Method for producing basic fibroblast growth factor using plants

Abstract

To provide methods for producing bFGF in a large amount and inexpensively using transient expression by plants or cultured plant cells, where the quality of the bFGF is usable in cell culture for regenerative medicine.SOLUTION: Disclosed is a method for producing a basic fibroblast growth factor comprising the following steps (1)-(3): (1) carrying out agroinfiltration of a plant or infection of a cultured plant cell with an Agrobacterium transformant having an introduced expression vector comprising a DNA encoding a fusion protein between a signal peptide and a basic fibroblast growth factor; (2) cultivating the agroinfiltrated plant or culturing the cultured plant cell; and (3) purifying the basic fibroblast growth factor from the plant or cultured plant cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a basic fibroblast growth factor using a plant. [Background technology]

[0002] Basic fibroblast growth factor (bFGF) is a member of the fibroblast growth factor (FGF) family and is also known as FGF-2. bFGF was discovered as a factor that promotes the proliferation of fibroblasts, and subsequent research has revealed that it is a growth factor involved in angiogenesis, wound healing, fetal development, etc. In recent years, it has also become clear that bFGF is a factor necessary for maintaining the undifferentiated state of pluripotent stem cells such as human induced pluripotent stem cells (human iPS cells).

[0003] For cell culture applications in regenerative medicine, a production system that is free of pathogenic and toxic substances is desired. Until now, animal cells and E. coli have been used to produce growth factors related to regenerative medicine, but production using animal cells has the problem of contamination by viruses that may be harmful to humans, and E. coli has the problem of post-translational modifications such as sugar chains not occurring properly.

[0004] Regarding the production of recombinant bFGF in plants, a method for producing it from genetically modified rice seeds has been reported (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] An N et al., Expression of a functional recombinant human basic fibroblast growth factor from transgenic rice seeds, Int. J. Mol. Sci. 2013, 14(2), 3556-3567 Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present invention is to provide a method for mass-produced, inexpensively, bFGF of a quality suitable for use in cell culture for regenerative medicine, by transient expression in plants or cultured plant cells. [Means for solving the problem]

[0007] In order to solve the above problems, the present invention includes the following inventions. [1] A method for producing a basic fibroblast growth factor, comprising the following steps (1) to (3): (1) agroinfiltrating an Agrobacterium transformant into a plant or infecting a plant culture cell with an expression vector containing DNA encoding a fusion protein of a signal peptide and a basic fibroblast growth factor, (2) cultivating the agroinfiltrated plant or culturing the infected plant culture cell, and (3) purifying the basic fibroblast growth factor from the plant or plant culture cell. [2] The method for producing according to [1] above, wherein the signal peptide is a signal peptide of human glucocerebrosidase. [3] The method according to [1] or [2] above, wherein the basic fibroblast growth factor is human basic fibroblast growth factor. [4] The method according to any one of [1] to [3] above, wherein the basic fibroblast growth factor is human basic fibroblast growth factor for xeno-free medium. [5] The method according to any one of [1] to [4] above, wherein in step (3), crude extraction is performed, followed by cation exchange chromatography and then affinity chromatography. [6] The method according to [5] above, wherein the crude extraction comprises ultrasonic treatment. [7] The method for producing a cation exchange column according to [5] or [6], further comprising washing the column with a volume of liquid at least twice the column volume after the crude extract is passed through the column and until all the green residue is washed away or until unnecessary cell-derived proteins are washed away. [8] The method according to any one of the above [5] to [7], wherein the cation exchange chromatography is sulfopropyl cation exchange chromatography. [9] The method according to any one of [5] to [8] above, wherein the affinity chromatography is heparin affinity chromatography.

[10] A method for purifying a basic fibroblast growth factor expressed in a plant, comprising the following steps (i) to (iii): (i) performing crude extraction; (ii) performing cation exchange chromatography; and (iii) performing affinity chromatography.

[11] The method according to

[10] above, further comprising ultrasonicating the crude extract in step (i).

[12] The method for producing a medicament according to

[10] or

[11] , further comprising washing the column with a volume of liquid at least twice the column volume after the crude extract is passed through the column in step (ii) until all the green residue has been washed away or until unnecessary cell-derived proteins have been washed away.

[13] The process according to any one of the above

[10] to

[12] , wherein in the step (ii), the cation exchange chromatography is sulfopropyl cation exchange chromatography.

[14] The method according to any one of

[10] to

[13] above, wherein in the step (iii), the affinity chromatography is heparin affinity chromatography.

[0101] A method for producing a basic fibroblast growth factor, comprising the following steps (1) to (3): (1) agroinfiltration into a plant of an Agrobacterium transformant into which an expression vector containing DNA encoding a fusion protein of a signal peptide and a basic fibroblast growth factor has been introduced, (2) cultivation of the agroinfiltrated plant, and (3) purification of the basic fibroblast growth factor from the plant.

[0102] A method for producing basic fibroblast growth factor, comprising the following steps (1) to (3): (1) infecting cultured plant cells with an Agrobacterium transformant into which an expression vector containing DNA encoding a fusion protein of a signal peptide and basic fibroblast growth factor has been introduced, (2) culturing the infected cultured plant cells, and (3) purifying basic fibroblast growth factor from the cultured plant cells. Effect of the Invention

[0008] The present invention provides a method for mass-producing bFGF at low cost and of a quality suitable for cell culture in regenerative medicine. The production method of the present invention can be carried out using a transient expression system in plants or plant culture cells without using genetically modified plants. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the structure of the bFGF expression vector pFK1-GCsp-bFGF. [Diagram 2] FIG. 1 shows the results of examining the effect of ultrasonic treatment of a crude extract on the yield of bFGF. [Diagram 3] FIG. 1 shows the results of examining the change in bFGF yield when a bFGF expression vector having a base sequence in which the codons of the human bFGF gene have been optimized to those of Nicotiana benthamiana, and when a bFGF expression vector having a base sequence of the human bFGF gene in which the codons have not been modified, is used. [Figure 4]FIG. 1 shows the results of SDS-PAGE of recombinant human bFGF in the final eluate obtained by purifying the crude extract through a two-stage purification process using sulfopropyl cation exchange chromatography and heparin affinity chromatography. [Diagram 5] FIG. 2 shows the results of a cell proliferation assay in which recombinant human bFGF produced by the production method of the present invention was added to the medium of CHO-K1 cells. [Figure 6] FIG. 2 shows the results of a cell proliferation assay in which recombinant human bFGF produced by the production method of the present invention was added to the culture medium of Hep-G2 cells. [Figure 7] FIG. 1 shows the results of investigating the recovery of bFGF using Triton X-100, Tween 20 and NP-40 as surfactants in the extraction buffer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] [Method for producing basic fibroblast growth factor using plants] The present invention provides a method for producing a basic fibroblast growth factor using a plant. The production method of the present invention may include the following steps (1) to (3): (1) A step of agroinfiltration into a plant or infection into a plant culture cell with an Agrobacterium transformant into which an expression vector containing DNA encoding a fusion protein of a signal peptide and basic fibroblast growth factor has been introduced. (2) Cultivating the agroinfiltrated plants or culturing the infected plant cell cultures. (3) Purifying basic fibroblast growth factor from a plant or cultured plant cells.

[0011] The production method of the present invention can produce basic fibroblast growth factor (hereinafter referred to as "bFGF") from various animals. The bFGF produced by the production method of the present invention may be bFGF from a mammal. Examples of mammals include, but are not limited to, humans, mice, rats, cows, pigs, etc. The bFGF produced by the production method of the present invention may be human bFGF.

[0012] The amino acid sequence information of bFGF of various animals and the nucleotide sequence information of the bFGF gene can be obtained from known databases (GenBank / EMBL / DDBJ, etc.). For example, an amino acid sequence of human bFGF is the amino acid sequence shown in SEQ ID NO: 1 (NCBI ACCESSION: NP_001348594.1). An example of the nucleotide sequence of the gene encoding human bFGF consisting of the amino acid sequence shown in SEQ ID NO: 1 is the nucleotide sequence shown in SEQ ID NO: 2 (345th to 812th in NCBI ACCESSION: NM_001361665.2). However, the nucleotide sequence is not limited to SEQ ID NO: 2, and a nucleotide sequence with modified codons may be used as long as it encodes the amino acid sequence shown in SEQ ID NO: 1. The present inventors have confirmed that even if the codons are modified, a recombinant human bFGF can be obtained with an expression level comparable to that of a human bFGF gene with no modified codons (see Examples).

[0013] Step (1) is a step of agroinfiltration into plants or infection into plant culture cells of Agrobacterium transformants into which an expression vector containing DNA encoding a fusion protein of a signal peptide and bFGF has been introduced. Since bFGF is a protein that does not have a signal peptide, it is expressed as a fusion protein with a signal peptide of a different protein. As the signal peptide, a signal peptide targeting the endoplasmic reticulum, a signal peptide targeting the chloroplast, a signal peptide targeting the peroxisome, a signal peptide targeting the mitochondria, a signal peptide targeting the plastid, etc. can be suitably used. For example, the signal peptide of a plant pathogen-induced protein, the signal peptide of a plant pathogen-induced protease, the signal peptide of a mammalian antibody, the signal peptide of rice-derived alpha-amylase, the signal peptide of rice-derived glutelin, the signal peptide of human glucocerebrosidase, the C-terminal KDEL sequence (SEQ ID NO: 3), etc. are included. The signal peptide of human glucocerebrosidase is preferable.

[0014] The amino acid sequence information and nucleotide sequence information of the signal peptide can be obtained from known databases (GenBank / EMBL / DDBJ, etc.). For example, the signal peptide of human glucocerebrosidase consists of the amino acid sequence shown in SEQ ID NO: 4 (1st to 39th in NCBI ACCESSION: BAA02545.1). An example of the nucleotide sequence of the gene encoding the signal peptide is the nucleotide sequence shown in SEQ ID NO: 5 (106th to 222nd in NCBI ACCESSION: D13286.1).

[0015] Depending on the signal peptide to be used, the signal peptide is linked to the N-terminus or C-terminus of bFGF. A DNA encoding a fusion protein of a signal peptide and bFGF can be prepared using known gene recombination techniques based on the above sequence information.

[0016] The expression vector is constructed to contain a promoter that enables expression in plant cells and DNA encoding a fusion protein of the signal peptide and bFGF. In the production method of the present invention, a binary vector or intermediate vector containing T-DNA derived from Ti plasmid or Ri plasmid of Agrobacterium can be used to introduce the expression vector into Agrobacterium. The binary vector containing T-DNA may be a pBI binary vector. Examples of pBI binary vectors include pBIG, pBIN19, pBI101, and pBI121.

[0017] Examples of promoters capable of expressing a gene introduced into a plant cell include the cauliflower mosaic virus 35S promoter (CaMV35S), various actin gene promoters, various ubiquitin gene promoters, nopaline synthase gene promoter, tobacco PR1a gene promoter, napin gene promoter, and oleosin gene promoter.

[0018] The expression vector may further contain other DNA segments. The other DNA segments are not particularly limited, but examples thereof include terminators, selection markers, enhancers, and the like. The transcription terminator is not particularly limited as long as it has a function as a transcription termination site, and known terminators can be suitably used. For example, a terminator derived from the HSP (Heat Shock Protein) gene can be used. For example, a drug resistance gene can be used as a transformant selection marker. Specific examples include drug resistance genes against hygromycin, bleomycin, kanamycin, gentamicin, chloramphenicol, and the like. To introduce an expression vector into Agrobacterium, known methods such as the calcium phosphate method, lipofection method, and electroporation method can be used.

[0019] For agroinfiltration, Agrobacterium transformants into which an expression vector for a fusion protein of a signal peptide and bFGF has been introduced can be suspended in a buffer solution suitable for infiltration into plant tissue. The turbidity of the suspension of the Agrobacterium transformants may be OD600 of 0.05 to 5, 0.1 to 2, or 0.2 to 1. The suspension of the Agrobacterium transformants may be composed of a solution containing a buffer solution whose components have been appropriately examined.

[0020] The plant to be used is not particularly limited as long as it is a plant that can be infected with Agrobacterium and can express bFGF, for example, Solanaceae plants such as tobacco, potato, tomato, etc., Brassicaceae plants such as arugula, komatsuna, mizuna, mustard, thailand purse, etc., Asteraceae plants such as chicory, endive, artichoke, etc., Leguminous plants such as alfalfa, mung bean, soybean, etc., Chenopodiaceae plants such as spinach, sugar beet, etc., Lamiaceae plants such as perilla, basil, etc., Umbelliferae plants such as mitsuba, etc., and Poaceae plants such as rice, wheat, barley, corn, etc.

[0021] Among them, solanaceae plants are preferred, and tobacco is more preferred. Examples of tobacco include Nicotiana tabacum, N. benthamiana, N. alata, N. glauca, N. longiflora, N. persica, N. rustica, and N. sylvestris, and N. benthamiana is preferred. Glycosylation-modified N. benthamiana is more preferred (Strasser R et al., Plant Biotechnol J. 2008 May;6(4):392-402).

[0022] The agroinfiltration method is well known in the art, and in the production method of the present invention, an appropriate agroinfiltration method can be selected and used from among known agroinfiltration methods.

[0023] The plant cultured cells may be cultured cells prepared from the above plants by known methods, such as the method described in "Mutation and Selection of Plant Cultured Cells (Biotechnology Series), April 1, 1985, edited by Yamada Yasuyuki, Kodansha, ISBN-10: 4061390058" and the method described in "Plant Biotechnology (Modern Chemistry, Supplementary Edition 5), April 25, 1986, edited by Yamada Yasuyuki and Okada Yoshimi, Tokyo Kagaku Dojin, ISBN-10: 4807902644".

[0024] The method of infecting a plant culture cell with an Agrobacterium transformant is not particularly limited, and a known method can be appropriately selected and used. Known methods include, for example, the method of Sukenik et al. (International Journal of Molecular Sciences 2018 Apr 16; 19(4): 1205), the method of Poborilova et al. (Plant Cell Reports https: / / doi.org / 10.1007 / s00299-020-02544-w), and the method of Rademacher et al. (Plant Biotechnology Journal (2019) 17, pp. 1560-1566). In addition, a method of co-culturing a plant culture cell with an Agrobacterium transformant and infecting it may be used.

[0025] In step (2), the period for cultivating the plant agroinfiltrated with the Agrobacterium transformant is not particularly limited, and may be 1 day or more, 2 days or more, 3 days or more, 4 days or more, or 5 days or more. The cultivation period may be 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, or 5 days or less. During this cultivation period, the fusion protein of the signal peptide and bFGF is transiently expressed in the agroinfiltrated plant.

[0026] The cultivation conditions are not particularly limited, and the cultivation may be performed under the same conditions as before agroinfiltration, or under conditions different from those before agroinfiltration. Usually, plants are cultivated in a greenhouse where temperature, illuminance, humidity, sunshine hours, etc. are controlled. The cultivation temperature may be 15 to 28°C, or may be 20 to 25°C. After agroinfiltration, it is preferable to apply a liquid fertilizer with a high nitrogen concentration, and it is preferable to change the control of temperature, sunshine hours, photon flux density, dense planting, light intensity, transpiration, etc.

[0027] In step (2), the period for culturing the cultured plant cells infected with the Agrobacterium transformant is not particularly limited, and may be 1 day or more, 2 days or more, 3 days or more, 4 days or more, or 5 days or more. The culture period may be 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, or 5 days or less. During this culture period, a fusion protein of signal peptide and bFGF is transiently expressed in the cultured plant cells infected with the Agrobacterium transformant.

[0028] The culture conditions are not particularly limited, and the culture may be performed under the same conditions as those before infection with the Agrobacterium transformant, or under conditions different from those before infection. Usually, cultured plant cells are cultured in an incubator in which the temperature, agitation state, medium components, etc. are controlled. The culture temperature may be 15 to 28°C, or may be 20 to 25°C. After infection with the Agrobacterium transformant, it is preferable to appropriately adjust or change the temperature, agitation state, etc.

[0029] Step (3) is a step of purifying basic fibroblast growth factor from a plant or cultured plant cells. The purification step may include the steps of crude extraction, cation exchange chromatography, and affinity chromatography. It is preferable that the steps of crude extraction, cation exchange chromatography, and affinity chromatography are performed in this order.

[0030] When a plant is used, in step (3), first, the plant that has undergone the cultivation period of step (2) is collected. The part of the plant to be collected is not particularly limited, and may be any of the whole plant, leaves, stems, roots, and flowers. The collected plant may be subjected to a treatment suitable for extraction. For example, fine cutting, crushing, freeze-pulverization, etc. may be included. Next, the collected plant is immersed in an extraction buffer to perform extraction, and a crude extract is obtained. The extraction buffer is not particularly limited, and may be appropriately selected from known buffers used to extract proteins from tissues. The extraction buffer may contain a protease inhibitor, a surfactant, glycerol, etc. Specific examples include, but are not limited to, 50 mM phosphate buffer pH 7.5 containing 1 mM EDTA, 150 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100, and 10% glycerol. As a surfactant, 0.5% Tween 20 may be used instead of 0.1% Triton X-100. The extraction time is not particularly limited and may be appropriately set based on preliminary studies, etc.

[0031] In the process of immersing the collected plant in an extraction buffer to obtain a crude extract, it is preferable to perform ultrasonic treatment. The present inventors have confirmed that when a crude extract is obtained by immersing the collected plant in an extraction buffer and then performing ultrasonic treatment, the yield is significantly increased compared to the case where ultrasonic treatment is not performed. When performing ultrasonic treatment, it is preferable to cool the crude extract to avoid an increase in temperature.

[0032] When cultured cells are used, in step (3), first, the cultured cells that have undergone the culture period in step (2) are collected. The collected cultured cells may be those from which the medium has been removed, or may contain the medium. The collected cultured cells may be subjected to a treatment suitable for extraction. For example, crushing, freeze-pulverization, etc. may be included. Next, the collected cultured cells are immersed in an extraction buffer solution to perform extraction, and a crude extract is obtained. The extraction buffer solution may be the same as that described above. In the process of immersing the collected cultured cells in the extraction buffer solution to obtain a crude extract, it is preferable to perform ultrasonic treatment. Specifically, for example, the collected cultured cells may be immersed in the extraction buffer solution, and then ultrasonic treatment and cooling may be repeated multiple times.

[0033] Next, the crude extract is subjected to cation exchange chromatography. As the carrier used for cation exchange chromatography, a carrier into which a sulfopropyl group has been introduced, a carrier into which a carboxymethyl group has been introduced, etc. can be suitably used. Cation exchange chromatography using a carrier into which a sulfopropyl group has been introduced (sulfopropyl cation exchange chromatography) is preferred. The carrier and column used for cation exchange chromatography can be commercially available products. When performing chromatography, an appropriate open column or the like may be filled with a carrier in an amount determined in consideration of the amount of protein contained in the crude extract and the protein adsorption performance of the carrier. In addition, the carrier may be equilibrated in advance with an appropriate buffer (Tris / hydrochloric acid buffer, glycine / sodium hydroxide buffer, phosphate buffer, etc.) before passing the crude extract through it.

[0034] When using plants, in the cation exchange chromatography of step (3), after the crude extract is passed through the column, it is preferable to wash the column until all the green residue is washed away, and further wash the column with a liquid volume of at least twice the column volume. By performing such a large-volume washing, the purity of the final purified product (recombinant bFGF) is improved. The washing liquid to be used is not particularly limited as long as it does not adversely affect the purification by cation exchange chromatography. Standard buffers (Tris / hydrochloric acid buffer, glycine / sodium hydroxide buffer, phosphate buffer, etc.) can be used. In addition, an extract for crude extraction may be used. The liquid volume for washing away all the green residue may be at least three times the column volume. It can be visually confirmed that all the green residue has been washed away. After washing the column, an appropriate elution liquid (for example, 50 mM phosphate buffer pH 6.5 containing 0.5 M sodium chloride, etc.) is used for elution, and fractions containing recombinant bFGF are collected.

[0035] When cultured cells are used, in the cation exchange chromatography in step (3), after the crude extract is passed through the column, the column is washed until the unnecessary cell-derived proteins are washed away, and it is preferable to further wash the column with a liquid volume of at least twice the column volume. By performing such a large-volume washing, the purity of the final purified product (recombinant bFGF) is improved. The washing liquid used is the same as above. The liquid volume in which the unnecessary cell-derived proteins are washed away may be a liquid volume of at least three times the column volume. The fact that the unnecessary cell-derived proteins have been washed away can be confirmed by measuring the protein amount of the washing liquid that has passed through the column. The protein amount of the washing liquid that has passed through the column can be measured, for example, by the absorbance at A280 of the washing liquid that has passed through the column or by using a known protein quantification method such as the Bradford method. After washing the column, the column is eluted with the same elution liquid as above, and a fraction containing recombinant bFGF is collected.

[0036] Next, affinity chromatography is carried out. As the affinity chromatography, heparin affinity chromatography and affinity chromatography using an anti-bFGF antibody can be suitably used.

[0037] Commercially available anti-bFGF antibodies, carriers, columns, etc. can be used. Heparin affinity chromatography of recombinant bFGF can be performed by appropriately selecting a known method (e.g., Seeger, A. and Rinas, U. Two-step chromatographic procedure for purification of basic fibroblast growth factor from recombinant Escherichia coli and characterization of the equilibrium parameters of adsorption J. Chromatogr. A 746 (1996) 17-24, etc.) or by appropriately modifying a known method.

[0038] The method of the present invention enables the mass production of high-purity recombinant bFGF at low cost. The recombinant human bFGF produced by the method of the present invention is suitable as bFGF to be added to a xeno-free medium for culturing cells to be used in regenerative medicine.

[0039] [Method for purifying bFGF expressed in plants] The present invention provides a method for purifying bFGF expressed in a plant. The production method of the present invention may comprise the following steps (i) to (iii): (i) A step of carrying out crude extraction (ii) performing cation exchange chromatography (iii) performing affinity chromatography

[0040] Steps (i), (ii) and (iii) of the purification method of the present invention correspond to the crude extraction step, cation exchange chromatography step and affinity chromatography step included in step (3) of the above-mentioned production method of the present invention, and can be carried out according to the explanation of the above-mentioned step (3). The purification method of the present invention is not limited to the purification of bFGF transiently expressed in a plant or cultured plant cells by the above-mentioned production method of the present invention, but can also be suitably applied to the purification of bFGF constitutively expressed in a genetically modified plant. EXAMPLES

[0041] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

[0042] Example 1: Production of recombinant human bFGF using Nicotiana benthamiana 1. Materials and Methods 1-1 Construction of bFGF expression vector (1) Synthesis of the gene encoding human bFGF The human bFGF gene was synthesized by GeneArt artificial gene synthesis service (Thermo Fisher Scientific) with a base sequence (SEQ ID NO: 2) of the human gene encoding human bFGF shown in SEQ ID NO: 1 optimized for the codons of Nicotiana benthamiana, which is used as the host plant. The synthesis of the original human bFGF gene was also outsourced at the same time. The synthetic gene was provided cloned into an ampicillin resistance plasmid (pMA) (pMA-OptbFGF and pMA-bFGF). In the base sequence optimized for codons in N. benthamiana, approximately 60% of the codons in the human bFGF gene have been modified.

[0043] (2) Fusion of the human glucocerebrosidase signal peptide gene and the human bFGF gene In order to link the gene (SEQ ID NO: 5) encoding the signal peptide (GCsp) of human glucocerebrosidase to the 5'-end of the human bFGF gene, inverse PCR was performed using a primer containing the GCsp nucleotide sequence with pMA-OptbFGF and pMA-bFGF as templates, respectively, to construct pMA-GCsp-OptbFGF and pMA-GCsp-bFGF, respectively. The nucleotide sequence of GCsp-bFGF is shown in SEQ ID NO: 7, and the nucleotide sequence of GCsp-OptbFGF is shown in SEQ ID NO: 8, respectively. Furthermore, in order to introduce restriction enzyme SpeI sites at both ends of GCsp-OptbFGF and GCsp-bFGF, PCR was performed using a primer having an SpeI sequence with pMA-GCsp-OptbFGF and pMA-GCsp-bFGF as templates, respectively. The amplified products were then digested with SpeI.

[0044] (3) Construction of pFK1-GCsp-OptbFGF and pFK1-GCsp-bFGF pFK1-bar is a plant expression vector using the kanamycin resistance bacterial selection marker KanR, which was created in our laboratory. pFK1-bar was digested with SpeI and treated with alkaline phosphatase. SpeI-digested pMA-GCsp-OptbFGF and pFK1-bar, and pMA-GCsp-bFGF and pFK1-bar were ligated with DNA ligase to construct pFK1-GCsp-OptbFGF and pFK1-GCsp-bFGF, respectively.

[0045] The structure of pFK1-GCsp-bFGF is shown in Figure 1. In Figure 1, 35sP is the cauliflower mosaic virus 35S promoter, HSPt is the Arabidopsis thaliana-derived heat shock protein terminator, Pnos is the nopaline synthase gene promoter, Bar is the Bialaphos resistance gene, pAg7 is the polyA sequence, RB is the right border, and LB is the left border.

[0046] 1-2 Preparation of Agrobacterium transformants The constructed pFK1-GCsp-OptbFGF and pFK1-GCsp-bFGF were introduced into Agrobacterium tumefaciens LBA4404 by electroporation to obtain transformants. The electroporation conditions were voltage 2.4 kV, resistance 200 ohms, and capacitance 25 μF. Transformation was confirmed by PCR amplification of the GCsp-OptbFGF gene or GCsp-bFGF gene and digestion of the plasmid extract of the transformant with SpeI restriction enzyme.

[0047] 1-3 Cultivation of Agrobacterium transformants and agroinfiltration Agrobacterium transformants were grown in LB medium containing streptomycin, rifampicin, and kanamycin for 2 days or until the OD600 reached 1. Agrobacterium were then washed and resuspended in infiltration buffer (10 mM MES, 10 mM MgSO4, pH 5.8, 0.56 mM ascorbic acid, and 0.03% Tween 20) as described in Zhao et al. (Zhao, H et al., An Improved Syringe Agroinfiltration Protocol to Enhance Transformation Efficiency by Combinative Use of 5-Azacytidine, Ascorbate Acid and Tween-20. Plants 2017, 6, 9) and adjusted to an OD600 of 0.5. N. benthamiana plants were exposed to light without flowers in a greenhouse for about one month, and then vacuum infiltrated twice with an Agrobacterium suspension to ensure infection with the Agrobacterium transformants, and then grown in the greenhouse for five days.

[0048] 2. Purification of Recombinant Human bFGF 2-1 Preparation of crude extract Recombinant human bFGF was extracted using the E. coli extraction protocol (Imsoonthornruksa S et al., Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture. J Mol Microbiol Biotechnol 2015;25:372-380) optimized for N. benthamiana extraction. Infected leaves were harvested 5 days after infection. 0.25 g (wet weight) of harvested leaves were frozen in liquid nitrogen, crushed, and immersed in extraction buffer (50 mM phosphate buffer pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100, and 10% glycerol) (1:3, w / v). Next, the extraction buffer containing the frozen and crushed leaves was soaked for 10 seconds and cooled on ice for 10 seconds, a cycle of which was repeated 10 times.The mixture was centrifuged, and the supernatant was used as a crude extract.

[0049] 2-2 Purification by sulfopropyl (SP) cation exchange chromatography The crude extract obtained above was directly semi-purified using an SP cation exchange column according to the protocol for E. coli by Seeger and Rinas (Seeger, A. and Rinas, U. Two-step chromatographic procedure for purification of basic fibroblast growth factor from recombinant Escherichia coli and characterization of the equilibrium parameters of adsorption J. Chromatogr. A 746 (1996) 17-24), with extensive modifications for N. benthamiana. The pH of the crude leaf extract was readjusted to 7.5 and applied to an SP650-m cation exchange column (Toyopearl). An initial wash with the extraction buffer was then performed for approximately three column volumes or until all the green residue had been washed off. The column was then washed with two column volumes of washing buffer (50 mM phosphate buffer pH 6.5 containing 0.25 M sodium chloride) and then eluted with elution buffer (50 mM phosphate buffer pH 6.5 containing 0.5 M sodium chloride).

[0050] 2-3 Purification by heparin affinity chromatography The semi-purified SP eluate was diluted with 50 mM phosphate buffer pH 6.5 to a sodium chloride concentration of 0.125 M. The eluate was passed through heparin affinity chromatography using the protocol described by Seeger and Rinas for E. coli with some modifications for N. benthamiana. After the semi-purified SP eluate was loaded, the column was washed with 2 column volumes of W1 (50 mM phosphate buffer pH 8.0, 500 mM sodium chloride) and then 3 column volumes of W2 (50 mM phosphate buffer pH 8.0, 1 M sodium chloride). After washing, purified recombinant human bFGF was eluted with elution buffer (50 mM phosphate buffer pH 8.0, 2 M sodium chloride). The resulting eluate was stored at -80°C.

[0051] 2-4 Protein analysis by Western blotting and Coomasie brilliant blue (CCB) staining All fractions were sampled and subjected to SDS-PAGE. The gels were stained with CCB after electrophoresis and analyzed for total protein content. The presence of bFGF was analyzed by Western blotting. Anti-bFGF mouse IgG (Santa Cruz Biotechnology) was used as the primary antibody, and HRP-conjugated anti-mouse IgG (GE Healthcare) was used as the secondary antibody. Luminata Forte Western HRP substrate (Millipore) was added to the membrane, and the resulting chemiluminescence was detected by exposure to X-ray film.

[0052] 3.Results 3-1 Examination of ultrasonic treatment of crude extract The bFGF yields were compared between the cases where the crude extract was ultrasonicated and not ultrasonicated in the crude extraction process. The results of Western blotting for both are shown in Figure 2. In the figure, (NS)EX indicates the crude extract not ultrasonicated, (S)EX indicates the crude extract that was ultrasonicated, and (-)EX indicates the crude extract from leaves that were not agroinfiltrated. The bFGF yield of the crude extract that was not ultrasonicated was 3.2 μg / g leaf, and the bFGF yield of the crude extract that was ultrasonicated was 10.1 μg / g leaf. In other words, the bFGF yield was increased by about three times by ultrasonicating the crude extract in the crude extraction process.

[0053] 3-2 Examination of the effect of codon modification on yield Crude extracts were prepared from plants agroinfiltrated with an expression vector (pFK1-GCsp-bFGF) containing the base sequence of the human bFGF gene, or with an expression vector (pFK1-GCsp-OptbFGF) containing the base sequence of the human bFGF gene with codons optimized for N. benthamiana, and subjected to SDS-PAGE and Western blotting.

[0054] The results are shown in Figure 3. The upper panel shows the results of Western blotting, and the lower panel shows the results of SDS-PAGE. In the figure, S1 is a crude extract from a plant agroinfiltrated with pFK1-GCsp-bFGF with no codon modification, S2 is a crude extract from a plant agroinfiltrated with pFK1-GCsp-OptbFGF with a modified codon, (-)EX is a crude extract from a leaf that was not agroinfiltrated, and (+) is a sample containing 20 ng of recombinant human bFGF produced in E. coli. The yield of S1 was 181 ng / g wet leaf, and that of S2 was 161 ng / g wet leaf, and it was confirmed that the yields were almost the same, although S1 was slightly higher.

[0055] 3-3 Two-step purification by SP cation exchange chromatography and heparin affinity chromatography The eluate finally obtained in the two-step purification was subjected to SDS-PAGE and stained with CBB, and the results are shown in Figure 4. The purity, recovery rate, and yield of the final purified product obtained from 42 plants were calculated as follows: Purity: over 99%, recovery rate: 46.5%, yield: 2.1 μg / g leaves. In addition, in the method of producing recombinant human bFGF from rice seeds described in Non-Patent Document 1, the purity was over 99%, recovery rate: 4.49%, and yield: 8.33 μg / g rice grain.

[0056] Example 2: Cell proliferation assay Cell proliferation assays were performed according to the method described by Liu and Wu (Liu, C., and Wu, K. Synergistic effects of basic fibroblast growth factor and insulin on Chinese hamster ovary cells under serum-free conditions. J Biosci Bioeng. 2009 Mar;107(3):312-7. doi: 10.1016 / j.jbiosc.2009.02.001.). CHO-K1 and Hep-G2 cells were seeded in 24-well plates and cultured for 24 h. CHO-K1 cells were cultured in Hams-F12 containing 10% fetal bovine serum, and Hep-G2 cells were cultured in high-glucose DMEM containing 10% fetal bovine serum (FBS). The medium was removed and the cells were washed three times with PBS and twice with each serum-free medium. CHO-K1 cells were incubated overnight at 37°C under 5% CO2 with serum-free Hams-F12. Hep-G2 cells were incubated overnight at 37°C under 5% CO2 with high glucose DMEM containing 0.1% FBS. 2 The cells were incubated overnight under conditions of 0.25% CO2 and 0.2% ethanol. Recombinant human bFGF purified from leaves infiltrated with Agrobacterium transformants carrying pFK1-GCsp-bFGF was added at 20 ng / mL or 200 ng / mL. The negative control was a cell culture without recombinant human bFGF. The number of viable cells was counted by trypan blue exclusion assay after 0, 2, 4, and 6 days for CHO-K1 cells and 0, 2, and 4 days for Hep-G2 cells.

[0057] The results for CHO-K1 cells are shown in Figure 5, and the results for Hep-G2 cells are shown in Figure 6. In both cells, the number of viable cells was significantly increased in a dose-dependent manner by conversion of recombinant human bFGF.

[0058] Example 3: Examination of surfactants used in extraction buffer In Example 1, 0.1% Triton X-100 was used as a surfactant in the extraction buffer, but in this example, 0.5% Tween 20 and 0.5% NP-40 were used in addition to 0.1% Triton X-100. The composition of the extraction buffer in this example was the same as in Example 1, 50 mM phosphate buffer pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol, and a surfactant. PBS without a surfactant was used as a control.

[0059] The Agrobacterium transformant prepared in Example 1 was agroinfiltrated into the leaves of N. benthamiana in the same manner as in Example 1, and cultivated in a greenhouse for 4 days. 0.25 g (wet weight) of the collected leaves were frozen and crushed, and a four-fold volume of extraction buffer was added to perform crude extraction. The samples were sonicated in the same manner as in Example 1, centrifuged to collect the supernatant, and the filtrate was obtained and used as the sample. The obtained sample was subjected to SDS-PAGE, and the gel after electrophoresis was stained with CCB. Recombinant human bFGF prepared in E. coli was used as a positive control. The presence of bFGF was analyzed by Western blotting. The antibodies and detection method used were the same as in Example 1.

[0060] The results are shown in Figure 7. When 0.5% Tween 20 was used as a surfactant, bFGF was detected as in the case of using 0.1% Triton X-100. On the other hand, when 0.5% NP-40 was used, bFGF was not detected. Therefore, it was found that 0.5% Tween 20 can be used as a surfactant in the extraction buffer. In addition, bFGF was detected when PBS containing no surfactant was used, indicating that PBS can be used as the extraction buffer.

[0061] The present invention is not limited to the above-mentioned embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in the different embodiments. In addition, all academic documents and patent documents described in this specification are incorporated herein by reference.

Claims

1. A method for producing a basic fibroblast growth factor, comprising the steps of: (1) agroinfiltration into a plant or infection into a plant culture cell with an Agrobacterium transformant into which an expression vector containing DNA encoding a fusion protein of a signal peptide of human glucocerebrosidase and a basic fibroblast growth factor has been introduced; (2) cultivating a plant agroinfiltrated with the Agrobacterium transformant in step (1) or cultivating cultured plant cells infected with the Agrobacterium transformant, thereby transiently expressing the fusion protein in the plant or in the cultured plant cells; and (3) recovering the plant in which the fusion protein has been transiently expressed by the cultivation of the step (2) or the cultured plant cells in which the fusion protein has been transiently expressed by the culture of the step (2), and purifying the basic fibroblast growth factor from the recovered plant or the cultured plant cells; In step (3), crude extraction, followed by cation exchange chromatography, followed by affinity chromatography, In the cation exchange chromatography, after the crude extract is applied to the column, the column is washed until all the green residue is washed away or until unnecessary cell-derived proteins are washed away, and the column is further washed with a liquid volume at least twice the column volume. Manufacturing method.

2. The method according to claim 1, wherein the basic fibroblast growth factor is human basic fibroblast growth factor.

3. 3. The method according to claim 1, wherein the basic fibroblast growth factor is human basic fibroblast growth factor for xeno-free medium.

4. The method according to any one of claims 1 to 3, wherein the crude extraction comprises ultrasonic treatment.

5. The method according to any one of claims 1 to 4, wherein the cation exchange chromatography is sulfopropyl cation exchange chromatography.

6. The method according to any one of claims 1 to 5, wherein the affinity chromatography is heparin affinity chromatography.

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