JP2984143B2 - Novel uricase gene, novel recombinant DNA and method for producing uricase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a uricase gene, a novel recombinant DNA and a method for producing uricase.

[0002]

BACKGROUND OF THE INVENTION Uricase is an enzyme that catalyzes the production of allantoin, hydrogen peroxide and carbon dioxide by hydrolyzing uric acid. It is used as a reagent for quantifying uric acid in blood or urine, and is used for biochemical diagnosis. Plays an important role. Conventionally, uricase is, for example, Candida utilis
(Candida utilis) is inoculated and cultured in a uric acid-containing medium, and uricase is collected from the culture (Japanese Patent Publication No. 42-5192).

[0003]

However, when the above-mentioned method for producing uricase is used, there have been problems such as an insufficient yield of uricase.

[0004]

Means for Solving the Problems Accordingly, the present inventors have
As a result of various studies to solve the above problems, the inventors succeeded in isolating and determining the structure of a uricase gene derived from Candida utilis for the first time, and furthermore, a recombinant DNA obtained by inserting a gene encoding uricase into a vector DNA. And use this recombinant as Eschericia (Escher
The present inventors have found that when a strain having uricase-producing ability contained in a strain belonging to the genus ichia) is cultured in a medium, uricase is efficiently produced without adding uric acid to the medium, and the like, and the present invention has been completed.

That is, a first aspect of the present invention is a novel uricase gene encoding the amino acid sequence of SEQ ID NO: 2 in the sequence listing, and a second aspect of the present invention is a novel uricase gene encoding the first novel uricase gene of the present invention. A third aspect of the present invention is a novel recombinant DNA characterized by being inserted into a vector DNA. The third aspect of the present invention relates to a genus Escherichia having the uricase-producing ability, comprising the second novel recombinant DNA of the present invention. A method for producing uricase, comprising culturing a microorganism belonging to a medium and collecting uricase from the culture.

Hereinafter, the present invention will be described in detail. The uricase gene of the present invention can be obtained, for example, as follows. First, Candida utiris
lis) ATCC9950 (International deposit No.AT based on the Budapest Treaty
CC74151), for example, Current Protocols in Molecular B
Chromosomal DNA is obtained from a strain obtained by culturing according to the method described in iology (WILEY Interscience, 1989) unit 13.11.

Next, the chromosomal DNA is partially digested with Sau3AI to obtain a chromosomal DNA fragment mixture. From the DNA fragment mixture thus obtained, for example, by ordinary agarose gel electrophoresis, a DNA fragment mixture having a size of preferably 10 to 20 kb is obtained, and after performing an alkaline phosphatase treatment or the like as necessary, Further, if necessary, for example, purification by purification means such as phenol extraction,
Furthermore, for example, a concentrated DNA fragment mixture (e.g., a DNA fragment containing a uricase-encoding gene is contained) is obtained by concentrating by means such as ethanol precipitation.

On the other hand, the vector DNA which can be used in the present invention includes, for example, bacteriophage vector DNA, plasmid vector DNA and the like, and specifically, λEMBL4 DNA (obtained from STRATAGENE) is preferable. To bring the bacteriophage vector DNA into a state capable of incorporating the Sau3AI fragment,
For example, a bacteriophage vector DNA capable of incorporating the Sau3AI fragment is obtained by digesting with BamHI (manufactured by Takara Shuzo Co., Ltd.) and, if necessary, performing ethanol precipitation.

Next, C.utilis obtained as described above is used.
A DNA fragment containing a gene encoding uricase derived from ATCC9950 and a bacteriophage vector DNA capable of incorporating the Sau3AI fragment obtained as described above are mixed, and, for example, T4 DNA ligase (manufactured by Boehringer Mannheim) is acted on. Recombinant phage D
Get NA. With the recombinant phage DNA thus obtained, phage particles can be formed by a usual in vitro packaging method.
The phage is, for example, E. coli P2 392 (STRATA
(Obtained from GENE) to obtain recombinant phage plaques carrying various DNA fragments.

To search for a recombinant phage having a DNA fragment containing the uricase gene from the plaques thus obtained, for example, oligos labeled with [γ- ³² P] ATP (obtained from Amersham Japan) Using the nucleotide as a probe, the Current Protocols in
Molecular Biology (WILEY Interscience, 1989) unit 6.
Plaque hybridization can be performed by the method described in 3.

To obtain purified phage DNA from the thus obtained recombinant phage (containing a DNA fragment containing a uricase gene), for example, see Molecular Cloning p.77- 85 (Cold Spring
Harbor Laboratory, 1982). Since unnecessary DNA other than the uricase-encoding gene is present in a large amount in the purified and purified recombinant phage DNA obtained as described above, the unnecessary DNA is removed by the following operation.

[0012] The thus obtained and purified recombinant DNA is digested with a restriction enzyme having no cleavage site on the uricase gene, such as StuI and EcoT22I, to obtain a DNA fragment mixture. Which DNA fragment in the DNA fragment mixture thus obtained contains the gene encoding uricase can be determined by Southern hybridization using the above-described radioactive oligonucleotide probe. Next, the DNA fragment mixture obtained by digestion as described above is subjected to ordinary agarose gel electrophoresis, for example, only the DNA fragments determined to contain the uricase-encoding gene as described above are subjected to: GENECLEAN II (Funakoshi
Purified using StuI-Eco
A T22I digested fragment (containing the gene encoding uricase) is obtained.

On the other hand, any vector DNA can be used in the present invention, for example, plasmid vector DNA, bacteriophage vector DNA, etc., and plasmids pUC118, pUC11
9 DNA and the like are preferred. The vector DNA is digested with a restriction enzyme such as SmaI or PstI (Takara Shuzo) to obtain a digested vector DNA.

Next, C.utilis obtained as described above is used.
A DNA fragment containing the uricase-encoding gene derived from ATCC9950 is mixed with the cut vector DNA, and the mixture is treated with, for example, T4 DNA ligase (Boehringer Mannheim) to obtain a recombinant DNA. Using this recombinant DNA, for example, E. coli K-12, preferably E. coli JM109 (obtained from Takara Shuzo), XL1-Blue (Funakoshi
, Etc.) to obtain the respective strains.
This transformation was performed using the method of DMMorrison (Methods in Enz
ymology, 68 , 326-331, 1979).

Using the DNA containing the uricase gene, the entire nucleotide sequence of the uricase gene is analyzed by the method shown in item (4) of the Examples, and then translated by the gene having the nucleotide sequence. The amino acid sequence of the polypeptide is determined. The gene encoding the amino acid sequence thus determined is the uricase gene of the present invention.

Next, since the uricase gene obtained as described above does not have a promoter for expression in Escherichia coli, the transformed strain having the recombinant DNA is:
It does not produce uricase, and a uricase-producing strain is obtained by the following operation. Based on the nucleotide sequence obtained by the above operation, an oligonucleotide of 30 bases corresponding to each of the 10 amino acids at the N-terminal and C-terminal of the uricase gene (the C-terminal side is a complementary strand) was synthesized, and this was used as a primer. Further, a polymerase chain reaction (hereinafter abbreviated as "PCR") is performed using the EcoRI-digested digest of the recombinant plasmid obtained above as a template to obtain a DNA consisting of only a region encoding uricase. The obtained DNA was subjected to a DNA sequence containing an expression regulatory region such as a promoter, an operator and a ribosome binding site derived from E. coli lactose operon (The Operon, p.227, Cold Spring Harbo
r Laboratory, 1980)
Insert into A. The vector DNA used may be a plasmid DNA or a bacteriophage DNA. Using the obtained recombinant DNA, for example, E. coli K-12, preferably E. coli JM109 (obtained from Takara Shuzo), E. coli XL1-Bleu,
Escherichia coli DH1 (ATCC33849) or the like is transformed or transduced to obtain each strain. This transformation can be performed by the method of DMMorrison (Methods in Enzymology, 68 , 326-331, 1979). Transduction was performed by the method of B. Hohn (Met
hods in Enzymology, 68 , 299-309, 1979).

Next, to produce uricase using the uricase-producing strain belonging to the genus Escherichia obtained as described above, a culture is obtained by culturing as described below. In order to culture the above microorganism, it may be cultured by a usual solid culture method, but it is preferable to employ a liquid culture method as much as possible.

The medium for culturing the above-mentioned microorganisms may be, for example, potassium dihydrogen phosphate, phosphorus extract or the like in one or more nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor or soybean or wheat koji leaching solution. One obtained by adding one or more inorganic salts such as dipotassium hydrogen oxyoxide, magnesium sulfate, ferric chloride, ferric sulfate, and manganese sulfate, and further appropriately adding a saccharide raw material, a vitamin, and the like, if necessary, is used.

It is appropriate that the initial pH of the medium is adjusted to 7-9. In addition, culture is performed at 30 to 42 ° C, preferably at 37 ° C.
It is preferable to carry out by aeration and stirring deep culture, shaking culture, stationary culture, etc. for 6 to 24 hours before and after. After completion of the culture, uricase can be collected from the culture using a conventional enzyme collecting means. The physicochemical properties of the uricase thus obtained are described in Agr. Biol. Chem., Vol. 31,
No. 11, p. 1256-1264, 1967.

[0020]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to the following Examples. Example (1) C.utilis ATCC9950 (International Deposit No. ATCC 74151 based on the Budapest Treaty) Preparation of Chromosomal DNA C.utilis ATCC9950 (International Deposit based on the Budapest Treaty N
o.ATCC 74151) was inoculated into 200 ml of YPD medium (1% yeast extract, 2% peptone, 2% glucose), cultured with shaking at 30 ° C. for 40 hours, and centrifuged at 3000 rpm for 10 minutes to collect the cells. The cells were added to a 25 ml sorbitol solution (0.9
M sorbitol, 0.1 M Tris-HCl pH 8.0, 0.1 M EDTA)
After suspension in Current Protocols in Molecular Biolo
According to the method described in gy (WILEY Interscience, 1989) unit 13.11, about 300 μg of chromosomal DNA was obtained.

(2) Preparation of cDNA Library 100 μg of the above chromosomal DNA was partially digested with Sau3AI (manufactured by Takara Shuzo) according to a conventional method, and then 10 μg of a DNA fragment of a 10-20 kb fraction was obtained by agarose gel electrophoresis. Bacteriophage vector λEMBL4 DNA (obtained from STRATAGENE) 1 μg of BamHI digest and C. utili obtained above
s Chromosomal DNA 2 μg of Sau3AI digest was added to T4 DNA
Ligase (Boehringer Maiheim Co., Ltd.) is connected by one unit, and in vitro packaging kit (Gigapack Gold, STRA
TAGENE), and package with E. coli (Ec
oli) P2392 (obtained from STRATAGENE) to obtain about 50,000 plaques. The obtained plaque is used as a nylon membrane filter Hybond-N ⁺ (manufactured by Amersham).
Was blotted according to the protocol of Amersham.

(3) Isolation of uricase gene by plaque hybridization Purified uricase from cultured cells of C. utilis ATCC9950 using DEAE cellulose, hydroxyapatite HPLC and reverse-phase HPLC, and the method of Gross and Witkop
(Journal of Biological Chemistry, 237 , 1856, 196
According to 2), the peptide was fragmented by treating with cyanogen bromide. After separating this by reverse phase HPLC, a protein sequencer (manufactured by Applied Biosystems, model 470) was used.
The amino acid sequence was determined according to A). Polynucleotide CCNCAA / GAAT / CCCNAAAA / GA corresponding to the obtained amino acid sequence Pro Gln Asn Pro Lys Lys
A (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 any of A, T, G, and C ) Was synthesized using a DNA synthesizer (Applied Biosystems, model 392). This synthetic DNA
1 μg was end-labeled with [γ- ³² P] ATP (manufactured by Amersham Japan) and T4 polynucleotide kinase (manufactured by Takara Shuzo) to use as a probe for plaque hybridization. Current Protocols in MolecularBiology
(WILEY Inter-science, 1989) Plaque hybridization was performed according to the method described in unit 6.3 to obtain one positive clone.

(4) Analysis of Uricase Gene Phage DNA of the isolated positive clone was obtained by a conventional method.
(Molecular Cloning, ed.Maniatis et al., P. 77-85,
Cold Spring Harbor Laboratory, USA, 1982), and Southern hybridization (J. Mol. Biol, 98 ,
503, 1975), it was found that the uricase gene was contained on a DNA fragment of about 1.6 kb cut out with StuI and EcoT22I. This fragment was subjected to agarose gel electrophoresis, and then GENECLEAN II (Funakoshi (strain) PUC118 and pU purified by PstI and SmaI
Inserted into C119. The resulting recombinant plasmid was transferred to Kilo-S
deletion kit for equence (purchased from Takara Shuzo) and 370 D
The nucleotide sequence was determined using NA Sequencing System (purchased from Applied Biosystems). The determined nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the polypeptide translated from the DNA is shown in SEQ ID NO: 2. The uricase gene had a coding region of 909 bases and encoded 303 amino acids.

(5) Preparation of Expression Vector pUTE100 The plasmid vector pBR322 DNA was digested with EcoRI and NruI, blunt-ended with a DNA Blunting kit (purchased from Takara Shuzo Co., Ltd.), and subjected to agarose gel electrophoresis according to a conventional method. Then, a DNA fragment of about 3.4 kb including a replication origin was obtained by GENECLEAN II (purchased from Funakoshi Co., Ltd.). The obtained DNA fragment was circularized with T4 DNA ligase, followed by EcoRI digestion to linearize it. Subsequently, a DNA sequence containing an HpaI cleavage site and an expression control region such as a promoter, an operator and a ribosome binding site derived from an E. coli lactose operon as shown below (The Operon, p.227, Cold Spring Harbor Laborator
y, 1980):

[0025]

[Outside 1]

The DNA Synthesizer Model 392 (Applied
Biosystems) and ligated with the EcoRI fragment obtained above to prepare an expression vector pUTE100. Oligonucleotides having the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 corresponding to the N-terminal and C-terminal amino acid sequences of uricase, respectively, were prepared using DNA Synthesizer Model 392 (Applied Biosystems).
Primers, and using the EcoRI digest of the recombinant plasmid DNA obtained above as a template,
neAmp DNA Amplification Reagent Kit with AmpliTaq
After only the uricase gene coating region was synthesized by the PCR method (purchased from Takara Shuzo), it was inserted into the HpaI site of the above expression plasmid vector pUTE100 DNA to obtain a recombinant plasmid pUOX101 DNA. DM
Morrison's method (Methods in Enzymology, 68 , 326-
331, 1979), the recombinant plasmid pUOX101 DN
A. is used to transform Escherichia coli JM109.
109 (pUOX101) was obtained. In addition, the strain is a microfabrication research institute No. 3842 to the Institute of Microbial Industry and Technology (FERM BP-3842)
Has been deposited as The obtained recombinant Escherichia coli JM10
9 (pUOX101) was added to a TY medium containing 1 mM isopropyl-β-D-galactoside (1% bacto-tryptone, 0.5%
Bacto-yeast extract, 0.5% NaCl, pH
7.0) at 37 ° C with shaking for 16 hours, and then the uricase activity was measured using Agri. Biol. Chem., Vol. 31, No. 11, p. 1256-126.
4, measured by the method described in 1967, 0.165U / ml
Met.

[0027]

According to the present invention, uricase can be efficiently produced without adding uric acid to the medium.

[0028]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 909 Sequence type: Nucleic acid number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin Organism: Candida utilis Strain name: ATCC 9950 Row: ATG TCA ACA ACG CTC TCA TCA TCC ACC TAC GGC AAG GAC AAC GTC AAG TTC CTC AAG GTC AAG AAG GAC CCG CAA AAC CCA AAG AAG CAG GAG GTT ATG GAG GCC ACC GTC ACG TGT CTG CTT GAA GGT GGG TTC GAC ACC TCG TAC ACG GAG GCT GAC AAC TCG TCC ATC GTG CCA ACA GAC ACC GTG AAG AAC ACC ATT CTC GTG TTG GCA AAG ACC ACG GAG ATT TGG CCA ATT GAG AGA TTT GCA GCC AAG CTG GCC ACG CAC TTT GTT GAG AAG TTAC TCG CATC GGC GTC TCC GTC AAG ATT GTC CAG GAC AGA TGG GTC AAG TAC GCC GTT GAT GGC AAG CCA CAC GAC CAC TCT TTT ATC CAC GAA GGT GGT GAG AAG AGA ATC ACT GAC CTG TAC TAC AAG AGA TCC GGT GAT TAC AAG CTG TCT ATC AAG GAC TTG ACG GTG CTG AAG TCC ACC GGC TCG ATG TTC TAC GGC TAC AAC AAG TGT GAC TTC ACC ACC TTG CAA CCA ACA ACT GAC AGA ATC TTG TCC ACC GAC GTC GAT GCC ACC TG G GTT TGG GAT AAC AAG AAG ATT GGC TCT GTC TAC GAC ATC GCC AAG GCT GCA GAC AAG GGA ATC TTT GAC AAC GTT TAC AAC CAG GCT AGA GAG ATC ACC TTG ACC ACC TTT GCT CTC GAG AAC TCT CCA TCT GTG CAG GCG ACG TTC AAC ATG GCT ACT CAG ATC TTG GAA AAG GCA TGC TCT GTC TAC TCG GTT TCA TAC GCC TTG CCA AAC AAG CAC TAC TTC CTC ATT GAC TTG AAA TGG AAA GGT TTG GAG AAC GAC AAC GAG TTG TTC TAC CCA TCT CCA CAT CCA ATC GGG TTG ATC AAG TGT ACT GTT GTC CGT AAG GAG AAG ACC AAG TTG SEQ ID NO: 2 Sequence length: 303 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence: Met Ser Thr Thr Leu Ser Ser Ser Ser Thr Tyr Gly Lys Asp Asn Val Lys Phe Leu Lys Val Lys Lys Asp Pro Gln Asn Pro Lys Lys Gln Glu Val Met Glu Ala Thr Val Thr Cys Leu Leu Glu Gly Gly Phe Asp Thr Ser Tyr Thr Glu Ala Asp Asn Ser Ser Ile Val Pro Thr Asp Thr Val Lys Asn Thr Ile Leu Val Leu Ala Lys Thr Thr Glu Ile Trp Pro Ile Glu Arg Phe Ala Ala Lys Leu Ala Thr His Phe Val Glu Lys Tyr Ser His Val Ser Gly Val Ser Va l Lys Ile Val Gln Asp Arg Trp Val Lys Tyr Ala Val Asp Gly Lys Pro His Asp His Ser Phe Ile His Glu Gly Gly Glu Lys Arg Ile Thr Asp Leu Tyr Tyr Lys Arg Ser Gly Asp Tyr Lys Leu Ser Ser Ala Ile Lys Asp Leu Thr Val Leu Lys Ser Thr Gly Ser Met Phe Tyr Gly Tyr Asn Lys Cys Asp Phe Thr Thr Leu Gln Pro Thr Thr Asp Arg Ile Leu Ser Thr Asp Val Asp Ala Thr Trp Val Trp Asp Asn Lys Lys Ile Gly Ser Val Tyr Asp Ile Ala Lys Ala Ala Asp Lys Gly Ile Phe Asp Asn Val Tyr Asn Gln Ala Arg Glu Ile Thr Leu Thr Thr Phe Ala Leu Glu Asn Ser Pro Ser Val Gln Ala Thr Met Phe Asn Met Ala Thr Gln Ile Leu Glu Lys Ala Cys Ser Val Tyr Ser Val Ser Tyr Ala Leu Pro Asn Lys His Tyr Phe Leu Ile Asp Leu Lys Trp Lys Gly Leu Glu Asn Asp Asn Glu Leu Phe Tyr Pro Ser Pro His Pro Asn Gly Leu Ile Lys Cys Thr Val Val Arg Lys Glu Lys Thr Lys Leu SEQ ID NO: 3 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Sequence characteristics: Synthetic DNA primer Sequence: ATG TCA ACA ACG GTC TCA TCA TCC ACC TAC SEQ ID NO: 4 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Sequence characteristics: Synthetic DNA primer sequence: TTA CAA CTT GGT CTT CTC CTT ACG GAC AAC

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI (C12N 9/06 C12R 1:19) (C12N 15/09 ZNA C12R 1:72) (58) Investigated field (Int.Cl. ⁶ , DB name) C12N 15/00-15/90 C12N 9/00-9/99 C12N 1/00-5/28 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A novel uricase gene encoding the amino acid sequence of SEQ ID NO: 2 in the sequence listing. 2. A novel recombinant DNA comprising the uricase gene according to claim 1 inserted into a vector DNA. 3. The novel recombinant DNA according to claim 2.
A method for producing uricase, comprising culturing a microorganism belonging to the genus Escherichia having uricase-producing ability in a medium and collecting uricase from the culture.