WO2013180208A1 - 糸状菌を用いた西洋ワサビ由来ペルオキシダーゼ組換えタンパク質の製造方法 - Google Patents
糸状菌を用いた西洋ワサビ由来ペルオキシダーゼ組換えタンパク質の製造方法 Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
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- D06L4/40—Bleaching fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods; Bleaching leather or furs using enzymes
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- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
Definitions
- the present invention relates to a method for producing a horseradish peroxidase recombinant protein using filamentous fungi. Specifically, a polynucleotide encoding a horseradish peroxidase polypeptide and capable of expressing the polypeptide in a filamentous fungus, an expression vector containing the polynucleotide, and a transformation obtained by introducing the expression vector into the filamentous fungus
- the present invention relates to a method for producing a horseradish peroxidase recombinant protein using the transformant, a horseradish peroxidase recombinant protein produced by the production method, a preparation containing the same, and uses thereof.
- Horseradish-derived peroxidase is widely used as an enzyme for detection in various tests such as enzyme immunoassay (Enzyme-Linked Immunosorbent Assay; ELISA), immunohistochemical staining, Southern blotting, Western blotting, and the like. In recent years, it has been widely used as an enzyme for clinical test kits.
- enzyme immunoassay Enzyme-Linked Immunosorbent Assay; ELISA
- ELISA Enzyme-Linked Immunosorbent Assay
- Western blotting Western blotting
- it has been widely used as an enzyme for clinical test kits.
- Peroxidase is commonly found in the plant kingdom, such as radish, sweet potato, wheat, Japanese horseradish, and horseradish. However, horseradish is preferred for industrial production because of its high peroxidase content. Yes.
- horseradish-derived peroxidase is composed of a plurality of enzymes such as acidic, neutral, and basic isozymes. Furthermore, the peroxidase content and the composition ratio of these isozymes vary significantly depending on the nature of the cultivated soil, the type and amount of fertilizer applied, the weather, the harvesting time, etc. Therefore, the quality of peroxidase purified from horseradish is not always constant. That is, most of the horseradish peroxidase products that are currently widely used are mostly a mixture of many isozymes, and the ratio is mostly different from lot to lot. When various measurements using such a horseradish-derived peroxidase product, for example, ELISA, are performed, there is a serious problem that dispersion occurs in each production lot and it is difficult to obtain a stable measurement result.
- enzymes such as acidic, neutral, and basic isozymes.
- the peroxidase content and the composition ratio of these isozymes vary significantly depending on the nature of the cultivated soil, the
- Peroxidase C1a which is one of the major isozymes in horseradish peroxidase, has been clarified in its DNA sequence and amino acid sequence (see Non-Patent Document 1).
- expression has been studied in E. coli, yeast and tobacco plant cells using a gene encoding peroxidase C1a.
- the expression level is 0.11 mg / L for Escherichia coli (see Non-Patent Document 2), 5.3 mg / L for yeast (see Non-Patent Document 3), and 3 mg / L for tobacco plant cells (see Non-Patent Document 4).
- the production is extremely low and impractical. From the above, it has been desired to develop a recombinant organism capable of mass production of horseradish peroxidase and a method for producing horseradish peroxidase using the same.
- the present invention has been made in view of the above-described problems of the prior art, and an object of the present invention is to provide a polynucleotide that enables efficient production of horseradish-derived peroxidase polypeptide in large quantities.
- the present inventors first introduced an expression vector containing a polynucleotide encoding wild-type horseradish-derived peroxidase (HRP) C1a into filamentous fungi, and HRP in the obtained transformant. The amount of peptide produced was examined. However, the production of HRP peptide from such a transformant cannot be confirmed, and even when a polynucleotide encoding a wild type HRP polypeptide is used, filamentous fungi cannot produce the HRP polypeptide. It was revealed.
- HRP horseradish-derived peroxidase
- the present inventors conducted intensive studies to highly express HRP polypeptides in filamentous fungi, and firstly considered codon usage in three species of Humicola, Aspergillus, and Trichoderma filamentous fungi. Next, a codon-modified HRP polynucleotide in which the frequency of codon appearance in the HRP polynucleotide was adapted to the obtained codon usage frequency was prepared. And when Humicola, Aspergillus, and Trichoderma were transformed using this codon-modified HRP polynucleotide, expression of HRP could be confirmed in Aspergillus and Trichoderma, although expression was not confirmed in Humicola. In particular, when Trichoderma was used, HRP was successfully produced at a concentration 100 times or more than before.
- the present inventors transformed Trichoderma using an HRP polynucleotide adapted only to the codon usage frequency of Trichoderma in order to express HRP at a higher level in Trichoderma.
- HRP polynucleotide adapted only to the codon usage frequency of Trichoderma in order to express HRP at a higher level in Trichoderma.
- only the host cell, Trichoderma was transformed with a modified polynucleotide taking into account the codon usage in three species of Humicola, Aspergillus and Trichoderma filamentous fungi.
- the productivity of HRP was remarkably higher than that obtained when transformation was performed using a polynucleotide adapted to the frequency of codon usage.
- HRP can be easily isolated and purified by a polypeptide fused to these HRPs. Further, the inventors have found that an HRP polypeptide produced by a filamentous fungus transformed with a codon-modified HRP polynucleotide can decolorize an anato dye in the presence of hydrogen peroxide, thereby completing the present invention. Specifically, the present invention provides the following in more detail.
- the usage frequency of GCC is 80% and the usage frequency of GCT is 20%;
- the usage frequency of CGC is 90%, the usage frequency of CGT is 10%;
- the amino acid encoded by the modified codon is asparagine, the frequency of use of AAC is 100%;
- the usage frequency of GAC is 95% and the usage frequency of GAT is 5%; If the amino acid encoded by the modified codon is cysteine, the usage frequency of TGC is 100%;
- the amino acid encoded by the modified codon is cysteine, the usage frequency of TGC is 100%;
- the horseradish-derived peroxidase C1a polypeptide is encoded, and has at least one characteristic selected from the group consisting of (i) to (ii) below: (1) to (3) (Ii) including the coding region of the nucleotide sequence described in SEQ ID NO: 1, and (ii) having a homology of 95% or more with the nucleotide sequence consisting of positions 91 to 1017 described in SEQ ID NO: 1.
- the horseradish peroxidase C1a polypeptide is encoded and described in any one of (1) to (3) having at least one characteristic selected from the group consisting of: (Ii) containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 26 (ii) having a homology of 95% or more with the nucleotide sequence consisting of positions 91 to 1017 described in SEQ ID NO: 26.
- An expression vector comprising the polynucleotide according to any one of (1) to (6).
- the transformant according to (8), wherein the filamentous fungus is a genus Trichoderma or Aspergillus.
- the transformant according to (8), wherein the filamentous fungus is Trichoderma viride or Aspergillus niger.
- the transformant according to (8), wherein the filamentous fungus is Trichoderma viride.
- the transformant according to any one of (8) to (11) is cultured, and the cultured transformant and / or a polypeptide expressed from the culture of the transformant is obtained.
- a method for decolorizing a dye wherein the polypeptide produced by the method according to (12) is allowed to act on the dye in the presence of hydrogen peroxide.
- a method for removing a phenolic compound which comprises reacting the phenolic compound with the polypeptide produced by the method according to (12) in the presence of hydrogen peroxide.
- a polynucleotide comprising a codon optimized for the expression of filamentous fungi encoding horseradish peroxidase was provided. Furthermore, it becomes possible to produce a horseradish peroxidase recombinant protein using Trichoderma viride or Aspergillus niger transformed with the polynucleotide.
- Filamentous fungi (Trichoderma) were transformed with an expression vector (pCB1-HRP_Native) containing a polynucleotide encoding wild-type horseradish peroxidase (HRP) C1a, and the culture supernatant of the resulting transformant was treated with anti-antibody.
- pCB1-HRP_Native an expression vector containing a polynucleotide encoding wild-type horseradish peroxidase (HRP) C1a
- HRP antibody HRP antibody
- FIG. 3 is a diagram showing the results of comparing a wild-type HRP polynucleotide and a polynucleotide of the present invention (codon-modified HRP polynucleotide) in the base sequence (positions 1 to 540).
- the upper sequence shows the base sequence of the wild type HRP polynucleotide (base sequence described in SEQ ID NO: 3), and the lower sequence shows the polynucleotide of the present invention (base sequence described in SEQ ID NO: 1). Show.
- FIG. 3 shows the base sequence of the wild type HRP polynucleotide (base sequence described in SEQ ID NO: 3)
- the lower sequence shows the polynucleotide of the present invention (base sequence described in SEQ ID NO: 1). Show.
- FIG. 3 is a view showing the results of comparing a wild-type HRP polynucleotide and the polynucleotide of the present invention (codon-modified HRP polynucleotide) in the base sequence (positions 541 to 1014).
- the upper sequence shows the base sequence of the wild type HRP polynucleotide (base sequence described in SEQ ID NO: 3), and the lower sequence shows the polynucleotide of the present invention (base sequence described in SEQ ID NO: 1). Show.
- Humicola was transformed with an expression vector containing the polynucleotide of the present invention (pNCE2-HRP-humicola), and the culture supernatant of the resulting transformant was analyzed by Western blot using an anti-His tag antibody. It is a photograph shown. In the figure, 1 lane shows the result of developing the molecular weight marker, and 2 lane shows the result of developing the culture supernatant of the transformant. Aspergillus was transformed with an expression vector containing the polynucleotide of the present invention (pAmyB-pyr-HRP-Aspergillus), and the culture supernatant of the resulting transformant was analyzed by Western blot using an anti-His tag antibody.
- Trichoderma was transformed with an expression vector (pCB1-HRP (Hisless) -tricho) containing the polynucleotide of the present invention, and the culture supernatant of the resulting transformant was analyzed by Western blot using an anti-HRP antibody. It is a photograph which shows a result. In the figure, 1 lane shows the result of developing the molecular weight marker, and 2 lane shows the result of developing the culture supernatant of the transformant. Trichoderma was transformed with the expression vector (pCB1-KR-HRP-tricho) containing the polynucleotide of the present invention, and the culture supernatant of the obtained transformant was analyzed by Western blot using an anti-HRP antibody.
- pCB1-HRP Hisless
- FIG. 3 is a diagram showing the results of comparing a wild-type HRP polynucleotide and a polynucleotide of the present invention (codon-modified HRP polynucleotide) in the base sequence (positions 1 to 540).
- the upper sequence shows the base sequence of the wild type HRP polynucleotide (base sequence described in SEQ ID NO: 3), and the lower sequence shows the polynucleotide of the present invention (base sequence described in SEQ ID NO: 26). Show.
- FIG. 3 shows the base sequence of the wild type HRP polynucleotide (base sequence described in SEQ ID NO: 3)
- the lower sequence shows the polynucleotide of the present invention (base sequence described in SEQ ID NO: 26).
- FIG. 3 is a view showing the results of comparing a wild-type HRP polynucleotide and the polynucleotide of the present invention (codon-modified HRP polynucleotide) in the base sequence (positions 541 to 1014).
- the upper sequence shows the base sequence of the wild type HRP polynucleotide (base sequence described in SEQ ID NO: 3)
- the lower sequence shows the polynucleotide of the present invention (base sequence described in SEQ ID NO: 26).
- FIG. 3 is a diagram showing the results of comparing the polynucleotides of the present invention in terms of their base sequences (positions 1 to 540).
- FIG. 3 is a diagram showing the results of comparing the polynucleotides of the present invention in terms of their base sequences (positions 541 to 1017).
- the upper sequence shows the base sequence described in SEQ ID NO: 1
- the lower sequence shows the base sequence described in SEQ ID NO: 26.
- Trichoderma was transformed with an expression vector (pCB1-HRP (Hisless) -tricho-3) containing the polynucleotide of the present invention, and the culture supernatant of the resulting transformant was obtained by Western blot using an anti-HRP antibody. It is a photograph which shows the result of analysis.
- 1 lane shows the result of developing the molecular weight marker
- 2 lane shows the result of developing the culture supernatant of the transformant.
- ⁇ Polynucleotide> As shown in Examples below, using a polynucleotide having a wild-type base sequence encoding horseradish-derived peroxidase polypeptide (HRP), the expression of HRP polypeptide is detected by Western blot analysis in filamentous fungi. However, when a modified polynucleotide having a nucleotide sequence different from that of the wild-type nucleotide sequence was used, the expression of HRP polypeptide could be detected in such filamentous fungi.
- HRP horseradish-derived peroxidase polypeptide
- the present invention provides the following polynucleotides.
- the HRP polypeptide means an enzyme extracted from horseradish and having an activity of oxidatively cleaving the peroxide structure to decompose it into two hydroxyl groups, such as HRP C1a and HRP C1b.
- HRP C1c, HRP C2, and HRP C3 isozymes are the isozymes with the highest content of horseradish peroxidase. Therefore, they are extracted from horseradish and are widely used as reagents. From the viewpoint that the property is most strongly reflected, it is preferably an HRP C1a polypeptide.
- the HRP C1a polypeptide is typically a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4. Furthermore, even in nature, amino acid sequences can be mutated. Therefore, the HRP polypeptide has an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 4 as long as it encodes the protein having the above activity.
- a polypeptide comprising up to the 30th alanine residue counting from the N-terminal methionine residue functions as an HRP signal peptide.
- a polypeptide means a molecule in which two or more amino acids are bound by peptide bonds. Therefore, this concept includes not only full-length proteins but also so-called oligopeptides.
- polypeptides are modified, for example, glycosylation, phosphorylation, palmitoylation, prenylation, methylation, acetylation, ubiquitination, SUMOylation, hydroxylation, amidation, etc. Also good.
- the “wild-type base sequence encoding an HRP polypeptide” is typically the base sequence described in SEQ ID NO: 3.
- “Codon” means a combination of three nucleotide bases encoding amino acids.
- the at least one codon having a base sequence different from the wild-type base sequence is preferably a codon that improves translation efficiency in filamentous fungi.
- This codon is preferably a codon into which a “degenerate mutation” without an amino acid sequence change is introduced.
- the codon can be determined as a “codon that improves translation efficiency in the filamentous fungus”.
- the number of codons to be modified is preferably at least 2 (eg, 3 or more, 5 or more), more preferably 10 or more (eg, 20 or more, 30 or more, 50 or more), The number is preferably 100 or more (for example, 120 or more, 150 or more, 180 or more), particularly preferably 200 or more (for example, 210 or more, 220 or more, 230 or more, 240 or more).
- the proportion of modified codons among all codons in the polynucleotide having the wild-type base sequence is preferably at least 10%, more preferably 30% or more, and particularly preferably 60%. This is the above (for example, 70% or more, 80% or more, 90% or more, 100%).
- the filamentous fungus means a fungus composed of hyphae, for example, Trichoderma spp., Aspergillus spp., Acremonium spp., Fusarium spp. Bacteria, genus Mycelioptora, genus Neurospora, genus Penicillium, genus Rhizomucor, genus Thermomyces, genus Thermopia thidium ) Genus.
- Trichoderma viride Trichoderma harzianum, Trichoderma koningii, Trichoderma kongii, Trichoderma ongi ragi (Trichoderma reesei).
- Aspergillus sp. Aspergillus niger (Aspergillus niger), Aspergillus awagi, Aspergillus foetidus, Aspergillus sp. Orise (Aspergillus oryzae) is mentioned.
- Trichoderma spp. And Aspergillus spp. Are preferable, and Trichoderma viride and Aspergillus niger are more preferable.
- the polypeptide to be encoded can be expressed means that the modified wild-type base sequence encoding the HRP polypeptide has a base sequence that differs in at least one codon.
- the filamentous fungus transformed by introducing the polynucleotide is cultured, and the concentration of HRP produced in the culture supernatant diluted so that the concentration of the transformant becomes 9 ⁇ 10 8 CFU / mL It means 0.001 mg / L or more, preferably 1 mg / L or more, more preferably 10 mg / L or more, further preferably 100 mg / L or more, and particularly preferably 300 mg / L or more.
- the codon usage frequencies shown in Table 1 shown below were calculated in consideration of the codon usage frequencies in three types of Humicola, Aspergillus and Trichoderma. Then, the frequency of codon occurrence in the wild-type HRP polynucleotide is adapted to the obtained codon usage frequency, and the base sequence of the wild-type HRP polynucleotide (base sequence described in SEQ ID NO: 3) is changed to SEQ ID NO: 1. The nucleotide sequence described was changed to the nucleotide sequence described in SEQ ID NO: 26.
- the filamentous fungus was transformed using the codon-modified HRP polynucleotide thus prepared, and the expression of the HRP polypeptide that could not be confirmed using the wild-type HRP polynucleotide was detected. We were able to.
- the polynucleotide of the present invention is a polynucleotide encoding an HRP polypeptide that has been modified so that the frequency of codon usage is the following percentage.
- GCC usage frequency is 80%, GCT usage frequency is 20%;
- the usage frequency of CGC is 90%, the usage frequency of CGT is 10%;
- the amino acid encoded by the modified codon is asparagine, the frequency of use of AAC is 100%;
- the usage frequency of GAC is 95% and the usage frequency of GAT is 5%;
- the usage frequency of TGC is 100%;
- the amino acid encoded by the modified codon is glutamine, the frequency of use of CAG is 100%;
- the usage frequency of GAG is
- “modifying the base sequence of wild-type HRP so that the frequency of codon usage is the percentage” not only matches the percentage value itself, but also adds 2.5 to the percentage value. It is intended to include adapting with a width of%. For example, when the amino acid encoded by the modified codon is serine, the AGC usage frequency is adjusted to 12.5 to 17.5% and the TCC usage frequency is adjusted to 82.5 to 87.5%. Therefore, it means that the frequency of use of the codon encoding serine in the nucleotide sequence of the polynucleotide encoding HRP polypeptide is corrected.
- At least one amino acid having the percentage of codon usage is sufficient, but preferably at least two (for example, three or more, five or more, seven or more), more Preferably there are 10 or more (for example, 12 or more, 15 or more, 17 or more), particularly preferably all 18 types.
- the present invention provides a polynucleotide encoding a horseradish peroxidase (HRP) C1a polypeptide and having at least one characteristic selected from the group consisting of the following (i) to (ii) ( i) includes a coding region of the base sequence described in SEQ ID NO: 1 (ii) has a homology of 95% or more with the base sequence consisting of positions 91 to 1017 described in SEQ ID NO: 1.
- HRP horseradish peroxidase
- the present invention also provides a polynucleotide encoding a horseradish peroxidase (HRP) C1a polypeptide and having at least one characteristic selected from the group consisting of the following (i) to (ii).
- HRP horseradish peroxidase
- (I) includes a coding region of the base sequence described in SEQ ID NO: 26
- (ii) has 95% or more homology with the base sequence consisting of positions 91 to 1017 described in SEQ ID NO: 26.
- the “base sequence consisting of positions 91 to 1017 described in SEQ ID NO: 1” and the “base sequence consisting of positions 91 to 1017 described in SEQ ID NO: 26” mean that the codon usage is the percentage described above.
- the base sequence encoding the HRP C1a polypeptide is further modified so that the base sequence encoding the signal sequence is excluded.
- homology for base sequences is used to mean the degree of coincidence of bases constituting each sequence among the sequences to be compared. Any numerical value of “homology” shown in the present specification may be a numerical value calculated using a homology search program known to those skilled in the art. For example, FASTA, BLAST, Smith-Waterman, etc. use the default (initial By using the parameter of (setting), it can be easily calculated.
- the polynucleotide of the present invention includes a polynucleotide encoding the signal sequence (SEQ ID NO: 1) from the HRP polynucleotide.
- the base sequence corresponding to positions 1 to 90 described above may be excluded.
- the polynucleotide of the present invention can be prepared by those skilled in the art by appropriately using known methods. For example, as shown in the examples described later, a base sequence that differs from the wild-type base sequence encoding the HRP polypeptide in at least one codon is designed, and a commercially available DNA synthesizer is used based on this base sequence information. Thus, the polynucleotide of the present invention can be chemically synthesized. The polynucleotide of the present invention can also be prepared by introducing a mutation (base substitution) into a wild-type HRP polynucleotide by a known site-directed mutagenesis method or the like. .
- an HRP polypeptide fused with a Trichoderma-derived CBH1 (cellobiohydrolase 1) polypeptide or His tag polypeptide is also used in filamentous fungi. It was also revealed that it can be expressed.
- the present invention provides a polynucleotide in which a polynucleotide encoding a desired polypeptide is further added to the codon-modified HRP polynucleotide.
- the desired polypeptide is not particularly limited.
- the desired polypeptide can be easily detected by adding the HRP polypeptide as a detection tag.
- a desired polypeptide may be added to the HRP polypeptide in order to purify the HRP polypeptide.
- the polypeptide used for purifying the HRP polypeptide include a polypeptide having a substrate adsorption ability, and more specifically, cellobiohydrolase (CBH), endoglucanase, ⁇ -Glucosidase, glucoamylase, albumin, antibody, Fab antibody, scFV antibody, His tag, GST tag, MBP tag, TAP tag, FLAG tag, Myc tag, HA tag, V5 tag, T7 tag.
- CBH cellobiohydrolase
- the addition frame does not shift the reading frame of these polypeptides, and it is a continuous fusion.
- Any polynucleotide can be used as long as it is translated as a polypeptide, and a polynucleotide encoding a desired polypeptide may be added to one or both of the 5 ′ side and 3 ′ side of the codon-modified HRP polynucleotide. Further, such addition may be direct or indirect.
- Examples of the indirect addition include a mode in which a polynucleotide encoding a linker polypeptide is inserted between the codon-modified HRP polynucleotide and a polynucleotide encoding a desired polypeptide.
- the length of such a linker polypeptide is usually 1 to 100 amino acids, preferably 1 to 50 amino acids, more preferably 1 to 30 amino acids, and particularly preferably 12 to 18 amino acids (for example, 15 amino acids).
- the present invention also provides an expression vector comprising the polynucleotide of the present invention.
- the expression vector of the present invention can be constructed on the basis of a self-replicating vector, that is, as an extrachromosomal independent substance, and its replication does not depend on chromosomal replication, for example, a plasmid.
- the expression vector when the present expression vector is introduced into a host filamentous fungus, the expression vector may be integrated into the genome of the filamentous fungus and replicated together with the chromosome into which it has been incorporated.
- the expression vector of the present invention introduces a filamentous fungus to express HRP and the like, a gene marker for selecting a polynucleotide that controls the expression, a transformant, and the like It is desirable to contain.
- polynucleotides that control such expression include promoters, leader sequences, and terminators.
- the promoter is not particularly limited as long as it exhibits transcriptional activity in filamentous fungi, and is used as a polynucleotide that controls the expression of a gene that encodes a polypeptide of the same or different species as the host or the same or different genera. Obtainable.
- promoters include ⁇ -amylase gene, glucoamylase gene, cellobiohydrolase gene, glyceraldehyde 3-phosphate dehydrogenase gene promoter.
- the terminator may be any sequence recognized by filamentous fungi to terminate transcription.
- TAKA amylase For example, TAKA amylase, glucoamylase, cellobiohydrolase gene, anthranilate synthase, ⁇ -glucosidase, trpC gene, trypsin-like protease gene Terminators.
- the leader sequence only needs to be an untranslated region of mRNA that can improve the translation efficiency by filamentous fungi. Examples thereof include leader sequences of TAKA amylase, triose phosphate isomerase, and glaA gene.
- a genetic marker for selecting a transformant it may be appropriately selected according to the method of selecting the transformant.
- a gene encoding drug resistance a gene complementary to auxotrophy is used.
- Such a vector can be designed and prepared appropriately by those skilled in the art by using a known genetic recombination technique.
- the present invention provides a transformant obtained by introducing the expression vector into a filamentous fungus.
- the filamentous fungi are preferably Trichoderma spp. (Especially Trichoderma viride) and Aspergillus spp. (Especially Aspergillus niger), from the viewpoint that the production amount of HRP polypeptide and the like is higher. More preferably, the genus is Trichoderma viride.
- the method for introducing the vector of the present invention is not particularly limited, and can be performed using a known method.
- known methods include protoplast method, calcium chloride method, electroporation method, competent method, heat shock method, spheroplast method, and lithium acetate method.
- a so-called co-transformation method may be used in which an expression vector containing the polynucleotide of the present invention and a vector containing the gene marker are introduced simultaneously.
- the polynucleotide of the present invention is obtained by culturing the transformant and collecting the expressed HRP polypeptide or the like from the cultured transformant and / or the culture of the transformant. Encoding polypeptides can be produced.
- the transformant according to the present invention can be cultured by appropriately selecting a medium, culture conditions and the like according to a conventional method.
- the term “culture” refers to a proliferated transformant obtained by culturing the transformant in an appropriate medium for filamentous fungi, a secreted product of the transformant, and the transformant. Medium containing metabolites, etc., including dilutions and concentrates thereof.
- Such a medium only needs to contain a substance that can be assimilated by filamentous fungi, such as carbon source, nitrogen source, sulfur source, inorganic salts, metal, peptone, yeast extract, meat extract, casein hydrolyzate, serum and the like. Listed as inclusions.
- a substance that can be assimilated by filamentous fungi such as carbon source, nitrogen source, sulfur source, inorganic salts, metal, peptone, yeast extract, meat extract, casein hydrolyzate, serum and the like.
- inclusions for example, an antibiotic corresponding to a drug resistance gene that can be encoded by the expression vector of the present invention, or a nutrient corresponding to a gene that complements the auxotrophy that can be encoded by the expression vector of the present invention. It may be added.
- the culture conditions according to the present invention may be any conditions as long as the transformant of the present invention can secrete and produce HRP polypeptide and the like in the medium.
- the temperature, the presence or absence of addition of air, the concentration of oxygen, the concentration of carbon dioxide, the pH of the medium, the culture temperature, the culture time, the humidity, and the like can be appropriately adjusted and set.
- the transformant is recovered (filtered, centrifuged, etc.) and extracted from the recovered transformant (grinding treatment). , Pressure crushing, etc.) and further purification (salting out method, solvent precipitation method, etc.).
- methods for collecting HRP polypeptide and the like expressed from the transformant include, for example, a method of removing filamentous fungi using a culture filter (for example, a filter having a pore size of 0.2 ⁇ m or less), extraction filtration, and centrifugation.
- a culture filter for example, a filter having a pore size of 0.2 ⁇ m or less
- extraction filtration and centrifugation.
- Known methods such as dialysis, concentration, drying, freezing, adsorption, desorption, and methods utilizing differences in solubility in various solutions (for example, precipitation, salting out, crystallization, recrystallization, transfer dissolution, chromatography), etc. It is done. Further, such methods may be used alone, or may be used in combination or repeatedly in any order.
- polypeptide encoded by the polynucleotide of the present invention contains a tag for purifying the aforementioned HRP, it can be purified using a substrate adsorbed by the tag.
- an HRP polypeptide By such a production method, an HRP polypeptide, an HRP polypeptide that does not contain a signal sequence, or a polypeptide in which a desired polypeptide is added to these HRP polypeptides can be obtained. Therefore, the present invention also provides these HRP polypeptides and the like.
- the sugar chain is added to the HRP C1a polypeptide extracted from horseradish by about 10 kDa, it is detected as a polypeptide having a molecular weight of about 40 kDa by analysis by SDS-PAGE or the like.
- the HRP C1a polypeptide obtained by the production method of the present invention is detected as a polypeptide having a molecular weight of about 32 kDa in analysis by SDS-PAGE or the like.
- the HRP C1a polypeptide extracted from horseradish is not cleaved by glycosidase F, but the sugar of the HRP C1a polypeptide obtained by the production method of the present invention.
- the present inventors have found that the chain is cleaved by the enzyme, and based on this finding, ⁇ 1,3-linked core fucose residues are added to the sugar chain of HRP C1a polypeptide extracted from horseradish. However, it has been clarified that ⁇ 1,3-linked core fucose residues are not added to the sugar chain of the HRP C1a polypeptide obtained by the production method of the present invention.
- the polypeptide of the present invention is It may be a polypeptide produced by a production method and having a sugar chain removed.
- the sugar chain can be removed using an enzyme that decomposes and removes the sugar chain. Examples of such enzymes include glycosidase F (glycopeptidase F) and endoglycosidase H.
- the present invention also provides a preparation containing the HRP polypeptide and the like produced by the production method.
- the preparation of the present invention only needs to contain the HRP polypeptide and the like produced by the production method described above, but other than the HRP polypeptide and the like of the present invention, other acceptable HRP polypeptides and the like are acceptable.
- Ingredients can be included. Examples of such other components include carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, stabilizers, preservatives, preservatives, and physiological saline.
- excipient lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used.
- starch starch, carboxymethylcellulose, calcium carbonate and the like can be used.
- Phosphate, citrate, acetate, etc. can be used as the buffer.
- emulsifier gum arabic, sodium alginate, tragacanth and the like can be used.
- suspending agent glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used.
- the stabilizer propylene glycol, diethylin sulfite, ascorbic acid or the like can be used.
- preservatives phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used.
- sodium azide, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
- the present invention provides a method for detecting a target molecule, which comprises binding a polypeptide produced by the production method of the present invention (polypeptide of the present invention) to the target molecule. provide.
- the target molecule detected by the method of the present invention is not particularly limited, and examples thereof include polypeptides, nucleic acids, sugars, and lipids.
- the polypeptide of the present invention in order to bind the polypeptide of the present invention and the target molecule, has a molecule (for example, an antibody) that specifically binds to the target molecule. Is preferably added.
- a polypeptide of the present invention to which a molecule that specifically binds to the molecule is also preferably used. Used.
- Such addition is not particularly limited, and may be addition at the gene level or chemical addition. As described above, addition at the gene level is achieved by using a codon-modified HRP polynucleotide to which a polynucleotide encoding the antibody or the like is added as the polynucleotide of the present invention.
- the chemical addition may be a covalent bond or a non-covalent bond.
- the “covalent bond” is not particularly limited. For example, an amide bond between an amino group and a carboxyl group, an alkylamine bond between an amino group and an alkyl halide group, a disulfide bond between thiols, a thiol group and a maleimide group or an alkyl halide. And a thioester bond with the group.
- the “non-covalent bond” include a biotin-avidin bond.
- Examples of the luminescent / chromogenic substrate used as a method for detecting the target molecule of the present invention include luminol, TMB, pyrogallol, guaiacol, and dianisidine.
- the present invention provides a method for decolorizing a dye, wherein the polypeptide produced by the method of the present invention is allowed to act on the dye in the presence of hydrogen peroxide.
- Examples of the dye that is decolorized by the method of the present invention include Anato, Orange II, Alizarin Red S, Troperion O, and Chalcone.
- the reaction conditions in the decolorization of the dye that is, the concentration of hydrogen peroxide, the temperature, the type and pH of the system (for example, buffer solution) for mixing the HRP polypeptide and the dye, and the like can be determined by those skilled in the art. It can be set as appropriate according to the type of pigment to be used.
- the HRP polypeptide by causing the HRP polypeptide to act as a catalyst, the phenol moiety in the phenolic compound is oxidized to a phenoxy radical, and the phenoxy radical self-polymerizes to form a water-insoluble multimer. It is known that the multimer can be easily removed as a precipitate.
- the present invention also provides a method for removing a phenolic compound, wherein the polypeptide produced by the method of the present invention is allowed to act on the phenolic compound in the presence of hydrogen peroxide. Can do.
- the phenolic compound removed by the method of the present invention is not particularly limited as long as it is a compound having a phenol moiety oxidized by peroxidase, as described above, and examples thereof include p-cresol and p-ethyl. Examples thereof include phenol and pn-propylphenol.
- the reaction conditions for removing such phenolic compounds that is, the concentration and temperature of hydrogen peroxide, the type and pH of the system (for example, buffer) in which the HRP polypeptide and the phenolic compound are mixed, are known to those skilled in the art. If it exists, it can be set appropriately according to the type of the phenolic compound.
- HRP_Native was ligated using TaKaRa DNA Ligation Kit Mighty Mix (Takara Shuzo Co., Ltd.) to prepare plasmid “pCB1-HRP_Native”. About the reaction conditions, such as an enzyme, the conditions of the description attached to a kit were followed. The sequence of the inserted DNA fragment cloned in the plasmid “pCB1-HRP_Native” was determined using Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and ABI PRISM Genetic Analyzer (Applied Biosystems). The determination was made according to the attached protocol. The plasmid “pCB1-HRP_Native” was constructed to express the HRP polypeptide in its host Trichoderma viride using its own start codon.
- Transformation of Trichoderma viride with plasmid “pCB1-HRP_Native” was carried out according to the method described in International Publication No. 2005/056787. Transformation was carried out by the co-transformation method using two strains of Trichoderma viride strain, which is a uracil biosynthetic gene (pyr4) deficient strain, and using the pyr4 gene of Neurospora crassa as a selection marker.
- pyr4 uracil biosynthetic gene
- Trichoderma viride strain 50 mL of cell formation medium 1% yeast extract, 1% malt extract, 2% polypeptone, 2.5% glucose, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate Heptahydrate, 0.0001% uridine (pH 7.0)
- cell formation medium 1% yeast extract, 1% malt extract, 2% polypeptone, 2.5% glucose, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate Heptahydrate, 0.0001% uridine (pH 7.0)
- the obtained microbial cells were washed with 0.5 mol / L sucrose and filtered through cotton.
- a protoplastizing enzyme solution (1 mg / mL ⁇ -glucuronidase, 0.3 mg / mL chitinase, 0.3 mg / mL zymolyce, 0.5 mol) / L sucrose).
- the mycelium was protoplasted by shaking at 30 ° C. for 60 minutes.
- the suspension was filtered and then centrifuged at 2500 rpm for 10 minutes to collect protoplasts.
- the SUTC buffer (0.5 mol / L sucrose, 10 mmol / L calcium chloride, 10 mmol / L tris-HCl (pH 7.5)) was collected. ).
- This protoplast was suspended in 100 ⁇ L of SUTC buffer, 7 ⁇ L of a DNA solution containing 7 ⁇ g of the plasmid “pCB1-HRP_Native” and 3 ⁇ L of a DNA solution containing the pyr4 gene were added, and allowed to stand in ice for 5 minutes. .
- 400 ⁇ L of PEG solution (60% PEG 4000, 10 mmol / L calcium chloride, 10 mmol / L tris-hydrochloric acid (pH 7.5)) was added and allowed to stand in ice for 20 minutes, then 10 mL of SUTC buffer was added and 2500 rpm And centrifuged for 10 minutes.
- the collected protoplasts are suspended in 1 mL of SUTC buffer, and 200 ⁇ L each is layered on a minimal medium containing 0.5 mol / L sucrose with soft agar. After culturing at 28 ° C. for 5 days, the grown colonies are transplanted again to the minimal medium. The colonies formed here were used as transformants.
- the obtained culture supernatant was subjected to electrophoretic separation using 12% gel SDS-PAGE mini (manufactured by Tefco), and PVDF membrane (manufactured by Millipore). Blotted on top.
- the blotted PVDF membrane was subjected to Western blotting using an anti-HRP antibody (manufactured by JIRL, product number: 123-055-021). The obtained results are shown in FIG.
- Example 1 Examination of HRP polypeptide expression in Humicola by HRP polynucleotide modified in consideration of codon usage of three types of filamentous fungi of Humicola, Aspergillus, and Trichoderma
- HRP polypeptide expression in filamentous fungi in order to highly express HRP polypeptide in filamentous fungi, Taking into account the codon usage of three types of filamentous fungi of Humicola, Aspergillus and Trichoderma, a modified polynucleotide having a base sequence different from the base sequence of the wild-type HRP gene is prepared in order to improve the translation efficiency of all three types did.
- Humicola Humicola insolens
- the expression of HRP polypeptide in the obtained transformant was examined. The results obtained by arranging these methods are shown below.
- codon table for optimizing the expression of all three types of Humicola, Aspergillus, and Trichoderma Considering the codon usage of the polypeptide whose expression was confirmed in Humicola, Aspergillus, Trichoderma, translation efficiency in all three types
- the codon usage frequency table shown in Table 1 was prepared. Specifically, among the three types of bacteria, the frequency of use of codons is extremely low (the frequency of use is less than 5%) even with one type of bacteria, and is set to “0%”. In addition, in all three types of bacteria, for codons with a usage frequency of 5% or more, the average value of the usage frequency of the three or two types of bacteria is calculated, and the average value is a multiple of 5. By changing, the codon usage table shown in Table 1 was prepared.
- HRP-humicola-F CCCGGATCCTGGGACAAGATCGCTCTCTCAGCTCCCTCC (SEQ ID NO: 5)
- HRP-humicola-R CCCGGATCCCTAGTGATGGTGATGATGGGTGTGGTGGGAGTTGAGTTGACGGACG (SEQ ID NO: 6).
- PCR was performed using these primers and using “pHRP” as a template.
- PCR was performed using Prime Starmax DNA polymerase (manufactured by Takara Bio Inc.). PCR was performed in 30 cycles of “98 ° C. for 10 seconds, 55 ° C. for 5 seconds, 72 ° C. for 10 seconds”.
- the amplified DNA fragment of about 1 kbp was cleaved with BamHI to obtain a gene fragment “HRP-humicola” of about 1 kbp.
- the plasmid “pJND-c5” was cleaved with BamHI to recover a fragment of about 8 kbp.
- HRP-humicola was ligated using TaKaRa DNA Ligation Kit Mighty Mix to prepare plasmid “pNCE2-HRP-humicola”.
- the sequence of the inserted DNA fragment cloned in the plasmid “pNCE2-HRP-humicola” was analyzed by the method described in Comparative Example 1 (2).
- the plasmid “pNCE2-HRP-humicola” was constructed to express the HRP polypeptide using its own start codon within the host Humicola insolens.
- the composition of (S) medium is 3.0% glucose, 2.0% yeast extract, 0.1% peptone, 0.03% calcium chloride, 0.03% magnesium chloride, pH 6.8.
- the obtained bacterial cells were washed with 0.5 M sucrose and filtered through a 0.45 ⁇ m filter, and the protoplastizing enzyme solution (3 mg / ml ⁇ -glucuronidase, 1 mg / ml chitinase, 1 mg / ml zymolyase, 0.5 M sucrose) ) Suspended in 10 ml. The hyphae were protoplasted by shaking at 30 ° C. for 60-90 minutes.
- the suspension was filtered, and centrifuged at 2500 rpm for 10 minutes to collect protoplasts, which were washed with SUTC buffer (0.5 M sucrose, 10 mM calcium chloride, 10 mM Tris-HCl (pH 7.5)).
- SUTC buffer 0.5 M sucrose, 10 mM calcium chloride, 10 mM Tris-HCl (pH 7.5)
- This protoplast was suspended in 1 mL of SUTC buffer, 10 ⁇ g of the plasmid “pNCE2-HRP-humicola” was added thereto, and the mixture was allowed to stand in ice for 5 minutes. Next, 400 ⁇ L of a PEG solution (60% PEG 4000, 10 mM calcium chloride, 10 mM Tris-HCl (pH 7.5)) was added and allowed to stand in ice for 20 minutes, then 10 mL of SUTC buffer was added, and 2500 rpm for 10 minutes. Centrifuged. The collected protoplasts were suspended in 1 mL of SUTC buffer, centrifuged at 4000 rpm for 5 minutes, and finally suspended in 100 ⁇ L of SUTC buffer.
- a PEG solution 50% PEG 4000, 10 mM calcium chloride, 10 mM Tris-HCl (pH 7.5)
- Protoplasts with the above treatments were added to regenerated YMG medium supplemented with hygromycin (200 ⁇ g / mL) (1% glucose, 0.4% yeast extract, 0.2% malt extract, 17.8% raffinose, 1% agar, pH 6 8) layered with soft agar and cultured at 37 ° C. for 5 days.
- the grown colonies were transplanted again into a regenerated YMG medium supplemented with hygromycin (200 ⁇ g / mL), and the grown colonies were used as transformants. .
- Example 2 Expression study of HRP polypeptide in Aspergillus using HRP polynucleotide modified in consideration of codon usage of three types of filamentous fungi of Humicola, Aspergillus and Trichoderma Next, the codon-modified HRP polynucleotide was used. Aspergillus (Aspergillus niger) was transformed, and the expression of HRP polypeptide in the obtained transformant was examined.
- Aspergillus transformation was performed using the Aspergillus niger pyr1 strain, which is a uracil biosynthetic gene (pyr4) deficient strain, as a host and using the Trichoderma viride-derived pyr4 gene as a selection marker in the following manner. Carried out.
- PCR was performed using the genomic DNA isolated and purified as described above as a template. PCR was performed using LA Taq polymerase (manufactured by Takara Bio Inc.). PCR was performed with a program that performed 30 cycles of 94 ° C. for 30 seconds, annealing for 30 seconds, and 72 ° C. for 2 minutes. The amplified DNA fragment of about 1100 bp was inserted into the pCR2.1-TOPO plasmid vector using the TOPO TA cloning kit (manufactured by Invitrogen) according to the attached protocol to obtain the plasmid “TOPO-PYR”.
- LA Taq polymerase manufactured by Takara Bio Inc.
- the sequence of the inserted DNA fragment cloned in the plasmid “TOPO-PYR” was analyzed by the method described in Comparative Example 1 (2). As a result of homology search of the base sequence obtained as a result, it showed homology with the PYR4 gene derived from Trichoderma. Therefore, this DNA fragment was judged to be a part of the PYR4 gene.
- This DNA fragment was amplified by PCR using the plasmid “TOPO-PYR” as a template in the same manner as described above, and the obtained PCR product was labeled using an ECL direct system (manufactured by Amersham Pharmacia Biotech) to obtain a probe.
- DNA preparation from positive clones was performed using DNA as a host E. coli according to the method of Maniatis et al. (J. Sambrook, EF Fritsch and T. Maniatls, Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). Was recovered.
- a band of about 0.8 kbp was detected in phage clone 1 and a band of about 2.2 kbp was detected in phage clone 2.
- pUC-PYR-clone1 includes the terminator side of the Pyr4 gene
- pUC-PYR-clone2 includes the promoter side.
- PUC-PYR-clone1 and pUC-PYR-clone2 were treated with PstI and subcloned into pUC118 in a ligated state to obtain a plasmid “pUC-Pyr4”.
- Tricho-pyr-N-xba GGTCTAGACTGCAGGCACTTCCAGCCA (SEQ ID NO: 9)
- Tricho-pyr-C-xba GGTCTAGAGCATGACGAATACATATCAAAC (SEQ ID NO: 10).
- plasmid “pAMY” (see WO 97/000944) was digested with XbaI to recover a fragment of about 8.3 kbp.
- Pyr4-xbaI was ligated using TaKaRa DNA ligation kit Mighty Mix to prepare plasmid “pAMY-Pyr4”.
- amyB-P-R5R and amyB-T-R5R were used as primers, PCR was performed using “pAMY-Pyr4” as a template, and Pyr4 derived from Trichoderma viride An expression vector "pAmyB-pyr” for Aspergillus niger containing the gene was obtained.
- amyB-P-R5R GATACTGTGGGGTTTATTGTCAGAGAA (SEQ ID NO: 11)
- amyB-T-R5R GATATCAGGGTGGAGATAGATATGATGGTA (SEQ ID NO: 12).
- HRP-Aspergillus-F GGCATTATGGCACTTCTCAGCTCCTCCA (SEQ ID NO: 13)
- HRP-Aspergillus-R CTAGTGATGGTGGATGGGTGGGTGTGGGGAGTGGAGTTGACGCG (SEQ ID NO: 14).
- PCR was performed using these primers and using pHRP as a template. PCR was performed using Prime Starmax DNA polymerase. The amplified DNA fragment of about 1 kbp was phosphorylated to obtain a gene fragment “HRP-Aspergillus” of about 1 kbp.
- the plasmid “pAmyB-pyr” prepared in Example 2 (1-2) was cleaved with EcoRV to recover a fragment of about 8.9 kbp.
- the gene fragment “HRP-Aspergillus” of about 1 kbp was ligated to this using TaKaRa DNA ligation kit Mighty Mix to produce a plasmid “pAmyB-pyr-HRP-Aspergillus”.
- the sequence of the inserted DNA fragment cloned in the plasmid “pAmyB-pyr-HRP-Aspergillus” was analyzed by the method described in Comparative Example 1 (2).
- the plasmid “pAmyB-pyr-HRP-Aspergillus” was constructed to express HRP in its host Aspergillus niger using its own start codon.
- a spore suspension of about 10 9 CFU / mL of Aspergillus niger NRRL337 strain was irradiated while gently mixing with a UV2 light at a height of 30 cm. This was applied to a selective medium and cultured at 30 ° C. for 7 days. The grown strain was selected to obtain Aspergillus niger pyr1 strain.
- This selective medium is a minimal medium supplemented with 10 ⁇ g / mL uridine and 4 mg / mL 5-fluoroorotic acid.
- Aspergillus niger pyr1 strain was cultured in (S) medium at 30 ° C., and after 24 hours, the cells were collected by centrifugation at 3000 rpm for 10 minutes.
- the composition of (S) medium is 3.0% glucose, 2.0% yeast extract, 0.1% peptone, 0.03% calcium chloride, 0.03% magnesium chloride, pH 6.8.
- the obtained microbial cells were washed with 4% sodium chloride and filtered with a 0.45 ⁇ m filter.
- Protoplastase solution (3 mg / ml ⁇ -glucuronidase, 1 mg / ml chitinase, 1 mg / ml zymolyase, 4% sodium chloride) 10 ml It was suspended in. The hyphae were protoplasted by shaking at 30 ° C. for 60-90 minutes. The suspension was filtered, and centrifuged at 2500 rpm for 10 minutes to collect protoplasts, which were washed with SUTC buffer (0.5 M sucrose, 10 mM calcium chloride, 10 mM Tris-HCl (pH 7.5)).
- SUTC buffer 0.5 M sucrose, 10 mM calcium chloride, 10 mM Tris-HCl (pH 7.5)
- This protoplast was suspended in 1 mL of SUTC buffer, 10 ⁇ g of the plasmid “pAmyB-pyr-HRP-Aspergillus” was added thereto, and the mixture was allowed to stand in ice for 5 minutes. Next, 400 ⁇ L of a PEG solution (60% PEG 4000, 10 mM calcium chloride, 10 mM Tris-HCl (pH 7.5)) was added, and the mixture was allowed to stand in ice for 20 minutes. Then, 10 ml of SUTC buffer was added, and 2500 rpm, 10 minutes. Centrifuged.
- a PEG solution 50% PEG 4000, 10 mM calcium chloride, 10 mM Tris-HCl (pH 7.5)
- the collected protoplasts were suspended in 1 mL of SUTC buffer, centrifuged at 4000 rpm for 5 minutes, and finally suspended in 100 ⁇ L of SUTC buffer.
- the protoplasts with the above treatments were layered with soft agar on a minimal medium containing 0.5 mol / L sucrose in 200 ⁇ L aliquots. After culturing at 30 ° C. for 5 days, the grown colonies were transplanted again to the minimal medium. The formed colonies were used as transformants.
- an HRP reagent (Wako: 169-10791) manufactured by Wako Pure Chemical Industries, Ltd. was used and diluted with milli-Q water to about 0.625 to 10 ng / mL.
- the HRP concentration in the culture supernatant of the transformant by pAmyB-pyr-HRP-Aspergillus was 0.004 mg / L.
- Example 3 Expression study of HRP polypeptide in Trichoderma using HRP polynucleotide modified in consideration of codon usage of three types of filamentous fungi of Humicola, Aspergillus and Trichoderma Next, the codon-modified HRP polynucleotide was used. Trichoderma (Trichoderma viride) was transformed, and the expression of HRP polypeptide in the obtained transformant was examined.
- HRP-tricho-F GGGAGGCCTGCCGCATCATGCACTTCTCAG (SEQ ID NO: 15)
- HRP-tricho-R CCCCCTCGAGCTAGGAGGTGGAGTTGACGAC (SEQ ID NO: 16)
- HRP-tricho-R (Hisless) CCCCTCGAGCTAGGAGTTGGAGTTGACGAC (SEQ ID NO: 17).
- PCR was performed using HRP-tricho-F and HRP-tricho-R, HRP-tricho-F and HRP-tricho-R (Hisless) as primer sets and pHRP as a template. PCR was performed using Prime Starmax DNA polymerase. The amplified DNA fragment of about 1 kbp was cleaved with StuI and XhoI to obtain gene fragments “HRP-N” and “HRP-N (Hisless)” of about 1 kbp, respectively. On the other hand, the plasmid “pCB1-Eg3X-hphless” was digested with StuI and XhoI, and a fragment of about 6 kbp was recovered.
- HRP-N and HRP-N (Hisless) were ligated to each other using TaKaRa DNA ligation kit Mighty Mix to prepare plasmids “pCB1-HRP-tricho” and “pCB1-HRP (Hisless) -tricho”.
- the sequence of the inserted DNA fragment cloned into the plasmids “pCB1-HRP-tricho” and “pCB1-HRP (Hisless) -tricho” was analyzed by the method described in Comparative Example 1 (2). Plasmids “pCB1-HRP-tricho” and “pCB1-HRP (Hisless) -tricho” were constructed to express HRP in their host Trichoderma viride using their start codons.
- the blotted PVDF membrane was subjected to Western blotting using an anti-His tag antibody for pCB1-HRP-tricho, and western blotting using an anti-HRP antibody for pCB1-HRP (Hisless) -tricho. The obtained results are shown in FIGS.
- a band (approximately 32 kDa HRP) could be detected.
- HRP is transformed from such a transformant. It has been found that polypeptides are produced.
- the HRP concentration in the culture supernatant of the transformant by pCB1-HRP-tricho was 123 mg / L
- the HRP concentration in the culture supernatant of the transformant by pCB1-HRP (Hisless) -tricho was 165 mg / L. Met.
- a culture supernatant in a jar fermenter culture of a transformant by “pCB1-HRP-tricho” or “pCB1-HRP (Hisless) -tricho” was obtained at a concentration of the transformant of 9 ⁇ 10 8 CFU / mL.
- the HRP concentration was measured according to Example 2 (6).
- the HRP concentration in the culture supernatant of the transformant by pCB1-HRP-tricho was 317 mg / L
- the HRP concentration in the culture supernatant of the transformant by pCB1-HRP (Hisless) -tricho was 525 mg / L. Met.
- guaiacol oxidation activity was measured using the culture supernatant in the flask culture of the transformant by “pCB1-HRP-tricho” or “pCB1-HRP (Hisless) -tricho”. That is, a culture supernatant liquid appropriately diluted with a phosphate buffer (pH 7.0) in 3.05 mL of 0.1 M phosphate buffer (pH 7.0) containing 1 ⁇ mol of guaiacol and 0.3 ⁇ mol of hydrogen peroxide. 0.05 mL was added, and the change in absorbance at a wavelength of 436 nm was measured for 10 to 15 minutes.
- the guaiacol oxidation activity was defined as the activity that oxidizes 1 ⁇ mol of guaiacol per minute, and was expressed as the activity per 1 mg of polypeptide contained in the culture supernatant (U / mg protein).
- U / mg protein the guaiacol oxidation activity in the transformant culture supernatant by pCB1-HRP-tricho was 1.54 U / mg protein
- the guaiacol oxidation activity in the transformant culture supernatant by pCB1-HRP (Hisless) -tricho was 7.60 U / mg protein.
- guaiacol oxidation activity was measured using the culture supernatant in the jar fermenter culture of the transformant by “pCB1-HRP-tricho” or “pCB1-HRP (Hisless) -tricho”.
- the guaiacol oxidation activity in the culture supernatant of the transformant by pCB1-HRP-tricho was 3.95 U / mg protein
- guaiacol in the culture supernatant of the transformant by pCB1-HRP (Hisless) -tricho The oxidation activity was 5.49 U / mg protein.
- Example 4 Expression study in Trichoderma of fusion polypeptide of HRP and Trichoderma-derived CBH1 using codon-modified HRP polynucleotide
- a fusion polypeptide of HRP and another polypeptide Whether or not can be highly expressed in filamentous fungi was examined by the following method.
- CBH1 co-expression vector “pCB1-KR” for Trichoderma viride TrichoCBH1HpaR and aTrichoPstF were inserted in order to insert the CBH1 cellulase binding site of plasmid “pCB1-Eg3X-hphless” and the HpaI and PstI sites.
- PCR was performed using pCB1-Eg3X-hphless as a template, and a CBH1 co-expression vector “pCB1-KR” was obtained.
- TrichoCBH1HpaR GGTTAACCTGAGTAGGGCCGGGAGAGA (SEQ ID NO: 18)
- aTrichoPstF GGCTGCAGATAAGGTACTCGAGCAAAAGCTT (SEQ ID NO: 19).
- HRP and CBH1 Fusion Polypeptide Expression Plasmid “pCB1-KR-HRP-tricho” The following primers were prepared based on the base sequence of codon-modified HRP polynucleotide.
- HRPHpaKR GCTATTGAGAAGCGCCAGCTCACCCCCTACTTCTACGAC (SEQ ID NO: 20)
- PERAspglaC CTAGGAGTTGGAGTTGACGAC (SEQ ID NO: 21).
- PCR was performed using pHRP as a template. PCR was performed using Prime Starmax DNA polymerase. The amplified DNA fragment of about 1 kbp was phosphorylated to obtain a gene fragment “HRP-Hpa” of about 1 kbp.
- the plasmid “pCB1-KR” was digested with HpaI, and a fragment of about 6 kbp was recovered. To this, HRP-Hpa was ligated using TaKaRa DNA Ligation Kit Mighty Mix to prepare plasmid “pCB1-KR-HRP-tricho”.
- the sequence of the inserted DNA fragment cloned in the plasmid “pCB1-KR-HRP-tricho” was analyzed by the method described in Comparative Example 1 (2).
- the plasmid “pCB1-KR-HRP-tricho” was constructed so as to be expressed as a fusion polypeptide of CBH1 and HRP in the host Trichoderma viride.
- Example 5 Purification study using a fusion polypeptide of HRP and His tag using codon-modified HRP polynucleotide (1) Purification by HisTrap HP column pCB1-HRP- cultured in Example 3 (3) 2 ml of the culture supernatant of the transformant by tricho was applied to a HisTrap HP column (manufactured by GE) equilibrated with 0.02 M Na 2 HPO 4 and 0.5 M NaCl (pH 7.5) buffer. The column was washed with the buffer used for equilibration and then eluted with 0.02 M Na 2 HPO 4 , 0.5 M NaCl, 0.5 M imidazole (pH 7.5) buffer.
- Example 6 Purification study using a fusion polypeptide of HRP and Trichoderma-derived CBH1 using a codon-modified HRP polynucleotide (1) Purification using binding activity to Avicel In (4) of Example 4 The cultured culture supernatant of pCB1-KR-HRP-tricho (10 ⁇ l) and 2% Avicel solution (90 ⁇ l) (20 mM acetate buffer (pH 5.0), 1M ammonium sulfate) are mixed well and mixed at 25 ° C. for 10 minutes. Left to stand.
- Example 7 Anato pigment degradation test of horseradish peroxidase recombinant polypeptide expressed in Trichoderma Using the culture supernatant of the transformant by pCB1-HRP-tricho cultured in Example 3 (3) Pigment degradation was measured. 10 ⁇ L of culture supernatant was added to 190 ⁇ L of 0.1 M phosphate buffer (pH 6.0) containing 1 ⁇ mol Anato dye (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.14 ⁇ mol of hydrogen peroxide. After standing for a period of time, the change in absorbance at 454 nm was measured. As a result, a decrease in absorbance was detected 30 minutes after the reaction, and it was revealed that HRP expressed from the codon-modified HRP gene exhibits anatomic pigment degradation activity.
- Example 8 Expression study of HRP polypeptide in Trichoderma using HRP polynucleotide adapted only to the codon usage frequency of Trichoderma Based on the above results, Trichoderma showed the highest HRP-producing ability among the three types of filamentous fungi Then, in order to further express the HRP polypeptide, a modified HRP polynucleotide having a base sequence adapted to the usage frequency of Trichoderma was prepared. Then, Trichoderma was transformed with this polynucleotide, and the expression of HRP polypeptide in the obtained transformant was examined. The method and the results obtained are shown below.
- the HRP polynucleotide was modified in order to highly express the HRP gene as an active protein in Trichoderma (Trichoderma viride). That is, based on the codon usage described in Table 2, the base sequence of the wild-type HRP gene (the base sequence described in SEQ ID NO: 3) is accompanied by a 29.9% base change, described in SEQ ID NO: 22. The base sequence was designed.
- PCR was performed using HRP-tricho-2-F and HRP-tricho-2-R (Hisless) as a primer set and pHRP-2 as a template. PCR was performed using Prime Star Max DNA polymerase. The amplified DNA fragment of about 1 kbp was cleaved with StuI and SalI to obtain a gene fragment “HRP-N (Hisless) -2” of about 1 kbp. On the other hand, the plasmid “pCB1-Eg3X-hphless” was digested with StuI and XhoI, and a fragment of about 6 kbp was recovered.
- HRP-N (Hisless) -2 was ligated using TaKaRa DNA Ligation Kit Mighty Mix to prepare plasmid “pCB1-HRP (Hisless) -tricho-2”.
- the sequence of the inserted DNA fragment cloned in the plasmid “pCB1-HRP (Hisless) -tricho-2” was analyzed by the method described in Comparative Example 1 (2).
- the plasmid “pCB1-HRP (Hisless) -tricho-2” was constructed to express HRP using its own start codon in the host Trichoderma viride.
- a polynucleotide in which Trichoderma is transformed using a modified polynucleotide is adapted only to the codon usage of Trichoderma. It was revealed that more HRP polypeptides can be produced than when Trichoderma was transformed using.
- Example 9 Examination of HRP polypeptide expression in Trichoderma using HRP polynucleotide modified in consideration of codon usage of three types of filamentous fungi of Humicola, Aspergillus and Trichoderma (2) Based on the above results, a sequence different from the codon-modified HRP polynucleotide described in Examples 1 to 3 was confirmed in order to confirm the effectiveness of the base sequence in consideration of the codon usage frequency of three types of filamentous fungi of Humicola, Aspergillus and Trichoderma. Taking into account the codon usage of the three types of filamentous fungi, modified polynucleotides were prepared. Then, Trichoderma was transformed with the polynucleotide, and the expression of HRP polypeptide in the obtained transformant was examined. The method and the results obtained are shown below.
- a modified HRP polynucleotide is artificially synthesized and inserted into pMA-T to obtain a plasmid “pHRP-3” into which the codon-modified HRP polynucleotide is inserted. It was.
- the sequence of the inserted DNA fragment cloned in the plasmid “pCB1-HRP (Hisless) -tricho-3” was analyzed by the method described in Comparative Example 1 (2).
- the plasmid “pCB1-HRP (Hisless) -tricho-3” was constructed to express HRP using its own start codon in the host Trichoderma viride.
- the codon has a nucleotide sequence that differs from the wild-type nucleotide sequence encoding horseradish-derived peroxidase (HRP) polypeptide in codons, and the frequency of use of the modified codon is Humicola, Aspergillus.
- HRP horseradish-derived peroxidase
- the polynucleotide of the present invention and the method for producing an HRP polypeptide using the polynucleotide are excellent in efficiently producing a large amount of homogeneous HRP isozyme, the enzyme immunoassay method, immunohistochemical staining method, Southern method, It is useful in the production of detection enzymes and clinical test kit enzymes in various tests such as blotting and Western blotting.
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Abstract
Description
(1) 西洋ワサビ由来ペルオキシダーゼポリペプチドをコードする野生型の塩基配列と少なくとも1個のコドンにおいて異なる塩基配列を有する、修飾されたポリヌクレオチドであって、コドンの使用頻度が下記百分率であり、糸状菌において、コードするポリペプチドを発現させることができるポリヌクレオチド
修飾されたコドンがコードするアミノ酸がアラニンである場合、GCCの使用頻度が80%、GCTの使用頻度が20%;
修飾されたコドンがコードするアミノ酸がアルギニンである場合、CGCの使用頻度が90%、CGTの使用頻度が10%;
修飾されたコドンがコードするアミノ酸がアスパラギンである場合、AACの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がアスパラギン酸である場合、GACの使用頻度が95%、GATの使用頻度が5%;
修飾されたコドンがコードするアミノ酸がシステインである場合、TGCの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がグルタミンである場合、CAGの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がグルタミン酸である場合、GAGの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がグリシンである場合、GGCの使用頻度が75%、GGTの使用頻度が25%;
修飾されたコドンがコードするアミノ酸がヒスチジンである場合、CACの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がイソロイシンである場合、ATCの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がロイシンである場合、CTCの使用頻度が80%、CTGの使用頻度が20%;
修飾されたコドンがコードするアミノ酸がリジンである場合、AAGの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がフェニルアラニンである場合、TTCの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がプロリンである場合、CCCの使用頻度が80%、CCTの使用頻度が20%;
修飾されたコドンがコードするアミノ酸がセリンである場合、AGCの使用頻度が15%、TCCの使用頻度が85%;
修飾されたコドンがコードするアミノ酸がスレオニンである場合、ACCの使用頻度が85%、ACGの使用頻度が15%;
修飾されたコドンがコードするアミノ酸がチロシンである場合、TACの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がバリンである場合、GTCの使用頻度が85%、GTGの使用頻度が5%、GTTの使用頻度が10%。
(2) 少なくとも2個のコドンが修飾されている(1)に記載のポリヌクレオチド。
(3) 少なくとも10%のコドンが修飾されている(1)又は(2)に記載のポリヌクレオチド。
(4) 西洋ワサビ由来ペルオキシダーゼC1aポリペプチドをコードし、下記(i)~(ii)からなる群から選択される少なくとも1の特徴を有する(1)~(3)のうちのいずれか一に記載のポリヌクレオチド
(i)配列番号:1に記載の塩基配列のコード領域を含む
(ii)配列番号:1に記載の91~1017位からなる塩基配列と95%以上の相同性を有する。
(5) 西洋ワサビ由来ペルオキシダーゼC1aポリペプチドをコードし、下記(i)~(ii)からなる群から選択される少なくとも1の特徴を有する(1)~(3)のうちのいずれか一に記載のポリヌクレオチド
(i)配列番号:26に記載の塩基配列のコード領域を含む
(ii)配列番号:26に記載の91~1017位からなる塩基配列と95%以上の相同性を有する。
(6) (1)~(5)のうちのいずれか一に記載のポリヌクレオチドに、さらに所望のポリペプチドをコードするポリヌクレオチドが付加されているポリヌクレオチド。
(7) (1)~(6)のうちのいずれか一に記載のポリヌクレオチドを含む発現ベクター。
(8) (7)に記載の発現ベクターを糸状菌に導入してなる形質転換体。
(9) 前記糸状菌が、トリコデルマ属菌又はアスペルギルス属菌である(8)に記載の形質転換体。
(10) 前記糸状菌が、トリコデルマ・ビリデ又はアスペルギルス・ニガーである(8)に記載の形質転換体。
(11) 前記糸状菌が、トリコデルマ・ビリデである(8)に記載の形質転換体。
(12) (8)~(11)のうちのいずれか一に記載の形質転換体を培養し、培養された形質転換体及び/又は該形質転換体の培養物から、発現させたポリペプチドを採取する工程を含む、(1)~(6)のうちのいずれか一に記載のポリヌクレオチドがコードするポリペプチドの製造方法。
(13) (12)に記載の方法によって製造されたポリペプチド。
(14) (12)に記載の方法によって製造され、糖鎖が除去されたポリペプチド。
(15) (12)に記載の方法によって製造されたポリペプチドを含む調製物。
(16) 標的分子を検出するための方法であって、(12)に記載の方法によって製造されたポリペプチドを前記標的分子に結合させることを特徴とする方法。
(17) 色素の脱色方法であって、該色素に(12)に記載の方法によって製造されたポリペプチドを過酸化水素存在下で作用させることを特徴とする方法。
(18) フェノール性化合物の除去方法であって、該フェノール性化合物に(12)に記載の方法によって製造されたポリペプチドを過酸化水素存在下で作用させることを特徴とする方法。
後述の実施例に示す通り、西洋ワサビ由来ペルオキシダーゼポリペプチド(HRP)をコードする野生型の塩基配列を有するポリヌクレオチドを用いては、糸状菌において、ウェスタンブロット分析により、HRPポリペプチドの発現を検出できなかったが、前記野生型の塩基配列とコドンにおいて異なる塩基配列を有する、修飾されたポリヌクレオチドを用いたところ、かかる糸状菌において、HRPポリペプチドの発現を検出することができた。
西洋ワサビ由来ペルオキシダーゼポリペプチドをコードする野生型の塩基配列と少なくとも1個のコドンにおいて異なる塩基配列を有する、修飾されたポリヌクレオチドであって、コドンの使用頻度が下記百分率であり、糸状菌において、コードするポリペプチドを発現させることができるポリヌクレオチド
修飾されたコドンがコードするアミノ酸がアラニンである場合、GCCの使用頻度が80%、GCTの使用頻度が20%;
修飾されたコドンがコードするアミノ酸がアルギニンである場合、CGCの使用頻度が90%、CGTの使用頻度が10%;
修飾されたコドンがコードするアミノ酸がアスパラギンである場合、AACの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がアスパラギン酸である場合、GACの使用頻度が95%、GATの使用頻度が5%;
修飾されたコドンがコードするアミノ酸がシステインである場合、TGCの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がグルタミンである場合、CAGの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がグルタミン酸である場合、GAGの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がグリシンである場合、GGCの使用頻度が75%、GGTの使用頻度が25%;
修飾されたコドンがコードするアミノ酸がヒスチジンである場合、CACの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がイソロイシンである場合、ATCの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がロイシンである場合、CTCの使用頻度が80%、CTGの使用頻度が20%;
修飾されたコドンがコードするアミノ酸がリジンである場合、AAGの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がフェニルアラニンである場合、TTCの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がプロリンである場合、CCCの使用頻度が80%、CCTの使用頻度が20%;
修飾されたコドンがコードするアミノ酸がセリンである場合、AGCの使用頻度が15%、TCCの使用頻度が85%;
修飾されたコドンがコードするアミノ酸がスレオニンである場合、ACCの使用頻度が85%、ACGの使用頻度が15%;
修飾されたコドンがコードするアミノ酸がチロシンである場合、TACの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がバリンである場合、GTCの使用頻度が85%、GTGの使用頻度が5%、GTTの使用頻度が10%。
修飾されたコドンがコードするアミノ酸がアラニンである場合、GCCの使用頻度が80%、GCTの使用頻度が20%;
修飾されたコドンがコードするアミノ酸がアルギニンである場合、CGCの使用頻度が90%、CGTの使用頻度が10%;
修飾されたコドンがコードするアミノ酸がアスパラギンである場合、AACの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がアスパラギン酸である場合、GACの使用頻度が95%、GATの使用頻度が5%;
修飾されたコドンがコードするアミノ酸がシステインである場合、TGCの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がグルタミンである場合、CAGの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がグルタミン酸である場合、GAGの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がグリシンである場合、GGCの使用頻度が75%、GGTの使用頻度が25%;
修飾されたコドンがコードするアミノ酸がヒスチジンである場合、CACの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がイソロイシンである場合、ATCの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がロイシンである場合、CTCの使用頻度が80%、CTGの使用頻度が20%;
修飾されたコドンがコードするアミノ酸がリジンである場合、AAGの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がフェニルアラニンである場合、TTCの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がプロリンである場合、CCCの使用頻度が80%、CCTの使用頻度が20%;
修飾されたコドンがコードするアミノ酸がセリンである場合、AGCの使用頻度が15%、TCCの使用頻度が85%;
修飾されたコドンがコードするアミノ酸がスレオニンである場合、ACCの使用頻度が85%、ACGの使用頻度が15%;
修飾されたコドンがコードするアミノ酸がチロシンである場合、TACの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がバリンである場合、GTCの使用頻度が85%、GTGの使用頻度が5%、GTTの使用頻度が10%。
(i)配列番号:1に記載の塩基配列のコード領域を含む
(ii)配列番号:1に記載の91~1017位からなる塩基配列と95%以上の相同性を有する。
(i)配列番号:26に記載の塩基配列のコード領域を含む
(ii)配列番号:26に記載の91~1017位からなる塩基配列と95%以上の相同性を有する。
本発明は、前記本発明のポリヌクレオチドを含む発現ベクターも提供する。本発明の発現ベクターとしては、自己複製ベクター、すなわち、染色体外の独立体として存在し、その複製が染色体の複製に依存しない、例えば、プラスミドを基本に構築することができる。また、本発現ベクターは、宿主である糸状菌に導入されたとき、その糸状菌のゲノム中に組み込まれ、それが組み込まれた染色体と一緒に複製されるものであってもよい。
本発明においては、前記形質転換体を培養し、培養された形質転換体及び/又は該形質転換体の培養物から、発現させたHRPポリペプチド等を採取することによって、本発明のポリヌクレオチドがコードするポリペプチドを製造することができる。本発明による形質転換体の培養は、常法に従って、培地、培養条件等を適宜選択することにより行うことができる。
HRPポリペプチドを触媒として作用させることにより、ルミノールやTMB(テトラメチルベンヂジン)等の発光・発色基質が酸化され、化学発光や発色が生じることが知られている。そして、HRPポリペプチドと、標的分子とを結合させることにより、前記化学発光等を指標として該標的分子を検出することができる。従って、本発明は、標的分子を検出するための方法であって、本発明の製造方法によって製造されたポリペプチド(本発明のポリペプチド)を前記標的分子に結合させることを特徴とする方法を提供する。
野生型西洋ワサビ由来ペルオキシダーゼ(HRP)の糸状菌での発現検討
先ず、下記方法にて、野生型HRPポリペプチドをコードする野生型の塩基配列を用いて、糸状菌(トリコデルマ・ビリデ)を形質転換し、得られた形質転換体におけるHRPポリペプチドの発現を調べた。
野生型HRP C1a遺伝子の配列として、「Eur.J.Biochem.、1988年、173巻、3号,681~687ページ」に記載の塩基配列(配列番号:3に記載の塩基配列)を用い、野生型HRP遺伝子を人工的に合成した。人工合成の際、開始コドンの上流の配列に制限酵素StuI認識サイトを、終始コドンの下流に制限酵素XhoI認識サイトを付加した。そして、合成して得られた野生型HRP遺伝子を、制限酵素SfiIにて処理したpMA-Tに挿入することにより、プラスミド「pHRP_Native」を得た。
プラスミド「pHRP_Native」をStuI及びXhoIで切断し、約1kbpの遺伝子断片「HRP_Native」を得た。一方、プラスミド「pCB1-Eg3X-hphless」(国際公開第2011/021616号 参照)をStuI及びXhoIで切断し、約6kbpの断片を回収した。これに「HRP_Native」をTaKaRa DNAライゲ―ションキットマイティミックス(宝酒造社製)を用いて連結し、プラスミド「pCB1-HRP_Native」を作製した。酵素等の反応条件についてはキットに添付の説明書の条件に従った。プラスミド「pCB1-HRP_Native」にクローニングされた挿入DNA断片のシークエンスは、ビッグダイターミネーターv3.1サイクルシークエンシングキット(アプライドバイオシステムズ社製)とABI PRISMジェネティックアナライザー(アプライドバイオシステムズ社製)とを用いて、添付のプロトコールに従って、決定した。プラスミド「pCB1-HRP_Native」は、宿主のトリコデルマ・ビリデ内にて、自身の開始コドンを用いてHRPポリペプチドを発現するように構築した。
プラスミド「pCB1-HRP_Native」によるトリコデルマ・ビリデの形質転換は、国際公開第2005/056787号に記載の方法に従い、実施した。ウラシル生合成遺伝子(pyr4)欠損株であるトリコデルマ・ビリデ株 2株を宿主とし、選択マーカーとしてニューロスポラ・クラッサ(Neurospora crassa)のpyr4遺伝子を用いたコートランスフォーメーション法により形質転換を実施した。トリコデルマ・ビリデ株 2株を50mLの菌体形成培地(1% イーストエキス、1% モルトエキス、2% ポリペプトン、2.5% グルコース、0.1% リン酸水素2カリウム、0.05% 硫酸マグネシウム7水和物、0.0001% ウリジン(pH7.0))において、28℃で24時間培養し、3000rpmで10分間遠心分離し、集菌した。得られた菌体を0.5mol/L シュークロースで洗浄し、綿で濾過したプロトプラスト化酵素溶液(1mg/mL β-グルクロニダーゼ、0.3mg/mL キチナーゼ、0.3mg/mL ザイモリエース、0.5mol/L シュークロース)に懸濁した。30℃で60分間振盪し、菌糸をプロトプラスト化させた。この懸濁液を濾過した後、2500rpmで10分間遠心分離してプロトプラストを回収し、SUTC緩衝液(0.5mol/L シュークロース、10mmol/L 塩化カルシウム、10mmol/L トリス塩酸(pH7.5))で洗浄した。
プラスミド「pCB1-HRP_Native」を導入し最少培地で生育した株を選抜し、国際公開第98/11239号の記載の方法に準じ、P培地(1.0%グルコース、4.0%ラクトース、2.0%大豆粕、1.0%イーストエキス、0.5%リン酸カリウム、0.2%硫酸アンモニゥム、0.2%炭酸カルシウム、0.03%硫酸マグネシウム)にて、フラスコを用い、28℃にて培養した。そして、HRPが発現しているか否かを確認するため、得られた培養上清液を12%ゲルSDS-PAGEミニ(テフコ社製)を用いて電気泳動分離を行い、PVDF膜(ミリポア社製)上にブロットした。ブロットしたPVDF膜について、抗HRP抗体(JIRL社製、製品番号:123-055-021)を用いたウェスタンブロットを行った。得られた結果を図1に示す。
フミコーラ、アスペルギルス及びトリコデルマ3種の糸状菌のコドン使用頻度を勘案して修飾したHRPポリヌクレオチドによる、フミコーラにおけるHRPポリペプチド発現の検討
前記結果を受け、糸状菌においてHRPポリペプチドを高発現させるべく、フミコーラ、アスペルギルス及びトリコデルマ3種の糸状菌のコドン使用頻度を勘案し、これら3種全てにおける翻訳効率を向上させるべく、野生型HRP遺伝子の塩基配列と異なる塩基配列を有する修飾されたポリヌクレオチドを作製した。そして、先ずは、このポリヌクレオチドを用いて、フミコーラ(フミコーラ・インソレンス)を形質転換し、得られた形質転換体におけるHRPポリペプチドの発現を調べた。以下にこれらの方法並び得られた結果について示す。
フミコーラ、アスペルギルス、トリコデルマで発現が確認できたポリペプチドのコドン使用頻度を考慮し、3種全てにおける翻訳効率を向上させるべく、表1に記載のコドン使用頻度表を作成した。具体的には、前記3種の菌において、1種類の菌でも使用頻度が極端に低い(使用頻度が5%未満である)コドンの使用頻度は「0%」と設定した。また、前記3種の菌全てにおいて、使用頻度が5%以上のコドンについては、3種又は2種の菌の使用頻度の平均値を算出し、さらに該平均値が5の倍数になるように変更することにより、表1に記載のコドン使用頻度表を作成した。
HRP遺伝子をフミコーラ、アスペルギルス及びトリコデルマ3種において、活性あるポリペプチドとして高発現させるために、HRPポリヌクレオチドの修飾を行った。すなわち、表1に記載のコドン使用頻度に基づき、野生型HRP遺伝子の塩基配列(配列番号:3に記載の塩基配列)から28.5%の塩基の変更を伴う、配列番号:1に記載の塩基配列を設計した(図2及び3 参照)。なお、このようにして設計した修飾HRPポリヌクレオチドの塩基配列においては、全コドン数338のうち246のコドンを変更、すなわち全コドンのうち72.8%のコドンが修飾(縮重変異)されている。そして、この塩基配列情報に基づき、pHRP_Native同様に、修飾HRPポリヌクレオチドを人工的に合成し、pMA-Tに挿入することにより、コドン修飾HRPポリヌクレオチドが挿入されたプラスミド「pHRP」を得た。
コドン修飾HRPポリヌクレオチドの塩基配列を基に、さらに該ポリヌクレオチドがコードするポリペプチドのC末端側にHisタグが付加されて発現されるように、以下のプライマーを設計し、作製した。
HRP-humicola-F:CCCGGATCCTGGGACAAGATGCACTTCTCCAGCTCCTCC(配列番号:5)
HRP-humicola-R:CCCGGATCCCTAGTGATGGTGATGATGGTGGTGGTGGGAGTTGGAGTTGACGACG(配列番号:6)。
「pNCE2-HRP-humicola」によるフミコーラ・インソレンスの形質転換は、国際公開第01/090375号に記載の方法に従い、実施した。フミコーラ・インソレンス MN200-1株を宿主とし、ハイグロマイシンを選択マーカーに用いて形質転換を実施した。フミコーラ・インソレンスMN200-1株を(S)培地中37℃で培養し、24時間後、3000rpm、10分間遠心分離により集菌した。(S)培地の組成は、3.0% グルコース、2.0% 酵母エキス、0.1% ペプトン、0.03% 塩化カルシウム、0.03% 塩化マグネシウム、pH6.8である。得られた菌体を0.5Mシュークロースで洗浄し、0.45μmのフィルターで濾過したプロトプラスト化酵素溶液(3mg/ml β-グルクロニダーゼ、1mg/ml キチナーゼ、1mg/ml ザイモリアーゼ、0.5Mシュークロース)10mlに懸濁した。30℃で60~90分間振盪し、菌糸をプロトプラスト化させた。この懸濁液を濾過した後、2500rpm、10分間遠心分離してプロトプラストを回収し、SUTC緩衝液(0.5Mシュークロース、10mM塩化カルシウム、10mMトリス塩酸(pH7.5))で洗浄した。
プラスミド「pNCE2-HRP-humicola」を導入しハイグロマイシン添加再生YMG培地で生育した株を選抜し、国際公開第01/090375号に記載の方法に準じて培養した。そして、HRPが発現しているか否かを確認するため、得られた培養上清液を12%ゲルSDS-PAGEミニを用いて電気泳動分離を行い、PVDF膜(ミリポア社製)上にブロットした。ブロットしたPVDF膜について、抗Hisタグ抗体(MBL社製、製品番号:D291-7)を用いたウェスタンブロットを行った。得られた結果を図4に示す。
次に、前記コドン修飾HRPポリヌクレオチドを用いて、アスペルギルス(アスペルギルス・ニガー)を形質転換し、得られた形質転換体におけるHRPポリペプチドの発現を調べた。
(1-1)選択マーカー用トリコデルマ・ビリデ由来pyr4発現プラスミド「pUC-Pyr4」の構築
先ず、下記方法にて、アスペルギルス・ニガーの形質転換において選択マーカーとして利用するトリコデルマ・ビリデ由来pyr4遺伝子のクローニングを行った。
トリコデルマ・ビリデの菌体より、堀内らの方法(H.Horiuchiら、J.Bacteriol.1988年、170巻、272~278ページ 参照)に従ってゲノムDNAを単離・精製した。単離したゲノムDNAを制限酵素Sau3AIにより部分消化した。これをファージベクターλEMBL3クローニングキット(ストラタジーン社製)のBamHIアームに、ライゲーションキットVer.2(宝酒造社製)を用いて連結させた。これをエタノール沈澱後、TE緩衝液に溶解した。連結混合物の全量をマックスプラックスλパッケージングキット(エピセンターテクノロジー社製)を用い、ファージ粒子を形成させ、大腸菌XL1-blue MRA(P2)株に感染させた。この方法により1.1×104個のファージから成るゲノムDNAライブラリーが得られた。
公開されているトリコデルマ・リセイの翻訳領域の配列を基に以下のプライマーを作製した。
PYRMET:ATGGCACCACACCCGACG(配列番号:7)
PYRSTOP:CTATCGCAGTAGCCGCTC(配列番号:8)。
前記にて作製したファージプラークは、ハイボンドN+ナイロントランスファーメンブラン(アマシャム社製)に転写し、0.4N水酸化ナトリウムでアルカリ処理し、メンブラン上の組換えファージDNAを1本鎖に変成後、5×SSC(1×SSC:15mMクエン酸3ナトリウム、150mM塩化ナトリウム)で洗浄し、風乾させDNAを固定した。その後、キットのマニュアルにしたがって、前記にて作製したプローブを用いてハイブリダイゼーションを行い、検出反応をし、FUJIメディカルX線フィルム(富士写真フィルム社製)に感光させ、2個の陽性クローンを得た。陽性クローンからのDNA調製は、Maniatisらの方法(J.Sambrook,E.F.Fritsch及びT.Maniat1s、Molecular Cloning,Cold Spring Harbor Laboratory Press、1989年)に従い、宿主大腸菌としてLE392を用いてファージDNAを回収した。以上のように調製したファージDNAをPstIで処理し、前記プローブを用いてハイブリダイゼーションを行った結果、ファージクローン1では約0.8kbpのバンドを検出し、ファージクローン2では約2.2kbpのバンドを検出した。
ファージクローン1の約0.8kbpのPstI断片及びファージクローン2の約2.2kbpのPstI断片をpUC118にクローン化し、それぞれプラスミド「pUC-PYR-clone1」及び「pUC-PYR-clone2」を得た。得られたプラスミドの塩基配列を実施例1-2記載の方法により解析した。その結果、「pUC-PYR-clone1」はPyr4遺伝子のターミネーター側が、「pUC-PYR-clone2」はプロモーター側が含まれていることが明らかになった。「pUC-PYR-clone1」及び「pUC-PYR-clone2」をPstI処理し、連結した状態でpUC118にサブクローニングし、プラスミド「pUC-Pyr4」を得た。
プラスミド「pUC-Pyr4」にXbaIサイトを付加するために、Tricho-pyr-N-xba及びTricho-pyr-C-xbaをプライマーとして使用し、「pUC-Pyr4」を鋳型にPCRを行った。増幅された約2.5kbpのDNA断片をXbaIで切断し、約2.5kbpの遺伝子断片「Pyr4-xbaI」を得た。
Tricho-pyr-N-xba:GGTCTAGACTGCAGGCACTTCCAGGCA(配列番号:9)
Tricho-pyr-C-xba:GGTCTAGAGCATGACGAATACATATCAAAC(配列番号:10)。
amyB-P-R5R:GATATCTGTGGGGTTTATTGTTCAGAGAA(配列番号:11)
amyB-T-R5R:GATATCAGGGTGGAGAGTATATGATGGTA(配列番号:12)。
次に、前記にて構築したプラスミド「pAmyB-pyr」に、前記コドン改変HRP遺伝子を挿入した。すなわち、先ず、コドン改変HRP遺伝子の塩基配列を基に、さらに該ポリヌクレオチドがコードするポリペプチドのC末端側にHisタグが付加されて発現されるように、以下のプライマーを設計し、作製した。
HRP-Aspergillus-F:GGCATTTATGCACTTCTCCAGCTCCTCCA(配列番号:13)
HRP-Aspergillus-R:CTAGTGATGGTGATGATGGTGGTGGTGGGAGTTGGAGTTGACGACG(配列番号:14)。
次に、ウラシル生合成遺伝子(pyr4)欠損株であるアスペルギルス・ニガー pyr1株を以下に示す方法にて作出した。
pyr4欠損株であるアスペルギルス・ニガー pyr1株を宿主とし、選択マーカーとしてpyr4遺伝子を用いた形質転換を実施した。
プラスミド「pAmyB-pyr-HRP-Aspergillus」を導入し最少再生培地で生育した株を選抜し、生産培地に植菌し30℃、4日間培養した。得られた培養上清液を12%ゲルSDS-PAGEミニを用いて電気泳動分離を行い、PVDF膜(ミリポア社製)上にブロットした。ブロットしたPVDF膜について、抗Hisタグ抗体を用いたウェスタンブロットを行った。得られた結果を図5に示す。
pAmyB-pyr-HRP-Aspergillusによる形質転換体の培養上清を適宜希釈し、該形質転換体の濃度が9×108CFU/mLとなるよう希釈した培養上清にテトラメチルベンヂジン試薬(コスモバイオ社製)を添加して、室温で10分間静置した。1N硫酸を添加し、反応を停止した後、波長450nmにおける吸光度を測定し、HRP量を算出した。なお、検量線には、和光純薬社製HRP試薬(和光:169-10791)を使用し、ミリQ水で0.625~10ng/mL程度に希釈したものを用いた。結果、pAmyB-pyr-HRP-Aspergillusによる形質転換体培養上清中のHRP濃度は0.004mg/Lであった。
次に、前記コドン修飾HRPポリヌクレオチドを用いて、トリコデルマ(トリコデルマ・ビリデ)を形質転換し、得られた形質転換体におけるHRPポリペプチドの発現を調べた。
コドン修飾HRPポリヌクレオチドの塩基配列を基に以下のプライマーを作製した。
HRP-tricho-F:GGGAGGCCTGCGCATCATGCACTTCTCCAG(配列番号:15)
HRP-tricho-R:CCCCTCGAGCTAGGAGTTGGAGTTGACGAC(配列番号:16)
HRP-tricho-R(Hisless):CCCCTCGAGCTAGGAGTTGGAGTTGACGAC(配列番号:17)。
プラスミド「pCB1-HRP-tricho」又は「pCB1-HRP(Hisless)-tricho」によるトリコデルマ・ビリデの形質転換は、比較例1(3)記載の方法により実施した。
プラスミド「pCB1-HRP-tricho」及び「pCB1-HRP(Hisless)-tricho」を各々導入し、最少培地で生育した株を選抜し、国際公開第98/11239号に記載の方法に準じ、P培地にて、フラスコ又はジャーファーメンターを用い、28℃にて培養した。HRPが発現しているか否かを確認するため、得られた培養上清液を12%ゲルSDS-PAGEミニを用いて電気泳動分離を行い、PVDF膜(ミリポア社製)上にブロットした。ブロットしたPVDF膜について、pCB1-HRP-trichoに関しては、抗Hisタグ抗体を用いたウェスタンブロットを行い、pCB1-HRP(Hisless)-trichoに関しては、抗HRP抗体を用いたウェスタンブロットを行った。得られた結果を図6及び7に示す。
「pCB1-HRP-tricho」又は「pCB1-HRP(Hisless)-tricho」による形質転換体のフラスコ培養における培養上清を、該形質転換体の濃度が9×108CFU/mLとなるよう適宜希釈し、HRP濃度の測定を実施例2(6)に従い、実施した。結果、pCB1-HRP-trichoによる形質転換体の培養上清中のHRP濃度は123mg/Lであり、pCB1-HRP(Hisless)-trichoによる形質転換体の培養上清中のHRP濃度は165mg/Lであった。同様に「pCB1-HRP-tricho」又は「pCB1-HRP(Hisless)-tricho」による形質転換体のジャーファーメンター培養における培養上清を、該形質転換体の濃度が9×108CFU/mLとなるよう適宜希釈し、HRP濃度の測定を実施例2(6)に従い、実施した。結果、pCB1-HRP-trichoによる形質転換体の培養上清中のHRP濃度は317mg/Lであり、pCB1-HRP(Hisless)-trichoによる形質転換体の培養上清中のHRP濃度は525mg/Lであった。
前記コドン修飾HRPポリヌクレチドを用いることにより、HRPと他のポリペプチドとの融合ポリペプチドも、糸状菌にて高発現させることができるかどうかを、以下に示す方法にて調べた。
プラスミド「pCB1-Eg3X-hphless」のCBH1セルラーゼ結合部位欠失、並びにHpaIサイト及びPstIサイトを挿入するために、TrichoCBH1HpaR及びaTrichoPstFをプライマーとして使用し、pCB1-Eg3X-hphlessを鋳型にPCRを行い、CBH1共発現用ベクター「pCB1-KR」を得た。
TrichoCBH1HpaR:GGTTAACCTGAGTAGGGCCGGGAGAGGA(配列番号:18)
aTrichoPstF:GGCTGCAGTAAGGTACTCGAGCAAAAGCTT(配列番号:19)。
コドン修飾HRPポリヌクレオチドの塩基配列を基に以下のプライマーを作製した。
HRPHpaKR:GCTATTGAGAAGCGCCAGCTCACCCCTACCTTCTACGAC(配列番号:20)
PERAspglaC:CTAGGAGTTGGAGTTGACGAC(配列番号:21)。
プラスミド「pCB1-KR-HRP-tricho」によるトリコデルマ・ビリデの形質転換は、比較例1(3)記載の方法により実施した。
プラスミド「pCB1-KR-HRP-tricho」を導入し最少培地で生育した株を選抜し、国際公開第98/11239号に記載の方法に準じ、P培地にて、フラスコを用い、28℃にて培養した。HRPが発現しているか否かを確認するため、得られた培養上清液を12%ゲルSDS-PAGEミニを用いて電気泳動分離を行い、PVDF膜(ミリポア社製)上にブロットした。ブロットしたPVDF膜について、抗HRP抗体を用いたウェスタンブロットを行った。得られた結果を図8に示す。
pCB1-KR-HRP-trichoによる形質転換体を、国際公開第98/11239号の記載の方法に準じ、P培地にて、フラスコを用い、28℃にて培養した。そして、得られた培養上清を、該形質転換体の濃度が9×108CFU/mLとなるよう適宜希釈し、HRP濃度の測定を実施例2(6)に従い、実施した。pCB1-KR-HRP-trichoによる形質転換体の培養上清中のHRP濃度は123mg/Lであった。
(1) HisTrap HPカラムによる精製
実施例3(3)で培養した、pCB1-HRP-trichoによる形質転換体の培養上清2mlを、0.02M Na2HPO4、0.5M NaCl(pH7.5)バッファーで平衡化したHisTrap HPカラム(GE製)に供した。平衡化に使用したバッファーを用いてカラムを洗浄後、0.02M Na2HPO4、0.5M NaCl、0.5M イミダゾール(pH7.5)バッファーにて溶出した。
(1) アビセルへの結合活性を用いた精製
実施例4の(4)にて培養した、pCB1-KR-HRP-trichoによる形質転換体培養上清10μlと2%濃度のアビセル溶液90μl(20mM酢酸バッファー(pH5.0)、1M硫安)とを良く混和し、25℃で10分間静置した。遠心後、上清を除去し、20mM酢酸バッファー(pH5.0)及び1M硫安を加え、アビセル溶液を洗浄した(2回実施)。最終の遠心後、上清を除去し、ミリQ水を混ぜ、37℃で10分間放置し、アビセルから剥離させた。剥離後の上清を回収し、上清中に含まれるHRP濃度の測定を実施例2(6)に従い、実施した。その結果、12.7ng/ml濃度のHRPを回収することが出来た。
実施例3(3)で培養した、pCB1-HRP-trichoによる形質転換体の培養上清を用いてアナトー色素分解を測定した。1μmolのアナトー色素(和光純薬社製)、0.14μmolの過酸化水素を含む0.1M リン酸塩緩衝液(pH6.0)190μLに培養上清液10μLを添加し、37℃において適当な時間静置した後、454nmの吸光度変化を測定した。その結果、反応30分後から吸光度の減少が検出され前記コドン改変HRP遺伝子から発現されたHRPは、アナトー色素分解活性を示すことが明らかになった。
上記結果を受け、3種類の糸状菌の中で最も高いHRP産生能を示したトリコデルマにおいて、HRPポリペプチドをさらに高発現させるべく、トリコデルマの使用頻度に適合させた塩基配列を有する修飾されたHRPポリヌクレオチドを作製した。そして、このポリヌクレオチドを用いてトリコデルマを形質転換し、得られた形質転換体におけるHRPポリペプチドの発現を調べた。以下にその方法及び得られた結果について示す。
トリコデルマ(トリコデルマ・ビリデ)で発現が確認できたポリペプチドのコドン使用頻度を考慮し、表2に記載のコドン使用頻度表を作成した。
HRP遺伝子をトリコデルマ(トリコデルマ・ビリデ)において、活性あるタンパク質として高発現させるために、HRPポリヌクレオチドの修飾を行った。すなわち、表2に記載のコドン使用頻度に基づき、野生型HRP遺伝子の塩基配列(配列番号:3に記載の塩基配列)から29.9%の塩基の変更を伴う、配列番号:22に記載の塩基配列を設計した。なお、このようにして設計した修飾HRPポリヌクレオチドの塩基配列においては、全コドン数338のうち242のコドンを変更、すなわち全コドンのうち71.6%のコドンが修飾(縮重変異)されている。そして、この塩基配列情報に基づき、pHRP_Native同様に、修飾HRPポリヌクレオチドを人工的に合成し、pMA-Tに挿入することにより、コドン修飾HRPポリヌクレオチドが挿入されたプラスミド「pHRP-2」を得た。
コドン修飾HRPポリヌクレオチドの塩基配列を基に以下のプライマーを作製した。
HRP-tricho-2-F:GGGAGGCCTGCGCATCATGCACTTCA(配列番号:24)
HRP-tricho-2-R(Hisless):CCCGTCGACGCTGTTGCTGTTGACGACGCGGCAGTT(配列番号:25)。
プラスミド「pCB1-HRP(Hisless)-tricho-2」によるトリコデルマ(トリコデルマ・ビリデ)の形質転換は、比較例1 (3)記載の方法により実施した。
プラスミド「pCB1-HRP(Hisless)-tricho-2」を導入し、最少培地で生育した株を選抜し、国際公開第98/11239号に記載の方法に準じ、P培地にて、フラスコを用い、28℃にて培養した。HRPが発現しているか否かを確認するため、得られた培養上清液を12%ゲルSDS-PAGE ミニを用いて電気泳動分離を行い、PVDF膜(ミリポア社製)上にブロットした。ブロットしたPVDF膜について、抗HRP抗体を用いたウェスタンブロットを行った。得られた結果を図9に示す。
「pCB1-HRP(Hisless)-tricho」による形質転換体のフラスコ培養における培養上清を、該形質転換体の濃度が9×108CFU/mLとなるよう適宜希釈し、HRP濃度の測定を実施例2 (6)に従い、実施した。
上記結果を受け、フミコーラ、アスペルギルス及びトリコデルマ3種の糸状菌のコドン使用頻度を勘案した塩基配列の有効性を確認するべく、実施例1~3に記載のコドン修飾HRPポリヌクレオチドとは異なる配列を有する、当該3種の糸状菌のコドン使用頻度を勘案し、修飾したポリヌクレオチドを作製した。そして、該ポリヌクレオチドを用いてトリコデルマを形質転換し、得られた形質転換体におけるHRPポリペプチドの発現を調べた。以下にその方法及び得られた結果について示す。
HRP遺伝子をトリコデルマにおいて活性あるタンパク質として高発現させるために、HRPポリヌクレオチドの修飾を行った。すなわち、表1に記載のコドン使用頻度に基づき、野生型HRP遺伝子の塩基配列(配列番号:3に記載の塩基配列)から28.0%の塩基の変更を伴う、配列番号:26に記載の塩基配列を設計した。
プラスミド「pHRP-3」及び「pCB1-Eg3X-hphless」をStuI及びXhoIで切断し、約1kbp及び約6kbpの断片を回収した。両者をTaKaRa DNAライゲーションキットマイティミックスを用いて連結し、プラスミド「pCB1-HRP(Hisless)-tricho-3」を作製した。プラスミド「pCB1-HRP(Hisless)-tricho-3」にクローニングされた挿入DNA断片のシークエンスを、比較例1 (2)記載の方法により解析した。プラスミド「pCB1-HRP(Hisless)-tricho-3」は、宿主のトリコデルマ・ビリデ内にて、自身の開始コドンを用いてHRPを発現するように構築した。
プラスミド「pCB1-HRP(Hisless)-tricho-3」によるトリコデルマ(トリコデルマ・ビリデ)の形質転換は、比較例1 (3)記載の方法により実施した。
プラスミド「pCB1-HRP(Hisless)-tricho-3」を導入し、最少培地で生育した株を選抜し、国際公開第98/11239号に記載の方法に準じ、P培地にて、フラスコを用い、28℃にて培養した。HRPが発現しているか否かを確認するため、得られた培養上清液を12%ゲルSDS-PAGE ミニを用いて電気泳動分離を行い、PVDF膜(ミリポア社製)上にブロットした。ブロットしたPVDF膜について、抗HRP抗体を用いたウェスタンブロットを行った。得られた結果を図14に示す。
「pCB1-HRP(Hisless)-tricho-3」による形質転換体を、国際公開第98/11239号に記載の方法に準じ、P培地にて、フラスコを用い、28℃にて培養した。そして、その培養上清を、該形質転換体の濃度が9×108CFU/mLとなるよう適宜希釈し、HRP濃度の測定を実施例2 (6)に従い、実施した。結果、pCB1-HRP(Hisless)-tricho-3による形質転換体の培養上清中のHRP濃度は200mg/Lであった。
<223> コドン使用頻度がトリコデルマ、フミコ―ラ及びアスペルギルスに適合されており、人工的に合成されたポリヌクレオチドの配列
配列番号:2
<223> 配列番号:1に記載の塩基配列からなる人工的に合成されたポリヌクレオチドがコードするポリペプチド
配列番号:5~21、24及び25
<223> 人工的に合成されたプライマーの配列
配列番号:22
<223> コドン使用頻度がトリコデルマに適合されており、人工的に合成されたポリヌクレオチドの配列
配列番号:23
<223> 配列番号:22に記載の塩基配列からなる人工的に合成されたポリヌクレオチドがコードするポリペプチド
配列番号:27
<223> 配列番号:26に記載の塩基配列からなる人工的に合成されたポリヌクレオチドがコードするポリペプチド
Claims (18)
- 西洋ワサビ由来ペルオキシダーゼポリペプチドをコードする野生型の塩基配列と少なくとも1個のコドンにおいて異なる塩基配列を有する、修飾されたポリヌクレオチドであって、コドンの使用頻度が下記百分率であり、糸状菌において、コードするポリペプチドを発現させることができるポリヌクレオチド
修飾されたコドンがコードするアミノ酸がアラニンである場合、GCCの使用頻度が80%、GCTの使用頻度が20%;
修飾されたコドンがコードするアミノ酸がアルギニンである場合、CGCの使用頻度が90%、CGTの使用頻度が10%;
修飾されたコドンがコードするアミノ酸がアスパラギンである場合、AACの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がアスパラギン酸である場合、GACの使用頻度が95%、GATの使用頻度が5%;
修飾されたコドンがコードするアミノ酸がシステインである場合、TGCの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がグルタミンである場合、CAGの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がグルタミン酸である場合、GAGの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がグリシンである場合、GGCの使用頻度が75%、GGTの使用頻度が25%;
修飾されたコドンがコードするアミノ酸がヒスチジンである場合、CACの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がイソロイシンである場合、ATCの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がロイシンである場合、CTCの使用頻度が80%、CTGの使用頻度が20%;
修飾されたコドンがコードするアミノ酸がリジンである場合、AAGの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がフェニルアラニンである場合、TTCの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がプロリンである場合、CCCの使用頻度が80%、CCTの使用頻度が20%;
修飾されたコドンがコードするアミノ酸がセリンである場合、AGCの使用頻度が15%、TCCの使用頻度が85%;
修飾されたコドンがコードするアミノ酸がスレオニンである場合、ACCの使用頻度が85%、ACGの使用頻度が15%;
修飾されたコドンがコードするアミノ酸がチロシンである場合、TACの使用頻度が100%;
修飾されたコドンがコードするアミノ酸がバリンである場合、GTCの使用頻度が85%、GTGの使用頻度が5%、GTTの使用頻度が10%。 - 少なくとも2個のコドンが修飾されている請求項1に記載のポリヌクレオチド。
- 少なくとも10%のコドンが修飾されている請求項1又は2に記載のポリヌクレオチド。
- 西洋ワサビ由来ペルオキシダーゼC1aポリペプチドをコードし、下記(i)~(ii)からなる群から選択される少なくとも1の特徴を有する請求項1~3のうちのいずれか一項に記載のポリヌクレオチド
(i)配列番号:1に記載の塩基配列のコード領域を含む
(ii)配列番号:1に記載の91~1017位からなる塩基配列と95%以上の相同性を有する。 - 西洋ワサビ由来ペルオキシダーゼC1aポリペプチドをコードし、下記(i)~(ii)からなる群から選択される少なくとも1の特徴を有する請求項1~3のうちのいずれか一項に記載のポリヌクレオチド
(i)配列番号:26に記載の塩基配列のコード領域を含む
(ii)配列番号:26に記載の91~1017位からなる塩基配列と95%以上の相同性を有する。 - 請求項1~5のうちのいずれか一項に記載のポリヌクレオチドに、さらに所望のポリペプチドをコードするポリヌクレオチドが付加されているポリヌクレオチド。
- 請求項1~6のうちのいずれか一項に記載のポリヌクレオチドを含む発現ベクター。
- 請求項7に記載の発現ベクターを糸状菌に導入してなる形質転換体。
- 前記糸状菌が、トリコデルマ属菌又はアスペルギルス属菌である請求項8に記載の形質転換体。
- 前記糸状菌が、トリコデルマ・ビリデ又はアスペルギルス・ニガーである請求項8に記載の形質転換体。
- 前記糸状菌が、トリコデルマ・ビリデである請求項8に記載の形質転換体。
- 請求項8~11のうちのいずれか一項に記載の形質転換体を培養し、培養された形質転換体及び/又は該形質転換体の培養物から、発現させたポリペプチドを採取する工程を含む、請求項1~6のうちのいずれか一項に記載のポリヌクレオチドがコードするポリペプチドの製造方法。
- 請求項12に記載の方法によって製造されたポリペプチド。
- 請求項12に記載の方法によって製造され、糖鎖が除去されたポリペプチド。
- 請求項12に記載の方法によって製造されたポリペプチドを含む調製物。
- 標的分子を検出するための方法であって、請求項12に記載の方法によって製造されたポリペプチドを前記標的分子に結合させることを特徴とする方法。
- 色素の脱色方法であって、該色素に請求項12に記載の方法によって製造されたポリペプチドを過酸化水素存在下で作用させることを特徴とする方法。
- フェノール性化合物の除去方法であって、該フェノール性化合物に請求項12に記載の方法によって製造されたポリペプチドを過酸化水素存在下で作用させることを特徴とする方法。
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Also Published As
Publication number | Publication date |
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US9540617B2 (en) | 2017-01-10 |
EP2857511A4 (en) | 2016-07-20 |
JP6110375B2 (ja) | 2017-04-05 |
EP2857511B1 (en) | 2018-07-04 |
EP2857511A1 (en) | 2015-04-08 |
DK2857511T3 (en) | 2018-08-27 |
JPWO2013180208A1 (ja) | 2016-01-21 |
US20150140574A1 (en) | 2015-05-21 |
ES2682271T3 (es) | 2018-09-19 |
TWI634208B (zh) | 2018-09-01 |
TW201408775A (zh) | 2014-03-01 |
CN104334723A (zh) | 2015-02-04 |
CN104334723B (zh) | 2018-05-22 |
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