JP2012147772A - Recombinant plasmid vector and method for producing protein by using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new vector capable of secreting and expressing a recombinant protein in a host in high efficiency, and to provide a method for producing the recombinant protein in high efficiency by the transformant obtained by using the vector.SOLUTION: The vector for expressing a protein originated from human beings contains a new oligonucleotide and an oligonucleotide encoding a signal peptide for allowing periplasm to secrete the protein. The transformant is obtained by transforming the host by using the vector. The method for producing the protein originated from the human beings includes a step of culturing the transformant.

Description

  The present invention relates to a novel vector for expressing a recombinant protein and a method for producing a protein using a transformant obtained by transforming a host using the vector.

  Many recombinant proteins produced using genetic engineering techniques are used in medicines, agricultural chemicals, research reagents and the like. Bacteria, yeast, filamentous fungi, animal cells and the like are mainly used as hosts for producing recombinant proteins. Among them, Escherichia coli Escherichia coli (hereinafter simply referred to as E. coli in the present specification) is typically used as a host for producing a recombinant protein, and there are many research examples. However, when a recombinant protein derived from a heterologous organism such as a human is to be expressed in the cytoplasm of E. coli, the recombinant protein is often expressed as an inactive inclusion body. In order to convert an inactive protein expressed as an inclusion body into an active protein, it is necessary to go through a refolding step, and the work becomes very complicated.

  Even a recombinant protein expressed as inclusion bodies in the cytoplasm may be expressed as a soluble active protein in the periplasm (Non-patent Document 1). In particular, when the recombinant protein to be produced has a disulfide bond, it is active by forming a disulfide bond in the periplasm where there is a protein involved in disulfide bond formation in an oxidative environment. It tends to be expressed as a type protein.

  In order to secrete and express the recombinant protein in the periplasm, it is necessary to express the recombinant protein in a state in which a signal peptide that promotes secretory expression to the periplasm is added to the N-terminal side. Since the signal peptide is excised from the recombinant protein by signal peptidase when the recombinant protein is secreted and expressed into the periplasm, a recombinant protein without the signal peptide is finally obtained. However, when a method for secretory expression in the periplasm is used, there is a problem that the production efficiency of the active recombinant protein is low.

JP 2000-083670 A JP-A-8-322586 JP 2001-061487 A JP 2008-245580 A

Choi, J .; H. and Lee, S .; Y. , Appl. Microbiol. Biotechnol. , 64, 625-635, 2004 Nucleic Acids Res. , 30, e43, 2002 Eur. J. et al. Biochem. , 267, 1565-1570, 2000

  When a recombinant protein is produced using a method for secretory expression to the periplasm of the host, methods for improving its efficiency include co-expression with a Dsb protein related to disulfide bond formation (Patent Document 1), co-expression with glutathione reductase. Methods for expression (Patent Document 2), methods for co-expression with molecular chaperones (Patent Document 3), and the like have been reported. However, each method requires complicated operations such as construction of a co-expression system and optimization of co-expression conditions.

  Therefore, the present invention is to provide a novel vector capable of secreting and expressing a recombinant protein in a host with high efficiency, and a method for producing a recombinant protein with high efficiency using a transformant obtained by using the vector.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have further inserted a novel oligonucleotide into a vector containing an oligonucleotide encoding a signal peptide for secreting the protein into the periplasm, whereby a recombinant protein is obtained. Has been found to be secreted with high efficiency in the host, and the present invention has been completed.

That is, the present invention includes the following embodiments:
(1) A vector for expressing a human-derived protein comprising an oligonucleotide having the base sequence described in any one of SEQ ID NOs: 1 to 6 and an oligonucleotide encoding a signal peptide that causes the periplasm to secrete the protein.

  (2) The vector according to (1), wherein the signal peptide that causes the periplasm to secrete a protein is an oligopeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 7 to 9.

  (3) A transformant capable of expressing a human-derived protein, obtained by transforming a host using the vector according to (1) or (2) into which a polynucleotide encoding a human-derived protein is inserted.

  (4) The transformant according to (3), wherein the host is Escherichia coli.

  (5) The transformant according to (3) or (4), wherein the human-derived protein is a human Fc-binding protein.

  (6) The transformant according to (3) or (4), wherein the human-derived protein is a human erythropoietin-binding protein.

  (7) A method for producing a human-derived protein, comprising a step of culturing the transformant according to (3) or (4).

  (8) A method for producing a human Fc-binding protein, comprising a step of culturing the transformant according to (5).

  (9) A method for producing a human erythropoietin-binding protein, comprising a step of culturing the transformant according to (6).

  Hereinafter, the present invention will be described in detail.

  The vector of the present invention includes an oligonucleotide consisting of the base sequence described in SEQ ID NO: 1 (5′-CTTTAAGAACGGAATATAC-3 ′), an oligonucleotide consisting of the base sequence described in SEQ ID NO: 2 (5′-CTTTAAGAAGAGAGACAT-3 ′), An oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 3 (5′-CTTTAAGAGAGAGATAATAATACAT-3 ′), an oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 4 (5′-CTTTAAGAAGGAGATATACATAATACAT-3 ′), SEQ ID NO: 5 (5 ′ -Oligonucleotide having the base sequence described in CTTTAAGAAGGAGATACATAATAATACAT-3 '), SEQ ID NO: 6 (5'-CTTTAAGAAACGAGATAT) And any of the oligonucleotide consisting of the nucleotide sequence of CATAATACAT-3 '), is characterized in that it contains an oligonucleotide encoding a signal peptide to secrete proteins into the periplasm, the. It should be noted that prior art was used by using a DNA sequence database (URL: http://blast.ncbi.nlm.nih.gov/Blast.cgi, access date: October 22, 2010) that was disclosed before the filing of the present application. As a result, a vector containing the nucleotide sequence of any one of SEQ ID NOS: 1 to 6 and an oligonucleotide encoding a signal peptide that secretes the protein into the periplasm has not been disclosed, and the vector of the present invention is novel. Confirm that it is a vector.

  The oligonucleotide (oligonucleotide A) comprising the base sequence described in any one of SEQ ID NOS: 1 to 6 and the oligonucleotide (oligonucleotide B) encoding a signal peptide that secretes the protein to the periplasm contained in the vector of the present invention Preferably contains oligonucleotide B directly added to the 3 ′ end of oligonucleotide A. More preferably, a restriction enzyme site is provided immediately after the 3 'end of the oligonucleotide B so that a polynucleotide encoding a human-derived protein to be expressed can be inserted into the site.

  All of the oligonucleotides comprising the nucleotide sequences set forth in SEQ ID NOs: 1 to 6 contained in the vector of the present invention control the translation efficiency of the expression of the polynucleotide encoding the human-derived protein on the 3 ′ end side thereof. It is an oligonucleotide having a function. The translation efficiency of the expression of the polynucleotide encoding the human-derived protein varies depending on which oligonucleotide is selected from the oligonucleotides having the base sequences described in SEQ ID NOs: 1 to 6. For example, when the host is Escherichia coli, they are SEQ ID NO: 3, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 6 in descending order of translation efficiency (see Reference Example). Among the oligonucleotides consisting of the nucleotide sequences set forth in SEQ ID NOs: 1 to 6, which oligonucleotide is selected depends on the translation efficiency of the expression of the polynucleotide encoding the human-derived protein to be expressed and the expression of the protein May be selected as appropriate in consideration of the host.

  The vector of the present invention may contain at least an oligonucleotide having the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 and an oligonucleotide for secreting the protein into the periplasm. In addition, a polynucleotide having a promoter function necessary for transcription initiation, a polynucleotide having a terminator function necessary for transcription termination, a polynucleotide having a replication origin function, and a function of a selection marker such as an antibiotic resistance gene. It is preferable that the polynucleotide further comprises a polynucleotide having a multiple cloning site having a plurality of restriction enzyme sites, and the like.

  The vector of the present invention may be any one of SEQ ID NOS: 1 to 6 at an appropriate location of a commercially available vector including the polynucleotide having the functions of the promoter, terminator, origin of replication, selection marker, multicloning site and the like described above. The polynucleotide comprising the nucleotide sequence described above and an oligonucleotide encoding a signal peptide that causes the periplasm to secrete a protein can be prepared by insertion using a genetic engineering technique. The commercially available vector may be appropriately selected as a preferred vector for the host to be transformed. When the host is Escherichia coli, plasmid vectors such as pUC, pET, pBBR, pBluescript, and pTrc99a can be exemplified. As an example of a specific method for producing the vector of the present invention, an oligo encoding a signal peptide that secretes a protein to the periplasm at the 3 ′ end side of a polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 6 An oligonucleotide to which nucleotides are directly bound is prepared by a PCR method, and the prepared oligonucleotide is used at a suitable position in a commercially available vector (for example, between a promoter and a terminator) using a commercially available restriction enzyme or a ligation reagent. The method of inserting in is mentioned.

  The signal peptide contained in the vector of the present invention that causes the periplasm to secrete the protein may be appropriately selected depending on the host that expresses the recombinant protein. When Escherichia coli is used as a host for producing a recombinant protein, a secretory pathway via a SecYEG complex and a secretory pathway via a twin arginine transport (Tat) system are known as periplasmic secretory pathways of E. coli (non-patented). From the literature 1), it is preferable to use a signal peptide that utilizes the above pathway. Specifically, a PelB signal peptide (MKYLLPTAAAGLLLLLAAQPAMA: UniProt No. P0C1C1 from the first to the 22nd region, SEQ ID NO: 7), a MalE signal peptide (MKIKTGARILALSALTMMFSASALA: UniProt No. P0AEX9 from the first to the 26th region). SEQ ID NO: 8), TorT signal peptide (MRVLLFLLLSLFMLPFAS: UniProt No. P38683, region 1 to 18; SEQ ID NO: 9).

  In order to produce a recombinant protein using the vector of the present invention, any linker peptide can be used directly on the 3 ′ end side of an oligonucleotide encoding a signal peptide that causes the periplasm to secrete the protein contained in the vector of the present invention. A transformant obtained by inserting a polynucleotide (recombinant protein gene) encoding a recombinant protein through a oligonucleotide encoding the gene and transforming the host using the vector into which the gene has been inserted is cultured. Can be manufactured.

  When the recombinant protein produced using the vector of the present invention is applied to a protein that is difficult to express as an active protein by conventional expression methods, such as a human-derived protein, or has a low expression level, the effect of the present invention can be obtained. It is preferable in that it can be exhibited. In addition, the recombinant protein produced using the vector of the present invention is a protein that is secreted and expressed extracellularly in the organism from which it is derived (for example, human), or a protein in which a part of the protein structure is exposed outside the cell after expression. However, it is preferable in that expression as an active protein can be expected in the periplasm of the host.

  Examples of human-derived proteins produced using the vector of the present invention include insulin, interferon, interleukin, antibody, erythropoietin, growth hormone, and their receptor proteins. The human-derived protein produced using the vector of the present invention may be a complete protein, a protein composed of only a part important for the function of the protein, or One or more amino acid residues constituting the protein may be deleted and / or inserted and / or substituted. In the case of a human Fc-binding protein, which is a preferred example of a human-derived protein produced using the vector of the present invention, the protein is the entire region of human FcγRI, which is one of human Fc-binding proteins (UniProt No. P12314). Or a partial region of human FcγRI (for example, a protein consisting of amino acids from the 16th glutamine to the 289th valine in the amino acid sequence described in SEQ ID NO: 76) Alternatively, it may be human FcγRI in which one or more amino acids in the whole region or a partial region of human FcγRI are deleted and / or inserted and / or substituted. In the case of a human erythropoietin-binding protein, which is another preferred example of a human-derived protein produced using the vector of the present invention, the protein is the entire region of human erythropoietin receptor (UniProt No. P19235). Or a partial region of the human erythropoietin receptor (eg, SEQ ID NO: 77), wherein one or more amino acids of the entire region or a partial region of the human erythropoietin receptor are deleted and / or inserted and It may be substituted. A tag sequence for facilitating simple purification and quantification may be added to the human-derived protein produced using the vector of the present invention. Examples of the tag sequence include oligopeptides frequently used in genetic engineering, such as polyhistidine tags and c-myc tags.

As a method for producing a polynucleotide encoding a human-derived protein to be inserted into the vector of the present invention,
(I) a method of converting a human-derived protein from an amino acid sequence to a base sequence, artificially synthesizing a polynucleotide containing the base sequence,
(II) A polynucleotide containing a whole or partial sequence of a human-derived protein is prepared directly or artificially or from a cDNA or the like of a human-derived protein using a DNA amplification method such as a PCR method, and the prepared polynucleotide is subjected to an appropriate method. The method of connecting and making with
Can be illustrated. In addition, when converting from an amino acid sequence to a base sequence, it is preferable to convert in consideration of the codon usage frequency in the host to be transformed. For example, when the host is E. coli, AGA / AGG / CGG / CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), proline ( In Pro), since CCC is used less frequently (because it is a so-called rare codon), it may be converted so as to avoid those codons. Analysis of codon usage frequency can also be performed by using a public database (for example, Codon Usage Database on the website of Kazusa DNA Research Institute).

  As a host to be transformed using a vector in which a polynucleotide encoding a human-derived protein is inserted into the vector of the present invention, a host suitable for the expression of the human-derived protein may be appropriately selected. COS cells and CHO cells Animal cells represented by genus Bacillus (including bacteria of the broad genus such as Brevibacillus genus and Paenibacillus genus) and bacteria typified by E. coli, Saccharomyces genus, Pichia genus and Schizosaccharomyces genus Yeast, and filamentous fungi typified by koji molds are preferable, but it is preferable to use E. coli which is easy to handle as a host. When the host is Escherichia coli, strains with known genotypes such as BL21 (DE3) strain, JM109 strain, and HB101 strain are particularly preferred.

  A method for transforming a host using a vector in which a polynucleotide encoding a human-derived protein is inserted into the vector of the present invention may be carried out according to a method commonly used in gene recombination techniques. Can be exemplified by the calcium chloride method, the electroporation method and the like. Thereby, a transformant capable of expressing the human-derived protein (hereinafter simply referred to as the transformant of the present invention) is obtained.

  The transformant of the present invention is well known in the art and may be grown in a medium suitable for culturing selected host cells. As an example, when the host is Escherichia coli, an example of a preferable medium is an LB (Luria-Bertani) medium supplemented with necessary nutrient sources. When a polynucleotide having a function of a selectable marker such as an antibiotic resistance gene is inserted into the vector of the present invention, the transformant of the present invention is obtained by culturing in a state where a drug corresponding to the selectable marker is added to the medium. Is preferable because it can be selectively proliferated. For example, when the vector of the present invention used for transformation has a kanamycin resistance gene inserted as a selection marker, the transformant of the present invention can be selectively grown by adding kanamycin to the medium. . In addition to carbon, nitrogen and inorganic salt sources, an appropriate nutrient source may be added to the medium. Further, if desired, it may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, and dithiothreitol. Further, a reagent such as glycine that promotes protein leakage from the host to the culture solution may be added to the medium.

  The culture temperature and the pH of the medium of the transformant of the present invention may be determined under conditions preferable for the host used for transformation. For example, when the host is Escherichia coli, the culture temperature is preferably in the range of 10 ° C. to 40 ° C., but in particular, the range of 30 ° C. to 38 ° C. is more preferable for growing the transformant. A range of 15 ° C. to 25 ° C. is more preferable for expression. The pH of the medium is preferably in the range of pH 6.5 to pH 7.5, and more preferably in the range of pH 6.8 to pH 7.2.

  When the host used for producing the transformant of the present invention is an aerobic organism or a facultative anaerobic organism, it is necessary to aerate the culture solution. As the method, when the host is a bacterium, a cotton plug is used. An example is a method of using a baffled conical flask with a breathable stopper such as a culture container and shaking the container during the culture to vent the culture solution.

When the vector of the present invention contains a polynucleotide having a function of an inducible promoter, it is preferable to induce and culture under conditions that allow efficient expression of a human-derived protein by the promoter. As an example, when the lac promoter or trc promoter is included in the vector of the present invention, induction may be performed by adding IPTG (isopropyl-β-D-thiogalactopyroside) to the medium. Specifically, the turbidity (Optical Density at 600 nm, OD ₆₀₀ ) of the culture solution is measured, and when it becomes about 0.5 to 1.0, an appropriate amount of IPTG is added and then cultured. The expression of human-derived protein can be induced. The addition concentration of IPTG may be appropriately selected from the range of 0.005 mM to 1.0 mM, but is preferably in the range of 0.01 mM to 0.05 mM. Various conditions relating to the IPTG induction may be performed under conditions well known in the art.

  In order to recover the human-derived protein from the culture broth of the transformant of the present invention, a general extraction method may be appropriately selected and used depending on the form of expression. For example, when a human-derived protein is expressed in the periplasm of a host cell, the host cell is collected by centrifugation, and then an enzyme or a surfactant is added, and the host cell is crushed by ultrasonic crushing treatment. What is necessary is just to extract a recombinant protein. On the other hand, when human-derived protein leaks from the cell periplasm into the culture supernatant, the host cell may be separated by centrifugation, and the human-derived protein may be extracted from the resulting culture supernatant.

  In order to separate and purify human-derived protein from the protein extract, a method known in the art may be used. An example is separation / purification using liquid chromatography. Examples of liquid chromatography include ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography and the like. A human-derived protein can be prepared with high purity by performing a purification operation by combining these chromatographies.

  The vector of the present invention comprises a novel oligonucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 6 and an oligonucleotide for allowing the periplasm to secrete a protein. It is said. The vector of the present invention can efficiently express a protein such as a human-derived protein even if the protein is difficult to express as an active protein by a conventional expression method or has a low expression level.

It is the figure which showed the outline | summary of the plasmid vectors pETmalE and pETmalE-p7, and its preparation process. It is the figure which showed the outline | summary of the recombinant plasmid vector pETmalEFcR-p7, and its preparation process. It is the figure which showed the outline | summary of the recombinant plasmid vector pETGFP-p7, and its preparation process. It is the schematic of the evaluation method of the translation efficiency which used GFP (green fluorescent protein) as a reporter protein. It is the figure which showed the result of having evaluated the amount of GFP expressed from the transformant by the fluorescence intensity per culture solution turbidity. It is the figure which showed the outline | summary of the plasmid vector pUC-EPOR, and its production process. It is the figure which showed the outline | summary of the plasmid vector pETmalE-p7-E, and its preparation process. It is the figure which showed the outline | summary of the plasmid vector pETmalE-p7-EPOR, and its preparation process.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example and a comparative example, this invention is not limited to these. The reference example does not constitute the present invention.

Example 1
A human Fc-binding protein expression vector containing an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 4 and a transformant obtained from the vector were prepared by the following method.
(1) A MalE signal peptide (SEQ ID NO: 8) was selected as a signal peptide for causing the periplasm to secrete proteins, and a polynucleotide encoding the signal peptide was prepared by the following two-step PCR.
(1-1) Based on the reaction solution composition shown in Table 1, the first stage PCR was carried out at 98 ° C. for 10 seconds for the first step, 55 ° C. for 5 seconds for the second step, and 72 ° C. for 1 minute for the third time. The reaction with one step as one cycle was performed by 5 cycles and then cooled to 4 ° C. The oligonucleotide is an oligonucleotide composed of the base sequence described in SEQ ID NO: 10 (5′-TATACATATGAAAATAAAAACAGGGTGCACCGCATCC-3 ′), an oligonucleotide composed of the base sequence described in SEQ ID NO: 11 (5′-GCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCCC-3 ′), An oligonucleotide consisting of the base sequence described in SEQ ID NO: 12 (5′-ATCGTCGTTAATGCGGATAATGCCGAGGATGCGTGCCACCTG-3 ′) and an oligonucleotide consisting of the base sequence described in SEQ ID NO: 13 (5′-TTGTCCCATGGCTTCTCTCGATTTGGCGAGAGCCG-3 ′).

(1-2) Based on the reaction solution composition shown in Table 2, the second-stage PCR was performed at 98 ° C for 10 seconds for the first step, 55 ° C for 5 seconds for the second step, and 72 ° C for 1 minute for the third time. The reaction with one step as one cycle was performed by cooling to 4 ° C. after 30 cycles. The first-stage PCR product was used as the template, and the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 10 and the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 13 were used as the PCR primers.

(2) A pET-26b (+) plasmid vector (Novagen) prepared by two-step PCR and digested with restriction enzymes NdeI and NcoI and then digested with restriction enzymes NdeI and NcoI in advance. The Escherichia coli BL21 (DE3) strain (Novagen) was transformed by using the calcium chloride method.
(3) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin, and then a plasmid was extracted from the cells, and this plasmid was designated as a plasmid vector pETmalE. An outline of the plasmid vector pETmalE and its production process are shown in FIG.
(4) The plasmid vector pETmalE (FIG. 1) as a template, the oligonucleotide consisting of the base sequence described in SEQ ID NO: 14 (5′-AGTAGTAGGTTGAGGCCGTTGAG-3 ′) as the PCR primer and SEQ ID NO: 15 (5′-TTTTCATATGTTATTGTTATCTCCTTTCTTAAA-3 ′ ) Using the oligonucleotides having the base sequences described in Table 2 under the reaction composition shown in Table 2, the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, and the second step at 72 ° C. for 1 minute. After performing 30 cycles of the reaction with the third step of 1 cycle, PCR was performed by cooling to 4 ° C.
(5) After the PCR product of (4) was digested with restriction enzymes SphI and NdeI, it was ligated to pETMalE previously digested with restriction enzymes SphI and NdeI, and this was used to transform E. coli BL21 (DE3) strain.
(6) After culturing the transformant of (5), a plasmid was extracted, and this plasmid was designated as a plasmid vector pETmalE-p7. The plasmid vector pETMalE-p7 contains an oligonucleotide having the base sequence described in SEQ ID NO: 4 and an oligonucleotide encoding the MalE signal peptide (SEQ ID NO: 8). An outline of the plasmid vector pETMalE-p7 and its production process are shown in FIG. In addition, the base sequence of the polynucleotide from the restriction enzyme BglII recognition site to the Bpu1102I recognition site in the plasmid vector pETmalE-p7 (FIG. 1) is shown in SEQ ID NO: 16. Of the nucleotide sequence set forth in SEQ ID NO: 16, the 88th to 115th nucleotides correspond to SEQ ID NO: 4, and the 116th to 193rd nucleotides are oligos encoding the MalE signal peptide (SEQ ID NO: 8). Corresponds to nucleotide.
(7) A recombinant plasmid vector pETMalEFcR-p7 and its transformant were prepared by the following method.
(7-1) A plasmid vector pECFcR containing a polynucleotide encoding human FcγRI whose codon was converted to E. coli was prepared according to the method disclosed in Japanese Patent Application Laid-Open No. 2008-245580 (Patent Document 4).
(7-2) A plasmid vector pECFcR as a template, an oligonucleotide consisting of a base sequence described in SEQ ID NO: 17 (5′-TCAGCCATGGGACAAGTAGATACACCACCAAGCTGTGATTA-3 ′) as a PCR primer and SEQ ID NO: 18 (5′-CCAAGCTTAATGATGATGATGGATGGATGGAGCGGGTGTGGAGGG Each of the oligonucleotides consisting of the described base sequences was used for the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, and the second step at 72 ° C. for 1 minute under the reaction solution composition shown in Table 2. 30 cycles of the reaction with the third step as one cycle, followed by PCR by cooling to 4 ° C. and adding a polyhistidine tag to the C-terminal side, human Fc A polynucleotide containing a base sequence encoding a polypeptide containing the extracellular region of γRI (the region from the 16th glutamine to the 289th valine in the amino acid sequence described in SEQ ID NO: 76) was prepared.
(7-3) After digesting the polynucleotide obtained in (7-2) with restriction enzymes NcoI and HindIII, ligation to the plasmid vector pETmalE-p7 (FIG. 1) previously digested with restriction enzymes NcoI and HindIII, Plasmid vector pETmalEFcR-p7 was constructed. FIG. 2 shows an outline of the plasmid vector pETmalEFcR-p7 and its production process.
(8) Escherichia coli BL21 (DE3) strain was transformed by the calcium chloride method using the plasmid vector pETmalEFcR-p7 (FIG. 2) to prepare a transformant of pETmalEFcR-p7.

  The recombinant protein expressed by the plasmid vector pETMalEFcR-p7 is an antibody-binding protein consisting of the extracellular region of human FcγRI. Hereinafter, this protein is abbreviated as rhFcγRI. The plasmid vector pETMalEFcR-p7 (FIG. 2) includes, from the 5 ′ end side, a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 4, an oligonucleotide encoding the MalE signal peptide (SEQ ID NO: 8), and a polynucleotide encoding rhFcγRI. The nucleotides and the oligonucleotides encoding the polyhistidine tags are arranged in this order. The nucleotide sequence from the restriction enzyme BglII recognition site to the HindIII recognition site in the plasmid vector pETMalEFcR-p7 (FIG. 2) is shown in SEQ ID NO: 19. Of the nucleotide sequence set forth in SEQ ID NO: 19, the 88th to 115th nucleotides correspond to SEQ ID NO: 4, and the 116th to 193rd nucleotides encode an MalE signal peptide (SEQ ID NO: 8). The nucleotides corresponding to nucleotides 215 to 1036 correspond to oligonucleotides encoding rhFcγRI (the region from the 16th glutamine to the 289th valine in the amino acid sequence shown in SEQ ID NO: 76). The 1054th nucleotide corresponds to the oligonucleotide encoding the polyhistidine tag.

Example 2
A human Fc-binding protein expression vector containing an oligonucleotide having the base sequence set forth in SEQ ID NO: 1 and a transformant obtained from the vector were prepared by the following method.
(1) The plasmid vector pETMalE-p7 (FIG. 1) prepared in Example 1 as a template was used as an oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 14 as a PCR primer and SEQ ID NO: 21 (5′-ATTTTCATATTATATCTCGTTCTAAA-3 ′) Each of the oligonucleotides consisting of the base sequences described in Table 1 was used, under the reaction solution composition shown in Table 2, for the first step at 98 ° C for 10 seconds, the second step at 55 ° C for 5 seconds, and 72 ° C for 1 minute. After performing 30 cycles of the reaction with the third step of 1 cycle, PCR was performed by cooling to 4 ° C.
(2) The PCR product was digested with restriction enzymes SphI and NdeI, and then ligated to a recombinant plasmid vector pETmalEFcR-p7 (FIG. 2) previously digested with restriction enzymes SphI and NdeI to prepare plasmid vector pETmalEFcR-rbs. In the plasmid vector pETmalEFcR-rbs, the oligonucleotide part consisting of the base sequence shown in SEQ ID NO: 4 contained in the plasmid vector pETmalEFcR-p7 (FIG. 2) was replaced with the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1. It is a structure.
(3) Escherichia coli BL21 (DE3) strain was transformed by the calcium chloride method using the plasmid vector pETmalEFcR-rbs to prepare a transformant of pETmalEFcR-rbs.

Example 3
A human Fc-binding protein expression vector containing an oligonucleotide having the base sequence set forth in SEQ ID NO: 2 and a transformant obtained from the vector were prepared by the following method.
(1) Example 2 except that an oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 14 and an oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 22 (5′-ATATACATAGTCCTCTCTTTAAAAGTT-3 ′) were used as PCR primers. A plasmid vector pETmalEFcR-m4 was prepared in the same manner as described above. In the plasmid vector pETmalEFcR-m4, the part of the oligonucleotide consisting of the base sequence described in SEQ ID NO: 4 contained in the plasmid vector pETmalEFcR-p7 (FIG. 2) was replaced with the oligonucleotide consisting of the base sequence described in SEQ ID NO: 2. It is a structure.
(2) Escherichia coli BL21 (DE3) strain was transformed with the plasmid vector pETmalEFcR-m4 by the calcium chloride method to prepare a pETmalEFcR-m4 transformant.

Example 4
A human Fc-binding protein expression vector containing an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 3 and a transformant obtained from the vector were prepared by the following method. (1) Example 2 except that an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 14 and an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 23 (5′-TTTTATACATGTATTTATACTCCCTTCTTAA-3 ′) were used as PCR primers. The plasmid vector pETMalEFcR-p4 was prepared by the same method as described above. In the plasmid vector pETMalEFcR-p4, the oligonucleotide part consisting of the base sequence described in SEQ ID NO: 4 contained in the plasmid vector pETMalEFcR-p7 (FIG. 2) was replaced with the oligonucleotide consisting of the base sequence described in SEQ ID NO: 3. It is a structure.
(2) Escherichia coli BL21 (DE3) strain was transformed by the calcium chloride method using the plasmid vector pETmalEFcR-p4 to prepare a pETmalEFcR-p4 transformant.

Example 5
A human Fc-binding protein expression vector containing an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 5 and a transformant obtained from the vector were prepared by the following method. (1) Example 2 except that an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 14 and an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 24 (5′-TTTTCATATGTATTATTTAGTTATCTCTCTCTCTA-3 ′) were used as PCR primers. A plasmid vector pETMalEFcR-p10 was prepared in the same manner as described above. In the plasmid vector pETmalEFcR-p10, the oligonucleotide part consisting of the base sequence described in SEQ ID NO: 4 contained in the plasmid vector pETmalAlFcR-p7 (FIG. 2) was replaced with the oligonucleotide consisting of the base sequence described in SEQ ID NO: 5. It is a structure.
(2) Escherichia coli BL21 (DE3) strain was transformed by the calcium chloride method using the plasmid vector pETmalEFcR-p10 to prepare a pETmalEFcR-p10 transformant.

Example 6
A human Fc-binding protein expression vector containing an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 6 and a transformant obtained from the vector were prepared by the following method.
(1) Example 2 except that the oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 14 and the oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 25 (5′-TTTTCATATGTATTATGTATATCTCGTTTCTTAAA-3 ′) were used as PCR primers. The plasmid vector pETMalEFcR-rbsp7 was prepared by the same method as described above. In the plasmid vector pETmalEFcR-rbsp7, the oligonucleotide part consisting of the base sequence described in SEQ ID NO: 4 contained in the plasmid vector pETmalAlFcR-p7 (FIG. 2) was replaced with the oligonucleotide consisting of the base sequence described in SEQ ID NO: 6. It is a structure.
(2) Escherichia coli BL21 (DE3) strain was transformed by the calcium chloride method using the plasmid vector pETmalEFcR-rbsp7 to prepare a transformant of pETmalEFcR-rbsp7.

Example 7
Using each transformant prepared in Example 1 to Example 6, rhFcγRI was expressed and the amount thereof was evaluated.
(1) pETmalEFcR-p7 transformant prepared in Example 1, pETmalEFcR-rbs transform prepared in Example 2, pETmalEFcR-m4 transformant prepared in Example 3, pETmalEFcR- prepared in Example 4 For the p4 transformant, the pETMalEFcR-p10 transformant prepared in Example 5, and the pETmalEFcR-rbsp7 transformant prepared in Example 6, respectively, the culture and expression of each transformant were carried out by the following method. Guidance was performed.
(1-1) Each transformant was inoculated into an LB medium containing 50 μg / mL kanamycin and cultured with shaking at 37 ° C. overnight to prepare a preculture solution.
(1-2) A LB medium newly containing 50 μg / mL kanamycin was inoculated with the preculture and cultured at 37 ° C. with shaking.
(1-3) When the value of the turbidity (OD ₆₀₀ ) of the culture solution becomes about 0.5, after culturing for 30 minutes at a culture temperature of 20 ° C., IPTG is cultured to a final concentration of 0.01 mM. Expression was induced by adding to the solution, followed by shaking culture at 20 ° C. for 22 hours.
(1-4) After cultivation, a soluble protein extract was prepared from the cells of each transformant obtained by centrifugation using BugBuster protein extraction Kit (manufactured by Novagen).
(2) The amount of rhFcγRI protein in the soluble protein extract was evaluated by the ELISA (Enzyme-Linked Immunosorbent Assay) method shown below.
(2-1) An antibody gamma globulin (manufactured by Chemo-Serum Therapy Laboratories) was fixed at a concentration of 1 μg / well (4 ° C., 18 hours) in a well of a 96-well microplate, and Starting Block Blocking Buffers (manufactured by PIERCE) ).
(2-2) After blocking, the soluble protein extract prepared in (1-4) was serially diluted and subjected to an ELISA reaction (30 ° C., 2 hours).
(2-3) Subsequently, after washing with Tris-HCl buffer (pH 8.0) containing 0.2% (w / v) Tween 20 and 150 mM NaCl, Horseradish Peroxidase anti-His-Tag antibody reagent (BETHYL) Made) and reacted at 30 ° C. for 2 hours.
(2-4) After the reaction, washing was performed with the buffer used in (2-3), TMB Peroxidase Substrate (manufactured by KPL) was added, and the absorbance at 450 nm was measured.
(2-5) Based on a commercially available rhFcγRI product having a known concentration (Recombinant Human FcγRI / CD64; manufactured by R & D systems), by calculating the protein concentration of rhFcγRI in the soluble protein extract from the measured absorbance, The expression level of rhFcγRI was calculated.

  As a result of measuring at n = 4, the expression amount of rhFcγRI per culture solution by each transformant was, in descending order, pETmalEFcR-p10 transformant (Example 5 / SEQ ID NO: 5: 823 ± 158 μg / L), pETmalEFcR-rbsp7 transformant (Example 6 / SEQ ID NO: 6: 700 ± 89 μg / L), pETmalEFcR-rbs transformant (Example 2 / SEQ ID NO: 539 ± 50 μg / L), pETMalEFcR-m4 transformant (Example 3 / SEQ ID NO: 490 ± 65 μg / L), pETMalEFcR-p7 transformant (Example 1 / SEQ ID NO: 466 ± 23 μg / L), pETMalEFcR-p4 transformant (Example 4 / sequence) No. 3: 410 ± 84 μg / L). Of the transformants prepared in Example 1 to Example 6, it can be seen that the pETMalEFcR-p10 transformant prepared in Example 5 can express rhFcγRI particularly efficiently.

Comparative Example 1
The oligonucleotide part consisting of the base sequence shown in SEQ ID NO: 4 contained in the plasmid vector pETMalEFcR-p7 prepared in Example 1 was derived from the plasmid vector pET-26b (+) consisting of the base sequence shown in SEQ ID NO: 26. Plasmid vector pETmalEFcR, which is a structure substituted with an existing oligonucleotide (5′-CTTTAAGAAGGGAATATAC-3 ′), and a transformant obtained from the vector were prepared by the following method.
(1) By digesting the recombinant plasmid vector pETMalEFcR-p7 (FIG. 2) prepared in Example 1 with restriction enzymes NdeI and HindIII, a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 20 is obtained. Polynucleotide fragments containing were made. In the amino acid sequence shown in SEQ ID NO: 20, the first methionine to the 26th alanine correspond to the MalE signal peptide (SEQ ID NO: 8), and the 34th glutamine to the 307th valine correspond to rhFcγRI ( In the amino acid sequence set forth in SEQ ID NO: 76, it corresponds to the region from the 16th glutamine to the 289th valine), and the 308th to 313th histidines correspond to the polyhistidine tag.
(2) The fragment prepared in (1) was ligated to the pET-26b (+) plasmid vector previously digested with restriction enzymes NdeI and HindIII, thereby preparing a plasmid vector pETmalEFcR.
(3) Escherichia coli BL21 (DE3) strain was transformed with the plasmid vector pETmalEFcR by the calcium chloride method to prepare a pETmalEFcR transformant.
(4) Using the pETMalEFcR transformant, rhFcγRI expression and expression level were evaluated in the same manner as in Example 7.

  As a result of measurement at n = 4, rhFcγRI expression level per culture solution by the pETmalEFcR transformant was 293 ± 10 μg / L, and the expression level of the present invention prepared in Examples 1 to 6 was The expression level of rhFcγRI by the transformant obtained using the vector was small. That is, in a transformant capable of expressing rhFcγRI, a transformant obtained by transforming with a plasmid vector containing the oligonucleotide having the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 6 (from Example 1) The transformant prepared in Example 6) is a transformant obtained by transforming with a plasmid vector (pETmalEFcR) that does not contain the oligonucleotide consisting of the nucleotide sequences set forth in SEQ ID NOS: 1 to 6 (prepared in this comparative example) It can be seen that rhFcγRI can be expressed with higher efficiency than the above-mentioned transformants.

Example 8
A human Fc-binding protein expression vector containing an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 4 and a transformant obtained from the vector were prepared by the following method.
(1) A PelB signal peptide (SEQ ID NO: 7) was selected as a signal peptide that causes the periplasm to secrete a protein, and a polynucleotide containing a base sequence encoding the signal peptide was prepared by the following two-step PCR.
(1-1) Based on the reaction solution composition shown in Table 1, the first stage PCR was carried out at 98 ° C. for 10 seconds for the first step, 55 ° C. for 5 seconds for the second step, and 72 ° C. for 1 minute for the third time. The reaction with one step as one cycle was performed by 5 cycles and then cooled to 4 ° C. In addition, as an oligonucleotide, an oligonucleotide having a base sequence described in SEQ ID NO: 27 (5′-TATACATATGAAATACCTATTGCCCTACGGCAGCCCCT-3 ′), an oligonucleotide having a base sequence described in SEQ ID NO: 28 (5′-ATTGTTATTACTCGCTGCCCCAACCAGCGATGGCC-3 ′), An oligonucleotide consisting of the base sequence described in SEQ ID NO: 29 (5′-GCAGCGGATAATAACAATCCAGCGGCTGCCCGTAGGCA-3 ′) and an oligonucleotide consisting of the base sequence described in SEQ ID NO: 30 (5′-TTTGGGTGATCTACTTGGGCCCATCGCTGGGT-3 ′) were used.
(1-2) Based on the reaction solution composition shown in Table 2, the second-stage PCR was performed at 98 ° C for 10 seconds for the first step, 55 ° C for 5 seconds for the second step, and 72 ° C for 1 minute for the third time. After performing 30 cycles of reaction with one step as a step, the reaction was performed by cooling to 4 ° C. to prepare a polynucleotide encoding the PelB signal peptide. The first-stage PCR product was used as the template, and the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 27 and the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 30 were used as the PCR primers.
(2) The plasmid vector pECFcR (FIG. 2) prepared in Example 1 as a template and the oligonucleotide consisting of the base sequence described in SEQ ID NO: 31 (5′-CAAGTAGATACCACCCAAAGCTGTGATTACGCTGCAACCACCGT-3 ′) as a PCR primer Each of the oligonucleotides consisting of the base sequences was used, under the reaction solution composition shown in Table 2, for the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, and the third step at 72 ° C. for 1 minute. After performing 30 cycles of the reaction with one step, a polynucleotide encoding rhFcγRI with a polyhistidine tag added to the C-terminal side was prepared by cooling to 4 ° C.
(3) The polynucleotide encoding rhFcγRI added with a polyhistidine tag on the C-terminal side prepared in (2) and the polynucleotide encoding the PelB signal peptide prepared in (1) by the two-step PCR shown below Connected.
(3-1) The first step of PCR was performed using the two polynucleotides and the first step at 98 ° C. for 10 seconds and the second step at 55 ° C. for 5 seconds based on the reaction solution composition shown in Table 3. The reaction was carried out by 5 cycles of a third step of 1 minute at 72 ° C., followed by cooling to 4 ° C.

(3-2) The second-stage PCR, the first-stage PCR product as a template, and the oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 27 and the oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 18 as PCR primers , Respectively, based on the reaction solution composition in Table 2, one cycle of 98 ° C. for 10 seconds first step, 55 ° C. for 5 seconds second step, 72 ° C. for 1 minute 1 step. The reaction was performed 30 cycles, and then cooled to 4 ° C.
By this two-step PCR, a polynucleotide (SEQ ID NO: 32) containing a base sequence encoding a series of polypeptides (SEQ ID NO: 33) consisting of a PelB signal peptide, rhFcγRI and a polyhistidine tag was prepared. Of the amino acid sequence shown in SEQ ID NO: 33, the first methionine to the 22nd alanine correspond to the PelB signal peptide (SEQ ID NO: 7), and the 23rd glutamine to the 296th valine correspond to rhFcγRI ( In the amino acid sequence shown in SEQ ID NO: 76, the region from the 16th glutamine to the 289th valine) corresponds to the histidine from the 297th to the 302nd, and corresponds to the polyhistidine tag.
(4) The plasmid vector pETMalE− prepared in Example 1 was digested with the restriction enzymes NdeI and HindIII and then digested with the restriction enzymes NdeI and HindIII in advance. The plasmid vector pETPelBFcR-p7 was prepared by ligation to p7 (FIG. 2). The plasmid vector pETPelBFcR-p7 includes, from the 5 ′ end, a polynucleotide consisting of the base sequence described in SEQ ID NO: 4, an oligonucleotide encoding the PelB signal peptide (SEQ ID NO: 7), a polynucleotide encoding rhFcγRI, and a polyhistidine The oligonucleotides encoding the tags are arranged in this order.
(5) A pETPelBFcR-p7 transformant was prepared by transforming E. coli BL21 (DE3) strain by the calcium chloride method using the plasmid vector pETPelBFcR-p7.

  Using the pETPelBFcR-p7 transformant, active rhFcγRI was expressed by the same method as in Example 7, and the expression level was evaluated. As a result of measurement at n = 3, the expression level of active rhFcγRI per culture solution by the pETPelBFcR-p7 transformant was 292 ± 36 μg / L.

Comparative Example 2
The oligonucleotide part consisting of the base sequence shown in SEQ ID NO: 4 contained in the plasmid vector pETPelBFcR-p7 prepared in Example 8 was derived from the plasmid vector pET-26b (+) consisting of the base sequence shown in SEQ ID NO: 26. Plasmid vector pETPelBFcR, which is a structure substituted with an existing oligonucleotide (5′-CTTTAAGAAGGAGAATACAT-3 ′), and a transformant obtained from the vector were prepared by the following method.
(1) The pET-26b (+) plasmid vector prepared in Example 8 was digested with the restriction enzymes NdeI and HindIII and then previously digested with the restriction enzymes NdeI and HindIII. Ligation produced a plasmid vector pETPelBFcR.
(2) Escherichia coli BL21 (DE3) strain was transformed by the calcium chloride method using the plasmid vector pETPelBFcR to prepare a pETPelBFcR transformant.
Using this pETPelBFcR transformant, rhFcγRI was expressed in the same manner as in Example 7, and the expression level was evaluated. As a result of measurement at n = 3, the expression level of rhFcγRI per culture solution by the pETPelBFcR transformant was 48 ± 3 μg / L, and the expression level was determined using the vector of the present invention prepared in Example 8. The amount of rhFcγRI expressed by the transformant thus obtained was small. That is, in a transformant capable of expressing rhFcγRI, a transformant obtained by transforming with a plasmid vector (pETPelBFcR-p7) containing an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 4 (produced in Example 8) A transformant obtained by transforming with a plasmid vector (pETPelBFcR) that does not contain an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 4 (the transformant prepared in this Comparative Example) and Comparison shows that rhFcγRI can be expressed with higher efficiency.

Example 9
A human Fc-binding protein expression vector containing an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 4 and a transformant obtained from the vector were prepared by the following method.
(1) A TorT signal peptide (SEQ ID NO: 9) was selected as a signal peptide that causes the periplasm to secrete a protein, and a polynucleotide containing a base sequence encoding the TorT signal peptide was prepared by the following two-step PCR.
(1-1) Based on the reaction solution composition shown in Table 1, the first stage PCR was carried out at 98 ° C. for 10 seconds for the first step, 55 ° C. for 5 seconds for the second step, and 72 ° C. for 1 minute for the third time. The reaction with one step as one cycle was performed by 5 cycles and then cooled to 4 ° C. In addition, as an oligonucleotide, an oligonucleotide consisting of the base sequence described in SEQ ID NO: 34 (5′-TATACATGCCGCGTACGCCTTTTTTAC-3 ′), an oligonucleotide consisting of the base sequence described in SEQ ID NO: 35 (5′-TTCTTTCCCTTTTCATGTTTGCCGGCATTTTCG-3 ′), An oligonucleotide consisting of the base sequence described in SEQ ID NO: 36 (5′-TGAAAAGGAGAAGAAGTAAAAAATAGCAGTACG-3 ′) and an oligonucleotide consisting of the base sequence described in SEQ ID NO: 37 (5′-TTTGGTGGTATCACTTGCGAAAATGCCGGCAACA-3 ′) were used.
(1-2) Based on the reaction solution composition shown in Table 2, the second-stage PCR was performed at 98 ° C for 10 seconds for the first step, 55 ° C for 5 seconds for the second step, and 72 ° C for 1 minute for the third time. After performing 30 cycles of reaction with one step as a step, the reaction was performed by cooling to 4 ° C. to prepare a polynucleotide encoding a TorT signal peptide. The first-stage PCR product was used as a template, and the oligonucleotide consisting of the sequence shown in SEQ ID NO: 34 and the oligonucleotide consisting of the sequence shown in SEQ ID NO: 37 were used as PCR primers.
(2) The polynucleotide encoding the TorT signal peptide and the polynucleotide encoding rhFcγRI with a polyhistidine tag added to the C-terminus prepared in Example 8 were ligated by the two-step PCR shown below.
(3-1) The first step of PCR was performed using the two polynucleotides and the first step at 98 ° C. for 10 seconds and the second step at 55 ° C. for 5 seconds based on the reaction solution composition shown in Table 3. The reaction was carried out by 5 cycles of a third step of 1 minute at 72 ° C., followed by cooling to 4 ° C.
(3-2) The second-stage PCR, the first-stage PCR product as a template, and the oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 34 and the oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 18 as a PCR primer , Respectively, based on the reaction solution composition in Table 2, one cycle of 98 ° C. for 10 seconds first step, 55 ° C. for 5 seconds second step, 72 ° C. for 1 minute 1 step. The reaction was performed 30 cycles, and then cooled to 4 ° C.
By this two-step PCR, a polynucleotide (SEQ ID NO: 38) containing a base sequence encoding a series of polypeptides (SEQ ID NO: 39) consisting of a TorT signal peptide, rhFcγRI and His tag peptide was prepared. Of the amino acid sequence shown in SEQ ID NO: 39, the first methionine to the 18th serine correspond to the TorT signal peptide (SEQ ID NO: 9), and the 19th glutamine to the 292nd valine correspond to rhFcγRI ( In the amino acid sequence described in SEQ ID NO: 76, the region from the 16th glutamine to the 289th valine) corresponds to the histidine from the 293th to the 298th, and corresponds to the polyhistidine tag.
(4) The plasmid vector pETmalE- produced in Example 1 was prepared by digesting the polynucleotide comprising the base sequence described in SEQ ID NO: 38 prepared in (3) with restriction enzymes NdeI and HindIII and then digesting with restriction enzymes NdeI and HindIII in advance. The plasmid vector pETTorTFcR-p7 was prepared by ligation to p7 (FIG. 2). The plasmid vector pETTorTFcR-p7 includes, from the 5 ′ end, a polynucleotide consisting of the base sequence described in SEQ ID NO: 4, an oligonucleotide encoding the TorT signal peptide (SEQ ID NO: 9), a polynucleotide encoding rhFcγRI, and a His tag Arranged in the order of oligonucleotides encoding peptides.
(5) Escherichia coli BL21 (DE3) was transformed by the calcium chloride method using the recombinant plasmid vector pETTorTFcR-p7 to prepare a pETTorTFcR-p7 transformant.
(6) rhFcγRI was expressed by the same method as in Example 7 using the pETTorTFcR-p7 transformant, and the expression level was evaluated. As a result of the measurement at n = 3, the expression level of rhFcγRI per culture solution by the transformant of pETTorTFcR-p7 was 320 ± 61 μg / L.

Comparative Example 3
The oligonucleotide part consisting of the base sequence set forth in SEQ ID NO: 4 contained in the plasmid vector pETTorTFcR-p7 prepared in Example 9 was derived from the plasmid vector pET-26b (+) consisting of the base sequence set forth in SEQ ID NO: 26. Plasmid vector pETTorTFcR, which is a structure substituted with an existing oligonucleotide (5′-CTTTAAGAAGGAGATACATA-3 ′), and a transformant obtained from the vector were prepared by the following method.
(6) The pET-26b (+) plasmid vector digested with the restriction enzymes NdeI and HindIII and then digested with the restriction enzymes NdeI and HindIII in advance, was prepared by digesting the polynucleotide comprising the base sequence set forth in SEQ ID NO: 38 prepared in Example 9. Ligation produced a plasmid vector pETTorTFcR.
(7) Escherichia coli BL21 (DE3) was transformed with the plasmid vector pETTorTFcR to prepare a pETTorTFcR transformant.
Using the pETTorTFcR transformant, rhFcγRI was expressed by the same method as in Example 7, and the expression level was evaluated. As a result of the measurement at n = 3, the expression level of active rhFcγRI per culture solution by the pETTorTFcR transformant was 13 ± 1 μg / L. The expression level was lower than the expression level of rhFcγRI by the transformant prepared in Example 9 and obtained using the vector of the present invention. That is, in a transformant capable of expressing rhFcγRI, a transformant obtained by transforming with a plasmid vector (pETTorTFcR-p7) containing an oligonucleotide having the base sequence described in SEQ ID NO: 4 (prepared in Example 9) A transformant obtained by transforming with a plasmid vector (pETTorTFcR) that does not contain an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 4 (the transformant prepared in this Comparative Example) and Comparison shows that rhFcγRI can be expressed with higher efficiency.

Reference Example This example merely evaluates the high translational efficiency of an oligonucleotide having the base sequences described in SEQ ID NOs: 1 to 6 when Escherichia coli is used as a host, and does not constitute the present invention. Absent.

Effect of the expression of the polynucleotide on the translation efficiency by making the oligonucleotide consisting of the nucleotide sequence of SEQ ID NOs: 1 to 6 and 26 adjacent to the 5 ′ end of the polynucleotide encoding a protein other than a human-derived protein evaluated. In this example, a green fluorescent protein (GFP) gene was used as the polynucleotide. GFP is known to be well expressed as an active form in the cytoplasm of an E. coli host.
(1) A polynucleotide (GFP gene) encoding GFP comprising an E. coli type codon was designed by the DNAworks method (Nucleic Acids Res., 30, e43, 2002: Non-Patent Document 2).
(2) In order to produce the GFP gene designed in (1), a total of 34 types of oligonucleotides comprising the sequences described in SEQ ID NOs: 40 to 73 were synthesized.
(3) A polynucleotide containing a GFP gene was prepared from the oligonucleotide synthesized in (2) by the following two-step PCR.
(3-1) First-stage PCR was carried out at 94 ° C. for 5 minutes, followed by a first step at 94 ° C. for 30 seconds, and a second step at 65 ° C. for 30 seconds under the composition of the reaction solution in Table 4. The reaction was carried out for 25 cycles, with the third step at 72 ° C. for 1 minute taken as one cycle, held at 72 ° C. for 7 minutes, and then cooled to 4 ° C. The DNA mix shown in Table 4 was obtained by collecting a fixed amount of each of the synthetic oligonucleotides (34 types in total) consisting of the sequences shown in SEQ ID NOs: 40 to 73 (concentration: 50 pmol / μL) and mixing them. Point to.

(3-2) The second-stage PCR was performed by heat treatment at 94 ° C. for 5 minutes, followed by the first step at 94 ° C. for 30 seconds, and the second step at 60 ° C. for 30 seconds based on the reaction solution composition shown in Table 5. Step, 25 cycles of the reaction in which the third step of 1 minute at 72 ° C. was performed for 1 cycle was carried out by holding at 72 ° C. for 7 minutes and then cooling to 4 ° C. The first-stage PCR reaction solution was used as a template, and the PCR primer was an oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 40 (5′-GCCTATATGAGCAAGGGGCGAAGAGCTGTTCACCGGCG-3 ′) and SEQ ID NO: 73 (5′-GAAAAGCTTTTCCAACCCCGCCTTATACGCTTTCATCATCAT An oligonucleotide having the base sequence described in 3 ′) was used. By this two-step PCR, a polynucleotide containing the GFP gene was prepared.

(4) A plasmid vector containing any one of the oligonucleotides having the base sequences described in SEQ ID NOs: 1 to 6 and 26 and the GFP gene, and a transformant using the vector were prepared by the following method.
(4-1) The plasmid vector pETMalEFcR-p7 prepared in Example 1 prepared by digesting the polynucleotide containing the GFP gene prepared in (3) with restriction enzymes NdeI and HindIII and then digesting with restriction enzymes NdeI and HindIII in advance (FIG. The plasmid vector pETGFP-p7 was produced by ligation to 2). The outline of the plasmid vector pETGFP-p7 and its production process are shown in FIG.
(4-2) Escherichia coli BL21 (DE3) was transformed with the recombinant plasmid vector pETGFP-p7 to prepare a pETGFP-p7 transformant. The plasmid vector pETGFP-p7 (FIG. 3) includes the GFP gene (SEQ ID NO: 75) with a polyhistidine tag added to the C-terminal side, and the 3 ′ end of the oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 4. The GFP gene was arranged in a continuous manner. SEQ ID NO: 74 shows the base sequence from the restriction enzyme BglII recognition site to the Bpu1102I recognition site in the plasmid vector pETGFP-p7. The polynucleotide consisting of the base sequence set forth in SEQ ID NO: 74 includes a GFP gene (SEQ ID NO: 75) with a His tag peptide added to the C-terminal side, and a base sequence set forth in SEQ ID NO: 4 on the 5 ′ end side. And an oligonucleotide.
(4-3) Ligation to the plasmid vector pETmalEFcR-rbs prepared in Example 2 in which the polynucleotide (SEQ ID NO: 74) containing the GFP gene described above was digested with restriction enzymes NdeI and HindIII and then digested with restriction enzymes NdeI and HindIII in advance. Thus, plasmid vector pETGFP-rbs was prepared. The plasmid vector pETGFP-rbs has a structure in which the oligonucleotide part consisting of the base sequence shown in SEQ ID NO: 4 contained in pETGFP-p7 is replaced with the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1. Thereafter, Escherichia coli BL21 (DE3) strain was transformed with the plasmid vector pETGFP-rbs to prepare a pETGFP-rbs transformant.
(4-4) Ligation to the plasmid vector pETmalEFcR-m4 prepared in Example 3 previously digested with the restriction enzymes NdeI and HindIII after digesting the polynucleotide containing the GFP gene (SEQ ID NO: 74) with the restriction enzymes NdeI and HindIII. Thus, plasmid vector pETGFP-m4 was prepared. The plasmid vector pETGFP-m4 has a structure in which the oligonucleotide part consisting of the base sequence shown in SEQ ID NO: 4 contained in pETGFP-p7 is replaced with the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2. Thereafter, Escherichia coli BL21 (DE3) strain was transformed with the plasmid vector pETGFP-m4 to prepare a pETGFP-m4 transformant.
(4-5) Ligation to the plasmid vector pETmalEFcR-p4 prepared in Example 4 which was previously digested with the restriction enzymes NdeI and HindIII after the polynucleotide containing the GFP gene (SEQ ID NO: 74) was digested with the restriction enzymes NdeI and HindIII. Thus, a plasmid vector pETGFP-p4 was prepared. The plasmid vector pETGFP-p4 has a structure in which the oligonucleotide part consisting of the base sequence shown in SEQ ID NO: 4 contained in pETGFP-p7 is replaced with the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 3. Thereafter, Escherichia coli BL21 (DE3) strain was transformed with the plasmid vector pETGFP-p4 to prepare a pETGFP-p4 transformant.
(4-6) Ligation to the plasmid vector pETmalEFcR-p10 prepared in Example 5 which was previously digested with the restriction enzymes NdeI and HindIII and then previously digested with the restriction enzymes NdeI and HindIII. Thus, a plasmid vector pETGFP-p10 was produced. The plasmid vector pETGFP-p10 has a structure in which the oligonucleotide part consisting of the base sequence shown in SEQ ID NO: 4 contained in pETGFP-p7 is replaced with the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5. Thereafter, Escherichia coli BL21 (DE3) strain was transformed with the plasmid vector pETGFP-p10 to prepare a pETGFP-p10 transformant.
(4-7) Ligation to the plasmid vector pETmalEFcR-rbsp7 prepared in Example 6 previously digested with the restriction enzymes NdeI and HindIII after the polynucleotide containing the GFP gene (SEQ ID NO: 74) was digested with the restriction enzymes NdeI and HindIII. Thus, a plasmid vector pETGFP-rbsp7 was produced. The plasmid vector pETGFP-rbsp7 has a structure in which the oligonucleotide part consisting of the base sequence shown in SEQ ID NO: 4 contained in pETGFP-p7 is replaced with the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 6. Then, Escherichia coli BL21 (DE3) strain was transformed with the plasmid vector pETGFP-rbsp7 to prepare a pETGFP-rbsp7 transformant.
(4-8) Ligation to the plasmid vector pETmalEFcR prepared in Comparative Example 1 previously digested with the restriction enzymes NdeI and HindIII and then previously digested with the restriction enzymes NdeI and HindIII after the polynucleotide containing the GFP gene (SEQ ID NO: 74) is digested with the restriction enzymes NdeI and HindIII. Thus, plasmid vector pETGFP was prepared. The plasmid vector pETGFP has a structure in which the oligonucleotide part consisting of the base sequence shown in SEQ ID NO: 4 contained in pETGFP-p7 is replaced with the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 26. Thereafter, Escherichia coli BL21 (DE3) strain was transformed with the plasmid vector pETGFP to prepare a pETGFP transformant.
(5) The influence of the polynucleotide consisting of the nucleotide sequences described in SEQ ID NOs: 1 to 6 and 26 on the translation efficiency of the expression of the GFP gene adjacent to the 3 ′ end was evaluated. FIG. 4 shows a schematic diagram of the method for evaluating the translation efficiency. The transcription efficiency was controlled by the amount of IPTG added to the culture solution using the pET system (manufactured by Novagen) so that the expression level of GFP reflects the translation efficiency. Since there is a correlation between the amount of GFP protein expressed and the fluorescence intensity (Scholz, O. et al., Eur. J. Biochem., 267, 1565-1570, 2000, Non-Patent Document 3), it depends on the fluorescence intensity of GFP. The expression level (translation efficiency) of GFP was evaluated.
(5-1) The prepared pETGFP transformant, pETGFP-rbs transformant, pETGFP-m4 transformant, pETGFP-p4 transformant, pETGFP-p7 transformant, pETGFP-p10 transformant, and pETGFP- The rbsp7 transformant was inoculated into an LB medium containing 50 μg / mL kanamycin and cultured overnight at 37 ° C. with shaking to obtain a preculture solution.
(5-2) 800 μL of LB medium containing 50 μg / mL kanamycin was placed in each well of a 96-well deep well plate, and 20 μL of the preculture solution of each transformant was inoculated. This was cultured with shaking at 1000 rpm at 37 ° C. for 1.5 hours using Bioshaker M · BR-022UP (manufactured by TAITEC).
(5-3) After 1.5 hours from the start of the culture, the temperature was changed to 20 ° C., and after shaking culture at 1000 rpm for 30 minutes, IPTG was added to induce expression. IPTG is added by adding 80 μL each of IPTG concentrate (dissolved in LB medium containing 50 μg / mL kanamycin) so that the final concentration of the culture solution is 0.01 mM, 0.05 mM, or 0.1 mM. It was done in.
(5-4) After addition of IPTG, the cells were further cultured with shaking at 20 ° C. and 1000 rpm for 22 hours. As a blank test, 80 μL of LB medium containing 50 μg / mL kanamycin is added instead of the IPTG concentrate, and the test is further performed by shaking culture at 20 ° C. and 1000 rpm for 22 hours.
(5-5) After 22 hours from the start of induction, the culture medium of each condition was collected, diluted 3-fold with 0.85% NaCl solution, put into a 96-well plate, 100 μL per well, and a fluorescent microplate reader (Infinite M200, The turbidity (OD ₆₀₀ ) of the culture solution and the GFP fluorescence intensity (excitation wavelength: 488 nm, fluorescence wavelength: 530 nm) of the culture solution were measured using n = 3.

  FIG. 5 shows the GFP fluorescence intensity per turbidity of the culture medium 22 hours after the start of induction. As shown in FIG. 5, the pETGFP transformant has a strong GFP fluorescence intensity per culture turbidity regardless of the IPTG concentration of 0.01 mM, 0.05 mM, or 0.1 mM. pETGFP-p4 transformant, pETGFP-m4 transformant, pETGFP-p7 transformant, pETGFP-p10 transformant, pETGFP-rbs transformant, and pETGFP-rbsp7 transformant in this order. A tendency of strong GFP fluorescence intensity was observed.

  From the result of FIG. 5, while the polynucleotide of the existing base sequence (SEQ ID NO: 26) derived from the pET-26b (+) plasmid vector controls the translation efficiency of the expression of the GFP gene adjacent to its 3 ′ end, It can be seen that all of the oligonucleotides consisting of the base sequences set forth in SEQ ID NOs: 1 to 6 have lower translation efficiency of the expression of the GFP gene than the oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 26. In addition, the high translation efficiency was SEQ ID NO: 3, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 6 in descending order.

Example 10
A human erythropoietin-binding protein expression vector containing an oligonucleotide having the base sequence set forth in SEQ ID NO: 4 and a transformant obtained from the vector were prepared by the following method.
(1) Synthesis of a polynucleotide encoding a human erythropoietin-binding protein with a polyhistidine tag added to the C-terminal side was performed by the two-step PCR shown below.
(1-1) The first-stage PCR is performed using EPOR cDNA (Human) (manufactured by OriGene Technologies, catalog number: SC125440) as a template and PCR primer described in SEQ ID NO: 78 (5′-CAGGATCCCCCCGGGAGAGCTCCCGCGCCCTAACCTC-3 ′) An oligonucleotide consisting of a base sequence with a restriction enzyme SacI recognition sequence and an oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 79 (5′-TTATCTAGATTTAATGATGGATGATGATGATGGTCCAGGTCGCTAGGG-3 ′) were used respectively under the reaction composition in Table 2. , A reaction consisting of a first step at 98 ° C. for 10 seconds, a second step at 55 ° C. for 5 seconds, and a third step at 72 ° C. for 44 seconds. 0 cycles were carried out. This PCR product was named EPOR1.
(1-2) Second-stage PCR is performed using EPOR1 as a template, an oligonucleotide having a nucleotide sequence set forth in SEQ ID NO: 78 as a PCR primer, and a restriction enzyme set forth in SEQ ID NO: 80 (5′-TTATCTAGACCTAGGTTAATGATGATGATGATGATG-3 ′) Oligonucleotides comprising a base sequence containing an XbaI recognition sequence were used, respectively, under the reaction composition shown in Table 2 for the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, The reaction was repeated 30 cycles with the third step of seconds as one cycle. This PCR product was named EPOR2.
(2) EPOR2 was digested with restriction enzymes SacI and XbaI, ligated to plasmid vector pUC19 (GenBank No. L09137) digested with restriction enzymes SacI and XbaI, and E. coli strain JM109 was transformed with it.
(3) After culturing the obtained transformant, a plasmid vector pUC-EPOR containing a polynucleotide encoding a human erythropoietin-binding protein having a polyhistidine tag added to the C-terminal side was prepared according to a conventional method. An outline of the plasmid vector pUC-EPOR and its production process are shown in FIG.
(4) An oligonucleotide consisting of a plasmid vector pUC-EPOR (FIG. 6) as a template and a nucleotide sequence containing the restriction enzyme BspHI recognition sequence described in SEQ ID NO: 81 (5′-CGGTCATGAAAGCTCCGCCGCCTAACCTCTCCGAC-3 ′) as a PCR primer and SEQ ID NO: 82 (5′-GAGCGGAATAACAATTTCACACAGGG-3 ′) was used, respectively, in accordance with the reaction composition of Table 2, according to the reaction composition of Table 2, the first step at 98 ° C. for 10 seconds and the second step at 55 ° C. for 5 seconds. PCR was carried out by performing 30 cycles of the step, which was a cycle of the third step of 44 seconds at 72 ° C., followed by cooling to 4 ° C. This PCR product was named EPOR3.
(5) The plasmid vector pETmalE described in Example 1 was digested with restriction enzymes NcoI and HindIII in advance after digesting EPOR3 with restriction enzyme BspHI (forms a protruding end capable of binding to a restriction enzyme NcoI digested fragment after digestion) and HindIII. -Ligated to p7 (Fig. 1) and used to transform E. coli JM109 strain.
(6) After culturing the obtained transformant, a plasmid vector pETmalE-p7-E was prepared according to a conventional method. An outline of the plasmid vector pETmalE-p7-E and its production process are shown in FIG.
(7) From a plasmid vector pUC-EPOR (FIG. 6) as a template, an oligonucleotide having the base sequence described in SEQ ID NO: 83 (5′-TCCGCTAGCGCTCTGCCGCCTCCCGCCGCCTAACCT-3 ′) as a PCR primer, and a base sequence described in SEQ ID NO: 82 Each oligonucleotide was used in accordance with the reaction composition of Table 2, and the first step at 98 ° C. for 10 seconds, the second step at 55 ° C. for 5 seconds, and the third step at 72 ° C. for 44 seconds was defined as one cycle. The reaction was performed for 30 cycles. This PCR product was named EPOR4.
(8) From the nucleotide sequence containing the restriction enzyme XbaI recognition sequence described in SEQ ID NO: 84 (5′-CCCTCTGAAAATATTTTGTTTAC-3 ′) as a PCR primer using the plasmid vector pETmalE-p7 (FIG. 1) described in Example 1 as a template. And the oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 85 (5′-GGCGGAGACGCGTAGCGGAAAAACATCATC-3 ′), respectively, in accordance with the reaction composition of Table 2, the first step at 98 ° C. for 10 seconds, 55 ° C. The reaction was carried out 30 cycles with the second step for 5 seconds and the third step for 44 seconds at 72 ° C. as one cycle. This PCR product was designated as PCRMalE.
(9) PCRMalE and EPOR4 were linked using PCR. Using a mixture of EPOR4 and PCRMalE as a template, and an oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 84 and an oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 82 as PCR primers, respectively, according to the reaction composition of Table 2. , 30 cycles of the reaction were performed with a first step at 98 ° C. for 10 seconds, a second step at 55 ° C. for 5 seconds, and a third step at 72 ° C. for 44 seconds as one cycle. This PCR product was designated as PCRMalE-EPOR.
(10) PCRMalE-EPOR is digested with restriction enzyme XbaI, then ligated to plasmid vector pETmalE-p7-E (FIG. 7) digested with restriction enzyme XbaI and dephosphorylated with alkaline phosphatase (FIG. 7), and the base described in SEQ ID NO: 4 A plasmid vector pETmalE-p7-EPOR, which is a human erythropoietin-binding protein expression vector containing an oligonucleotide consisting of a sequence, was prepared.
(11) Escherichia coli BL21 (DE3) strain was transformed using plasmid vector pETmalE-p7-EPOR to prepare a pETmalE-p7-EPOR transformant. FIG. 8 shows an outline of the plasmid vector pETmalE-p7-EPOR and its production process.

  In the plasmid vector pETmalE-p7-EPOR (FIG. 8), from the 5 ′ end side, a polynucleotide comprising the nucleotide sequence described in SEQ ID NO: 4, an oligonucleotide encoding the MalE signal peptide (SEQ ID NO: 8), and human erythropoietin binding The polynucleotide encoding the sex protein (SEQ ID NO: 77) and the oligonucleotide encoding the polyhistidine tag are arranged in this order. Of the plasmid vector pETmalE-p7-EPOR (FIG. 8), the base sequence from the restriction enzyme BglII recognition site to the HindIII recognition site is shown in SEQ ID NO: 86. Of the nucleotide sequence set forth in SEQ ID NO: 86, nucleotides from 88th to 115th correspond to SEQ ID NO: 4, and nucleotides from 116th to 193rd encode an MalE signal peptide (SEQ ID NO: 8). The nucleotides from 194th to 868th correspond to the oligonucleotide encoding human erythropoietin-binding protein (SEQ ID NO: 77), and the 869th to 886th nucleotides correspond to the oligonucleotide encoding the polyhistidine tag. Equivalent to. SEQ ID NO: 87 shows the amino acid sequence of the polypeptide obtained by translation from the 116th to 886th nucleotides of the base sequence set forth in SEQ ID NO: 86.

Example 11
A human erythropoietin-binding protein expression vector containing an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 1 and a transformant obtained from the vector were prepared by the following method.
(1) After digesting pETmalE-p7-EPOR (FIG. 8) prepared in Example 10 with restriction enzymes NdeI and HindIII, a polynucleotide fragment containing an oligonucleotide encoding a human erythropoietin-binding protein (SEQ ID NO: 77) is obtained. The plasmid vector pETMalE-rbs-EPOR was ligated to the plasmid vector pETMalEFcR-rbs prepared in Example 2 previously digested with restriction enzymes NdeI and HindIII. The plasmid vector pETMalE-rbs-EPOR is a portion of the oligonucleotide consisting of the base sequence described in SEQ ID NO: 4 contained in the plasmid vector pETmalE-p7-EPOR (FIG. 8). It is a structure substituted with nucleotides.
(2) Escherichia coli BL21 (DE3) strain was transformed with the plasmid vector pETmalE-rbs-EPOR to prepare a pETmalE-rbs-EPOR transformant.

Example 12
A human erythropoietin-binding protein expression vector containing an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 2 and a transformant obtained from the vector were prepared by the following method.
(1) After digesting pETmalE-p7-EPOR (FIG. 8) prepared in Example 10 with restriction enzymes NdeI and HindIII, a polynucleotide fragment containing an oligonucleotide encoding a human erythropoietin-binding protein (SEQ ID NO: 77) is obtained. The plasmid vector pETmalE-m4-EPOR was prepared by ligation to the plasmid vector pETmalEFcR-m4 prepared in Example 3 previously digested with restriction enzymes NdeI and HindIII. The plasmid vector pETmalE-m4-EPOR is a portion of the oligonucleotide consisting of the base sequence described in SEQ ID NO: 4 contained in the plasmid vector pETmalE-p7-EPOR (FIG. 8). It is a structure substituted with nucleotides.
(2) Escherichia coli BL21 (DE3) was transformed with the plasmid vector pETmalE-m4-EPOR to prepare a pETmalE-m4-EPOR transformant.

Example 13
A human erythropoietin-binding protein expression vector containing an oligonucleotide having the base sequence set forth in SEQ ID NO: 3 and a transformant obtained from the vector were prepared by the following method.
(1) After digesting pETmalE-p7-EPOR (FIG. 8) prepared in Example 10 with restriction enzymes NdeI and HindIII, a polynucleotide fragment containing an oligonucleotide encoding a human erythropoietin-binding protein (SEQ ID NO: 77) is obtained. The plasmid vector pETMalE-p4-EPOR was prepared by ligation to the plasmid vector pETMalEFcR-p4 prepared in Example 4 previously digested with restriction enzymes NdeI and HindIII. The plasmid vector pETmalE-p4-EPOR is a portion of the oligonucleotide consisting of the base sequence described in SEQ ID NO: 4 contained in the plasmid vector pETmalE-p7-EPOR (FIG. 8), and the oligo consisting of the base sequence described in SEQ ID NO: 3. It is a structure substituted with nucleotides.
(2) Escherichia coli BL21 (DE3) strain was transformed with the plasmid vector pETmalE-p4-EPOR to prepare a pETmalE-p4-EPOR transformant.

Example 14
A human erythropoietin-binding protein expression vector containing an oligonucleotide having the base sequence set forth in SEQ ID NO: 5 and a transformant obtained from the vector were prepared by the following method.
(1) After digesting pETMalE-p7-EPOR prepared in Example 10 with restriction enzymes NdeI and HindIII, a polynucleotide fragment containing an oligonucleotide encoding a human erythropoietin-binding protein (SEQ ID NO: 77) is preliminarily obtained by restriction enzyme NdeI. And ligated to the plasmid vector pETMalEFcR-p10 prepared in Example 5 digested with HindIII to prepare a plasmid vector pETmalE-p10-EPOR. The plasmid vector pETmalE-p10-EPOR is a portion of the oligonucleotide consisting of the base sequence shown in SEQ ID NO: 4 contained in the plasmid vector pETmalE-p7-EPOR (FIG. 8), and the oligo consisting of the base sequence shown in SEQ ID NO: 5. It is a structure substituted with nucleotides.
(2) Escherichia coli BL21 (DE3) strain was transformed with the plasmid vector pETmalE-p10-EPOR to prepare a pETmalE-p10-EPOR transformant.

Example 15
A human erythropoietin-binding protein expression vector containing an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 6 and a transformant obtained from the vector were prepared by the following method.
(1) After digesting pETmalE-p7-EPOR (FIG. 8) prepared in Example 10 with restriction enzymes NdeI and HindIII, a polynucleotide fragment containing an oligonucleotide encoding a human erythropoietin-binding protein (SEQ ID NO: 77) is obtained. The plasmid vector pETMalE-rbsp7-EPOR was prepared by ligation to the plasmid vector pETMalEFcR-rbsp7 prepared in Example 6 previously digested with restriction enzymes NdeI and HindIII. In the plasmid vector pETmalE-rbsp7-EPOR, the oligonucleotide part consisting of the base sequence described in SEQ ID NO: 4 contained in the plasmid vector pETmalE-p7-EPOR was replaced with the oligonucleotide consisting of the base sequence described in SEQ ID NO: 6. It is a structure.
(2) Escherichia coli BL21 (DE3) strain was transformed with the plasmid vector pETmalE-rbsp7-EPOR to prepare a pETmalE-rbsp7-EPOR transformant.

Comparative Example 4
A human erythropoietin-binding protein expression vector containing an existing oligonucleotide (5′-CTTTAAGAAGGAGAATATAC-3 ′) derived from a plasmid vector pET-26b (+) having the base sequence set forth in SEQ ID NO: 26, and a trait obtained by the vector A converter was prepared by the following method.
(1) A polynucleotide fragment containing an oligonucleotide encoding a human erythropoietin-binding protein (SEQ ID NO: 77) after digesting the plasmid vector pETMalE-p7-EPOR (FIG. 8) prepared in Example 10 with restriction enzymes NdeI and HindIII Was ligated to a pET-26b (+) plasmid vector previously digested with restriction enzymes NdeI and HindIII to prepare a plasmid vector pETmalE-EPOR. The plasmid vector pETMalE-EPOR is a portion of the oligonucleotide consisting of the base sequence described in SEQ ID NO: 4 contained in the plasmid vector pETmalE-p7-EPOR (FIG. 8) into the oligonucleotide consisting of the base sequence described in SEQ ID NO: 26. It is a substituted structure.
(2) Escherichia coli BL21 (DE3) strain was transformed with the plasmid vector pETmalE-EPOR to prepare a pETmalE-EPOR transformant.

Example 16
Using each transformant prepared in Examples 10 to 15 and Comparative Example 4, human erythropoietin-binding protein was expressed and the amount thereof was evaluated.
(1) pETmalE-p7-EPOR transformant prepared in Example 10, pETmalE-rbs-EPOR transformant prepared in Example 11, pETmalE-m4-EPOR transformant prepared in Example 12, Example PETMalE-p4-EPOR transformant prepared in Example 13, pETMalE-p10-EPOR transformant prepared in Example 14, pETmalE-rbsp7-EPOR transformant prepared in Example 15, and Comparative Example 4 The pETmalE-EPOR transformant was inoculated into LB medium containing 50 μg / mL of kanamycin, respectively, and cultured with shaking at 37 ° C. overnight to prepare a preculture solution.
(2) The culture solution of (1) was inoculated into LB medium newly containing 50 μg / mL kanamycin, and cultured at 37 ° C. with shaking.
(3) When the turbidity value (OD ₆₀₀ ) of the culture solution becomes about 0.5, the culture temperature is changed from 37 ° C. to 20 ° C., and after further incubation for 30 minutes, the IPTG becomes a final concentration of 0.01 mM. Thus, the expression of human erythropoietin-binding protein was induced by adding to the culture solution as described above, followed by shaking culture at 20 ° C. for 22 hours.
(4) A soluble protein extract fraction was prepared from the cells of each transformant obtained by centrifuging the culture solution using BugBuster protein extraction Kit (manufactured by Novagen).
(5) The amount of human erythropoietin-binding protein in the soluble protein extract fraction was measured by the ELISA method shown below.
(5-1) Anti-erythropoietin receptor antibody diluted with 50 mM Tris-HCl buffer (pH 8.0) (Monoclonal Anti-human EPO R Antibody) (manufactured by R & D Systems, Inc.) Catalog number MAB3071) was fixed at a concentration of 0.1 μg / well (4 ° C., 18 hours), and TBS buffer (20 mM Tris-HCl, 0.5% (w / v) bovine serum albumin (BSA) was added. It was blocked with 137 mM NaCl, 2.68 mM KCl, pH 7.5).
(5-2) After blocking, the soluble protein extract fraction prepared in (4) was serially diluted with a TBS buffer containing 0.5% BSA and subjected to an ELISA reaction (30 ° C., 2 hours).
(5-3) After washing with Tris-HCl buffer (pH 7.5) containing 0.2% (w / v) Tween 20 and 150 mM NaCl, Horseradish Peroxidase Anti-His-Tag Antibody Reagent (manufactured by BETHYL) And reacted at 30 ° C. for 2 hours.
(5-4) After the reaction, washing was performed with the buffer used in (5-3), TMB Peroxidase Substrate (manufactured by KPL) was added, and the absorbance at 450 nm was measured.
(5-5) Human erythropoietin-binding protein in a soluble protein extract fraction based on measured absorbance based on a known concentration of human erythropoietin-binding protein (Recombinant Human EPO Receptor / EPOR) (manufactured by Sino Biological, catalog number 10707-H08H) The expression level of the human erythropoietin-binding protein per culture solution was calculated by calculating the protein concentration of the sex protein.

  The expression level of human erythropoietin-binding protein per culture solution by each transformant was pETmalE-p10-EPOR transformant (Example 14 / SEQ ID NO: 5: 2.07 ± 0.33 mg / L), pETmalE-p7. -EPOR transformant (Example 10 / SEQ ID NO: 4: 1.22 ± 0.09 mg / L), pETmalE-p4-EPOR transformant (Example 13 / SEQ ID NO: 3: 0.97 ± 0.01 mg / L) L), pETmalE-rbsp7-EPOR transformant (Example 15 / SEQ ID NO: 6: 0.89 ± 0.15 mg / L), pETmalE-rbs-EPOR transformant (Example 11 / SEQ ID NO: 1: 0. 65 ± 0.11 mg / L), pETmalE-m4-EPOR transformant (Example 12 / SEQ ID NO: 0.32 ± 0.01 mg / L), pE MalE-EPOR transformants (Comparative Example 4 / SEQ ID NO 26: 0.17 ± 0.01mg / L) was. From this result, it was found that the human erythropoietin-binding protein expression vector containing the existing oligonucleotide consisting of the base sequence described in SEQ ID NO: 26 derived from the plasmid vector pET-26b (+) was used. It has been found that a human erythropoietin-binding protein can be prepared in a larger amount by using a human erythropoietin-binding protein expression vector containing an oligonucleotide having any one of the nucleotide sequences described above. In addition, it turns out that the pETMalE-p10-EPOR transformant produced in Example 14 can express human erythropoietin binding protein especially efficiently among the transformants produced in Example 10 to Example 15.

Claims (9)

A vector for expressing a human-derived protein, comprising an oligonucleotide having the base sequence of any one of SEQ ID NOs: 1 to 6 and an oligonucleotide encoding a signal peptide that causes the periplasm to secrete the protein. The vector according to claim 1, wherein the signal peptide that causes the periplasm to secrete a protein is an oligopeptide having the amino acid sequence of any one of SEQ ID NOs: 7 to 9. A transformant capable of expressing a human-derived protein, obtained by transforming a host with the vector according to claim 1 or 2, into which a polynucleotide encoding a human-derived protein has been inserted. The transformant according to claim 3, wherein the host is Escherichia coli. The transformant according to claim 3 or 4, wherein the human-derived protein is a human Fc-binding protein. The transformant according to claim 3 or 4, wherein the human-derived protein is a human erythropoietin-binding protein. A method for producing a human-derived protein, comprising a step of culturing the transformant according to claim 3 or 4. A method for producing a human Fc-binding protein, comprising a step of culturing the transformant according to claim 5. A method for producing a human erythropoietin-binding protein comprising a step of culturing the transformant according to claim 6.

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