TW201602131A - Protein composition that enhances cell proliferation via EGF receptor - Google Patents

Abstract

Disclosed is a novel protein composition having an activity to promote cell proliferation via EGF receptor, which can be utilized instead of conventional EGF protein products. The protein composition according to the present invention is obtained by introducing a nucleic acid encoding the amino acid sequence of epidermal growth factor into a plant cell to express a protein, and extracting and purifying the protein in the plant cell. The composition has an activity to promote cell proliferation, and shows three peaks within the range of m/z 5000 to 5500 in mass spectrometry.

Description

Protein composition that promotes cell proliferation through the EGF receptor

The present invention relates to a novel protein composition having an activity of promoting cell proliferation through an epidermal growth factor (EGF) receptor.

The cell proliferating factor binds to the corresponding receptor, and promotes the proliferation of the cells by inducing the metabolic system activities required for the proliferation of the cells via the intracellular signaling system. Epithelial cell proliferative factor (EGF) is one of the cell proliferation factors. As a physiological activity of EGF, in addition to cell proliferation, gastric acid secretion inhibition, antiulcer action, gastrointestinal mucosal protection, deoxyribonucleic acid synthesis promotion, corneal repair, wound healing are known. Promoting action, anti-inflammatory effect, analgesic effect, etc.

EGF was originally discovered and purified from the submandibular gland of mice by Cohen et al. (Non-Patent Document 1). Thereafter, it is purified from human urine as a substance having a gastric acid secretion inhibiting action (Non-Patent Document 2). Human EGF is synthesized in vivo in the form of a precursor protein comprising 1207 amino acids, which is then processed to contain 53 amino acids with a molecular weight of about 6 kDa and 3 disulfide bonds in the molecule. (Non-Patent Document 3).

Cell proliferation factors such as EGF are used as medicines, cosmetics, etc. One of the ingredients. Regarding the effective production methods of EGF, the industry has also conducted research, and various methods for producing EGF by genetic recombination have been reported. As an example of such reports, production of coliforms (Non-Patent Document 4), production using yeast (Non-Patent Documents 5 to 7), production using plants (Patent Document 1, Non-Patent Document 8), and the like are available.

The EGF produced by genetic recombination receives various modifications in the cells depending on the species used. EGF currently used commercially is the use of EGF produced by coliforms and EGF produced by yeast. In the technique of producing EGF in bacteria, in order to closely monitor and control the fine culture conditions, an expensive system must be introduced, and thus the price of recombinant EGF and various products using the same is still expensive. If the plant can be mass-produced, it may be provided at a low cost, but it has not been established to be put into practical use by utilizing the production method of the plant. For example, in the method described in Patent Document 1, recombinant EGF is stably produced in barley in the genome of barley, and recombinant EGF is produced in barley. However, in this method, management of genetically modified plants must be performed, and the obstacles to large-scale production are large. Non-Patent Document 8 describes a method of producing a virus in which a human EGF gene is incorporated, infecting tobacco with the virus, and transiently expressing human EGF, thereby producing EGF. However, the method described in Non-Patent Document 8 has a problem in terms of yield and cannot be regarded as a practical method. Therefore, it has not yet been clarified which recombinant EGF protein is available in the case of using plants to mass produce EGF.

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] WO2011/083500

[Non-patent literature]

[Non-Patent Document 1] S. Cohen, J. Biol. Chem., 237, 1555 (1962)

[Non-Patent Document 2] H. Gregory, Nature, 257, 325 (1975)

[Non-Patent Document 3] G. I. Bell, et al., Nucleic Acids Research 14 (21), 8427 1986)

[Non-Patent Document 4] F. Kishimoto et al., Gene, 45, 311 (1986)

[Non-Patent Document 5] M. S. Urdea, et al., Proc. Natl. Acad. Sci. USA, 80 7461 (1983)

[Non-Patent Document 6] J. M. Clements, et al., Gene 106, 267 (1991)

[Non-Patent Document 7] Z. Hambali, Journal of International Studies, 10, 36 (2009)

[Non-Patent Document 8] Sonin Wirth, et al., Molecular Breeding 13: 23-35, 2004

It is an object of the present invention to provide a novel protein composition having an activity of promoting cell proliferation through an EGF receptor which can be effectively used in place of a conventional EGF protein preparation.

The inventors of the present invention conducted an effort to study, and as a result, successfully developed a technique for efficiently producing bioactive EGF from a plant by a transient expression system. The composition of the protein composition containing recombinant EGF recovered from plant leaves by this technique was confirmed by molecular weight determination by time-of-flight mass spectrometry (TOF-MS) and amino acid analysis. As a result, it was confirmed that it was the No. 48 of the amino acid sequence of 53 residues of mature human EGF. The invention of the present invention was completed by a mixture of three protein fragments of a sequence in which the amino acid to the C-terminal region was deleted.

That is, the present invention provides a protein composition obtained by introducing a nucleic acid encoding an amino acid sequence of an epithelial cell proliferation factor into a plant cell to express a protein, and extracting and purifying the protein in the plant cell. It has the activity of promoting cell proliferation, and exhibits three peaks in the range of m/z 5000~5500 during mass spectrometry.

According to the present invention, a novel protein composition having cell proliferation activity produced in plant cells based on the amino acid sequence of mature human EGF can be provided. Plants and humans belong to the multicellular organisms of eukaryotes, and the cells of both have very similar mechanisms of protein expression. The protein composition of the present invention is advantageous in terms of production cost, equipment investment, and production expandability as compared with the recombinant EGF protein produced by bacteria which is currently commercially used. According to the protein composition of the present invention, a conventional cosmetic or pharmaceutical product using recombinant EGF can be provided at a lower price.

Fig. 1 is a structural diagram of a tobacco mosaic virus (TMV) Tobacco Mosaic Virus expression vector UB001 used in the examples.

2 is a result of silver staining of EGF extracted from purified TMV infected tobacco leaves by SDS-PAGE, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis. Figure. Zone 1: molecular marker, zone 2: standard, zone 3: with C-terminal Endoplasmic reticulum message KDEL (5 times concentrated), zone 4: no C-terminal endoplasmic reticulum message (5 times concentrated), zone 5: C-terminal endoplasmic reticulum message KDEL, zone 6: none C-terminal endoplasmic reticulum message, zone 7: molecular marker.

Fig. 3 shows the results of measurement of biological activity (proliferation promoting action) of recombinant protein samples extracted and purified from recombinant TMV infected tobacco leaves. A. Human fibroblast PDL3. B. From mouse embryonic cell BALB/3T3.

Figure 4 shows the results of measurement of the biological activity (proliferation promoting action) of a commercially available human EGF protein standard. A. Human fibroblast PDL3. B. From mouse embryonic cell BALB/3T3.

Figure 5 is the result of mass spectrometry analysis of recombinant protein samples extracted from purified TMV infected tobacco leaves.

The protein composition of the present invention can be obtained by introducing a nucleic acid encoding an amino acid sequence (SEQ ID NO: 2) identical to the mature human EGF into a plant cell, and then extracting and purifying the protein in the plant cell. . The protein composition is composed of three kinds of fragments of the protein encoded by the above nucleic acid as a main component, and exhibits three peaks in the range of m/z 5000 to 5500 during mass spectrometry (m/z 5113±5, m/z). 5200 ± 5 and m / z 5315 ± 5) (Figure 5). By further detailed analysis, the three protein fragments contained in the protein composition were identified as fragments of the region containing the amino acid No. 1 to No. 47 in the amino acid sequence represented by SEQ ID NO: A fragment containing a region of the amino acid No. 2 to No. 47, and a fragment containing a region of the amino acid No. 3 to No. 47 (refer to the following examples). It is considered that a protein expressed in a plant cell is subjected to modification such as cleavage by an enzyme in a cell to produce such a protein fragment. Reported on the use of coliform to produce EGF protein The protein composition obtained in the case of a substance has an amino acid composition and an amino acid sequence which are substantially identical to the 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 a characteristic significantly different from EGF derived from Escherichia coli.

The protein composition of the present invention has an activity of promoting cell proliferation. This cell proliferative activity is obtained by agonist activity against the EGF receptor. That is, the protein composition of the present invention binds to the EGF receptor to operate the downstream message delivery system, thereby promoting cell proliferation. Among the above three kinds of protein fragments, Cys No. No. 42 Cys (He-Shu Lu, et al., J. Biol), which is known as a region important for the biological activity of a mature human EGF molecule, is known. . Chem., 276, 34913-34917 (2001); H. Ogiso et al., Cell, 110, 775-787 (2002)), with the protein composition of the present invention having agonist activity against EGF receptor The situation is the same. The cell proliferative activity can be confirmed, for example, by the method described in G. Carpenter and J. Zendegui, Analytical Biochemistry, 153, 279 (1985). In the following examples, a method of confirming the cell proliferation activity of a protein composition using a human fibroblast cell line will be described in detail. Further, the binding energy to the EGF receptor can be investigated, for example, by the method described in R. L. Ladda, et al., Analytical Biochemistry, 93, 286 (1979).

Hereinafter, a method for producing the protein composition of the present invention will be described.

The plant cell used in the production of the protein composition of the present invention may be a cultured cell or a plant individual, but it is preferred to obtain a protein composition by recovering the protein expressed in the plant individual.

Introduced into nucleic acids in plant cells, encoded with mature humans The same amino acid sequence of EGF. The human EGF gene is registered in the Gene Center of the National Center of Biotechnology Information (NCBI) with the Gene ID of 1950, and its sequence is, for example, in GenBank, accession No.NM_001963. Registration. SEQ ID NOs: 1 and 2 of the Sequence Listing represent the base sequence of the region encoding the mature EGF in the natural human EGF gene, and the amino acid sequence of the mature EGF.

In the nucleic acid, the base sequence encoding the mature human EGF sequence is preferably a base sequence in which a codon is changed in such a manner that the protein can be efficiently expressed in a plant cell. It is known that 18 amino acids other than Met and Trp are encoded by 2 to 6 synonymous codons, but the frequency of synonymous codons is different depending on the species of the 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. Acids Res. 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 et al. report the frequency of use of codons in plants (Murray et al., (1989) Nucl. Acids Res. 17, 477-498), which can be used as a reference to improve the efficiency of expression in plant cells. The codons of the nucleic acids introduced into the plant cells are altered.

Among the synonymous codons of the 18 amino acids, as the codons having a high frequency of use in plants (especially dicotyledons), those shown in Table 1 can be cited. These codons are codons that are preferably used in plants. In the present invention, when the base sequence encoding the mature human EGF sequence is changed, it is based on the human EGF gene. For the base sequence, a part or all of the codons encoding the respective amino acids may be replaced with the codons shown in Table 1.

The nucleotide sequence shown in SEQ ID NO: 3 is an example of a nucleotide sequence encoding a mature human EGF sequence which is optimized by codon expression in a plant cell. This sequence can be particularly preferably used in the production of the protein composition of the present invention. This base sequence is a sequence in which a 38-base in a natural mature human EGF coding sequence (SEQ ID NO: 1) is changed to change a codon. This sequence can be particularly preferably used in the production of the protein composition of the present invention. Further, not only the base sequence identical to the sequence number 3 but also a few bases is not used. The sequence of the same degree can be preferably used in the same manner as the nucleotide sequence of SEQ ID NO: 3, as long as it is a sequence using a codon having a higher frequency of use in plants as exemplified in Table 1 above. The "minority" referred to herein may be, for example, one to fifteen, one to twelve, one to several, one to five, or one to three. When it is expressed by the identity (%) of SEQ ID NO: 3, the identity of the base sequence system and SEQ ID NO: 3 can be changed to 90% or more, 92% or more, 95%. Above, or 98% or more.

Here, the "identity" of the base sequence is expressed by a percentage in such a manner that the bases of the two base sequences to be compared are aligned as much as possible, and the number of identical bases is divided by the total number of bases. The base is derived. At the time of the above-mentioned sorting, a gap is appropriately inserted in one of the two sequences to be compared or both. The sorting of such a sequence can be performed, for example, using a well-known program such as a Basic Local Alignment Search Tool (BLAST), a FASTA (Database Similarity Search Program), and a CLUSTAL W (Multiple Sequence Alignment Software). . In the case of inserting a gap, the total number of bases described above is the number of bases counted by using one gap as one base. When the total number of bases counted in the above manner differs between the two sequences to be compared, the identity (%) is calculated by dividing the number of identical bases by the total number of bases of the longer sequence. However, when the sequence to be compared is in a state of being connected to any other sequence (for example, a state incorporated in a performance vector, etc.), only a comparable region is extracted and the sequence is compared, and the identity is calculated. For example, when comparing base sequences encoding mature human EGF, only the region corresponding to the region encoding the mature human EGF is extracted and the sequence is compared.

In the nucleic acid encoding the mature EGF sequence, a nucleic acid encoding a useful signal peptide for intracellular delivery or localization of the expressed protein can be linked. News Polypeptides usually contain about 3 to 60 residues of amino acid, indicating the structure of the transport and localization of proteins synthesized in the cytoplasm.

In the present invention, as the message peptide for intracellular delivery, a message peptide of extensin which is possessed by Tobacco N. plumbaginifolia can be preferably used. The extensin message peptide can be used by being linked to the N-terminal side of the mature EGF sequence. The extensin gene sequence of the tobacco leaf tobacco is registered in the gene bank with accession No. M34371, and the base sequence of the message peptide coding region visible in the registration sequence is represented by SEQ ID NO: 4, and the amine of the message peptide is represented by SEQ ID NO: 5. Base acid sequence.

The base sequence encoding the message peptide can also be used by changing the codon in such a manner that it is efficiently expressed in plant cells in the same manner as the nucleic acid encoding the mature EGF sequence. The nucleotide sequence represented by SEQ ID NO: 6 is an example of a nucleotide sequence encoding an extensin message peptide sequence for optimizing a codon using a performance in a plant cell of a transient expression system. This base sequence is a sequence in which a codon is changed by changing 29 bases in the native tobacco extensin message peptide coding sequence (SEQ ID NO: 6). In the production of the protein composition of the present invention, the sequence can be particularly preferably used as a sequence encoding a message peptide for intracellular delivery. Further, not only the sequence identical to the sequence number 6 but also a few sequences (for example, one to fifteen, one to twelve, one to several, one to five, or one) can be preferably used. The sequence of ~3 bases different from each other, particularly the sequence using the codon which is frequently used in plants as exemplified in Table 1 above, can also be preferably used in the same manner as the nucleotide sequence of SEQ ID NO: 6. . When it is expressed by the identity (%), the identity of the base sequence system and the sequence number 6 can be changed in the same manner as in the SEQ ID NO: 6 to be 90% or more, 92% or more, 95% or more, or 98%. the above.

Further, in the present invention, as the message peptide for intracellular localization, for example, an endoplasmic reticulum retention message containing a sequence of KDEL (SEQ ID NO: 7) can be preferably used. As the nucleotide sequence encoding the reservation message, for example, a nucleotide sequence represented by SEQ ID NO: 8 can be preferably used, but a sequence similar to the sequence encoding the mature human EGF or the sequence encoding the extensin message peptide can be used. It is not limited to the sequence identical to SEQ ID NO: 8.

The nucleotide sequence shown in SEQ ID NO: 9 is linked to a codon-optimized base encoding the extensin message peptide sequence on the upstream side of the nucleotide sequence (SEQ ID NO: 3) encoding the codon-optimized EGF sequence. The sequence (SEQ ID NO: 6) and the base sequence encoding the nucleotide sequence of the endoplasmic reticulum retention message KDEL (SEQ ID NO: 8) are ligated to the downstream side. This base sequence is particularly preferable as the base sequence of the nucleic acid introduced into the plant cell in the production of the protein composition of the present invention. It is believed that if the foreign protein is expressed in the plant cell by fusion with the extensin message peptide and the endoplasmic reticulum retention message, it accumulates in the cytoplasm of the cell, especially the endoplasmic reticulum.

A nucleic acid comprising the altered base sequence of SEQ ID NO: 9 can be prepared by chemical synthesis by conventional methods. Alternatively, it can also be prepared by synthesizing a complementary deoxyribonucleic acid (cDNA) having a naturally occurring sequence by a well-known genetic engineering method, using the cDNA as a mold, and appropriately using an appropriate primer. Import variations. Alternatively, a plurality of nucleic acid fragments may be chemically synthesized by dividing the nucleic acid to be prepared into a plurality of shorter regions, and each fragment may be ligated by a known fusion polymerase chain reaction (fusion PCR).

In the manufacture of the protein composition of the present invention, the performance of the protein in the plant cell is preferably transient. The so-called "transient expression" of proteins, And means that the nucleic acid encoding the protein is expressed in the plant cell in a state in which it is not incorporated into the genome of the host plant cell. Such transient performance can be performed by using a viral vector. Various plant virus vectors for expressing a desired foreign gene in a plant cell are known (for example, refer to "protein nucleic acid enzyme", vol. 45, p. 607-613, etc.), and various commercially available products are also available. Among the plant viral vectors, the most active and widely used researcher is the Tobacco Mosaic Virus (TMV) vector, and the TMV vector can also be preferably used in the present invention.

When a viral vector is used to transiently express a foreign protein in a plant, the nucleic acid construct encoding the foreign protein is an appropriate RNA polymerase necessary for synthesizing recombinant viral RNA (Ribonucleic Acid) by transcription reaction. A promoter (for example, a phage polymerase promoter such as a T7 promoter), a viral replicase cDNA, a mobile protein cDNA, and an overhang protein cDNA linkage. In the case of using a Tobamovirus vector such as TMV, the [RNA polymerase promoter]-[RNA replicase cDNA]-[mobile protein cDNA]-[nucleic acid construct]-[outside The order of the protein-coated cDNA] can be linked. Typically, such genetic constructs take the form of plastid DNA.

Specific examples of the TMV-based transient performance system include BSG1037 and BSG1057 described in Japanese Patent No. 4750285, and GENEWARE (registered trademark) of Kentucky BioProcessing Co., Ltd. in a commercial product. The isoform system can express the plastid of recombinant TMV which is systemically infectious, and contains an RNA polymerase promoter, a TMV replicase (RNA-dependent RNA polymerase) cDNA, a mobile protein cDNA, and an overcoated protein cDNA. Inserting the desired foreign gene into the mobile protein cDNA and the overlying protein cDNA, and synthesizing it as a mold to synthesize the RNA having a cap at the 5' end, thereby obtaining an internal cell surface Systemically infectious TMV viral RNA of the gene is now required. The synthesis of capped RNA can be easily carried out using a commercially available kit (for example, Life Technologies' mMESSAGE mMACHINE (registered trademark) kit, etc.).

Further, in the present invention, in the case of a virus "replicase", "mobile protein", or "overcoat protein", in addition to the sequence having the same factor as that of a natural plant virus, Included are sequences that include artificial changes that are preferred for transient performance. For example, in the TMV expression vector BSG1057 described in Japanese Patent No. 4750285, a change is made to the replicase cDNA and the mobile protein cDNA, and a replicaase and a mobile protein having slightly different sequences than the native TMV are produced, but such application has Sequence changers can also be used in the manufacture of the protein compositions of the present invention.

The systemic infectious TMV viral RNA incorporating a nucleic acid encoding a mature EGF sequence can be inoculated directly into the plant under the condition of the naked virus genome, or can be mixed with the purified protein to form a protein coat, and is set as a virus. The state of the particles is inoculated into the plants. In the present invention, the term "recombinant virus" includes both a naked virus genome and virions obtained by forming a protein coat. The inoculation of the virus may be carried out by mechanically damaging the leaves of the plants by using fine particles such as carbon powder or diatomaceous earth, and bringing them into contact with the inoculum.

In plants infected with recombinant viruses, the virus proliferates and produces a large amount of foreign proteins in plant cells. The protein composition of the present invention can be obtained by cultivating the leaves and extracting the purified protein after culturing for about 10 to 15 days after virus inoculation.

Extraction and purification of a target protein from a plant tissue infected with a recombinant virus can basically be carried out using a conventional 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, for example, it may be carried out as follows.

The tissue of the infected plant is added to a buffer containing a proteolytic enzyme inhibitor, and the protein is extracted by homogenization by a stirrer. After centrifuging the liquid, the supernatant is recovered, and the impure protein in the supernatant is removed by ultrafiltration, and if necessary, concentrated by an ultrafiltration membrane to obtain a crude purified product. The ultrafiltration system can be carried out, for example, by filtering with a filter such as Miracloth (CALBIOCHEM) or glass fiber filter paper, and then filtering the filtrate by cross flow filtration using a film having a molecular weight of about 10 to 30 kDa.

The crude purified material obtained was further purified more than once using a hydrophobic interaction chromatography carrier. The carrier is not particularly limited, and may be any of known carriers which are generally used in hydrophobic interaction chromatography such as gels or resins. As the hydrophobic interaction chromatography carrier, a phenyl group as a functional group such as a phenyl-sepharose carrier can be preferably used. After adding a final concentration of 1 to 3 M ammonium sulfate or the like to the salt, the salt concentration is increased, and then contacted with a hydrophobic interaction chromatography carrier to adsorb the target protein to the carrier. Then, the adsorbed protein is eluted by a buffer such as sodium phosphate having a concentration of about several mM (using a pH near neutral).

Further, if necessary, the protein in the eluate is adsorbed to the strong anion exchange chromatography carrier, and is dissolved by a glycine buffer containing sodium chloride adjusted to be alkaline (about pH 9 to 11). A protein composition of the present invention comprising three protein fragments as a main component. The protein composition can be subjected to further treatment such as desalination treatment.

The protein composition of the present invention can be effectively used in place of the conventional EGF protein preparation. For example, it can be used as a cosmetic (lotion, lotion, beauty) Liquid, cream, foundation, makeup base, lipstick, concealer, eye shadow, etc. Moreover, it can be used as a component of a pharmaceutical product (a mouthpiece such as a tablet, a capsule, a granule, or a serum preparation, and a parenteral preparation such as an injection, a drip, or a skin external preparation). A cosmetic or pharmaceutical preparation comprising the protein composition of the present invention can be produced by a conventional method known in various fields, in addition to the use of the protein composition of the present invention in place of the conventional EGF protein preparation.

[Examples]

Hereinafter, the present invention will be more specifically described based on examples. However, the invention is not limited to the following examples.

1. Preparation of recombinant TMV expression vector

The embedded DNA (SEQ ID NO: 11) containing the DNA containing the altered base sequence (SEQ ID NO: 9) was commissioned by GeneArt (now Life Technologies) to be produced by chemical synthesis linked to tobacco stretch. The protein message peptide sequence, the 53-residue mature human EGF sequence, and the endoplasmic reticulum retention message Kdel amino acid sequence (SEQ ID NO: 10).

On the other hand, the plastid UB001 (Fig. 1) produced by the well-known plastid BSG1057 (Japanese Patent No. 4750285) which can express the recombinant tobacco mosaic virus is digested by PacI and XhoI and linearized using agarose The gel was purified from gel extraction after electrophoresis.

The above embedded DNA was ligated with linearized UB001 (37 ° C, 20 min) using T4 DNA ligase (Invitrogen). The obtained plastid DNA was introduced into Escherichia coli NEB5-α (New England BioLabs) by heat shock, and a single colony (Single colony) was proliferated by using a medium containing ampicillin, using mini The prep kit (Qiagen) extracts and purifies the plastid DNA from the coliform cells, thereby obtaining a recombinant TMV expressing plastid inserted with DNA embedded between the MP region and the CP region of the UB001 tobacco mosaic virus. The plastids were digested with PacI and XhoI and the size of the insert was confirmed.

2. Preparation of recombinant TMV particles

The recombinant TMV obtained as described above was expressed as a mold, and a transcription reaction was carried out using an Ambion mMessage mMachine kit (Life Technologies). The composition of the reaction liquid was prepared in the following manner.

The reaction solution was incubated at 37 ° C for 1 to 3 hours to prepare a recombinant TMV RNA which was expressed by fusing the extensin message peptide and the endoplasmic reticulum retention message peptide to the same amino acid sequence as the mature human EGF. Protein. 0.5 μL of the reaction mixture was subjected to electrophoresis using a 1% agarose gel to confirm the transcription product.

19.5 μL of the reaction mixture was mixed 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 overcoated protein (10 mg/mL, extracted and purified from TMV infected tobacco leaves). The protein coat formation reaction was carried out by culturing at room temperature for 2 hours to one night. Thereby, a recombinant TMV particle which expresses a protein obtained by fusing an extensin message peptide and an endoplasmic reticulum retention message peptide to the same amino acid sequence as the mature human EGF (SEQ ID NO: 10) is obtained.

3. Preparation of the original inoculation

The inoculum was prepared using the reaction solution after the protein coat formation reaction. The buffer (GENEWARE (registered trademark) Inoculation Buffer (GIB)) used for the preparation of the vaccination was prepared in the following manner.

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 were added to the beaker. 10H ₂ O, stirred with a stirrer for 15 minutes or more. The dissolved solution was transferred to a measuring cylinder having a volume of 500 mL, and adjusted to 300 mL with 18.2 MΩ of water. Transfer it to an autoclavable container, add 3.00 g of bentonite and 3.00 g of diatomaceous earth, rotate and stir to sufficiently wet the bentonite and diatomaceous earth, and then autoclave (121 ° C, 20 minutes). . After cooling, it was stored at 4 ° C (steadily stored at 4 ° C for 1 year).

A liquid diluted with a stock solution of the protein coat forming reaction solution or a suitable nuclease-free water is mixed with an equal amount of GIB and used as an inoculum. For each of the two true leaves of tobacco (Nicotiana benthamiana), 30 μL of vaccination per leaf is added, and the leaves are damaged by rubbing with a gloved finger, thereby making tobacco Infected with recombinant TMV.

4. Recovery of EGF from tobacco leaves (1)

After 12 days of cultivation after inoculation, tobacco leaves were harvested. The 500 g of the harvested leaves were placed on ice for about 45 minutes until extraction. Extraction buffer (50 mM Tris-hydroxymethane buffer (pH 8.5), 10 mM Ethylenediamine Tetraacetic Acid, 1 mM Phenylmethylsulfonyl fluoride, (PMSF, Phenylmethylsulfonyl fluoride, As a protease inhibitor), 0.1% Triton X-100) was stored at 4 ° C until cryopreservation. 500 mL of the extraction buffer was added to 500 g of the leaves, and the mixture was pulverized and extracted by a juicer, placed in a glass bottle, and stored in an ice liquid. The liquid was centrifuged at 10,000 g for 10 minutes to serve as a material for subsequent filtration. The supernatant was centrifuged to remove most of the protein in the filtrate by using Miracloth and a 30 kDa polyethersulfone Kvick flow ultrafiltration (UF, Ultra filtration) cartridge (0.1 m ² ) to concentrate and recombine. Protein (30kDa filtrate). Further, the 30 kDa filtrate was further concentrated to 90 mL ( ² kDa filtrate) using a ² kDa Hydrosart Sartorius cassette (0.1 m ² ). Further, each of the filtration residues was washed with 7 times the amount of the extraction buffer (total 1,600 mL), and the washing liquid (30 kDa residue and 2 kDa residue) was collected.

For each sample of tobacco leaf pulverization, centrifugation supernatant, 30 kDa filtrate, 30 kDa residue, 2 kDa filtrate, and 2 kDa residue, protein detection was carried out by Western blotting using antibodies specific for human EGF. As a result, in addition to the pulverization liquid and the centrifugation supernatant, a recombinant protein of about 5.5 kDa was detected in the 30 kDa filtrate sample and the 2 kDa filtration residue sample. The recombinant protein produced by the recombinant TMV in the tobacco leaf cells was translated under the condition that the message peptide was added, but the signal peptide was removed by the enzyme reaction in the plant cell, and thus the result was detected at the above size.

5. Recover EGF from tobacco leaves (2)

After 12 days of cultivation after inoculation, tobacco leaves were harvested. The harvested 1500 g of leaves were placed on ice for about 45 minutes until extraction. Extraction buffer (50 mM Tris-hydroxymethane buffer (pH 8.5), 10 mM EDTA, 1 mM PMSF (benzylsulfonate, as a protease inhibitor), 0.1% Triton X-100) until extraction It was stored at 4 ° C in the refrigerator. 1500 mL of the extraction buffer was added to 1500 g of the leaves, and the mixture was pulverized and extracted by a juicer, placed in a glass bottle, and stored in an ice liquid. The liquid was centrifuged at 10,000 g for 10 minutes to serve as a material for subsequent filtration. The recombinant protein was concentrated by filtering the supernatant after centrifugation using Miracloth and a 30 kDa PES Kvick flow UF cassette (0.1 m ² ) to remove most of the protein in the filtrate. Ammonium sulfate was added to the 30 kDa filtrate to 1.5 M to be adsorbed on a Phenyl Sepharose FF column, and then eluted with a 5 mM sodium phosphate buffer (pH 7.0). Further, ammonium sulfate was added to the liquid so as to be 2.5 M, and the mixture was adsorbed on a phenyl sepharose FF column, and then eluted with a 5 mM sodium phosphate buffer (pH 7.0). The liquid was passed through a NuviaQ column to adsorb the recombinant protein, and eluted with a glycine buffer (pH 10.0) supplemented with 500 mM sodium chloride. This was used as a recombinant protein sample, and the measurement of the purity by SDS-PAGE and silver staining and the measurement of the biological activity were carried out.

The results of silver staining are shown in Fig. 2. It was confirmed that the recombinant protein in the purified tobacco leaf can be recovered in a higher purity by the above method.

6. Determination of biological activity of recombinant proteins derived from tobacco

As a biological activity of the recombinant protein, cell proliferation activity was measured. Human fibroblast PDL3 was added in 5000 wells in each well of a 96-well microplate. The recombinant protein sample was diluted from 20 pg/mL to 2000 pg/mL in 11 stages, and 0.5 mL of each diluted sample was added to each well (6 wells) in a well of 11 rows. One row of 0.5 mL of each of the culture solutions used in the culture in which PDL3 was added was prepared as a negative control group. The microplate was cultured for 4 days using a carbon dioxide cell culture incubator. After the culture, the culture solution was removed, and 1 mL of the tetrazolium-based living cell staining solution was added for staining for 3 hours. The absorbance at 490 nm was measured using a microdisk reader.

Further, in addition to PDL3, biological activity was measured in the same manner using BALB/c derived from mouse embryonic cells BALB/3T3. As a reference, biological activity was measured in the same manner using a commercially available human EGF protein standard (Promega) in place of the above recombinant protein sample.

The results of the biological activity measurement of the recombinant protein sample are shown in Fig. 3. Any of PDL3 cells (Fig. 3A) and BALB/3T3 cells (Fig. 3B) promoted cell proliferation based on the added concentration of the recombinant protein sample, which is inferior to the commercially available human EGF protein (Fig. 4A, B). result. Thereby, it was confirmed that the recombinant protein sample purified and recovered from the tobacco leaves by the above method has biological activity.

7. TOF-MS analysis of recombinant protein samples

50 μL of the recombinant protein sample was desalted by ZipTip C18 (Millipore) and concentrated to 10 μL. 1 μL of this solution was mixed with 4 μL of Matrix CCA (α-cyano-4-hydroxycinnamic acid), and 1 μL of the solution was supplied to a Bruker REFLEXTM matrix-assisted laser desorption mass spectrometer (MALDI-TOF MS, Matrix-Assisted Laser Desorption Ionization). Time-of-Flight Mass Spectrometer).

The obtained mass spectrum is shown in Fig. 5. Three peaks of m/z 5113.687, 5208.814, and 5316.018 were confirmed.

8. Analysis of amino acid sequence of recombinant protein sample

The analysis of the amino acid sequence of the peptide contained in the recombinant protein sample obtained above was carried out by Toray Research Center Co., Ltd. N-terminal amino acid sequence analysis by Edman degradation, confirmed from N-terminal to 10 The amino acid sequence of one residue. As a result, three sequences of the predicted N-terminal sequence (SEQ ID NO: 2), the N-terminal one residue-deficient sequence, and the N-terminal two-residue defect sequence were confirmed in the protein sample.

Further, based on the measurement of the molecular weight by TOF-MS and the analysis of the amino acid derived from the N-terminus, the structure of the peptide was estimated.

Based on the above data, it is presumed that the peptide contained in the protein sample is a peptide having a C-terminal end as L (leucine).

<110> Youyou Biological Co., Ltd.

<120> Protein composition that promotes cell proliferation through the EGF receptor

<130> PF516-PCT

<160> 11

<170> PatentIn version 3.1

<210> 1

<211> 159

<212> DNA

<213> Modern race

<400> 1

<210> 2

<211> 53

<212> PRT

<213> Modern race

<400> 2

<210> 3

<211> 159

<212> DNA

<213> Artificial

<220>

<223> Altered sequence encoding mature human EGF

<400> 3

<210> 4

<211> 78

<212> DNA

<213> Wrinkled Tobacco

<400> 4

<210> 5

<211> 26

<212> PRT

<213> Wrinkled Tobacco

<400> 5

<210> 6

<211> 78

<212> DNA

<213> Artificial

<220>

<223> The sequence of the altered tobacco extensin message peptide

<400> 6

<210> 7

<211> 4

<212> PRT

<213> Bentobacillus

<400> 7

<210> 8

<211> 12

<212> DNA

<213> Artificial

<220>

<223> Sequence of altered encoded endoplasmic reticulum retention messages

<400> 8

<210> 9

<211> 249

<212> DNA

<213> Artificial

<220>

<223> Altered base sequence encoding SP-EGF-KDEL

<220>

<221> CDS

<222> (1)..(249)

<223>

<400> 9

<210> 10

<211> 83

<212> PRT

<213> Artificial

<220>

<223> Altered base sequence encoding SP-EGF-KDEL

<400> 10

<210> 11

<211> 275

<212> DNA

<213> Artificial

<220>

<223> Insertion sequence, SP-EGF-KDEL

<400> 11

Claims (14)

A protein composition obtained by introducing a nucleic acid encoding an amino acid sequence of an epithelial cell proliferating factor into a plant cell to express a protein, extracting and purifying the protein in the plant cell, and promoting cell proliferation It is active and exhibits three peaks in the range of m/z 5000~5500 during mass spectrometry. The protein composition of claim 1, wherein the purification of the extracted protein comprises the step of contacting the hydrophobic interaction chromatography carrier with the protein solution to dissolve the protein adsorbed to the carrier. The protein composition of claim 2, wherein the hydrophobic interaction chromatography carrier is a phenyl-sepharose. The protein composition according to any one of claims 1 to 3, wherein the amino acid sequence of the epithelial cell proliferator is an amino acid of the mature human epithelial cell proliferative factor of 53 residues represented by SEQ ID NO: Base acid sequence. The protein composition according to any one of claims 1 to 4, wherein the amino acid sequence of the amino acid sequence No. 1 to No. 47 in the amino acid sequence represented by SEQ ID NO: 2 is contained, and comprises A fragment of the region of the amino acid No. 2 to No. 47, and a fragment containing a region of the amino acid No. 3 to No. 47. The protein composition according to any one of claims 1 to 5, wherein the plant is tobacco. The protein composition according to any one of claims 1 to 6, wherein the expression of the protein in the plant cell is transient. The protein composition of claim 7, wherein the introduction of the nucleic acid into the plant cell and the expression of the protein are carried out by infecting the plant with a recombinant virus, and the recombinant virus is a virus in which the nucleic acid is incorporated Expression vector preparation. The protein composition of claim 8, wherein the viral expression vector is a tobacco mosaic virus expression vector. 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 in the form of a protein to which at least one message peptide is added. The protein composition of claim 10, wherein the protein encoded by the nucleic acid is a tobacco extensin message peptide attached to the N-terminal side of the amino acid sequence of the mature human epithelial cell proliferator cell, and The form of the protein to which the endoplasmic reticulum retention message KDEL is attached to the terminal side is expressed in plant cells. The protein composition of claim 11, wherein the nucleic acid of the nucleotide sequence of SEQ ID NO: 8 in the Sequence Listing is introduced into a plant cell. A cosmetic comprising the protein composition of any one of claims 1 to 12. A pharmaceutical composition comprising the protein composition of any one of claims 1 to 12.