plasmid pEMR473 was partially digested with the enzyme XbaI and totally digested with the enzyme Mlul.

The large XbaI-MluI fragment was purified. This fragment contains especially the sequences of the origin of replication and the locus STB of the 2p fragment, the LEU2d gene, the ampicillin resistance gene AmpR, the origin of replication of pBR322 and the expression cassette for urate oxidase. On the other hand, it contains neither the URA3 gene nor that part of the 2p fragment which is between the XbaI and NheI sites.

The large XbaI-MluI fragment was recircularized via the following sequence adapter containing MluI and modified XbaI sticky ends: modified XbaI

VCTAGGCTAGCGGGCCCGCATGCA

CGATCGCCCGGGCGTACGTGCGCA

Plasmid pEMR515 obtained in this way has only one of the three components of the target FRT site of the recombinase coded for by the FLP gene of the 2 J fragment.

Plasmids pEMR469, pEMR473 and pEMR515 possess the gene coding for urate oxidase, which has the following sequence: 57 ATGTCTGCTG TTAAGGCTGC CAAGGTTCAC AAGGACGAGA CCGTCTGTGT GCTTCTGGAG GACAACAGCG FCATTGTCGC CACCGCCAAG CAGAACCCCG TGGGCACACA CTTCATTGAG AACPTTGTCT GCCACCGCTG CCCTCACTCC TTCATCCGCG ACGFGGTCCA GGGCAAGGGC ACCGTGCTGA AGAGCACCAA GTACACCACA CTTAAGGAGA ATGCCACTTG GCAGTIGGAAC CACGTGCCTA AGTTCCATGC GA GACTTTT GCTGAAGATA AGATOCCACA GCAAATCCTG, TACTCGITC CTAACAAC GjccrCCAA AACACCC-GCA ACCCCAA!CGG- TCTCUATCAAG

AAATTC-.

TAGATACGGT

AG AC CGCCTGT

GGTGAGATTG

AACCGACTCC

TTACTCCTCC

A AG TA CAAC C

GACCCGCATG

AC AGC GAG GA A TC GATATC A CTCG CAGTTC

CCTGGSACCG

AATTTCAGTC

TACCTGGCC

A CAG TG C CAG CC CCC CCAGC

CTATTTCGAA

ACG ,AAC (11CCG A TGTACCGTC3 A AGG A CAAC G C C AGA CG G TG A GA CCTCT TA ATTA AGA A CA C GAG CTGTTC

ACATCCATGC

GACATTGACG

GAAGCGGAAT

AGTCGTCTCT

V CGG CT TCC TAT CCT GAGCC

GACTCCAGGA

ACTGCTCGCG

CGCTG C AGGC C AG C CTG GAT C G A AT CCA C CT CA C G TCT-rTCCC T GCCC T CCT C

TTACACTCTA

TACCAGATGA

CACCAAGCC

C CATT TAC AT CC CT C CATC C

CGCTCACCTC

C CAAG CCACA C TG CAGGT CC GTCCGCC CTC

TCCGTGACCA

AC CGACG C C CC ICCCGCTCG AC CTC ACT CT

ACTATCTACA

'A CTGTCG AC C CTGGCCCA AA

CCTCACTCGG

T CT GA ACTCT EXAMELE 11: Tjzanaformajion of the EMY761 yeast strain by _VIazmnii TpEMR469. IFMR473 and DEMP5-5- Transformation of the ~M5Q0 and GRFIB yeast .atraina by olasmnjAE- R51 nrjati~n w -ih election either for th prototrophy of uracil or for the oDrototrorhy of leucine Three non-isogenic strains of Saccharomyces cerevisiae were used as recipient strains: the EMY761 strain (Mata, leu2, ura3, his3, gal) the EMY500 strain (Mata, leu2, ura3, pep4) the GRF18 strain (Mata, leu2, his3) The GRF18 strain is well known to those skilled in the art (Gerry FINK, MIT, USA). The EMY761 and EMY500 58 strains are related to the GRF18 strain. They were obtained by successively crossing the GRF18 strain with a ur-3 strain derived from the FL100 strain (deposited in the ATCC under no 28 383) and with the 20B12 strain (Mata, tspl, pep4) described by E.W. JONES JONES et al. (1977) Genetics, 85, 23).

The GRF18 strain can be obtained by curing plasmid pEMR515 of the GRF18 pEMR515 (leu-) strain deposited in the CNCM under reference no 1-920 on 28 December 1989, and the EMY500 strain can be obtained by curing plasmid pEMR515 of the EMY500 pEMR515 (leu strain deposited in the CNCM under reference no 1-919 on 28 December 1989.

These strains contain mutations (leu2 and ura3) capable of being complemented by the LEU2d defective selection marker and the URA3 selection marker, which are present in each of plasmids pEMR469 and pEMR473.

1) Tranformation with selection for the prototrohv of A colony of the EMY761 strain was used to inoculate 100 ml of a medium called liquid YPG medium (cf.

Table III below). When the cell density had reached 107 cells per ml, the cells were treated with lithium acetate 0.2 M for transformation by a technique well known to those skilled in the art and described by ITO et al. (ITO et al., 1983, J. Bacteriology 153, 163-168).

The EMY761 cells were transformed in parallel with about 1 pg of each of plasmids pEMR469 and pEMR473.

The transformed cells are selected for the auxotrophic character of uracil on a medium called uracil-free solid medium (cf. TablL III below). An EMY761 pEMR469 (ura-) transformed strain and an EMY761 pEMR473 (ura-) transformed strain were thus retained.

59 2) Transformation with selection for the prototrophv of eJ.ucijne.

The transformation technique used is a variant of that described by Beggs et al. (Beggs et al. (1978) Nature 275, 104-109). It consists in subjecting yeasts to a protoplastization treatment in the presence of an osmotic stabilizer, namely sorbitol at a concentration of 1 M.

The precise transformation protocol is specified below: a) 200 ml of liquid YPG medium (cf. Table III) are inoculated with about 5 x 106 cells of a culture in the stationary phase, and the culture inoculated in this way is agitated overnight at 30 0

C.

b) When the density of the culture reaches about 107 cells per ml, the cells are centrifuged at 4000 rpm for 5 min and the residue is washed with sorbitol 1 M.

c) The cells are suspended in 5 ml of sorbitol solution 1 M containing 25 mM EDTA and 50 mM dithiothreitol, and are incubated for 10 min at d) The cells are washed once with 10 ml of sorbitol 1 M and suspended in 20 ml of sorbitol. Zymolase-lOOT (a preparation obtained by partial purification of A crthbaer ut.us culture supernatant on an affinity column and containing 3-1,3-glucan laminaripentahydrolase, marketed by SEYKAGAKU KOGYO Co. Ltd.) is added up to a final concentration of 20 ug/ml and the suspension is incubated at room temperature for about 15 min.

e) The cells are resuspended in 20 ml of a medium containing sorbitol, called sorbitol YPG medium (cf.

Table III below) and incubated for 20 min at 30°C, with gentle agitation.

f) The cells are centrifuged for 3 min at 2500 rpm.

g) The cells are resuspended in 9 ml of trans- 60 formation buffer (sorbitol 1 M, Tris-HC1 10 mM pH 7.5 and CaC12 10 mM).

h) 0.1 ml of cells and 5 pl of DbIA solution (about 5 gg) are added and The suspensicn obtained is left for 10 to 15 min at room temperature.

i) 1 ml of the following sclution is added: polyethylene glycol PEG 4000 20%, Tris-HCl 10 mM pH and CaCl2 10 mM.

j) 0.1 ml of the suspension obtained in i) is poured into a tube containing leucine-free solid regeneration medium (cf. Table III below) which has been melted beforehand and kept liquid at about 45 0 C. The suspension is poured into a Petri dish containing a solidified layer of 15 nl of leucine-free solid regeneration medium.

k) Step j) is repeated with the remainder of the cell Lsspension obtained in i).

The transformed strains start to appear after three cays.

The EMY761 pEMR469 EMY761 pEMR473 (leu-), EMY762 pEMR515 GRF18 pEMR515 (leu and EMY500 pEMR515 (leu transformed strains were thus retained.

TABLE=11 Principal mdia used in Examle11, 12. 13 and 14 uracil-free solid medium 6.7 g of Yeast nitrogen base without Amino Acids (from

DIFCO)

g of casein hydrolyzate (Casamino acids from DIFCO) 10 g of glucose g of agar Mix all the ingredients in distilled water and make up the final volume to 1 1 with distilled water. Autoclave for 15 min at 1200C.

nril-free i qdm 2= Use the formulation o- the uracil-free solid medium without the agar. Autoclave for 15 min at 120 0

C.

61 leucine-free solid medium 6.7 g of Yeast nitrogen base without Amino Acids (from

DIFCO)

mg of adenine mg of uracil mg of 1-tryptophan 20 mg of 1-histidine mg of 1-arginine mg of 1-methionine mg of 1-tyrosine mg of 1-isoleucine mg of 1-lysine mg of 1-phenylalanine 100 mg of 1-glutamic acid 150 mg of 1-valine 400 mg of 1-leucine g of glucose g of agar Mix all the ingredients in distilled water. Make up the final volume to 1 1 with distilled water. Autoclave for 15 min at 120°C. After autoclaving, add 200 mg of 1-threonine and 100 mg of l-aspartic acid.

leucine-free solid regeneration medium Use the formulation of the leucine-free solid medium, mixing in 30 g of agar instead of 20 g and adding 182 g of sorbitol to the mixture.

0 leucine-free liguid medium Use the formulation of the leucine-free solid medium without the agar. Autoclave for 15 min at 120 0

C.

After autoclaving, add 200 mg of 1-threonine and 100 mg of 1-aspartic acid.

liquid YP medium g of yeast extract (Bacto-yeast extract from DIFCO) 20 g of peptone (Bacto-peptone from DIFCO) Mix the ingredients in distilled water. Make up the final volume to 1 1 with distilled water. Autoclave for 15 min at 120°C.

liquid YPG medium Use the formulation of the liquid YP medium, adding, S after autoclaving, glucose at a concentration of g/1.

sorbitol YPG medium Use the formulation of the liquid YPG medium, adding, after autoclaving, sorbitol at a concentration of 1 M.

62 ethanol-glvcerol YP medium Use the formulation of the liquid YP medium. After autoclaving, add 10 ml of ethanol 100% final concentration) and 30 g of glycerol.

ethanol-glvcerol-galactose YP medium Use the formulation of the liquid YP medium. After autoclaving, add 10 ml of ethanol 100%, 30 g of glycerol and 30 g of galactose.

EXAMPLE 12: Expression, in an Erlenmever flask, of urate oxidase cDNA by the EMY761 pEMR469 (ura-+.

EMY761 pEMR473 EMY761 pEMR469 (leu and EMY761 pEMR473 (leu+) strains Immunodetection by Western blot Assay of the urate oxidase activity and the soluble proteins 1) Expression of urate oxidase cDNA a) Str selected on uracil-free medium A colony of each of the EMY761 pEMR469 (ura-) and EMY761 pEMR473 (ura-) strains was cultured in 20 ml of uracil-free liquid medium (cf. Table III, Example 11).

After one night at 30°C, with agitation, the two cultures were centrifuged for 10 min at 7000 rpm. The residues were taken up in 10 ml of sterile distilled water and centrifuged again for 10 min at 7000 rpm. Expression of the urate oxidase was induced by taking up the cells in 20 ml of ethanol-glycerol YP medium (cf. Table III, Example 11) for the EMY761 pEMR469 (ura-) strain and in ml of ethanol-glycerol-galactose YP medium (cf. Table III, Example 11) for the EMY761 pEMR473 (ura-) strain.

The cultures were incubated again at 30C for 22 h, with agitation.

b) Strains selected on leucine-free medium In a first stage, a colony of each of the EMY761 pEMR469 (leu-) and EMY761 pEMR473 (leu-) strains was cultured in 20 ml of leucine-free liquid medium (cf.

Table III, Example 11). This made it possible to obtain 63 and maintain a large number of copies of plasmids by carrying out the selection for complementation of the leu2 mutation by the LEU2d gene carried by plasmids pEMR469 and pEMR473.

After one night at 30 0 C, with agitation, the two cultures were centrifuged for 10 min at 7000 rpm. The residues were taken up in 10 ml of sterile distilled water and centrifuged again for 10 min at 7000 rpm.

Expression of the urate oxidase was induced by taking up the cells in 20 ml of ethanol-glycerol YP medium for the EMY761 pEMR469 (leu-) strain and in 20 ml of ethanolglycerol-galactose YP medium (cf. Table III, Example 11) for the EMY761 pEMR473 (leu-) strain. The cultures were incubated again at 30°C for 22 h, with agitation.

c) Control strain The non-transformed EMY761 strain, i.e. the EMY761 strain without plasmid, was cultivated as above.

It was subjected on the one hand to induction in 10 ml of ethanol-glycerol liquid YP medium and on the other hand to induction in 10 ml of ethanol-glycerol-galactose YP medium.

2) Preparation of the samples a) The cells cultivated in la), Ib) and Ic) were centrifuged and the supernatant was removed. The residues were taken up in 10 ml of distilled water and centrifuged for 10 min at 7000 rpm. The residues washed in this way were taken up in about 1 ml of triethyleneamine buffer, TEA, of pH 8.9. About 300 Pl of cells taken up in said buffer were lyzed in the presence of glass beads (from 400 to 500 pm in diameter), representing about half the final volume. This mixture was agitated vigorously in a Vortex 4 times for 1 min, the samples being placed in ice for 30 s between grinding operations. The liquid was withdrawn from the tubes with a Pasteur pipette and transferred to a microtube. The 64 glass beads were washed once with about 200 il of TEA buffer of pH 8.9. The beads were agitated in a Vortex once for 1 min and the liquid was withdrawn with a Pasteur pipette and added to the above lyzate. The lyzate was then centrifuged in a microtube for 5 min at 7000 rpm. The supernatant was cautiously withdrawn and stored at -20°C for Western blot, assay of the urate oxidase activity and assay of the proteins. The residue of the lyzed cells was stored separately at -20 0 C for Western blot (cf. 3) below).

Furthermore, samples of the cultures prepared in la) and Ib) were taken in the following manner before induction: 2 ml of culture were centrifuged for 10 min at 7000 rpm. The residues were taken up in 500 pl of distilled water and centrifuged again for 5 min at 7000 rpm.

The residues were taken up in about 200 il of TEA buffer of pH 8.9 and lyzed as above in the presence of glass beads. The supernatants and the residues of the lyzed cells were stored separately at -20 0

C.

3) Immunodetection of the urate oxidase by Western blot a) Procedure The residues and the supernatants of the different samples were subjected to a Western blot a technique well known to those skilled in the art which comprises the following steps: solubilization of the residue by boiling for 10 min in a buffer, called a loading buffer, consisting of Tris- HC1 0.125 M pH 6.8, SDS bromophenol blue 0.002%, glycerol 20%, 3-mercaptoethanol 10% (according to the protocol described by LAEMMLI LAEMMLI, Nature, 227 (1970) 680-685)); electrophoretic separation of the different proteins contained in the solubilizate, according to the protocol described by LAEMMLI LAEMMLI, Nature, 227 (1970) 680-685); and 65 transfer of said proteins contained in the gel on to a nitrocellulose filter (according to the technique of H.

TOWBIN et al., Proc. Natl. Acad. Sci. USA 7I (1979) 4350-4354).

Immunodetection, performed according to the technique of BURNETTE BURNETTE, Ana. Biochem. 112 (1981) 195- 203), involves the following successive operations: Srinsing the nitrocellulose filter for 10 min with a buffer A (Tris-HCl 10 mM, NaC1 170 mM, KC1 1 mM); bringing the nitrocellulose filter into contact with a buffer B (buffer A with bovine serum albumin added at a rate of 3 g per 100 ml) for 30 min at 37 0

C;

Sbringing the nitrocellulose filter into contact with an immune serum (polyclonal antibodies recognizing A fLavuo urate oxidase) for 1 h at 370C; *rinsing the nitrocellulose filter with buffer B; bringing the nitrocellulose filter into contact with a solution of protein G, labeled with iodine 125 at a rate of 0.1 microcurie/ml, for 1 h at 370C; rinsing the filter with buffer A; Sdrying the filter between two absorbent sheets; Sbringing the filter into contact with an X-ray film; and *developing the film.

b) Eaujlta It is found that the EMY761 pEMR469 (ura-), EMY761 pEMR473 EMY761 pEMR469 (leu-) and EMY761 pEMR473 (leu-) strains produce a protein with an apparent molecular weight of about 33 kDa, which is recognized by antibodies directed against A. flavus urate oxidaae and which is absent from the control strain.

It is also found that the non-induced strains produce none or very little of the protein described above.

Comparison between the amounts of this protein 66 for the residues and the supernatants makes it possible to deduce that about 80% of said protein is in soluble form in the lyzate.

4) Assay of the urate oxidase activity The urate oxidase activity was measured on the supernatants of the lyzed cells according to the procedure described in Example 9 above.

The results obtained are collated in Table IV below, which specifies the urate oxidase activity in U/ml for each strain induced by glycerol-ethanol, each strain induced by glycerol-ethanol-galactose and each noninduced strain.

TABLEIV

Strain/Inducer Urate oxidase activity (U/ml) EMY761/YP ethanol-glycerol-galactose 0.1 EMY761/YP ethanol-glycerol 0.1 EMY761 pEMR469 (ura )/(non-induced) 0.4 EMY761 pEMR469 (ura-)/YP ethanol-glycerol 12 EMY761 pEMR469 (leu-)/(non-induced) 0.17 EMY761 pEMR469 (leu+)/YP ethanol-glycerol 36 EMY761 pEMR473 (ura-)/(non-induced) 0.1 EMY761 pEMR473 (ura-)/YP ethanol-glycerolgalactose 12.5 EMY761 pEMR473 (leu-)/(non-induced) 0.1 EMY761 pEMR473 (leu+)/YP ethanol-glycerolgalactose 15.3 The above Table clearly shows that the yeast cells transformed by these plasmids pEMR469 and pEMR473 are capable of producing urate oxidase activity after induction.

Assay of the total soluble proteins in the lyzates The protein assay kit from BIORAD was 'ised for assaying the total proteins present in the supernatant of S the lyzed cells. It is based on the observation that the 67 maximum absorbance of an acid solution of Coomassie brilliant blue g-250 changes from 465 nm to 595 nm when proteins become attached thereto Reisner et al., Anal. Biochem., 64, 509 (1975)).

a) Procedure The following volumes are introduced into the cell of a spectrophotometer set to 595 nm: 10 pl of sample to which 790 il of distilled water have been added 200 pl of concentrated Dye reagent (Biorad).

The ingredients are mixed and the optical density is read off at 595 nm. A calibration range with increasing concentrations of BSA (bovine serum albumin) was prepared in this way. The unknown concentration of the total proteins in the lyzates is read off on the calibration curve obtained.

b) Results The main results obtained are collated in Table V below, which specifies the amount (in n:g/ml) of total soluble proteins and the percentage of urate oxidase in the total soluole proteins for each strain induced by glycerol-ethanol, each strain induced by glycerolethanol-galactose and each non-induced strain (it is assumed here that the specific activity of the recombinant protein is identical to that of the urate oxidase obtained from A. flavus: 30 U/mg).

68-

TABLEV

Total of urate Strain/Inducer soluble oxidase in the proteins total soluble Mg/Mi proteins EMY76l/glyoerol-ethanol 5.3 <0.05 EtY761/glycerol-ethanol-galactose 5.8 0.05 EMY761 pEM-R469 (ura-)/non-induced 8.5 0.25 EMY761 pEt4R469 (ura-l)/glycerol-ethanol 5.3 4.7 EM'Y761 PEMR469 (leu-)/non-induced 1-7 0.3 E11Y761 PEMR469 (leu-)/glycerol-ethanol 5.9 EMY'761 pEMR473 (ura-)/non-induced 10.3 0.05 EM1Y761 pEMR473 (ura-)/glycerol-ethanolgalactose 6.5 6.4 EMY761 pE11R473 (leu-)/non-induced 0.5 0.05 EIhY761 pEMR473 (leu-I)/glycerol-ethanolgalactose 3.9 13 It is found that the production rate of urate oxidase varies from 5 to 20% according to the transformants and the mode of selection of the transformed strains (leu'-j.

KXAM1LE 1 Exporession, in a 2. 5 1 femne.of urate oxidase cDNA by t~he 1DEME 7 (ra-) strin 1) Fermentat'h Dr~tQOl a M~Dia Inoculum medium A colony of the EMY761 pEMR473 (ura-) strain was cultured in 200 ml of uracil-free liquid medium (of.

Table III, Example 11). Culture is continued overnight, with agitation, until the OD is about 3.

69 Culture medium A for 1 1 of purified water on an apparatus of the Milli-Q type glucose 30 g glycerol 30 g casein hydrolyzate (Casamino acids from DIFCO) 30 g Yeast Nitrogen Base (from DIFCO) 15 g Yeast extract (from DIFCO) 2.5 g K2HPO4 3 g MgS04.7H20 0.5 g Acditional medium B for 100 ml of purified water on an apparatus of the Milli-Q type glycerol 30 g peptone hydrolyzate (Primatone from G. Sheffield) 30 g Yeast Nitrogen Base (from DIFCO) 15 g Yeast extract (from DIFCO) 5 g K2HPO4 3 g MgSO4.7H20 0.5 g b) Fermentation parameters Bioreactor of total volume 2.5 1, equipped with two turbines Temperature pH Q. Oxygen partial pressure 30 mm Hg 70 Air flow rate 1 1/min The bioreactor is filled with 1.5 1 of medium A and inoculated with 150 ml of the inoculum.

Once the glucose has been exhausted at OD 2.5 to about OD 17, induction is effected by the addition of a volume of 150 ml of galactose at 20% weight/volume.

Growth is continued and additional medium B is then added at about OD Growth continues for about another fifteen hours and the product was harvested at OD 104.

2) Preparation and analysis of the samples The samples were prepared as described in Example 9 2) a) from the culture in the fermenter. Two samples were taken: the first after 7 h of induction and the second after 22 h of induction.

The following tests, described in Example 9, were performed on these two lyzates obtained after lysis of the cells: immunodetection by Western blot assay of the biological activity assay of the total proteins The following results were obtained: a) Immunodetection by Western blot It is found that the EMY761 pEMR473 (ura-) strain, cultivated in a 2 1 fermenter, produces a protein with an apparent molecular weight o' 33 kDa, which is recognized by antibodies directed against A. flavus urate oxidase (said antibodies being prepared in rabbits by techniques well known to those skilled in the art: q.v.

VAITUKAITIS et al. (1981) "Methods in Enzymology", Academic Press, New York, vol. 73, p. 46) and which is absent from the control strain.

b) Aay of the biological activity The results obtained are collated in Table VI below: 71 TABLE VI Strain/Induction time U/ml EMY761 pEMR473 (ura-)/7 h 9 EMY761 pEMR473 (ura-)/22 h 12.5 It is found that the EMY761 pEMR473 (ura-) strain, cultivated in a fermenter, is capable of producing urate oxidase activity after induction.

c) Assay of the total soluble proteins The results are collated in Table VII below: TABLEJLIl Strain/Induction time Total of urate soluble oxidase in the proteins total soluble mg/ml proteins EMY761 pEMR473 (ura-)/7 h 5.2 5.7 EMY761 pEMR473 (ura-)/21 h 6.2 6.6 These results indicate that the rate of synthesis of urate oxidase by the EMY761 pEMR473 (ura-) strain, cultivated in a fermenter, is about 5% of the total proteins of the cell after 7 h and 21 h of induction.

EXAMLE 14: Expression, in an Erlenmever flask, of urate oxidase cDNA by the EMY761 pEMR515 (leu).

EMY500 pEMR515 (leu-) and GRF18 pEMR515 (leul+ strains A colony of each of the above three strains was cultured in 20 ml of leucine-free liquid medium.

After one night at 30°C, with agitation, the three cultures were centrifuged for 10 min at 7000 rpm.

The cell residues were taken up in 10 ml of sterile _1 -72distilled water and centrifuged again for 10 min. Expression of the urate oxidase was induced by taking up the cells in 20 ml of ethanol-glycerol-galactose YP medium (cf. Table I, Example The cultures were incubated again at 30°C for about 20 h, with agitation.

The non-transformed host strains were each cultured as controls.

The cells of each of the six cultures are separated out again by centrifugation and the supernatant is removed. The residues were taken up in 10 ml of distilled water and centrifuged for 10 min at 7000 rpm.

The residues washed in this way were taken up in about 1 ml of TEA buffer of pH 8.9 and the grinding and removal of the particles by centrifugation were carried out as described in Example 9, The supernatant of each culture is used, as previously, for assaying the urate oxidase and the total proteins. The main results obtained are collated in Table VIII below: I 20 TABLE II Strain/Culture Urate oxidase Total soluble of urate conditions activity proteins oxidase in (U/ml) (mg/ml) the soluble proteins GRF18 pEMR515 0.1 2.2 0.05 EMY500 pEMR515 0.1 0.9 0.05 EMY761 pEMR515 0.1 1.8 0.05 GRF18 pEMR515 38 5.4 23 EMY500 pEMR515 20 2.5 26 EMY761 pEMR515 33 4.2 26 the strains are cultivated in the presence of glucose (noninduction conditions) the strains are cultivated in the absence of glucose and in the presence of galactose (induction) These results show that a high level of expression of urate oxidase can be obtained with three non- -73isogenic recipient strains transformed by the expression vector according to the invention.

EXAMPLE 15: Expression in a 2.5 1 fermenter of the cDNA of urate oxidase for the EMY500 pEMR515 strain. Purification and partial characterization of the recombinant urate oxidase: 1) Culture in a 2.5 1 fermenter of the EMY500 pEMR515 strain: The culture of the EMY500 pEMR515 strain is carried out in the following manner: a) Preculture stage in erlenmeyer A 500 ml erlenmeyer containing 90 ml of a growth medium MCPA, (sterilizable by autoclave) complemented with 1.28 g of MES N-morpholino/-ethanesulfonic acid Sigma no M8250) and 10 ml of a growth medium MCPF (sterilized by ultra filtration) is seeded with 1 ml of a solution of the EMY500 pEMR515 strain in a medium containing 20% glycerol with a number of cells corresponding to an Optical Density of 2.35. The compositions of the media MCPA and MCPF are given hereinafter. After 24 hours of incubation, under stirring at 30 0 C, the Optical Density of the culture is about 7.

b) Culture phase in fermenter The above culture is used for seeding a 2.5 1 fermenter containing the culture medium having the following composition: 900 ml of MCPA 200 ml of MCPF The pH of the culture is regulated by the fermenter to the given value of 5.5. After 6-7 hours of culture at 30 0 C, 72 ml of a 500 g/1 glucose solution is linearly added over a period of 9 hours (namely a total of 36 g of glucose).

74 c) Expression stage To the previously described mixture, 100 ml of the expression medium MEPA (sterilizable by autoclave) and 150 ml of the expression medium MEPF (sterilized by ultra filtration) having the following compositions, are added. The culture is then continued for 5 hours. Then 150 ml of a solution containing 30 g of galactose, 15 g of glycerol and 36 g of ethanol are linearly added for 20 hours. An optical density of about 160 is then obtained.

CHEMICAL COMPOSITION OF THE GROWTH AND EXPRESSION MEDIA Growth medium MCPA (sterilizable by autoclave) For total 900 ml NTA (nitrilotriacetic acid) 1.2 g Yeast extract (DIFCO) 6 g K2SO 4 1.2 g NaC1 0.6 g MgSO 4 7H20 1.2 g CaC1 2H20 840 mg FeC13 108 mg glutamic acid 4.44 g HYCASE SF (Sheffield Products) 30 g leucine 2.16 g histidine 600 mg methionine 1.2 g oligoelements I (see hereinafter) 5 ml uracil 1.2 g 75 List of oligoelements I for 1 1 of ultra purified water CuSO4, 5H20 H3BO3 ZnSO 4 7H 2 0

KI

MnSO,4 2H 2 0 Na2MO 4 2H20 FeC1 3 6 H2 0 780 mg 5 g 3 g Sg 3.5 g 2 g 4.8 g Add 100 ml of adjust to 1,000 Growth medium concentrated hydrochloric acid to the solution and ml.

MCPF (sterilized by ultra filteration) for total 200 ml of ultra purified water

KH

2 P0 4 Tryptophane Vitamin I (see hereinafter) glucose 4.8 g 420 mg 5 ml 36 g Heat to dissolve, return to ambient temperature, add and filter through 0.2 pm filter.

the vitamins I -76- List of vitamins I for total 100 ml of ultra purified water biotine folic acid niacine (nicotinic acid) pyridoxine. HC1 thiamine. HC1 calcium pantothenate mesoinositol 1.2 mg 1 mg 144 mg 60 mg 240 mg 1.2 g 2.4 g Fill to 100 ml after dissolving Sterile filter, cold, at 0.2 ym Expression medium MEPA (sterilizable by autoclave) for total 100 ml of ultra purified water NTA 1.2 g K2SO 4 2.08 g glutamic acid 6 g HYCASE SF (Sheffield Products) 24 g leucine 2.16 g histidine 600 mg methionine 1.2 g MgSO 4 7H 2 0 720 mg CaC1 2 2H 2 0 840 mg FeCl 3 6H 2 0 108 mg oligoelements I 5 ml uracil 1.2 g L~ L 77 Adjust the pH to 5.5 with concentrated H2SO 4 or concentrated KOH Autoclave for 20 mins at 1200C Expression medium MEPF (sterilized by ultra filtration) for total 150 ml of ultra purified water

KH

2

PO

4 2.4 g tryptophane 420 mg vitamins I 5 ml glycerol 36 g galactose 45 g Heat to dissolve, return to ambient temperature, add the vitamins and filter.

2) Grinding of the cells After 20 hours of induction, the OD of the culture, measured at 600 nm, is 98. 800 g of the fermentation wort are centrifugated for 5 minutes at 10,000 g and the cell cake is taken up in 80 ml of a lysis buffer (glycine 20 mM pH The cells are then ground twice at 40C, for 2.5 minutes in a grinding device (Vibrogenic Zellmihle mill V14) in the presence of a volume of beads (0.50 mm in diameter) equal to that of the solution of cells to be lysed. After grinding, the supernatant is taken up and the beads are washed twice with 80 ml of a lysis buffer. 210 ml of a lysate are recovered; said lysate has a total protein content of about 3 mg/ml and a urate oxydase activity of about 7.7 U/ml (namely a urate oxidase percentage towards the total protein of about 8.5 considering a specific activity of that protein of U/mg).

-78 3) Purification of the recombinant urate oxidase a) Purification protocol The abrve lysate is submitted to the two-step purification protocol disclosed hereinafter.

Step 1 Anionic chromatography Support DEAE (diethylaminosulphate) sepharose fast flow (Pharmacia ref.

17.07.09.91) The compressed gel occupies a volume of 70 ml.

The separation is carried out at ambient temperature, the recovered fractions being preserved at 0 0

C.

Separation conditions A gradient of a chloride ionic force between buffer 1 (sodium borate 10 mM, pH 9.2) and buffer 2 (sodium borate 10 mM, sodium chloride 1M) is used. The buffers are previously degased and preserved at 00C during the elution. In each buffer 0.02 of azide are added.

The raw extract is deposited (10 ml) and eluted with buffer 1 up to the complete recovery of the urate oxidase (by fractions of 10 ml) which is not retained on the column.

The pigments and the contaminating proteins are thereafter removed by an elution with buffer 2.

The purification is followed by measuring of the OD of the eluate at 214 nm.

Step 2 High pressure and inverse phase liquid chromatography Support: Grafted C8 silica column, Aquapore OD-300 (100 x 2.1 mm) -79 (Brownlee-Applied Biosystems) Operating conditions Eluent 1 ultrapurified water (filtered through a Millipore system) containing 0.1 of trifluoroacetic acid.

Eluent 2 Acetonitrile (of spectrophotometric quality or similar) containing 0.08 of trifluoroacetic acid.

Flow rate 0.3 ml/min.

The gradient is of 35 of acetonitrile/TFA to 70 of acetonitrile/TFA for 20 minutes and is maintained at 70 for minutes. The injected quantity is of 1 ml per run.

Recovery of the fractions The separation is followed by measurement of the optical density at 218 nm. The acetonitrile is evaporated during the centrifugation under vacuum.

b) Results The sample before and after the first step of purification was analysed by liquid chromatography on a grafted C8 silica column, the Aquapore OD-300 previously disclosed with the same gradient, with an injected quantity of 50 pl. Purified urate oxidase from A flavus is used as an external control.

In the starting lysate, the urate oxidase represents 63 of the total proteins. After the first step of purification, the urate oxidase represents 84 of the total proteins.

The whole sample obtained after step 2 was used for the following partial characterization. Said sample certainly contains more than 84 of urate oxidase.

4) Partial characterization of the recombinant urate oxidase a) Analysis of the amino acids The analysis of the amino acids of the acid hydrolysate of the purified recombinant urate oxidase was carried out in an analyser from Applied Biosystems model 420-130A. The distribution of the quantified amino acids is compatible (there exists no significant difference) with the supposed sequence. The same result was observed for the purified urate oxidase extracted from A. flavus (obtained in example 4) b) Tryptic peptidic map A tryptic peptidic map was established for the purified recombinant urate oxidase and for the purified urate oxidase extract obtained in example 4) under the following conditions A urate oxidase solution having a concentration of 1 mg/ml is prepared. Extemporaneously a trypsin solution having a concentration of 1 mg/ml is prepared.

The two solutions are mixed together in a proportion of 1/30 enzyme/substrate for 8 hours at ambient temperature. The tryptic hydrolysate is then chromatographied (liquid phase chromatography) on a C18 grafted silica column (5 pm; lichrosorb 250 x 4.0 mm Hichrom-ref. RP 18-5-250A) provided with a UV detector coupled with a recorder. The gradient applied is of 1 acetonitrile/TFA to 60 acetonitrile/TFA for 120 minutes and then the gradient is maintained at 60 for 5 minutes.

The peptidic maps obtained have a very narrow profile.

5) Determination of the blocked character of the amino-terminal sequence The amino-terminal sequence was analysed by means of the sequencer, Applied Biosystem model 470A, coupled with an analyser of phenylthiohydantoic derivatives, Applied Biosystems model 120A.

The purified recombinant urate oxidase (200 pmoles detected by analysis of the amino acids) was put on the sequencer in the presence of 20 pmoles of 1 -lactoglobuline (control protein).

No amino-terminal sequence corresponding to the sequence of the urate oxidase was detected, whereas the amino-terminal sequence of -81 the control protein was detected.

Therefore, the recombinant urate oxidase of the invention, as well as the urate oxidase extract, has a blocked amino-terminal end.

EXAMPLE_ 1: Construction of an expression vector for urate oxidase cDNA in animal cells: plasmid pSV860 This vector was obtained by ligation of the small AccI-SnaBI fragment containing a sequence coding for urate oxidase with the exception of the first 16 amino acids, said fragment being derived from plasmid p466 (an expression vector for A. flavus urate oxidase in E. coli, available in the laboratory and described below), with a synthetic HindIII-AccI fragment, which made it possible to obtain a HindIII-SnaBI fragment containing a complete sequence coding for A. flavus urate oxidase and a non-translated 5' sequence favoring expression in animal cells; and insertion of the HindIII-SnaBI fragment between the HindIII and SnaBI sites of the multiple cloning site (also called polylinker) of the expression vector for animal cells, namely plasmid pSEi.

The following account will successively describe the construction of plasmid p 4 6 6 plasmid pSEi and plasmid pSV860.

1) Construction of plasmid p466 Plasmid p466, an expression vector for urate oxidase cDNA in E. coli, was prepared. It comprises a fragment of pBR327 including the origin of replication and the ampicillin resistance gene; it also comprises a synthetic promoter of E. coli RODRIGUEZ and M.

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

This plasmid was constructed from an expression 82 plasmid for hGH in E. coli (p462) by replacing a fragment carrying the hGH gene with urate oxidase cDNA.

The construction of plasmid p466 was described in detail in Example 7 above.

2) Construction of an expression vector for animal cells: plasmid pSEi The strategy employed uses fragments obtained from pre-existing plasmids available to the public, and fragments prepared synthetically by the techniques now in common use. The cloning techniques employed are those described by T. MANIATIS, E.F. FRITSCH and J. SAMBROOK in "Molecular Cloning, a laboratory manual" (Cold Spring Harbor Laboratory, 1984). The oligonucleotides are synthesized with the aid of a Biosearch 4600 DNA synthesizer.

The following description will be understood more clearly with reference to Figure 13, which shows a restriction map of plasmid pSEi, the sites which have disappeared due to ligation being indicated in brackets.

The symbols used in this Figure will be specified in the description below.

This plasmid was constructed by successive ligations of the fo-lowing components: 1) a PvuII-PvuII fragment symbolized by in Figure 13 of 2525 bp, obtained by complete digestion of plasmid pTZ18R (Pharmacia) with the restriction enzyme PvuII. This fragment contains the origin of replication of phage F1 (denoted by ORI F1 in Figure 13), a gene (denoted by AmpR in Figure 13) carrying ampicillin res...tance, and the origin of replication (denoted by ORI pBR322 in Figure 13) permitting the replication of this plasmid in E. coli. The first PvuII blunt site disappears on ligation with the EcoRV blunt site (which also disappears) of the fragment described in 7).

2) a PvuII-HpaI fragment symbolized by =gg in 83 Figure 13 of 1060 bp, of type 5 adenovirus DNA between position 11299 (PvuII restriction site) and position 10239 (HpaI restriction site) (DEKKER VAN ORMONDT, Gene 2Z, 1984, 115-120), containing the information for VA-I and VA-II RNA's. The HpaI blunt site disappears on ligation with the PvuII blunt site (which also disappears) of the fragment described in 3).

3) a PvuII-HindIII fragment symbolized by in Figure 13 of 344 bp, derived from SV40 virus DNA and obtained by complete digestion with the restriction enzymes PvuII and HindIII. This fragment contains the origin of replication and the early promoter of virus DNA (ref. B.J. BYRNE et al., PNAS-USA (1983) 721-725).

The HindIII site disappears on ligation with the site binding to HindIII of the fragment described in 4).

4) a synthetic "site binding to HindIII"-HindIII fragment symbolized by in Figure 13 of 419 bp, whose sequence, given below, is similar to the nontranslated 5' sequence of the HTLV1 virus (ref. WEISS et al., "Molecular Biology of Tumor Viruses" part 2 2nd edition 1985 Cold Spring Harbor Laboratory p.

1057).

84 site binding to HindlII

AGCTGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCC

CCGAGCGTAGAGAGGAAGTGCGCGGGCGGCGGGATGGACTCCGGCGGTAGGTGCGG

GGTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTA

CCACTCAGCGCAAGACGGCGGAGGGCGGACACCACGGAGGACTTGACGCAGGCGGCAGAT

GGTAGGCTCCAAGGGAGCCGGACAAAGGCCCGGTCTCGACCTGACCTCTAAACTTACCTA

CCATCCGAGGTTCCCTCGGCCTGTTTCCGGGCCAGAGCTGGACTCGJAGATTTGAATGGUAT

GACTCAS3CCGGCTCTCCACGCTTTGCCTGACCCTGC iTGCTCAACTCTACGTCTTTGTTT

CTGAGTCGGCCGAGAGGTGCGAAACGGACTGGGACGAACGAGTTGAGATGCAGAAACAAA

CGTTTTCTGTTCTGCGCCGTTACAACTTCAAGGTATGCGCTGGGACCTGGCAGGCGGCAT

GCAAAAGACAAGACGCGGCAATGTTGAAGTTCCATACGCGACCCTGGACCGTCCGCCGTA

C TG GGA CCCC TAG GAAG G GCT TGG GG G TCCT CCT G CC C AAG GC AG GGAA C ATAGTG G TC C GAC CCTG G GGA TC CTTC C CGA AC CC C CAG GAG CA CG GG T TCC G TC CCTT G TAT CA C CAG G

CAGGAAGGGGAGCAGAGGCATCAGGGTGTCCACTTTGTCTCCGCAGCTCCTGAGCCTGCA

GTCCTTCCCCTCGTCTCCGTAGTCCCACAGGTGAAACAGAGGCGTCGAGGACTCGGACGT

GA

CTTCGA

HindlII 85 a synthetic HindIII-"site binding to BamHI" fragment symbolized by XXXX in Figure 13 containing the promoter of the RNA polymerase of phage T7 and also a polylinker containing the SmaI cloning site.

AGCTTGTCGACTAATACGACTCACTATAGGGCGGCCGCGGGCCCCTGCAGGAATTC

ACAGCTGATTATGCTGAGTGATATCCCGCCGGCGCCCGGGGACGTCCTTAAG

HindIII Smal site binding to BamHI V

V

GGATCCCCCGGGTGACTGACT

CCTAGGGGGCCCACTGACTGACTAG

6) a BamHI-BcII fragment of 240 bp represented by F in Figure 13 which is a small fragment obtained by complete digestion of the SV40 virus with the enzymes BcII and BamHI and containing the late polyadenylation site of said virus FITZGERALD et al., Cell, 24, 1981, 251-260). The BamHI and BcII sites disappear on ligation respectively with the site binding to BamHI of the fragment described in 5) and the BamHI site (which also disappears) of the fragment described in 7).

7) a BamHI-EcoRV fragment symbolized by C10 0 in Figure 13 of 190 bp, which is a small fragment derived from plasmid pBR322 after complete digestion with the enzymes EcoRV and BamHI.

3) Construction of plasmid pSV860 Plasmid p466 (cf. Figure 9) was completely digested with the enzymes AccI and SnaBI. The small AccI- SnaBI fragment, which contains a DNA sequence coding for urate oxidase with the exception of the first 18 aminoterminal acids, was purified and ligated with the synthetic HindIII-AccI fragment having the following sequence: 86 HindlII Acci V If

AGCTTGCCGCCACTATGTCCGCAGTAAAAGCAGCCCGCTACGGCAAGGACAATGTCCGCGT

ACGGCGGTGAT4 CAGGCGTCATTTTCGTCGGGCGATGCCGTTCCTGTTACAGGCGCAGA This ligation makes it possible to obtain the HindIII-SnaBI fragment containing a sequence, coding for urate oxidase, which is identical to that of clone 9C and a non-translated 5' sequence favoring expression in animal cells (KOZAK, Nuol. Acids Res., 12, 2, 1984, 857-872).- The HindIII-SnaBI fragment contains the following sequence:

-AGCTTGCCG

CAATGTCCGC

CGGTGTACGA

TCTTACACCA

G AA CACCA TT

TGTTCGGCTC

C ATG CCG CTC

TGACGGCAAG

GGAATGTGCA

TCTCTGTCCG

CTTCCTGCGT

TG AG CAC CGA C AGG AGG TC C

TCGCGAGGTC

AGGCCAC TAT

ATCGAGACTG

CCTGAGCTGG

TCGCTCCTCA

TCCTCTCTGA

CCACTATGTC

GTCTACAAGG

GA TGA C CGTC

AGGCCGACAA

TA CA TCA CC G

CATCCTGGGC

ACGTCAACAT

CCACACCCTC

GGTGGACGTG

GCCTGACCGT

GACGAGTACA

C GTC GA TGC C

GCTCGCACGT

ACTCTGAAGA

G TA CAAG ATG

TCGAGTACTC

C ACAAGGGC C GTC GGAC CC C

AGTCTAAATT

CGCAGTAAAA

TTCACAAGGA

TGTGTGCTTC

C AG CG TC ATT

CCAAGCAGAA

ACACACTTCA

TGTCTGCCAC

ACTCCTTCAT

GTCGAGGGCA

GCTGAAGAGC

CCACACTTAA

ACTTGGCAGT

GCCTAAGTTC

CTTTTGCTGA

GC AG AG CAAA

GTTGCCTAAC

TCCAAAACAC

AACGGTCTGA

GCAGCCCGCT

CGCAGAAG AC C

TGGAGGGTGA

GTCGCAACCG

C CCC G TTAC T

TTGAGAAGTA

CGCTGGACCC

CCGCGCACAGC

ACG GGCAT CGA

ACCAACTCGC

GGAGA C CTGG

GGPAAGAATTT

GA TG CTAC CT AG ATAA CAG T

TCCTGGCGCG

AAGCACTATT

CGGCAAGAAC

TCAAGTGTAC

AC GGC AAGGA CC TGT C CAGA

GATTGAGACC

ACTCCATTAA

CCTCU'CGAGC

CAACCACATC

GGA TGGACA T G AGGA GA AG C

TATCAAGTCG

AGTTCTGGGG

GACCGTATCC

C AG TG GA CTC

GGGCCACTGC

GCCAGCGTGC

C CAGC AG CTG

TCGAAATCGA

GCCGACGTCT

CGTCGGCCGG

87 The HindIII-SnaBI fragment was then inserted into vector pSEi, which had first been incubated with the enzymes HindIII and SmaI. This gave plasmid pSV860 shown in Figure 14, in which the symbols have the same meanings as in Figure 13, the novel HindIII-SnaBI fragment being symbolized by (The SnaBI and Smal sites disappeared on ligation.) EXAMPLE 7 :Transient expression of urate oxidase cDNA in COS cells Assay of the urate oxidase activity in the cell lvzate COS cells are monkey kidney cells expressing the T-antigen of the SV40 virus (Gluzman, Cell 23, 1981, 175-182). These cells, which permit the replication of vectors containing the origin of replication of virus DNA, are preferred hosts for studying the expression of genes in animal cells.

1) Transfection of COS cells and transient expression of urate oxidase cDNA 4.105 COS cells are plated out in a Petri dish of diameter 6 cm (Corning) in 5 ml of Dulbecco's modified Eagle's medium (from Gibco), hereafter called DMEM, which contains 0.6 g/l of glutamine and 3.7 g/l of NaHCOs and is complemented with fetal calf serum (GIBCO) at a rate of After about 16 h of culture at 370C in an atmosphere containing 5% of carbon dinxide, the culture medium is sucked off and the cells are washed with 3 ml of PBS (phosphate buffered saline from GIBCO). The following mixture is then added: 1000 pl of (DMEM 10% of fetal calf serum (GIBCO)), 110 il of diethylaminoethyldextran of average molecular weight 500,000 at a concentration of 2 mg/ml (Pharmacia), 1.1 jil of chloroquine 100 mM (Sigma) and 3 pg of DNA of either plasmid pSV860 or plasmid pSEi (for the control). After incubation for 5 h at 37*C in an atmosphere containing 5% of carbon dioxide, the mixture is withdrawn from the cells. 2 ml of PBS containing 88 of dimethyl sulfoxide (spectroscopic grade, Merck) are then added. After incubation for 1 min at room temperature, the mixture is withdrawn and the cells are washed twice with PBS. 5 ml of DMEM complemented with fetal calf serum at a rate of 2% are added. Incubation is continued for 4 days at 37°C under an atmosphere containing 5% of carbon dioxide.

2) Preparation of the samples The culture medium is sucked off and the COS cells are rinsed twice with 3 ml of PBS. The cells are then collected by scratching with a rubber spatula (policeman) in 1 ml of PBS. After scratching, the dish is rinsed with 1 ml of PBS. The two cell suspensions are combined and centrifuged for 10 min at 1000 rpm. The supernatant is removed and the cell residue is resuspended in 1 ml of triethylanmmonium (TEA) 0.05 M of pH 8.9/ EDTA buffer.

The cells are lyzed by sonication (on ice) by means of 10 s pulses with a sonicator (Vibra Cell from Sonics and Materials Inc. USA) set to a power of 12 W.

The cell lyzate is centrifuged for 10 min at 10,000 rpm and the supernatant is recovered for assay of the urate oxidase.

3) Assay of the urate oxidase activity The urate oxidase activity was assayed as described in Example 9.

The results are collated in the Table below: COS cells transfected Urate oxidase activity by U/ml pSV860 0.105 pSEi 0.01 It is found that the COS cells transfected by 89plasmid pSV860 carrying urate oxidase cDNA express an appreciable level of urate oxidase activity, whereas no urate oxidase activity is detectable in the control.

There is therefore expression of urate oxidase cDNA.

Claims (4)

1. A protein possessing a specific urate oxidase activity of at least 16 U/mg and having the following sequence Ser Ala Val Lys ALa Ala Arg Val Cys Ser Lys His Cys Phe GLy Ser Lys Trp Asp Asp Leu His Lys Cys Tyr GLy Lys Asp Asn Val GLy Val Gin Thr Val Tyr ILe GLu Thr Ser fyr Thr Ser ILe Lys Asn Thr Ile Pro GLu Leu Phe GLy Ser His ILe His Ala Ala His Asp ILe Asp Gly Lys Pro Lys Arg Asn Val Gin Val Ser Ser Leu Ser GLy Leu GLy Phe Leu Arg Asp GLu Leu Ser Thr Asp Val Asp GLn GLu VaL Arg Ser His Arg GLu Val Tir Leu Lys Ala Thr Met Tyr Lys Met GLu Thr VaL GLu Tyr Ser Ser Trp His Lys GLy Leu Pro Gin Ser Asp Pro Asn Leu Lys Ser Lys Leu Val Gly Arg Ser preceded if appropriate, by a methionine degFe-ee-hmoe-l-wjah--t-seq.uenc-. 4 i y &W0-Iqbv--I-- n

2. A protein according to claim 1, possessing a specific urate oxidase activity of at least 30 U/mg.

3. A protein according to claim I or 2, which presents, by analysis on a bidimensional gel, a spot of molecular mass of about

33.5 kDa a-an-nse--e -po-i-t--a-re a4-83.0-, representing at least 90 of the protein mass. 4. A protein according to anyone of claims I to 3, having a purity degree, determined by liquid chromatography on a C8 grafted silica column, higher than 80 A protein according to anyone of claims I to 4, having an 3k, isoelectric point around 91 6. A protein according to anyone of claims I to 4, which carries a blocking group on the amino-terminal serine having preferably a molecular mass around 43 units of atomic mass. 7. A drug containing a protein according to anyone of claims 1 to 6. 8. A recombinant gene which has the DNA sequence coding for the protein having the following sequence Met Tyr Met Ala ILe Leu Asn His Ser Ala Val Lys Ala ALa Arg Tyr GLy Lys Asp Asn Val Arg Lys Val His Lys Asp GLu Lys Thr GLy VaL GLn Thr Vat Tyr Thr Val Cys Val Leu Leu Glu Gly GLu ILe Glu Thr Ser Tyr Thr Asp Asn Ser Val ILe Val Ala Thr Asp Ser ILe Lys Asn Thr ILe Thr ALa Lys GLn Asn Pro Val Thr Pro Pro Glu Leu Phe Gly Ser GLy Thr His Phe ILe GLu Lys Tyr Asn His ILe His Ala ALa His ILe Val Cys His Arg Trp Thr Arg Met Asp ILe Asp Gly Lys Pro His Ser Phe ILe Arg Asp Ser Glu GLu Lys Arg Asn VaL VaL Asp Val Va GLu GLy Lys GLy ILe Asp ILe Lys Ser Ser Leu Ser GLy Leu Thr Val Leu Lys Ser Thr Asn Ser GLn Phe Trp GLy Phe Leu Arg Asp Glu Tyr Thr Thr Leu Lys GLu Thr Trp Asp Arg ILe Leu Ser Thr Asp Val Asp Ala Thr Trp GLn Trp Lys Asn Phe Ser GLy Leu GLn Glu Val Arg Ser His VaL Pro Lys Phe Asp Ala Thr Trp ALa Thr Ala Arg GLu Val Thr Leu Lys Thr Phe ALa GLu Asp Asn Ser Ala Ser Val GLn Ala Thr Met Tyr Lys Met Ala Glu GLn ILe Leu Ala Arg GLn GLn Leu ILe Glu Thr Val Glu Tyr Ser Leu Pro Asn Lys His Tyr Phe GLu ILe Asp Leu Ser Trp His Lys Gly Leu GLn Asn Thr Gly Lys Asn Ala GLu Val Phe Ala Pro GLn Ser Asp Pro Asn GLy Leu ILe Lys Cys Thr Val GLy Arg Ser Ser Leu Lys Ser Lys Leu 9. A recombinant gene according to claim 8, which permits the expression in the prokaryotic microorganisms. 10. A recombinant gene according to claim 9, wherein the DNA sequence contains the followings sequence 92 ATGTCTGCGG CAAGGTTCAC CCGTCTGTGT GACAACAGCG CACCGCCAAG TGGGCACACA AACATTGTCT CCCTCACTCC ACGTGGTCGA ACCGTGCTGA GTACACCACA ATGCCACTTG CACGTGCCTA GAAGACTTTT AGATGGCAGA TACTCGTTGC GGGCCTCCAA ACCCCAACGG AAATTG. TAAAAGCAGC AAGGACGAGA GCTTCTGGAG TCATTGTCGC CAGAACCCCG CTTCATTGAG GCCACCGCTG TTCATCCGCG GGGCAAGGGC AGAGCACCAA CTTAAGG AGA GCAGTGGAAG AGTTCGATGC GCTGAAGATA GCAAATCCTG CTAACAAGCA AACACCGGCA TCTGATCAAG GCGCTACGGC AGACCGGTGT GGTGAGATTG AACCGACTCC TTACTCCTCC AAGTACAACC GACCCGGATG ACAGCGAGGA ATCGATATCA CTCGCAGTTC CCTGGGACCG AATTTCAGTG TACCTGGGCC ACAGTGCCAG GCGCGCCAGC CTATTTCGAA AGAACGCCGA Tb T-IACGTCC AAGGACAATG CCAGACGGTG AGACCTCTTA ATTAAGAACA CGAGCTGTTC ACATCCATGC GACATTGACG GAAGCGGAAT AGTCGTCTCT TGGGGCTTCC TATCCTGAGC GACTCCAGGA ACTGCTCGCG CGTGCAGGCC AGCTGATCGA ATCGACCTGA GGTCTTCGCT GCCGGTCCTC TTCGCGTCTA TACGAGATGA CACCAAGOCC CCATTTACAT OGCTCCATCC CGCTCACGTC OCAAGCCACA GTGCAGGTGG GTCCGGCCTG TGCGTGACGA ACCGACGTCG GGTCCGC.TCG AGGTCACTCT ACTATOTACA GACTGTCGAG GCTGGCACAA CCTCAGTCGG TCTGAAGTCT II. A recombinant gene according to claim 8, which permits the expression in the eukaryotic cells. 12. A recombinant gene according to claim II, wherein the DNA sequence contains the following sequence 93 ATGTCTGCTG CAAGGTTCAC CC G TCT G T G GACAACAGCG C AC CGC CAAG T GG G CACAC A AACATTGTCT CC CTCACTC C ACGTGGTCGA ACCGTGCTGA G TA CA CC AC A ATGCCACTTG CA CGTG C CTA GAAGACTTTT AGATGGCAGA TACTCGTTGC GGGCCTCCAA AC CC CAAC GG AAATTG. TTA AGGC TG C AAGGACGAGA GCTTCTGGAG TCATTGTCGC C AGAAC C CCG CTTCATTGAG GCCACCGCTG TTCATCCGCG GGGCAAGGGC AGAGCACCAA CTTAAGGAGA SC AGTGG AAG A GTT CGATG C GCTGAAGATA GCAAATCCTG CTAACAAGCA AA CA C CGGCA TCTGATCAAG T AG ATA C GGT AGACCGGTGT GGTGAGATTG AACC'GACTCC TTACTCCTCC AAG TA CA AC C GACCCGGATG ACAGCGAGGA ATCGATATCA CTCGCAGTTC CCTGGGACC,,j A ATTT CAG TG TACCTGGGCC AC AGTG C CAG GCGCGCCAGC CTATTTCGAA AGAACGCCGA TG TA C CGTCG A AG GA CAAC G CCAGACGGTG AGACCTCTTA AT TAAGAA CA C GAG CTGTTC ACATCCATGC GAC ATTG AC G GAAGCGGAAT AGTCGTCTCT TGGGGCTTCC TATCCTGAGC GACTCCAGGA ACTGCTCGCG C GTGC AGGC C AGCTGATCGA ATCGACCTGA GGTCTTCGCT GCCGGTCCTC TT AG A C TCTA TACGAGATGA CACCAAGGCC C CATT TAC AT GGCTCCATCC CGCTCACGTC GCAAGCCACA GTGCAGGTGG GTCCGGCCTG TGCGTGACGA ACCGACGTCG GGTCCGCTCG AG GTC A CTC T AC TATG TA CA GACTGTCGAG GCTGGCACAA C CTC AGTC GG TCTGAAGTCT 13. A recombinant gene according to claim 8, which permits the expression in the animal cells. 14. A recombinant gene according to claim 13, wherein the DNA sequence contains the following sequence: d4 CAATGTCCGC CGGTGTACGA TCTTACACCA GAACACCATT TGTTCGGCTC CATGCCGCTC TGACGGCAAG GGAATGTGCA TCTCTGTCCG CTTCCTGCGT TGAGCACCGA CAGGAGGTCC TCGCGAGGTC AGGCCACTAT ATCGAGACTG CCTGAGCTGG TCGCTCCTCA TCCTCTCTGA -ATGTC GTCTACAAGG GATGACCGTC AGGCCGACAA TACATCACCG CATCCTGGGC ACGTCAACAT CCACACCCTC GGTGGACGTG GCCTGACCGT GACGAGTACA CGTCGATGCC GCTCGCACGT ACTCTGAAGA GTACAAGATG TCGAGTACTC CACAAGGGCC GTCGGACCCC AGTCTAAATT CGCAGTAAAA TTCACAAGGA TGTGTGCTTC CAGCGTCATT CCAAGCAGAA ACACACTTCA TGTCTGCCAC ACTCCTTCAT GTCGAGGGCA GCTGAAGAGC CCACACTTAA ACTTGGCAGT GCCTAAGTTC CTTTTGCTGA GCAGAGCAAA GTTGCCTAAC TCCAAAACAC AACGGTCTGA GCAGCCCGCT CGAGAAGACC TGGAGGGTGA GTCGCAACCG CCCCGTTACT TTGAGAAGTA CGCTGGACCC CCGCGACAGC AGGGCATCGA ACCAACTCGC GGAGACCTGG GGAAGAATTT GATGCTACCT AGATAACAGT TCCTGGCGCG AAGCACTATT CGGCAAGAAC TCAAGTGTAC ACGGCAAGGA GGTGTCCAGA GATTGAGACC ACTCCATTAA CCTCCCGAGC CAACCACATC GGATGGACAT GAGGAGAAGC TATCAAGTCG AGTTCTGGGG GACCGTATCC CAGTGGACTC GGGCCACTGC GCCAGCGTGC CCAGCAGCTG TCGAAATCGA GCCGAGGTCT CGTCGGCCGG preceded by a non-translated 5' sequence favoring expression in nnimal cells. Recombinant gene according to claim 14, wherein the non-translated 5' sequence favoring expression in animal cells comprises the sequence AGCTTGCCGCCACT, located immediately upstream from the sequence described in claim 14. 16. An expression vector carrying a recombinant gene according to any one of claims 8 to 15 with the means necessary for its expression. 17. An expression vector according to claim 16, which carries at least one selection marker. 18. An expression vector according to claim 17, which has the characteristics of one of plasmids pEMR469, pEMR473, and pEMR 515. 19. Prokaryotic microorganisms which are transformed by an expression vector according to claim 16, carrying a recombinant gene according to claim 9. 95 Eukaryotic cells which are transformed by one of the expression vectors according to any one of claims 16 to 18, carrying the recombinant gene according to claim 11. 21. A strain of Saccharomyces cerevisiae which is transformed by one of the expression vectors according to any one of claims 16 to 18. 22. A strain according to claim 21, which carries a mutation on at least one of the genes responsible for the synthesis of leucine or uracil. 23. A strain according to claim 22, which carries o mutation on at least oneof the LEU2 and URA3 genes. 24. A process for producing a recombinant urate oxidase which comprises the steps of 1) cultivating a strain according to claims 21 to 23 2) lysing the cells 3) isolating and purifying the recombinant urate oxidase contained in the lysate. Animal cells containing a recombinant gene according to claim 13 with the means necessary for its expression. 26. Animal cells containing an expression vector according to claim 16, carrying a recombinant gene according to claim 14. DATED this THIRTEENTH day of MARCH 1991 Sanofi by DAVIES COLLISON Patent Attorneys for the Applicant