WO2014163176A1 - Ny-eso-1タンパク質の製造方法 - Google Patents
Ny-eso-1タンパク質の製造方法 Download PDFInfo
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- WO2014163176A1 WO2014163176A1 PCT/JP2014/059928 JP2014059928W WO2014163176A1 WO 2014163176 A1 WO2014163176 A1 WO 2014163176A1 JP 2014059928 W JP2014059928 W JP 2014059928W WO 2014163176 A1 WO2014163176 A1 WO 2014163176A1
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- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- the present invention relates to a recombinant Escherichia coli used for production of NY-ESO-1 protein, and a method for producing NY-ESO-1 protein using the strain.
- the recombinant Escherichia coli of the present invention is useful for the production of hydrophobic proteins that tend to form inclusion bodies such as NY-ESO-1 protein.
- Hosts that produce proteins from different organisms include prokaryotic cells such as bacteria (eg, E. coli, Bacillus subtilis), and eukaryotic cells such as fungi (eg, filamentous fungi, yeast), insects, and animal and plant cells.
- prokaryotic cells such as bacteria (eg, E. coli, Bacillus subtilis), and eukaryotic cells such as fungi (eg, filamentous fungi, yeast), insects, and animal and plant cells.
- bacteria eg, Escherichia coli have many advantages such as a high growth rate, a large amount of growth on an inexpensive medium, and progress in genetic research and the use of various vectors according to the purpose.
- eukaryotic proteins are heterologously expressed in a prokaryotic bacterium, some expressed proteins cannot take the correct three-dimensional structure even if they are synthesized in the microbial cells, and are insoluble and physiologically insoluble. It is a problem that it accumulates in the fungus body as an
- NY-ESO-1 protein which is a cancer / testis antigen
- NY-ESO-1 protein is a useful protein that is specific to cancer, is expressed in a wide range, has high immunity induction ability, and has been confirmed to be clinically effective as a cancer vaccine.
- Its amino acid sequence (SEQ ID NO: 4) Is registered as Genbank accession number CAA05908 and NCBI reference sequence NP_001318 (see also Non-Patent Document 1).
- SEQ ID NO: 4 amino acid sequence
- NY-ESO-1 protein is highly hydrophobic, in order to purify the protein from E. coli, inclusion bodies from E.
- Non-Patent Documents 2 to 4 Even after such many steps, it still contains a lot of impurities, and it was difficult to obtain NY-ESO-1 protein with high purity.
- An object of the present invention is to provide a method for producing NY-ESO-1 protein with high purity, and a strain system useful for carrying out the method.
- the present invention is a system for producing NY-ESO-1 protein, which is difficult to mass-produce with high purity, using an Escherichia coli strain excellent in growth rate, protein production amount, etc. It is an object to establish a system with few impurities in a product.
- the present inventors have used (NY-ESO-1) in a purified product from a cell disruption by using E. coli in which the gatZ gene has been deleted or inactivated. It has been found that the amount of E. coli-derived impurities (other than proteins) is significantly reduced, and the present invention has been completed. Specifically, when a gene encoding the NY-ESO-1 protein is introduced into E. coli in which the gatZ gene has been deleted or inactivated, the cell disruption product is compared with that before deletion or inactivation. It was found that the amount of contaminating E. coli-derived protein was significantly reduced without reducing the amount of NY-ESO-1 protein in the purified product.
- E. coli in which the gatZ gene has been deleted or inactivated and the NY-ESO-1 gene has been introduced.
- a method for producing NY-ESO-1 protein comprising the step of extracting NY-ESO-1 protein from E. coli according to [1].
- An E. coli host for producing NY-ESO-1 protein in which the gatZ gene is deleted or inactivated.
- the present invention further relates to the following inventions.
- Escherichia coli obtained by a step of deleting or inactivating the gatZ gene in the genome of E. coli and a step of introducing a gene encoding NY-ESO-1 protein into the E. coli.
- the NY-ESO-1 protein can be obtained with high purity by using the recombinant Escherichia coli of the present invention.
- FIG. 1A shows the result of staining the protein in the separated gel
- FIG. 1B shows the result of Western blotting with an anti-NY-ESO-1 antibody.
- the recombinant Escherichia coli of the present invention is characterized in that the gatZ gene is deleted or inactivated, and is useful for producing NY-ESO-1 protein with high purity. Therefore, the present invention provides a novel use of a recombinant Escherichia coli in which the gatZ gene is deleted or inactivated as an E. coli host for producing NY-ESO-1 protein.
- the recombinant E. coli for obtaining NY-ESO-1 protein with high purity means recombinant E. coli modified so that the amount of impurities in the purified NY-ESO-1 protein is reduced. .
- high purity means that the concentration of a host-derived protein other than NY-ESO-1 protein in the purified NY-ESO-1 protein is less than 50 ng / mg, preferably 40 ng at most. means less than / mg, more preferably less than 30 ng / mg, most preferably less than 20 ng / mg.
- Methods for measuring the concentration of host-derived protein are known in the art, and E. coli Host Cell Protein ELISA Kit (Cygnus Technologies, product code F410) for measuring E. coli-derived protein concentration is known. ) And other immunoassay kits are commercially available.
- the E. coli gatZ gene is a gene encoding an enzyme gatZ (also known as tagatose-6-phosphate kinase) that phosphorylates tagatose-6-phosphate to form tagatose-6-diphosphate.
- gatZ also known as tagatose-6-phosphate kinase
- the nucleotide sequence (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO: 6) of the Escherichia coli gatZ gene are registered with GenBank under accession number X79837, and Jeong H et al. Genome sequences of Escherichia coli B strain REL606 and BL21 (DE3 ). J Mol Biol, 2009 (4): 644-52.
- the “gatZ gene” refers to a protein consisting of the amino acid sequence of SEQ ID NO: 6, or 70% or more, preferably 80% or more, more preferably 90% or more, and still more preferably 95%. As mentioned above, it means a polynucleotide that encodes a protein having an amino acid sequence identity of 98% or more and having an activity to phosphorylate tagatose-6-phosphate.
- a method for evaluating the phosphorylation activity of a protein or polypeptide is known in the art. For example, tagatose-6-phosphate is incubated under conditions suitable for phosphorylation of tagatose-6-phosphate. By detecting the amount of tagatose-6-diphosphate, the activity to phosphorylate tagatose-6-phosphate can be determined.
- the gene (gatZ gene) to be deleted or inactivated from the E. coli host for the production of NY-ESO-1 protein more specifically, DNA comprising the sequence described in SEQ ID NO: 5, or A polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 is exemplified.
- a person skilled in the art can know the latest sequence information from a public database or the like as appropriate based on the gene name.
- the biological activity is “an equivalent polypeptide” as compared to the biological activity of the comparison target, preferably 30% or more, preferably It means a polypeptide having a biological activity of 50% or more, more preferably 70% or more, still more preferably 80% or more, even more preferably 90% or more, particularly preferably 95% or more.
- the gatZ gene can be inactivated by a method such as inserting another DNA fragment into the gatZ gene or giving a mutation to the transcription or translation initiation region of the gatZ gene. It is more desirable to physically delete the gatZ gene.
- a method by homologous recombination may be used. That is, a circular recombinant plasmid obtained by cloning a DNA fragment containing a part of the gatZ gene into an appropriate plasmid vector is incorporated into the parent strain, and the parent E. coli genome is homologously recombined in a part of the gatZ gene. It is possible to disrupt and inactivate the above gatZ gene.
- E. coli Type of host E. coli
- E. coli C41 strain, C43 strain, BL21 Strains, B834 strains, and HMS174 strains can be used as the recombinant Escherichia coli of the present invention. It is not limited.
- the protein produced using the recombinant Escherichia coli of the present invention is preferably NY-ESO-1 protein, which is a hydrophobic protein that is difficult to produce with high purity in a normal E. coli host.
- the highly purified NY-ESO-1 protein purified product produced by the production method of the present invention is particularly useful in medical applications such as cancer vaccines. Therefore, the NY-ESO-1 protein produced using the recombinant Escherichia coli of the present invention may be a peptide fragment (for example, 80 amino acids or more) having immunogenicity equivalent to a protein having a full-length amino acid sequence, A fusion protein to which a known tag peptide is added may be used as necessary. Note that methods for evaluating the immunogenicity of a protein or polypeptide are known in the art.
- NY-ESO-1 protein refers to a protein consisting of the amino acid sequence of SEQ ID NO: 4, or 70% or more, preferably 80% or more, more preferably 90% or more, and still more preferably the protein. It means a protein having amino acid sequence identity of 95% or more, particularly preferably 98% or more, or a peptide fragment having equivalent immunogenicity, and expressed as a cancer / testis antigen having such an amino acid sequence. You can also.
- “NY-ESO-1 protein” (I) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4; (Ii) a polypeptide comprising an amino acid sequence having 80% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 4, and having an immunogenicity equivalent to that of the polypeptide comprising the amino acid sequence of SEQ ID NO: 4 A polypeptide having; (Iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 in which several (eg, a maximum of 10) amino acids are substituted, deleted, or added, and is equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 A polypeptide having the immunogenicity of (Iv) a polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4, and has the same immunity as the polypeptide consisting of the amino acid
- stringent (hybridization) conditions refers to conditions in which a nucleic acid molecule hybridizes to its target sequence in a nucleic acid mixture, but does not hybridize to other sequences to a detectable extent. (That is, conditions for specifically hybridizing with the target sequence). Stringent conditions depend on the sequence, but generally longer sequences specifically hybridize at higher temperatures, and appropriate hybridization conditions can be routinely selected by those skilled in the art.
- Exemplary stringent hybridization conditions include: 50% formamide, 5 ⁇ SSC, and 1% SDS, 42 ° C. incubation, or 5 ⁇ SSC, 1% SDS, 65 ° C. And 0.2 ⁇ SSC and 0.1% SDS, wash at 50 ° C.
- Suitable hybridization conditions include “rapid-hyb buffer” (GE Healthcare) for 30 minutes or longer at 68 ° C., addition of labeled probe, and 1 hour at 68 ° C. Further warming can also be included.
- the washing step can be performed, for example, under low stringency conditions.
- exemplary low stringency conditions include, for example, 42 ° C., 2 ⁇ SSC, 0.1% SDS, or preferably 50 ° C., 2 ⁇ SSC, 0.1% SDS.
- exemplary high stringency conditions include, for example, 2 ⁇ SSC at room temperature, 3 washes for 20 minutes in 0.01% SDS, followed by 1 ⁇ SSC at 37 ° C. in 0.1% SDS 3 washings for 20 minutes at 2 and 2 washings for 20 minutes in 1 ⁇ SSC, 0.1% SDS at 50 ° C. are included.
- temperature and salt concentration can affect the stringency of hybridization, one skilled in the art can appropriately select these factors to obtain the required stringency.
- the gene encoding NY-ESO-1 protein is upstream of the regulatory region involved in transcription and translation of the gene, that is, the transcription initiation regulatory region including the promoter and transcription initiation site, and / or the ribosome binding site and initiation codon. It is desirable to be functionally linked to a translation initiation region containing
- a gene encoding the NY-ESO-1 protein into E. coli is carried out by incorporating a recombinant plasmid in which a DNA fragment containing the gene and an appropriate plasmid vector are combined into a host E. coli cell by a general transformation method. Can be implemented.
- the recombinant E. coli of the present invention can also be obtained by using a DNA fragment obtained by binding an appropriate homologous region to the host E. coli genome to the DNA fragment and directly integrating it into the host E. coli genome.
- the production of NY-ESO-1 protein using the recombinant Escherichia coli of the present invention is performed by inoculating the strain into a medium containing an assimilable carbon source, nitrogen source and other essential components, and cultivating by a normal E. coli culture method. Then, after completion of the culture, the produced NY-ESO-1 protein may be recovered and purified.
- the produced NY-ESO-1 protein is preferably produced in a host cell. Therefore, in the production method of the present invention, NY-ESO-1 protein is recovered by crushing or dissolving E. coli that produces NY-ESO-1 protein, and recovering NY-ESO-1 protein from the disrupted product or lysate. .
- NY-ESO-1 protein is highly hydrophobic, so it is easily included in inclusion bodies when synthesized in a host cell. Therefore, in order to purify the NY-ESO-1 protein, it is preferable to purify the inclusion body from the crushed host E. coli and solubilize it. Methods for purifying and dissolving inclusion bodies are known in the art, and for example, inclusion bodies recovered by centrifugation or the like can be dissolved with urea or the like.
- the column to be used is not particularly limited as long as it is useful in the purification, and one or more types of columns can be used.
- NY-ESO-1 protein is expressed as a fusion protein with a tag peptide that is widely used for purification of recombinant proteins in this technical field, use an affinity column that utilizes the affinity with the tag peptide. Is preferred.
- a His tag (a tag peptide comprising about 6 consecutive histidine (His) residues) and a metal ion (such as nickel) immobilization column, a biotin tag and an avidin immobilization Columns, GST (glutathione-S-transferase) tags and glutathione-immobilized columns are well known and can be used as appropriate in the production method of the present invention.
- GST glutathione-S-transferase tags and glutathione-immobilized columns
- glutathione-immobilized columns are well known and can be used as appropriate in the production method of the present invention.
- the NY-ESO-1 protein contains a highly hydrophobic region, it is preferable to use a column utilizing hydrophobic interaction.
- a column using a resin such as phenyl sepharose or butyl sepharose can be used.
- Example 1 Preparation of NY-ESO-1-producing Escherichia coli strain (conventional type) pET9a vector (Novagen, catalog # 69431-3) pET-9a24a vector lacking the repressor binding site and lacI gene A cDNA expressing the His-NY-ESO-1 protein shown in Fig. 1 was incorporated to prepare a His-NY-ESO-1 expression vector.
- This expression vector and pRARE vector (Novagen) are introduced into Escherichia coli competent cell C41 (DE3) (COSMO BIO INC., Catalog # 60341), and the Escherichia coli strain His-NY-ESO is expressed.
- -1 / C41 (DE3) was produced. After culturing the obtained strain in LB medium, the cells were collected, the medium was replaced with LB medium without antibiotics, glycerol was added to a final concentration of 40%, and Master Cell Bank (Master Cell Bank: MCB).
- Example 2 Preparation of NY-ESO-1-producing Escherichia coli strain (gatZ deletion type) Preparation of gatZ gene-deficient E. coli GatZ gene-deficient E. coli C41 (DE3) ⁇ gatZ was prepared using the Red / ET homologous recombination method (Patent No. 4139561). Specifically, for the C41 (DE3) E. coli strain, the gatZ region shown in SEQ ID NO: 2 is replaced with the gene sequence shown in SEQ ID NO: 3, whereby the E. coli strain C41 (DE3) ⁇ gatZ lacking the gatZ gene. Was made.
- Example 3 Production and purification of NY-ESO-1 protein Culturing
- 60 mL of the culture solution was added and cultured at 37 ° C. and 250 rpm.
- OD600 reached about 50
- IPTG was added to a final concentration of 0.5 mM to induce protein expression, and the culture was terminated about 12 hours after the start of induction.
- Extraction The following extraction operation is performed at 20 ° C or lower.
- the collected culture solution is centrifuged (about 15,000 g, 30 minutes), and the precipitated cells are 200 mL of homogenate buffer (50 mM Tris-HCl, 0.1 M NaCl, 1 mM MgSO 4 , 5 mM DTT, pH 8.0) Then, 5 L of a homogenate buffer was added and suspended again.
- a homogenate solution was prepared by crushing the cells at about 1,000 Bar using a high-pressure homogenizer. The homogenate solution was centrifuged (about 15,000 g, 30 minutes) to precipitate inclusion bodies containing NY-ESO-1 protein.
- washing buffer 1 50 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, 5 mM DTT, pH 8.0.
- the suspension was centrifuged again (about 15,000 g, 30 minutes), and the resulting precipitate was suspended in 5 L of homogenate buffer 2 (100 mM sodium phosphate, 0.5 M NaCl, 5 mM DTT, pH 7.5).
- the suspension is centrifuged again (about 15,000 g, 30 minutes), and the resulting precipitate is suspended in 10 L of solubilization buffer (100 mM sodium phosphate, 7.5 M urea, 5 mM DTT, pH 7.5).
- the solubilization treatment was performed by leaving for about 4 hours.
- the lysate is then pre-filtered with Opticap XLT20 Milligard 1.2 / 0.5 ⁇ m (Millipore), then filtered through a MaxiCap filter, 0.45 / 0.2 ⁇ m (Sartorius), and His-NY-ESO -1 protein extract was obtained.
- the obtained extract was stored in a refrigerator.
- the eluate was concentrated using Sartocon (registered trademark) PESU Cassette (Sartorius) and then replaced with AEC equilibration buffer (20 mM phosphate, 7.5 M urea, 5 mM DTT, pH 7.5).
- Example 4 SDS-PAGE ( Figure 1A) The purified His-NY-ESO-1 protein obtained in Example 3 for the NY-ESO-1-producing E. coli strain (conventional type) was subjected to SDS-PAGE on a 4-12% precast gradient gel (Invitrogen). did. The gel was then stained with SYPRO® Ruby Protein Stain (Molecular Probes) to visualize the signal. As a result, bands with various molecular weights were detected, including bands corresponding to NY-ESO-1 monomer and gatZ.
- Example 5 SDS-PAGE, Western blot (FIG. 1B) After performing SDS-PAGE as in Example 4, the gel was immersed in a transfer buffer (Invitrogen) and transferred to a nitrocellulose membrane using a blotting apparatus. After immersing the transferred nitrocellulose membrane in water, anti-NY-ESO-1 monoclonal antibody (clone E978) (Santa Cruz) as the primary antibody and HRP-labeled anti-mouse IgG antibody (GE Healthcare) as the secondary antibody The reaction was allowed to visualize the signal using Western blot detection reagent (GE Healthcare).
- a transfer buffer Invitrogen
- anti-NY-ESO-1 monoclonal antibody (clone E978) (Santa Cruz) as the primary antibody
- HRP-labeled anti-mouse IgG antibody GE Healthcare
- Example 6 Two-dimensional electrophoresis (FIG. 2) The purified His-NY-ESO-1 protein obtained in Example 3 for the NY-ESO-1 producing E. coli strain (conventional type) and the NY-ESO-1 producing E. coli strain (gatZ deletion type) was obtained using Immobiline DryStrip ( It was subjected to the first-dimensional electrophoresis using GE Health Science. Next, second-dimensional electrophoresis was performed using 7.5% SDS-PAGE gel. The gel was then stained with SYPRO® Ruby Protein Stain (Molecular Probes) to visualize the signal (FIG. 2). As a result, the E.
- Example 7 Measurement of host-derived impurities His-NY-ESO- obtained in Example 3 for NY-ESO-1-producing E. coli strains (conventional type) and NY-ESO-1 producing E. coli strains (gatZ deletion type) 1 Host protein concentration in the purified protein was measured using E. coli Host Cell Protein ELISA Kit (Cygnus Technologies, product code F410). As a result, the concentration of the host-derived residual protein was 50.5 ng / mg in the wild type, whereas it was 18.7 ng / mg in the gatZ deletion type.
- Example 8 Two-dimensional electrophoresis and Western blot using anti-NY-ESO-1 antibody (FIG. 3)
- the IPG First-dimensional electrophoresis was performed using ReadyStrip gel (17 cm, pH 3-10NL, BIO-RAD). After equilibrating the IPG gel, it was set on a 10/16% gradient gel (19 ⁇ 17 cm) and subjected to second-dimensional electrophoresis (SDS-PAGE).
- the obtained gel was stained with SYPRO Ruby Protein Stain (Molecular Probes) or blotted onto a PVDF membrane at 400 mA per gel for 2 hours.
- NY-ESO-1 protein was detected from the PVDF membrane after transfer using an anti-NY-ESO-1 antibody (clone E978, Santa Cruz Biotechnology, Inc.) and Western Breeze kit (Invitrogen).
- Anti-NY-ESO-1 antibody reactive spots were observed in both the NY-ESO-1-producing E. coli strain (conventional type, Fig. 3A) and the NY-ESO-1 producing E. coli strain (gatZ deletion type, Fig. 3B) (Inside the dotted box) these were considered to be His-NY-ESO-1.
- coli strains ( gatZ deletion type) is recognized in a narrow range of pH7 to pH10, and the purity of His-NY-ESO-1 as a molecular species may be improved in NY-ESO-1 producing E. coli strains (gatZ deletion type) Sex was also suggested.
- deletion of gatZ in the E. coli strain that expresses the NY-ESO-1 protein significantly reduces the amount of host-derived impurities in the resulting purified NY-ESO-1 protein. Became clear.
- the present invention provides a method for producing NY-ESO-1, which is a hydrophobic protein that is difficult to produce recombinantly, with high purity, and a recombinant Escherichia coli useful for the method.
- Such production method and NY-ESO-1 protein produced by recombinant Escherichia coli are particularly useful in applications such as medical use where production with high purity is desirable.
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Abstract
Description
〔1〕gatZ遺伝子が欠失または不活性化されており、かつNY-ESO-1遺伝子を導入した大腸菌。
〔2〕〔1〕の大腸菌からNY-ESO-1タンパク質を抽出する工程を含む、NY-ESO-1タンパク質の生産方法。
〔3〕gatZ遺伝子が欠失または不活性化されている、NY-ESO-1タンパク質生産用大腸菌宿主。
〔4〕NY-ESO-1タンパク質の生産において用いるための、gatZ遺伝子が欠失または不活性化されている大腸菌。
〔5〕大腸菌ゲノム中のgatZ遺伝子を欠失または不活性化させる工程、および該大腸菌にNY-ESO-1タンパク質をコードする遺伝子を導入する工程によって得られる、大腸菌。
本明細書において、「高純度」とは、NY-ESO-1タンパク質精製物における、NY-ESO-1タンパク質以外の宿主由来タンパク質の濃度が、多くても50 ng/mg未満、好ましくは40 ng/mg未満、より好ましくは30 ng/mg未満、最も好ましくは20 ng/mg未満であることを意味する。宿主由来タンパク質の濃度を測定する方法(例えば免疫学的測定法)は当技術分野において公知であり、大腸菌由来タンパク質濃度を測定するためのE. coli Host Cell Protein ELISA Kit(シグナステクノロジーズ、商品コードF410)などの様々な免疫学的測定キットが市販されている。
大腸菌gatZ遺伝子は、タガトース-6-リン酸をリン酸化してタガトース-6-二リン酸を形成する酵素gatZ(別名:タガトース-6-リン酸キナーゼ)をコードする遺伝子である。大腸菌gatZ遺伝子のヌクレオチド配列(配列番号5)およびアミノ酸配列(配列番号6)は、GenBankにアクセッション番号X79837で登録されており、Jeong H et al. Genome sequences of Escherichia coli B strain REL606 and BL21(DE3). J Mol Biol, 2009 (4):644-52にも記載されている。
したがって、本明細書において、「gatZ遺伝子」とは、配列番号6のアミノ酸配列からなるタンパク質、または当該タンパク質と70%以上、好ましくは80%以上、より好ましくは90%以上、さらに好ましくは95%以上、特に好ましくは98%以上のアミノ酸配列同一性を有するタンパク質であって、タガトース-6-リン酸をリン酸化する活性を有するタンパク質をコードするポリヌクレオチドを意味する。なお、タンパク質またはポリペプチドが有するリン酸化活性を評価する方法は当技術分野において公知であり、例えば、タガトース-6-リン酸のリン酸化に適した条件下でタガトース-6-リン酸をインキュベートし、タガトース-6-二リン酸の量を検出することによって、タガトース-6-リン酸をリン酸化する活性を決定することができる。
本発明は、gatZ遺伝子中に他のDNA断片を挿入する、または、gatZ遺伝子の転写もしくは翻訳開始領域に変異を与える等の方法によってgatZ遺伝子を不活性化することができるが、好適には、gatZ遺伝子を物理的に欠失させることがより望ましい。gatZ遺伝子を欠失または不活性化するには、例えば相同組換えによる方法を用いればよい。すなわち、gatZ遺伝子の一部を含むDNA断片を適当なプラスミドベクターにクローニングして得られる環状の組換えプラスミドを親菌株内に取り込ませ、gatZ遺伝子の一部の領域における相同組換えによって親大腸菌ゲノム上のgatZ遺伝子を分断して不活性化することが可能である。
本発明の組換え大腸菌を構築するための親菌株としては、目的タンパク質を組換え生産するのに利用可能な大腸菌宿主として多くの菌株が知られており、例えば、大腸菌C41株、C43株、BL21株、B834株、およびHMS174株、ならびにそれらのDE3溶原菌(Lucigen社、コスモバイオ社、フィルジェン社、フナコシなどで購入可能)が本発明の組換え大腸菌として利用可能であるが、これらに限定されるものではない。
本発明の組換え大腸菌を用いて生産させるタンパク質としては、通常の大腸菌宿主では高純度で生産することが困難な疎水性タンパク質であるNY-ESO-1タンパク質が好ましい。本発明の生産方法により生産される高純度のNY-ESO-1タンパク質精製物は、がんワクチンなどの医療用途において特に有用である。したがって、本発明の組換え大腸菌を用いて生産させるNY-ESO-1タンパク質は、全長アミノ酸配列を有するタンパク質と同等の免疫原性を有するペプチド断片(例えば、80アミノ酸以上)であってもよく、必要に応じて公知のタグペプチドを付加した融合タンパク質とすることもできる。なお、タンパク質またはポリペプチドの免疫原性を評価する方法は、当技術分野において公知である。
(i)配列番号4のアミノ酸配列を含むポリペプチド;
(ii)配列番号:4のアミノ酸配列に対して80%以上のアミノ酸配列同一性を有するアミノ酸配列を含むポリペプチドであって、配列番号4のアミノ酸配列からなるポリペプチドと同等の免疫原性を有するポリペプチド;
(iii)数個(例えば、最大10個)のアミノ酸が置換、欠失、または付加された配列番号4のアミノ酸配列を含むポリペプチドであって、配列番号4のアミノ酸配列からなるポリペプチドと同等の免疫原性を有するポリペプチド;
(iv)配列番号4のアミノ酸配列からなるポリペプチドをコードするポリヌクレオチドに対してストリンジェントな条件下でハイブリダイズするポリヌクレオチドであって、配列番号4のアミノ酸配列からなるポリペプチドと同等の免疫原性を有するポリペプチドをコードするポリヌクレオチド;
からなる群より選択されるポリペプチド、またはそれらと同等の免疫原性を有するペプチド断片である。
NY-ESO-1タンパク質をコードする遺伝子は、その上流において、当該遺伝子の転写や翻訳に関わる制御領域、すなわち、プロモーターおよび転写開始点を含む転写開始制御領域、ならびに/またはリボソーム結合部位および開始コドンを含む翻訳開始領域と機能的に連結されていることが望ましい。
NY-ESO-1タンパク質をコードする遺伝子の大腸菌への導入は、当該遺伝子を含むDNA断片と適当なプラスミドベクターを結合させた組換えプラスミドを、一般的な形質転換法によって宿主大腸菌細胞に取り込ませることによって実施することができる。また、当該DNA断片に宿主大腸菌ゲノムとの適当な相同領域を結合したDNA断片を用い、宿主大腸菌ゲノムに直接組み込むことによっても、本発明の組換え大腸菌を得ることができる。
本発明の組換え大腸菌を用いたNY-ESO-1タンパク質の生産は、当該菌株を同化性の炭素源、窒素源、その他の必須成分を含む培地に接種し、通常の大腸菌培養法にて培養し、培養終了後、産生されたNY-ESO-1タンパク質を回収・精製することにより行えばよい。
NY-ESO-1タンパク質は疎水性が高いため、宿主細胞内で合成されると封入体に包含されやすい。そのため、NY-ESO-1タンパク質を精製するためには、宿主大腸菌の破砕物から封入体を精製し、それを可溶化することが好ましい。封入体の精製・溶解方法は当技術分野において公知であり、例えば遠心分離などにより回収した封入体を尿素などによって溶解することができる。
大腸菌破砕物または封入体可溶化物からNY-ESO-1タンパク質を精製するには、硫酸アンモニウムまたはエタノール沈殿、酸抽出、逆相カラムクロマトグラフィー、陽イオン交換クロマトグラフィー、陰イオン交換クロマトグラフィー、ホスホセルロースクロマトグラフィー、疎水性相互作用クロマトグラフィー、アフィニティクロマトグラフィー、等電点クロマトグラフィー、サイズ排除クロマトグラフィー、ヒドロキシルアパタイトクロマトグラフィーおよびレクチンクロマトグラフィーを含めた公知の方法が好適に用いられ得る。
クロマトグラフィーによりNY-ESO-1タンパク質を精製する場合、用いるカラムは当該精製において有用なものであれば特に限定されるものではなく、1種類または複数種類のカラムを用いることができる。
例えば、NY-ESO-1タンパク質が、当技術分野において組換えタンパク質の精製に汎用されているタグペプチドとの融合タンパク質として発現する場合、当該タグペプチドとの親和性を利用したアフィニティーカラムを用いることが好ましい。このようなタグペプチドとアフィニティーカラムとの組み合わせとしては、Hisタグ(6個程度の連続するヒスチジン(His)残基からなるタグペプチド)と金属イオン(ニッケルなど)固定化カラム、ビオチンタグとアビジン固定化カラム、およびGST(グルタチオン-S-トランスフェラーゼ)タグとグルタチオン固定化カラムなどがよく知られており、本発明の生産方法において適宜用いることができる。
また、NY-ESO-1タンパク質は疎水性が高い領域を含有するため、疎水性相互作用を利用したカラムを用いることが好ましい。このようなカラムとしては、フェニルセファロースやブチルセファロースなどの樹脂を用いたカラムが利用可能である。
pET9aベクター(Novagen、カタログ# 69431-3)のリプレッサー結合部位及びlacI遺伝子を欠失させたpET-9a24aベクターに、配列番号1で示すHis-NY-ESO-1タンパク質を発現するcDNAを組み込んで、His-NY-ESO-1発現ベクターを作製した。この発現ベクターおよびpRAREベクター(Novagen)を大腸菌コンピテントセルC41(DE3)(コスモバイオ株式会社、カタログ#60341)に導入し、His-NY-ESO-1タンパク質を発現する大腸菌株His-NY-ESO-1/C41(DE3)を作製した。得られた菌株をLB培地で培養した後、菌体を集めて培地を抗生物質不含LB培地に交換し、終濃度が40%になるようにグリセロールを添加してマスターセルバンク(Master Cell Bank:MCB)とした。
1.gatZ遺伝子欠失型大腸菌の作製
Red/ET相同組換え法(特許第4139561号)を用いてgatZ遺伝子欠失型大腸菌C41(DE3)ΔgatZを作製した。具体的には、C41(DE3)大腸菌株に対して、配列番号2に示すgatZ領域を、配列番号3に示す遺伝子配列に置換することにより、gatZ遺伝子を欠失した大腸菌株C41(DE3)ΔgatZを作製した。
作製したC41(DE3)ΔgatZに、実施例1で作製したHis-NY-ESO-1タンパク質のcDNAを含む発現ベクターを導入し、NY-ESO-1産生・gatZ欠失型大腸菌His-NY-ESO-1/C41(DE3)ΔgatZを作製した。
1.培養
実施例1及び2で作製したNY-ESO-1産生大腸菌株のMCB溶液0.1mLをLB培地100mLに添加し、37℃、250rpmで約12時間前培養を行い、LB培地12Lに対して前培養液を60mL添加して37℃、250rpmで培養を行った。OD600が約50になったところでIPTGを終濃度0.5mMになるよう添加してタンパク質の発現を誘導し、誘導開始から約12時間後に培養を終了した。
以下の抽出操作は20℃以下で行う。回収した培養液を遠心操作(約15,000g、30分)にかけ、沈殿させた菌体を200mLのホモジネートバッファー(50 mM Tris-HCl, 0.1 M NaCl, 1 mM MgSO4, 5 mM DTT, pH 8.0)に懸濁した後、5Lのホモジネートバッファーを添加して再度懸濁した。高圧ホモジナイザーを用いて約1,000 Bar にて菌体を破砕してホモジネート液を調製した。ホモジネート液を遠心操作(約15,000g、30分)にかけてNY-ESO-1タンパク質を含む封入体を沈殿させた。遠心操作の後、上清を除き、沈殿を5Lの洗浄バッファー1(50 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, 5 mM DTT, pH 8.0)に懸濁した。懸濁液を再度遠心操作(約15,000g、30分)にかけ、得られた沈殿物を5Lのホモジネートバッファー2 (100 mM リン酸ナトリウム, 0.5 M NaCl, 5 mM DTT, pH 7.5)に懸濁した。懸濁液を再度遠心操作(約15,000g、30分)にかけ、得られた沈殿物を10Lの可溶化バッファー(100 mM リン酸ナトリウム, 7.5 M 尿素, 5 mM DTT, pH 7.5)に懸濁し、約4時間放置して可溶化処理を行った。その後、可溶化液をオプティキャップ(Opticap) XLT20 ミリガード(Milligard) 1.2/0.5 μm(Millipore)で前ろ過を行った後、MaxiCapフィルター, 0.45 / 0.2 μm(Sartorius)でろ過し、His-NY-ESO-1タンパク質抽出液を得た。得られた抽出液は冷蔵にて保管した。
(1)アフィニティークロマトグラフィーによる精製
得られた抽出液は、まずNickel Sepharose 6FF樹脂(GEヘルスケア)を用いて精製した。カラムをIMAC平衡化バッファー (100 mM リン酸ナトリウム, 7.5 M 尿素, 5 mM DTT, pH 7.5) で平衡化した後、抽出液を添加し、75mMのイミダゾールを含むIMAC平衡化バッファーで洗浄した後、イミダゾール濃度を75mMから500mMまで漸増してHis-NY-ESO-1タンパク質を溶出させた。溶出液はSartocon(登録商標)PESU Cassette (Sartorius) を用いて濃縮した後、AEC平衡化バッファー(20 mM リン酸塩, 7.5 M 尿素, 5 mM DTT, pH 7.5) に置換した。
次に、Q Sepharose XL樹脂(GEヘルスケア)を用いて精製した。カラムをAEC平衡化バッファー (20 mM リン酸塩, 7.5 M 尿素, 5 mM DTT, pH 7.5) で平衡化した後、前記アフィニティーカラムの溶出液を添加し、AEC平衡化バッファーで洗浄して、His-NY-ESO-1タンパク質を含むフロースルーを回収した。
続いて、Phenyl Sepharose HP樹脂(GEヘルスケア)を用いて精製を行った。カラムをHIC平衡化バッファー(100 mM リン酸ナトリウム, 7.5 M 尿素, 1 M 硫酸アンモニウム, 5 mM DTT, pH 7.5)で平衡化した後、前記イオン交換カラムで回収したフロースルー液に等量のHIC調整バッファー(180 mM リン酸ナトリウム, 7.5 M 尿素, 2 M 硫酸アンモニウム, 5 mM DTT, pH 7.5)を添加した溶液を添加し、HIC平衡化バッファーで洗浄した後、硫酸アンモニウム濃度を0.9Mから0.1Mまで漸減してHis-NY-ESO-1タンパク質を溶出させた。溶出液はSartocon(登録商標)PESU Cassette (Sartorius) を用いて濃縮した後、最終バッファー(100 mM リン酸ナトリウム, 145 mM 塩化ナトリウム, 4 M 尿素, 50 mM グリシン, pH 6.5) に置換し、His-NY-ESO-1タンパク質精製物とした。
NY-ESO-1産生大腸菌株(従来型)について実施例3で得られたHis-NY-ESO-1タンパク質精製物を4-12%のプレキャストグラジェントゲル(Invitrogen)でのSDS-PAGEに供した。その後、ゲルをSYPRO(登録商標)Ruby Protein Stain(Molecular Probes)で染色してシグナルを可視化した。その結果、NY-ESO-1モノマーやgatZに相当するバンドをはじめとして様々な分子量のバンドが検出された。
実施例4と同様にSDS-PAGEを行った後、ゲルを転写用緩衝液(Invitrogen)に浸漬し、ブロッティング装置を用いてニトロセルロース膜に転写した。転写後のニトロセルロース膜を水に浸漬した後、1次抗体として抗NY-ESO-1モノクローナル抗体(クローンE978)(Santa Cruz)、2次抗体としてHRP標識抗マウスIgG抗体(GEヘルスケア)と反応させ、ウェスタンブロット検出試薬(GEヘルスケア)を用いてシグナルを可視化した。
NY-ESO-1産生大腸菌株(従来型)およびNY-ESO-1産生大腸菌株(gatZ欠失型)について実施例3で得られたHis-NY-ESO-1タンパク質精製物を、Immobiline DryStrip(GEヘルスサイエンス)を用いた一次元目の電気泳動に供した。次いで、7.5%のSDS-PAGEゲルを用いて2次元目の電気泳動を行った。その後、ゲルをSYPRO(登録商標)Ruby Protein Stain(Molecular Probes)で染色してシグナルを可視化した(図2)。その結果、従来型の菌株から得られた精製サンプルでは観察された大腸菌由来タンパク質が、gatZ欠失型の菌株から得られた精製サンプルでは、ほとんど観察されなかった。さらに、従来型の菌株から得られた精製サンプルに含まれていた大腸菌由来のタンパク質のシグナルのいくつかが明らかに消失していることが確認された。
NY-ESO-1産生大腸菌株(従来型)およびNY-ESO-1産生大腸菌株(gatZ欠失型)について実施例3で得られたHis-NY-ESO-1タンパク質精製物における宿主タンパク質の濃度を、E. coli Host Cell Protein ELISA Kit(シグナステクノロジーズ、商品コードF410)を用いて測定した。その結果、宿主由来の残存タンパク質の濃度が、野生型では50.5 ng/mgであったのに対し、gatZ欠失型では18.7 ng/mgであった。
NY-ESO-1産生大腸菌株(従来型)およびNY-ESO-1産生大腸菌株(gatZ欠失型)について実施例3で得られたHis-NY-ESO-1タンパク質精製物を使用し、IPG ReadyStrip ゲル (17 cm, pH3-10NL, BIO-RAD) を用いて一次元目の泳動 (等電点電気泳動) を行った。IPG ゲルを平衡化した後、10/16% グラジエントゲル (19 × 17 cm) にセットし、二次元目の泳動 (SDS-PAGE) を行った。得られたゲルについて、SYPRO Ruby Protein Stain(Molecular Probes)で染色するか、もしくはゲル1枚あたり400mAで2時間の条件でPVDF膜へのブロッティングを実施した。転写後のPVDF膜に対し、抗NY-ESO-1抗体(クローンE978,Santa Cruz Biotechnology, Inc.)とWesternBreeze kit(Invitrogen)を用いてNY-ESO-1タンパク質の検出を行った。NY-ESO-1産生大腸菌株(従来型,図3A)およびNY-ESO-1産生大腸菌株(gatZ欠失型,図3B)の双方で抗NY-ESO-1抗体に反応性のスポットが認められ(点線のボックス内)、これらはHis-NY-ESO-1であると考えられた。抗NY-ESO-1抗体には反応しないが、SYPRO Ruby染色で検出できるスポットも認められ、これらは宿主由来不純物と考えられた。宿主由来不純物のスポットは、NY-ESO-1産生大腸菌株(従来型)に比べてNY-ESO-1産生大腸菌株(gatZ欠失型)で大幅に減少している傾向が確認された。また、抗NY-ESO-1抗体反応性スポットがNY-ESO-1産生大腸菌株(従来型)ではpH5.5~pH10の広い範囲で認められるのに対し、NY-ESO-1産生大腸菌株(gatZ欠失型)ではpH7~pH10の狭い範囲で認められ、NY-ESO-1産生大腸菌株(gatZ欠失型)ではHis-NY-ESO-1の分子種としての純度も向上している可能性も示唆された。
Claims (3)
- gatZ遺伝子が欠失または不活性化されており、かつNY-ESO-1遺伝子を導入した大腸菌。
- 請求項1記載の大腸菌からNY-ESO-1タンパク質を抽出する工程を含む、NY-ESO-1タンパク質の生産方法。
- gatZ遺伝子が欠失または不活性化されている、NY-ESO-1タンパク質生産用大腸菌宿主。
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JP2007312794A (ja) * | 2001-05-29 | 2007-12-06 | Kyowa Hakko Kogyo Co Ltd | 工業的生産に有用な微生物 |
WO2009116566A1 (ja) * | 2008-03-18 | 2009-09-24 | 協和発酵キリン株式会社 | 工業的に有用な微生物 |
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JP2007312794A (ja) * | 2001-05-29 | 2007-12-06 | Kyowa Hakko Kogyo Co Ltd | 工業的生産に有用な微生物 |
WO2009116566A1 (ja) * | 2008-03-18 | 2009-09-24 | 協和発酵キリン株式会社 | 工業的に有用な微生物 |
Non-Patent Citations (2)
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LOWE, A, J. ET AL.: "Expression and purification of cGMP grade NY -ESO-1 for clinical trials", BIOTECHNOLOGY PROGRESS, vol. 27, no. 2, March 2011 (2011-03-01), pages 435 - 441 * |
PIATESI, A. ET AL.: "Directed evolution for improved secretion of cancer-testis antigen NY ESO-1 from yeast", PROTEIN EXPRESSION AND PURIFICATION, vol. 48, 2006, pages 232 - 242 * |
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CN116751801A (zh) * | 2023-07-21 | 2023-09-15 | 中国人民解放军海军军医大学 | 以pGEX6P1作为载体制备肿瘤相关抗原NY-ESO-1的方法及应用 |
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