HK1000099A1 - Protein with urate oxidase activity, 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

The invention relates to a new protein with urate oxidase activity, a drug containing it and the genetic engineering tools to produce this protein, including a recombinant gene, a vector of expression carrying this gene, prokaryotic microorganisms and eukaryotic cells transformed by this vector of expression.

Uratine oxidase (EC 1.7.3.3) is an enzyme in the purine degradation pathway, which is not found in primates (such as humans), birds, some reptiles, or most insects, and is also absent in some dogs (such as the Dalmatian).

In humans, the pure bases adenine and guanine are converted to xanthine. Xanthine is oxidized by xanthine oxidase to form uric acid according to the reaction: ${\text{xanthine + H}}_{\text{2}}{\text{O + O}}_{\text{2}}{\text{→ acide urique + O}}_{\text{2}}{\text{}}^{\text{-}}\text{.}$

The O2 radical, a substrate of superoxide dismutase, is converted by superoxide dismutase to hydrogen peroxide.

Uric acid, a metabolite in the blood, is normally found mainly in the form of soluble monosodium salt. However, it can happen that in some people, uric acid precipitates and forms stones. Hyperuricemia, an increase in the amount of uric acid circulating in the blood, causes the deposition of uric acid in the cartilage tissues, which leads to gout. Hyperuricemia can also have consequences on the kidneys: an excess of uric acid in the urine and in the kidneys can lead to uric acid nephrolithiasis, that is, the accumulation of very painful uric stones that can damage the kidneys. These stones are possibly associated with uric acid and oxalates.

The overproduction of uric acid can have various origins: congenital metabolic defects, Lesch-Nyhan syndrome, excessive intake of purine or protein, treatment with uricosuric drugs, treatment of haemopathies, particularly cancerous ones, with cytolytics (chemotherapy) or radiotherapy, etc. (Gutman, AB and YU, T.F. (1968) Am. J. Med. 45 - 756-779).

Urat oxidases, an enzyme that catalyzes the breakdown of uric acid into allantoin (a compound much more soluble than uric acid, which does not crystallize at concentrations in biological liquids), are therefore of therapeutic interest. When used as an injection, they have many advantages in the treatment of hyperuricemia and nephrolithiasis: rapid hypo-uricemic effect (decrease of about 50% of hyperuricemia in less than 24 h), better protection of the kidney against other drugs such as allopurinol (a lithium-oxanthine oxidase inhibitor), etc. This enzyme is currently mainly used as an adjuvant to chemotherapeutic cytotherapy.

The urea oxidase currently used as a medicine is obtained by a process involving the culture of a mycelium of Aspergillus flavus and the isolation of urea oxidase by extraction from the culture medium and several steps of partial purification of this protein. This process, which produces urea oxidase with a specific urate oxidase activity of about 8 U/mg, free of toxic contaminants, has however some disadvantages. The physiology and especially the genetics of A. flavus are not easily worked (WOLOSHUK et al, (1989) Applied. microbiol. vol. 55, p. 86-90). It is therefore not possible to obtain strains that sometimes produce this enzyme in large quantities.

There is therefore a need for purer A. flavus urate oxidase and genetic engineering tools and techniques to overcome these drawbacks.

The applicant purified the urate oxidase extracted from A. flavus, hereinafter 'extractive urate oxidase', which is of a purity much higher than that already known for that protein, determined its partial sequence and constructed two pools of labelled probes capable of hybridising with the nucleotides coding for two portions of that protein. It then constructed an expression vector containing that DNA, transformed an E. coli K12 strain with that DNA, cultured that DNA and verified that the lysate of the cells contained a recombinant protein of the expected molecular mass, which has urate oxidase activity (capacity to degrade uric acid into allantoin).

The applicant also constructed several vectors of expression in eukaryotic cells containing a recombinant gene coding for urate oxidase whose sequence contains variations from the isolated cDNA, introduced with the aim of establishing codons common to eukaryotic cells, transformed different eukaryotic cells using these vectors, cultured them in a low volume as well as in a larger volume (fermenter) and found that the lysates of the cells contained a high proportion of the expected molecular weight recombinant protein with urate oxidase activity.

It purified this recombinant protein and partially characterized it, in a comparative manner with extractive urate oxidase.

The invention therefore concerns a new protein characterised by a specific urate oxidase activity of at least 16 U/mg, having the following sequence: possibly preceded by a methionine.

Preferably, this protein has a specific urate oxidase activity of about 30 U/mg.

A protein of this type is considered to be one which, when analysed on a two-dimensional gel, has a molecular mass spot of approximately 33.5 kDa and a nearby isoelectric point of 8.0 representing at least 90% of the protein mass.

Preferably the purity of this protein determined by liquid chromatography on a C8 grafted silica column is greater than 80%.

An interesting protein of this type is one with an isoelectric point of about 8.0. It is appreciated that the amino-terminal serine carries a blocking group, preferably of mass close to 43 atomic mass units, such as the acetyl group.

The invention also relates to a medicinal product containing the protein defined above in a pharmaceutically acceptable vehicle, which can be used as an advantageous substitute for extractive urate oxidase with a specific urate oxidase activity of about 8 U/mg, sold as an injectable preparation under the brand name Uricozyme (Vidal 1990).

The invention also relates to a recombinant gene characterised by the presence of a DNA sequence encoding the following sequence protein:

Because of the degeneration of the genetic code, there are a large number of DNA sequences coding for a protein whose sequence meets the formula given above.

Another preferred DNA sequence particularly suitable for expression in eukaryotic cells, such as yeast, is:

Another preferred DNA sequence that is particularly suitable for expression in animal cells is the following: preceded by a 5′ untranslated sequence favorable for expression in animal cells. Such a preferred untranslated 5′ sequence is one that includes the sequence : AGCTTGCCGCCACT, immediately upstream of the sequence explained above.

It should be noted that the protein encoded by the cDNA sequences given above may be matured by methyonyl-amino-peptidase, which cleavethis from its amino-terminal methionine residue.

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

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

For expression in eukaryotic cells, the expression vector of the invention carries the recombinant gene defined above with the means necessary for its expression, replication in eukaryotic cells and selection of the transformed cells. Preferably, this vector carries a selection marker, chosen for example to complement a mutation in receptive eukaryotic cells, which allows the selection of cells that have integrated the recombinant gene in a high number of copies either in their genome or in a multicopy vector.

The sequence around the initiating ATG is preferably chosen based on the consensus sequence described by KOZAK (M. KOZAK (1978) Cell 15, 1109-1123). An intronic sequence, e.g. the DHBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 322-derived pBR 323-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 324-derived pBR 32

For an expression in eukaryotic cells such as yeast, e.g. Saccharomyces cerevisiae, the coding sequence must be inserted between, on the one hand, sequences recognized as effective promoters and, on the other hand, a transcription terminator. The promoter-coding-terminator sequence set, called an expression cassette, is either cloned into a plasmid vector (monocopy or polycopy for yeast) or integrated in multicopy into the yeast genome.

The invention also relates to eukaryotic cells transformed by the previous vector of expression, including strains of Saccharomyces cerevisiae, in particular those with a mutation in one of the genes responsible for the synthesis of leucine or uracil, e.g. the LEU2 or URA3 genes.

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

The invention also concerns a process for the production of recombinant urate oxidase which includes the steps of: 1) culture of a strain as defined above;2) cell lysis;3) isolation and purification of the recombinant urate oxidase contained in the lysate.

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The test chemical is a chemical that is used to test the presence of a substance in the blood.

The urea oxidase-producing strain of A. flavus was grown under urea oxidase-producing conditions, i.e. in a medium containing uric acid of the following composition: glucose 15 g/l, MgSO4,7H2O 1 g/l, KH2PO4 0,75 g/l, CaCO3 1,2 g/l, uric acid 1,2 g/l, KOH 0,5 g/l, soybean oil 0,66 ml/l, FeSO4,7H2O 10 mg/l, CuSO4,5H2O 1 mg/l, Zn4,7H2O 3 mg/l, Mn4,H2O 1 mg/l.

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

In a 5 1 cm field, 1.5 l of medium seed is sown with about 1 to 3 107 spores.

The culture is incubated for approximately 40 h at 30°C under agitation (120 rpm) and the mycelium is recovered by filtration on gauze, washed with water and frozen in liquid nitrogen.

15 g of mycelium (wet weight) are thawed and resuspended in 45 ml of lysine pad and then taken up in the same volume of beads (0.45 μm diameter). The lysine pad is made up of 4 M guanidine thiocyanate, Tris-HCl 10 mM pH 7.6, EDTA 10 mM, β-mercaptoethanol 50 ml/l. The mycelium suspension is ground in the Zellmühle (vibrogen) mill for 5 min.

The crusher is recovered and the balls are settled, the surfactant is removed (about 45 ml) and reduced to a final 3 M in lithium chloride and stored at 0 °C.

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

Add proteinase K (SIGMA), 40 μg/ml SDS (0.1% P/V) and EDTA at 20 mM. Incubate at 37°C for 3 h. Precipitate with 2 volumes of ethanol, then wash with 70% ethanol. Re-insert the coating in 0.5 ml TE buffer (tris HCl 10 mM EDTA, 1 mM pH 7.5), extract twice with chloroform, then precipitate with ethanol. RNA is stored at -80°C in alcohol.

EXAMPLE 2 Purification of the poly A+ fraction of RNA

Approximately 1 mg of RNA is precipitated for 20 min at 4°C (15 000 rpm) and washed with 70% ethanol and dried.

The test substance is then collected into 1 ml of TE buffer and resuspended by vortex agitation. The dT cellulose trace 3 (marketed by Collaborative Research Inc, Biomedicals Product Division) is prepared according to the manufacturer's recommendations. The RNA is deposited on the dT trace, gently agitated to resuspend the beads, and then heated for 1 min at 65°C.

The suspension is adjusted to 0.5 M NaCl and gently shaken for 10 min. The suspension is then centrifuged for 1 min at 1000 rpm, the surgical solution is removed, the collet is washed twice with 1 ml of TE buffer containing 0.5 M NaCl. The surgical solutions are removed. The polyadenylated fraction of RNA (made up of messenger RNA) is obtained by suspending the balls in 1 ml of TE buffer, then heating this suspension to 60°C for 1 min, followed by a pre-shaking for 10 min on a basaltic plate. The following centrifugation is done 1 min to 1000 rpm, which allows the pre-shaking of the RNA containing 1 ml of the buffer and the usual cellulose-free solution.

EXAMPLE 3: Establishment of the DNA bank

From the isolated messenger RNAs as described in the previous example, a cDNA bank was made in the vector pTZ19R (marketed by PHARMACIA).

The cloning technique used is that described by Caput et al., (Primer-adapter technique: (Caput et al., Proc. Natl. Acad. Sci. (U.S.A.) (1986) 83, 1670-1674)).

The corresponding fragment of the vector is purified on a column of Sepharose CL4B (Pharmacia). It thus comprises a polydC tail at one end, the other end being a cohesive one, of the BamHI type. On the other hand, messenger RNAs are subjected to reverse transcription from an amorphous sequence of 5'<GATCCGCCCT12) <3.

Err1:Expecting ',' delimiter: line 1 column 479 (char 478)

EXAMPLE 4 Purification of the extractive oxidase urate from A. flavus and characterisation of the extractive oxidase 1) Purification of extractive urea oxidase from A. flavus.

A preparation of urate oxidase extracted from A. flavus (uricozyme - Clin Midy Laboratories) with a specific urate oxidase activity of 8 U/mg (specific urate oxidase activity is the ratio of urate oxidase activity measured by the test described in Example 9 to total protein mass measured by the Bradford method: Anal. Biochem., 72, 248-254) was purified by chromatography on a column of Redagarose 120 grafted agarose (SIGMA), concentration by ultrafiltration and filtration on a polyacrylamide-after-garose gel Ultrogel A 44 (CAIBF), according to the cyclic agaric protocol:

Step 1: Affinity chromatography on grafted agarose

Temperature: 4° Column: Pharmacy K50/30 Resin: Red 120 Agarose (3 000 CL/R-0503 SIGMA) (gel volume = 410 ml, gel height = 20 cm) Balancing buffer: glycine/NaOH 20 mM pH 8.3 Elution buffer: glycine/NaOH 20 mM, NaCl 2M pH 8.3 Conditioning rate: 250 ml.h-1 Operating rate: 160 ml.h-1 Elution rate: 160 ml.h-11) Place the Uricozyme solution on the column head with a constant flow pump2) After adsorption, wash the column by 2 times its volume in the balancing buffer3) Eluate by a gradient of i with the following diametric force: The following substances are to be classified in the same category as the active substance: The total volume of the gradient is equal to 10 times the volume of the column, halved in each of the constituents. Chromatographic recording is performed at λ = 280 nm; the urate oxidase pool is collected after grouping together fractions with a specific urate oxidase activity of 16 U/mg or greater.

Step 2: Concentration of the urate oxidase pool by ultrafiltration using a Biopass system with a 10 kDa ultrafiltration membrane.

Step three:

Temperature: 4° column: Pharmacy K 50/100 The following is the list of active substances in the active substance: The concentration of the gel = 1.6 l/gel height = 80 cmBalancing buffer: glycine/NaOH 20 mM pH 8.3Conditioning rate:40 ml.h-1Running rate:24 ml.h-11) Place the concentrated urate oxidase pool at the head of the column using a constant flow pump.2) After the sample has been placed, continue to feed the column with glycine/NaOH buffer 20 mM pH 8.3.3) After chromatography, wash with NaCl 2M until a U.V. absorbance value (λ = 280 nm) < 0.05. Storage in NaCl 2 M at 4 °C. The chromatographic recording shall be performed at λ = 280 nm; The urate oxidase pool is collected after grouping together fractions presenting: a specific urate oxidase activity equal to or greater than 20 U/mg.2 bands only by electrophoresis under denaturing conditions (presence of SDS) and exposure to silver nitrate (Biorad staining kit), with: . a major band of 33-34 kDa. a minor band of 70-71 kDa

(2) Characterisation of purified extractive urea oxidase from A. flavus: (a) partial sequencing

In order to obtain information on the amino acid sequence of purified extractive urate oxidase, which would allow the synthesis of the probes needed for cloning cDNA, a direct amino-terminal sequencing of the protein was attempted, but this was not successful due to amino-terminal blockade of the protein (see f below).

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

(a) Hydrolysis of urate oxidase by trypsin, purification and sequencing of peptides:

Urat oxidases at 9 mg/ml in a 100 mM ammonium carbonate buffer pH 8.9 were digested by trypsin (Worthington, TPCK) with a urate oxidases/trypsin ratio of 30/1 by weight at 30°C for 24 h. After tryptic hydrolysis, 60 μg of digested urate oxidases were directly injected into an HPLC reverse phase column of Brownlee G18 grafted silica (column 10 x 0,2 cm), balanced in acetonitrile 1 % (v/v), trifluoroacetic acid 0,1 % (v/v) in water. The peptides were then ejected by a linear gradient of acetonitrile in a solution of trifluoroacetic acid (0,1 %/v) in water with a debitrate of 60 μm/min at a peptide density of 150 μm, then were devitrified at a peptide density of 60 μm/min at a peptide debitrate of 1 μm/min.

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

Each peak was collected and stored at -20°C until the time of analysis on a protein sequencer (Applied Biosystems Model 470 A) equipped with a chromatograph (Applied Biosystems Model 430 A) which continuously analyses the phenylethylidenthoic derivatives formed after each degradation cycle. Table (1) below shows the peptide sequences of the 9 identified peaks.

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

Uratide oxidase, at a concentration of 2 mg/ml in a 100 mM ammonium acetate buffer pH 6.8, was digested by Staphylococcus aureus (Boehringer-Mannheim) protease V8 with a urate oxidase/protease ratio of 60/1 to 30°C for 72 h. 160 μg of digested urate oxidase was then injected into a HPLC column of Brownlee G18 grafted silica reverse phase (10 x 0.2 cm column; 7 x 0.03 μm) particles balanced in 218 % acetrylonitrile, 0.1% (v/v) trifluoroacetic acid in water. The peptides were then elicited by a linear gradient of acetrylonitrile in a trifluoroacetic acid solution (0.1 μm/v) in water with a peptide density of 60 μm/min, at a peptide defect rate of 60 μm/v, at a peptide defect rate of 1 μm/min.

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

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

The following table (1) shows the peptide sequences of the 5 identified peaks.

(b) Specific activity:

Purified extractive urate oxidase has a specific activity of approximately 30 U/mg.

(c) Electrophoresis under denaturing conditions

Electrophoresis of purified extractive urate oxidase on polyacrylamide gel in the presence of SDS (sodium dodecyl sulfate), followed by silver exposure, shows 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:

Operating mode: Using ready-to-use gels, the LKB Ampholines Gel Plates from Pharmacia of pH ranges: (3.5-9.5) and (5-8).Deposit of 10 μl of LKB control proteins (control protein isoelectric point range: 3.5-9.5) and purified urate oxidase 4 μg and 8 μg (on two different tracks).Run 1 h 30, 12 V, 6°C.Then colouration in Commassian blue (0.1%) in (25 % Ethanol, 8 % Acetic Acid) to colour the proteins, each with a following discoloration using a solution containing 25 % Ethanol and 8 % Acetic Acid (to eliminate background noise).Results: Observation on two strips of two rapidly moving isoelectric strips (two isoelectric strips) and 8,1 points each.

(e) Analysis by two-dimensional gel

The two-dimensional gel analysis allows proteins to be separated firstly by their isoelectric points and secondly by their molecular masses.

The Protocol

Sample: purified extractive urea oxidase solution in a glycine buffer of 20 mM pH 8,3.

Preparation of the sample

Two samples of 5 μg and 10 μg of urea oxidase; vacuum centrifuge drying, withdrawn in 5 μl from the lysis buffer of the following composition: urea 2.5 M, / 3- (cholamidopropyl) dimethylammonium) 1-propane sulphonate CHAPS (Sigma) / 2% (v/v), / Amphoteric Ampholines (LKB) pH range 5-8 and 3.5-9.5 / 0.4% and β mercaptoethanol 5%.

The following shall be indicated in the column 'C' of the column:

Preparation of a solution containing: urea 9.5 M, CHAPS 5%, Ampholines LKB (pH (3.5-9.5) 1 %; pH (5-8) 1 %), Acrylamide/bisacrylamide (28.4%/1,7%) 3.5% final, H2O.Solution filtered and degassed and then added 0.075 % of tetramethylethylene diamine Temed (Pharmacia) and 0.015 % of ammonium persulfate.Solution poured into tubes (16 x 0.12 cm) - polymerization overnight at 20°C.Cathodic solution: NaOH 0.1 Modified.Anodic solution: H3PO4 25 M.Pre-run 45 min 4 mA (voltage 300 000 V) V.Sample at the level of the SDS; 19 000 mR 1 D; 1 D in a cathode at 20°C and 0.8 MT at pH 20 °C.

The following information is provided for the purpose of this Annex:

Preparation of a solution containing: Acrylamide/bisacrylamide (30%/0.8%) 15%, final tris-HCl (pH 8.8) 0.375 M, H2O.Filtered and degassed solution followed by addition of SDS (0.1%), ammonium persulfate 0.05% and Temed 0.05%.Polymerization overnight at 4°C (16 x 20 x 0.15 cm gel).The isoelectrofocusing gel after balancing is deposited on the surface of the PAGE/SDS gel which is sealed with agarose.Electrophoresis buffer: (Tris-HCl 25 mM 8.3% pH, glycine 0.192 M, SDS 0.1 m).Reflective 100A - 6 h at 6°C.The presence of molasses is then determined in a 50 per cent molasses, 10 per cent colour between the surface of the silver and the silver.The presence of nitrosamine is then measured in a plasma scanner (Blumethyl and H.Kodamide, 1987), allowing a measurement of the density of the protein in the surface of each sample.

The result:

Two spots of close molecular mass of 33.5 kDa are observed, one with a majority of a close isoelectric point of 8.0 of intensity 5.2 (representing about 93 per cent of the protein mass), the other with a minority of a close isoelectric point of 7.4 of intensity 0.41 (representing about 7 per cent of the protein mass).

(f) Determination of the amino-terminal sequence and the mass of the blocking amino-terminal group:

(a) highlighting the blocked nature of the amino-terminal sequence: The amino-terminal sequence was analysed using an Applied Biosystem model 470 A sequencer coupled with an Applied Biosystem model 120A phenylethylohydantoic derivatives analyser. Purified urate oxidase (200 pmoles controlled by amino acid analysis) was deposited on the sequencer in the presence of 20 pmoles of beta-lactoglobulin, the control protein. - What? No amino-terminal sequence corresponding to a urate oxidase sequence was detected (the amino-terminal sequence of the control protein was detected, therefore the sequencer works). - What? The urea oxidase of A. flavus therefore has the amino-terminal end blocked. (b) determination of the sequence of an amino-terminal peptide of 32 amino acids and the mass of the blocking amino-terminal group:

Method: Digestion by cyanogen bromide

The purified extractive urate oxidase is subjected to a filtration gel on a gel obtained by cross-linking dextran with epichlorohydrin, Sephadex G25 (PD10 - Pharmacia), balanced with a solution containing 7% formic acid, which allows the removal of salts and the change of buffer. By vacuum centrifugation, the formic acid concentration is increased to 70%. Cyanogen bromide is then added at final 0,2 M and left to react for 20 h under argon, in the absence of light, at room temperature.

- Chromatographic separation of ion exchange peptides from the digestion of protein with cyanogen bromide

The peptides were separated on an ion exchange column based on hydrophilic mono S resin (Pharmacia). The test chemical is used to determine the concentration of ammonium acetate in the test medium. The test chemical is used to determine the concentration of ammonium acetate in the test medium. Flow rate: 0.6 ml/min, peak detection by optical density measurement at 278 nm Gradients: from 0 to 100% B in 30 min - collection of 1 ml fractions.

The fractions from the ion exchange step were analysed by PAGE/SDS gel using the method described by Schagger and Von Jagow (1987) Anal. Biochem 166 - p. 368-379.

- Purification of the amino-terminal peptide by reverse phase HPLC and analysis by mass spectrometry of the latter

The peptide from the ion exchange step with a molar mass of about 4 000 Da (on PAGE/SDS gel) was purified on a C18 reverse phase HPLC column based on grafted silica, Beckman Altex C18 column (250 x 2.1 mm) Flow rate: 0.3 ml/min, peak detection by optical density measurement at 218 nm The following shall be used for the test: The following information is provided for the purpose of the analysis: Gradient of 1 to 50% of B in 60 minutes.

The peptide collected after a first step of reverse phase HPLC was repurified on the same reverse phase HPLC column but with a different gradient. Gradient of 1 to 50% of B in 10 minutes. The peak collected was analysed by rapid atom bomb mass spectrometry (FAB/MS) with a glycerol + thioglycerol matrix.

- Digestion by chymotrypsin of the amino-terminal peptide and amino acid analysis of the chymotryptic peptides separated by reverse phase HPLC

In order to establish the sequence of the reverse phase HPLC purified peptide, it was digested by chymotrypsin. Flow rate 0,3 ml/min, peak detection by optical density measurement at 218 nm, The following shall be used for the calculation of the maximum level of the buffer: The following table shows the results of the analysis: Gradient from 1% B to 50% B in 60 min - peak collection.

The chemotryptic peptides were identified by amino acid analysis on Applied Biosystem Analyzer (model 420-130A).

Results:

The results presented below, obtained after the determination of the cDNA sequence of A. flavus urate oxidase and the derived amino acid sequence (see example 6), can only be understood in the light of these. Analysis by mass spectrometry of the amino-terminal peptide.

A difference of about 42 atomic mass units is observed between the two molecular masses determined by mass spectrometry, 3684 and 3666, and the theoretical molecular masses determined from the following sequence (amino acid sequence derived from the cDNA of the urate oxidase of A. flavus with cleavage of the amino-terminal methionine group and peptide cut by cyanogen bromide after the first methionine residue): with a carboxyterminal methionine residue modified by the action of cyanogen bromide into either homosine, 3642, or homosine, 3624.

There is therefore a blocking group on the amino-terminal serine which confers an additional mass of about 42 atomic mass units, probably corresponding to an acetylation of the latter (mass of CH3CO-mass H = 42 atomic mass units). Amino acid analysis of chymotryptide peptides:

This analysis has shown unequivocally that the sequence of the amino-terminal peptide obtained by digestion with cyanogen bromide includes the sequence (1) explained above.

The complete amino acid sequence of urate oxidase is given below:

EXAMPLE 5: Screening of bacteria 1) Preparation of the marked probes

Two pools of probes derived from amino acid sequences of the protein were synthesized using a Biosearch 4600 DNA synthesizer. The first pool corresponds to the His-Tyr-Phe-Glu-Ile-Asp residue sequence (part of the T27 sequence), i.e. 5′ to 3′: This pool is actually made up of 24 x 3 = 48 different oligonucleotides representing all possible combinations. The second pool corresponds to the sequence of amino acid residues Gln-Phe-Trp-Gly-Phe-Leu (part of the V5 sequence), i.e. 5′ to 3′: This pool consists of 24 x 4 = 64 combinations. The probes are marked with the terminal deoxynucleotide transferase (TdT) (marketed by IBI, Inc.).

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2) Hybridisation and detection of the cDNA-containing colonies of urate oxidase:

Approximately 40,000 colonies are screened by the in situ hybridization technique developed by Grunstein and Hogness (1975, U.S.A., 72 3961). Approximately 6,000 bacteria are spread on Petri dishes to produce 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 probe pools, so that all the resulting colonies are tested with the 2 probe pools in parallel.

The filters are then hybridized with one of two pools of probes in a buffer containing 6 x SSC, 10 x Denhardt's and 100 μg/ml of sonicated and denatured salmon sperm DNA (SIGMA). The hybridization temperature is 42°C and the duration is 16 h. The 6 x SSC solution is obtained by dilution of a 20 x SSC solution. The preparation of the 20 x SSC buffer is described in Maniatis, Fritsch and Sambrook (op. cit.).

After washing in 6 x SSC solution at 42°C (3 h with 5 bath changes), the filters are wiped with Joseph paper and placed on autoradiography.

Five of these colonies were taken and purified, and the plasmid DNA from each of these colonies was prepared and this DNA was analysed by digestion with either BamHI or HindIII or both BamHI and HindIII.

After analysis on agarose gel, it was found that the 5 plasmids obtained were linearized by BamHI and HindIII. The double digestion allows the release of a fragment corresponding to the entire cloned cDNA. The size of this fragment is about 1.2 Kb in 3 cases, about 0.9 Kb in the other 2 cases. For the determination below one of the 0.9 Kb fragments and one of the 1.2 Kb fragments that were recloned were selected (see example 6 below).

EXAMPLE 6 Determination of the DNA sequence of urate oxidase

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The nucleotide sequence of clone 9C is shown in Figure 3 which also indicates by an arrow the beginning of clone 9A and by a nucleotide symbol with an asterisk * the nucleotides sequenced from clone 9A which are not identical to those from clone 9C (when matching the two sequences and the AccI and BamHI restriction sites used in subsequent constructions (see example 10).

It is noted that: The nucleotide sequence of the longest fragment (clone 9C) overlaps that of the shortest fragment (clone 9A), with two differences (see Figure 3). One of the differences is silent, the other corresponds to a change from a tryptophan residue to a glycine residue. These differences may be due either to differences in the isolated messenger RNAs (see example 2 above) or to errors in the reverse transcriptase used in the constitution of the cDNA bank (see example 3 above).

In the case of the longest fragment, an ATG codon (at position 109 in Figure 3) opens an open phase corresponding to a 302-amino acid polypeptide with a molecular weight of approximately 34240 Da, the sequence of which corresponds to the partial sequence of purified A. flavus urate oxidase (see example 4).

The DNA sequence opened by the ATG codon and the coded polypeptide, and the arrows in front of the coded polypeptide, represent the sequence of the peptides (see example 4) obtained by hydrolysis of A. flavus urate oxidase with trypsin and protease V8.

The polypeptide sequence is found to end in the Ser-Lys-Leu triplet, typical of peroxisomally localized enzymes (Gould S.J. et al, J. Cell, Biology 108 (1989) 1657-1664).

EXAMPLE 7: Construction of a DNA expression vector from urate oxidase

Err1:Expecting ',' delimiter: line 1 column 315 (char 314)

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

The structure of the p466 plasmid will now be described in more detail in the following paper, which will refer to Figures 5, 6, 7, 8, 9.

Figure 5 shows a restriction map of the plasmid p163.1 The different restriction segments are arbitrarily marked with the following caption: = DNA segment from the plasmid pBR322 = Location of origin of replication (ORI) = DNA segment containing the sequence coding for a natural precursor of hGH = DNA segment of the fd phage containing a transcription terminator = DNA segment containing a tryptophan-lactose UV5 hybrid promoter-operator = DNA segment coding for β-lactamase (ApR: resistance to ampicillin)

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

Figure 7 represents the restriction map of the plasmid p373.2 The different restriction segments are arbitrarily marked with the following caption: = PvuI-BamHI sequence from the plasmid pBR327 = PvuI-XhoI sequence from the plasmid p163,1 = XhoI-HincII sequence from the plasmid p163,1 Fragment 4 described below = Fragment 3 described below = DNA segment of the fd phage 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 the p466 plasmid, the NdeI-KpnI fragment containing the urea oxidase gene being represented by:

1) Construction of the plasmid p373.2

The strategy used is to use fragments obtained from pre-existing plasmids available to the public and fragments prepared by synthesis using the techniques now commonly used.The cloning techniques used are those described by T. MANIATIS EF, FRITSCH and J. SAMBROOK, Cold Spring Harbor Laboratory (1982).Oligonucleotide synthesis is carried out using a Biosearch 4600 DNA synthesizer.

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

The plasmid pBR327, well known to the public, was also subjected to digestion by the PvuI and BamHI enzymes (see SOBERON, X et al., Gene, 9 (1980) 287-305). Hereafter fragment 2 - containing the replication source has been purified.

Then we prepared fragment 3, which is a synthetic BamHI fragment containing the lac i gene and its promoter, the sequence of which is as follows, where the two ends of the strand are marked with the numbers 1 and 2 to specify the orientation of the fragment in the plasmids described in Figures 6 and 7.

Fragment 3

Fragments 1, 2 and 3 were then linked to produce the plasmid p160 shown in Figure 6.

This plasmid was partially digested by the restriction enzymes HincII and PstI. The large fragment HincII-PstI, containing the replication origin and shown in Figure 6, was then bound to fragment 4, shown below, which is a synthetic DNA fragment containing a sequence coding for the first 44 amino acids of a natural HGH precursor and upstream of this sequence of regulatory signals.

Fragment 4

In this fragment, the amino acids are denoted by letters according to the following code: - What?

A = Alanine	M = Méthionine
C = Cystéine	N = Asparagine
D = Acide aspartique	P = Proline
E = Acide glutamique	Q = Glutamine
F = Phénylalanine	R = Arginine
G = Glycine	S = Sérine
H = Histidine	T = Thréonine
I = Isoleucine	V = Valine
K = Lysine	W = Tryptophane
L = Leucine	Y = Tyrosine

The sequences -35 (TTGCTT) and -10 (TATAAT) of the promoter sequence, and the sequence of Shine and Dalgarno well known to the man of art, are successively highlighted in this fragment.

The plasmid p380,1 was thus obtained.

The plasmid p380,1 was then digested by the ClaI and NdeI restriction enzymes to remove the small ClaI-NdeI fragment from the above fragment 4 and replace it with the ClaI-NdeI fragment as follows:

The resulting plasmid is the plasmid p373,2 (Figure 7).

2) Construction of the p466 plasmid:

The plasmid p373,2 was subjected to double digestion by the enzymes BglII and HindIII. The large fragment resulting from this digestion was purified and bound with a synthetic DNA fragment, the sequence of which is given below to reconstitute the end of the hGH gene followed by 3′ of the KpnI and SnaBI cloning sites.

This fragment comprises the cohesive ends BglII and HindIII. The new plasmid thus formed p462 (see Fig. 8) thus includes a KpnI site and a NdeI site which will be used to clone the fragment carrying the urate oxidase cDNA in the expression vector.

The pTZ19R-derived hybrid plasmid with cDNA of about 1.2 Kb (clone 9C), urate oxidase (see example 3) contains a single KpnI site, located a few base pairs downstream of the cDNA cloning site.

The resulting synthetic fragment has one NdeI and one AccI end. The fragment and the synthetic sequence were linked to the expression vector cut by KpnI and by III. This three-part linkage produced the expression vector p466, the Ndeurate oxidase for E. (Fig. 9). This plasmid was subjected to a series of enzymatic hydrolysis by restriction enzymes, which allowed the presence of specific sites for the detection of the gene that carries the restriction, particularly those encoded by the Ndeurate coliase.

The plasmid p466 therefore contains by construction a gene coding for urea oxidase of the following sequence: (Different nucleotides from the nucleotides of the cDNA isolated from A. flavus are highlighted in the sequence above. These differences have been introduced onto the synthetic fragment AccI-KpnI so as to have a nucleotide sequence downstream of the ATG more in line with those usually found in a prokaryotic gene).

EXAMPLE 8 DNA expression of urate oxidase

The E. coli K12 RR1 (Bethesda research lab. Inc.) strain was modified for ampicillin resistance with the plasmid p466 and a negative control plasmid pBR322. Ampicillin-resistant colonies were obtained in both cases. 1 colony of each type was cultured in the medium (LB + ampicillin 100 μg/ml). After a night at 37°C under agitation, both cultures were diluted 100 times in the medium (LB + ampicillin 100 μg/ml). After 1 hour of culture, 1 mM of IPTG (Isopropyl β-DThiogalactoside) is added for 3 hours. Immunodetection of urate oxidase by Western blot:

1) Mode of operation:

The culture medium obtained after 3 h of induction in the IPTG is taken with an aliquot fraction corresponding to 0.2 ml at DO = 1. Solubilize the coating by boiling for 10 min in a buffer called a load buffer consisting of Tris HCl 0,125 M pH 6,8 SDS 4%, bromophenol blue 0,002%, glycerol 20%, β-mercaptoethanol 10% (according to the protocol described by LAEMMLI (U.K. LAEMMLI, Nature, 227 (1970),680-685),electrophoretic separation of the various proteins contained in the solubilisate according to the protocol described by LAEMMLI (U.K. LAEMMLI, Nature, 227 (1970), 680-685),transfer of the said proteins contained in the gel onto a nitrocellulose filter (according to the technique of H. TOWBIN et al. Natl. Acad. Sci. USA 76 (1979) 4350-4354), Immunodetection, carried out according to the technique of BURNETTE (W.W. BURNETTE Ana. Biochem. 112 (1981) 195-203), involves successively: . Rinse the nitrocellulose filter for 10 min with a buffer A (Tris-HCl 10 mM, NaCl 170 mM, KCl 1 mM).. Contacting the nitrocellulose filter for 1 hour at 37°C with an immunoserum (polyclonal antibodies that recognize the urate oxidase of A. flavus).. Rinsing the nitrocellulose filter with the B-pad.. Contacting the nitrocellulose filter for 1 hour at 37°C with a G-protein solution labeled at 125 μc/ml iodine.. Rinsing the filter with the A-pad.. Drying the filter between two absorbent sheets.. Contacting an X-ray film.. Revealing the film.

2) Results:

The strain transformed by the p466 plasmid is found to overproduce a protein of apparent molecular weight of approximately 33 KDa which is recognized by antibodies directed against A. flavus urate oxidase and which is absent from the control strain.

EXAMPLE 9 - Dosage of urate oxidase activity

The culture medium obtained after 3 h of induction of IPTG under the culture conditions described in the previous example is taken with an aliquot fraction corresponding to the equivalent of 0.5 ml at DO = 1. The latter is centrifuged and the surfactant is removed. The cells are collected in 1 ml of TEA (Triethanolamine) buffer 0.05 M pH 8.9. The cell suspension is sonicated twice for 30 s in the ice with an ultrasonic sonicator W10 (regulated at 8 power and intensity 4). The extracts are centrifuged at 10 000 g for 10 min. The surfactants are used for dosing.

The above operations are performed for four randomly selected colonies of E. coli K12 transformed by the plasmid p466 (colonies A1, B1, C1 and D1) and one colony transformed by the plasmid pBR322).

The first principle

The transformation of uric acid into allantoin is followed by the decrease in absorbance at 292 nm. The reaction is as follows: - What?

2) Reagents

(a) TEA buffer 0,05 M pH 8,9/EDTA is used Dissolve 7.5 g of TEA (analytical reagent - Prolabo reference 287.46.266) in 400 ml of distilled water,dissolve 0.372 g of Complexon III (Merck reference 8418) in 50 ml of distilled water,mix the two solutions and make up to 500 ml (solution 1),adjust the pH of this solution to 8.9 by HCl 0.2N,make up to 1 000 ml with distilled water (solution 2).b) Uric acid Stock solution Dissolve 100 mg of uric acid (Carbiochem reference 6671) in 50 ml of solution 1, adjust by HCl 0,2N to pH 8,9, complete to 100 ml with distilled water. The resulting solution may be stored for one week at 4°C. (c) Uric acid Solution Substrate

Take 1.5 ml of stock solution of uric acid (carbiochem reference 6671) and dilute to 100 ml with TEA buffer/reagent for analysis-Prolabo reference 287.46.266).

This solution should be used during the day.

3) Method of operation

The following volumes are introduced into the quartz tank of a spectrophotometer set at 292 nm and thermostated at 30 °C: 600 μl of uric acid substrate solution (preheated at 30°C),100 μl of the above supernatants to which 200 μl of TEA pH 8.9 (preheated at 30°C) have been added.

The change in optical density is mixed and read every 30 seconds for 5 minutes.

4) Results:

The enzyme urate oxidase activity A expressed in U/ml DO 1 is calculated from ΔE measurement using the formula: $\text{A =}\frac{\text{ΔE x Vr x d}}{{\text{I x V}}_{\text{PE}}}$ where the symbols Vr, d, I and VPE represent the reaction volume (0,9 ml), the dilution rate (2), the uric acid extinction coefficient at 292 nm (12,5) and the test volume (0,3 ml) respectively.

The results obtained are summarised in Table II below: TABLEAU (II)

Souche d'E. coli K12 transformée par		Activité urate oxydase (U/ml DO 1)
pBR322		< 0,001
p466		0,086
		0,119
		0,135
		0,118

It is clear from the above table that E. coli cells transformed by the plasmid p466 are capable of producing urate oxidase activity in the presence of IPTG.

EXAMPLE 10 Construction of three vectors for the expression of urate oxidase cDNA in yeast, the plasmids pEMR469, pEMR473 and pEMR515.

Err1:Expecting ',' delimiter: line 1 column 372 (char 371)

Err1:Expecting ',' delimiter: line 1 column 425 (char 424)

1) Construction of the pEMR469 plasmid:

This plasmid was constructed from the E. coli yeast pEMR414 shuttle vector, constructed by successive ligations of the following elements: the PstI-HindIIIo fragment - symbolised by in Figure 10 - of the plasmid pJDB207 (BEGGS, 1978: Gene cloning in yeast-p. 175-203 in: Genetic Engineering vol 2 - WILLIAMSON - Academic Press - London UK) comprising the upstream portion of the ampicillin resistance gene AmpR of pBR322 (Sutcliffe 1979 Cold Spring Symp. Quart. Biol. 43, 779) and a 2 μ endogenous B-form fragment containing the LEU2 gene of S. cerevisiae partially derived from its promoter (called LEU2d),The HindIII-SmaI fragment - represented by the yeast chromosome V fragment containing the URA3 gene with its promoter (ROSE et al., 1984, Gene, 29, p. 113-124). This HindIII-SmaI fragment is derived from the plasmid pFL1 (CHEVALLIER et al., 1980, Gene 11, 11-19)). The HindIII-SmaI fragment of this plasmid was made free by the action of the Kleenow polymerase.a SamI-BamhI fragment - represented by in Figure 10 - containing a synthetic version of the ADH2 gene promoter which differs only from the natural version described by RUSSEL and SMITH (RUSSEL et al., 1983). J. Biol. Chem. 258, 2674-2682) by a few base pairs for introducing restriction sites. (The natural sequence could be used with slightly different results). The sequence of this fragment is given as follows: the BgIII-HindIII fragment - represented by in Figure 10 - bearing the 3′ end of the yeast PGK gene. This fragment is obtained by complete digestion by BgIIIIII of the yeast DNA chromosomal fragment,The PGK gene described by HITZEMAN et al. (1982 Nucleic Acids Res., 10, 7791-7808) which has only one BgIII site. This digestion results in two HindIII-BgIII fragments, the smallest of which, about 0.4 Kb, which carries the 3′ end of the yeast PGK gene, being retained. The sequence of this latter fragment is described by HITZEMANN et al. (reference cited above). The BgIII site is cloned in the BamHI site of the previous fragment (thus BamHI and BgIII sites disappear) and the HindIII site, which is rendered free by action of Kleenow polymerases, is cloned in the PIIP site of the PBR22 fragment of the PBR22 fragment as described below.the PvuII-PstI fragment - symbolised by in Figure 10 - of pBR322 containing the replication origin and the downstream part of the ampicillin resistance gene AmpR.

The resulting pEMR414 plasmid therefore has the following components: a replication origin and an ampicillin resistance gene AmpR allowing replication and selection of the plasmid in E. coli cells. These elements allow transformation in E. coli cells. a replication origin for yeast (ARS), the STB locus and the LEU2 gene of S. cerevisiae without a promoter and the URA3 gene with its promoter from S. cerevisiae. These elements allow replication and selection of the plasmid in S. cerevisiae cells and sufficient partition efficiency in cells containing the endogenous 2μ plasmid.

The plasmid pEMR414 was completely digested by the NheI and ClaI restriction enzymes. The small NheI-ClaI fragment containing the URA3 gene, hereinafter referred to as fragment A, was purified.

The plasmid pEMR414 was completely digested by the enzymes NheI and BamHI. The large NheI-BamHI fragment containing in particular the LEU2d gene and the replication origin of the plasmid pBR322, hereinafter referred to as fragment B, was purified.

The synthetic ClaI-AccI fragment containing the beginning of a gene coding for the protein derived from the urate oxidase cDNA sequence (clone 9C) was prepared, which contains modifications from clone 9C, introduced with the aim of establishing the usual codons in yeast (see SHARP et al., 1986, Nucl. Ac. Res. Vol. 14, 13, pp. 5125-5143) without changing the encoded amino acids.

The plasmid from the 9C clone (see Figure 3) was subjected to digestion by the enzymes AccI and BamHI. The AccI-BamHI fragment containing the end of the urea oxidase cDNA, hereinafter referred to as fragment D, was purified. This fragment has the following sequence:

The fragments A, B, C and D were linked to produce the plasmid pEMR469, as shown in Figure 11, in which the symbols have the same meaning as in Figure 10, with the new fragments ClaI-AccI and AccI-BamHI being represented by

2) Construction of the plasmid pEMR473:

The large MluI-SphI fragment containing the urate oxidase gene was then linked to the synthetic fragment, sequence given below, corresponding to a portion (200 pb) of the upstream sequence of the TATA element of the GAL 7 promoter of S. cerevisiae, which includes upstream activation sequences (UAS).

The resulting pEMR473 plasmid is shown in Figure 12, where the symbols have the same meaning as in Figure 11, with the newly introduced MluI-SphI fragment represented by

3) Construction of the plasmid pEMR515:

The large XbaI-MluI fragment was purified, containing the sequences of the replication origin and the 2μ STB locus, the LEU2d gene, the ampicillin resistance gene AmpR, the replication origin of pBR322, and the urate oxidase expression cassette. It does not contain the URA3 gene, nor the 2μ part between the sites of UBAI and NheI X.

The large XbaI-MluI fragment was recirculated via the following sequence adapter with modified XbaI and MluI cohesive ends:

The resulting plasmid pEMR515 has only one of the three elements of the target FRT site of the recombinant encoded by the 2μ FLP gene.

The plasmids pEMR 469, pEMR 473 and pEMR 515 have the following urate oxidase gene sequence:

EXAMPLE 11 - Transformation of the yeast strain EMY761 by the plasmids pEMR469, pEMR473 and pEMR515 - Transformation of the yeast strains EMY500 and GRF18 by the plasmid pEMR515 - Transformation by selection for either uracil or leucine prototrophy

Three non-isogenic Saccharomyces cerevisiae strains were used as receptor strains: The following substances are to be used in the manufacture of the test chemical:

The GRF18 strain is well known to the public (Gerry FINK, MIT, USA). The EMY761 and EMY500 strains are related to the GRF18 strain. They were obtained by successive crosses of the GRF18 strain with a ura3 strain derived from the FL100 strain (filled with the ATCC under No. 28383) and with the 20B12 (Mata, tspl, pep4) strain described by E.W. JONES (E.W. JONES et al. (1977) Genetics, 85, 23).

The GRF18 strain can be obtained by curing the pEMR515 plasmid of the GRF18 pEMR515 (light+) strain, which was registered with the CNCM under reference No I-920 on 28 December 1989, and the EMY500 strain by curing the pEMR515 plasmid of the EMY500 pEMR515 (light+) strain, which was registered with the CNCM under reference No I-919 on 28 December 1989.

These strains contain mutations (Leu2 and ura3), which may be complemented by the defective selection marker LEU2d and the selection marker URA3, present in each of the pEMR469 and pEMR473 plasmids.

1) Selective transformation for the prototrophy of the uracil:

A colony of the EMY761 strain was used to inseminate 100 ml of a medium called liquid YPG medium (see Table III below). At a cell density of 107 cells per ml, the cells were treated with 0.2 M lithium acetate for processing using a well-known technique described by ITO et al. (ITO et al., 1983, J. Bacteriology 153, 163-168).

The EMY761 cells were transformed in parallel with approximately 1 μg of each of the plasmids pEMR469 and pEMR473 The transformed cells were selected for uracil (ura+) auxotrophy on a medium called a uracil-free solid medium (see Table III below) resulting in a transformed EMY761 pEMR469 (ura+) and a transformed EMY761 pEMR473 (ura+).

2) Selective transformation for the prototrophy of leucine:

The transformation technique used is a variant of that described by Beggs et al. (Beggs et al. (1978), Nature 275, 104-109).

The precise processing protocol is specified below: (a) 200 ml of liquid YPG medium (see Table III) are inoculated with approximately 5 x 106 cells of a stationary culture and the inoculated culture is placed overnight under agitation at 30°C. (b) When the culture reaches approximately 107 cells per ml, the cells are centrifuged at 4000 rpm for 5 min and the coating is washed with sorbitol 1 M. (c) The cells are suspended in 5 ml of sorbitol 1 M solution containing 25 mM EDTA and 50 mM dithiothretol and incubated for 10 min at 30°C.Zymolyase-100T (a preparation obtained by partial purification on an affinity column of the culture surfactant Arthobacter luteus and containing β-1,3-glucan-laminaripentahydrolase, marketed by SEYKAGAKU KOGYO Co. Ltd) is added to a final concentration of 20 μg/ml and the suspension is incubated at room temperature for approximately 15 min.e) The cells are resuspended in 20 ml of a sorbitol-containing medium called YPG sorbitol (see Table III below) and incubated for 20 min. at 30°C.f. under gentle agitation for 500 g/min.e) for 2 to 2 hours.(h) Add 0.1 ml of cells and 5 μl of DNA solution (approximately 5 μg) and leave the resulting suspension for 10 to 15 min at room temperature. (i) Add 1 ml of the solution: PEG 4000 glycol 20%, tris-HCl 7.5 10 mM and CaCl2 10 mM. (j) Pour 0.1 ml of the resulting suspension in (i) into a tube containing the leucine-free regenerating solid medium (see Table III below) which has been previously melted and kept liquid at approximately 45°C.Pour the suspension onto a petri dish containing a 15 ml solidified layer of leucine-free solid regenerative medium. (k) Repeat step j) with the remainder of the cell suspension obtained in i.

The transformed ones start to appear after three days.

The transformed EMY761 pEMR469 (light +), EMY761 pEMR473 (light +), EMY761 pEMR515 (light +), GRF18 pEMR515 (light +) and EMY500 pEMR515 (light +) were therefore selected.

EXAMPLE 12 - Expression of urate oxidase cDNA in the urine by the strains EMY761 pEMR469 (ura+), EMY761 pEMR473 (ura+), EMY761 pEMR469 (leak+) and EMY761 pEMR473 (leak+) - Immunodetection by Western Blot, measurement of urate oxidase activity and soluble proteins 1) DNA expression of urate oxidase: (a) Strains selected from a medium without uracil

A colony of each of EMY761 pEMR469 (ura+) and EMY761 pEMR473 (ura+) strains was cultured in 20 ml of a liquid medium without uracil (see Table III, example 11). After a night at 30°C under agitation, both cultures were centrifuged for 10 min at 7000 rpm. The cultures were recovered in 10 ml of sterile distilled water and re-centrifuged for 10 min at 7000 rpm. The expression of urate oxidase was induced by recovering the cells in 20 ml of YP ethanol-glycerol (see Table III, example 11) The cultures for EMY761 pEMR469 (ura+) and 20 ml of egalactol-pEMR470 (ura+) were recovered during the agitation (see Table III, example 11) at 30°C.

(b) Strains selected on leucine-free medium

Initially, a colony of each of the EMY761 pEMR469 (leukine+) and EMY761 pEMR473 (leukine+) strains was cultured in 20 ml of leucine-free liquid medium (see Table III, example 11). This allowed a high number of plasmid copies to be obtained and maintained by selection for Leu2 mutation complementation by the LEU2d gene carried by the pEMR469 and pEMR473 plasmids.

After an overnight agitation at 30°C, both cultures were centrifuged for 10 min at 7 000 rpm. The cells were then taken back into 10 ml of sterile distilled water and centrifuged again for 10 min at 7 000 rpm. Uratine oxidase expression was induced by taking back the cells into 20 ml of YP ethanol-glycerol medium for EMY761 pEMR469 (light) and 20 ml of YP ethanol-glycerol-galactose medium (see III, example 11) for EMY761 pEMR473 (light). The cultures were replaced at 30°C agitation for 22 h.

(c) Control strain

The unprocessed, i.e. plasmid-free strain EMY761 was cultured as above, induced in 10 ml of YP ethanol-glycerol-galactose liquid medium and induced in 10 ml of YP ethanol-glycerol-galactose medium.

2) Sample preparation:

(a) The cells cultured in 1a, 1b and 1c) were centrifuged and the surfactant removed. The cells were collected in 10 ml of distilled water and centrifuged for 10 min at 7000 rpm. The cells thus washed were collected in about 1 ml of pH 8.9 TEA triethanolamine buffer. About 300 μl of the cells thus collected were liquefied in the presence of glass balls (diameter 400 to 500 μm) representing about half the final volume. This mixture was vigorously vorticized for 1 min, 4 times, with the samples placed in the ice for 30 s each time.The liquid was removed from the tubes with a pasteur pipette and transferred into a microtube. The glass beads were washed 1 time with about 200 μl of pH 8.9 TEA buffer. The beads were vorticized for 1 min, 1 time, and the liquid was removed from the pasteur pipette to be added to the previous lysate. The lysate was then centrifuged in a microtube for 5 min at 7000 rpm. The supernatant was carefully removed and stored at -20°C for the Western Blot, measuring urate oxidase activity and protein dosing.The lysed cell culture was stored separately at -20°C for the Western Blot (see 3 below).

In addition, culture samples were taken in 1a) and 1b) as follows before induction: 2 ml of culture were centrifuged for 10 min at 7000 rpm. The cocoons were collected in 500 μl of distilled water and centrifuged again for 5 min at 7000 rpm. The cocoons were collected in about 200 μl of pH 8.9 TEA buffer and lysed as above in the presence of glass beads. The overswimmers and the cocoons of the lysed cells were kept at -20°C separately.

3) Immunodeception of urate oxidase by Western Blot: (a) Method of operation

The cocoons and overflows of the various samples were subjected to a Western Blot, a technique well known to the art world, which consists of the following steps: Solubilization of the collet by boiling for 10 min in a buffer called a charge buffer consisting of tris-HCl 0,125 M pH 6,8 SDS 4%, bromophenol blue 0,002% glycerol 20%, β-mercaptoethanol 10% (according to the protocol described by LAEMMLI (U.K. LAEMMLI, Nature, 227 (1970), 680-685)), (step implemented for collets only), electrophoresis separation of the various proteins contained in the solubilisate according to the protocol described by LAEMMLI (U.K. LAEMMLI, Nature, 227 (1970), 680-685), transfer of proteins contained in a nitrocellulose gel (USA Procellon, Nature, H.W. and Acad. SciB. 7650-44), and 43-450 (USA Procellon, H.W. and Acad. 4350-449),

Immunodetection, carried out according to the BURNETTE technique (W.W. BURNETTE Ana. Biochem. 112 (1981) 195-203), involves successively: rinsing the nitrocellulose filter for 10 min with a tampon A (tris-HCl 10 mM, NaCl 170 mM, Kl 1 mM). Contacting the nitrocellulose filter for 30 min at 37°C with a tampon B (ampoule A with added bovine serum albumin at a ratio of 3 g per 100 ml). Contacting the nitrocellulose filter for 1 ml at 37°C with a tampon immuno-sampler (polyclonal antibodies recognizing A. flavus). Rinsing the nitrocellulose filter with the tampon B. During the rinsing the nitrocellulose film is in contact with a film of absorbent proteins at a ratio of 1 g/m30. The film is in contact with a film of absorbent oxygen at a temperature of 125°C. The filter is then rinsed with a film of 0.1 g/m30.

(b) Results

The strains EMY761 pEMR469 (ura+), EMY761 pEMR473 (ura+), EMY761 pEMR469 (light+) and EMY761 pEMR473 (light+) are found to produce a protein of apparent molecular weight of approximately 33 KDa which is recognised by antibodies directed against A. flavus urate oxidase and which is absent from the control strain.

It is also found that non-induced strains do not produce or very little of the protein described above.

Comparison of the amounts of this protein for coelacanths and superswimmers shows that about 80% of this protein is in lysate-soluble form.

4) Dose of urate oxidase activity:

The urate oxidase activity was measured on the lysed cell supernatants using the procedure described in example 9 above.

The results obtained are summarised in Table IV below, which gives the urate oxidase activity in U/ml for each glycerol-ethanol-induced, glycerol-ethanol-galactose-induced or non-induced strain. TABLEAU IV

Souche / Inducteur	Activité urate oxydase (U/ml)
EMY761/YP éthanol-glycérol-galactose	< 0,1
EMY761/YP éthanol-glycérol	< 0,1
	0,4
	12
	0,17
	36
	< 0,1
	12,5
	< 0,1
	15,3

It is clear from the above table that yeast cells transformed by these plasmids pEMR469 and pEMR473 are capable of induction-induced urate oxidase activity.

5) Dose of total proteins soluble in lysates:

The BIORAD protein assay kit was used to measure total proteins in lysed cell supernatant, based on the observation that the maximum absorbance for a bright blue acidic solution of Coomassie g-250 increases from 465 nm to 595 nm when proteins are attached (see Reisner et al., Anal Biochem, 64, 509-(1975)).

(a) Method of operation

Err1:Expecting ',' delimiter: line 1 column 212 (char 211)

The optical density is mixed and read at 595 nm. A standard range with increasing concentrations of BSA (Bovine Serum Albumin) is obtained.

(b) Results

The main results obtained are summarized in Table V below, which gives for each glycerol-ethanol-induced, glycerol-ethanol-galactose-induced or non-induced strain the amount (in mg/ml) of total soluble proteins and the percentage of urate oxidase in total soluble proteins (here the specific activity of the recombinant protein is assumed to be identical to that of the urate oxidase obtained from A. flavus: 30 U/mg). - What? TABLEAU V

Souche / Inducteur	Protéines totales solubles mg/ml	% d'urate oxydase dans les protéines totales solubles
EMY761/glycérol-éthanol	5,3	< 0,05
EMY761/glycérol-éthanol-galactose	5,8	< 0,05
	8,5	0,25
	5,3	4,7
EMY761 pEMR69 (leu)/non induit	1,7	0,3
	5,9	20
	10,3	< 0,05
	6,5	6,4
	0,5	< 0,05
	3,9	13

The rate of production of urate oxidase varies from 5 to 20% depending on the processors and the method of selection of the processors (leu+).

EXAMPLE 13 Expression of urate oxidase cDNA in a 2,5 l fermenter for the EMY761 pEMR473 strain (ura+) 1) Fermentation protocol: (a) Middle class Innoculum medium

A colony of EMY761 pEMR473 (ura+) strain was cultured in 200 ml of uracil-free liquid medium (see Table III, example 11).

Crops in the environment

	pour 1 l d'eau purifiée sur un appareil de type Milli-q
glucose	30 g
glycérol	30 g
hydrolysat de caséine (Casamino-acids de DIFCO)	30 g
base azotée de levure (Yeast Nitrogen Base de DIFCO)	15 g
extrait de levure (Yeast extract de DIFCO)	2,5 g
	3 g
	0,5 g

Additional environment B

(b) Parameters of fermentation

Bioreactors with two turbines the temperature = 30°C pH is 5 Oxygen partial pressure = 30 mmHg The air flow rate is 1 l/min.

The bioreactor is filled with 1.5 l of medium A and is seeded with 150 ml of inoculum.

Once the glucose is depleted at DO 2.5 to DO 17, induction is performed by adding a volume of 150 ml of galactose at 20% by weight/volume.

The growth takes about 15 hours and the harvest was carried out at a DO 104.

2) Sample preparation and analysis:

The samples were prepared as described in Example 9 (2) (a) from the fermentation culture and two samples were taken: the first after 7 h of induction, the second after 10 h of induction.

On these two lysates obtained after cell lysis, the following tests described in Example 9 were performed: Western Blood immunoassay of biological activity of total proteins.

The results are as follows.

(a) Immunodetection by Western Blot

Err1:Expecting ',' delimiter: line 1 column 372 (char 371)

(b) Dose of biological activity

The results obtained are summarised in Table VI below: - What? TABLEAU VI

Souche / Temps d'induction	U/ml
	9
	12,5

The EMY761 pEMR473 (ura+) strain, cultured in a fermenter, is found to be capable of producing urate oxidase activity after induction.

(c) Dosage of total soluble proteins

The results are summarised in Table VII below: - What? TABLEAU VII

Souche / Temps d'induction	Protéines totales solubles mg/ml	% d'urate oxydase dans les protéines totales solubles
	5,2	5,7
	6,2	6,6

These results indicate that the rate of urate oxidase synthesis of the EMY761 pEMR473 (ura+) strain cultured in a fermenter is approximately 5% of total cell proteins after 7 h and 21 h of induction.

EXAMPLE 14: Expression of urate oxidase cDNA in the urine by the EMY761 pEMR515 (leucine +), EMY500 pEMR515 (leucine +) and GRF18 pEMR515 (leucine +) strains

A colony of each of the above three strains was cultured in 20 ml of leucine-free liquid medium.

After a night at 30°C agitation, the three cultures were centrifuged for 10 min at 7000 rpm. The cell cultures were collected in 10 ml of sterile distilled water and centrifuged again for 10 min. The expression of urate oxidase was induced by collecting the cells in 20 ml of YP ethanol-glycerol-galactose medium (see Table I, example 8). The cultures were re-substituted at 30°C agitation for about 20 h. As a control, a culture of each unprocessed host strain was performed.

The cells of each of the six cultures were recut by centrifugation and the surfactant removed. The cells were collected in 10 ml of distilled water and centrifuged for 10 min at 7000 rpm. The cells washed were collected in about 1 ml of pH 8.9 TEA buffer and the milling and removal of particles by centrifugation were carried out as described in example 9.2. The surfactant from each culture is used as before for a dosage of the oxidase sump and total proteins. The main results obtained are summarized in Table VIII below: TABLEAU VIII

Souche/Conditions de culture	Activité urate oxydase (U/ml)	Protéines totales solubles (mg/ml)	% d'urate oxydase dans les protéines solubles
	< 0,1	2,2	< 0,05
	< 0,1	0,9	< 0,05
	< 0,1	1,8	< 0,05
	38	5,4	23 %
	20	2,5	26 %
	33	4,2	26 %

TABLEAU VIII

a) : les souches sont cultivées en présence de glucose (conditions de non-induction)

b) : les souches sont cultivées en absence de glucose et en présence de galactose (induction).

These results show that a high level of urate oxidase expression can be obtained with three non-isogenic receptor strains transformed by the expression vector of the invention.

EXAMPLE 15 Expression of the urate oxidase cDNA in a 2.5 l fermenter for EMY500 pEMR515 strain. 1) 2.5 litre fermenter culture of the EMY500 pEMR515 strain:

The culture of EMY500 pEMR515 strain shall be carried out in a fermenter and shall be carried out as follows:

(a) Preculture phase of the erlenmeyer

From 1 ml of solution in a medium containing 20% glycerol of the EMY500 pEMR515 strain with a cell count corresponding to a DO of 2.35, a 500 ml seed pot containing 90 ml of MCPA autoclave phase growth medium supplemented with 1.28 g of MES (2/N-morpholino/-ethanesulfonic acid: Sigma no. M 8250) and 10 ml of MCPF filtered phase growth medium is sown. The compositions of the MCPA and MCPF media are specified below. After 24 hours of incubation, agitated at 30°C, the optical density of the culture is approximately 7.

(b) Fermentation phases

The above culture is used to sow a 2,5 l fermenter containing the following culture medium:

The pH of the culture is regulated by the fermenter to the recommended value of 5.5 After 6 to 7 hours of culture at 30°C, 72 ml of a 500 g/l glucose solution is added linearly for 9 hours, for a total of 36 g of glucose.

(c) Expression phase

To the mixture described above, add 100 ml of MEPA autoclavable phase expression medium and 150 ml of MEPF filtered phase expression medium (the compositions of which are described below) and continue the culture for 5 h. Then add 150 ml of a solution containing 30 g galactose, 15 g glycerol and 36 g ethanol linearly for 20 h. This results in a D.O. close to 160.

CHEMICAL COMPOSITION of the environment in which they grow and express themselves

- What? - Autoclavable growth medium phase MCPAList of trace elements 1

	pour 1 l d'eau ultrapurifiée
	780 mg
	5 g
	3 g
KI	1 g
	3,5 g
	2 g
	4,8 g

Add 100 ml of concentrated hydrochloric acid to the solution. - Filtered phase growth medium MCPF

	pour 200 ml final d'eau ultrapurifiée
	4,8 g
tryptophane	420 mg
vitamine I (Cf. ci-après)	5 ml
glucose	36 g

Heat to dissolve, temper, add the vitamins I and filter to 0,2 μ. List of vitamins I

	pour 100 ml final d'eau ultrapurifiée
biotine	1,2 mg
acide folique	1 mg
niacine	144 mg
(acide nicotinique pyridoxine. HCl	60 mg
thiamine. HCl	240 mg
pantothénate de calcium	1,2 g
mésoinositol	2,4 g

Make up to 100 ml after reconstitution. Filter cold sterile to 0,2 μ. - Means of expression MEPA autoclavable phase

	pour 100 ml d'eau ultrapurifiée
NTA	1,2 g
	2,08 g
acide glutamique	6 g
HYCASE SF (Sheffield Products)	24 g
leucine	2,16 g
histidine	600 mg
méthionine	1,2 g
	720 g
	840 mg
	108 mg
oligoéléments liste 1	5 ml
uracile	1,2 g

Adjust the pH to 5.5 with concentrated H2SO4 or concentrated KOH. Autoclave for 20 minutes at 120°C. - Medium of expression phase filtered MEPF

	pour 150 ml final d'eau ultrapurifiée
	2,4 g
tryptophane	420 mg
vitamines I	5 ml
glycérol	36 g
galactose	45 g

Heat to dissolve, temper, add the vitamins and filter.

2) Grinding of cells

After 20 hours of induction, the optical density, measured at 600 nm, of the culture is 98.800 g of fermentation must are centrifuged 5 min at 10,000 g and the cell culot is taken up into 80 ml of lysine buffer (glycine 20 mM pH 8.5). The cells are then ground at 4°C, 2 times for 2.5 min in a grinder (Vibrogen Zellmühle V14) in the presence of a volume of 0.5 mm diameter balls, equal to the volume of the lyser cell solution. After grinding, the surfactant is taken up and the balls are washed twice with 80 ml of lysine buffer. 210 ml of total lysine is recovered with a total protein content of about 3 mg/ml and a specific urate activity of about 7.7 U/ml (U/ml) with a percentage of oxytocin activity of about 8.5% (U/ml) compared to the last U of 30 mg/ml.

3) Purification of recombinant urate oxidase (a) Purification protocol

The previous lysate is subjected to the two-stage purification protocol described below.

Step one: The following are the characteristics of the product: Support:

The Commission shall, by means of implementing acts, lay down the rules for the application of this Regulation. The gel, when pressed, has a volume of 70 ml. Separation is carried out at room temperature, the collected fractions are stored at 0°C.

Conditions for separation:

A chloride ionic force gradient is used between buffer 1 (sodium borate 10 mM, pH 9.2) and buffer 2 (sodium borate 10 mM, sodium chloride 1 M). The buffers are degassed and kept at 0°C during the elution. The equivalent of 0.02% azide has been added to each buffer.

The raw extract is deposited (10 ml) and selected with buffer 1 until complete collection of the urea oxidase (in fractions of 10 ml), which is not retained on the column.

The contaminating pigments and proteins are then removed by buffer 2 elution.

The purification is followed by measurement of the optical density of the eluate at 214 nm.

Step two: High pressure liquid chromatography with reverse phase: Support:

The following information is provided for the purpose of the assessment of the use of the product:

Operating conditions:

Eluant 1 is ultrapurified water (passed through a Millipore system) with 0.1% trifluoroacetic acid. Eluant 2 : Acetonitrile (of spectrophotometric quality or equivalent) with 0,08% trifluoroacetic acid. The flow rate: 0,3 ml/min. The gradient is 35 per cent acetonitrile/ TFA to 70 per cent acetonitrile/ TFA in 20 min, maintained at 70 per cent for 5 min.

Collecting fractions:

The separation process is monitored by measuring optical density at 218 nm. The acetonitrile is evaporated during vacuum centrifugation.

(b) Results:

The sample before and after the first purification step was analysed by liquid chromatography on a C8 grafted silica column, Aquapore OD-300 described above with the same gradient, with an injected amount of 50 μl. The purified urate oxidase from A. flavus is used as an external control.

In the starting lysate, urea oxidase accounts for 63% of total proteins.

The entire sample obtained at the end of the second step, which certainly contains more than 84% urea oxidase, was used for the partial characterisation described below.

4) Partial characterization of recombinant urate oxidase. (a) Amino acid analysis

The amino acid analysis of the purified acid hydrolysate of recombinant urate oxidase was performed on an Applied Biosystems model 420-130A analyzer. The amino acid distribution quantified is consistent (no significant difference) with the assumed sequence. The same result was observed for purified extractive urate oxidate of A. flavus (obtained in example 4).

(b) Tryptic peptide map

A tryptic peptide map was established for purified recombinant oxidase urate and purified extractive oxidase urate (obtained in example 4) under the following conditions:

A solution of urea oxidase at a concentration of about 1 mg/ml and a solution of trypsin at a concentration of 1 mg/ml are prepared extemporaneously.

The two solutions are placed in an enzyme/substrate ratio of 1/30 for 8 hours at room temperature. The tryptic hydrolysate is then chromatographed in liquid phase on a C18.5 μm grafted silica column, lichrosorb (250 x 4.6 mm) (Hichrom-ref.RP 18-5-250A), equipped with a 218 UV detector coupled to a recorder. The gradient applied is 1 % acetonitrile/TFA to 60 % acetonitrile/TFA in 120 min, then maintained at 60 % for 5 min.

The resulting peptide maps have a very close profile.

3) Evidence of the blocked nature of the amino-terminal sequence:

The amino-terminal sequence was analysed using an Applied Biosystem Model 470A sequencer coupled with an Applied Biosystem Model 120A phenylethylidantoic derivatives analyser. Purified recombinant oxidase urate (200 pmol controlled by amino acid analysis) was deposited on the sequencer in the presence of 20 pmol of beta-lactoglobulin, the control protein.

No amino-terminal sequence corresponding to a urate oxidase sequence was detected (the amino-terminal sequence of the control protein was detected).

The recombinant urate oxidase therefore has, like the extractive urate oxidase, the amino-terminal end blocked.

Example 16: Construction of a vector for the expression of urate oxidase cDNA in animal cells, plasmid PSV860.

This vector was obtained by: the small AccI-SnaBI fragment containing a sequence coding for urate oxidase except the first 16 amino acids, derived from the plasmid p466 : vector for expression of urate oxidase from A. flavus in E. coli available in the laboratory and described below, to a synthetic HindIII-AccI fragment, resulting in a HindIII-SnaBI fragment containing a complete sequence coding for urate oxidase from A. flavus and a 5′ untranslated sequence favourable for expression in animal cells.Insertion of the HindIII-SnaBI fragment between the sites of HindIII and the cloning polysite (also called polylinker) SnaBI vector for expression in animal cells, the plasmid pSE1.

The following paper will show the construction of the plasmid p466, the construction of the plasmid pSE1 and the assembly of the plasmid pSV860 in succession.

1) Construction of the p466 plasmid

Err1:Expecting ',' delimiter: line 1 column 343 (char 342)

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

The structure of the p466 plasmid has been described in detail in Example 7 above.

2) Construction of an expression vector for animal cells, the pSE1 plasmid

Err1:Expecting ',' delimiter: line 1 column 372 (char 371)

The following description will be better understood by reference to Figure 13, which is a map of the assembly of the pSE1 plasmid, with the sites which have disappeared by ligation being indicated in parentheses.

This plasmid was constructed by successive ligations of the following elements: 1) a PvuII-PvuII fragment - symbolised by in Figure 13 - of 2525 pb, obtained by complete digestion of the plasmid pTZ18R (Pharmacia) with the PvuII restriction enzyme. This fragment contains the replication origin of the F1 phage (noted ORI F1 in Figure 13), a gene (noted AmpR in Figure 13) carrying ampicillin resistance and the replication origin (noted ORI pBR322 in Figure 13) allowing the replication of this plasmid in E. coli. The PvuII free site disappears by binding to the EcoRV free site (which also disappears) of the first fragment described in Figure 7).- A PvuII-HpaI fragment - symbolized by in Figure 13 - of 1060 pb of adenovirus type 5 DNA between positions 11299 (PvuII restriction site) and 10239 (HPAI restriction site) (DEKKER * VAN ORMONDT, Gene 27, 1984, 115-120) containing information for VA-I and VA-II RNAs. The HpaI free site disappears by binding to the PvuII free site (which also disappears) of the fragment described in Figure 3).3) - A PvuII-HindIII fragment - symbolized by in Figure 13 - of 344 pb, of SV40 virus DNA obtained by complete restriction digestion with the PvuII and HindIII release enzymes.Err1:Expecting ',' delimiter: line 1 column 323 (char 322)6) - A BamHI-BcII fragment of 240 pb - represented by Figure 13 - small fragment obtained by complete digestion with the BcII and BamHI enzymes of SV40 virus containing the site of late polyadenylation of SV40 virus (M. FITZGERALD et al. Cell, 24, 1981, 251-260).

3) Construction of the plasmid pSV860

The p466 plasmid (see Figure 9) was completely digested by the enzymes AccI and SnaBI. The small fragment AccI-SnaBI, which contains a DNA sequence coding for urate oxidase except for the first 16 amino-terminal acids, was purified and bound to the synthetic fragment HindIII-AccI of the following sequence:

This binding results in the HindIII-SnaBI fragment, which contains a urate oxidase coding sequence identical to that of clone 9C and a 5′ untranslated sequence favorable for expression in animal cells (KOZAK, M., Nucl. Acids Res., 12, 2, 1984, 857-872).

The HindIII-SnaBI fragment contains the following sequence:

The HindIII-SnaBI fragment was then inserted into the pSE1 vector previously incubated with the enzymes HindIII and SmaI, resulting in the plasmid pSV860 as shown in Figure 14, in which the symbols have the same meaning as in Figure 13, the new HindIII-SnaBI fragment being symbolised by (SnaBI and SmaI sites disappeared by ligation).

Example 17: Transitional expression of urate oxidase cDNA in COS cells.

COS cells are monkey kidney cells expressing the SV40 virus T-antigen (Gluzman, Y. Cell 23, 1981, 175-182). These cells, which allow the replication of vectors containing the origin of SV40 virus DNA replication, are hosts of choice for studying gene expression in animal cells.

1) Transfection of COS cells and transient expression of urate oxidase cDNA.

4.105 COS cells are placed in a 6 cm diameter Petri dish (Corning) in 5 ml of Dulbecco modified Eagle medium (hereinafter referred to as DMEM) containing 0.6 g/l glutamine, 3.7 g/l NaHCO3 and supplemented with 5% GIBCO. After about 16 h of culture at 37°C in an atmosphere containing 5% carbon dioxide, the culture medium is removed by aspiration and the cells are washed with 3 ml of PBS (GIBCO Buffer Salphine buffer) and the following mixture is added: 1 000 μl (DMEM + 10% GIBCO),110 μl diethylaminoethyl dextrane of mean molecular weight 500 000, at a concentration of 2 mg/ml (Pharmacia), 1.1 μl chloroquine 100mM (Sigma) and 3 μg DNA from either plasmid pSV860 or plasmid pSE1 (for control). After 5 hours incubation at 37°C in an atmosphere containing 5% carbon dioxide, the mixture is removed from the cells. 2 ml of PBS buffer containing 10% dimethyl sulfoxide (quality Spectroscopy, Merck) is then added. After 1 min incubation at room temperature, the mixture is removed and the cells are washed twice with the buffer. 5 ml of DMEM supplemented with 2% veetal serum are added.The incubation period is continued for 4 days at 37°C in an atmosphere containing 5% carbon dioxide.

(2) Preparation of samples.

Err1:Expecting ',' delimiter: line 1 column 227 (char 226)

The cells are lysed by sonication (on ice) by pulses of 10 s with a sonicator (the Sonics and Materials Inc. USA Vibra Cell) set to a power of 12 W. The cell lysate is centrifuged for 10 min at 10,000 rpm and the surfactant is recovered for the dosing of the urate oxidase.

3) Dose of urate oxidase activity.

The measurement of urate oxidase activity was performed as described in Example 9.

The results are summarised in the following table: - What?

Cellules COS transfectées par	Activité urate oxydase U/ml
pSV860	0,105
	<0,01

COS cells transfected by the plasmid pSV860 carrying the urate oxidase cDNA are found to express a significant level of urate oxidase activity, whereas no urate oxidase activity is detectable on the control.

Claims (26)

1. Recombinant protein, characterized in that it has a specific urate oxidase activity of at least 16 U/mg and in that it has the following sequence: preceded, optionally, by a methionine.
2. Recombinant protein according to claim 1, characterized in that it has a specific urate oxidase activity of approximately 30 U/mg.
3. Recombinant protein according to one of claims 1 and 2, characterized in that it has, by bidimensional gel analysis, a spot of molecular mass of approximately 33.5 kDa which represents at least 90% of the proteic weight.
4. Recombinant protein according to any one of claims 1 to 3, characterized in that its purity degree, determined by liquid chromatography on a C8 grafted silica column, is higher than 80%.
5. Recombinant protein according to any one of claims 1 to 4, characterized in that it has an isoelectric point around 8.0.
6. Recombinant protein according to any one of claims 1 to 4, characterized in that it carries a blocking group, preferably of molecular mass around 43 units of atomic mass, on the amino-terminal serine.
7. Medicament, characterized in that it contains the recombinant protein according to any one of claims 1 to 6.
8. Recombinant gene, characterized in that it comprises a DNA sequence coding for the protein which has the following sequence:
9. Recombinant gene according to claim 8, characterized in that it permits expression in prokaryotic microorganisms.
10. Recombinant gene according to claim 9, characterized in that said DNA sequence contains the following sequence:
11. Recombinant gene according to claim 8, characterized in that it permits expression in eukaryotic cells.
12. Recombinant gene according to claim 11, characterized in that said DNA sequence contains the following sequence:
13. Recombinant gene according to claim 8, characterized in that it permits expression in animal cells.
14. Recombinant gene according to claim 13, characterized in that said DNA sequence comprises the following sequence: preceded by a 5'-non-translated sequence favoring expression in animal cells.
15. Recombinant gene according to claim 14, characterized in that the 5'-non-translated sequence favoring expression in animal cells comprises the sequence AGCTTGCCGCCACT, located immediately upstream from the sequence described in claim 14.
16. Expression vector, characterized in that it carries a recombinant gene according to any one of claims 8 to 15 with the means necessary for its expression.
17. Expression vector according to claim 16, characterized in that it carries at least one selection marker.
18. Expression vector according to claim 17, characterized in that it has the characteristics of one of plasmids pEMR469, pEMR473, described in Figures 11 and 12, or pEMR515 derived from pEMR473 by deletion of the gene URA3 and of the fragment XbaI-NheI of the 2 micron.
19. Prokaryotic microorganisms, characterized in that they are transformed by an expression vector according to claim 16, carrying a recombinant gene according to claim 9.
20. Eukaryotic cells, characterized in that they 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. Strain of Saccharomyces cerevisiae, characterized in that it is transformed by one of the expression vectors according to any one of claims 16 to 18.
22. Strain according to claim 21, characterized in that it carries a mutation on at least one of the genes responsible for the synthesis of leucine or uracil.
23. Strain according to claim 22, characterized in that it carries a mutation on at least one of the LEU2 and URA3 genes.
24. Method for obtaining the recombinant urate oxidase according to claim 1, characterized in that it comprises the following steps:

    1/ culturing of a strain according to any one of claims 21 to 23;

    2/ lysis of cells;

    3/ isolation and purification of the recombinant urate oxidase contained in the lyzate.
25. Animal cells, characterized in that they contain a recombinant gene according to claim 13 with the means necessary for its expression.
26. Animal cells, characterized in that they contain an expression vector according to claim 16, carrying a recombinant gene according to claim 14.