WO2015050005A1 - Protein composition for promoting cell proliferation via egf receptor - Google Patents
Protein composition for promoting cell proliferation via egf receptor Download PDFInfo
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- WO2015050005A1 WO2015050005A1 PCT/JP2014/074841 JP2014074841W WO2015050005A1 WO 2015050005 A1 WO2015050005 A1 WO 2015050005A1 JP 2014074841 W JP2014074841 W JP 2014074841W WO 2015050005 A1 WO2015050005 A1 WO 2015050005A1
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- protein
- protein composition
- composition according
- amino acid
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
Definitions
- the present invention relates to a novel protein composition having an activity of promoting cell proliferation via an EGF receptor.
- EGF Epidermal growth factor
- Non-patent Document 1 Human EGF is a protein with a molecular weight of about 6 kDa consisting of 53 amino acids synthesized in vivo as a precursor protein consisting of 1207 amino acids and then processed, and has 3 disulfide bonds in the molecule (Non-patent literature). 3).
- Non-Patent Document 4 Production by Escherichia coli
- Non-Patent Documents 5 to 7 production by yeast
- Patent Document 1 Non-Patent Document 8
- EGF produced by genetic recombination undergoes various modifications in cells depending on the species used.
- EGF currently commercially used is EGF produced in E. coli and EGF produced in yeast.
- EGF EGF produced in bacteria
- the EGF gene is stably recombined in the barley genome and recombinant EGF is produced in barley.
- Non-Patent Document 8 describes a method of producing EGF by preparing a virus incorporating a human EGF gene, infecting tobacco with this virus, and transiently expressing human EGF.
- the method described in Non-Patent Document 8 has a problem in yield and cannot be said to be a practical method. Therefore, it is still unclear what composition of recombinant EGF protein can be obtained when EGF is mass-produced in plants.
- An object of the present invention is to provide a novel protein composition having an activity of promoting cell proliferation via an EGF receptor, which can be used in place of a conventional EGF protein product.
- the inventors of the present application have succeeded in developing a technique for efficiently producing EGF having biological activity by a transient expression system in a plant body.
- the protein composition containing recombinant EGF recovered from plant leaves by the technique was subjected to molecular weight confirmation and amino acid analysis by TOF-MS, and its composition was confirmed.
- the present invention relates to a protein composition obtained by introducing a nucleic acid encoding an amino acid sequence of an epidermal growth factor into a plant cell to express the protein, and then extracting and purifying the protein in the plant cell.
- a protein composition is provided that has an activity of promoting cell growth and exhibits three peaks in mass spectrometry in the range of m / z 5000-5500.
- a novel protein composition having cell proliferation activity produced in plant cells based on the amino acid sequence of mature human EGF Plants, like humans, are multicellular organisms belonging to eukaryotes, and both cells have very similar protein expression mechanisms. Compared to recombinant EGF protein produced by bacteria currently used commercially, the protein composition of the present invention is advantageous from the viewpoint of production cost, capital investment and production expandability. According to the protein composition of the present invention, it is possible to provide products such as cosmetics and pharmaceuticals using conventional recombinant EGF at a lower price.
- FIG. 1 is a structural diagram of TMV expression vector UB001 used in Examples.
- FIG. It is a figure which shows the result of having silver-stained EGF extracted and refined from the tobacco leaf of recombinant TMV infection after SDS-PAGE.
- Lane 1 molecular marker
- lane 2 standard
- lane 3 C-terminal endoplasmic reticulum signal with KDEL (5-fold enrichment)
- lane 4 no C-terminal endoplasmic reticulum signal (5-fold enrichment)
- lane 5 C-terminal endoplasmic reticulum With signal KDEL
- lane 6 no C-terminal endoplasmic reticulum signal
- lane 7 molecular marker.
- the protein composition of the present invention introduces a nucleic acid encoding the same amino acid sequence (SEQ ID NO: 2) as mature human EGF into a plant cell to express the protein, and then extracts and purifies the protein in the plant cell. Can be obtained.
- the protein composition is composed mainly of three types of fragments of the protein encoded by the nucleic acid, and has three peaks (m / z 5113 ⁇ 5, m / z) in the range of m / z 5000-5500 in mass spectrometry. 5200 ⁇ 5 and m / z 5315 ⁇ 5) (FIG. 5).
- the three types of protein fragments contained in the protein composition are the fragments consisting of the region of amino acids 1 to 47 in the amino acid sequence shown in SEQ ID NO: 2, and the 2nd to 47th amino acids. It has been identified as a fragment consisting of the amino acid region and a fragment consisting of the 3rd to 47th amino acid region (see Examples below). It is considered that such a protein fragment is generated as a result of modification of the protein expressed in the plant cell such as enzymatic cleavage in the cell. It has been reported that the protein composition obtained when EGF protein is produced in E. coli has almost the same amino acid composition and amino acid sequence as natural human-derived EGF protein (T. Oka, et al. Proc. Natl. Acad. Sci., 82, 7212-7216, 1985), the protein composition of the present invention has distinctly different characteristics from EGF derived from E. coli.
- the protein composition of the present invention has an activity of promoting cell proliferation. This cell proliferating activity is brought about by an agonistic activity on the EGF receptor. That is, the protein composition of the present invention binds to the EGF receptor and activates a downstream signal transduction system, thereby promoting cell proliferation.
- the three types of protein fragments described above include the 6th to 42nd Cys (He-Shu Lu, et al, J. Biol), which is known as an important region for the mature human EGF molecule to exert biological activity. .Chem., 276, 34913-34917 (2001); H. Ogiso et al. Cell, 110,775-787 (2002)), and the protein composition of the present invention is an agonist for the EGF receptor. Consistent with having activity.
- Cell proliferation activity can be confirmed by, for example, the method described in G. Carpenter and J. Zendegui, Analytical Biochemistry, 153, ⁇ ⁇ ⁇ 279 ⁇ ⁇ ⁇ (1985).
- the following examples detail the method for confirming the cell growth activity of a protein composition using a human fibroblast cell line.
- the ability to bind to the EGF receptor can be examined by the method described in, for example, R.RL. Ladda, et al, Analytical Biochemistry, 93, 286 (1979).
- the plant cell used for the production of the protein composition of the present invention may be a cultured cell or a plant individual, but it is preferable to obtain a protein composition by recovering the protein expressed in the plant individual.
- the nucleic acid to be introduced into plant cells encodes the same amino acid sequence as mature human EGF.
- the human EGF gene is registered in the NCBI database Gene with a Gene ID of 1950, and the sequence is registered in GenBank as accession No. NM_001963, for example.
- SEQ ID Nos. 1 and 2 in the sequence listing show the base sequence of the region encoding mature EGF and the amino acid sequence of the mature EGF in the natural human EGF gene.
- the base sequence encoding the mature human EGF sequence is preferably a base sequence in which codons are modified so that the protein can be efficiently expressed in plant cells.
- Eighteen amino acids other than Met and Trp are encoded by 2 to 6 synonymous codons, but it is known that the frequency of use of synonymous codons varies depending on the type of organism (Grantham, R. (1980) Trends Biochem. Sci. 5, 327-331 .; Grantham, R., Gautier, C. and Gouy, M. (t980) Nucl. Acids Res. 8, 1893-1912 .; Grantham, R. , Gautier, C., Gouy, M., Mercier, R. and Pave, A. (1980) Nucl.
- codons frequently used in plants include those shown in Table 1. These codons can be preferably used in plants.
- Table 1 when a base sequence encoding a mature human EGF sequence is modified, a part or all of the codons encoding each amino acid is shown in Table 1 based on the base sequence of the human EGF gene. Should be replaced.
- the base sequence shown in SEQ ID NO: 3 is an example of a base sequence encoding a mature human EGF sequence that has been subjected to codon optimization so that it can be efficiently expressed in plant cells.
- the sequence can be particularly preferably used.
- This base sequence is a sequence obtained by changing the codon by changing 38 bases in the natural mature human EGF coding sequence (SEQ ID NO: 1).
- the sequence can be particularly preferably used. Further, not only a completely identical base sequence to SEQ ID NO: 3, but also a codon frequently used in plants as exemplified in Table 1 above, as long as the sequence is such that a small number of bases are different.
- the “minority” here may be, for example, 1 to 15, 1 to 12, 1 to several, 1 to 5, or 1 to 3.
- identity % with SEQ ID NO: 3
- the modified base sequence that can be used in the same manner as SEQ ID NO: 3 has an identity with SEQ ID NO: 3 of 90% or more, 92% or more, 95% or more, or It can be 98% or more.
- the “identity” of the base sequences is a percentage obtained by aligning both sequences so that the bases of the two base sequences to be compared match as much as possible, and dividing the number of matched bases by the total number of bases. It is represented by.
- a gap is appropriately inserted in one or both of the two sequences to be compared as necessary.
- sequence alignment can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like.
- the total number of bases is the number of bases obtained by counting one gap as one base.
- the identity is calculated by dividing the total number of bases of the longer sequence and dividing the number of matched bases.
- the sequence to be compared is linked to any other sequence (for example, incorporated into an expression vector), only the corresponding region is taken out and the sequences are compared, calculate. For example, when comparing base sequences encoding mature human EGF, only the region corresponding to the region encoding mature human EGF is taken out and the sequences are compared.
- the nucleic acid encoding a mature EGF sequence can be linked to a nucleic acid encoding a signal peptide useful for intracellular transport and localization of the expressed protein.
- a signal peptide usually consists of amino acids of about 3 to 60 residues, and is a structure that directs the transport and localization of a protein synthesized in the cytoplasm.
- the signal peptide of extensin possessed by tobacco N. plumbaginifolia can be preferably used as a signal peptide for intracellular transport.
- the extensin signal peptide can be used by linking to the N-terminal side of the mature EGF sequence.
- the extensin gene sequence of N. plumbaginifolia is registered with GenBank as accession No. M34371.
- the nucleotide sequence of the signal peptide coding region found in this registered sequence is SEQ ID NO: 4, and the amino acid sequence of the signal peptide is sequenced. The number 5 is shown.
- the base sequence encoding the signal peptide can also be used by modifying the codon so that it can be efficiently expressed in plant cells, like the nucleic acid encoding the mature EGF sequence.
- the base sequence shown in SEQ ID NO: 6 is an example of a base sequence encoding an extendin signal peptide sequence in which a codon is optimized for expression in a plant cell using a transient expression system.
- This base sequence is a sequence obtained by changing the codons by changing 29 bases in the natural tobacco extensin signal peptide coding sequence (SEQ ID NO: 6).
- the sequence can be particularly preferably used as a sequence encoding a signal peptide for intracellular transport.
- a sequence having a different base can be preferably used in the same manner as the base sequence of SEQ ID NO: 6.
- identity a modified base sequence that can be used in the same manner as SEQ ID NO: 6 has 90% or more, 92% or more, 95% or more, or 98% or more identity with SEQ ID NO: 6. obtain.
- an endoplasmic reticulum retention signal comprising, for example, the KDEL (SEQ ID NO: 7) sequence can be preferably used as a signal peptide for intracellular localization.
- the base sequence encoding the retention signal for example, the base sequence shown in SEQ ID NO: 8 can be preferably used, but it can be used in the same manner as the sequence encoding mature human EGF and the sequence encoding extensin signal peptide.
- Such a sequence is not limited to a sequence that is completely identical to SEQ ID NO: 8.
- the base sequence shown in SEQ ID NO: 9 is a codon-optimized base sequence (SEQ ID NO: 6) encoding an extensin signal peptide sequence upstream of the codon-optimized base sequence (SEQ ID NO: 3) encoding the mature EGF sequence.
- This base sequence is a particularly preferred example as a base sequence of a nucleic acid to be introduced into a plant cell in the production of the protein composition of the present invention.
- a foreign protein fused with an extensin signal peptide and an endoplasmic reticulum retention signal is considered to accumulate in the cytoplasm of the cell, particularly in the endoplasmic reticulum.
- a nucleic acid containing a modified base sequence such as SEQ ID NO: 9 can be prepared by conventional chemical synthesis. Alternatively, it can also be prepared by synthesizing a cDNA having a naturally occurring sequence by a well-known genetic engineering technique, and then introducing appropriate mutations using this cDNA as a template and using appropriate primers. Alternatively, the nucleic acid to be prepared can be divided into several short regions, a plurality of nucleic acid fragments can be chemically synthesized, and each fragment can be ligated by a known fusion PCR method.
- the expression of the protein in the plant cell is preferably a transient expression.
- Transiently expressing a protein means that the nucleic acid encoding the protein is expressed in the plant cell in a state where it is not integrated into the genome of the host plant cell.
- Such transient expression can be performed by using a viral vector.
- Various plant viral vectors for expressing a desired foreign gene in plant cells are known (for example, see “Protein Nucleic Acid Enzyme”, vol.45, p.607-613), and commercially available products are also available. There are various things.
- plant virus vectors the most actively studied and widely used is the tobacco mosaic virus (TMV) vector, and the TMV vector can be preferably used in the present invention.
- TMV tobacco mosaic virus
- the nucleic acid construct encoding the foreign protein is an appropriate RNA polymerase promoter (for example, required for synthesizing recombinant viral RNA by a transcription reaction) , Phage polymerase promoter such as T7 promoter), viral replicase cDNA, transfer protein cDNA and coat protein cDNA. If a tobamovirus vector such as TMV is used, it may be ligated in the order of [RNA polymerase promoter]-[RNA replicase cDNA]-[transition protein cDNA]-[nucleic acid construct]-[coat protein cDNA]. Usually, these genetic constructs take the form of plasmid DNA.
- TMV-based transient expression systems include BSG1037 and BSG1057 described in Japanese Patent No. 4750285, and commercially available products such as GENEWARE (registered trademark) of Kentucky Bioprocessing.
- These plasmids are those capable of expressing systemic infectious recombinant TMV, and include RNA polymerase promoter, TMV replicase (RNA-dependent RNA polymerase) cDNA, transfer protein cDNA and coat protein cDNA.
- RNA polymerase promoter TMV replicase (RNA-dependent RNA polymerase) cDNA
- transfer protein cDNA transfer protein cDNA
- coat protein cDNA coat protein cDNA
- a whole body capable of expressing a desired foreign gene in a plant cell by inserting the desired foreign gene between the transfer protein cDNA and the coat protein cDNA and synthesizing RNA having a cap at the 5 ′ end using this as a template.
- Infectious TMV viral RNA can be obtained.
- the replicase cDNA and the transfer protein cDNA are modified to produce a replicase and transfer protein having slightly different sequences from that of natural TMV. Those with sequence modifications can also be used to produce the protein composition of the present invention.
- Systemic infectious TMV viral RNA incorporating a nucleic acid encoding the mature EGF sequence may be inoculated directly into plants in the form of a naked viral genome, or mixed with purified coat protein to form capsids. It is possible to inoculate a plant after making it into a state of virus particles.
- the term “recombinant virus” includes both naked virus genomes and virus particles obtained by encapsidation. Inoculation with the virus may be performed by mechanically scratching the leaves of the plant with fine particles such as carbon powder or celite and bringing them into contact with the inoculum as usual.
- the virus grows and a large amount of foreign protein is produced in the plant cell.
- the protein composition of the present invention can be obtained by harvesting leaves and extracting and purifying the protein.
- the extraction and purification of the target protein from the plant tissue infected with the recombinant virus is basically performed by a general protein purification method (C. D. Mount et al., Archives of Biochemistry and Biophysics, 240 33 (1985) , U. H. Gregory and I. R. Willshire, Hoppe-Sayler's Z. Physiol. Chem.,. 356 1765 (1975)). Specifically, it may be performed as follows, for example.
- the obtained crude product is further purified once or more using a hydrophobic interaction chromatography carrier.
- the carrier is not particularly limited, and may be any known carrier that is generally used for hydrophobic interaction chromatography, such as a gel or a resin.
- the hydrophobic interaction chromatography carrier those having a phenyl group as a functional group, for example, a phenyl-sepharose carrier are preferably used.
- the protein is adsorbed on a strong anion exchange chromatography carrier and eluted with a glycine buffer containing sodium chloride adjusted to basic (pH 9 to 11), so that three types of proteins can be obtained.
- the protein composition of the present invention containing a fragment as a main component can be obtained. This protein composition may be subjected to further treatment as desired, such as desalting treatment.
- a plasmid UB001 (Fig. 1) prepared by modifying a known plasmid BSG1057 (patent No. 4750285) capable of expressing a recombinant tobacco mosaic virus was linearized by digestion with PacI and XhoI, and was run on an agarose gel. It was later extracted and purified from the gel.
- the above insert DNA and linearized UB001 were ligated with T4 DNA ligase (Invitrogen) (37 ° C., 20 minutes).
- the obtained plasmid DNA was introduced into E. coli NEB5- ⁇ (New England BioLabs) by heat shock, screened with ampicillin-containing medium, single colonies were grown, and plasmid DNA was used from this E. coli cell using mini prep kit (Qiagen).
- plasmid DNA was used from this E. coli cell using mini prep kit (Qiagen).
- the insert size was confirmed by digesting the plasmid with PacI and XhoI.
- the reaction solution was incubated at 37 ° C. for 1 to 3 hours to prepare a recombinant TMV RNA capable of expressing a protein in which an extensin signal peptide and an endoplasmic reticulum retention signal peptide were fused to the same amino acid sequence as mature human EGF.
- 0.5 ⁇ L of the reaction solution was electrophoresed on a 1% agarose gel to confirm the transcription product.
- a stock solution of a capsid formation reaction solution or a solution diluted appropriately with nuclease-free water was mixed with an equal amount of GIB and used as an inoculum.
- Recombinant TMV can be applied to tobacco by dripping 30 ⁇ L of the inoculum per leaf onto two true leaves per tobacco (Nicotiana benthamiana) and rubbing the leaves lightly with a gloved finger. Infected.
- the supernatant after centrifugation was filtered with Miracloth and 30 KDa PES Kvick flow UF cassette (0.1 m 2 ) to remove most of the protein in the filtrate and concentrate the recombinant protein (30 KDa filtrate).
- the 30 KDa filtrate was further concentrated to 90 mL with a 2 KDa Hydrosart Sartorius cassette (0.1 m 2 ) (2 KDa filtrate).
- each filtration residue was washed with 7 times the amount of extraction buffer (total 1,600 mL), and the washings were collected (30 KDa residue and 2 KDa residue).
- the supernatant after centrifugation was filtered with Miracloth and 30 KDa PES Kvick flow UF cassette (0.1 m 2 ) to remove most of the protein in the filtrate and concentrate the recombinant protein.
- Ammonium sulfate was added to the 30 KDa filtrate to 1.5 M and adsorbed on a Phenyl Sepharose FF column, followed by elution with 5 mM sodium phosphate buffer (pH 7.0). Further, ammonium sulfate was added to this solution to 2.5 M and adsorbed on a Phenyl Sepharose FF column, followed by elution with 5 mM sodium phosphate buffer (pH 7.0).
- This solution was passed through a NuviaQ column to adsorb the recombinant protein, and eluted with glycine buffer (pH 10.0) supplemented with 500 mM sodium chloride. Using this as a recombinant protein sample, purity measurement and biological activity measurement were performed by SDS-PAGE and silver staining.
- biological activity was measured in the same manner using BALB / c mouse embryo-derived cells BALB / 3T3.
- the biological activity was similarly measured using a commercially available human EGF protein standard (Promega) instead of the above recombinant protein sample.
- TOF-MS analysis of recombinant protein sample 50 ⁇ L of recombinant protein sample was desalted with ZipTip C18 (Millipore) and concentrated to 10 ⁇ L. Mix 1 ⁇ L of this solution with 4 ⁇ L of Matrix CCA (Alpha-cyano-4-hydroxy cinnamic acid), and use the 1 ⁇ L for Bruker REFLEXTM Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometer (MALDI-TOF MS) analysis. Provided.
- Matrix CCA Alpha-cyano-4-hydroxy cinnamic acid
- the structure of the peptide was estimated from the results of molecular weight measurement by TOF-MS and amino acid analysis from the N-terminus.
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Abstract
In the present invention, disclosed is a novel protein composition, which has activity to promote cell proliferation via an EGF receptor and which can be used in place of a conventional EGF protein preparation. This protein composition is obtained by introducing to the inside of plant cells the nucleic acid that encodes the amino acid sequence of epidermal cell growth factor to induce protein expression, and then extracting and purifying the protein inside the plant cells. The composition has activity to promote cell proliferation, and displays three peaks within a range of m/z 5000 to 5500 by mass analysis.
Description
本発明は、EGF受容体を介して細胞増殖を促進する活性を有する新規タンパク質組成物に関する。
The present invention relates to a novel protein composition having an activity of promoting cell proliferation via an EGF receptor.
細胞増殖因子は、対応する受容体に結合して、細胞内シグナル伝達系を経て細胞の増殖に必要な代謝系活動を誘発し、細胞の増殖を促す。上皮細胞増殖因子（EGF）は細胞増殖因子のひとつである。EGFの生理活性として、細胞増殖作用の他、胃酸分泌抑制作用、抗潰瘍作用、消化器粘膜保護作用、DNA合成促進作用、角膜修復作用、創傷治癒促進作用、抗炎症作用、鎮痛作用などが知られている。
Cell growth factor binds to the corresponding receptor, induces metabolic system activities necessary for cell growth via the intracellular signal transduction system, and promotes cell growth. Epidermal growth factor (EGF) is one of the cell growth factors. Known physiological activities of EGF include cell growth, gastric acid secretion suppression, anti-ulcer, digestive mucosal protection, DNA synthesis, corneal repair, wound healing, anti-inflammatory and analgesic. It has been.
EGFは、最初Cohenらがマウスの顎下腺より発見し精製された(非特許文献１)。その後、ヒトの尿より胃酸分泌抑制作用のある物質として精製されている（非特許文献２）。ヒトEGFは、生体内では、アミノ酸1207個から成る前駆タンパク質として合成され、その後処理された53個のアミノ酸から成る分子量約6kDaのタンパク質で、分子内に３カ所のジスルフィド結合をもっている（非特許文献３）。
EGF was first discovered and purified from the submandibular gland of a mouse by Cohen et al. (Non-patent Document 1). Thereafter, it has been purified from human urine as a substance having an inhibitory action on gastric acid secretion (Non-patent Document 2). Human EGF is a protein with a molecular weight of about 6 kDa consisting of 53 amino acids synthesized in vivo as a precursor protein consisting of 1207 amino acids and then processed, and has 3 disulfide bonds in the molecule (Non-patent literature). 3).
EGF等の細胞増殖因子は、医薬品及び化粧品等の成分の一つとして利用されている。EGFの効率的な生産方法についても研究が進められており、遺伝子組み換えによるEGFの生産方法が種々報告されている。それらの報告の一例として、大腸菌による生産（非特許文献４）、酵母による生産（非特許文献５～７）、植物による生産（特許文献１、非特許文献８）等がある。
Cell growth factors such as EGF are used as one of ingredients of pharmaceuticals and cosmetics. Research is also progressing on an efficient production method of EGF, and various production methods of EGF by genetic recombination have been reported. Examples of such reports include production by Escherichia coli (Non-Patent Document 4), production by yeast (Non-Patent Documents 5 to 7), and production by plants (Patent Document 1, Non-Patent Document 8).
遺伝子組み換えにより生産されたEGFは、使用する生物種に応じて細胞内で様々な修飾を受ける。現在商業的に利用されているEGFは、大腸菌で生産したEGFと酵母で生産したEGFである。細菌内でEGFを生産する技術では、微細な培養条件を精密に監視して制御するために高価なシステムを導入する必要があるため、組換えEGF及びこれを用いた各種製品の価格は依然として高価である。植物での大量生産が可能になれば低コストで提供できる可能性があるが、植物を利用した生産方法で実用化可能なものは未だ確立していない。例えば、特許文献１記載の方法では、大麦のゲノムにEGF遺伝子を安定的に組み換えて大麦に組換えEGFを生産させるが、この方法では遺伝子組み換え植物の管理が必須となり、大規模生産のハードルは高い。非特許文献８には、ヒトEGF遺伝子を組み込んだウイルスを作製し、このウイルスをタバコに感染させ、ヒトEGFを一過性に発現させることによりEGFを産生する方法が記載されている。しかしながら、非特許文献８記載の方法は収量に問題があり、実用的な方法とはいえない。従って、植物でEGFを大量生産した場合にどのような組成の組換えEGFタンパク質を得ることができるかも依然として不明である。
EGF produced by genetic recombination undergoes various modifications in cells depending on the species used. EGF currently commercially used is EGF produced in E. coli and EGF produced in yeast. In the technology to produce EGF in bacteria, it is necessary to introduce an expensive system to precisely monitor and control fine culture conditions, so the price of recombinant EGF and various products using it is still expensive. It is. If mass production with plants becomes possible, it may be possible to provide at low cost, but no practical production method using plants has been established yet. For example, in the method described in Patent Document 1, the EGF gene is stably recombined in the barley genome and recombinant EGF is produced in barley. In this method, however, the management of genetically modified plants is essential, and the hurdle for large-scale production is high. Non-Patent Document 8 describes a method of producing EGF by preparing a virus incorporating a human EGF gene, infecting tobacco with this virus, and transiently expressing human EGF. However, the method described in Non-Patent Document 8 has a problem in yield and cannot be said to be a practical method. Therefore, it is still unclear what composition of recombinant EGF protein can be obtained when EGF is mass-produced in plants.
本発明は、従来のEGFタンパク質製品に代えて活用することができる、EGF受容体を介して細胞増殖を促進する活性を有する新規なタンパク質組成物を提供することを目的とする。
An object of the present invention is to provide a novel protein composition having an activity of promoting cell proliferation via an EGF receptor, which can be used in place of a conventional EGF protein product.
本願発明者らは、鋭意研究の結果、植物体内において一過性発現系により生物活性のあるEGFを効率よく製造する技術の開発に成功した。当該技術により植物葉から回収した、組換えEGFを含むタンパク質組成物について、TOF-MSによる分子量の確認及びアミノ酸分析を行ない、その組成を確認したところ、成熟型ヒトEGFの５３残基のアミノ酸配列のうち第４８番アミノ酸～C末端の領域が欠失した配列を有する３種類のタンパク質断片の混合物であることを確認し、本願発明を完成した。
As a result of earnest research, the inventors of the present application have succeeded in developing a technique for efficiently producing EGF having biological activity by a transient expression system in a plant body. The protein composition containing recombinant EGF recovered from plant leaves by the technique was subjected to molecular weight confirmation and amino acid analysis by TOF-MS, and its composition was confirmed. The amino acid sequence of 53 residues of mature human EGF Of these, it was confirmed that this was a mixture of three types of protein fragments having sequences deleted from the 48th amino acid to the C-terminal region, and the present invention was completed.
すなわち、本発明は、上皮細胞増殖因子のアミノ酸配列をコードする核酸を植物細胞内に導入してタンパク質を発現させた後、該植物細胞内のタンパク質を抽出及び精製することにより得られるタンパク質組成物であって、細胞増殖を促進する活性を有し、質量分析においてm/z 5000～5500の範囲で３つのピークを呈する、タンパク質組成物を提供する。
That is, the present invention relates to a protein composition obtained by introducing a nucleic acid encoding an amino acid sequence of an epidermal growth factor into a plant cell to express the protein, and then extracting and purifying the protein in the plant cell. A protein composition is provided that has an activity of promoting cell growth and exhibits three peaks in mass spectrometry in the range of m / z 5000-5500.
本発明により、成熟型ヒトEGFのアミノ酸配列に基づいて植物細胞内で製造された、細胞増殖活性を有する新規タンパク質組成物が提供される。植物はヒトと同じく真核生物に属する多細胞生物であり、両者の細胞は非常によく似たタンパク質の発現機構を有している。現在商業的に利用されている細菌で生産された組換えEGFタンパク質と比べ、本発明のタンパク質組成物は生産コスト、設備投資、生産拡張性の観点から有利である。本発明のタンパク質組成物によれば、従来の組換えEGFを利用した化粧品や医薬品等の製品をより低価格で提供することが可能になる。
According to the present invention, there is provided a novel protein composition having cell proliferation activity produced in plant cells based on the amino acid sequence of mature human EGF. Plants, like humans, are multicellular organisms belonging to eukaryotes, and both cells have very similar protein expression mechanisms. Compared to recombinant EGF protein produced by bacteria currently used commercially, the protein composition of the present invention is advantageous from the viewpoint of production cost, capital investment and production expandability. According to the protein composition of the present invention, it is possible to provide products such as cosmetics and pharmaceuticals using conventional recombinant EGF at a lower price.
本発明のタンパク質組成物は、成熟型ヒトEGFと同じアミノ酸配列（配列番号２）をコードする核酸を植物細胞内に導入してタンパク質を発現させた後、該植物細胞内のタンパク質を抽出及び精製することにより得られる。当該タンパク質組成物は、上記の核酸にコードされるタンパク質の３種類の断片を主成分とし、質量分析においてm/z 5000～5500の範囲で３つのピーク（m/z 5113±5、m/z 5200±5及びm/z 5315±5）を呈する（図５）。さらなる詳細な解析により、当該タンパク質組成物に含まれる３種類のタンパク質断片は、配列番号２に示すアミノ酸配列中の第１番～第４７番アミノ酸の領域からなる断片、第２番～第４７番アミノ酸の領域からなる断片、及び第３番～第４７番アミノ酸の領域からなる断片と同定されている（下記実施例参照）。植物細胞内で発現されたタンパク質が、細胞内で酵素による切断等の修飾を受けた結果、このようなタンパク質断片が生じるものと考えられる。大腸菌でEGFタンパク質を生産した場合に得られるタンパク質組成物は、天然のヒト由来EGFタンパク質とほぼ同一のアミノ酸組成及びアミノ酸配列を有していることが報告されており（T. Oka, et al. Proc. Natl. Acad. Sci., 82, 7212-7216, 1985）、本発明のタンパク質組成物は大腸菌由来のEGFとは明らかに異なる特徴を有する。
The protein composition of the present invention introduces a nucleic acid encoding the same amino acid sequence (SEQ ID NO: 2) as mature human EGF into a plant cell to express the protein, and then extracts and purifies the protein in the plant cell. Can be obtained. The protein composition is composed mainly of three types of fragments of the protein encoded by the nucleic acid, and has three peaks (m / z 5113 ± 5, m / z) in the range of m / z 5000-5500 in mass spectrometry. 5200 ± 5 and m / z 5315 ± 5) (FIG. 5). According to further detailed analysis, the three types of protein fragments contained in the protein composition are the fragments consisting of the region of amino acids 1 to 47 in the amino acid sequence shown in SEQ ID NO: 2, and the 2nd to 47th amino acids. It has been identified as a fragment consisting of the amino acid region and a fragment consisting of the 3rd to 47th amino acid region (see Examples below). It is considered that such a protein fragment is generated as a result of modification of the protein expressed in the plant cell such as enzymatic cleavage in the cell. It has been reported that the protein composition obtained when EGF protein is produced in E. coli has almost the same amino acid composition and amino acid sequence as natural human-derived EGF protein (T. Oka, et al. Proc. Natl. Acad. Sci., 82, 7212-7216, 1985), the protein composition of the present invention has distinctly different characteristics from EGF derived from E. coli.
本発明のタンパク質組成物は細胞増殖を促進する活性を有する。この細胞増殖活性は、EGF受容体に対するアゴニスト活性によりもたらされる。すなわち、本発明のタンパク質組成物は、EGF受容体に結合して下流のシグナル伝達系を作動させ、それにより細胞の増殖を促進する。上記した３種類のタンパク質断片には、成熟型ヒトEGF分子が生物活性を発揮するために重要な領域として知られる第６番Cys～第４２番Cys（He-Shu Lu, et al, J. Biol. Chem., 276, 34913-34917 (2001); H. Ogiso et al. Cell, 110,775-787 (2002)）に該当する領域が含まれており、本発明のタンパク質組成物がEGF受容体に対するアゴニスト活性を有することと合致する。細胞増殖活性は、例えばG. Carpenter and J. Zendegui, Analytical Biochemistry, 153, 279 (1985)に記載される方法で確認することができる。下記実施例には、ヒト線維芽細胞株を用いてタンパク質組成物の細胞増殖活性を確認する方法が詳述されている。また、EGF受容体への結合能は、例えばR. L. Ladda, et al, Analytical Biochemistry, 93, 286 (1979)に記載される方法で調べることができる。
The protein composition of the present invention has an activity of promoting cell proliferation. This cell proliferating activity is brought about by an agonistic activity on the EGF receptor. That is, the protein composition of the present invention binds to the EGF receptor and activates a downstream signal transduction system, thereby promoting cell proliferation. The three types of protein fragments described above include the 6th to 42nd Cys (He-Shu Lu, et al, J. Biol), which is known as an important region for the mature human EGF molecule to exert biological activity. .Chem., 276, 34913-34917 (2001); H. Ogiso et al. Cell, 110,775-787 (2002)), and the protein composition of the present invention is an agonist for the EGF receptor. Consistent with having activity. Cell proliferation activity can be confirmed by, for example, the method described in G. Carpenter and J. Zendegui, Analytical Biochemistry, 153, 例 え ば 279 例 え ば (1985). The following examples detail the method for confirming the cell growth activity of a protein composition using a human fibroblast cell line. The ability to bind to the EGF receptor can be examined by the method described in, for example, R.RL. Ladda, et al, Analytical Biochemistry, 93, 286 (1979).
以下、本発明のタンパク質組成物の製造方法について説明する。
Hereinafter, a method for producing the protein composition of the present invention will be described.
本発明のタンパク質組成物の製造に用いる植物細胞は、培養細胞でも植物個体でもよいが、植物個体内で発現させたタンパク質を回収してタンパク質組成物を得ることが好ましい。
The plant cell used for the production of the protein composition of the present invention may be a cultured cell or a plant individual, but it is preferable to obtain a protein composition by recovering the protein expressed in the plant individual.
植物細胞内に導入する核酸には、成熟型ヒトEGFと同一のアミノ酸配列がコードされている。ヒトEGF遺伝子はNCBIのデータベースGeneに1950のGene IDで登録されており、その配列は例えばGenBankにaccession No. NM_001963で登録されている。配列表の配列番号１及び２には、天然のヒトEGF遺伝子中の、成熟型EGFをコードする領域の塩基配列、及び該成熟型EGFのアミノ酸配列を示す。
The nucleic acid to be introduced into plant cells encodes the same amino acid sequence as mature human EGF. The human EGF gene is registered in the NCBI database Gene with a Gene ID of 1950, and the sequence is registered in GenBank as accession No. NM_001963, for example. SEQ ID Nos. 1 and 2 in the sequence listing show the base sequence of the region encoding mature EGF and the amino acid sequence of the mature EGF in the natural human EGF gene.
該核酸において、成熟型ヒトEGF配列をコードする塩基配列は、植物細胞内で効率よくタンパク質を発現できるようにコドンを改変した塩基配列であることが好ましい。Met及びTrp以外の１８種のアミノ酸は２～６種の同義語コドンにコードされているが、生物の種類に応じて同義語コドンの使用頻度が異なっていることが知られている（Grantham, R. (1980) Trends Biochem. Sci. 5, 327-331.; Grantham, R., Gautier, C. and Gouy, M. (t980) Nucl. Acids Res. 8, 1893-1912.; Grantham, R., Gautier, C., Gouy, M., Mercier, R. and Pave, A. (1980) Nucl. AcidsRes. 8, r49-r62.; Grantham, R., Gautier, C., Gouyt, M., Jacobzone, M. and Mercier, R. (1981) Nucd.Acids Res. 9, r43-r74.; Aota, S.-I., Gojobori, T., Ishibashi, F., Maruvama, T. and Ikemura, T. (1988) Nucl.Acids Res. 16, r315-r402.）。Murrayらは植物におけるコドンの使用頻度について報告しており（Murray et al. (1989) Nucl. AcidsRes. 17, 477-498）、当該報告を参考にして、植物細胞に導入する核酸のコドンを植物細胞内での発現効率が高まるように改変することが可能である。
In the nucleic acid, the base sequence encoding the mature human EGF sequence is preferably a base sequence in which codons are modified so that the protein can be efficiently expressed in plant cells. Eighteen amino acids other than Met and Trp are encoded by 2 to 6 synonymous codons, but it is known that the frequency of use of synonymous codons varies depending on the type of organism (Grantham, R. (1980) Trends Biochem. Sci. 5, 327-331 .; Grantham, R., Gautier, C. and Gouy, M. (t980) Nucl. Acids Res. 8, 1893-1912 .; Grantham, R. , Gautier, C., Gouy, M., Mercier, R. and Pave, A. (1980) Nucl. AcidsRes. 8, r49-r62 .; Grantham, R., Gautier, C., Gouyt, M., Jacob , M. and Mercier, R. (1981) Nucd.Acids Res. 9, r43-r74 .; Aota, S.-I., Gojobori, T., Ishibashi, F., Maruvama, T. and Ikemura, T. (1988) Nucl. Acids Res. 16, 315r315-r402.). Murray et al. Reported the frequency of codon usage in plants (Murray et al. (1989) Nucl. AcidsRes. 17, 477-498). It can be modified to increase the expression efficiency in the cell.
１８種のアミノ酸の同義語コドンのうち、植物（特に双子葉植物）で使用頻度の高いコドンとしては、表１に示すものを挙げることができる。これらのコドンは植物において好ましく使用できるコドンである。本発明において、成熟型ヒトEGF配列をコードする塩基配列を改変する場合には、ヒトEGF遺伝子の塩基配列をもとに、各アミノ酸をコードするコドンの一部又は全部を表１に示したコドンに置き換えればよい。
Among the 18 synonymous codons of amino acids, examples of codons frequently used in plants (particularly dicotyledonous plants) include those shown in Table 1. These codons can be preferably used in plants. In the present invention, when a base sequence encoding a mature human EGF sequence is modified, a part or all of the codons encoding each amino acid is shown in Table 1 based on the base sequence of the human EGF gene. Should be replaced.
配列番号３に示す塩基配列は、植物細胞内で効率よく発現できるようにコドンの最適化を施した、成熟型ヒトEGF配列をコードする塩基配列の一例である。本発明のタンパク質組成物を製造する際には、当該配列を特に好ましく用いることができる。この塩基配列は、天然の成熟型ヒトEGFコード配列（配列番号１）のうちの３８個の塩基を変更してコドンを改変した配列である。本発明のタンパク質組成物を製造する際には、当該配列を特に好ましく用いることができる。また、配列番号３と完全に同一の塩基配列だけではなく、少数の塩基が相違する程度の配列であれば、とりわけ上記表１に例示したような植物で使用頻度の高いコドンを利用することになる配列であれば、配列番号３の塩基配列と同様に好ましく使用可能である。ここでいう「少数」とは、例えば１個～１５個、１個～１２個、１個～数個、１個～５個、又は１個～３個であり得る。配列番号３との同一性（％）で表現した場合、配列番号３と同様に使用可能な改変塩基配列は、配列番号３との同一性が９０％以上、９２％以上、９５％以上、又は９８％以上であり得る。
The base sequence shown in SEQ ID NO: 3 is an example of a base sequence encoding a mature human EGF sequence that has been subjected to codon optimization so that it can be efficiently expressed in plant cells. In producing the protein composition of the present invention, the sequence can be particularly preferably used. This base sequence is a sequence obtained by changing the codon by changing 38 bases in the natural mature human EGF coding sequence (SEQ ID NO: 1). In producing the protein composition of the present invention, the sequence can be particularly preferably used. Further, not only a completely identical base sequence to SEQ ID NO: 3, but also a codon frequently used in plants as exemplified in Table 1 above, as long as the sequence is such that a small number of bases are different. In the same manner as the base sequence of SEQ ID NO: 3. The “minority” here may be, for example, 1 to 15, 1 to 12, 1 to several, 1 to 5, or 1 to 3. When expressed in terms of identity (%) with SEQ ID NO: 3, the modified base sequence that can be used in the same manner as SEQ ID NO: 3 has an identity with SEQ ID NO: 3 of 90% or more, 92% or more, 95% or more, or It can be 98% or more.
ここで、塩基配列の「同一性」とは、比較すべき２つの塩基配列の塩基ができるだけ多く一致するように両配列を整列させ、一致した塩基数を、全塩基数で除したものを百分率で表したものである。上記整列の際には、必要に応じ、比較する２つの配列の一方又は双方に適宜ギャップを挿入する。このような配列の整列化は、例えばBLAST、FASTA、CLUSTAL W等の周知のプログラムを用いて行なうことができる。ギャップが挿入される場合、上記全塩基数は、１つのギャップを１つの塩基として数えた塩基数となる。このようにして数えた全塩基数が、比較する２つの配列間で異なる場合には、同一性（％）は、長い方の配列の全塩基数で、一致した塩基数を除して算出される。ただし、比較すべき配列が他の任意の配列と連結された状態にある場合には（例えば、発現ベクターに組み込まれた状態など）、相当する領域のみを取り出して配列を対比し、同一性を算出する。例えば、成熟型ヒトEGFをコードする塩基配列を比較するときには、当該成熟型ヒトEGFをコードする領域に相当する領域のみを取り出して配列を対比する。
Here, the “identity” of the base sequences is a percentage obtained by aligning both sequences so that the bases of the two base sequences to be compared match as much as possible, and dividing the number of matched bases by the total number of bases. It is represented by. In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary. Such sequence alignment can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like. When gaps are inserted, the total number of bases is the number of bases obtained by counting one gap as one base. When the total number of bases counted in this way differs between the two sequences to be compared, the identity (%) is calculated by dividing the total number of bases of the longer sequence and dividing the number of matched bases. The However, when the sequence to be compared is linked to any other sequence (for example, incorporated into an expression vector), only the corresponding region is taken out and the sequences are compared, calculate. For example, when comparing base sequences encoding mature human EGF, only the region corresponding to the region encoding mature human EGF is taken out and the sequences are compared.
成熟型EGF配列をコードする核酸には、発現させたタンパク質の細胞内輸送や局在化等のために有用なシグナルペプチドをコードする核酸が連結され得る。シグナルペプチドは、通常、３～６０残基程度のアミノ酸からなり、細胞質内で合成されたタンパク質の輸送及び局在化を指示する構造である。
The nucleic acid encoding a mature EGF sequence can be linked to a nucleic acid encoding a signal peptide useful for intracellular transport and localization of the expressed protein. A signal peptide usually consists of amino acids of about 3 to 60 residues, and is a structure that directs the transport and localization of a protein synthesized in the cytoplasm.
本発明においては、細胞内輸送のためのシグナルペプチドとして、タバコN. plumbaginifoliaが有するエクステンシンのシグナルペプチドを好ましく用いることができる。エクステンシンシグナルペプチドは、成熟型EGF配列のN末端側に連結して用いることができる。N. plumbaginifoliaのエクステンシン遺伝子配列は、GenBankにaccession No. M34371で登録されており、この登録配列中に見られるシグナルペプチドコード領域の塩基配列を配列番号４に、当該シグナルペプチドのアミノ酸配列を配列番号５に示す。
In the present invention, the signal peptide of extensin possessed by tobacco N. plumbaginifolia can be preferably used as a signal peptide for intracellular transport. The extensin signal peptide can be used by linking to the N-terminal side of the mature EGF sequence. The extensin gene sequence of N. plumbaginifolia is registered with GenBank as accession No. M34371. The nucleotide sequence of the signal peptide coding region found in this registered sequence is SEQ ID NO: 4, and the amino acid sequence of the signal peptide is sequenced. The number 5 is shown.
シグナルペプチドをコードする塩基配列も、成熟型EGF配列をコードする核酸と同様に、植物細胞内で効率よく発現されるようにコドンを改変して用いることができる。配列番号６に示す塩基配列は、一過性発現系を用いた植物細胞内での発現のためにコドンを最適化したエクステンシンシグナルペプチド配列をコードする塩基配列の一例である。この塩基配列は、天然のタバコエクステンシンシグナルペプチドコード配列（配列番号６）のうちの２９個の塩基を変更してコドンを改変した配列である。本発明のタンパク質組成物を製造する際には、細胞内輸送のためのシグナルペプチドをコードする配列として、当該配列を特に好ましく用いることができる。また、配列番号６と完全に同一の配列だけではなく、少数（例えば１個～１５個、１個～１２個、１個～数個、１個～５個、又は１個～３個）の塩基が相違する配列、とりわけ上記表１に例示したような植物で使用頻度の高いコドンを利用することになる配列であれば、配列番号６の塩基配列と同様に好ましく使用可能である。同一性（％）で表現した場合、配列番号６と同様に使用可能な改変塩基配列は、配列番号６との同一性が９０％以上、９２％以上、９５％以上、又は９８％以上であり得る。
The base sequence encoding the signal peptide can also be used by modifying the codon so that it can be efficiently expressed in plant cells, like the nucleic acid encoding the mature EGF sequence. The base sequence shown in SEQ ID NO: 6 is an example of a base sequence encoding an extendin signal peptide sequence in which a codon is optimized for expression in a plant cell using a transient expression system. This base sequence is a sequence obtained by changing the codons by changing 29 bases in the natural tobacco extensin signal peptide coding sequence (SEQ ID NO: 6). When producing the protein composition of the present invention, the sequence can be particularly preferably used as a sequence encoding a signal peptide for intracellular transport. In addition, not only the sequence completely identical to SEQ ID NO: 6, but also a small number (for example, 1 to 15, 1 to 12, 1 to several, 1 to 5, or 1 to 3) A sequence having a different base, particularly a sequence that uses a codon frequently used in plants as exemplified in Table 1 above, can be preferably used in the same manner as the base sequence of SEQ ID NO: 6. When expressed in terms of identity (%), a modified base sequence that can be used in the same manner as SEQ ID NO: 6 has 90% or more, 92% or more, 95% or more, or 98% or more identity with SEQ ID NO: 6. obtain.
また、本発明では、細胞内局在化のためのシグナルペプチドとして、例えばKDEL（配列番号７）の配列からなる小胞体保留シグナルを好ましく用いることができる。当該保留シグナルをコードする塩基配列としては、例えば配列番号８に示す塩基配列を好ましく用いることができるが、成熟型ヒトEGFをコードする配列やエクステンシンシグナルペプチドをコードする配列と同様に、使用可能な配列は配列番号８と完全に同一の配列にのみ限定されるものではない。
In the present invention, an endoplasmic reticulum retention signal comprising, for example, the KDEL (SEQ ID NO: 7) sequence can be preferably used as a signal peptide for intracellular localization. As the base sequence encoding the retention signal, for example, the base sequence shown in SEQ ID NO: 8 can be preferably used, but it can be used in the same manner as the sequence encoding mature human EGF and the sequence encoding extensin signal peptide. Such a sequence is not limited to a sequence that is completely identical to SEQ ID NO: 8.
配列番号９に示す塩基配列は、成熟型EGF配列をコードするコドン最適化した塩基配列（配列番号３）の上流側にエクステンシンシグナルペプチド配列をコードするコドン最適化した塩基配列（配列番号６）を、下流側に小胞体保留シグナルKDELをコードする塩基配列（配列番号８）をそれぞれ連結した塩基配列である。該塩基配列は、本発明のタンパク質組成物の製造において植物細胞内に導入する核酸の塩基配列として特に好ましい一例である。エクステンシンシグナルペプチド及び小胞体保留シグナルと融合させた外来タンパク質は、植物細胞内で発現させると、該細胞の細胞質内、特に小胞体に集積すると考えられる。
The base sequence shown in SEQ ID NO: 9 is a codon-optimized base sequence (SEQ ID NO: 6) encoding an extensin signal peptide sequence upstream of the codon-optimized base sequence (SEQ ID NO: 3) encoding the mature EGF sequence. Is a base sequence in which a base sequence (SEQ ID NO: 8) encoding an endoplasmic reticulum retention signal KDEL is linked to the downstream side. This base sequence is a particularly preferred example as a base sequence of a nucleic acid to be introduced into a plant cell in the production of the protein composition of the present invention. When expressed in a plant cell, a foreign protein fused with an extensin signal peptide and an endoplasmic reticulum retention signal is considered to accumulate in the cytoplasm of the cell, particularly in the endoplasmic reticulum.
配列番号９のような改変された塩基配列を含む核酸は、常法の化学合成により調製することができる。あるいは、周知の遺伝子工学的手法により、天然に存在する配列を有するcDNAを合成した後、このcDNAを鋳型として、適当なプライマーを用いて適宜変異を導入することにより調製することもできる。あるいはまた、調製すべき核酸をいくつかの短い領域に分けて複数の核酸断片を化学合成し、公知のfusion PCR法により各断片を連結させて調製することもできる。
A nucleic acid containing a modified base sequence such as SEQ ID NO: 9 can be prepared by conventional chemical synthesis. Alternatively, it can also be prepared by synthesizing a cDNA having a naturally occurring sequence by a well-known genetic engineering technique, and then introducing appropriate mutations using this cDNA as a template and using appropriate primers. Alternatively, the nucleic acid to be prepared can be divided into several short regions, a plurality of nucleic acid fragments can be chemically synthesized, and each fragment can be ligated by a known fusion PCR method.
本発明のタンパク質組成物の製造において、植物細胞内でのタンパク質の発現は、一過性の発現であることが好ましい。タンパク質を「一過性に発現させる」とは、該タンパク質をコードする核酸が宿主植物細胞のゲノムに組み込まれない状態で該植物細胞内で発現させることをいう。そのような一過性発現は、ウイルスベクターを用いることで実施できる。植物細胞内で所望の外来遺伝子を発現させるための植物ウイルスベクターは各種のものが知られており（例えば、「蛋白質 核酸 酵素」、vol.45、p.607-613など参照）、市販品も各種のものが存在する。植物ウイルスベクターの中でも、最も活発に研究され広く利用されているのはタバコモザイクウイルス（TMV）ベクターであり、本発明でもTMVベクターを好ましく用いることができる。
In the production of the protein composition of the present invention, the expression of the protein in the plant cell is preferably a transient expression. “Transiently expressing” a protein means that the nucleic acid encoding the protein is expressed in the plant cell in a state where it is not integrated into the genome of the host plant cell. Such transient expression can be performed by using a viral vector. Various plant viral vectors for expressing a desired foreign gene in plant cells are known (for example, see “Protein Nucleic Acid Enzyme”, vol.45, p.607-613), and commercially available products are also available. There are various things. Among plant virus vectors, the most actively studied and widely used is the tobacco mosaic virus (TMV) vector, and the TMV vector can be preferably used in the present invention.
ウイルスベクターを用いて植物体内で外来タンパク質を一過性に発現させる場合、外来タンパク質をコードする核酸構築物は、転写反応により組換えウイルスRNAを合成する際に必要となる適当なRNAポリメラーゼプロモーター（例えば、T7プロモーター等のファージポリメラーゼプロモーター）、ウイルスのレプリカーゼcDNA、移行タンパク質cDNA及び外被タンパク質cDNAと連結される。TMV等のトバモウイルスベクターを用いる場合であれば、[RNAポリメラーゼプロモーター]－[RNAレプリカーゼcDNA]－[移行タンパク質cDNA]－[核酸構築物]－[外被タンパク質cDNA]の順で連結すればよい。通常、こうした遺伝子構築物は、プラスミドDNAの形態をとる。
When a foreign protein is transiently expressed in a plant using a viral vector, the nucleic acid construct encoding the foreign protein is an appropriate RNA polymerase promoter (for example, required for synthesizing recombinant viral RNA by a transcription reaction) , Phage polymerase promoter such as T7 promoter), viral replicase cDNA, transfer protein cDNA and coat protein cDNA. If a tobamovirus vector such as TMV is used, it may be ligated in the order of [RNA polymerase promoter]-[RNA replicase cDNA]-[transition protein cDNA]-[nucleic acid construct]-[coat protein cDNA]. Usually, these genetic constructs take the form of plasmid DNA.
TMVに基づく一過性発現系の好ましい具体例を挙げると、特許第4750285号に記載されるBSG1037及びBSG1057や、市販品ではケンタッキーバイオプロセシング社のGENEWARE（登録商標）などがある。これらのプラスミドは、全身感染性の組換えTMVを発現可能なプラスミドであり、RNAポリメラーゼプロモーター、TMVのレプリカーゼ（RNA依存性RNAポリメラーゼ）cDNA、移行タンパク質cDNA及び外被タンパク質cDNAが含まれている。所望の外来遺伝子を移行タンパク質cDNAと外被タンパク質cDNAの間に挿入し、これを鋳型として5'末端にキャップを有するRNAを合成することで、所望の外来遺伝子を植物細胞内で発現可能な全身感染性のTMVウイルスRNAを得ることができる。キャップが付加されたRNAの合成は、市販のキット（例えばライフテクノロジーズ社のmMESSAGE mMACHINE（登録商標）kitなど）を用いて容易に行うことができる。
Specific examples of TMV-based transient expression systems include BSG1037 and BSG1057 described in Japanese Patent No. 4750285, and commercially available products such as GENEWARE (registered trademark) of Kentucky Bioprocessing. These plasmids are those capable of expressing systemic infectious recombinant TMV, and include RNA polymerase promoter, TMV replicase (RNA-dependent RNA polymerase) cDNA, transfer protein cDNA and coat protein cDNA. A whole body capable of expressing a desired foreign gene in a plant cell by inserting the desired foreign gene between the transfer protein cDNA and the coat protein cDNA and synthesizing RNA having a cap at the 5 ′ end using this as a template. Infectious TMV viral RNA can be obtained. The synthesis of RNA with a cap can be easily performed using a commercially available kit (for example, mMESSAGE mMACHINE (registered trademark) kit manufactured by Life Technologies).
なお、本発明において、ウイルスの「レプリカーゼ」、「移行タンパク質」、「外被タンパク質」といった場合には、天然の植物ウイルスが有するこれら因子と同一の配列を有するもののほか、一過性発現のために好適化された、人為的な改変を含む配列を有するものも包含される。例えば、特許第4750285号に記載されるTMV発現ベクターBSG1057では、レプリカーゼcDNA及び移行タンパク質cDNAに改変が加えられ、天然のTMVとは配列が若干異なるレプリカーゼと移行タンパク質が産生されるが、このような配列の改変が加えられたものも本発明のタンパク質組成物の製造に使用可能である。
In the present invention, in the case of viral “replicase”, “transition protein”, “coating protein”, in addition to those having the same sequence as those of natural plant viruses, Also included are those having sequences that contain artificial modifications that are suitable for. For example, in the TMV expression vector BSG1057 described in Japanese Patent No. 4750285, the replicase cDNA and the transfer protein cDNA are modified to produce a replicase and transfer protein having slightly different sequences from that of natural TMV. Those with sequence modifications can also be used to produce the protein composition of the present invention.
成熟型EGF配列をコードする核酸を組み込んだ全身感染性のTMVウイルスRNAは、そのまま裸のウイルスゲノムの状態で植物に接種してもよいし、あるいは、精製した外被タンパク質と混合してカプシド形成させ、ウイルス粒子の状態にしてから植物に接種してもよい。本発明において、「組換えウイルス」という語には、裸のウイルスゲノムと、カプシド形成させて得られるウイルス粒子の両者が包含される。ウイルスの接種は、常法の通り、植物の葉にカーボン粉末やセライト等の微粒子等を用いて機械的に傷をつけて接種原と接触させることにより行なえばよい。
Systemic infectious TMV viral RNA incorporating a nucleic acid encoding the mature EGF sequence may be inoculated directly into plants in the form of a naked viral genome, or mixed with purified coat protein to form capsids. It is possible to inoculate a plant after making it into a state of virus particles. In the present invention, the term “recombinant virus” includes both naked virus genomes and virus particles obtained by encapsidation. Inoculation with the virus may be performed by mechanically scratching the leaves of the plant with fine particles such as carbon powder or celite and bringing them into contact with the inoculum as usual.
組換えウイルスを感染させた植物体内では、ウイルスが増殖し、植物細胞内で大量の外来タンパク質が生産される。ウイルス接種後１０～１５日間程度栽培した後、葉を収穫してタンパク質を抽出・精製すれば、本発明のタンパク質組成物を得ることができる。
In the plant body infected with the recombinant virus, the virus grows and a large amount of foreign protein is produced in the plant cell. After cultivation for about 10 to 15 days after virus inoculation, the protein composition of the present invention can be obtained by harvesting leaves and extracting and purifying the protein.
組換えウイルスを感染させた植物組織からの目的タンパク質の抽出及び精製は、基本的には一般的なタンパク質精製方法（C. D. Mount et al., Archives of Biochemistry and Biophysics, 240 33 (1985), U. H. Gregory and I. R. Willshire, Hoppe-Sayler's Z. Physiol. Chem., 356 1765 (1975)）を用いて行なうことができる。具体的には、例えば以下のようにして行えばよい。
The extraction and purification of the target protein from the plant tissue infected with the recombinant virus is basically performed by a general protein purification method (C. D. Mount et al., Archives of Biochemistry and Biophysics, 240 33 (1985) , U. H. Gregory and I. R. Willshire, Hoppe-Sayler's Z. Physiol. Chem.,. 356 1765 (1975)). Specifically, it may be performed as follows, for example.
タンパク分解酵素阻害剤を含む緩衝液に感染した植物の組織を加え、ミキサーによって均一化してタンパク質を抽出する。この液を遠心後、上清を回収し、限外濾過で上清中の不純なタンパク質を除去して、必要に応じて更に限外膜による濃縮を行い、粗精製物を得る。限外濾過は、例えば、Miracloth（CALBIOCHEM社）やガラス繊維ろ紙等のフィルターで濾過後、濾液を分画分子量10～30 kDa程度のメンブレンでクロスフロー濾過することにより実施できる。
∙ Add infected plant tissue to a buffer containing a protease inhibitor and homogenize with a mixer to extract the protein. After centrifuging this solution, the supernatant is recovered, and impure proteins in the supernatant are removed by ultrafiltration, and if necessary, further concentrated with an ultramembrane to obtain a crude product. The ultrafiltration can be performed, for example, by filtering with a filter such as Miracloth (CALBIOCHEM) or glass fiber filter paper, and then cross-flow filtering the filtrate with a membrane having a molecular weight cut off of about 10 to 30 kDa.
得られた粗精製物は、疎水性相互作用クロマトグラフィー担体を用いてさらに１回以上精製する。担体は特に限定されず、ゲルや樹脂など、疎水性相互作用クロマトグラフィーに一般的に用いられている公知の担体のいずれであってもよい。疎水性相互作用クロマトグラフィー担体としては、官能基としてフェニル基を有するもの、例えばフェニル－セファロース担体が好ましく用いられる。粗精製物に終濃度1～3 Mの硫酸アンモニウム等を添加して塩濃度を高めた後、疎水性相互作用クロマトグラフィー担体と接触させ、目的タンパク質を担体に吸着させる。次いで、数mM程度の濃度のリン酸ナトリウム等の緩衝液（pHは中性付近のものを用いる）で吸着したタンパク質を溶出させる。
The obtained crude product is further purified once or more using a hydrophobic interaction chromatography carrier. The carrier is not particularly limited, and may be any known carrier that is generally used for hydrophobic interaction chromatography, such as a gel or a resin. As the hydrophobic interaction chromatography carrier, those having a phenyl group as a functional group, for example, a phenyl-sepharose carrier are preferably used. After adding salt sulfate or the like at a final concentration of 1 to 3 M to the crude product to increase the salt concentration, the protein is brought into contact with a hydrophobic interaction chromatography carrier to adsorb the target protein onto the carrier. Next, the adsorbed protein is eluted with a buffer solution such as sodium phosphate having a concentration of about several mM (the pH is near neutral).
さらに、必要に応じ、溶出液を強陰イオン交換クロマトグラフィー担体にタンパク質を吸着させ、塩基性（pH9～11程度）に調整した塩化ナトリウムを含むグリシン緩衝液で溶出させることにより、３種類のタンパク質断片を主成分として含む本発明のタンパク質組成物を得ることができる。このタンパク質組成物は、脱塩処理等、所望によりさらなる処理に付してよい。
In addition, if necessary, the protein is adsorbed on a strong anion exchange chromatography carrier and eluted with a glycine buffer containing sodium chloride adjusted to basic (pH 9 to 11), so that three types of proteins can be obtained. The protein composition of the present invention containing a fragment as a main component can be obtained. This protein composition may be subjected to further treatment as desired, such as desalting treatment.
以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。
Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
１．組換えTMV発現ベクターの調製
タバコエクステンシンシグナルペプチド配列と、５３残基の成熟型ヒトEGF配列と、小胞体保留シグナルKDELとを連結させたアミノ酸配列（配列番号１０）をコードする、改変された塩基配列からなるDNA（配列番号９）を含むインサートDNA（配列番号１１）は、GeneArt社（現Life Technologies社）に依頼し、化学合成により作製した。 1. Preparation of Recombinant TMV Expression Vector Modified to encode an amino acid sequence (SEQ ID NO: 10) linked to tobacco extensin signal peptide sequence, 53-residue mature human EGF sequence, and endoplasmic reticulum retention signal KDEL The insert DNA (SEQ ID NO: 11) containing the DNA consisting of the base sequence (SEQ ID NO: 9) was prepared by chemical synthesis at the request of GeneArt (currently Life Technologies).
タバコエクステンシンシグナルペプチド配列と、５３残基の成熟型ヒトEGF配列と、小胞体保留シグナルKDELとを連結させたアミノ酸配列（配列番号１０）をコードする、改変された塩基配列からなるDNA（配列番号９）を含むインサートDNA（配列番号１１）は、GeneArt社（現Life Technologies社）に依頼し、化学合成により作製した。 1. Preparation of Recombinant TMV Expression Vector Modified to encode an amino acid sequence (SEQ ID NO: 10) linked to tobacco extensin signal peptide sequence, 53-residue mature human EGF sequence, and endoplasmic reticulum retention signal KDEL The insert DNA (SEQ ID NO: 11) containing the DNA consisting of the base sequence (SEQ ID NO: 9) was prepared by chemical synthesis at the request of GeneArt (currently Life Technologies).
その一方で、組換えタバコモザイクウイルスを発現可能な公知のプラスミドBSG1057（特許第4750285号）を改変して作製したプラスミドUB001（図１）をPacI及びXhoIで消化してリニア化し、アガロースゲルで泳動後にゲルから抽出・精製した。
On the other hand, a plasmid UB001 (Fig. 1) prepared by modifying a known plasmid BSG1057 (patent No. 4750285) capable of expressing a recombinant tobacco mosaic virus was linearized by digestion with PacI and XhoI, and was run on an agarose gel. It was later extracted and purified from the gel.
上記のインサートDNAとリニア化UB001をT4 DNAリガーゼ（インビトロジェン）でライゲーションした（３７℃、２０分）。得られたプラスミドDNAをヒートショックにより大腸菌NEB5-α（New England BioLabs）に導入し、アンピシリン含有培地でスクリーニングしてシングルコロニーを増殖させ、この大腸菌細胞からmini prepキット（キアゲン）を用いてプラスミドDNAを抽出・精製することにより、UB001のタバコモザイクウイルスMP領域とCP領域との間にインサートDNAが挿入された組換えTMV発現プラスミドを得た。PacIとXhoIでプラスミドを消化してインサートサイズを確認した。
The above insert DNA and linearized UB001 were ligated with T4 DNA ligase (Invitrogen) (37 ° C., 20 minutes). The obtained plasmid DNA was introduced into E. coli NEB5-α (New England BioLabs) by heat shock, screened with ampicillin-containing medium, single colonies were grown, and plasmid DNA was used from this E. coli cell using mini prep kit (Qiagen). Was extracted and purified to obtain a recombinant TMV expression plasmid in which an insert DNA was inserted between the tobacco mosaic virus MP region and CP region of UB001. The insert size was confirmed by digesting the plasmid with PacI and XhoI.
２．組換えTMV粒子の調製
上記で得た組換えTMV発現プラスミドDNAを鋳型として、Ambion mMessage mMachine kit（Life Technologies）を用いて転写反応を行なった。反応液組成は以下の通りとした。 2. Preparation of Recombinant TMV Particles Using the recombinant TMV expression plasmid DNA obtained above as a template, a transcription reaction was performed using Ambion mMessage mMachine kit (Life Technologies). The reaction solution composition was as follows.
上記で得た組換えTMV発現プラスミドDNAを鋳型として、Ambion mMessage mMachine kit（Life Technologies）を用いて転写反応を行なった。反応液組成は以下の通りとした。 2. Preparation of Recombinant TMV Particles Using the recombinant TMV expression plasmid DNA obtained above as a template, a transcription reaction was performed using Ambion mMessage mMachine kit (Life Technologies). The reaction solution composition was as follows.
反応液を３７℃で１～３時間インキュベートし、成熟型ヒトEGFと同一のアミノ酸配列にエクステンシンシグナルペプチドと小胞体保留シグナルペプチドを融合させたタンパク質を発現可能な組換えTMV RNAを調製した。反応液のうち0.5μLを１％アガロースゲルで電気泳動し、転写産物を確認した。
The reaction solution was incubated at 37 ° C. for 1 to 3 hours to prepare a recombinant TMV RNA capable of expressing a protein in which an extensin signal peptide and an endoplasmic reticulum retention signal peptide were fused to the same amino acid sequence as mature human EGF. 0.5 μL of the reaction solution was electrophoresed on a 1% agarose gel to confirm the transcription product.
反応液19.5μLを、1.58mLのヌクレアーゼフリー水、0.2mLの1Mリン酸ナトリウムpH7.0、及び0.2mLの精製TMV外被タンパク質（10mg/mL、TMV感染タバコ葉より抽出精製したもの）と混合し、室温で２時間～一晩インキュベートすることで、カプシド形成反応を行なった。これにより、成熟型ヒトEGFと同一のアミノ酸配列にエクステンシンシグナルペプチドと小胞体保留シグナルペプチドを融合させたタンパク質（配列番号１０）を発現可能な組換えTMV粒子を得た。
Mix 19.5 μL of the reaction solution with 1.58 mL of nuclease-free water, 0.2 mL of 1 M sodium phosphate pH 7.0, and 0.2 mL of purified TMV coat protein (10 mg / mL, extracted and purified from TMV-infected tobacco leaves) The capsid formation reaction was carried out by incubating at room temperature for 2 hours to overnight. As a result, recombinant TMV particles capable of expressing a protein (SEQ ID NO: 10) in which an extensin signal peptide and an endoplasmic reticulum retention signal peptide were fused to the same amino acid sequence as mature human EGF were obtained.
３．接種原の調製
カプシド形成反応後の反応液を用いて接種原を調製した。接種原の調製に用いるバッファー（GENEWARE（登録商標）Inoculation Buffer（GIB））は、以下の通りに調製した。 3. Preparation of inoculum An inoculum was prepared using the reaction solution after the capsid formation reaction. A buffer (GENEWARE (registered trademark) Inoculation Buffer (GIB)) used for preparing the inoculum was prepared as follows.
カプシド形成反応後の反応液を用いて接種原を調製した。接種原の調製に用いるバッファー（GENEWARE（登録商標）Inoculation Buffer（GIB））は、以下の通りに調製した。 3. Preparation of inoculum An inoculum was prepared using the reaction solution after the capsid formation reaction. A buffer (GENEWARE (registered trademark) Inoculation Buffer (GIB)) used for preparing the inoculum was prepared as follows.
ビーカーに250mLの18.2 MΩ水、2.25gのグリシン、3.14gのK2HPO4、3.00gのNa4P2O7・10H2Oを入れ、スターラーで１５分以上撹拌した。溶解後の溶液を500mL容のメスシリンダーに移し、18.2 MΩ水にて300mLに調整した。これをオートクレーブ可能な容器に移し替え、3.00gのベントナイトと3.00gのセライトを加え、回旋撹拌してベントナイトとセライトを十分に湿らせた後、オートクレーブ処理（121℃、20分）した。冷却後は４℃で保存した（４℃で１年間安定）。
In a beaker, 250 mL of 18.2 MΩ water, 2.25 g of glycine, 3.14 g of K 2 HPO 4 and 3.00 g of Na 4 P 2 O 7 · 10H 2 O were added, and the mixture was stirred with a stirrer for 15 minutes or more. The solution after dissolution was transferred to a 500 mL graduated cylinder and adjusted to 300 mL with 18.2 MΩ water. This was transferred to a container capable of autoclaving, 3.00 g of bentonite and 3.00 g of celite were added, and the mixture was swirled to sufficiently wet the bentonite and celite, and then autoclaved (121 ° C., 20 minutes). After cooling, it was stored at 4 ° C. (stable at 4 ° C. for 1 year).
カプシド形成反応液の原液又は適宜ヌクレアーゼフリー水で希釈した液を等量のGIBと混合し、接種原として用いた。タバコ（Nicotiana benthamiana）１個体につき２枚の本葉に、葉１枚当たり30μLの接種原を滴下し、手袋をはめた指で軽く擦って葉に傷をつけることにより、組換えTMVをタバコに感染させた。
A stock solution of a capsid formation reaction solution or a solution diluted appropriately with nuclease-free water was mixed with an equal amount of GIB and used as an inoculum. Recombinant TMV can be applied to tobacco by dripping 30 μL of the inoculum per leaf onto two true leaves per tobacco (Nicotiana benthamiana) and rubbing the leaves lightly with a gloved finger. Infected.
４．タバコ葉からのEGFの回収(1)
接種後12日間栽培した後、タバコ葉を収穫した。収穫した500gの葉は、抽出までの間、約45分間氷上に置いた。抽出用緩衝液（50mMトリス緩衝液（pH8.5）、10mM EDTA、1mM PMSF（Phenylmethylsulfonyl fluoride、プロテアーゼ阻害剤として）、0.1% Triton X-100）は、抽出作業まで4℃に冷蔵保存した。500g の葉に500mLの抽出用緩衝液を加えて、ジューサーで破砕抽出して、ガラス瓶に入れて氷液に保存した。この液を10,000gで10分間遠心して、その後の濾過の材料とした。遠心後の上清をMiracloth 及び30KDa PES Kvick flow UFカセット(0.1m2)で濾過することにより、濾液中の大部分のタンパク質を除去して組換えタンパク質を濃縮した（30KDa濾過液）。また、この30KDa濾過液を2KDa Hydrosart Sartorius カセット(0.1m2)でさらに90mLに濃縮した（2KDa濾過液）。さらに、各濾過残渣を7倍量の抽出用緩衝液（計1,600mL）で洗浄し、洗浄液を回収した（30KDa残渣、及び2KDa残渣）。 4). Recovery of EGF from tobacco leaves (1)
After cultivation for 12 days after inoculation, tobacco leaves were harvested. Harvested 500 g leaves were placed on ice for about 45 minutes until extraction. Extraction buffer (50 mM Tris buffer (pH 8.5), 10 mM EDTA, 1 mM PMSF (Phenylmethylsulfonyl fluoride, as protease inhibitor), 0.1% Triton X-100) was refrigerated at 4 ° C. until extraction. 500 mL of extraction buffer was added to 500 g of leaves, crushed and extracted with a juicer, placed in a glass bottle, and stored in ice solution. This solution was centrifuged at 10,000 g for 10 minutes to obtain a material for subsequent filtration. The supernatant after centrifugation was filtered with Miracloth and 30 KDa PES Kvick flow UF cassette (0.1 m 2 ) to remove most of the protein in the filtrate and concentrate the recombinant protein (30 KDa filtrate). The 30 KDa filtrate was further concentrated to 90 mL with a 2 KDa Hydrosart Sartorius cassette (0.1 m 2 ) (2 KDa filtrate). Furthermore, each filtration residue was washed with 7 times the amount of extraction buffer (total 1,600 mL), and the washings were collected (30 KDa residue and 2 KDa residue).
接種後12日間栽培した後、タバコ葉を収穫した。収穫した500gの葉は、抽出までの間、約45分間氷上に置いた。抽出用緩衝液（50mMトリス緩衝液（pH8.5）、10mM EDTA、1mM PMSF（Phenylmethylsulfonyl fluoride、プロテアーゼ阻害剤として）、0.1% Triton X-100）は、抽出作業まで4℃に冷蔵保存した。500g の葉に500mLの抽出用緩衝液を加えて、ジューサーで破砕抽出して、ガラス瓶に入れて氷液に保存した。この液を10,000gで10分間遠心して、その後の濾過の材料とした。遠心後の上清をMiracloth 及び30KDa PES Kvick flow UFカセット(0.1m2)で濾過することにより、濾液中の大部分のタンパク質を除去して組換えタンパク質を濃縮した（30KDa濾過液）。また、この30KDa濾過液を2KDa Hydrosart Sartorius カセット(0.1m2)でさらに90mLに濃縮した（2KDa濾過液）。さらに、各濾過残渣を7倍量の抽出用緩衝液（計1,600mL）で洗浄し、洗浄液を回収した（30KDa残渣、及び2KDa残渣）。 4). Recovery of EGF from tobacco leaves (1)
After cultivation for 12 days after inoculation, tobacco leaves were harvested. Harvested 500 g leaves were placed on ice for about 45 minutes until extraction. Extraction buffer (50 mM Tris buffer (pH 8.5), 10 mM EDTA, 1 mM PMSF (Phenylmethylsulfonyl fluoride, as protease inhibitor), 0.1% Triton X-100) was refrigerated at 4 ° C. until extraction. 500 mL of extraction buffer was added to 500 g of leaves, crushed and extracted with a juicer, placed in a glass bottle, and stored in ice solution. This solution was centrifuged at 10,000 g for 10 minutes to obtain a material for subsequent filtration. The supernatant after centrifugation was filtered with Miracloth and 30 KDa PES Kvick flow UF cassette (0.1 m 2 ) to remove most of the protein in the filtrate and concentrate the recombinant protein (30 KDa filtrate). The 30 KDa filtrate was further concentrated to 90 mL with a 2 KDa Hydrosart Sartorius cassette (0.1 m 2 ) (2 KDa filtrate). Furthermore, each filtration residue was washed with 7 times the amount of extraction buffer (total 1,600 mL), and the washings were collected (30 KDa residue and 2 KDa residue).
タバコ葉破砕液、遠心上清、30KDa濾過液、30KDa残渣、2KDa濾過液及び2KDa残渣の各試料について、ヒトEGFに特異的な抗体を用いたウエスタンブロットによりタンパク質の検出を行なった。その結果、破砕液、遠心上清のほか、30kDa濾過液試料と2kDa濾過残渣試料中に約5.5kDaの組換えタンパク質が検出された。タバコ葉細胞内で組換えTMVから産生された組換えタンパク質は、シグナルペプチドが付加された状態で翻訳されるが、その後に植物細胞内の酵素反応によってシグナルペプチドが外されるので、上記したサイズで検出される結果となった。
For each sample of tobacco leaf crushing solution, centrifugal supernatant, 30 KDa filtrate, 30 KDa residue, 2 KDa filtrate and 2 KDa residue, protein was detected by Western blot using an antibody specific for human EGF. As a result, about 5.5 kDa recombinant protein was detected in the 30 kDa filtrate sample and the 2 kDa filtration residue sample in addition to the disrupted solution and the centrifugal supernatant. Recombinant protein produced from recombinant TMV in tobacco leaf cells is translated with the signal peptide added, but the signal peptide is subsequently removed by an enzymatic reaction in the plant cell. As a result, it was detected.
５．タバコ葉からのEGFの回収(2)
接種後12日間栽培した後、タバコ葉を収穫した。収穫した1500gの葉は、抽出までの間、約45分間氷上に置いた。抽出用緩衝液（50mMトリス緩衝液（pH8.5）、10mM EDTA、1mM PMSF（Phenylmethylsulfonyl fluoride、プロテアーゼ阻害剤として）、0.1% Triton X-100）は、抽出作業まで4℃に冷蔵保存した。1500g の葉に1500mLの抽出用緩衝液を加えて、ジューサーで破砕抽出して、ガラス瓶に入れて氷液に保存した。この液を10,000gで10分間遠心して、その後の濾過の材料とした。遠心後の上清をMiracloth 及び30KDa PES Kvick flow UFカセット(0.1m2)で濾過することにより、濾液中の大部分のタンパク質を除去して組換えタンパク質を濃縮した。この30KDa濾過液に1.5Mになるように硫酸アンモニウムを加えてPhenyl Sepharose FFカラムに吸着させた後、5mMリン酸ナトリウム緩衝液（pH7.0）で溶出した。更に、この液に2.5Mになるように硫酸アンモニウムを加えてPhenyl Sepharose FFカラムに吸着させた後、5mMリン酸ナトリウム緩衝液（pH7.0）で溶出した。この液をNuviaQカラムを通し組換えタンパク質を吸着させて、500mM塩化ナトリウムを加えたグリシン緩衝液（pH 10.0）で溶出した。これを組換えタンパク質試料として、SDS-PAGE・銀染色による純度の測定及び生物活性の測定を実施した。 5. Recovery of EGF from tobacco leaves (2)
After cultivation for 12 days after inoculation, tobacco leaves were harvested. Harvested 1500 g leaves were placed on ice for about 45 minutes until extraction. Extraction buffer (50 mM Tris buffer (pH 8.5), 10 mM EDTA, 1 mM PMSF (Phenylmethylsulfonyl fluoride, as protease inhibitor), 0.1% Triton X-100) was refrigerated at 4 ° C. until extraction. 1500 g of extraction buffer was added to 1500 g of leaves, crushed and extracted with a juicer, and stored in an ice solution in a glass bottle. This solution was centrifuged at 10,000 g for 10 minutes to obtain a material for subsequent filtration. The supernatant after centrifugation was filtered with Miracloth and 30 KDa PES Kvick flow UF cassette (0.1 m 2 ) to remove most of the protein in the filtrate and concentrate the recombinant protein. Ammonium sulfate was added to the 30 KDa filtrate to 1.5 M and adsorbed on a Phenyl Sepharose FF column, followed by elution with 5 mM sodium phosphate buffer (pH 7.0). Further, ammonium sulfate was added to this solution to 2.5 M and adsorbed on a Phenyl Sepharose FF column, followed by elution with 5 mM sodium phosphate buffer (pH 7.0). This solution was passed through a NuviaQ column to adsorb the recombinant protein, and eluted with glycine buffer (pH 10.0) supplemented with 500 mM sodium chloride. Using this as a recombinant protein sample, purity measurement and biological activity measurement were performed by SDS-PAGE and silver staining.
接種後12日間栽培した後、タバコ葉を収穫した。収穫した1500gの葉は、抽出までの間、約45分間氷上に置いた。抽出用緩衝液（50mMトリス緩衝液（pH8.5）、10mM EDTA、1mM PMSF（Phenylmethylsulfonyl fluoride、プロテアーゼ阻害剤として）、0.1% Triton X-100）は、抽出作業まで4℃に冷蔵保存した。1500g の葉に1500mLの抽出用緩衝液を加えて、ジューサーで破砕抽出して、ガラス瓶に入れて氷液に保存した。この液を10,000gで10分間遠心して、その後の濾過の材料とした。遠心後の上清をMiracloth 及び30KDa PES Kvick flow UFカセット(0.1m2)で濾過することにより、濾液中の大部分のタンパク質を除去して組換えタンパク質を濃縮した。この30KDa濾過液に1.5Mになるように硫酸アンモニウムを加えてPhenyl Sepharose FFカラムに吸着させた後、5mMリン酸ナトリウム緩衝液（pH7.0）で溶出した。更に、この液に2.5Mになるように硫酸アンモニウムを加えてPhenyl Sepharose FFカラムに吸着させた後、5mMリン酸ナトリウム緩衝液（pH7.0）で溶出した。この液をNuviaQカラムを通し組換えタンパク質を吸着させて、500mM塩化ナトリウムを加えたグリシン緩衝液（pH 10.0）で溶出した。これを組換えタンパク質試料として、SDS-PAGE・銀染色による純度の測定及び生物活性の測定を実施した。 5. Recovery of EGF from tobacco leaves (2)
After cultivation for 12 days after inoculation, tobacco leaves were harvested. Harvested 1500 g leaves were placed on ice for about 45 minutes until extraction. Extraction buffer (50 mM Tris buffer (pH 8.5), 10 mM EDTA, 1 mM PMSF (Phenylmethylsulfonyl fluoride, as protease inhibitor), 0.1% Triton X-100) was refrigerated at 4 ° C. until extraction. 1500 g of extraction buffer was added to 1500 g of leaves, crushed and extracted with a juicer, and stored in an ice solution in a glass bottle. This solution was centrifuged at 10,000 g for 10 minutes to obtain a material for subsequent filtration. The supernatant after centrifugation was filtered with Miracloth and 30 KDa PES Kvick flow UF cassette (0.1 m 2 ) to remove most of the protein in the filtrate and concentrate the recombinant protein. Ammonium sulfate was added to the 30 KDa filtrate to 1.5 M and adsorbed on a Phenyl Sepharose FF column, followed by elution with 5 mM sodium phosphate buffer (pH 7.0). Further, ammonium sulfate was added to this solution to 2.5 M and adsorbed on a Phenyl Sepharose FF column, followed by elution with 5 mM sodium phosphate buffer (pH 7.0). This solution was passed through a NuviaQ column to adsorb the recombinant protein, and eluted with glycine buffer (pH 10.0) supplemented with 500 mM sodium chloride. Using this as a recombinant protein sample, purity measurement and biological activity measurement were performed by SDS-PAGE and silver staining.
銀染色の結果を図２に示す。上記の方法でタバコ葉内の組換えタンパク質を高い純度で回収精製できていることが確認された。
The result of silver staining is shown in FIG. It was confirmed that the recombinant protein in tobacco leaves could be recovered and purified with high purity by the above method.
６．タバコ由来組換えタンパク質の生物活性の測定
組換えタンパク質の生物活性として、細胞増殖活性を測定した。96穴マイクロプレートの各ウェルにヒト線維芽細胞PDL3を5000個になるように入れた。上記組換えタンパク質試料を20pg/mLから2000pg/mLまで１１段階で希釈し、各希釈試料0.5mLをウェル一列（６ウェル）ずつ、計１１列のウェルにそれぞれ加えた。PDL3の培養に用いた培養液を0.5mLずつ加えたウェル一列をネガティブコントロールとして準備した。このマイクロプレートを炭酸ガス細胞培養器にて4日間培養した。培養後、培養液を除き、テトラゾリウム系生細胞染色液を1mLを加えて3時間染色して、マイクロプレートリーダーを用いて490nmの吸光度を測定した。 6). Measurement of biological activity of tobacco-derived recombinant protein As a biological activity of the recombinant protein, cell proliferation activity was measured. 5000 human fibroblasts PDL3 were added to each well of a 96-well microplate. The recombinant protein sample was diluted in 11 steps from 20 pg / mL to 2000 pg / mL, and 0.5 mL of each diluted sample was added to each well in a total of 11 rows, one well (6 wells). One row of wells each containing 0.5 mL of the culture solution used for PDL3 culture was prepared as a negative control. The microplate was cultured in a carbon dioxide cell incubator for 4 days. After culturing, the culture solution was removed, 1 mL of a tetrazolium-based viable cell staining solution was added and stained for 3 hours, and the absorbance at 490 nm was measured using a microplate reader.
組換えタンパク質の生物活性として、細胞増殖活性を測定した。96穴マイクロプレートの各ウェルにヒト線維芽細胞PDL3を5000個になるように入れた。上記組換えタンパク質試料を20pg/mLから2000pg/mLまで１１段階で希釈し、各希釈試料0.5mLをウェル一列（６ウェル）ずつ、計１１列のウェルにそれぞれ加えた。PDL3の培養に用いた培養液を0.5mLずつ加えたウェル一列をネガティブコントロールとして準備した。このマイクロプレートを炭酸ガス細胞培養器にて4日間培養した。培養後、培養液を除き、テトラゾリウム系生細胞染色液を1mLを加えて3時間染色して、マイクロプレートリーダーを用いて490nmの吸光度を測定した。 6). Measurement of biological activity of tobacco-derived recombinant protein As a biological activity of the recombinant protein, cell proliferation activity was measured. 5000 human fibroblasts PDL3 were added to each well of a 96-well microplate. The recombinant protein sample was diluted in 11 steps from 20 pg / mL to 2000 pg / mL, and 0.5 mL of each diluted sample was added to each well in a total of 11 rows, one well (6 wells). One row of wells each containing 0.5 mL of the culture solution used for PDL3 culture was prepared as a negative control. The microplate was cultured in a carbon dioxide cell incubator for 4 days. After culturing, the culture solution was removed, 1 mL of a tetrazolium-based viable cell staining solution was added and stained for 3 hours, and the absorbance at 490 nm was measured using a microplate reader.
また、PDL3の他に、BALB/cマウス胎仔由来細胞BALB/3T3を用いて同様に生物活性を測定した。参考データとして、上記の組換えタンパク質試料に代えて市販のヒトEGFタンパク質標準品（Promega社）を用いて同様に生物活性を測定した。
In addition to PDL3, biological activity was measured in the same manner using BALB / c mouse embryo-derived cells BALB / 3T3. As reference data, the biological activity was similarly measured using a commercially available human EGF protein standard (Promega) instead of the above recombinant protein sample.
組換えタンパク質試料の生物活性測定結果を図３に示す。PDL3細胞（図3A）及びBALB/3T3細胞（図3B）いずれも、組換えタンパク質試料の添加濃度に応じて細胞の増殖が促進されており、市販のヒトEGFタンパク質（図4A, B）と遜色のない結果であった。これにより、上記の方法でタバコ葉から回収精製された組換えタンパク質試料が生物活性を有していることが確認された。
The results of measuring the biological activity of the recombinant protein sample are shown in FIG. Both PDL3 cells (Fig. 3A) and BALB / 3T3 cells (Fig. 3B) promote cell growth depending on the concentration of the recombinant protein sample added, and are comparable to commercially available human EGF protein (Fig. 4A, B). There was no result. Thereby, it was confirmed that the recombinant protein sample collected and purified from tobacco leaves by the above method has biological activity.
７．組換えタンパク質試料のTOF-MS分析
組換えタンパク質試料50μLをZipTip C18(Millipore社)によって脱塩して10μLに濃縮した。この溶液1μLとMatrix CCA(Alpha-cyano-4-hydroxy cinnamic acid) 4μLとを混合して、その1μLを，Bruker REFLEXTM Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometer (MALDI-TOF MS)解析に供した。 7). TOF-MS analysis of recombinant protein sample 50 μL of recombinant protein sample was desalted with ZipTip C18 (Millipore) and concentrated to 10 μL.Mix 1 μL of this solution with 4 μL of Matrix CCA (Alpha-cyano-4-hydroxy cinnamic acid), and use the 1 μL for Bruker REFLEXTM Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometer (MALDI-TOF MS) analysis. Provided.
組換えタンパク質試料50μLをZipTip C18(Millipore社)によって脱塩して10μLに濃縮した。この溶液1μLとMatrix CCA(Alpha-cyano-4-hydroxy cinnamic acid) 4μLとを混合して、その1μLを，Bruker REFLEXTM Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometer (MALDI-TOF MS)解析に供した。 7). TOF-MS analysis of recombinant protein sample 50 μL of recombinant protein sample was desalted with ZipTip C18 (Millipore) and concentrated to 10 μL.
得られたマススペクトルを図５に示す。m/z5113.687、5200.814、5316.018の３つのピークが確認された。
The obtained mass spectrum is shown in FIG. Three peaks of m / z 5113.687, 5208.814, and 5316.018 were confirmed.
８．組換えタンパク質試料のアミノ酸配列の分析
上記で得られた組換えタンパク質試料に含まれるペプチドのアミノ酸配列の分析は、株式会社東レリサーチセンターに委託して実施した。Edman分解によるN末端アミノ酸配列分析により、N末端より１０残基のアミノ酸配列を確認した。その結果、当該タンパク質試料には、予想されるN末端配列（配列番号２）、N末端１残基欠損配列、及びN末端２残基欠損配列の３種類の配列が確認された。 8). Analysis of the amino acid sequence of the recombinant protein sample The analysis of the amino acid sequence of the peptide contained in the recombinant protein sample obtained above was commissioned to Toray Research Center. The amino acid sequence of 10 residues from the N-terminal was confirmed by N-terminal amino acid sequence analysis by Edman degradation. As a result, three types of sequences were confirmed in the protein sample: an expected N-terminal sequence (SEQ ID NO: 2), an N-terminal 1 residue deletion sequence, and an N-terminal 2 residue deletion sequence.
上記で得られた組換えタンパク質試料に含まれるペプチドのアミノ酸配列の分析は、株式会社東レリサーチセンターに委託して実施した。Edman分解によるN末端アミノ酸配列分析により、N末端より１０残基のアミノ酸配列を確認した。その結果、当該タンパク質試料には、予想されるN末端配列（配列番号２）、N末端１残基欠損配列、及びN末端２残基欠損配列の３種類の配列が確認された。 8). Analysis of the amino acid sequence of the recombinant protein sample The analysis of the amino acid sequence of the peptide contained in the recombinant protein sample obtained above was commissioned to Toray Research Center. The amino acid sequence of 10 residues from the N-terminal was confirmed by N-terminal amino acid sequence analysis by Edman degradation. As a result, three types of sequences were confirmed in the protein sample: an expected N-terminal sequence (SEQ ID NO: 2), an N-
さらに、TOF-MSによる分子量の測定及びN末端からのアミノ酸分析の結果より、ペプチドの構造の推定を行なった。
Furthermore, the structure of the peptide was estimated from the results of molecular weight measurement by TOF-MS and amino acid analysis from the N-terminus.
上記のデータより、タンパク質試料中に含まれるペプチドは、いずれもC末端をL(ロイシン)とするペプチドであると推定された。
From the above data, it was estimated that all the peptides contained in the protein sample were peptides having C (terminal) L (leucine).
Claims (12)
- 上皮細胞増殖因子のアミノ酸配列をコードする核酸を植物細胞内に導入してタンパク質を発現させた後、該植物細胞内のタンパク質を抽出及び精製することにより得られるタンパク質組成物であって、細胞増殖を促進する活性を有し、質量分析においてm/z 5000～5500の範囲で３つのピークを呈する、タンパク質組成物。 A protein composition obtained by introducing a nucleic acid encoding an amino acid sequence of an epidermal growth factor into a plant cell to express the protein, and then extracting and purifying the protein in the plant cell. A protein composition that has an activity of promoting oxidization and exhibits three peaks in the range of m / z 5000 to 5500 in mass spectrometry.
- 抽出されたタンパク質の精製は、疎水性相互作用クロマトグラフィー担体をタンパク質溶液と接触させ、担体に吸着したタンパク質を溶出する工程を含む、請求項１記載のタンパク質組成物。 2. The protein composition according to claim 1, wherein the purification of the extracted protein includes a step of contacting the hydrophobic interaction chromatography carrier with the protein solution and eluting the protein adsorbed on the carrier.
- 疎水性相互作用クロマトグラフィー担体がフェニル－セファロースである、請求項２記載のタンパク質組成物。 The protein composition according to claim 2, wherein the hydrophobic interaction chromatography carrier is phenyl-sepharose.
- 上皮細胞増殖因子のアミノ酸配列は、配列番号２に示す５３残基の成熟型ヒト上皮細胞増殖因子のアミノ酸配列である、請求項１ないし３のいずれか１項に記載のタンパク質組成物。 The protein composition according to any one of claims 1 to 3, wherein the amino acid sequence of the epidermal growth factor is the amino acid sequence of the 53-residue mature human epidermal growth factor shown in SEQ ID NO: 2.
- 配列番号２に示すアミノ酸配列中の第１番～第４７番アミノ酸の領域からなる断片、第２番～第４７番アミノ酸の領域からなる断片、及び第３番～第４７番アミノ酸の領域からなる断片を含有する、請求項１ないし４のいずれか１項に記載のタンパク質組成物。 It consists of a fragment consisting of the 1st to 47th amino acid region, a fragment consisting of the 2nd to 47th amino acid region, and a 3rd to 47th amino acid region in the amino acid sequence shown in SEQ ID NO: 2. The protein composition according to any one of claims 1 to 4, comprising a fragment.
- 前記植物がタバコである、請求項１ないし５のいずれか１項に記載のタンパク質組成物。 The protein composition according to any one of claims 1 to 5, wherein the plant is tobacco.
- 植物細胞内でのタンパク質の発現は一過性の発現である、請求項１ないし６のいずれか１項に記載のタンパク質組成物。 The protein composition according to any one of claims 1 to 6, wherein the protein expression in the plant cell is a transient expression.
- 植物細胞内への核酸の導入及びタンパク質の発現は、前記核酸が組み込まれたウイルス発現ベクターから調製された組換えウイルスを植物体に感染させることにより行われる、請求項７記載のタンパク質組成物。 The protein composition according to claim 7, wherein the introduction of the nucleic acid into the plant cell and the expression of the protein are performed by infecting a plant with a recombinant virus prepared from a virus expression vector in which the nucleic acid is incorporated.
- ウイルス発現ベクターがタバコモザイクウイルス発現ベクターである、請求項８記載のタンパク質組成物。 The protein composition according to claim 8, wherein the virus expression vector is a tobacco mosaic virus expression vector.
- 前記核酸にコードされるタンパク質は、少なくとも１種のシグナルペプチドが付加されたタンパク質として植物細胞内で発現される、請求項１ないし９のいずれか１項に記載のタンパク質組成物。 The protein composition according to any one of claims 1 to 9, wherein the protein encoded by the nucleic acid is expressed in a plant cell as a protein to which at least one signal peptide is added.
- 前記核酸にコードされるタンパク質は、成熟型ヒト上皮細胞増殖因子のアミノ酸配列のN末側にタバコエクステンシンシグナルペプチドを、C末側に小胞体保留シグナルKDELを付加したタンパク質として植物細胞内で発現される、請求項１０記載のタンパク質組成物。 The protein encoded by the nucleic acid is expressed in plant cells as a protein with the tobacco extensin signal peptide added to the N-terminal side of the amino acid sequence of mature human epidermal growth factor and the endoplasmic reticulum retention signal KDEL added to the C-terminal side. The protein composition according to claim 10.
- 配列表の配列番号８に示される塩基配列の核酸が植物細胞に導入される、請求項１１記載のタンパク質組成物。 The protein composition according to claim 11, wherein the nucleic acid having the base sequence represented by SEQ ID NO: 8 in the sequence listing is introduced into plant cells.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001029242A2 (en) * | 1999-10-21 | 2001-04-26 | Monsanto Company | Post-translational modification of recombinant proteins produced in plants |
| US20030228612A1 (en) * | 2002-04-30 | 2003-12-11 | Kenward Kimberly D. | Production of recombinant epidermal growth factor in plants |
| WO2006015057A2 (en) * | 2004-07-29 | 2006-02-09 | Large Scale Biology Corporation | C-terminally truncated interferon |
| US20120302733A1 (en) * | 2011-05-23 | 2012-11-29 | Padgett Hal S | Selection and characterization of novel plant-derived recombinant human interferons with broad spectrum activity |
| WO2014097733A1 (en) * | 2012-12-21 | 2014-06-26 | 株式会社ＵｎｉＢｉｏ | Method for manufacturing human epidermal cell growth factor |
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2014
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001029242A2 (en) * | 1999-10-21 | 2001-04-26 | Monsanto Company | Post-translational modification of recombinant proteins produced in plants |
| US20030228612A1 (en) * | 2002-04-30 | 2003-12-11 | Kenward Kimberly D. | Production of recombinant epidermal growth factor in plants |
| WO2006015057A2 (en) * | 2004-07-29 | 2006-02-09 | Large Scale Biology Corporation | C-terminally truncated interferon |
| US20120302733A1 (en) * | 2011-05-23 | 2012-11-29 | Padgett Hal S | Selection and characterization of novel plant-derived recombinant human interferons with broad spectrum activity |
| WO2014097733A1 (en) * | 2012-12-21 | 2014-06-26 | 株式会社ＵｎｉＢｉｏ | Method for manufacturing human epidermal cell growth factor |
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