JP5923995B2 - Alkaline phosphatase - Google Patents

Description

  The present invention relates to a bacterial alkaline phosphatase.

  Alkaline phosphatase (EC 3.1.3.1, hereinafter also referred to as AP) is an enzyme that catalyzes the reaction of hydrolyzing phosphate monoesters to produce alcohol and inorganic phosphate. It is known to be widely distributed regardless. In addition to being used as an enzyme for genetic engineering, AP is widely used as a labeling enzyme in enzyme immunoassay (EIA). At present, most of the AP used for this EIA is bovine small intestine derived AP (CIAP). One of the reasons why CIAP comes in handy is its high specific activity. The specific activity of commercially available CIAP varies depending on the manufacturer and grade, but when p-nitrophenyl phosphate is used as a substrate, there is a high specific activity type exceeding 6000 U / mg-protein. A number of various high-sensitivity luminescent substrates for CIAP containing 1,2-dioxetane comb and acridan in the basic skeleton are also on the market, realizing high measurement sensitivity in immunoassay.

  One of the problems with the known CIAP is poor stability. On the other hand, bacteria-derived AP typified by Escherichia coli is more stable than CIAP, but is significantly inferior to CIAP in terms of specific activity. For example, Non-Patent Document 1 and Non-Patent Document 2 report APs derived from the genus Shewanella, which have relatively high specific activities, but the specific activities are both less than 2000 U / mg. The AP described in Non-Patent Document 1 has been registered as an industrially useful patent (Patent Document 1), but this is not suitable for genetic engineering because most enzymes are inferior in thermal stability to E. coli-derived AP. It is an application that is useful, and its usefulness as a labeling enzyme for immunoassay is not even mentioned. Patent Document 2 describes an AP derived from Bacillus, but this enzyme has a specific activity of about 3000 U / mg, which is not sufficient, and is a reaction with 1,2-dioxetane or acridan luminescent substrates. It was not practical and was not practical. Non-patent document 1 describes a bacterial AP having a specific activity exceeding 10,000 U / mg, but approximately 20% of the inactivation occurred after treatment at 30 ° C. for 15 minutes, and almost at 50 ° C. for 15 minutes. Compared with CIAP, such as complete loss of activity, the stability is remarkably low and it is not practical.

  Another problem with the known CIAP is a problem such as handling due to having a sugar chain. A protein having a sugar chain often has a non-uniform structure and size of the sugar chain, which may hinder purification, such as the target peak becomes broad in purification by gel filtration. In addition, the presence of sugar chains on the surface of the molecule can also be a physical barrier when the enzyme contacts the substrate in solution. Patent Document 3 describes a method for reducing sugar chains by deglycosylating CIAP expressed in yeast. However, sugar chains are not completely removed, and such steps are added. This leads to cost increase.

  Bacteria-derived AP does not have a sugar chain, so the above problems can be solved, and it can be easily expressed in an economical host system such as Escherichia coli, so that it can achieve cost reduction. Until now, no substance has been known that can sufficiently satisfy the necessary conditions in immunodiagnosis including reactivity to existing substrates. For this reason, CIAP continues to be used as a labeling enzyme in the field of immunodiagnosis while having the above problems.

Japanese Patent No. 3507890 Japanese Patent No. 4035738 Japanese Patent No. 4303283

Cloning and expression of a highly active recombinant alkaline phosphatase from psychrotrophic Cobetia marina, Biotechnol Lett (2011), Nasu et al.

  An object of the present invention is to provide an AP that has higher stability than CIAP, does not have a sugar chain on the enzyme surface, and has a specific activity at least equivalent to CIAP.

  As a result of intensive studies, the present inventors have succeeded in recombinant production of AP by obtaining an AP gene from a bacterium belonging to the genus Shewanella, and culturing a microorganism transformed with the gene, The inventors have found that recombinant AP has higher thermal stability than CIAP, has a specific activity comparable to CIAP, and has sufficient reactivity with various luminescent substrates, thereby completing the present invention.

That is, the present invention has the following configuration.
[Claim 1]
An alkaline phosphatase comprising the polypeptide of any one of (A) to (C) below.
(A) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.
(B) A polypeptide having an alkaline phosphatase activity consisting of a sequence obtained by deleting, substituting, inserting or adding one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 2.
(C) A polypeptide comprising an amino acid sequence having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and having alkaline phosphatase activity.
[Section 2]
DNA of any one of (A) to (F) below.
(A) DNA consisting of the base sequence set forth in SEQ ID NO: 1.
(B) DNA encoding the amino acid sequence set forth in SEQ ID NO: 2.
(C) DNA comprising a base sequence having a homology with the base sequence set forth in SEQ ID NO: 1 of 80% or more and encoding a polypeptide having alkaline phosphatase activity;
(D) a DNA that hybridizes under stringent conditions to a base sequence complementary to the base sequence represented by SEQ ID NO: 1 and encodes a polypeptide having alkaline phosphatase activity;
(E) DNA encoding a polypeptide having alkaline phosphatase activity, which is a base sequence in which one or several bases are substituted, deleted, inserted or added in the base sequence described in SEQ ID NO: 1;
(F) DNA encoding a polypeptide comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 and having alkaline phosphatase activity .
[Section 3]
A vector incorporating the DNA of Item 2.
[Claim 4]
A transformant in which a host cell is transformed with the vector according to Item 3.
[Section 5]
Item 5. The transformant according to Item 4, wherein the host cell is E. coli.
[Claim 6]
A method for producing alkaline phosphatase, comprising culturing a microorganism having the ability to produce alkaline phosphatase according to Item 1, and collecting a protein having alkaline phosphatase activity from the obtained culture.
[Claim 7]
Item 7. The method for producing alkaline phosphatase according to Item 6, wherein the microorganism having the ability to produce alkaline phosphatase according to Item 1 is derived from the genus Shewanella.
[Section 8]
Item 6. The method for producing alkaline phosphatase according to Item 6, wherein the microorganism having the ability to produce alkaline phosphatase according to Item 1 is the transformant according to Item 4 or 5.
[Claim 9]
A conjugate formed by labeling the alkaline phosphatase according to Item 1.

  According to the present invention, alkaline phosphatase useful as a labeling enzyme for immunoassay, alkaline phosphatase labeled antibody, and the like can be provided.

The stability at each temperature of AP derived from Shewanella fodinae and CIAP is shown.

1. Alkaline phosphatase 1-1. Polypeptides In a preferred embodiment of the invention, the invention is an alkaline phosphatase consisting of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, or one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 2. An alkaline phosphatase consisting of a polypeptide having an alkaline phosphatase activity, consisting of a sequence obtained by deleting, substituting, inserting or adding amino acid residues. The amino acid sequence of SEQ ID NO: 2 is derived from Shewanella fodinae as described later.

When the alkaline phosphatase of the present invention consists of a polypeptide having one or more amino acid substitutions, additions, deletions or insertions in the amino acid sequence shown in SEQ ID NO: 2, the number and type of such amino acid mutations are: There is no particular limitation as long as it does not affect the enzyme properties such as alkaline phosphatase activity and thermal stability described above. Preferably, the specific number of mutations is 1 to 30, more preferably 1 to 15, further preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 3. .

When the alkaline phosphatase of the present invention is composed of a polypeptide in which one or more amino acids are substituted in the amino acid sequence shown in SEQ ID NO: 2, the substitution of the amino acid is particularly effective as long as the alkaline phosphatase activity and the enzyme properties described above are not impaired. Although not limited, it is preferably substituted by a similar amino acid. Examples of similar amino acids include the following.
Aromatic amino acids: Phe, Trp, Tyr
Aliphatic amino acids: Ala, Leu, Ile, Val
Polar amino acids: Gln, Asn
Basic amino acids: Lys, Arg, His
Acidic amino acids: Glu, Asp
Amino acids having a hydroxyl group: Ser, Thr

  In a preferred embodiment of the present invention, the present invention is an alkaline phosphatase comprising a polypeptide having an alkaline phosphatase activity, comprising an amino acid sequence having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 2. The identity with the amino acid sequence described in SEQ ID NO: 2 is more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, 99% or more.

  As used herein, “identity” refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm uses a sequence of sequences for optimal alignment). The ratio of the same amino acid residue to the total overlapping amino acid residues (in which one or both of the gaps can be considered).

  Algorithms for determining amino acid sequence identity include, for example, Karlin et al. , Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 ( 1997))].

  In the amino acid sequence identity search by the identity calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Alignment Search Tool), the known sequence that shows the highest identity with the sequence shown in SEQ ID NO: 2 is Shewanella e. ) A sequence deduced from an ORF annotated as AP present in the OS185 strain genome, and its identity is 71%. That is, there is no known AP that shows 80% or more identity with SEQ ID NO: 2. In addition, the sequences of APs derived from the genus Shewanella described in Non-Patent Documents 1 and 2 have 67% and 70% identity, respectively, and the specific activity differs from these APs by 2 times or more as described above. However, the AP of the present invention is clearly distinguished from the known AP. One of the criteria for clearly defining the AP of the present invention is the high degree of identity with this SEQ ID NO: 2, and the preferred identity is specifically at least 85% or more, more preferably 90% or more, more Preferably it is 95% or more, More preferably, it is 98% or more, Most preferably, it is 99% or more.

1-2. Origin The source of the AP of the present invention is not particularly limited as long as it has the above-mentioned characteristics, but is preferably derived from bacteria, more preferably from the genus Shewanella, even more preferably Shewanella fodinae. fdinae), and most preferably derived from a strain distributed by the National Institute for Product Evaluation Technology as NBRC105216 strain.
The AP of the present invention may be obtained from a culture solution obtained by culturing the genus Shewanella bacterium, or obtained by introducing a gene into a host organism different from the bacterium from which it is derived and expressing it. Also good.

1-3. Thermal Stability The present invention is preferably an AP having a high thermal stability compared to CIAP, more preferably an AP that can maintain an activity of 80% or more after a heat treatment at 60 ° C. for 60 minutes, and still more preferably Is an AP that can maintain an activity of 80% or more after heat treatment at 65 ° C. for 60 minutes.

  The heat stability described in the present invention means that AP is dissolved in 50 mM triethanolamine, 1 mM magnesium chloride, 0.1 mM zinc sulfate (pH 7.0) so that the protein amount becomes 0.01 mg / mL. It is defined as the residual ratio of AP activity after heating to AP activity before heating when the solution is heated for 60 minutes. The heating treatment described in the present invention is performed under the above conditions. The method for measuring the activity is as described below.

1-4. Specific activity The specific activity described in the present invention is determined by the method described in “Examples of protein quantification and calculation of specific activity” described later. The specific activity of the AP of the present invention is at least 5000 U / mg or more, preferably 5500 U / mg or more. According to Non-Patent Document 1, the specific activity of a known Shewanella genus-derived AP is estimated to be 1880 U / mg at a high temperature of 50 ° C. and lower at 37 ° C. In addition, the strain described in Non-Patent Document 2 has 1500 U / mg at an optimum condition of 70 ° C. and pH 10.6, and approximately 37 U / mg at 37 ° C. The AP of the present invention is superior to the known AP of the genus Shewanella in terms of specific activity, and is clearly distinguished from these APs.

2. In a preferred embodiment of the present invention, the present invention comprises culturing a microorganism having the ability to produce alkaline phosphatase, and collecting a protein having alkaline phosphatase activity from the resulting culture. A method for producing alkaline phosphatase.

The AP of the present invention was engineered to express (1) a method of extracting and purifying the enzyme-producing cells as raw materials, (2) a method of chemically synthesizing, and (3) a gene recombination technique. It can be obtained by appropriately using a method of purifying from cells, or (4) a method of biochemical synthesis from a nucleic acid encoding AP using a cell-free transcription / translation system.
Hereinafter, the methods (1) to (4) will be sequentially described.

2-1. (1) Method of Extracting and Purifying the Cell Producing the Enzyme as a Raw Material The microorganism having the ability to produce the AP of the present invention is not particularly limited. For example, natural cells include microorganisms derived from the genus Shewanella. For example, the Shewanella fodinae NBRC105216 strain is exemplified, and can be purchased from (Germany) Product Evaluation Technology Infrastructure, and the strain of the present invention can be produced by culturing and growing the strain according to a conventional method.

  Isolation and purification of AP from natural AP-producing cells can be performed, for example, as follows. AP-producing cells are homogenized in an appropriate buffer solution, and cell extracts are obtained by sonication, surfactant treatment, etc., and purified by appropriately combining separation techniques conventionally used for protein separation and purification. can do. Such separation techniques include, for example, methods utilizing differences in solubility such as salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, non-denaturing polyacrylamide electrophoresis (PAGE), sodium dodecyl sulfate-poly A method using molecular weight difference such as acrylamide electrophoresis (SDS-PAGE), a method using charge such as ion exchange chromatography and hydroxyapatite chromatography, a method using specific affinity such as affinity chromatography, and the like Examples include, but are not limited to, a method utilizing a difference in hydrophobicity such as phase high performance liquid chromatography and a method utilizing a difference in isoelectric point such as isoelectric focusing.

2-2. (2) Method of chemically synthesizing AP is manufactured by chemical synthesis, for example, by synthesizing all or part of the sequence using a peptide synthesizer based on the amino acid sequence shown in SEQ ID NO: 2. It can be carried out. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. When the partial peptide or amino acid that can constitute the AP of the present invention is condensed with the remaining portion, and the product contains a protecting group, the protecting group is eliminated, whereby the target protein can be produced. Here, the condensation and the removal of the protecting group are carried out according to a method known per se, for example, the method described in the following (1) and (2).
(1) M.M. Bodanszkyand M.M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)

The AP of the present invention thus obtained can be purified and isolated by a known purification method. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
When the AP obtained by the above method is a free form, the free form can be converted into an appropriate salt by a known method or a method analogous thereto, and conversely, when a protein is obtained as a salt. The salt can be converted into a free form or other salt by a known method or a method analogous thereto.

2-3. (3) Method for purification from cells engineered to express AP by gene recombination technology 2-3-1. DNA encoding alkaline phosphatase
The AP of the present invention is preferably produced by cloning (or chemically synthesizing) a nucleic acid encoding the protein and isolating and purifying it from a transformant culture containing an expression vector carrying the nucleic acid. be able to.

In a preferred embodiment, the gene encoding the AP of the present invention is the following DNA. That is, it is a DNA having a base sequence encoding the amino acid shown in SEQ ID NO: 2, and a more specific example is a DNA consisting of the base sequence shown in SEQ ID NO: 1.
In addition, as long as the protein to be expressed has alkaline phosphatase activity, the approximate DNA is preferably the amino acid described in SEQ ID NO: 2 as long as the properties of AP are equal to or higher than those described in the present application. The sequence may be DNA encoding an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added, or one or more bases in the base sequence shown in SEQ ID NO: 1 May be substituted, deleted, added, or inserted DNA.
In another embodiment, the approximate DNA is a DNA that hybridizes under stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 1, and a polypeptide having alkaline phosphatase activity. It may be DNA that encodes. The nucleic acid that hybridizes with the base sequence shown in SEQ ID NO: 1 under stringent conditions is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, with the base sequence shown in SEQ ID NO: 1, Particularly preferred is a nucleic acid containing a base sequence having 90% or more identity, most preferably 95% or more identity.
In another embodiment, the approximate DNA is a DNA encoding a polypeptide having a base sequence having a homology of 80% or more with the base sequence set forth in SEQ ID NO: 1 and having alkaline phosphatase activity. May be.

The identity of the base sequences in this specification is determined using the following condition (expectation value = 10; allow gap; filtering = ON; match) using the identity calculation algorithm NCBI BLAST (National Center for Biotechnology Information Local Alignment Search Tool) Score = 1; mismatch score = -3).
Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning, 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can be preferably performed according to stringent conditions.

In the above, “stringent conditions” means that only a base sequence having a transcription termination region function equivalent to the base sequence shown in SEQ ID NO: 1 is hybridized with a base sequence complementary to SEQ ID NO: 1 (so-called specific conditions). A base sequence that forms a hybrid) and does not have an equivalent function means a condition that does not form a hybrid (so-called non-specific hybrid) with a base sequence complementary to SEQ ID NO: 1. Those skilled in the art can easily select such conditions by changing the temperature during the hybridization reaction and washing, the salt concentration of the hybridization reaction solution and the washing solution, and the like. Specifically, 42 ° C. in 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0.2 M NaH 2 PO 4,20 mM EDTA) · 2Na, pH 7.4) Examples of the stringent conditions of the present invention include, but are not limited to, the conditions in which the cells are hybridized with and washed with 0.5 × SSC at 42 ° C.
The stringent conditions are preferably highly stringent conditions. High stringent conditions are conditions in which washing is performed at a salt concentration corresponding to, for example, 0.1 × SSC and 0.1% SDS at 60 ° C. )

  The DNA encoding the AP of the present invention can be obtained from the genomic DNA or RNA (cDNA) of a Shewanella genus bacterium, but it is possible to chemically synthesize a DNA strand or to synthesize a partially overlapping oligo DNA short. It is also possible to construct a DNA encoding the full length of AP by connecting the strands using the PCR method. The advantage of constructing full-length DNA in combination with chemical synthesis or PCR is that the codons used can be designed over the entire length of the gene in accordance with the host into which the gene is introduced. A plurality of codons encoding the same amino acid are not used uniformly, and the frequency of use varies depending on the species. In general, codons contained in genes that are highly expressed in a given species contain many codons that are frequently used in that species, and conversely, the presence of codons that are used infrequently becomes a bottleneck for genes with low expression levels. There are many examples that prevent high expression. In the expression of a heterologous gene, many examples have been reported so far in which the expression level of the heterologous protein has been increased by replacing the gene sequence with a codon that is frequently used in the host organism. It is expected to be effective in increasing the expression level of heterologous genes.

  For the above reasons, it is desirable to modify the DNA encoding the AP of the present invention into a codon more suitable for the host cell into which it is introduced (ie, a codon frequently used in the host). The codon usage frequency of each host is defined by the ratio of each codon used in all genes present on the genome sequence of the host organism, and is expressed, for example, by the number of times used per 1000 codons. In addition, the codon usage frequency can be approximately calculated from the sequence of a plurality of typical genes in an organism whose entire genome sequence has not been elucidated. The data on the frequency of codon usage in the host organism to be recombined uses, for example, the genetic code frequency database published on the website of Kazusa DNA Research Institute (http://www.kazusa.or.jp). Or you can refer to the literature describing the codon usage in each organism, or you can determine the codon usage data for the host organism you are using. By referring to the obtained data and the gene sequence to be introduced and replacing the codon used in the gene sequence that is not frequently used in the host organism with a codon that encodes the same amino acid and is frequently used Good.

2-3-2. Host cell The host cell into which the DNA encoding the AP of the present invention is introduced is not particularly limited as long as a recombinant expression system is established, but preferably bacteria such as Escherichia coli and Bacillus subtilis, actinomycetes, and bacilli. And microbial hosts such as yeast, insect cells, animal cells, higher plants and the like, more preferably E. coli (for example, K12 strain, B strain, etc.). For example, in the case of the K12 strain, codons frequently used in E. coli are GGT or GGC for Gly, GAA for Glu, GAT for Asp, GTG for Val, GCG for Ala, GCG for Arg, CGT or CGC, Ser for AGC, Lys for AAA, Ile for ATT or ATC, Thr for ACC, Leu for CTG, Gln for CAG, Pro for CCG and the like.
As the DNA encoding the AP substituted with the codon frequently used in the host as described above, for example, the DNA encoding the AP derived from the genus Shewanella bacterium encodes the same amino acid sequence as the AP, and the Escherichia coli K12 strain And DNAs substituted with frequently used codons.

2-3-3. Vector The present invention also provides a recombinant vector comprising DNA encoding the AP of the present invention. The recombinant vector of the present invention is not particularly limited as long as it can be replicated and maintained or autonomously propagated in various prokaryotic and / or eukaryotic host cells, and includes plasmid vectors and viral vectors. The recombinant vector is simply a known cloning vector or expression vector available in the technical field, and the DNA encoding the above AP is converted to an appropriate restriction enzyme and ligase, or, if necessary, a linker or adapter DNA. Can be prepared by ligation using In addition, gene fragments amplified using a DNA polymerase that adds a single base to the amplification end, such as Taq polymerase, can be connected to a vector by TA cloning.

  Examples of vectors include plasmids derived from E. coli, such as pBR322, pBR325, pUC18, pUC19, pBluescript SK (-), pBluescript KS (+), etc., yeast-derived plasmids such as pSH19, pSH15, etc. pUB110, pTP5, pC194, etc. are mentioned. Moreover, bacteriophage such as λ phage, papovirus such as SV40, bovine papilloma virus (BPV), retrovirus such as Moloney murine leukemia virus (MoMuLV), adenovirus (AdV), adeno-associated virus (AAV), Illustrative are animal and insect viruses such as vaccinia virus, baculovirus.

In particular, the present invention provides an AP expression vector in which DNA encoding AP is placed under the control of a promoter functional in the intended host cell. As a vector to be used, a promoter region that functions in various prokaryotic and / or eukaryotic host cells and can control transcription of a gene located downstream thereof (for example, trp promoter when the host is E. coli, When the host is Bacillus subtilis, such as lac promoter, lectA promoter, etc. When the host is a mammalian cell, such as SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. SV40-derived early promoter, MoMuLV-derived long terminal repeat, adenovirus-derived early promoter, and the like, and a transcription termination signal of the gene, that is, a terminator region, The terminator region is not particularly limited as long as it is linked via a sequence containing at least one restriction enzyme recognition site, preferably a unique restriction site that cleaves the vector only at that site. It further contains a selection marker gene for selection of transformants (a gene that confers resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, a gene that complements auxotrophic mutations, etc.) It is preferable. Further, when the DNA encoding the AP to be inserted does not contain a start codon and a stop codon, the start codon (ATG or GTG) and the stop codon (TAG, TGA, TAA) are respectively located downstream of the promoter region and the terminator region. A vector contained upstream of is preferably used.
When a bacterium is used as a host cell, the expression vector generally needs to contain a replicable unit capable of autonomous replication in the host cell in addition to the promoter region and terminator region described above. The promoter region includes an operator and a Shine-Dalgarno (SD) sequence in the vicinity of the promoter.
When yeast, animal cells, or insect cells are used as the host, the expression vector preferably further includes an enhancer sequence, 5 ′ and 3 ′ untranslated regions of AP mRNA, a polyadenylation site, and the like.

  Host organisms into which the prepared recombinant vector is introduced include bacteria such as Escherichia coli and Bacillus subtilis for which recombinant expression systems have been established, microbial hosts such as actinomycetes, bacilli, and yeast, insect cells, animal cells, higher plants, etc. Among them, it is preferable to use Escherichia coli having excellent protein expression ability. In addition, it is also preferable to use an E. coli host from the viewpoint that the problem seen in CIAP can be solved by using an AP having no sugar chain. As a method for introducing the recombinant plasmid, in addition to introduction by electroporation, introduction by heat shock is possible if the host is made competent by chemical treatment such as calcium chloride. Selection for the presence or absence of transfer of the target recombinant plasmid into the host vector may be performed by searching for microorganisms that simultaneously express markers represented by various drug resistance genes of the vector holding the target DNA and AP activity, For example, a microorganism that grows in a selective medium based on a drug resistance marker and expresses AP may be selected.

2-3-4. Culture The AP of the present invention can be produced by culturing a transformant containing the AP expression vector prepared as described above in a medium, and collecting and collecting AP from the resulting culture.

  The medium to be used preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, and sucrose. Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, large Examples include soybean cake and potato extract. Further, other nutrients [for example, inorganic salts (for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.)] may be included as desired. .

Culturing is performed by methods known in the art. Specific media and culture conditions used according to the host cells are exemplified below, but the culture conditions in the present invention are not limited to these.
When the host is a bacterium, actinomycetes, yeast, filamentous fungus or the like, for example, a liquid medium containing the above nutrient source is suitable. Preferably, the medium has a pH of 5-9. When the host is Escherichia coli, LB medium and M9 medium [Miller. J. et al. , Exp. Mol. Genet, p. 431, Cold Spring Harbor Laboratory, New York (1972)]. Culturing can be performed usually at 14 to 43 ° C. for about 3 to 72 hours with aeration and stirring as necessary. When the host is Bacillus subtilis, it can be carried out usually at 30 to 40 ° C. for about 16 to 96 hours with aeration and stirring as necessary. When the host is yeast, as a medium, for example, Burkholder minimal medium [Bostian. K. L. et al, Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)], and the pH is preferably 5-8. The culture is usually carried out at about 20 to 35 ° C. for about 14 to 144 hours, and if necessary, aeration and agitation can be performed.
When the host is an animal cell, the medium is, for example, a minimal essential medium (MEM) containing about 5 to 20% fetal calf serum [Science, 122, 501 (1952)], Dulbecco's modified Eagle medium (DMEM) [Virology, 8 , 396 (1959)], RPMI 1640 medium [J. Am. Med. Assoc. , 199, 519 (1967)], 199 medium [Proc. Soc. Exp. Biol. Med. , 73, 1 (1950)] or the like. A metal salt for stabilizing AP may be added to these media, and as such a metal salt, a magnesium salt and / or a zinc salt is preferably used. These metal salts may be set within a range not toxic to the cells to be cultured. The final concentration range is 0.001 mM to 10 mM for a magnesium salt, and 0.001 mM to 1 mM for a zinc salt. Although illustrated, it is not limited to this range. The pH of the medium is preferably about 6 to 8, and the culture is usually carried out at about 30 to 40 ° C. for about 15 to 72 hours, and if necessary, aeration and stirring can be performed.
When the host is an insect cell, for example, Grace's medium containing fetal calf serum [Proc. Natl. Acad. Sci. USA, 82, 8404 (1985)], etc., and the pH is preferably about 5-8. The culture is usually performed at about 20 to 40 ° C. for 15 to 100 hours, and if necessary, aeration and stirring can be performed.

2-3-5. Purification Purification of AP can be performed by appropriately combining various commonly used separation techniques depending on the fraction in which AP activity exists.
The AP present in the culture medium is obtained by centrifuging or filtering the culture to obtain a culture supernatant (filtrate), for example, salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration. By appropriately selecting a known separation method such as non-denaturing PAGE, SDS-PAGE, ion exchange chromatography, hydroxylapatite chromatography, affinity chromatography, reverse phase high performance liquid chromatography, isoelectric focusing, etc. Can be obtained.

  On the other hand, AP present in the cytoplasm collects cells by centrifuging or filtering the culture and suspending it in an appropriate buffer, for example, sonication, lysozyme treatment, freeze-thawing, osmotic shock, and / or triton. -After disrupting (dissolving) the cells and the organelle membrane by treatment with a surfactant such as -X100, the debris is removed by centrifugation or filtration to obtain a soluble fraction. It can be isolated and purified by treating with a method.

As a means for obtaining recombinant AP quickly and conveniently, an amino acid sequence (for example, a base such as histidine, arginine, lysine, etc.) that can be adsorbed to a metal ion chelate at a certain part (preferably N or C terminal) of the AP coding sequence A DNA sequence encoding an amino acid sequence (preferably a sequence consisting of histidine) is added by genetic manipulation and expressed in a host cell, and the metal ion chelate is immobilized from the AP active fraction of the cell culture. A method of separating and recovering AP by affinity with the carrier thus obtained is preferably exemplified. The DNA sequence encoding the amino acid sequence that can be adsorbed to the metal ion chelate is obtained by, for example, using a hybrid primer in which the DNA sequence is linked to the base sequence encoding the C-terminal amino acid sequence of AP in the process of cloning the DNA encoding AP The DNA can be introduced into the AP coding sequence by performing PCR amplification or by inserting the DNA encoding AP in frame into an expression vector containing the DNA sequence before the stop codon. The metal ion chelate adsorbent used for purification contains transition metals such as cobalt, copper, nickel, iron divalent ions, or iron, aluminum trivalent ions, etc., preferably cobalt or nickel divalent ions. The solution is prepared by contacting a ligand, such as an iminodiacetic acid (IDA) group, a nitrilotriacetic acid (NTA) group, a tris (carboxymethyl) ethylenediamine (TED) group, etc., in contact with the ligand. The matrix part of the chelate adsorbent is not particularly limited as long as it is a normal insoluble carrier.
Alternatively, affinity purification can be performed using glutathione-S-transferase (GST), maltose binding protein (MBP), HA, FLAG peptide, or the like as a tag.

  In the purification step, treatment such as membrane concentration, concentration under reduced pressure, addition of an activator and a stabilizer can be performed as necessary. The solvent used in these steps is not particularly limited, but various buffer solutions represented by K-phosphate buffer solution, Tris-HCl buffer solution, GOOD buffer solution and the like having a buffer capacity in the range of about pH 6-9 are preferable. . In order to ensure the stability of the present AP, a metal salt may be added to these buffers, and as such a metal salt, a magnesium salt and / or a zinc salt is preferably used. These metal salts may be set within a range effective for stabilizing AP, and the final concentration is 0.001 mM to 10 mM for magnesium salts, and 0.001 mM to 1 mM for zinc salts. However, it is not limited to this range.

  When the AP thus obtained is a free form, the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the protein is obtained as a salt, a method known per se Alternatively, the salt can be converted into a free form or other salt by a method analogous thereto.

  The purified enzyme is liquid and can be used for industrial use, but can also be pulverized or further granulated. The liquid enzyme is pulverized by lyophilization by a conventional method.

2-4. (4) Method of biochemical synthesis from nucleic acid encoding AP using a cell-free transcription / translation system Further, the AP of the present invention is a rabbit reticulocyte lysate using RNA corresponding to the DNA encoding the template as a template. It can also be synthesized by in vitro translation using a cell-free protein translation system consisting of wheat germ lysate, E. coli lysate and the like.
The RNA encoding the AP of the present invention is purified from the host cell expressing the RNA using a conventional method, or the RNA encoding the AP of the present invention is used as a template and RNA polymerase is used as a template. It can be obtained by preparing cRNA using a cell-free transcription system. As the cell-free protein transcription / translation system, a commercially available one can be used, or a method known per se, specifically, an Escherichia coli extract can be obtained from Pratt J. M.M. et al. “Transcribation and Translation”, Hames B .; D. and Higgins S. J. et al. eds. , IRL Press, Oxford 179-209 (1984). Commercially available cell lysates include those derived from E. coli. Examples include E. coli S30 extract system (Promega) and RTS 500 Rapid Translation System (Roche), and those derived from rabbit reticulocytes are from Rabbit Reticulocyte Lysate System (Promega) PROTEIOS ^™ (manufactured by TOYOBO) and the like. Of these, those using wheat germ lysate are preferred. As a method for producing wheat germ lysate, for example, Johnston F. et al. B. et al. , Nature, 179: 160-161 (1957) or Erickson A. et al. H. et al. , Meth. Enzymol. 96: 38-50 (1996).

As a system or apparatus for protein synthesis, a batch method [Pratt, J. et al. M.M. et al. (1984) above] and a continuous cell-free protein synthesis system that continuously supplies amino acids, energy sources, and the like to the reaction system [Spirin A. et al. S. et al. , Science, 242: 1162-1164 (1988)], dialysis method (Kigawa et al., 21st Japan Molecular Biology Society, WID6), or multi-layer method (PROTEIOS ^™ Heat germ cell-free protein synthesis instructions) : Manufactured by TOYOBO). Furthermore, a method of supplying template RNA, amino acid, energy source and the like to the synthesis reaction system when necessary, and discharging the synthesized product and degradation product when necessary (Japanese Patent Laid-Open No. 2000-333673) can be used.

2-5. Conjugates Another aspect of the present invention is a conjugate that is labeled with the above-mentioned alkaline phosphatase, and the substance to be labeled is, for example, a nucleic acid probe, a biological substance such as biotin, a polypeptide, an avidin, or an antibody. These proteins are preferably selected. As the labeling method, methods such as a maleimide method, a pyridyl disulfide method, and a glutaraldehyde method can be applied, and the labeling method, the functional group to be used, the purpose of use, etc. can be used properly. For methods for preparing AP-labeled antibodies and AP-labeled antigens used in ELISA and immunodiagnostic reagents, and immunoassay methods using them, “Ultrasensitive Enzyme Immunoassay” (Ishikawa Yuji, published by Academic Publishing Center), etc. Be familiar with.

  In an example of a typical immunoassay method, first, a solution containing a primary antibody of a substance to be measured is added to and incubated on a solid phase. This solid phase may be a container used as a reaction layer, or may be magnetic beads prepared separately from the reaction layer. After adsorbing the primary antibody, remove the solution and rinse several times with wash buffer to remove non-adsorbed material. As the washing buffer, one having a buffering ability in a neutral pH range where the antibody can stably exist can be used, and a surfactant may be included to enhance the washing ability. The washed solid phase is further immersed in a solution containing a protein such as bovine serum albumin or inactivated AP, and blocked by incubation. The solid phase after blocking is washed with the above-described washing buffer, then brought into contact with the sample to be measured, and incubated for a fixed time, thereby adsorbing the measurement target to the primary antibody. After completely removing the sample solution and washing with the above washing buffer, adding a solution containing an AP-labeled secondary antibody and incubating for a certain period of time, the object to be measured captured by the primary antibody on the solid phase is subjected to AP. A labeled secondary antibody is adsorbed. After completely removing the solution and washing with the above washing buffer, AP substrate is added to detect the activity. As an AP substrate, p-nitrophenyl phosphate or 5-bromo-4-chloro-3-indolyl phosphate is used when the activity detection method is a colorimetric method, and 4-methylumbellite is used when the method is a fluorescence method. If ferryl phosphate is a luminescence method, various luminescent substrates composed of 1,2-dioxetane or acridan luminescent substrates can be used. Among these, the AP of the present invention is particularly excellent in reactivity with a luminescent substrate, and a method using this is more preferably selected. Examples of the luminescent substrate include, but are not limited to, AMPPD, CSPD, CDP-star, Lumigen PPD, Lumi-Phos530, and APS-5. The substance to be measured is quantified from a calibration curve prepared in advance using a standard solution for the substance to be measured.

  An example of the configuration of a typical immunoassay reagent is as follows: a reaction layer, a solid phase on which a primary antibody is immobilized and blocked with a protein such as bovine serum albumin or inactivated AP, and a standard solution of an antigen to be measured , A secondary antibody labeled with AP, a washing solution for washing after reacting a sample or secondary antibody in a reaction layer, a substrate solution of AP, and a use manual. As an AP substrate, p-nitrophenyl phosphate or 5-bromo-4-chloro-3-indolyl phosphate is used when the activity detection method is a colorimetric method, and 4-methylumbellite is used when the method is a fluorescence method. If ferryl phosphate is a luminescence method, various luminescent substrates composed of 1,2-dioxetane or acridan luminescent substrates can be used. Among these, the AP of the present invention is particularly excellent in reactivity with a luminescent substrate, and a method using this is more preferably selected. Examples of the luminescent substrate include, but are not limited to, AMPPD, CSPD, CDP-star, Lumigen PPD, Lumi-Phos530, and APS-5.

3. Activity measurement examples The AP activity described in the present invention is measured by the following method unless otherwise specified.
First, the following solutions A and B are prepared.
A: 1M diethanolamine buffer (pH 9.8)
B: 0.67M p-nitrophenyl phosphate (dissolves in solution A)
Prepare 2.9 ml of solution A and 0.1 ml of solution B in a cuvette (optical path length = 1.0 cm), and preheat at 37 ° C. for 5 minutes. Add 0.1 ml of AP solution and mix gently. Record the change in absorbance at 405 nm for 3 to 5 minutes using a spectrophotometer controlled at 37 ° C. with water as a control, and change the absorbance per minute from the linear portion. (ΔODTEST). In the blind test, 0.1 ml of a buffer in which the enzyme is dissolved is added instead of the enzyme, and the change in absorbance per minute is similarly determined (ΔODBLANK). Using these values, the AP activity is obtained from the following formula.

AP activity (U / ml) = {(ΔODTEST−ΔODBLANK) × 3.1} / {18.2 × 1.0 × 0.1}

3.1: Amount of reaction solution after addition of AP solution (ml)
18.2: Molar molecular extinction coefficient (cm ² / μmol) of p-nitrophenol under the above measurement conditions
1.0: Optical path length (cm)
0.1: Amount of enzyme solution added (ml)

4). Example of calculation of protein quantification and specific activity The amount of protein described in the present invention is measured by measuring absorbance at 280 nm. In other words, the enzyme solution was diluted with distilled water so that the absorbance at 280 nm was in the range of 0.1 to 1.0, and the absorbance at 280 nm (Abs) was obtained using a zero point correction using distilled water. Measure. The protein concentration described in the present invention approximates 1 Abs≈1 mg / ml, and is expressed by a value obtained by multiplying the absorbance measurement and the measured solution dilution rate. The specific activity described in the present invention is the activity (U / mg) of AP per mg as the amount of protein according to this measurement method, and the AP activity at this time is obtained by measuring according to the above activity measurement example. Value.

  EXAMPLES Hereinafter, although this invention is shown as an Example concretely, this invention is not limited to a following example.

Cloning of AP gene
Shewanella fodinae NBRC105216 strain was inoculated into 5 ml LB medium in a test tube and cultured with shaking at 30 ° C. for 24 hours. The culture solution was placed in a 1.5 ml Eppendorf tube, centrifuged at 12000 rpm for 5 minutes with a cooling centrifuge, and the supernatant was removed by suction to obtain bacterial cells. From this bacterial cell, genomic DNA was obtained using a genomic DNA extraction kit (manufactured by TOYOBO, NPK-1) according to the manual attached to the kit. This genomic DNA was digested with restriction enzymes BamHI or BglII and purified using a DNA purification kit (manufactured by TOYOBO, NPK-6) to remove the restriction enzymes. This DNA fragment was mixed with pBR322 that had been digested with BamHI and purified, and an equal amount of ligation solution (Ligation High, manufactured by TOYOBO) was added to the mixture and incubated at 16 ° C. overnight. This ligation solution was added to Escherichia coli JM109 strain competent cells (manufactured by TOYOBO, competent high JM109), and the plasmid was transformed by heat shock to prepare a genomic DNA library of the T3-3 strain. This library was inoculated into an LB agar medium containing 50 μg / ml BCIP and 100 μg / ml ampicillin and cultured at 30 ° C. to form transformed colonies. Of the formed colonies, the blue colony was attached with a toothpick, inoculated into 5 ml LB medium (containing 100 μg / ml ampicillin) in a test tube, and cultured with shaking at 30 ° C. for 16 hours. From this culture solution, a plasmid (pLPP105216-1) containing the NBRC105216 strain-derived AP gene was extracted and purified using a plasmid extraction kit (manufactured by TOYOBO, NPK-3). The obtained plasmid had an insert of about 6 kb, and by sequencing this sequence, the sequence of the full length AP gene and its adjacent region was determined. The determined base sequence of the AP gene is shown in SEQ ID NO: 1, and the amino acid sequence deduced from this base sequence is shown in SEQ ID NO: 2.

Expression of AP in E. coli host The plasmid (pLPP105216-1) obtained in Example 1 was introduced into E. coli strain C600 by electroporation, applied to an LB agar medium containing 100 μg / ml ampicillin, and cultured at 30 ° C. overnight. By doing so, a transformed colony was formed. This transformed colony was inoculated with one platinum loop in 60 ml LB medium (containing 100 μg / ml ampicillin) in a 500 ml Sakaguchi flask and cultured overnight at 30 ° C. and 180 rpm. The total amount of this culture solution was added to a 6 L production medium (1.2% peptone, 2.4% yeast extract, 0.1% NaCl, 0.1 mM zinc sulfate, 100 μg / ml ampicillin, pH 7.0 in a 10 L jar fermenter. ), And aerated with aeration at an aeration rate of 2 L / min, agitation of 380 rpm, and a temperature of 30 ° C. for 48 hours. This produced 4000 U / ml AP.

Purification of E. coli recombinant AP The culture solution obtained in Example 3 was dispensed into a 500 ml centrifuge tube, centrifuged at 8000 rpm for 30 minutes with a high-speed cooling centrifuge, and the supernatant was removed by decantation to obtain bacterial cells. The cells were suspended in 1.5 L of 20 mM Tris-HCl buffer (pH 7.5) and crushed with a French press crusher at a pressure of 80 MPa. 4% of 5% (w / v) polyethyleneimine was added to the crushed liquid, and the resulting solid content was removed by centrifugation at 8000 rpm for 30 minutes in a high-speed cooling centrifuge. 0.15 saturated ammonium sulfate was dissolved in this solution, and the resulting solid content was removed by centrifuging at 8000 rpm for 30 minutes with a high-speed cooling centrifuge. Further, ammonium sulfate was added and dissolved so that the final concentration became 0.55 saturation, and the mixture was centrifuged at 8000 rpm for 30 minutes with a high-speed cooling centrifuge, and the supernatant was removed by decantation to obtain a precipitate containing AP. This precipitate was dissolved by adding 90 ml of 20 mM Tris-HCl buffer (pH 7.5, containing 1 mM magnesium chloride). This solution was desalted using G-25 Sepharose gel (manufactured by GE Healthcare) buffered with 20 mM Tris-HCl buffer (pH 7.5 and containing 1 mM magnesium chloride). This solution is adsorbed on DEAE Sepharose gel (manufactured by GE Healthcare) buffered with 20 mM Tris-HCl buffer (pH 7.5 and containing 1 mM magnesium chloride), and the NaCl concentration is increased to 0.5 M with the same buffer. Gradient elution was performed. Fractions having AP activity were collected, and ammonium sulfate was dissolved so as to be 0.2 saturated. This solution was applied to Phenyl Sepharose gel (manufactured by GE Healthcare) buffered with 20 mM Tris-HCl buffer (pH 7.5, containing 0.2 saturated ammonium sulfate and 1 mM magnesium chloride), and the same buffer was continuously passed through. The non-adsorbed fraction was collected, and gradient elution was performed by lowering the ammonium sulfate concentration to 0 with the same buffer. The AP-containing fractions were pooled and gel filtered on a Sephadex 200 column buffered with 20 mM triethanolamine (pH 7.5, containing 0.3 M sodium chloride, 1 mM magnesium chloride and 0.1 mM zinc sulfate). Fractions containing were collected. The specific activity of this AP solution was measured and found to be 5670 U / mg.

Thermal stability of T3-3 strain-derived recombinant AP The AP prepared in Example 4 was adjusted to a concentration of 10 μg / mL using 50 mM triethanolamine, 1 mM magnesium chloride, 0.1 mM zinc sulfate (pH 7.0). Diluted. This solution was incubated at 60 ° C., 65 ° C., and 70 ° C. for 1 hour, and the AP activity before and after incubation was compared. Moreover, the same process was performed also about CIAP with a specific activity of 6000 U / mg as a comparison object. Each incubation temperature and the activity remaining rate after incubation are shown in FIG. The AP of the present invention maintained an activity of 90% or more even at 65 ° C. On the other hand, CIAP has a residual rate of 29% after treatment at 60 ° C. and 2% at 65 ° C., which indicates that the AP of the present invention has higher thermal stability than CIAP.

Reactivity of AP to Luminescent Substrate For the AP prepared in Example 4, 0.5 U / ml or 0.2 μm each using 20 mM Tris-HCl buffer (containing pH 7.5, 1 mM magnesium chloride and 0.1 mM zinc sulfate). Diluted to 1 U / ml. 5 μL of this AP dilution was dispensed into a 96-well ELISA plate. Here, 50 μL of any of APS-5, Lumifos 530 (manufactured by Lumigen) and CDP-star (Roche) is added as a luminescent substrate, and a multi-label plate counter (Perkin Elmer, Wallac 1420 ARVO MX) is added. The emission intensity was measured. The results are shown in Table 1. It was confirmed that the AP of the present invention has sufficient reactivity with a general-purpose luminescent substrate.

  The alkaline phosphatase of the present invention is useful not only as a labeling enzyme for immunoassay but also as a labeling enzyme for probe hybridization and western blotting. Furthermore, the alkaline phosphatase of the present invention can be used as an enzyme for genetic engineering for dephosphorylating a DNA fragment.

Claims (9)

An alkaline phosphatase comprising the polypeptide of any one of (A) to (C) below.
(A) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.
In the amino acid sequence set forth in (B) SEQ ID NO: 2, one or several amino acid residues deleted, substituted, inserted, or added so composed sequences, have a alkaline phosphatase activity, the specific activity 5000U / Mg or more of the polypeptide.
(C) consists of the amino acid sequence at least 90% amino acid sequence identity with that set forth in SEQ ID NO: 2, have a alkaline phosphatase activity, a polypeptide specific activity is 5000 U / mg or more. DNA shown in any one of the group consisting of the following (A), (B), (C), (E) and (F) .
(A) DNA consisting of the base sequence set forth in SEQ ID NO: 1.
(B) DNA encoding the amino acid sequence set forth in SEQ ID NO: 2.
(C) identity to the nucleotide sequence described in SEQ ID NO: 1 is the nucleotide sequence is 90% or more and have a alkaline phosphatase activity, encoding a polypeptide specific activity is 5000 U / mg or more DNA to do ;
In the nucleotide sequence described in (E) SEQ ID NO: 1, substitution of one or several bases, deletion, a nucleotide sequence which is inserted or added, have a alkaline phosphatase activity, the specific activity 5000 U / DNA encoding a polypeptide that is greater than or equal to mg ,
(F) in the amino acid sequence set forth in SEQ ID NO: 2, one or several amino acid residues are substituted, deleted, inserted or added amino acid sequence, and, have a alkaline phosphatase activity, the specific activity DNA encoding a polypeptide of 5000 U / mg or more . A vector incorporating the DNA according to claim 2. A transformant in which a host cell is transformed with the vector according to claim 3. The transformant according to claim 4, wherein the host cell is Escherichia coli. A method for producing alkaline phosphatase, comprising culturing a microorganism having the ability to produce alkaline phosphatase according to claim 1, and collecting a protein having alkaline phosphatase activity from the obtained culture. The method for producing alkaline phosphatase according to claim 6 , wherein the microorganism having the ability to produce alkaline phosphatase according to claim 1 is derived from the genus Shewanella. The method for producing alkaline phosphatase according to claim 6, wherein the microorganism having the ability to produce alkaline phosphatase according to claim 1 is the transformant according to claim 4 or 5. A conjugate formed by labeling the alkaline phosphatase according to claim 1.