JP6548293B2 - Method of purifying bFGF and method of producing bFGF - Google Patents

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 fibroblast growth factor. In recent years, it has been revealed that the bFGF is a factor necessary for maintaining the undifferentiated state of pluripotent stem cells such as human induced pluripotent stem cells. The demand for bFGF is expected to increase along with the development of regenerative medicine, but when purifying bFGF by column chromatography, it is necessary to carry out a large number of column chromatography, which is troublesome. For this reason, there is a need for a method for purifying bFGF that can easily purify bFGF.

  Therefore, the present invention aims 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 according to the present invention comprises the steps of: subjecting a sample containing bFGF to cation exchange chromatography to obtain a crude purified sample containing bFGF;
Adjusting the salt concentration of the crude purified sample;
Subjecting the crude purified sample after the preparation to hydrophobic interaction chromatography to obtain purified bFGF.
It is characterized in that the salt concentration in the crude purified sample after the adjustment exceeds 1.5 mol / L.

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

  According to the present invention, a simple method for purifying bFGF and a method for producing bFGF can be provided.

FIG. 1 is a photograph showing the results of SDS-PAGE of purified samples. FIG. 2 is a photograph showing the results of SDS-PAGE of a sample of 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 of the cation exchange chromatography is a sulfopropyl group.

  In the purification method of the present invention, for example, the hydrophobic ligand of the filler of the hydrophobic interaction chromatography 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 crude purified sample after the adjustment,
In the step of obtaining 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 disrupted E. coli strain expressing bFGF, or a liquid fraction of a disrupted E. coli strain expressing bFGF.

The purification method of the present invention further comprises, for example, culturing E. coli transduced with the bFGF gene, and causing the E. coli to express bFGF;
And b. Preparing a sample containing bFGF from E. coli expressing the obtained bFGF.

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

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

  In the present invention, isoforms of bFGF (basic fibroblast growth factor, FGF-2) are not particularly limited. Examples of the isoform include isoforms such as 16.5 kDa, 18 kDa, 22 kDa, 22.5 kDa, 24 kDa, 34 kDa and the like. The origin of the bFGF is not particularly limited, and examples thereof include humans and non-human animals. Examples of the non-human animals include mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, guinea pigs, cattle, pigs and the like. 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 fibroblasts are, for example, Balb / 3T3 clone A31 cells.

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

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

bFGF isoform 16.5 kDa (SEQ ID NO: 1)
MPALPEDGGSGAFPPGHFKDPKRLYCKNGGFLRI HPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNSRSYTSKYVALKRTGQYKLGSKTPGQKAILFLPMSAKS

bFGF isoform 18 kDa (SEQ ID NO: 2)
MAAGSITTLPALPEDGGGAFPPGHFKDPKRLYCKNGFGFRIHPDGR V GVREKDPPICLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERENSNNYNSRTYTSRKYVALKRTGQYKLGGTPGQQKAILFLPMSAKS

bFGF isoform 22 kDa (SEQ ID NO: 3)
LGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALPEDGSGAFFPPGHFKDPKRLYCKGLFLIPHDGRDVRVRAEDPDPLEQLQAEERGVVSIKGKCANRYL MAKECGRAGSCECFFFELLES NNY RSRKYTSWYVALKRTQQYKLGSKTG

bFGF isoform 22.5 kDa (SEQ ID NO: 4)
LPGGRLGGGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMA AG SITTLAPPALPED GSGFPFPHFKDPKRLYCKGLFRI HPDGRDVRQDLEQDQLQQ AEERG VVS IKGG CANRYL MAKED GRL LAS CLT CFF FER NSONYRSRKYTSWYVALKRTGQYGPLCIG

bFGF isoform 24 kDa (SEQ ID NO: 5)
LGDRGRGRALPGGRLGRAGRVGRAGRGGRAPERVGGRGRGTAAPRAAGAGAGAGTAGPAAGSITTLPALPEDGGGAFPPGHFKDPKRLYCKGLFRIKDVRDVREKDPDQLQLQQAEG LQ MQRY LY MAKEDGRL CLK VT LES FF LG LG LG LG LG LG LG NT NT NT NT NT NT LG LG LG LG LG

bFGF isoform 34 KDa (SEQ ID NO: 6)
A portion is a part of the UT UT, A, A, A, A, A, A, A, A, A, A, A, B IV UT, A, A, A, A, A, B IV UT UT;

  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 by default parameters using, for example, analysis software such as BLAST and FASTA (the same applies hereinafter).

  The bFGF sample may include the bFGF. Examples of the bFGF sample include transformants transduced with bFGF, cells expressing bFGF, tissues, organs, cultured cells and the like. The host for producing the transformant is not particularly limited, and examples thereof include microorganisms, animal cells, insect cells, non-human hosts such as cultured cells thereof, isolated human cells or cultured cells thereof and the like. can give. Specifically, examples of the transformant include cells transduced with bFGF or cultured cells, and E. coli transduced with bFGF. The bFGF sample is, for example, preferably a crushed product such as the transformant, since the recovery rate of bFGF can be improved, and a liquid fraction of the crushed product such as the transformant is more preferable. As a specific example, the bFGF sample may be, for example, a disrupted product of bFGF-expressing E. coli (bFGF-expressing E. coli), preferably a liquid fraction of the bFGF-expressing E. coli disrupted product. The bFGF sample may contain, for example, one bFGF, or two or more bFGFs.

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

  The bFGF sample preferably contains a reducing agent, for example, because it can maintain the activity of the bFGF at the time of purification. 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 type may be used alone, or two or more types 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, for example, in an equilibration solution, a washing solution, and an elution solution described later.

  The salt is not particularly limited, and examples thereof include salts usable for salting out. Examples of the salt include citrate, tartrate, sulfate, acetate, chloride and the like. Specific examples of the salt include sodium chloride, ammonium sulfate and the like, with preference given to ammonium sulfate. The salts may be used alone or in combination of two or more.

  The packing material for the cation exchange chromatography (first packing material) is not particularly limited, and known packing materials for cation exchange chromatography can be used. The first filler contains, 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 (eg, sepharose), silica, dextran, polymer resin and the like. 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 phosphoric acid group, a sulfo group, a sulfomethyl group, a sulfoethyl group, a sulfopropyl group and the like, 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), etc. Can be mentioned.

  The hydrophobic interaction chromatography filler (second filler) is not particularly limited, and known fillers for hydrophobic interaction chromatography can be used. The second filler includes, for example, a carrier and a hydrophobic ligand. As the carrier, for example, the description of the carrier in the above-mentioned first filler can be used. The hydrophobic ligand is not particularly limited. For example, a linear or branched alkyl group such as methyl group, ethyl group, propyl group, isopropyl group, butyl group, tert-butyl group, hexyl group, octyl group, phenyl group And an aryl group such as polypropylene glycol group, and the like, and preferably a 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 Corp.), Butyl-S Sephalose Fast Flow (manufactured by GE Healthcare) Etc.).

  Hereinafter, each step of the purification method of the present invention will be described. The reagents and the like used in each step may be, for example, reagents not containing animal-derived components, so-called animal free grade reagents.

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

  The bFGF-transduced E. coli can be obtained, for example, by transducing the bFGF gene into E. coli. Therefore, the purification method of the present invention may include, for example, the step of transducing the bFGF gene into the E. coli.

  The isoform of bFGF encoded by the bFGF gene is not particularly limited, and may be, for example, any isoform of bFGF described above. 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 the following (b1) to (b6) and (b7). In the following (b1) to (b6) and (b7), the fibroblasts are, for example, Balb / 3T3 clone A31 cells.

(B1) A polynucleotide consisting of 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 comprising an inserted and / or added nucleotide sequence and encoding a protein having a fibroblast proliferation activity (b3) consisting of a nucleotide sequence having a predetermined identity to the nucleotide sequence of (b1) above A polynucleotide encoding a protein having a proliferative activity of fibroblasts (b4) a nucleotide sequence complementary to a polynucleotide hybridizing under stringent conditions with a polynucleotide consisting of the nucleotide 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, Polynucleotide (b7) consisting of SEQ ID NOs: 1 to 5 consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added and which has a fibroblast proliferation activity A polynucleotide comprising an amino acid sequence having a predetermined identity to at least one amino acid sequence selected from the group, and encoding a protein having fibroblast proliferation activity

  The nucleotide 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 the coding sequences of the bFGF isoform consisting of the amino acid sequences of SEQ ID NOs: 1 to 5 and 6, respectively. The above (b1) can be obtained, for example, from human.

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

  In the above (b3), the “predetermined identity” may be, for example, a range in which the protein encoded by the polynucleotide of the above (b3) has the proliferation activity of the fibroblasts. 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, relative to the base sequence of (b1). It is 99% or more.

In the above (b4), the "hybridizable polynucleotide" is, for example, a polynucleotide which 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 The methods described in Press (1989)] can also be adopted.

In the above (b4), the "stringent conditions" may be, for example, low stringency conditions, medium stringency conditions, or high stringency conditions. "Low stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, 32 ° C. The "moderately stringent conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, 42 ° C. “Highly stringent conditions” are, for example, 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, for example, by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, and time. "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)] can also be adopted.

  The polynucleotide (b5) may be, for example, a base sequence in which a protein encoded by the polynucleotide (b5) has a proliferation activity of the fibroblast. 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” relating to the amino acid sequence may be, for example, a range in which the protein encoded by the polynucleotide of the above (b6) has the proliferation activity of the fibroblast. The “one 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, for example. 1 to 12, 1 to 9, 1 to 6, 1 to 3, 1 or 2;

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

  The method for introducing the bFGF gene into E. coli is not particularly limited, and can be carried out by a known method. The introduction method includes, for example, introduction method by 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. Direct injection method using an aqueous solution, cationic liposome method, a method using a transfer aid, and the like.

  The bFGF gene may be introduced into the E. coli by, for example, the 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 known vectors can be used. The vector preferably has, for example, a regulatory sequence. Examples of the regulatory sequences include promoters, terminators, enhancers, polyadenylation signal sequences, replication origin sequences (ori) and the like. When transformation is performed on bacteria such as E. coli, examples of the vector include pETDuet-1 vector (Novagen), pET-3a (Novagen), pQE-80L vector (QIAGEN), etc.

  The culture of E. coli can be performed, for example, by culturing in a medium. As the medium used for culturing the above-mentioned E. coli, conventionally known media such as solid media and liquid media can be used appropriately. The medium may include, for example, a commercially available medium. The commercially available medium is not particularly limited, and examples thereof include LB medium, super broth medium, M9 medium and the like. One of these may be used alone, or two or more may be used in combination. The pH of the medium is not particularly limited, and is, for example, pH 6 to 8, pH 6.5 to 7.5. The medium may contain, for example, the above-mentioned bFGF expression inducer. The expression-inducing agent is not particularly limited, and can be appropriately determined according to the promoter of the bFGF gene. As a specific example, when using a lac promoter as the said promoter, the said expression inducer can use isopropyl- (beta)-thiogalactopyranoside (IPTG) etc., for example.

  The method for culturing the above-mentioned E. coli is not particularly limited, and when the solid medium is used, for example, a plate culture method, a slope culture method and the like can be mentioned. Moreover, when using the said liquid culture medium, it does not restrict | limit in particular, For example, a stationary culture method, aeration culture method, a shaking culture method etc. are mention | raise | lifted.

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 preparation method of the sample containing the bFGF is not particularly limited. In the preparation method, for example, the bFGF-expressing E. coli may be prepared as the bFGF sample, or a fragment obtained by crushing the bFGF-expressing E. coli may be prepared as the bFGF sample, or The liquid fraction of the debris may be prepared as the bFGF sample. The crushing method of the bFGF-expressing E. coli is not particularly limited, and known crushing methods can be used, for example, crushing methods such as crushing with glass beads, French press, sonication, lysonium treatment, freeze-thaw treatment and the like. When the liquid fraction is collected, the method of collecting the liquid fraction is not particularly limited, and for example, a known solid-liquid separation method may be performed to collect the obtained liquid fraction. Examples of the solid-liquid separation method include treatment methods such as filtration treatment, centrifugation treatment, precipitation treatment, membrane separation treatment, adsorption treatment, freeze-drying treatment and the like. The bFGF-expressing E. coli may contain, for example, a medium in which the E. coli has been 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, in the crude purification step, for example, the bFGF sample is brought into contact with the first filler, and the bFGF and the first filler are combined; And washing the bound product with the first filler, and dissociating the bFGF from the bound product with the first eluate to obtain a crude purified sample containing bFGF.

  The contact between the bFGF sample and the packing material can be performed, for example, by packing the first packing material into a first column and loading the bFGF sample into the first column. The volume, material, and the like of the first column are not particularly limited, and can be determined as appropriate according to, for example, the type, filling amount, and the like of the first filler. The loading amount of the first filler to be loaded 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. A first column containing the first packing may, for example, equilibrate the first packing with a first equilibration solution prior to loading of the bFGF sample. The equilibration is performed using, for example, the first equilibration solution at 2 to 10 ° C. and 4 to 8 ° C. under room temperature (eg, around 25 ° C.) conditions. The said equilibration liquid can be suitably determined, for example according to the kind of said 1st filler. The pH of the first equilibration solution is, for example, pH 6 to 8, pH 7 to 7.5. The first equilibration solution is, for example, a buffer, preferably a phosphate buffer. The concentration of the buffer solution is, for example, 0.01 to 0.1 mol / L and 0.01 to 0.05 mol / L. The first equilibration solution may contain, for example, the salt. The salt is, for example, sodium chloride. The salt concentration in the first washing solution is, for example, 0.1 to 0.4 mol / L and 0.2 to 0.4 mol / L.

  After loading the bFGF sample, for example, the first washing solution is loaded on the first column to wash the conjugate of the bFGF and the first filler. The washing is performed using, for example, the first washing solution at 2 to 10 ° C. and 4 to 8 ° C. under room temperature (eg, around 25 ° C.) conditions. The first cleaning solution can be appropriately determined, for example, according to the type of the first filler. The pH of the first washing solution is, for example, pH 6 to 8, pH 7 to 7.5. The first washing solution is, for example, a buffer, preferably a phosphate buffer. The number of moles of the buffer solution is, for example, 0.01 to 0.1 mol / L and 0.01 to 0.05 mol / L. The first cleaning solution may contain, for example, the salt. The salt is, for example, sodium chloride. The salt concentration in the first washing solution may be, for example, the same concentration as the salt concentration in the first equilibration solution, and 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, and the bFGF is dissociated from the conjugate to obtain a crude purified sample containing bFGF. The crudely purified sample may obtain, for example, part or all of the solution eluted with the first eluate. 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 crude purified sample. The crude purified sample is obtained, for example, at room temperature (eg, around 25 ° C.) using the first eluate at 2 to 10 ° C. and 4 to 8 ° C. The first eluate can be appropriately determined, for example, according to the type of the first filler. The pH of the first eluate is, for example, pH 6 to 8, pH 7 to 7.5. The first eluate is, for example, a buffer, preferably a phosphate buffer. The number of moles of the buffer solution is, for example, 0.01 to 0.1 mol / L and 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, a concentration higher than the salt concentration in the first washing solution, specifically, 0.5 to 2 mol / L, 0.6 to 0.8 mol / L. is there.

  Next, in the salt concentration adjusting step, as described above, the salt concentration of the crudely purified sample is adjusted. The adjustment is not particularly limited, and can be performed by, for example, addition of the salt to the crude purified sample, dilution of the crude purified sample, and 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 adjusting step, or the concentration of the salt derived from both the salt present in the crude purified sample before the adjustment and the salt added in the adjustment. May be. The salt concentration in the adjusted sample may be more than 1.5 mol / L, for example, preferably 2.5 mol / L or less, because the contaminants can be efficiently precipitated and bFGF can be purified with higher yield. More than 1.5 mol / L, and more preferably 2.5 mol / L or less, because it is 2.3 mol / L or less, the impurities can be deposited more efficiently, and the bFGF can be purified with a higher yield. It is 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 concentration. As a specific example, when ammonium sulfate is added as the salt, the concentration of ammonium sulfate in the adjustment sample is, for example, 1.8 to 2.3 mol / L, 1.8 to 2.1 mol / L, 1.8 to 2 mol / l. L Moreover, when the adjustment sample contains sodium chloride and ammonium sulfate as the salt, the concentration of sodium chloride in the adjustment sample is, for example, 0.2 to 0.4 mol / L, and the concentration of ammonium sulfate is, for example, It is 8-2.3 mol / L, 1.8-2.1 mol / L, 1.8-2 mol / L.

  Next, in the bFGF acquisition step, as described above, the adjusted sample is subjected to hydrophobic interaction chromatography to obtain purified bFGF. Specifically, in the bFGF obtaining step, for example, the step of bringing the adjusted sample into contact with the second filler to bind bFGF with the second filler, and using a second washing solution with the bFGF. And washing the conjugate with the second filler, and dissociating the bFGF from the conjugate with the second eluate to obtain purified bFGF.

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

  After loading the conditioned sample, for example, the second washing solution is loaded on the second column to wash the conjugate of the bFGF and the second filler. The washing is performed using, for example, the second washing solution at 10 to 30 ° C. and 15 to 25 ° C. under room temperature (eg, around 25 ° C.) conditions. The second cleaning solution can be appropriately determined, for example, according to the type of the second filler. The pH of the second washing solution is, for example, pH 6 to 8, pH 7 to 7.5. The second washing solution is, for example, a buffer, preferably a phosphate buffer. The number of moles of the buffer solution is, for example, 0.01 to 0.1 mol / L and 0.01 to 0.05 mol / L. The second cleaning solution may contain, for example, the salt. The salt is, for example, ammonium sulfate. The salt concentration in the second washing solution is not particularly limited, and is, for example, a salt concentration equal to or less than the salt concentration of the second equilibration solution, specifically, more than 1.5 mol / L, 2.3 mol / L or less, 1.8 to 2.3 mol / L, and 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 conjugate to obtain a purified bFGF. The purified bFGF may, for example, acquire a part of the solution eluted with the second eluate or may acquire the whole. In the former case, for example, the solution eluted with the eluate is fractionated into a plurality of fractions, and the fraction containing the bFGF is obtained as the purified bFGF. The purified bFGF is obtained, for example, at room temperature (eg, around 25 ° C.) using the second eluate at 10 to 30 ° C. and 15 to 25 ° C. The second eluate can be appropriately determined, for example, according to the type of the second filler. The pH of the second eluate is, for example, pH 6 to 8, pH 7 to 7.5. The second eluate is, for example, a buffer, preferably a phosphate buffer. The number of moles of the buffer solution is, for example, 0.01 to 0.1 mol / L and 0.01 to 0.05 mol / L. The second eluate may contain, for example, the salt. 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 washing solution, specifically, 0.9 to 1.4 mol / L, 1 to It is 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, for example, by SDS-PAGE, reverse phase high performance liquid chromatography (RP-HPLC), mass spectrometry and the like. When measuring purity using said SDS-PAGE, it can calculate, for example by densitometry. Moreover, when measuring purity using said RP-HPLC and said mass spectrometry, it can calculate based on the integral value (peak area) of the obtained chromatograph chart, for example.

  In the purification method of the present invention, in the adjusted sample, for example, contaminants other than bFGF contained in the crude 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, since the purity of the bFGF can be further increased, and in the step of obtaining the purified bFGF, the liquid fraction It may be subjected to hydrophobic interaction chromatography to obtain the purified bFGF. The method for recovering the liquid fraction from the adjusted sample is not particularly limited, and, for example, the description of the method for recovering the liquid fraction described above can be used. In addition, 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>
The method for producing bFGF of the present invention (hereinafter also referred to as "production method") includes the step of purifying purified bFGF from a sample containing bFGF as described above, and the purification step is the step of purifying bFGF of the present invention It is characterized in that it is 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 the other steps and conditions are not particularly limited. According to the production method of the present invention, it is possible to easily produce purified bFGF. In the production method of the present invention, the description of the purification method of the present invention can be used, for example, as the purification step.

  In the production method of the present invention, the purity of bFGF obtained in the purification step is not particularly limited. For example, the above description can be used as the purity and the method of measuring the purity.

  Below, the Example of this invention is described. The present invention is not limited by the following examples.

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

(1) Preparation of bFGF sample After amplification of 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 using human derived cDNA as a template, lac promoter The vector was ligated to a 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 competitor E. Coli (High Efficiency), NEW ENGLAND Labs Inc.) by the heat shock method. The resulting transformants were cultured at 37 ° C. using LB medium containing ampicillin until OD ₆₀₀ = 0.6 or more. Furthermore, IPTG was added to 0.2 mmol / L and cultured at 37 ° C. for 3 hours. Cultured E. coli was harvested, suspended in lysis buffer and sonicated. The ultrasonication was performed twice on ice for 10 minutes by using an ultrasonic crusher (UD-201, manufactured by TOMY) under the condition of output 6 and a duty ratio of 80%. In addition, the lysis buffer may be a buffer solution (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. Then, centrifugation was performed under the conditions of 20000 g and 4 ° C. for 20 minutes, and the obtained supernatant was recovered. Furthermore, the bFGF sample was prepared by filtering the supernatant through a DISMIC 0.8 μm cellulose acetate membrane (manufactured by ADVANTEC).

(2) Cation Exchange Chromatography TOYOPEARL (registered trademark) SP-550C was used as a packing material (first packing material) used for cation exchange chromatography. 100 mL of the first filler was washed three times with purified water, and then loaded on an Econo column (5.0 × 10 cm, manufactured by 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 (pH 7.2) containing 20 mmol / L sodium dihydrogen phosphate. The first column was then loaded with the bFGF sample. After the loading, 800 mL of the first wash was loaded onto the first column and washed. The first washing solution was a buffer (pH 7.2) containing 20 mmol / L sodium dihydrogen phosphate and 0.2 mol / L NaCl. Furthermore, 800 mL of the first eluate was loaded on the first column, and the total volume of the eluted solution was obtained as the crude purified sample. The first eluate was a buffer (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) Adjustment of salt concentration A buffer solution (pH 7.2) containing equal amounts of 20 mmol / L sodium dihydrogen phosphate and 4 mol / L ammonium sulfate is added to the crude purified sample, and 2 mol / L ammonium sulfate is contained as a salt. A conditioned sample was prepared. After addition of the buffer, the conditioned sample was stirred at 4 ° C. for 1 hour. Next, the conditioned sample was centrifuged at 20000 g at 4 ° C. for 20 minutes, and the obtained supernatant was recovered. Furthermore, 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 As a filler (second filler) used for hydrophobic interaction chromatography, TOYOPEARL® Butyl-650M was used. The second filler was washed three times with purified water, and then packed in the Econo column (second column). The second column was loaded with 200 mL of a second equilibration buffer to equilibrate the column. The second equilibration buffer was a buffer (pH 7.2) containing 20 mmol / L sodium dihydrogen phosphate and 2 mol / L ammonium sulfate. Next, the second column was loaded with the liquid fraction of the conditioned sample. The liquid fraction obtained from the second column after loading of the liquid fraction was collected as a flow-through fraction. After the loading, 800 mL of the second wash 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 on the second column, and the whole of the eluted solution was obtained as purified bFGF. The second eluate was a buffer (pH 7.2) containing 20 mmol / L sodium dihydrogen phosphate and 1.3 mol / L ammonium sulfate. Then, after concentration by ultrafiltration, the concentration of the purified bFGF was adjusted to 1 mg / mL bFGF (purified sample) based on the absorbance at 280 nm. The 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 volume of sample buffer and boiled for 5 minutes. After the boiling, 2 μL of the boiled purified sample was electrophoresed using a 17.5% SDS-PAGE gel. Next, the gel was stained for 30 minutes using a CBB staining solution. The stained gel was decolorized with a decolorizing solution. After the color removal, the gel was immersed in purified water and incubated for 1 hour to detect a band. Also, in place of the purified sample, detection of bands is similarly performed except that the bFGF sample, the crude purified sample, the liquid fraction of the adjusted sample, the flow-through fraction, and the recombinant human-derived bFGF are used. I did. The sample buffer comprises 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 containing β-mercaptoethanol.

  The results are shown in FIG. FIG. 1 is a photograph showing the results of SDS-PAGE of the purified sample. In FIG. 1, the left side and the right side 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), the crude purification It is a result of a sample (C), a liquid fraction (A) of the said adjustment sample, the said flow through fraction (F), the said purification sample (E), and a molecular weight marker (M).

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

(Example 2)
Adjustment of the salt concentration of the crudely purified sample confirmed that impurities were precipitated.

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

  The results are shown in FIG. FIG. 2 is a photograph showing the results of SDS-PAGE of a 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), the crude purified sample ( C) Results of the purified bFGF (I) and the sample of the solid fraction (A).

  As shown in FIG. 2, in the purified bFGF and the recombinant bFGF, bands were observed around about 16.5 KDa, confirming that the bFGF can be purified by the purification method of the present invention. In addition, when the bFGF sample and the crude purified sample are compared, the ratio of the band occupied in the vicinity of about 16.5 KDa in the lane of the crude purified sample is increased, and it is confirmed that the crude purification can be performed by cation exchange chromatography. The Furthermore, when the bFGF sample, the crude purified sample, and the sample of the solid fraction are compared, no band is observed around about 16.5 KDa in the sample of the solid fraction; The proportion of bands in the region of From these things, it was confirmed that impurities other than the bFGF are precipitated by adjusting the salt concentration of the crude purified sample.

  Although the present invention has been described above with reference to the embodiments and the examples, the present invention is not limited to the above embodiments and the examples. The configurations and details of the present invention can be modified in various ways that can be understood by those skilled in the art within the scope of the present invention.

  According to the present invention, bFGF can be purified conveniently. Therefore, the present invention can be said to be useful, for example, in the life science field using bFGF, the medical field, and the like.

Claims (12)

subjecting the sample containing bFGF to cation exchange chromatography to obtain a crude purified sample containing bFGF;
Adjusting the salt concentration of the crude purified sample;
Subjecting the crude purified sample after the preparation to hydrophobic interaction chromatography to obtain purified bFGF.
The concentration of ammonium sulfate in crude samples after adjustment, characterized in that it is a 2 mol / L, the purification method of bFGF. The method for purifying bFGF according to claim 1, wherein the concentration of sodium chloride in the crude purified sample after adjustment is 0.3 mol / L. The purification method of bFGF according to claim 1 or 2 , wherein the functional group of the cation exchange chromatography filler is a sulfopropyl group. The purification method of bFGF according to any one of claims 1 to 3 , wherein the hydrophobic ligand of the filler of the hydrophobic interaction chromatography is a butyl group. The method for purifying bFGF according to any one of claims 1 to 4 , wherein the sample containing bFGF contains a reducing agent. Recovering the liquid fraction from the crude purified sample after the adjustment;
The method for purifying bFGF according to any one of claims 1 to 5 , wherein in the step of obtaining the 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 6 , wherein the sample containing bFGF is a disrupted E. coli expressing bFGF. The method for purifying bFGF according to any one of claims 1 to 6 , wherein the sample containing bFGF is a liquid fraction of a disrupted E. coli expressing bFGF. culturing E. coli transduced with the bFGF gene, and expressing the bFGF in the E. coli;
And b. Preparing a sample containing bFGF from E. coli expressing the obtained bFGF, and purifying the bFGF according to any one of claims 1 to 8 . The method for purifying bFGF according to any one of claims 1 to 9 , comprising the step of transducing bFGF gene into E. coli. obtaining a purified bFGF from a sample containing bFGF,
A method for producing bFGF, wherein the purification step is carried out by the method for purifying bFGF according to any one of claims 1 to 10 . The method for producing bFGF according to claim 11 , wherein the purity of bFGF obtained in the purification step is 96% or more.