JP2016168051A - Modified alkaline phosphatases - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an alkaline phosphatase that maintains its activity in immunological assays at the same level before and after the modification thereof, more specifically, to provide an alkaline phosphatase of which activity will not lower after the introduction of a maleimide group using various maleimide reagents used for the preparation of alkaline phosphatase conjugates.SOLUTION: The invention provides an alkaline phosphatase modified by substituting the lysine residue near the active center with another amino acid residue.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a modified alkaline phosphatase and a method for modifying 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). Currently, most of the APs used for this EIA are bovine small intestine derived APs (hereinafter also referred to as CIAPs). 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 with acridan as the basic skeleton are also on the market, realizing high measurement sensitivity in immunoassay.

  One of the important issues in immunoassay is to achieve high sensitivity. As attempts to achieve high sensitivity, progress has been made so far by increasing the number of labeled enzyme molecules adsorbed per antigen molecule and developing a highly sensitive substrate for the labeled enzyme. However, the sensitivity of today's immunodiagnostic methods does not always satisfy the requirements. For example, in the case of influenza detection kits by immunodiagnostics, even if the result is negative, the suspicion of infection may not be completely eliminated. That is also true. Depending on the measurement target, the concentration in the sample is often 1 pM or less, and for the purpose of shortening the time required for diagnosis, it can be said that improvement of sensitivity is an eternal theme.

For the purpose of increasing the sensitivity, CIAP having a plurality of isozymes so far is isolated in the process of purification, or a high specific activity CIAP gene is identified and recombinantly produced. Furthermore, those skilled in the art respond to the demand for high sensitivity by increasing specific activity by introducing site-specific amino acid mutations important for realizing high specific activity (Patent Document 1, etc.).
On the other hand, the inventors have found an AP derived from a bacterium belonging to the genus Shewanella, having a high specific activity comparable to CIAP, and high reactivity to various luminescent substrates widely used in immunoassays. The inventors have also succeeded in producing AP by obtaining a gene encoding the AP and culturing a microorganism transformed with the gene. These inventions have been internationally filed as PCT / JP2012 / 053924.

  In immunoassays, AP is often used as an enzyme for labeling a protein that can become an antibody or antigen including forms such as reduced IgG and Fab. As the labeling method, the glutaraldehyde method, the periodic acid method, and the maleimide method are preferably used. For the labeling of the antibody, the IgG hinge portion that does not substantially affect the antigen-antibody reaction is labeled. The maleimide method that can be used as a target is most preferably used. In this method, AP is first modified with a maleimide group introduction reagent such as N- (6-maleimidocaproyloxy) succinimide (hereinafter also referred to as EMCS) or N- (4-maleimidobutyryloxy) succinimide (hereinafter also referred to as GMBS). Then, it is converted into maleimide and coupled with the SH group to be labeled to give a conjugate. At this time, it has been pointed out that the AP activity as a conjugate is significantly lower than that of unlabeled AP, and the ratio varies depending on the literature, but is also said to be an activity remaining rate of 50 to 60%. (Non-Patent Document 1). However, there has been no report on the cause of such a decrease in activity and a means for solving it.

Japanese Patent No. 4295386

"Ultra-sensitive enzyme immunoassay" (Ishikawa Yuji, published by the Academic Publishing Center, 1993)

  An object of the present invention is to provide an alkaline phosphatase that can have an activity equivalent to that before modification even after modification in immunoassay. More specifically, it is to provide an alkaline phosphatase that does not cause a decrease in activity even after introduction of a maleimide group with various maleimidating reagents used for preparing an alkaline phosphatase conjugate in immunoassay.

  As a result of intensive studies, the present inventors have found that an alkali whose activity does not decrease even after maleimidation treatment for enzyme labeling by substituting a lysine residue located in the vicinity of the AP active center with another amino acid residue. Successful production of phosphatase has led to the completion of the present invention.

That is, the present invention has the following configuration.
(1)
A modified alkaline phosphatase obtained by substituting a lysine residue located near the active center of alkaline phosphatase with another amino acid residue.
(2)
The modified alkaline phosphatase according to (1), wherein the alkaline phosphatase is derived from bacteria.
(3)
The modified alkaline phosphatase according to (2), wherein the bacterium is of the genus Shewanella.
(4)
The modified alkaline phosphatase according to (1), wherein the alkaline phosphatase is derived from a mammal.
(5)
The modified alkaline phosphatase according to (1), wherein the alkaline phosphatase is derived from bovine small intestine (6)
The modified alkaline phosphatase according to (1), wherein in the amino acid sequence shown in SEQ ID NO: 2, the amino acid corresponding to the 161st lysine and / or the 184th lysine is substituted with another amino acid.
(7)
The modified alkaline phosphatase according to (1), wherein an amino acid corresponding to the 161st lysine and / or the 184th lysine of the amino acid sequence shown in SEQ ID NO: 2 is substituted with serine or arginine.
(8)
The modified alkaline phosphatase according to (1), wherein the amino acid corresponding to the 161st lysine and / or the 184th lysine in the amino acid sequence shown in SEQ ID NO: 2 is substituted with serine.
(9)
The modified alkaline phosphatase according to (1), wherein the amino acid corresponding to the 100th lysine in the amino acid sequence shown in SEQ ID NO: 11 is substituted with another amino acid.
(10)
The modified alkaline phosphatase according to (1), wherein the amino acid corresponding to the 81st lysine in the amino acid sequence shown in SEQ ID NO: 12 is substituted with another amino acid.
(11)
The modified alkaline phosphatase according to (1), wherein the amino acid corresponding to the 100th lysine in the amino acid sequence shown in SEQ ID NO: 13 is substituted with another amino acid.
(12)
The modified alkaline phosphatase according to (1), wherein an amino acid corresponding to the 100th lysine and / or the 127th lysine is substituted with another amino acid in the amino acid sequence shown in SEQ ID NO: 14.
(13)
The modified alkaline phosphatase according to (1), wherein the amino acid corresponding to the 157th lysine and / or the 180th lysine of the amino acid sequence shown in SEQ ID NO: 16 is substituted with another amino acid.
(14)
The modified alkaline phosphatase according to (1), wherein the amino acid corresponding to the 157th lysine and / or the 180th lysine of the amino acid sequence shown in SEQ ID NO: 18 is substituted with another amino acid.
(15)
The modified alkaline phosphatase according to (1), wherein an amino acid corresponding to the 81st lysine and / or the 87th lysine in the amino acid sequence shown in SEQ ID NO: 10 is substituted with another amino acid.
(16)
A conjugate formed by labeling the alkaline phosphatase according to any one of (1) to (15).
(17)
An immunoassay method using the conjugate according to (16).
(18)
An immunoassay reagent comprising the conjugate according to (16).
(19)
Hereinafter, a method for producing a modified alkaline phosphatase, including the steps (A) to (D).
(A) A step of identifying or estimating a lysine residue located near the active center of alkaline phosphatase.
(B) A step of replacing a codon on the alkaline phosphatase gene encoding the lysine residue specified or estimated in (A) with a codon encoding another amino acid.
(C) A step of transforming the gene prepared in (B) into a host cell and culturing the transformant to express the gene.
(D) A step of purifying the modified alkaline phosphatase that is the expression product in (C).
(20)
Hereinafter, the production method of the modified alkaline phosphatase according to (19), including the steps (A) to (D).
(A) An amino acid residue corresponding to the 161st lysine and / or the 184th lysine of the amino acid sequence shown in SEQ ID NO: 2 is obtained by aligning the amino acid sequence of alkaline phosphatase to be modified with the amino acid sequence shown in SEQ ID NO: 2 Estimating process.
(B) A step of replacing the codon on the alkaline phosphatase gene encoding the lysine residue estimated in (A) with a codon encoding another amino acid.
(C) A step of transforming the gene prepared in (B) into a host cell and culturing the transformant to express the gene.
(D) A step of purifying the modified alkaline phosphatase that is the expression product in (C).

  According to the present invention, alkaline phosphatase useful as a labeling enzyme for immunoassay and an alkaline phosphatase-labeled antibody capable of detecting a target substance with high sensitivity can be provided.

The relationship between the number of maleimide group introduction | transductions and a specific activity residual rate at the time of maleimidizing the wild type and modified enzyme of AP derived from T3-3 strain is shown. The relationship between the number of maleimide group introductions and the reactivity to the luminescent substrate per unit protein amount when T3-3 strain-derived mutant AP and bovine small intestine-derived AP (CIAP) are maleimidized is shown.

(1)
The present invention is an alkaline phosphatase obtained by substituting a lysine residue located near the active center with another amino acid.

(1-1) Alkaline phosphatase Alkaline phosphatase to be mutated is not particularly limited as long as it can be used for immunoassay. Examples thereof include those derived from mammals and bacteria.
Preferable examples of the mammal-derived one include bovine-derived alkaline phosphatase and pink lobster-derived alkaline phosphatase. Among bovine origin, alkaline phosphatase isolated from calf small intestine is a preferred example.
Examples of those derived from bacteria include alkaline phosphatase derived from Bacillus bacteria, alkaline phosphatase derived from Shewanella bacteria, and the like. Among them, an alkaline phosphatase derived from a genus Shewanella is also a more preferable example. As a bacterium belonging to the genus Shewanella, Shewanella fodinae (for example, NBRC105216 strain), Shewanella chillikensis (for example, NBRC105217 strain), Shewanella baltica (for example, OS185 strain), Shewanella sp. T3-3 strain, Shewanella sp. W3-18-1 strain Etc. can be exemplified. Among these, those with NBRC numbers are strains stored in the Biotechnology Center, Biotechnology Center, National Institute of Technology and Evaluation, and may be sold for sale through a prescribed procedure. it can.

  Examples of other organisms from which APs to be mutated are derived include microorganisms present in water systems such as soil, rivers, and lakes or in the ocean, microorganisms that are resident on the surface or inside various animals and plants, and the like. Microorganisms that grow in a low temperature environment, a high temperature environment such as a volcano, an oxygen-free / high-pressure / no-light environment such as the deep sea, and a special environment such as an oil field may be used as the isolation source.

  APs to be mutated include not only APs directly isolated from microorganisms, but also those in which the isolated APs have been modified by amino acid sequences or the like by protein engineering methods, or by genetic engineering methods. included. For example, the enzyme obtained by modifying the enzyme obtained from the above-mentioned genus Shewanella or the like and further adding the mutation of the present invention may be used.

  The reason why the present invention does not limit the origin of alkaline phosphatase is as follows. EMCS and GMBS, which are maleimidation reagents, are introduced onto the enzyme surface by coupling lysine residues on the enzyme surface with active esters of these reagents. As a cause of the above-described decrease in activity, the inventors do not occur because the lysine residue in the vicinity of the active center couples with the maleimidating reagent, and the introduced side chain prevents the substrate from contacting the active center. Estimated. As shown in the examples described later, it was shown that the mutant AP in which the lysine residue near the active center was substituted has a higher activity retention rate after maleimidation than the wild-type AP. This result strongly suggests that the above estimation is correct. Such a decrease in activity after maleimidation can theoretically occur in any AP if a lysine residue is present in the vicinity of the active center. The avoidance of the decrease in activity is considered to be effective for any AP as well. There has been no mention of the possibility of measures to avoid such a decrease in the activity of alkaline phosphatase after modification and labeling.

(1-2) Identification of lysine residue located near the active center of alkaline phosphatase In principle, the specification of lysine residue located near the active center of alkaline phosphatase is known in this document for the three-dimensional structure and the active center. This is performed using the 3D structure information of the AP. An example of such an AP is a Shewanella sp. AP-1 derived AP (ProteinDataBank registration number: 3A52).

  In this case, first, an amino acid residue in the vicinity of the active center is specified for an AP having a known three-dimensional structure, and then an amino acid sequence is aligned between the AP having the known three-dimensional structure and the AP to introduce a mutation. Thus, an amino acid residue located in the vicinity of the active center of the AP into which a mutation is to be introduced can be estimated. If lysine is present among these residues, that is, it can be assumed that it is a lysine residue near the active center.

The primary sequence alignment can be calculated using various algorithms used to calculate amino acid identity, for example, using a commercially available analysis tool available through a telecommunication line (Internet). .
In this document, the National Biotechnology Information Center (NCBI) homology algorithm BLAST (Basic local alignment search tool) http: // www. ncbi. nlm. nih. Alignment is performed using default (initial setting) parameters in gov / BLAST /.

  A method for identifying an amino acid located near the active center from a three-dimensional structure has various methods capable of calculating the distance between atoms from a PDB file including coordinate information of all atoms constituting the AP and inorganic phosphate as a substrate analog. Software can be used, and in this book, MolFeat manufactured by Firelux is used. Judgment that it is in the vicinity of the active center is based on the fact that at least a part of the amino acid residue is located within 15 angstroms from the phosphorus atom constituting the phosphate group of inorganic phosphate as a substrate analog. It is presumed that the 184th lysine of AP derived from Shewanella sp. T3-3 strain shown in SEQ ID NO: 2 is just 15 angstroms away from the substrate, and after modification by substituting this residue as shown in the Examples The activity remaining rate of is improved. From this, it can be understood that lysine residues at a distance of at least 15 angstroms can be expected to have an effect of avoiding a decrease in activity due to maleimidation by substitution with other amino acids.

  If the lysine residue located near the active center cannot be identified by the above method, it can be identified from the three-dimensional structure information of the AP to be mutated. Crystallization of a high-purity AP product to be mutated plus inorganic phosphoric acid or a substrate analog according to conventional methods, and X-ray crystal structure analysis yields three-dimensional structure information. The lysine residue in the vicinity of the active center can be identified from the coordinates of the functional group corresponding to the acid group or phosphate group and the coordinates of the atoms constituting the AP. It is also possible to predict the structure of the three-dimensional structure from the amino acid sequence of the AP to be mutated using various structure prediction software, and to estimate the lysine residue located near the active center from this information. is there. Examples of available 3D structure prediction software include Discovery Studio (manufactured by Accelrys), MOE (manufactured by Chemical Computing Group), Desert Scientific Software (manufactured by Infocom SWIFI, etc.), or Swiss Institute SMO Is exemplified.

  If any of the above methods can identify a lysine residue located near the active center of an AP, this time, identify a lysine residue located near the active center of another AP. I can do it.

Specific examples of lysine residues that are located on the alkaline phosphatase sequence and are specified near the active center are shown below.
In the case of the AP derived from Shewanella sp. T3-3 strain (SEQ ID NO: 2) described in PCT / JP2012 / 053924 described above, the 161st and 184th lysines are applicable. As shown in Example 5 to be described later, this was identified by structural comparison with AP derived from the Chewanella sp. AP1 strain having a known three-dimensional structure and active center.

In the sequence having high identity with the T3-3 strain such as AP derived from other genus Shewanella or closely related to the genus Shewanella, as a result of the primary sequence alignment with the AP sequence derived from the T3-3 strain, these 161st positions This corresponds to the lysine residue corresponding to the 184th lysine. For example, in the AP derived from the Shewanella fodinae NBRC105216 strain shown in SEQ ID NO: 16, the 157th and 180th lysines, and in the same way, the AP derived from the Shewanella chillensis NBRC105217 strain shown in SEQ ID NO: 18 is also used. Is estimated to be located near the active center.
Further, for example, in the case of bovine small intestine-derived alkaline phosphatase shown in SEQ ID NOs: 11 to 14, the 100th lysine in CIAP I of SEQ ID NO: 11 and the 81st lysine in CIAP II shown in SEQ ID NO: 12, In CIAP III of SEQ ID NO: 13, the 100th lysine is specified, and in CIAP IV of SEQ ID NO: 14, the 100th and 127th lysines are located near the active center.
Furthermore, in the human alkaline phosphatase shown in SEQ ID NO: 10, the 81st and 87th lysines are specified to be located in the vicinity of the active center.
In the AP having high identity with the amino acid sequence of human or bovine small intestine-derived AP, as a result of primary sequence alignment with the human or bovine small intestine-derived AP sequence, a lysine residue corresponding to the lysine corresponds to this. .

(1-3) Substituted amino acids Other amino acids that substitute lysine residues in the vicinity of the active center are not particularly limited as long as they are other than lysine and can maintain alkaline phosphatase activity, but alanine, valine, leucine, Examples include aliphatic amino acids such as isoleucine, hydrophilic amino acids such as glycine, serine, threonine, methionine and proline, acidic amino acids such as glutamic acid and aspartic acid, and basic amino acids other than lysine such as arginine and histidine. Among these, arginine, which is the same basic amino acid as a glycine, alanine, serine, or lysine residue with low hydrophobicity and a small side chain, and which is presumed not to have a large effect on properties, is given as a suitable example. It is done. Among these, serine and arginine are further preferable examples, and serine is the most preferable example among them.

(2) Method for producing modified alkaline phosphatase Another aspect of the present invention is a method for producing modified alkaline phosphatase. The method is
(A) identifying a lysine residue located near the active center of alkaline phosphatase,
(B) substituting the codon on the alkaline phosphatase gene encoding the lysine residue specified or estimated in (A) with a codon encoding another amino acid,
(C) transforming the gene prepared in (B) into a host cell, culturing the transformant to express the gene,
(D) purifying the modified alkaline phosphatase that is the expression product in (C),
Is included.

(2-1)
The step of specifying a lysine residue located in the vicinity of the active center of alkaline phosphatase can be performed by the method already described in (1-1).

(2-2)
Replacing a codon on the alkaline phosphatase gene encoding the specified lysine residue with a codon encoding another amino acid replaces the codon encoding the above-mentioned lysine residue on the alkaline phosphatase gene sequence with another codon. The gene can be transformed into a host cell, the transformant is cultured to express the gene, and the modified alkaline phosphatase that is the expression product is purified. Such a mutant alkaline phosphatase gene can be produced by chemical synthesis of the designed modified AP gene full-length sequence, and DNA is replicated using a wild-type alkaline phosphatase gene as a template and a mismatch primer. This is also possible.

(2-3)
The AP of the present invention preferably comprises an expression vector carrying a gene encoding the protein, or is isolated and purified from a transformant culture obtained by inserting the gene into genomic DNA. Can be manufactured.

  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 has been established as will be described later, but preferably bacteria such as Escherichia coli and Bacillus subtilis, actinomycetes And microbial hosts such as gonococci and yeast, insect cells, animal cells, higher plants and the like.

  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, it is preferable to use an AP expression vector in which a DNA encoding AP is placed under the control of a promoter functional in the target host cell. Examples of the vector used include prokaryotic and / or eukaryotic cells. Promoter region that functions in various host cells and can control transcription of a gene located downstream thereof (for example, trp promoter, lac promoter, recA promoter when the host is Escherichia coli, SPO1 when the host is Bacillus subtilis, etc. Promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. When the host is a mammalian cell, SV40-derived early promoter, MoMuLV-derived long terminal repeat, adenovirus A viral promoter such as an early promoter) and a transcription termination signal of the gene, that is, a terminator region, and the promoter region and the terminator region contain at least one restriction enzyme recognition site, preferably the vector only at that site. There is no particular limitation as long as it is ligated via a sequence containing a unique restriction site that cleaves at <RTIgt;, </ RTI> but a selection marker gene for selection of transformants (tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin) It is preferable to further contain a gene that imparts resistance to a drug such as a gene that complements an auxotrophic mutation. 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. 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.

  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 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, examples of the medium include Burkholder's 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. 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.

  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.

(2-4)
The purification of AP can be carried out 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. . Moreover, in order to ensure the stability of 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. Moreover, when providing in liquid form, it is preferable to contain a buffer, a metal salt, and an antiseptic | preservative, and it is preferable to contain an antifreezing agent, surfactant, etc. as needed. As the buffer, phosphate, tris (hydroxymethyl) aminomethane hydrochloride, triethanolamine hydrochloride, GOOD buffer, and the like are preferably selected, but not limited thereto. The concentration of the buffer is not particularly limited as long as the pH of the solution can be kept constant, but is preferably in the range of 1 mM to 500 mM, more preferably in the range of 2 mM to 200 mM. Moreover, it is preferable that the pH of a solution is the range which can maintain the activity of AP stably, Preferably it is the range of pH 6-9. Further, magnesium salt and / or zinc salt is preferably used as the metal salt, and the concentration may be set within a range effective for stabilizing AP, and if magnesium salt, final concentration of 0.001 mM to 10 mM, zinc salt If so, 0.001 mM to 1 mM is exemplified as a preferable range of addition amount, but is not limited to this range. Examples of preservatives include, but are not limited to, azides, antibiotics, proclin 150, proclin 300, and the like. Further, as the antifreezing agent, non-protein agents such as glycerin and dimethylsulfoxide are preferable. Further, when adding a surfactant, Triton X-100, Tween 20, Tween 80, Emulgen A60, Emulgen 430, Brij35, etc. are preferably selected.

(3) Conjugate formed by labeling modified alkaline phosphatase, immunoassay method using the conjugate, immunoassay reagent containing the conjugate Further, another aspect of the present invention is labeled with the above-mentioned alkaline phosphatase For example, a nucleic acid probe, a biological material such as biotin, a protein such as a polypeptide, avidin, or an antibody is preferably selected as the substance to be labeled. As the labeling method, the maleimide method is preferably selected. 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.

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. (ΔOD _TEST ). In the blind test, 0.1 ml of a buffer in which an enzyme is dissolved is added instead of the enzyme, and the change in absorbance per minute is similarly determined (ΔOD _BLANK ). Using these values, the AP activity is obtained from the following formula.

AP activity (U / ml) = {(ΔOD _TEST− ΔOD _BLANK ) × 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)

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.

Example of maleimide group quantification method The method of quantifying maleimide groups in the present invention is as follows.
First, 50 μl of maleimidated AP is mixed with 400 μl of 0.1 M sodium phosphate buffer (pH 6.0), and 50 μl of 0.5 mM 2-MEA is added and incubated at 30 ° C. for 20 minutes. As a blind test, add 50 μl of 0.5 mM 2-MEA to 450 μl of 0.1 M sodium phosphate buffer (pH 6.0) and incubate at 30 ° C. for 20 minutes. Further, 20 μl of 5 mM 4-PDS is added, and after incubation at 30 ° C. for 10 minutes, the absorbance at 324 nm is measured (A324 _sample ). In the blind test, 50 μl of 0.5 mM 2-MEA is added to 450 μl of 0.1 M sodium phosphate buffer (pH 6.0), and the same operation is performed to measure the absorbance at 324 nm (A324 _blank ). Using these values, the maleimide group concentration is determined by the following formula.

Maleimide group concentration (mM) = (A324 _blank −A324 _sample ) × 520 / (19.8 × 50)

520: Volume of total liquid (μl)
19.8: millimolar molecular extinction coefficient 50: sample volume (μl)
The number of maleimide groups per AP molecule is determined by measuring the protein concentration of the AP solution used for the measurement by the method described above, calculating the AP mmol concentration from the molecular weight of AP of 100,000, and dividing the maleimide group concentration by the AP mmol concentration. Can be determined.

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

[Acquisition of AP-producing strain]
An underwater sediment sample collected from the coast of Tsuruga Bay in Tsuruga City, Fukui Prefecture was applied to an LB agar medium (pH 7.5) containing 50 μg / ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP), Cultured at 25 ° C. Among colonies formed on the culture medium, colonies showing a blue color due to hydrolysis of the phosphate ester of BCIP were purified. A part of the DNA region constituting the 16S rRNA sequence was amplified from this strain by colony direct PCR using 10F / 800R primers described in the Japanese Pharmacopoeia “Method for Rapid Identification of Microorganisms by Genetic Analysis”. When this sequence was searched with NCBI-BLAST, it was presumed to be a bacterium belonging to the genus Shewanella, so this strain was identified as Shewanella sp. It was named T3-3 strain.

[Cloning of wild-type AP gene]
Shewanella sp. The T3-3 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 (pBRT3-3LPP) containing the AP gene derived from the T3-3 strain 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 wild-type AP in E. coli host]
Primers were prepared so as to cover the entire length of the AP gene and the sequence including the promoter region of the T3-3 strain AP gene and to have a BamHI site at each 5 ′ end (SEQ ID NOs: 3 and 4). PCR was performed using the plasmid (pBRT3-3LPP) as a template. The amplified DNA fragment is applied to a TAE gel containing 1% agarose and subjected to electrophoresis, and the band of the amplified fragment is excised while irradiating with UV, and the DNA fragment from the gel is extracted using a DNA purification kit (NPK-6). Extraction and purification were performed. This genomic DNA fragment was digested with the restriction enzyme BamHI and ligated to pBluescprSK (−) treated with the restriction enzyme to prepare an expression plasmid (pBST3-3LPP). The ligated plasmid 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 to form transformed colonies. This transformed colony was inoculated with one platinum ear in 60 ml LB medium (containing 100 μg / ml ampicillin) in a 500 ml Sakaguchi flask, and cultured with shaking at 30 ° C. and 180 rpm overnight. 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 800 U / ml AP.

[Purification of E. coli recombinant wild-type 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. To the crushed liquid, 5% (w / v) polyethyleneimine was added to 3% of the 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. Furthermore, ammonium sulfate was added and dissolved so that the final concentration became 0.55 saturation, and 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 0.05 saturated. This solution was applied to Octyl Sepharose gel (manufactured by GE Healthcare) buffered with 20 mM Tris-HCl buffer (pH 7.5, containing 0.05 saturated ammonium sulfate and 1 mM magnesium chloride), and the same buffer was continuously passed through. The non-adsorbed fraction was collected. Phenyl Sepharose gel in which ammonium sulfate was further dissolved in this solution to a final concentration of 0.2 and buffered with 20 mM Tris-HCl buffer (pH 7.5, containing 0.2 saturated ammonium sulfate and 1 mM magnesium chloride). (GE Healthcare) was applied, and gradient elution was performed by lowering the ammonium sulfate concentration to 0 with the same buffer. Fractions containing AP were pooled and desalted on G-25 Sepharose gel buffered with 20 mM triethanolamine (pH 7.5, containing 1 mM magnesium chloride and 0.1 mM zinc sulfate) and purified T3-3. Strain-derived recombinant AP. The specific activity of this AP solution was measured and found to be 6090 U / mg.

[Estimation of lysine residues located near the active center of AP derived from T3-3 strain]
From sulfuric acid (used as an analog of phosphate ester as a substrate) located at the active center of the three-dimensional structure information (Protein Data Bank accession number: 3A52) of AP derived from Shewanella sp. AP-1 Amino acid residues present within angstroms are identified by MolFeat v. The search was performed using 3.6 (Fiarax). Subsequently, for the amino acid sequence shown in SEQ ID NO: 2 which is the sequence of AP derived from the T3-3 strain, primary sequence alignment with the AP amino acid sequence derived from Shewanella sp. AP-1 shown in SEQ ID NO: 9 Thus, residues on the T3-3 strain-derived AP sequence corresponding to the amino acid residues located in the vicinity of the active center of AP1-derived AP were determined. Among these deduced residues, lysine residues are residues that are effective for AP modification by substitution. That is, in SEQ ID NO: 2, the 161st and 184th lysines were presumed to be lysine residues located near the active center.

[Production of modified alkaline phosphatase substituted with a lysine residue presumed to be located near the active center]
First, in order to substitute the 184th lysine with serine, a replication reaction was performed using pBST3-3LPP as a template and a mismatch primer shown in SEQ ID NOs: 5 and 6 and a PCR kit (KOD plus manufactured by Toyobo). The reaction solution composition and reaction conditions followed the recommended conditions for normal PCR described in the manual attached to the kit. 2 μL of the restriction enzyme DpnI is added to 50 μL of the reaction solution containing the replication product and treated at 37 ° C. for 2 hours to digest the template pBST3-3LPP, and the digested product is heat shocked to E. coli strain JM109 (competent high JM109 manufactured by TOYOBO). Then, an SOC medium was added, shaken at 37 ° C. for 1 hour, applied to an LB agar medium containing 100 μg / ml ampicillin, and cultured at 30 ° C. overnight to form transformed colonies. The transformed colony was inoculated with a toothpick, inoculated into 5 ml of LB medium containing 100 μg / ml ampicillin, and cultured with shaking at 30 ° C. overnight. From this culture solution, a plasmid was extracted and purified using a plasmid extraction kit (manufactured by TOYOBO, NPK-3). As a result of analyzing the sequence of the AP gene in the plasmid, the codon AAG encoding the 184th lysine was converted to TCG encoding serine (that is, the 550th A in the nucleotide sequence shown in SEQ ID NO: 1 was changed to T, the 551st A was converted to C, and this plasmid was named pBST3-3LPP184S. Furthermore, in order to replace the 161st lysine with serine, a replication reaction was performed using pBSpBST3-3LPP184S as a template and a mismatch primer shown in SEQ ID NOs: 7 and 8 and a PCR kit (KOD plus manufactured by Toyobo). The reaction solution composition and reaction conditions followed the recommended conditions for normal PCR described in the manual attached to the kit. 2 μL of the restriction enzyme DpnI is added to 50 μL of the reaction solution containing the replication product and treated at 37 ° C. for 2 hours to digest the template pBST3-3LPP, and the digested product is heat shocked to E. coli strain JM109 (competent high JM109 manufactured by TOYOBO). Then, an SOC medium was added, shaken at 37 ° C. for 1 hour, applied to an LB agar medium containing 100 μg / ml ampicillin, and cultured at 30 ° C. overnight to form transformed colonies. The transformed colony was inoculated with a toothpick, inoculated into 5 ml of LB medium containing 100 μg / ml ampicillin, and cultured with shaking at 30 ° C. overnight. From this culture solution, a plasmid was extracted and purified using a plasmid extraction kit (manufactured by TOYOBO, NPK-3). As a result of analyzing the sequence of the AP gene in the plasmid, the codon AAA encoding the 161st lysine was converted to TCG encoding serine (that is, the 481st A in the nucleotide sequence shown in SEQ ID NO: 1 was changed to T, the 482nd A was converted to C, and the 483rd A was converted to G), and this plasmid was named pBST3-3LPP161S184S. Using these plasmids, transformants were prepared and cultured according to the methods described in Examples 3 and 4, and AP was purified to produce K184S single mutation and K161S + K184S double mutant enzymes as modified APs.

[Comparison of residual activity of wild-type and modified AP after maleimidation]
The wild-type and mutant AP prepared in Example 4 and Example 6 were respectively maleimidated in the following manner. First, 500 μL of a solution containing 2 mg of each AP is put into a cellophane tube, put into a mug containing dialysis buffer (50 mM sodium borate, 1 mM magnesium chloride, 0.1 mM zinc chloride, pH = 7.6), and the buffer is several times. Dialyzed overnight with replacement. 10 μL of EMCS solution was added to the dialysate and incubated at 30 ° C. for 30 minutes. At this time, the EMCS concentration was changed in the range of 0.2 to 1.0 mM in order to change the maleimide group introduction efficiency of the AP molecule. This solution was replaced with 0.1 M Tris hydrochloride, 1 mM magnesium chloride, 0.1 mM zinc chloride, pH = 7.6 using a desalting spin column to obtain a maleimidated AP solution. Specific activity and the number of maleimide groups introduced were measured for this maleimidated AP. The results are shown in FIG. In the wild type AP, the specific activity is reduced by maleimidation, in the K184S single mutant enzyme, the specific activity remaining rate of the AP after maleimidation is higher than in the wild type, and in the K161S + K184S double mutant enzyme, it is almost completely after the maleimidation. It was shown that the specific activity of was maintained. That is, it was shown that the decrease in activity after maleimide group introduction was suppressed by substituting a lysine residue near the active center.

[Comparison of reactivity of modified AP with commercially available bovine small intestine-derived AP after maleimidation]
Maleimidation was performed for the K161S + K184S double mutant enzyme and bovine small intestine-derived AP (Native enzyme, specific activity 6000 U / mg) prepared in Example 6 according to the procedure of Example 7. However, the EMCS concentration during the maleimidation reaction was changed in the range of 0.25 to 1 mM. Dilute with dilution buffer (30 mM triethanolamine hydrochloride, 1 mM magnesium chloride, 0.1 mM zinc chloride, pH = 7.6) to a concentration of 10 μg / ml before and after maleimidation. Inject 10 μL each into a 96-well ELISA plate (nunc 96F MAXISORP BLACK MICROWELL SH) coated with BSA, add 50 μL of luminescent substrate solution (Lumigen Lumi-phos 530) pre-warmed at 37 ° C. Measurements were made using a luminometer (Wallac 1420 ARVO MX from PerkinElmer). FIG. 2 shows a plot of the number of maleimide groups introduced and the relative luminescence intensity of each AP (relative value when the luminescence intensity when using a non-maleimidating enzyme of bovine small intestine derived AP is 100). In the AP derived from bovine small intestine, which is currently widely used as a labeling enzyme, the amount of luminescence per unit protein decreases as the number of maleimide groups introduced increases, whereas in K161S + K184S double mutant enzymes, the number of maleimide groups introduced depends on the number of maleimide groups introduced. It showed a certain amount of luminescence.

[Identification or estimation of lysine residues near the active center in mammalian AP]
Present within 15 Å of the phosphate group (used as an analog of the phosphate ester substrate) located in the active center of the three-dimensional structure information of human-derived AP (Protein Data Bank registration number: 3A52). Amino acid residues to be converted into MolFeat v. The search was performed using 3.6 (Fiarax). The lysine residues located near the active center in the human-derived AP shown in SEQ ID NO: 10 are the 81st lysine and the 87th lysine. Further, among amino acid residues of human-derived AP, amino acid residues located within 15 angstroms from the active center are specified, and the amino acid sequence is aligned with various bovine small intestine-derived AP amino acid sequences (SEQ ID NOs: 11 to 14). Thus, amino acid residues located near the active center were estimated, and lysine residues were picked up among them. The lysine residues in the vicinity of the active center in the bovine small intestine-derived AP thus estimated are the 100th lysine in SEQ ID NOS: 11 and 13 and the 81st lysine in SEQ ID NO: 12 in SEQ ID NO: 14. In this case, the 100th lysine and the 127th lysine correspond to this. By substituting these lysine residues with other amino acids, it is possible to change to AP that can maintain the activity before modification even after modification such as maleimidation via lysine residues.

[Acquisition of alkaline phosphatase gene from other Shewanella bacteria and estimation of lysine residues near the active center]
From the National Institute for Product Evaluation and Technology, buy the Shewanella Fodinae NBRC105216 and Shewanella Chirikensis NBRC105217 strains, extract genomic DNA from the cells according to the procedure in Example 2, respectively, create a library, and include the AP gene plasmid And the AP gene sequence was determined. The determined base sequence of the AP gene derived from Shewanella fodinae NBRC105216 strain is shown in SEQ ID NO: 15, and the amino acid sequence of the polypeptide encoded by the approximate AP gene is shown in SEQ ID NO: 16. In addition, the determined base sequence of the AP gene derived from Shewanella chilikensis NBRC105217 strain is shown in SEQ ID NO: 17, and the amino acid sequence of the polypeptide encoded by the approximate AP gene is shown in SEQ ID NO: 18. For these sequences, lysine residues located in the vicinity of the active center were estimated according to the procedure of Example 5. The residues estimated in this way are the 157th and 180th lysines in the AP derived from the Shewanella fodinae NBRC105216 strain shown in SEQ ID NO: 16, and the AP derived from the Shewanella chilikensis NBRC105217 strain shown in the SEQ ID NO: 18. Similarly, the 157th and 180th lysines correspond to this. Similar to the T3-3 strain, by substituting these amino acid residues, it is possible to change to AP that can maintain the activity before modification even after modification such as maleimidation via a lysine residue.

  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.

Claims (5)

  A modified alkaline phosphatase in which the 184th lysine is substituted with serine in the amino acid sequence shown in SEQ ID NO: 2, or the 161st lysine and 184th lysine are substituted with serine. A conjugate formed by labeling the alkaline phosphatase according to claim 1.   An immunoassay method using the conjugate according to claim 2.   An immunoassay reagent comprising the conjugate according to claim 2. Hereinafter, a method for producing a modified alkaline phosphatase, including the steps (A) to (C).
(A) Of the amino acid sequence shown in SEQ ID NO: 2, the codon encoding 184th lysine is a codon encoding serine, or the codons encoding 161st lysine and 184th lysine are codons encoding serine, The step of replacing.
(B) A step of transforming the gene prepared in (A) into a host cell and culturing the transformant to express the gene.
(C) A step of purifying alkaline phosphatase which is an expression product in (B).