DE4316505A1 - DNA coding for Candida utilis uricase enzyme - for prodn. of recombinant uricase used for quantitative determn. of uric acid in blood or urine - Google Patents

Abstract

A nucleotide sequence (I) (a) a DNA sequence defined in the specification, or part of this sequence; (b) a DNA sequence coding for a defined sequence of 303 amino acids given in the specification, or part of this sequence; (c) a DNA sequence which is an allelic variant of (a) or (b); or (d) a DNA sequence corresp. to (a)-(c) on the basis of the degeneracy of the genetic code. Also claimed are: (1) a recombinant DNA mol. contg. (I); (2) a host cell contg. recombinant DNA mol. (1); and (3) uricase encoded by (I) or produced by culturing host cell (2). (I) is derived from C utilis. The recombinant DNA mol. contains (I) under the control of regulatory elements ensuring its expression in a desired host cell. The host cell is a bacterial, yeast insect, plant or mammalian cell, esp. E. coli JM109(pUOX101) (FERM BP-3842). USE/ADVANTAGE - Uricase is useful as a reagent for quantitative determn. of uric acid in blood or urine. E. coli bacteria transformed with the Candida utilis uricase gene are capable of producing uricase in culture media not contg. uric acid (cf. C. utilis itself: JA5192/67).

Description

The invention relates to a uricase gene, a recombinant te DNA and a method for producing the uricase.

Uricase is an enzyme that prevents the formation of allantoin, Hydrogen peroxide and carbon dioxide through hydrolysis of urine acid catalyzed. This enzyme is used as a reagent for quantita tive determination of uric acid in the blood or urine and plays an important role in biochemical slide gnose.

Usually Uricase was e.g. B. by inoculating Candida utilis in a medium containing uric acid, subsequent Cultivation of the microorganism and isolation of the uricase from the Culture Generates (Japanese Patent Publication No. 5,192 / 67).

The invention relates to an isolated uricase gene that that in the sequence listing under SEQ ID NO. 2 defined Encoded amino acid sequence; the invention further relates to a recombinant DNA that isolated the above Contains uricase gene inserted into a vector DNA; the he The invention also relates to a method for producing the vrica se, in which a microorganism of the Genus Escherichia, the recombinant mentioned above Contains DNA and the ability to produce uricase sits, breeds and then the uricase from the culture isolated.  

According to the invention, uricase can be used efficiently without the addition of Uric acid can be generated to a medium.

In the prior art method described above There have been some problems with the technique for producing uricase such as insufficient yield of uricase, etc.

As a result of extensive research to solve the For the first time, the inventors have problems successfully Candida utilis-derived uricase gene isolated and its Structure determined; they received a recombinant DNA, at inserting the gene encoding the uricase into a vector DNA is. The inventors have found that uricase is efficient without adding uric acid to a medium by growing one Bacterial strain of the genus Escherichia with the ability to Production of uricase in which the recombinant gene is inserted has been generated.

The uricase gene according to the invention can, for example, how described below.

Chromosomal DNA is grown from microorganisms of the strain Candida utilis ATCC 9950 (deposited according to the Budapest contract under the deposit number ATCC 74151) after the z. B. in Section 13.11 of Current Protocols in Molecular Biology (WILEY Interscience, 1989) Procedure received.

Then the chromosomal DNA with Sau3AI becomes partial cleaved, creating a mixture of chromosomal DNA fragments ten is obtained. From the mixture of DNA Fragments will be a mixture of DNA fragments with one before drawn length of 10-20 kb z. B. by conventional agarose obtained gel electrophoresis. This may be the case treated DNA mixture with alkaline phosphatase etc. and can be cleaned further, e.g. B. by cleaning procedure of phenol extraction etc., and is then z. B. with  concentrated ethanol precipitation etc., whereby a purified Mixture of DNA fragments (containing the DNA fragments with the gene for uricase) is obtained.

Examples of the vector DNA which ver can be used are bacteriophage vector DNA, plasmid Vector DNA, etc., preferably λEMBL4 DNA (available from Strata gene) as a specific example.

The above bacteriophage vector DNA is e.g. B. with BamHI (manufactured by Takara Shuzo Co., Ltd.) split, if necessary then precipitated with ethanol etc., so that bacteriophage vector DNA capable of incorporation of a Sau3AI-cleaved fragment is obtained.

Subsequently, the DNA fragments with the C. utilis ATCC 9950 derived gene encoding uricase with bacteriophage vector DNA capable of incorporation of a Sau3AI-cleaved fragment, then mixed under Ver application z. B. T4 DNA ligase (manufactured by Boehringer Mannheim GmbH) ligated, resulting in recombinant phage DNAs be preserved. The recombinant phage DNAs thus obtained can in phage particles according to the conventional in vitro Packaging processes are packed. E. coli P2 392 (related von Stratagene) for example is infected with the phages, resulting in a wide variety of recombinant phage plaques of DNA fragments can be obtained.

To search for recombinant phages carrying DNA fragments containing uricase genes, among the plaques thus obtained, the plaque hybridization can be carried out according to the method described in section 6.3 of Current Protocols in Molecular Biology (WILEY Interscience, 1989); a with z. B. [γ- ³² P] ATP labeled oligonucleotide (available from Amersham Japan) was used as the probe.

A z. B. Molecular Cloning, pp. 77-85 (Cold Spring  Harbor Laboratory, 1982) Purification of the phage DNAs (which contain the uricase gene) the recombinant phage thus obtained can be used.

In the purified recombinant phage In addition to the gene encoding uricase, DNAs are large quantities redundant DNAs present and should, as below described, removed.

The purified recombinant DNAs thus obtained are with restriction enzymes (e.g. StuI and EcoT22I) without a gap place cleaved in the uricase gene so that a mixture of DNA fragments is obtained. The ones containing the uricase gene DNA fragments can be taken after Southern hybridization Use of the radioactive oligonucleo mentioned above tidsonde be identified. The cleaved DNA fragments are then a conventional agarose gel electrophoresis un thrown. Only the DNA fragment that contains uricase genes, as described above is identified under Using GENECLEAN II (available from Funakoshi K.K.) cleaned, whereby a StuI and EcoT22I-split, cleaned nigt DNA fragment (containing the uricase gene) obtained becomes.

Any vector DNA can be used in the present invention and examples are plasmid vector DNA, bacteriophage Vector DNA, etc., preferably plasmid pUC118 and pUC119 DNAs Etc . . The above vector DNA is used with restriction enzymes, e.g. B. SmaI and PstI (manufactured by Takara Shuzo Co., Ltd.), cleaved to give cleaved vector DNAs.

The DNA fragment thus obtained which contains the uricase gene contains and is derived from C. utilis ATCC 9950, with the cleaved vector DNAs are mixed and then z. B. with T4 DNA ligase (manufactured by Boehringer Mannheim GmbH) ligated, whereby a recombinant DNA is obtained.  

The recombinant DNA thus obtained is z. B. in E. coli K-12, preferably E. coli JM109 (available from Takara Shuzo Co., Ltd.), XL1-Blue (available from Funakoshi K.K.) trans forms, whereby a transformant originating in the respective bacterial strain is obtained. The transformation can after the D.M. Morrison method (Methods in Enzymology, 68, 326-331, 1979).

After a z. B. in point (4) of the examples below explained method is the entire nucleotide sequence of the Uricase gene by analysis of the above DNAs, which the Uricase gene contained, determined, and the amino acid sequence of the poly translated from the gene with this nucleotide sequence peptide is determined. The gene that the amino acid so determined encoded sequence is the uricase gene according to the invention.

Since the uricase obtained as described above Gene has no promoter for expression in E. coli, produ adorns a transformant that contains only the recombinant DNA, no uricase. Therefore, a uricase producing strain obtained by the following procedures.

Based on the ermit in the above procedure The nucleotide sequence becomes an oligonucleotide of 30 bases of the uricase gene (in which the C-terminal side of its compl entärstrang is) corresponding to 10 amino acids, each on N-terminus and at the C-terminus are present as primers syn thetized, and the recombinant plasmid obtained above is split as a matrix with EcoRI. The polymerase chain action (PCR) is performed using this primer and this Template performed so that the DNA exclusively from the the uricase coding region is obtained. The so get ne DNA is inserted into a vector DNA that a DNA Se quenz contains the expression regulating elements, such as a promoter, an operator, a ribosome binding site etc. derived from the E. coli lactose operon etc. (see The Operon, p. 227, Cold Spring Harbor Laboratory,  1980). The vector DNA used can be a plasmid DNA or be a bacteriophage DNA. The recombinant so obtained DNA is e.g. B. in E. coli K-12, preferably E. coli JM109 (get from Takara Shuzo Co., Ltd.), E. coli XL1-Blue, E. coli DH1 (ATCC 33849), etc. transformed or transduced, whereby a transformant originating in the respective bacterial strain is obtained. According to the D.M. Morrison Methods (Methods in Enzymology, 68, 326-331, 1979) be performed. The transduction can be done according to B. Hohn-Ver drive (Methods in Enzymology, 68, 299-309, 1979) will.

The bacterium thus obtained is used to generate the uricase strain of the genus Escherichia with the ability to become a pro production of uricase as described below, and the culture is isolated.

The transformant can be grown in a conventional manner solid culture, preferably in a liquid culture be performed.

A medium becomes the culture medium for the transformant used in which one or more inorganic salts, such as Potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate, iron (III) chloride, iron (III) sulfate and manganese sulfate fat, and, if necessary, sugar, vitamins, etc. one or several nitrogen sources, such as yeast extract, peptone, meat extract, corn steep liquor and bean or buckwheat always solution are added.

The initial pH of the medium is preferred to be one Value set from 7-9. Breeding is preferably 6-24 Hours at 30-42 ° C, preferably around 37 ° C, in a spinner Submerged culture with aeration, a shake culture, one stationary culture, etc. performed. After completing the The uricase can be cultivated from the culture according to a conventional method be isolated methods for the isolation of an enzyme.  

The physico-chemical properties of the so obtained uricase correspond to those described in Agr. Biol. Chem. Vol. 31, No. 11, pp. 1256-1264, 1967.

The following examples are intended to illustrate the invention explain without restricting them.

Examples 1) Generation of a chromosomal DNA from C. utilis ATCC 9950 (deposited in accordance with the Budapest Treaty under Hin accession number ATCC 74151).

C. utilis ATCC 9950 (deposited according to the Budapest Contract under the deposit number 74151) was in 200 ml of a YPD medium (1% yeast extract, 2% peptone and 2% Glucose) inoculated, ge at 30 ° C with shaking for 40 h and then by centrifugation for 10 minutes 3000 rpm isolated. The microorganisms thus obtained were in 25 ml sorbitol solution (0.9 M sorbitol, 0.1 M Tris-HCl, pH 8.0, 0.1 M EDTA) suspended. About 300 µg was made from this suspension chromosomal DNA according to the method described in Section 13.11 of Current Protocols in Molecular Biology (WILEY Interscience, 1989) method described.

2) Creation of a DNA bank.

According to a conventional method, 100 µg of above chromosomal DNA with Sau3AI (manufactured by Takara Shuzo Co., Ltd.) partially split and then one Subjected to agarose gel electrophoresis, whereby 10 ug DNA Frag elements of 10-20 kb were obtained. Using 1 T4 DNA ligase unit (manufactured by Boehringer Mannheim GmbH) were 2 µg of the chromosomal DNA cleaved with Sau3AI from C. utilis with 1 µg of the bacteriophage vector λEMBL4-DNA (obtained from Stratagene) that had been digested with BamHI, ligated, and then using an in vitro  Packing kits (Gigapack Gold, manufactured by Stratagene) packed, whereby the phage particles were obtained. E. coli P2 392 (obtained from Stratagene) was made with the phage particles infected, resulting in approximately 50,000 plaques. Blot the plaques on a Hybond-N + nylon membrane filter (manufactured by Amersham) was manufactured according to the instructions of Amersham performed.

3) Isolation of the uricase gene by plaque hybridization.

The uricase was purified from the cultured microorganism C. utilis ATCC 9950 using DEAE cellulose, hydroxyapatite HPLC and reverse phase HPLC. According to the method described by Gross and Witkop (Journal of Biological Chemistry, 237, 1856, 1962), the uricase was treated with cyanogen bromide and fragmented into peptides. The peptide fragments were fractionated by reverse phase HPLC, and the amino acid sequence of the uricase was determined in a protein sequencer (Model 470A, manufactured by Applied Biosystems Inc.). The polynucleotide CCNCAA / GAAT / CCCNAAA / GAA (where A is adenine, C is cytosine, G is guanine, T is thymine, A / G is adenine or guanine, T / C is thymine or cytosine, and N is A, T , G or C), corresponding to the determined amino acid sequence Pro-Gln-Asn-Pro-Lys-Lys, was synthesized using a DNA synthesizer (Model 392, manufactured by Applied Biosystems Inc.). As a probe for the plaque hybridization, 1 µg of synthetic DNA was terminally labeled with [λ- ³² P] ATP (manufactured by Amersham Japan) and T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.). Using this probe, plaque hybridization was performed according to the procedure described in Section 6.3 of Current Protocols in Molecular Biology (WILEY Interscience, 1989), whereby 1 positive clone was obtained.

4) Analysis of the uricase gene.

The phage DNA of the isolated positive clone was  according to the conventional method (Molecular Cloning, ed. von Maniatis et al. Pp. 77-85, Cold Spring Harbor Laboratory, USA, 1982) isolated and then Southern hybridization under Using the synthetic oligonucleotide as a probe (J. Mol. Biol. 98, 503, 1975). As a result, found that the uricase gene is contained in an approximately 1.6 kb DNA Fragment that had been cleaved by StuI and EcoT22I was included. Therefore, this fragment became an agarose gel subjected to electrophoresis, then with GENECLEAN II (obtained from Funakoshi K.K.) cleaned and then in pUC118 and pUC119, the had been digested with PstI and SmaI. That so Recombinant plasmid obtained was used to determine the Nu cleotide sequence using a kilo sequence deletion Kits (obtained from Takara Shuzo Co., Ltd.) and the 370 DNA Sequencing systems (obtained from Applied Biosystems Inc.) analyzed. The determined nucleotide sequence and the amino acid The sequence of the polypeptide translated by the DNA is shown in SEQ ID NOs. 1 and 2 respectively. The uricase gene has one coding region of 909 bases encoding 303 amino acids.

5) Generation of the expression vector pUTE100.

After cleaving the DNA of the plasmid vector pBR322 with EcoRI and NruI were blunt ended using a DNA Blunting kits (obtained from Takara Shuzo Co. Ltd.); then the DNA was subjected to agarose gel electrophoresis after a subject to usual procedures; an approximately 3.4 kb long DNA Fragment with a replication original was used obtained from GENECLEAN II (obtained from Funakoshi K.K.). That so DNA fragment obtained was circularized with T4 DNA ligase and then linearized by cleavage with EcoRI. Separately from it was a DNA sequence (SEQ ID NO. 5 in the sequence listing), which regulates an HpaI cleavage site and expression Elements such as a promoter, an operator, a ribosome Binding site, etc, contained and from E. coli lactose operon etc. (see The Operon, p. 227, Cold Spring Harbor Laboratory, 1980) using a DNA syn  thesis devices (Model 392, manufactured by Applied Biosystems Inc.) synthesized. The DNA thus synthesized was transferred to the fragment obtained above, knotted with EcoRI, whereby the expression vector pUTE100 was produced.

The oligonucleotides with the nucleotide sequences SEQ ID NOs. 3 and 4, the N-terminal and C-terminal amino acid, respectively sequences corresponding to uricase were used as primers with ei DNA synthesizer (Model 392, manufactured by Applied Biosystems Inc.). The one obtained above recombinant plasmid DNA was spotted as a template with EcoRI In the presence of this primer and this template, the Polymerase chain reaction using a GeneAmp DNA Amplification reagent kits with AmpliTaq (purchased from Takara Shuzo Co., Ltd.) performed, whereby only the coding Region of the uricase gene was synthesized. The so obtained DNA was inserted into the HpaI site of the expression plasmid Vector DNA pUTE100 inserted so that the recombinant Plas mid-DNA pUOX101 was obtained. The recombinant plasmid DNA pUOX101 was developed in E. coli JM109 after the D.M. Morrison Ver drive (Methods in Enzymology, 68, 326-331, 1979) transfor mated, whereby the transformant E. coli JM109 (pUOX101) received was. This transformant has been deposited number FERM BP-3842 at the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan. The recombinant E. coli strain JM109 (pUOX101) was at 37 ° C With shaking in a TY medium (1% Bacto- Trypton, 0.5% Bacto yeast extract, 0.5% NaCl, pH 7.0), included tend 1 mM isopropyl-β-D-galactoside, grown. The Urica seak Activity in the culture was determined according to the Agri. Biol. Chem., Vol. 31, No. 11, pp. 1256-1264, 1967 and was 0.165 U / ml.

Claims (10)

1. nucleotide sequence encoding a protein with the biological activity of a uricase, which

• a) the DNA sequence of SEQ ID NO. 1 or part of it, or
• b) a DNA sequence which contains the amino acid sequence of SEQ ID NO. 2 encoded, or part of it, or
• c) a DNA sequence which is an allelic derivative of the sequence of (a) or (b), or
• d) a DNA sequence which is degenerated to a DNA sequence according to one of the sequences according to (a) to (c) on the basis of the genetic code.

2. The nucleotide sequence of claim 1, which is from Candida utilis is derived. 3. Recombinant DNA molecule containing a DNA sequence after Claim 1 or 2. 4. A recombinant DNA molecule according to claim 3, wherein the DNA Sequence is under the control of regulatory elements that allow its expression in a desired host cell. 5. Host cell containing the recombinant DNA molecule after Claim 4. 6. Host cell according to claim 5, which is a bacterial cell, a yeast cell, an insect cell, a plant cell or is a mammalian cell.   7. A host cell according to claim 6, which is Escherichia coli JM109 (pUOX101), deposited under the deposit number FERM BP- 3842. 8. A method for producing a uricase comprising the breed tion of the host cell according to one of claims 5 to 7 under for the expression of the DNA sequence suitable conditions and the Isolation of the protein from the culture. 9. uricase encoded by the nucleotide sequence of claim 1 or 2. 10. uricase produced by the method of claim 8.