JP2017066082A - Purification and production methods of bfgf - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide simple purification and production methods of bFGF.SOLUTION: The invention provides a method for purifying bFGF comprising the steps of: subjecting a sample containing bFGF to cationic ion exchange chromatography to obtain a crude sample containing bFGF; adjusting the salt concentration of the crude sample; subjecting the adjusted crude sample to hydrophobic interaction chromatography to obtain purified bFGF, where the salt concentration of the crude sample after adjustment is higher than 1.5 mol/L.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for purifying bFGF and a method for producing bFGF.

  bFGF (basic fibroblast growth factor, FGF-2) is a protein identified as a growth factor for fibroblasts. In recent years, it has been clarified that the bFGF is a factor necessary for maintaining an undifferentiated state of pluripotent stem cells such as human induced pluripotent stem cells. Although the demand for bFGF is expected to increase with the development of regenerative medicine, there is a problem that when bFGF is purified by column chromatography, it is necessary to perform column chromatography many times, which is complicated. Therefore, there is a need for a method for purifying bFGF that can easily purify bFGF.

  Accordingly, an object of the present invention is to provide a simple method for purifying bFGF and a method for producing bFGF.

In order to solve the problems of the present invention, the method for purifying bFGF of the present invention includes a step of subjecting a sample containing bFGF to cation exchange chromatography to obtain a crude purified sample containing bFGF,
Adjusting the salt concentration of the roughly purified sample;
Subjecting the prepared crude purified sample to hydrophobic interaction chromatography to obtain purified bFGF,
The salt concentration in the roughly purified sample after the adjustment exceeds 1.5 mol / L.

The method for producing bFGF of the present invention comprises a step of purifying purified bFGF from a sample containing bFGF,
The purification step is performed by the method for purifying bFGF of the present invention.

  According to the present invention, a simple bFGF purification method and bFGF production method can be provided.

FIG. 1 is a photograph showing the results of SDS-PAGE of a purified sample. FIG. 2 is a photograph showing the results of SDS-PAGE of a sample of the solid fraction.

  In the purification method of the present invention, for example, the salt concentration is 2.5 mol / L or less, preferably 1.8 to 2.5 mol / L.

  In the purification method of the present invention, for example, the salt is ammonium sulfate.

  In the purification method of the present invention, for example, the functional group of the filler in the cation exchange chromatography is a sulfopropyl group.

  In the purification method of the present invention, for example, the hydrophobic ligand of the hydrophobic interaction chromatography filler is a butyl group.

  In the purification method of the present invention, for example, the sample containing bFGF contains a reducing agent.

The purification method of the present invention includes, for example, a step of recovering a liquid fraction from the prepared crude purified sample,
In the obtaining step of the purified bFGF, the liquid fraction is subjected to the hydrophobic interaction chromatography to obtain the purified bFGF.

  In the purification method of the present invention, for example, the sample containing bFGF may be a crushed E. coli expressing bFGF, or a liquid fraction of a crushed E. coli expressing bFGF.

The purification method of the present invention further includes, for example, a step of culturing Escherichia coli transduced with the bFGF gene, and expressing bFGF in the E. coli.
Preparing a sample containing bFGF from the obtained Escherichia coli expressing bFGF.

  The purification method of the present invention further includes, for example, a step of transducing the bFGF gene into E. coli.

  In the production method of the present invention, for example, the purity of bFGF obtained in the purification step is 96% or more.

<Method for purifying bFGF>
As described above, the method for purifying bFGF of the present invention (hereinafter also referred to as “purification method”) includes subjecting a sample containing bFGF (hereinafter also referred to as “bFGF sample”) to cation exchange chromatography, and bFGF. A step of obtaining a crudely purified sample (crude purification step), a step of adjusting the salt concentration of the crudely purified sample (salt concentration adjusting step), and a crude purified sample after adjustment (hereinafter referred to as “adjusted sample”) And a step of obtaining purified bFGF (bFGF obtaining step), and the salt concentration in the adjusted crude purified sample exceeds 1.5 mol / L. And The purification method of the present invention is characterized in that the salt concentration in the prepared sample exceeds 1.5 mol / L, and other steps and conditions are not particularly limited.

  As a result of intensive studies, the present inventors have found that impurities other than bFGF in the roughly purified sample can be efficiently precipitated by setting the salt concentration in the adjusted sample to a concentration exceeding 1.5 mol / L. It was. For this reason, the purification method of the present invention can purify bFGF having the purity described later, for example, in two steps of the crude purification step and the bFGF acquisition step as a purification step using chromatography. Therefore, according to the purification method of the present invention, bFGF can be easily purified. Moreover, since the purification method of the present invention can purify bFGF easily, for example, the time required for purification can be shortened. For example, the activity of bFGF decreases with the passage of time from the start of purification. However, according to the purification method of the present invention, for example, the time required for purification is shortened, so that bFGF in a more active state can be purified. . The purification method of the present invention can be purified without using, for example, the heparin that binds to the bFGF. Since the heparin has an anticoagulant action, for example, when bFGF containing the heparin enters a living body, a side effect due to the heparin occurs. However, since the purification method of the present invention can be purified without using heparin, for example, side effects due to the contamination of the heparin can be eliminated.

  In the present invention, the isoform of bFGF (basic fibroblast growth factor, FGF-2) is not particularly limited. Examples of the isoform include isoforms such as 16.5 KDa, 18 KDa, 22 KDa, 22.5 KDa, 24 KDa, and 34 KDa. The origin of the bFGF is not particularly limited, and examples thereof include humans and non-human animals. Examples of the non-human animal include mouse, rat, rabbit, dog, sheep, horse, cat, goat, monkey, guinea pig, cow and pig. As a specific example, the bFGF includes, for example, at least one protein selected from the group consisting of the following (B1), (B2), and (B3). In the following (B1), (B2), and (B3), the fibroblast is, for example, a Balb / 3T3 clone A31 cell.

(B1) A protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 and 6 (B2) In the amino acid sequence of (B1), one or several amino acids are deleted, substituted, or inserted And / or a protein having a fibroblast proliferating activity (B3) consisting of an added amino acid sequence and an amino acid sequence having a predetermined identity with respect to the amino acid sequence of (B1), and proliferating fibroblasts Active protein

  The amino acid sequences of SEQ ID NOs: 1 to 6 in (B1) are as follows. Said (B1) can be obtained from a human, for example.

bFGF isoform 16.5 KDa (SEQ ID NO: 1)
MPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS

bFGF isoform 18 KDa (SEQ ID NO: 2)
MAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS

bFGF isoform 22KDa (SEQ ID NO: 3)
LGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQQFLSKYPM

bFGF isoform 22.5 KDa (SEQ ID NO: 4)
LPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSSAKTSWYVALKRTGGKL

bFGF isoform 24 KDa (SEQ ID NO: 5)
LGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESQNYVALSTGTGPG

bFGF isoform 34 KDa (SEQ ID NO: 6)
MVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS

  The “one or several” is, for example, 1 to 58, 1 to 44, 1 to 29, 1 to 15, 1 to 12, 1 to 9, 1 to 6, 1 to 3 One or two.

  The “predetermined identity” is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more. The identity can be calculated with default parameters using analysis software such as BLAST and FASTA (hereinafter the same).

  The bFGF sample may include the bFGF. Examples of the bFGF sample include a transformant transduced with bFGF, a cell expressing bFGF, a tissue, an organ, or a cultured cell. The host for producing the transformant is not particularly limited, and examples thereof include microorganisms, animal cells, insect cells, or non-human hosts such as cultured cells thereof, isolated human cells or cultured cells thereof. can give. Specifically, examples of the transformant include cells or cultured cells transduced with bFGF, and Escherichia coli transduced with bFGF. Since the bFGF sample can improve the recovery rate of bFGF, for example, a crushed material such as the transformant is preferable, and a liquid fraction of the crushed material such as the transformant is more preferable. As a specific example, the bFGF sample includes, for example, a crushed material of E. coli expressing bFGF (bFGF-expressing E. coli), and preferably a liquid fraction of the crushed material of bFGF-expressing E. coli. The bFGF sample may include, for example, one type of bFGF or two or more types of bFGF.

  The bFGF sample is preferably a liquid sample, for example. As the bFGF sample, for example, an undiluted solution of the bFGF sample may be used as a liquid sample as it is, or a diluted solution obtained by suspending, dispersing or dissolving the bFGF sample in a medium may be used as a liquid sample. Good. When the bFGF sample is solid, for example, a diluted solution obtained by suspending, dispersing, or dissolving the bFGF sample in the medium may be used as the liquid sample. The medium is not particularly limited, and examples thereof include water and a buffer solution. The buffer is not particularly limited, and examples thereof include Tris buffer, phosphate buffer, imidazole buffer, acetate buffer, borate buffer, citrate buffer, and various Good buffers. The concentration of the buffer solution is not particularly limited and is, for example, 0.01 to 0.1 mol / L.

  The bFGF sample preferably contains a reducing agent because, for example, the activity of the bFGF during purification can be maintained. The reducing agent is not particularly limited, and known reducing agents for proteins can be used, and examples thereof include dithiothreitol (DTT), β-mercaptoethanol, cysteine, glutathione and the like. As the reducing agent, for example, one kind may be used alone, or two or more kinds may be used in combination. The concentration of the reducing agent is, for example, 0.001 to 0.005 mol / L. The reducing agent may be contained in, for example, an equilibration liquid, a cleaning liquid, and an eluent described later.

  The salt is not particularly limited, and examples thereof include salts that can be used for salting out. Examples of the salt include citrate, tartrate, sulfate, acetate, chloride salt and the like. Specific examples of the salt include sodium chloride and ammonium sulfate, and preferably ammonium sulfate. For example, one type of the salt may be used alone, or two or more types may be used in combination.

  The filler for the cation exchange chromatography (first filler) is not particularly limited, and a known filler for cation exchange chromatography can be used. The first filler includes, for example, a carrier and a functional group. The carrier is not particularly limited and can be appropriately set according to the type of the functional group. Examples of the carrier include styrene beads, agarose beads (for example, sepharose), silica, dextran, and polymer resin. The diameter of the carrier is, for example, 20 to 300 μm. The functional group is not particularly limited, and examples thereof include a carboxymethyl group, a carboxyl group, a phosphate group, a sulfo group, a sulfomethyl group, a sulfoethyl group, a sulfopropyl group, and preferably a sulfopropyl group. As a specific example, the first filler is, for example, TOYOPEARL (registered trademark) SP-550, TOYOPEARL (registered trademark) SP-650 (manufactured by Tosoh Corporation), SP Sepharose Fast Flow (manufactured by GE Healthcare) or the like. Can be given.

  The hydrophobic interaction chromatography packing material (second packing material) is not particularly limited, and known packing materials for hydrophobic interaction chromatography can be used. The second filler includes, for example, a carrier and a hydrophobic ligand. For the carrier, for example, the description of the carrier in the first filler described above can be used. The hydrophobic ligand is not particularly limited, and examples thereof include a straight chain or branched alkyl group such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a tert-butyl group, a hexyl group, and an octyl group, a phenyl group. And aryl groups such as polypropylene glycol group, and the like, preferably butyl group. As a specific example, the second filler is, for example, TOYOPEARL (registered trademark) Butyl-600, TOYOPEARL (registered trademark) Butyl-650 (manufactured by Tosoh Corporation), Butyl-S Sephalose Fast Flow (manufactured by GE Healthcare) ) Etc.

  Hereinafter, each step of the purification method of the present invention will be described. As the reagent used in each step, for example, a reagent that does not contain animal-derived components, that is, a so-called animal-free grade reagent may be used.

  The purification method of the present invention may include a step of preparing a bFGF sample to be used in the rough purification step (sample preparation step) prior to the rough purification step. The sample preparation step includes, for example, culturing Escherichia coli transduced with the bFGF gene, allowing the E. coli to express bFGF, and preparing a sample containing bFGF from the obtained Escherichia coli expressing bFGF. .

  The Escherichia coli transduced with bFGF can be obtained, for example, by transducing the bFGF gene into Escherichia coli. For this reason, the purification method of the present invention may include, for example, a step of transducing the bFGF gene into the E. coli.

  The bFGF isoform encoded by the bFGF gene is not particularly limited, and may be, for example, any of the aforementioned bFGF isoforms. The origin of the bFGF gene is not particularly limited, and for example, the description of the bFGF can be used. As a specific example, the bFGF gene includes, for example, at least one polynucleotide selected from the group consisting of (b1) to (b6) and (b7) below. In the following (b1) to (b6) and (b7), the fibroblast is, for example, a Balb / 3T3 clone A31 cell.

(B1) a polynucleotide comprising at least one base sequence selected from the group consisting of SEQ ID NOs: 7 to 11 and 12 (b2) in the base sequence of (b1), one or several bases are deleted, substituted, A polynucleotide encoding a protein having fibroblast proliferation activity (b3) consisting of a base sequence having a predetermined identity to the base sequence of (b1) A polynucleotide encoding a protein having fibroblast proliferation activity (b4) a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising the base sequence of (b1) above And a polynucleotide (b5) sequence encoding a protein having fibroblast proliferation activity A polynucleotide encoding a protein consisting of at least one amino acid sequence selected from the group consisting of Nos. 1 to 5 and 6 (b6) in at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 and 6, A polynucleotide (b7) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added, and encoding a protein having fibroblast proliferative activity, consisting of SEQ ID NOs: 1 to 5 and 6 A polynucleotide encoding a protein having an amino acid sequence having a predetermined identity to at least one amino acid sequence selected from the group and having fibroblast proliferation activity

  The base sequences of SEQ ID NOs: 7 to 11 and 12 are as follows. The nucleotide sequences of SEQ ID NOs: 7 to 11 and 12 are coding sequences of bFGF isoforms consisting of the amino acid sequences of SEQ ID NOs: 1 to 5 and 6, respectively. Said (b1) can be obtained from a human, for example.

bFGF isoform 16.5 KDa cDNA (SEQ ID NO: 7)
5'-atgcccgccttgcccgaggatggcggcagcggcgccttcccgcccggccacttcaaggaccccaagcggctgtactgcaaaaacgggggcttcttcctgcgcatccaccccgacggccgagttgacggggtccgggagaagagcgaccctcacatcaagctacaacttcaagcagaagagagaggagttgtgtctatcaaaggagtgtgtgctaaccgttacctggctatgaaggaagatggaagattactggcttctaaatgtgttacggatgagtgtttcttttttgaacgattggaatctaataactacaatacttaccggtcaaggaaatacaccagttggtatgtggcactgaaacgaactgggcagtataaacttggatccaaaacaggacctgggcagaaagctatactttttcttccaatgtctgctaagagctga-3 '

bFGF isoform 18 KDa cDNA (SEQ ID NO: 8)
5'-atggcagccgggagcatcaccacgctgcccgccttgcccgaggatggcggcagcggcgccttcccgcccggccacttcaaggaccccaagcggctgtactgcaaaaacgggggcttcttcctgcgcatccaccccgacggccgagttgacggggtccgggagaagagcgaccctcacatcaagctacaacttcaagcagaagagagaggagttgtgtctatcaaaggagtgtgtgctaaccgttacctggctatgaaggaagatggaagattactggcttctaaatgtgttacggatgagtgtttcttttttgaacgattggaatctaataactacaatacttaccggtcaaggaaatacaccagttggtatgtggcactgaaacgaactgggcagtataaacttggatccaaaacaggacctgggcagaaagctatactttttcttccaatgtctgctaagagctga-3 '

bFGF isoform 22 KDa cDNA (SEQ ID NO: 9)
5 '-3'

bFGF isoform 22.5 KDa cDNA (SEQ ID NO: 10)
5 '-3'

bFGF isoform 24 KDa cDNA (SEQ ID NO: 11)
5 '-3'

bFGF isoform 34 KDa cDNA (SEQ ID NO: 12)
5 '-3'

  In the above (b2), “1 or several” may be within a range in which the protein encoded by the polynucleotide of (b2) has the fibroblast proliferation activity, for example. In the base sequence of (b1), “1 or several” of (b2) is, for example, 1 to 174, 1 to 131, 1 to 87, 1 to 43, 1 to 35, -27, 1-18, 1-9, 1-4, 1-3, 1 or 2.

  In the (b3), the “predetermined identity” may be, for example, within a range in which the protein encoded by the polynucleotide of the (b3) has the fibroblast proliferation activity. The identity of (b3) is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more with respect to the base sequence of (b1), 99% or more.

In (b4), the “hybridizable polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (b1). The hybridization can be detected by, for example, various hybridization assays. The hybridization assay is not particularly limited, for example, Zanburuku (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) " [(Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.

In the above (b4), “stringent conditions” may be, for example, low stringent conditions, medium stringent conditions, or high stringent conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 32 ° C. “Medium stringent conditions” are, for example, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. The degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, time, and the like. "Stringent conditions" are, for example, Zanburuku previously described (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) " [(Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.

  The polynucleotide (b5) only needs to be a base sequence in which the protein encoded by the polynucleotide (b5) has the fibroblast proliferation activity, for example. The base sequence of the polynucleotide (b5) can be designed, for example, by replacing it with the corresponding codon based on the amino acid sequences of SEQ ID NOs: 1 to 5 and 6.

  In the above (b6), “one or several” regarding the amino acid sequence may be within a range in which the protein encoded by the polynucleotide of (b6) has the fibroblast proliferation activity. The “1 or several” of (b6) is, for example, 1 to 58, 1 to 44, 1 to 29, 1 to 15 in the amino acid sequences of SEQ ID NOs: 1 to 5 and 6, 1 to 12, 1 to 9, 1 to 6, 1 to 3, 1, or 2.

  In the (b7), the “predetermined identity” regarding the amino acid sequence may be, for example, within a range in which the protein encoded by the polynucleotide of the (b7) has the fibroblast proliferation activity. The identity of (b7) is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more with respect to the amino acid sequences of SEQ ID NOs: 1 to 5 and 6. 98% or more, 99% or more.

  The method for introducing the bFGF gene into E. coli is not particularly limited and can be performed by a known method. Examples of the introduction method include introduction method using a gene gun such as particle gun, calcium phosphate method, polyethylene glycol method, lipofection method using liposome, electroporation method, ultrasonic nucleic acid introduction method, DEAE-dextran method, micro glass tube, etc. For example, a direct injection method using a cation, a cationic liposome method, a method using an introduction aid, and the like.

  The bFGF gene may be introduced into the E. coli by, for example, a polynucleotide encoding the bFGF, or may be introduced into the E. coli by a vector containing the polynucleotide encoding the bFGF. The vector is not particularly limited, and a known vector can be used. The vector preferably has a regulatory sequence, for example. Examples of the regulatory sequence include a promoter, terminator, enhancer, polyadenylation signal sequence, origin of replication sequence (ori) and the like. When transforming bacteria such as E. coli, examples of the vector include pETDuet-1 vector (Novagen), pET-3a (Novagen), pQE-80L vector (QIAGEN) and the like.

  The E. coli can be cultured, for example, by culturing in a medium. As the medium used for culturing the E. coli, a conventionally known medium such as a solid medium or a liquid medium can be appropriately used. The medium may include a commercially available medium, for example. The commercial medium is not particularly limited, and examples thereof include LB medium, super broth medium, and M9 medium. These may be used alone or in combination of two or more. The pH of the medium is not particularly limited, and is, for example, pH 6-8, pH 6.5-7.5. The medium may contain, for example, the bFGF expression inducer. The expression inducer is not particularly limited and can be appropriately determined according to the promoter of the bFGF gene. As a specific example, when the lac promoter is used as the promoter, for example, isopropyl-β-thiogalactopyranoside (IPTG) can be used as the expression inducer.

  The method for culturing the E. coli is not particularly limited, and examples thereof include a plate culture method and a slope culture method when the solid medium is used. Further, when the liquid medium is used, there is no particular limitation, and examples thereof include a stationary culture method, an aeration culture method, and a shake culture method.

The culture temperature of the E. coli is not particularly limited, and examples thereof include 15 to 40 ° C. and 30 to 37 ° C. The culture time of the E. coli is not particularly limited, and can be determined, for example, by the absorbance of the culture solution containing the E. coli. The absorbance is, for example, OD ₆₀₀ = 0.4 to 0.8.

  The method for preparing the sample containing bFGF is not particularly limited. In the preparation method, for example, the bFGF-expressing E. coli may be prepared as the bFGF sample, a crushed material obtained by crushing the bFGF-expressing E. coli may be prepared as the bFGF sample, A liquid fraction of the crushed material may be prepared as the bFGF sample. The crushing method of the bFGF-expressing E. coli is not particularly limited, and a known crushing method can be used. In the case of collecting the liquid fraction, the method for collecting the liquid fraction is not particularly limited. For example, the liquid fraction can be collected by performing a known solid-liquid separation method and collecting the obtained liquid fraction. Examples of the solid-liquid separation method include filtration, centrifugation, precipitation, membrane separation, adsorption, lyophilization, and the like. The bFGF-expressing E. coli may include, for example, a medium in which the E. coli is cultured.

  Next, in the crude purification step, as described above, the bFGF sample is subjected to cation exchange chromatography to obtain a crude purified sample containing bFGF. Specifically, the rough purification step includes, for example, bringing the bFGF sample into contact with the first filler and binding the bFGF and the first filler; And a step of washing a bound substance of the first filler and a step of dissociating the bFGF from the bound substance with a first eluate to obtain a crudely purified sample containing bFGF.

  The contact between the bFGF sample and the filler can be performed by, for example, filling the first column with the first filler and loading the bFGF sample onto the first column. The capacity, material, and the like of the first column are not particularly limited, and can be appropriately determined according to, for example, the type of the first filler, the packing amount, and the like. The amount of the first filler packed in the first column is not particularly limited, and can be appropriately determined according to, for example, the amount of the bFGF sample, the binding capacity of the first filler, and the like. The first column containing the first filler may equilibrate the first filler with a first equilibration liquid, for example, prior to loading the bFGF sample. The equilibration is performed, for example, using the first equilibration liquid at 2 to 10 ° C. and 4 to 8 ° C. under room temperature (for example, around 25 ° C.) conditions. The equilibration liquid can be appropriately determined according to, for example, the type of the first filler. The pH of the first equilibration liquid is, for example, pH 6-8, pH 7-7.5. The first equilibration solution is, for example, a buffer solution, and preferably a phosphate buffer solution. The concentration of the buffer solution is, for example, 0.01 to 0.1 mol / L, 0.01 to 0.05 mol / L. The first equilibration liquid may contain, for example, the salt. The salt is, for example, sodium chloride. The salt concentration in the first cleaning liquid is, for example, 0.1 to 0.4 mol / L, 0.2 to 0.4 mol / L.

  After loading the bFGF sample, for example, the first washing solution is loaded on the first column, and the combined product of the bFGF and the first packing is washed. The washing is performed, for example, using the first washing liquid at 2 to 10 ° C. and 4 to 8 ° C. under room temperature (for example, around 25 ° C.) conditions. The first cleaning liquid can be appropriately determined according to, for example, the type of the first filler. The pH of the first cleaning liquid is, for example, pH 6-8, pH 7-7.5. The first cleaning solution is, for example, a buffer solution, and preferably a phosphate buffer solution. The number of moles of the buffer solution is, for example, 0.01 to 0.1 mol / L, 0.01 to 0.05 mol / L. The first cleaning liquid may contain, for example, the salt. The salt is, for example, sodium chloride. The salt concentration in the first cleaning solution may be, for example, the same concentration as the salt concentration in the first equilibration solution, specifically 0.1 to 0.4 mol / L, 0.2 to 0.4 mol. / L.

  After the washing, for example, the first eluate is loaded onto the first column, the bFGF is dissociated from the bound product, and a crude purified sample containing bFGF is obtained. For example, a part of the solution eluted with the first eluate may be obtained as the crudely purified sample, or the whole may be obtained. In the former case, for example, the solution eluted by the first eluate is fractionated into a plurality of fractions, and the fraction containing the bFGF is obtained as the roughly purified sample. The acquisition of the crudely purified sample is performed, for example, using the first eluate at 2 to 10 ° C. and 4 to 8 ° C. under room temperature (for example, around 25 ° C.) conditions. The first eluate can be appropriately determined according to, for example, the type of the first filler. The pH of the first eluate is, for example, pH 6-8, pH 7-7.5. The first eluate is, for example, a buffer solution, and preferably a phosphate buffer solution. The number of moles of the buffer solution is, for example, 0.01 to 0.1 mol / L, 0.01 to 0.05 mol / L. The first eluate may contain, for example, the salt. The salt is, for example, sodium chloride. The salt concentration in the first eluate is, for example, higher than the salt concentration in the first cleaning solution, specifically 0.5 to 2 mol / L, 0.6 to 0.8 mol / L. is there.

  Next, in the salt concentration adjustment step, as described above, the salt concentration of the roughly purified sample is adjusted. The adjustment is not particularly limited, and can be performed, for example, by adding the salt to the roughly purified sample, diluting the roughly purified sample, or the like. The addition of the salt may be, for example, addition of a solution containing the salt. The salt concentration may be, for example, the concentration of the salt derived from the salt added in the salt concentration adjustment step, or the concentration of the salt derived from both the salt present in the roughly purified sample before the adjustment and the salt added at the time of adjustment. But you can. The salt concentration in the adjusted sample only needs to exceed 1.5 mol / L. For example, since the contaminants can be efficiently precipitated and bFGF can be purified with higher yield, preferably 2.5 mol / L or less, Since it is 2.3 mol / L or less, the impurities can be precipitated more efficiently, and the bFGF can be purified with higher yield, more preferably more than 1.5 mol / L and 2.5 mol / L or less. They are 1.8-2.5 mol / L, 1.8-2.3 mol / L, 2.1-2.3 mol / L, 1.8-2.1 mol / L. The salt concentration may be, for example, the salt concentration of one type of salt or the total salt concentration of two or more types of salt. As a specific example, when ammonium sulfate is added as the salt, the concentration of ammonium sulfate in the prepared sample is, for example, 1.8 to 2.3 mol / L, 1.8 to 2.1 mol / L, 1.8 to 2 mol / L. Moreover, when the said adjustment sample contains sodium chloride and ammonium sulfate as said salt, the density | concentration of sodium chloride in the said adjustment sample is 0.2-0.4 mol / L, for example, and the density | concentration of ammonium sulfate is 1. They are 8-2.3 mol / L, 1.8-2.1 mol / L, 1.8-2 mol / L.

  Next, in the bFGF obtaining step, as described above, the prepared sample is subjected to hydrophobic interaction chromatography to obtain purified bFGF. Specifically, the bFGF obtaining step includes, for example, a step of bringing the adjusted sample into contact with the second filler and binding bFGF and the second filler; Washing a bound substance with the second filler, and dissociating the bFGF from the bound substance with a second eluate to obtain purified bFGF.

  The contact between the adjusted sample and the filler can be performed, for example, by filling the second column with the second filler and loading the adjusted sample on the second column. The volume, material, and the like of the second column are not particularly limited, and can be appropriately determined according to, for example, the type of the second filler, the packing amount, and the like. The amount of the second filler packed in the second column is not particularly limited, and can be appropriately determined according to, for example, the amount of the adjusted sample, the binding capacity of the second filler, and the like. The second column containing the second filler may equilibrate the second filler with a second equilibration liquid, for example, prior to loading the conditioning sample. The equilibration is performed, for example, using the second equilibration liquid at 10 to 30 ° C. and 15 to 25 ° C. under a room temperature (for example, around 25 ° C.) condition. The second equilibration liquid can be appropriately determined according to, for example, the type of the second filler. The pH of the second equilibration liquid is, for example, pH 6-8, pH 7-7.5. The second equilibration solution is, for example, a buffer solution, and preferably a phosphate buffer solution. The concentration of the buffer solution is, for example, 0.01 to 0.1 mol / L, 0.01 to 0.05 mol / L. The second equilibration liquid may contain the salt, for example. The salt is, for example, ammonium sulfate. The salt concentration in the second equilibration liquid is, for example, 1.5 to 2.5 mol / L, 1.9 to 2.1 mol / L.

  After the adjustment sample is loaded, for example, the second washing liquid is loaded onto the second column, and the combined product of the bFGF and the second packing material is washed. The washing is performed, for example, using the second washing liquid at 10 to 30 ° C. and 15 to 25 ° C. under room temperature (for example, around 25 ° C.) conditions. The second cleaning liquid can be appropriately determined according to, for example, the type of the second filler. The pH of the second cleaning liquid is, for example, pH 6-8, pH 7-7.5. The second cleaning solution is, for example, a buffer solution, and preferably a phosphate buffer solution. The number of moles of the buffer solution is, for example, 0.01 to 0.1 mol / L, 0.01 to 0.05 mol / L. The second cleaning liquid may contain the salt, for example. The salt is, for example, ammonium sulfate. The salt concentration in the second cleaning solution is not particularly limited, and is, for example, a salt concentration equal to or lower than the salt concentration of the second equilibration solution, specifically, more than 1.5 mol / L and 2.3 mol. / L or less, 1.8 to 2.3 mol / L, 1.8 to 2 mol / L.

  After the washing, for example, the second eluate is loaded onto the second column, and the bFGF is dissociated from the bound substance to obtain purified bFGF. For the purified bFGF, for example, a part of the solution eluted with the second eluate may be obtained, or the whole may be obtained. In the former case, for example, the solution eluted by the eluate is fractionated into a plurality of fractions, and the fraction containing the bFGF is obtained as the purified bFGF. Acquisition of the purified bFGF is carried out, for example, using the second eluate at 10 to 30 ° C. and 15 to 25 ° C. under room temperature (for example, around 25 ° C.) conditions. The second eluate can be appropriately determined according to, for example, the type of the second filler. The pH of the second eluate is, for example, pH 6-8, pH 7-7.5. The second eluate is, for example, a buffer solution, and preferably a phosphate buffer solution. The number of moles of the buffer solution is, for example, 0.01 to 0.1 mol / L, 0.01 to 0.05 mol / L. The second eluate may contain the salt, for example. The salt is, for example, ammonium sulfate. The salt concentration in the second eluate is not particularly limited, and is, for example, a salt concentration less than the salt concentration of the second cleaning solution, specifically 0.9 to 1.4 mol / L, 1 to 1.3 mol / L.

  In this way, purified bFGF can be purified. The purity of bFGF obtained by the purification method of the present invention is, for example, 96% or more, 97% or more, 98% or more, 99% or more. The purity can be measured by, for example, SDS-PAGE, reverse phase high performance liquid chromatography (RP-HPLC), mass spectrometry and the like. When the purity is measured using the SDS-PAGE, for example, it can be calculated by densitometry. Moreover, when measuring purity using the said RP-HPLC and the said mass spectrometry, it can calculate based on the integrated value (peak area) of the obtained chromatograph, for example.

  In the purification method of the present invention, in the adjusted sample, for example, impurities other than bFGF contained in the roughly purified sample are efficiently precipitated. Therefore, the purification method of the present invention includes, for example, a step of recovering a liquid fraction from the adjusted sample because the purity of the bFGF can be further increased. In the step of obtaining the purified bFGF, the liquid fraction is The purified bFGF may be obtained by subjecting to hydrophobic interaction chromatography. The method for collecting the liquid fraction from the adjusted sample is not particularly limited, and for example, the description of the method for collecting the liquid fraction can be cited. Moreover, the method for obtaining the purified bFGF from the liquid fraction can be carried out, for example, by using the liquid fraction in place of the adjustment sample in the description of the bFGF step.

<Method for producing bFGF>
As described above, the method for producing bFGF of the present invention (hereinafter also referred to as “manufacturing method”) includes a step of purifying purified bFGF from a sample containing bFGF, and the purification step includes the step of purifying bFGF of the present invention. It is characterized by being carried out by a purification method. The production method of the present invention is characterized in that the purification step is carried out by the purification method of the present invention, and other steps and conditions are not particularly limited. According to the production method of the present invention, bFGF purified easily can be produced. In the production method of the present invention, for the purification step, for example, the description of the purification method of the present invention can be used.

  In the production method of the present invention, the purity of bFGF obtained in the purification step is not particularly limited. The above-mentioned explanation can be used for the purity and the measuring method of purity, for example.

  Next, examples of the present invention will be described. In addition, this invention is not restrict | limited by the following Example.

Example 1
It was confirmed that bFGF can be purified by the purification method of the present invention.

(1) Preparation of bFGF sample Using human-derived cDNA as a template, the following human bFGF 16.5 KDa cDNA (SEQ ID NO: 7) encoding 16.5 KDa isoform (SEQ ID NO: 1) of human-derived bFGF is amplified, and then the lac promoter is amplified. Ligated to the vector (pET-3a, manufactured by Novagen) to prepare a bFGF expression vector. The vector contains an ampicillin resistance gene.

The bFGF expression vector was transduced into E. coli (T7 Express Lys Y Competent E. Coli (High Efficiency), NEW ENGLAND Labs Inc.) by the heat shock method. The obtained transformant was cultured at 37 ° C. using LB medium containing ampicillin until OD ₆₀₀ = 0.6 or more. Furthermore, IPTG was added so that it might become 0.2 mmol / L, and it culture | cultivated at 37 degreeC for 3 hours. The cultured E. coli was collected, suspended in lysis buffer, and sonicated. The ultrasonic crushing was carried out twice on ice for 10 minutes using an ultrasonic crusher (UD-201, manufactured by TOMY) under the conditions of output 6 and duty ratio 80%. The lysis buffer includes a buffer (pH 7.2) containing 20 mmol / L sodium dihydrogen phosphate, 0.1 mmol / L NaCl, 2 mmol / L DTT, and 1 mmol / L PMSF (phenylmethylsulfonyl fluoride). did. And it centrifuged on 20000 g and 4 degreeC and the conditions for 20 minutes, and collect | recovered the obtained supernatant liquids. Further, a bFGF sample was prepared by filtering the supernatant with a DISMIC 0.8 μm cellulose acetate membrane (manufactured by ADVANTEC).

(2) Cation Exchange Chromatography TOYOPEARL (registered trademark) SP-550C was used as a filler (first filler) used for cation exchange chromatography. 100 mL of the first filler was washed with purified water three times, and then packed into an Econo column (5.0 × 10 cm, Bio-rad, first column). The first column was loaded with 200 mL of the first equilibration buffer to equilibrate the first column. The first equilibration buffer was a buffer solution (pH 7.2) containing 20 mmol / L sodium dihydrogen phosphate. Next, the bFGF sample was loaded onto the first column. After the loading, 800 mL of the first washing solution was loaded on the first column and washed. The first washing solution was a buffer solution (pH 7.2) containing 20 mmol / L sodium dihydrogen phosphate and 0.2 mol / L NaCl. Further, 800 mL of the first eluate was loaded onto the first column, and the entire amount of the eluted solution was obtained as the crudely purified sample. The first eluate was a buffer solution (pH 7.2) containing 20 mmol / L sodium dihydrogen phosphate and 0.6 mol / L NaCl. Cation exchange chromatography was performed at room temperature (25 ° C.).

(3) Salt concentration adjustment A buffer solution (pH 7.2) containing an equal amount of 20 mmol / L sodium dihydrogen phosphate and 4 mol / L ammonium sulfate is added to the roughly purified sample, and 2 mol / L ammonium sulfate is contained as a salt. A conditioned sample was prepared. After the addition of the buffer, the prepared sample was stirred at 4 ° C. for 1 hour. Next, the prepared sample was centrifuged under the conditions of 20000 g, 4 ° C. and 20 minutes, and the resulting supernatant was recovered. Further, the supernatant was filtered through a 0.42 μm cellulose acetate membrane (manufactured by ADVANTEC) to prepare a liquid fraction of the adjusted sample.

(4) Hydrophobic interaction chromatography TOYOPEARL (registered trademark) Butyl-650M was used as a filler (second filler) used for hydrophobic interaction chromatography. The second filler was washed three times with purified water and then packed into the Econo column (second column). The second column was loaded with 200 mL of second equilibration buffer to equilibrate the column. The second equilibration buffer was a buffer solution (pH 7.2) containing 20 mmol / L sodium dihydrogen phosphate and 2 mol / L ammonium sulfate. Next, the liquid fraction of the adjusted sample was loaded on the second column. The liquid fraction obtained from the second column after loading the liquid fraction was collected as a flow-through fraction. After the loading, 800 mL of the second washing solution was loaded onto the second column and washed. The second washing solution was a buffer solution (pH 7.2) containing 20 mmol / L sodium dihydrogen phosphate and 1.8 mol / L ammonium sulfate. Furthermore, 800 mL of the second eluate was loaded onto the second column, and the entire amount of the eluted solution was obtained as purified bFGF. The second eluate was a buffer solution (pH 7.2) containing 20 mmol / L sodium dihydrogen phosphate and 1.3 mol / L ammonium sulfate. The purified bFGF was concentrated by ultrafiltration, and the concentration was adjusted to 1 mg / mL bFGF (purified sample) based on the absorbance at 280 nm. Hydrophobic interaction chromatography was performed at room temperature (25 ° C.).

(5) Purification confirmation A purified sample containing 0.5 μg of bFGF was suspended in an equal amount of sample buffer and boiled for 5 minutes. After the boiling, 2 μL of the purified sample after boiling was electrophoresed using a 17.5% SDS-PAGE gel. Next, the gel was stained for 30 minutes using a CBB staining solution. The dyed gel was decolorized with a decolorizing solution. After the decolorization, the gel was immersed in purified water and incubated for 1 hour to detect the band. In addition, in place of the purified sample, detection of bands was performed in the same manner except that the bFGF sample, the crudely purified sample, the liquid fraction of the adjusted sample, the flow-through fraction, and the recombinant human-derived bFGF were used. I did it. The sample buffer was 62.5 mmol / L Tris-HCl (pH 6.8), 2% (w / v) SDS, 25% (w / v) glycerol, 0.01% bromophenol blue and 5% (w / V) Buffer solution containing β-mercaptoethanol.

  The result is shown in FIG. FIG. 1 is a photograph showing the results of SDS-PAGE of the purified sample. In FIG. 1, the left and right sides of the photograph show the molecular weight of the molecular weight marker, and each lane shows, from the left, the recombinant human-derived bFGF (S), the molecular weight marker (M), the bFGF sample (L), and the crude purification. It is the result of the sample (C), the liquid fraction (A) of the adjusted sample, the flow-through fraction (F), the purified sample (E), and the molecular weight marker (M).

  As shown in FIG. 1, in the bFGF sample and the roughly purified sample, many bands were observed in addition to the band around about 16.5 KDa. Further, in the liquid fraction of the prepared sample, almost no bands were observed other than the band around 16.5 KDa. In contrast, a band was observed in the vicinity of about 16.5 KDa in the purified sample and the recombinant bFGF. These bands approximated the molecular weight of the bFGF 16.5 KDa isoform. Furthermore, in the flow-through fraction, a band around about 16.5 KDa was hardly observed, and it was confirmed that almost all bFGF was adsorbed on the column in the hydrophobic interaction chromatography. From these results, it was confirmed that bFGF can be purified with high yield by the purification method of the present invention.

(Example 2)
It was confirmed that impurities were precipitated by adjusting the salt concentration of the roughly purified sample.

  In the same manner as in Example 1, a prepared sample was prepared and then centrifuged. After the centrifugation, the solid fraction was collected and suspended in the sample buffer to prepare a solid fraction sample of the adjusted sample. Then, the band was detected in the same manner except that the sample of the solid fraction was used instead of the purified sample. In addition, bands were detected in the same manner except that the bFGF sample, the recombinant human-derived bFGF, the crude purified sample, and the purified bFGF were used instead of the purified sample.

  The result is shown in FIG. FIG. 2 is a photograph showing the results of SDS-PAGE of the sample of the solid fraction. In FIG. 2, the left side of the photograph shows the molecular weight of the molecular weight marker, and each lane shows, from the left, the recombinant human-derived bFGF (S), the molecular weight marker (M), the bFGF sample (L), and the crudely purified sample ( C) Results of the purified bFGF (I) and the sample (A) of the solid fraction.

  As shown in FIG. 2, a band was observed in the vicinity of about 16.5 KDa in the purified bFGF and the recombinant bFGF, and it was confirmed that bFGF could be purified by the purification method of the present invention. In addition, when the bFGF sample and the crudely purified sample were compared, the proportion of the band increased in the vicinity of about 16.5 KDa in the lane of the crudely purified sample, and it was confirmed that the crude purification was possible by cation exchange chromatography. It was. Further, when the bFGF sample, the crudely purified sample, and the solid fraction sample are compared, no band is observed in the vicinity of about 16.5 KDa in the solid fraction sample. The proportion of bands in the area increased. From these results, it was confirmed that impurities other than the bFGF were precipitated by adjusting the salt concentration of the roughly purified sample.

  Although the present invention has been described with reference to the exemplary embodiments and examples, the present invention is not limited to the above exemplary embodiments and examples. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

  According to the present invention, bFGF can be easily purified. For this reason, it can be said that the present invention is useful in, for example, the life science field and the medical field using bFGF.

Claims (14)

subjecting the sample containing bFGF to cation exchange chromatography to obtain a crude purified sample containing bFGF;
Adjusting the salt concentration of the roughly purified sample;
Subjecting the prepared crude purified sample to hydrophobic interaction chromatography to obtain purified bFGF,
The method for purifying bFGF, wherein the salt concentration in the crudely purified sample after the adjustment exceeds 1.5 mol / L. The method for purifying bFGF according to claim 1, wherein the salt concentration is 2.5 mol / L or less. The method for purifying bFGF according to claim 1 or 2, wherein the salt concentration is 1.8 to 2.5 mol / L. The method for purifying bFGF according to any one of claims 1 to 3, wherein the salt is ammonium sulfate. The purification method of bFGF as described in any one of Claim 1 to 4 whose functional group of the filler of the said cation exchange chromatography is a sulfopropyl group. The method for purifying bFGF according to any one of claims 1 to 5, wherein the hydrophobic ligand of the packing for hydrophobic interaction chromatography is a butyl group. The method for purifying bFGF according to any one of claims 1 to 6, wherein the sample containing bFGF contains a reducing agent. A step of recovering a liquid fraction from the adjusted crude purified sample,
The method for purifying bFGF according to any one of claims 1 to 7, wherein, in the step of obtaining purified bFGF, the liquid fraction is subjected to the hydrophobic interaction chromatography to obtain the purified bFGF. The method for purifying bFGF according to any one of claims 1 to 8, wherein the sample containing bFGF is a crushed E. coli expressing bFGF. The method for purifying bFGF according to any one of claims 1 to 8, wherein the sample containing bFGF is a liquid fraction of a crushed Escherichia coli expressing bFGF. culturing Escherichia coli transduced with bFGF gene and expressing bFGF in the E. coli;
The method for purifying bFGF according to any one of claims 1 to 10, further comprising a step of preparing a sample containing bFGF from the obtained Escherichia coli expressing bFGF. The method for purifying bFGF according to any one of claims 1 to 11, comprising a step of transducing the bFGF gene into E. coli. purifying purified bFGF from a sample containing bFGF,
The method for producing bFGF, wherein the purification step is performed by the method for purifying bFGF according to any one of claims 1 to 12. The method for producing bFGF according to claim 13, wherein the purity of bFGF obtained in the purification step is 96% or more.