KR20190026725A - A method of high level expression of target proteins as RbcS fusion proteins and method of preparing composition for oral delivery of the RbcS-fused target proteins expressed in leaf tissues as protein drugs - Google Patents

Abstract

The present invention provides a plant expressing a target protein, a method for producing the plant, and a method for producing the target protein using the plant. A target protein expression system using plant cells according to the present invention solves problems of conventional culturing methods of plant cells, and mass production of the target protein containing a biopharmaceutical protein enables commercialization of biopharmaceuticals such as plant-derived protein products, thereby being very effective.

Description

A method of high level expression of target proteins as RbcS fusion proteins and method of preparing composition for oral delivery of the RbcS-fused target proteins expressed in leaf tissues as protein drugs}

The present invention relates to a plant expressing a protein of interest, a method for producing the same, and a method for producing a protein of interest using the same, and more specifically, fusion of a target protein to RbcS (ribulose bisphosphate carboxylase/oxygenase small subunit), a protein present in chloroplasts. And it relates to a technology for mass production by introducing it into a plant, and a composition for oral administration of biopharmaceuticals using the same.

Biopharmaceutical refers to the use of substances existing in the living body as pharmaceuticals, and in a broader sense, it can be defined as pharmaceuticals produced based on bioengineering technologies such as high-tech biotechnology such as gene recombination, cell fusion, and cell culture. have. These biopharmaceuticals are classified as protein drugs, therapeutic antibodies, vaccines, gene therapy products, and cell therapy products.

Currently, most of the recombinant proteins are produced using higher cells such as animal cells and insect cells as hosts, or through microorganisms such as yeast and bacteria. However, production of recombinant proteins through animal cell culture has disadvantages that the medium cost is high, the possibility of virus contamination that can infect humans is high, and a separate purification process is required to remove them due to the possibility of influx of bovine serum-derived proteins. . In addition, in the case of bacteria or yeast, while there is an advantage in that mass production of recombinant proteins is easy, there is a problem that the modification process does not occur at all after protein synthesis or is not suitable for the production of glycoproteins because they are very different from humans.

Accordingly, plant cell culture has recently emerged as an alternative production system for recombinant proteins. Plant cells are considered a safe production system because they are not infected with animal-derived viruses or pathogens, and there is no concern about mixing of animal-derived substances.

However, plant cell culture shows a relatively low protein expression level and a slow growth rate compared to other hosts including animal cells. Therefore, in order to commercialize plant-derived biopharmaceuticals, when a recombinant protein is produced from a plant, it is urgent to develop a technology to increase the expression efficiency of the transgene.

On the other hand, protein medicines are most widely used to be administered to the human body in an injection formulation, and protein medicines are the most effective way to work, but there is a problem that it may cause pain to patients. In particular, in the case of metabolic diseases such as diabetes, the patient's pain is enormous because it is administered through regular injections for a long period of time. For this reason, there is an urgent need to develop technology for oral administration of protein drugs. In the case of biopharmaceuticals produced from plants, it can be a revolutionary method because it is possible to administer orally without separating and purifying proteins, but on the other hand, when a protein drug is administered orally, pepsin secreted from the stomach and intestines There is a problem that it is decomposed by secreted trypsin.

Accordingly, the present inventors have found that the RbcS gene, which is known to be stably present by forming a protein complex in the chloroplast, increases the expression of the target protein in plant cells, and the fusion protein of the target protein and RbcS binds to RbcL to form a macromolecule. The present invention was completed by confirming that the resistance to protein degradation by pepsin was greatly increased.

Accordingly, it is an object of the present invention to provide an RbcS gene fragment and a gene construct for high expression of a protein of interest.

Another object of the present invention is to provide a recombinant expression vector for high expression of a protein of interest.

Another object of the present invention is to provide a transgenic plant that highly expresses a protein of interest.

Another object of the present invention is to provide a method for producing a transgenic plant that highly expresses a target protein.

Another object of the present invention is to provide a method for producing an expression vector for high expression of a target protein in plant cells and using it to mass-produce a target protein from a plant body.

Another object of the present invention is to provide a fusion protein in which a protein of interest and an RbcS peptide fragment are bound.

Another object of the present invention is to provide a method of making a large complex having a size of 500 ~ 800 kD in a state in which the protein of interest and the RbcS peptide fragment are bound to have resistance to proteolytic enzymes present in the digestive tract.

Another object of the present invention is to provide a pharmaceutical formulation composition for oral administration comprising a fusion protein in which a target protein and an RbcS peptide fragment are bound as an active ingredient.

In order to achieve the object of the present invention as described above, the present invention is a small subunit of ribulose bisphosphate carboxylase/oxygenase, characterized in that it enhances the expression level of the protein of interest located downstream thereof in plant cells. (RbcS, ribulose bisphosphate carboxylase/oxygenase small subunit) gene fragment is provided.

In order to achieve the above object, the present invention provides a gene construct for high expression of a protein of interest in which (a) the RbcS gene and (b) the gene encoding the target protein are sequentially operably linked.

According to a preferred embodiment of the present invention, the RbcS gene may be a polynucleotide sequence represented by SEQ ID NO: 1.

According to another preferred embodiment of the present invention, the target protein is an antigen, antibody, antibody fragment, structural protein, regulatory protein, transcription factor, toxin protein, hormone, hormone analogue, cytokine, enzyme, enzyme inhibitor, transport protein, It may be any one or more selected from the group consisting of a receptor, a fragment of a receptor, a biological defense inducer, a storage protein, a movement protein, an explosive protein, and a reporter protein, but is not limited thereto.

According to a preferred embodiment of the present invention, the gene construct may further include a promoter gene at the 5'end of the RbcS gene, and the promoter is a 35S promoter derived from cauliflower mosaic virus, cauliflower mosaic It may be selected from a 19S RNA promoter derived from a virus (cauliflower mosaic virus), a plant actin protein promoter, and a ubiquitin protein promoter, but is not limited thereto.

According to another preferred embodiment of the present invention, a protein tag gene may be further included at the 3'end of the gene encoding the target protein, and the protein tag is Avi tag, Calmodulin tag, polyglutamate tag, E tag, FLAG Group consisting of tag, HA tag, His tag, Myc tag, S tag, SBP tag, IgG-Fc tag, CTB tag, Softag 1 tag, Softag 3 tag, Strep tag, TC tag, V5 tag, VSV tag, and Xpress tag It may be any one selected from, but is not limited thereto.

The present invention also provides a recombinant expression vector comprising the gene construct.

In order to achieve another object of the present invention, the present invention provides a transgenic plant that is transformed with the recombinant expression vector and highly expresses a target protein.

According to a preferred embodiment of the present invention, the plant is a food crop including rice, wheat, barley, corn, beans, potatoes, wheat, red beans, oats, sorghum; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; Specialty crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, and rapeseed; Fruit trees including apple trees, pear trees, jujubes, peaches, grapes, tangerines, persimmons, plums, apricots, and bananas; It may be selected from flowers including roses, carnations, chrysanthemums, lilies, and tulips, but is not limited thereto.

In order to achieve another object of the present invention, the present invention provides a method for producing a transgenic plant that highly expresses a target protein comprising the step of preparing a recombinant expression vector and introducing the recombinant expression vector into a plant. .

In a preferred embodiment of the present invention, the step of introducing the recombinant expression vector into the plant is Agrobacterium sp.-mediated method, particle gun bombardment, silicon carbide whisker method ( silicon carbide whiskers), sonication, electroporation, and polyethylenglycol (PEG)-mediated transformation methods, but are not limited thereto.

In order to achieve another object of the present invention, the present invention comprises the steps of (a) preparing the recombinant expression vector, (b) introducing the recombinant expression vector into a plant to prepare a transgenic plant, (c) the It provides a method for producing a protein of interest comprising the step of culturing a transgenic plant and (d) separating and purifying the target protein from the transgenic plant or culture medium.

In a preferred embodiment of the present invention, the recombinant expression vector is introduced into a plant in which the RbcS gene present in the genome is deleted to prepare a transgenic plant, and a target protein can be effectively produced therefrom.

In order to achieve another object of the present invention, the present invention provides a fusion protein comprising a protein of interest and an RbcS peptide fragment bound to the 5′ end of the protein of interest.

In a preferred embodiment of the present invention, the RbcS peptide fragment may be an amino acid sequence represented by SEQ ID NO: 2.

In a preferred embodiment of the present invention, the fusion protein may be a large complex of 500 ~ 800 kD. The size of the fusion protein may vary depending on the size of the target protein bound to the RbcS peptide fragment.

In a preferred embodiment of the present invention, the fusion protein is characterized in that it has resistance to digestive enzymes.

In order to achieve another object of the present invention, the present invention provides a pharmaceutical formulation composition for oral administration comprising a fusion protein in which an RbcS peptide fragment is bound to the 5` end of the target protein as an active ingredient.

In order to achieve another object of the present invention, the present invention provides a pharmaceutical formulation composition for oral administration comprising as an active ingredient a transgenic plant expressing a fusion protein in which an RbcS peptide fragment is bound to the 5` end of the target protein. .

In a preferred embodiment of the present invention, the RbcS peptide fragment may be an amino acid sequence represented by SEQ ID NO: 2.

Accordingly, the present invention provides a plant expressing a target protein, a method for producing the same, and a method for producing a target protein using the same. Through the present invention, high-efficiency, high-expression target proteins can be obtained from plants, so they can be used for mass production of industrial or medical proteins using plants. It can be used to prepare a pharmaceutical formulation composition for administration.

1 is a schematic diagram of an RbcS fusion construct prepared according to an embodiment of the present invention.
Figure 2 is a Western blot photograph confirming that the target protein is highly expressed in plant cells due to RbcS fusion prepared according to an embodiment of the present invention.
3 is a Western blot photograph confirming that the expression of the RbcS fusion protein prepared according to an embodiment of the present invention is higher in the RbcS-deficient mutant than in the normal plant.
Figure 4 is a Western blot photograph confirming the expression of the RbcS fusion protein in a transgenic plant prepared according to an embodiment of the present invention.
5 is an experiment result of pepsin resistance effect by concentration of a target protein (RbcS-FL:leptin:HA) in which RbcS protein is fused according to an embodiment of the present invention.
6 is an experiment result of the pepsin resistance effect of the RbcS protein-fused target protein (RbcS-FL:leptin:HA) according to an embodiment of the present invention by reaction time.
7 is a Blue-Native PAGE result showing the result of confirming the formation of a complex of a target protein fused with RbcS protein according to an embodiment of the present invention.
8 is a result of confirming the stability in the digestive tract of RbcS-FL:leptin:HA according to an embodiment of the present invention.
9 is a result of confirming the high expression of the fusion protein RFeEx4fG15 according to an embodiment of the present invention.
10 is a result of confirming the formation of a Rubisco complex of the fusion protein RFeEx4fG15 according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

As described above, in order to devise a method for mass production of a target protein in plant cells, the present inventors pay attention to protein domain fusion capable of stabilizing a target protein in a cell, and form a protein complex in the Arabidopsis chloroplast. The RbcS gene, which is known to be stably present, was isolated, and it was confirmed that the fusion of the RbcS gene increases the expression of the target protein in plant cells (see Example 2).

Accordingly, the present invention provides an RbcS gene fragment, characterized in that it enhances the expression level of a protein of interest located downstream thereof in a plant cell.

The RbcS gene may preferably consist of the nucleotide sequence of SEQ ID NO: 1. In addition, variants of the base sequence are included within the scope of the present invention. Specifically, the gene is a base sequence having a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, most preferably 95% or more, respectively, with the nucleotide sequence of SEQ ID NO: 1 Can include. The “% of sequence homology” for a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) compared to (not including).

In addition, the present invention (a) RbcS (ribulose bisphosphate carboxylase/oxygenase small subunit) gene; And (b) provides a gene construct for high expression of the protein of interest to which the genes encoding the protein of interest are operably sequentially linked.

The gene construct of the present invention may further include a promoter gene at the 5'end of the RbcS gene.

The gene construct of the present invention may further include a protein tag gene at the 3'end of the gene encoding the target protein.

The gene construct of the present invention comprises: (a) a promoter gene; (b) rbcS (ribulose bisphosphate carboxylase/oxygenase small subunit) gene; (c) a gene encoding a protein of interest and (d) a protein tag gene may be operably linked sequentially.

In the above, the promoter is not particularly limited as long as it can express the inserted gene in the plant, but preferably the promoter is a 35S promoter derived from cauliflower mosaic virus, cauliflower mosaic virus. It may be selected from the 19S RNA promoter derived from, the plant actin protein promoter, and the ubiquitin protein promoter.

The RbcS gene may be a gene encoding the ribulose bisphosphate carboxylase/oxygenase subunit subunit, and most preferably may be an RbcS gene represented by SEQ ID NO: 1 derived from a baby pole.

In the above, the target protein is a term that refers to a protein to be produced, and is not limited to a specific protein. Specifically, target proteins include antigens, antibodies, antibody fragments, structural proteins, regulatory proteins, transcription factors, toxin proteins, hormones, hormone analogs, cytokines, enzymes, enzyme inhibitors, transport proteins, receptors, receptor fragments, and biodefensive inducers. , A storage protein, a movement protein, an explosive protein, and a reporter protein.

In one embodiment of the present invention, parathyroid hormone (PTH) or leptin was used as the target protein. However, the protein of interest of the present invention is not limited to Leptin or PTH. The present invention can also be used for mass expression of various small peptide hormones such as human growth hormone, GLP1, Exendin-4, and the like. The protein thus expressed can be purified purely by being separated from RbcS by introducing a specific proteolytic site at the fusion site with RbcS.

In addition, the protein tag gene of the present invention is not limited thereto so long as it is a tag that enables identification to separate and purify proteins. Specifically, protein tags are Avi tag, Calmodulin tag, polyglutamate tag, E tag, FLAG tag, HA tag, His tag, Myc tag, S tag, SBP tag, IgG-Fc tag, CTB tag, Softag 1 tag, Softag 3 tag. , Strep tag, TC tag, V5 tag, VSV tag, and Xpress tag may be any one or more selected from the group consisting of.

The present invention also provides a recombinant expression vector comprising the gene construct.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. Recombinant cells may express genes or gene segments that are not found in the natural form of the cell in either a sense or antisense form. In addition, recombinant cells can express genes found in cells in their natural state, but the genes are modified and reintroduced into cells by artificial means.

In the present invention, the RbcS gene sequence may be inserted into a recombinant expression vector. The term “recombinant expression vector” refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus or other vector. In general, any plasmid and vector can be used as long as they can replicate and stabilize in the host. An important characteristic of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element.

Expression vectors containing the RbcS gene sequence and appropriate transcription/translational control signals can be constructed by methods well known to those of skill in the art. The method includes in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology.

The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Suitable vectors for expressing the gene encoding the DNA fragment and the protein of interest according to the present invention in plant cells include pUC19-based plasmid or Ti plasmid vector.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a part of itself, a so-called T-region, to plant cells when present in a suitable host such as Agrobacterium tumafaciens. Other types of Ti-plasmid vectors are currently being used to transfer hybrid DNA sequences into plant cells, or protoplasts from which new plants can be produced that properly insert hybrid DNA into the plant's genome. A particularly preferred form of Ti-plasmid vector is a so-called binary vector as claimed in EP 0120 516 B1 and in US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are double-stranded plant viruses (e.g., CaMV) and viral vectors such as those that can be derived from single-stranded viruses, gemini viruses, etc. For example, it can be selected from incomplete plant viral vectors. The use of such vectors can be advantageous, especially when it is difficult to properly transform a plant host.

Examples of the suitable vector are not limited thereto, but it is preferable to use a binary vector such as 326 GFP or pCAMBIA series, and those skilled in the art can express the gene encoding the DNA fragment and the protein of interest according to the present invention in plant cells. Any vector that can be used can be selected and used.

More specifically, the recombinant vector uses an existing vector used for protein expression as a basic skeleton, and the DNA fragment according to the present invention that enhances the expression level of a promoter and a protein of interest and a gene encoding a target protein can be sequentially operated. It is a recombinant expression vector that is closely linked.

In addition, the expression vector may include a ribosome binding site and a transcription terminator as a translation initiation site.

The expression vector will preferably contain one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by a chemical method, and all genes capable of distinguishing a transformed cell from a non-transgenic cell correspond to this. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Such as antibiotic resistance gene, aadA gene, etc., but is not limited thereto.

In the recombinant vector of the present invention, the promoter may be CaMV 35S, actin, ubiquitin, pEMU, MAS, histone promoter, or Clp promoter, but is not limited thereto. The term "promoter" refers to a region upstream of DNA from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in a plant cell. “Constitutive promoter” is a promoter that is active under most environmental conditions and developmental states or cell differentiation. Since the selection of transformants can be made by various tissues at various stages, a constitutive promoter is used in the present invention. It may be desirable, therefore, constitutive promoters do not limit the possibility of selection.

In the recombinant vector of the present invention, a conventional terminator can be used, examples of which include nopaline synthase (NOS), rice amylase RAmy1 A terminator, paseolin terminator, Agrobacterium tumafaciens octopine gene The terminator of E. coli and the rrnB1/B2 terminator of Escherichia coli are, but are not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

Host cells capable of continuously cloning and expressing the vector of the present invention while being stable in prokaryotic cells may be any host cell known in the art. For example, E. coli JM109, E. coli BL21, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringensis, and other strains of the genus Bacillus, and Salmonella typhimurium, Serratia marsessons, and various Pseudomonas Intestinal bacteria and strains such as species are included.

In addition, when the vector of the present invention is transformed into a eukaryotic cell, as a host cell, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (eg, CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines), and plant cells. The host cell is preferably a plant cell, and the plant is a food crop including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats, and sorghum; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; Specialty crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, and rapeseed; Fruit trees including apple trees, pear trees, jujubes, peaches, grapes, tangerines, persimmons, plums, apricots, and bananas; It may be selected from flowers including roses, carnations, chrysanthemums, lilies, and tulips.

The method of transporting the vector of the present invention into a host cell is, when the host cell is a prokaryotic cell, the CaCl ₂ method, one method (Hanahan, D., J. Mol. Biol., 166:557-580 (1983)). And an electroporation method. In addition, when the host cell is a eukaryotic cell, the vector can be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment. I can.

In order to achieve another object of the present invention, the present invention provides a transgenic plant that is transformed with the recombinant expression vector and highly expresses a target protein.

According to another preferred embodiment of the present invention, the plant is a food crop including rice, wheat, barley, corn, beans, potatoes, wheat, red beans, oats, sorghum; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; Specialty crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, and rapeseed; Fruit trees including apple trees, pear trees, jujubes, peaches, grapes, tangerines, persimmons, plums, apricots, and bananas; It may be selected from flowers including roses, carnations, chrysanthemums, lilies, and tulips.

In order to achieve another object of the present invention, the present invention comprises the steps of constructing a recombinant expression vector; And it provides a method for producing a transgenic plant highly expressing a target protein comprising the step of introducing the recombinant expression vector into the plant.

The introduction of the plant expression vector into plant cells or plants is Agrobacterium sp.-mediated method, particle gun bombardment, sonication, electroporation, and Any one selected from the group consisting of a PEG (polyethylen glycol)-mediated transformation method may be used. In an embodiment of the present invention, a PEG-mediated transformation method was used for transformation of Arabidopsis thaliana protoplasts, and Agrobacterium sp.-mediated method was used to produce Arabidopsis thaliana transformants.

In order to achieve another object of the present invention, the present invention provides a method for mass-producing a protein of interest using a plant transformed with a recombinant vector.

Specifically, the method of producing a protein of interest of the present invention comprises the steps of: (a) preparing the recombinant expression vector; (b) introducing the recombinant expression vector into a plant to prepare a transgenic plant; (c) culturing the transformed plant; And (d) separating and purifying the target protein from the transformed plant or culture medium.

In the method for producing a protein of interest of the present invention, the plant may be a plant in which the RbcS gene present in the genome is deleted.

The method for producing a protein of interest from the transformed plant cell can be obtained from the transformed cell after transforming the plant cell with the recombinant vector according to the present invention and then incubating for an appropriate time so that the protein of interest is expressed. . At this time, any method known in the art may be used as a method for expressing the target protein.

In the above method, the transformant was produced as Arabidopsis thaliana, but is not limited thereto, and both protoplasts isolated from dicotyledonous plants or monocotyledonous plants can be used in the method of the present invention.

In order to achieve another object of the present invention, the present invention provides a fusion protein comprising a protein of interest and an RbcS peptide fragment bound to the 5′ end of the protein of interest.

The fusion protein of the present invention is characterized in that it has resistance to proteolytic enzymes in the digestive organs by forming a large complex of 500 to 800 kD.

In order to achieve another object of the present invention, the present invention provides a pharmaceutical formulation composition for oral administration comprising a fusion protein in which an RbcS peptide fragment is bound to the 5` end of the target protein as an active ingredient.

In order to achieve another object of the present invention, the present invention provides a pharmaceutical formulation composition for oral administration comprising as an active ingredient a transgenic plant expressing a fusion protein in which an RbcS peptide fragment is bound to the 5` end of the target protein. .

In a preferred embodiment of the present invention, the RbcS peptide fragment may be an amino acid sequence represented by SEQ ID NO: 2.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1. Recombinant expression vector construction

The present inventors used a gene construct operably linked [35S promoter]-[RbcS gene]-[target protein gene]-[HA tag] as an expression vector (FIG. 1). When the gene encoding the target protein is cloned into an expression vector and then expressed in plant cells, RbcS is expressed in a fused state, transferred to the chloroplast and accumulated.

In one embodiment of the present invention, parathyroid hormone (PTH) was used as the target protein, and the method of constructing the expression vector is as follows. PCR reaction using a forward primer consisting of'XbaI-RbcS N-termianl sequence' (SEQ ID NO: 6) and a reverse primer consisting of'BamHI-RbcS C-terminal sequence' (SEQ ID NO: 7) using Arabidopsis genomic DNA as a template To amplify the RbcS gene. In addition, a forward primer consisting of'BamHI-PTH N-termianl sequence' (SEQ ID NO: 8) and a reverse primer consisting of'XhoI-stop codon-HA C-terminal sequence' (SEQ ID NO: 9) using plasmid DNA containing PTH as a template. Using a PCR reaction was performed to amplify the PTH gene in which the HA-tag was fused. The amplified PCR products were digested and purified with XbaI/BamHI (first PCR product) and BamHI/XhoI (second PCR product), respectively. These two PCR products were cloned between the cauliflower mosaic virus 35S promoter (SEQ ID NO: 3) and the NOS terminator in the 326 GFP (Arabidopsis Biological Resource Center, Ohio State University, Ohio, USA) plasmid digested with XbaI/XhoI, and an expression vector RbcS:PTH:HA was prepared.

Example 2. RbcS Comparison of protein expression effects of fusion vectors

After preparing a transformant by introducing the expression vector prepared in Example 1 into a protoplast isolated from Arabidopsis thaliana leaves by a PEG-mediated transformation method, the amount of RbcS fusion protein expressed therefrom was determined by Western blot (Western blot) analysis method.

Specifically, 30 µg of the cell lysate was mixed with the SDS sample buffer, heated, and electrophoresed on a 10% SDS-PAGE gel. The separated protein was transferred to a PVDF membrane and blocked with 5% skim milk. After that, it was reacted with anti-HA antibody (1:1000 dilution), and reacted with a secondary antibody to which horseradish peroxidase (HRP) was bound. Thereafter, the ECL solution was treated according to the manufacturer's method.

Example 3. Within the plant cell RbcS Analysis of protein expression level due to fusion

In order to confirm the increase in protein expression due to RbcS fusion, a plasmid gene installed with the entire RbcS gene (full-length, SEQ ID NO: 1) on the top of PTH:HA was used, and as a control, the RbcS transition instead of the entire RbcS gene. A plasmid gene installed on top of PTH:HA with a peptide (RbcS transit peptide, SEQ ID NO: 10) was used. These genes were introduced into protoplasts isolated from leaves of Arabidopsis thaliana by a PEG-mediated transformation method, and after culturing for 24 hours, the protoplasts were collected, dissolved and quantified, followed by Western blot analysis.

As a result, as shown in FIG. 2, the expression of PTH:HA obtained by fusion of the entire RbcS gene in Arabidopsis protoplast was higher than that of PTH:HA obtained by fusion of the control RbcS transition peptide. From this, it was found that the use of the RbcS gene as a fusion partner has the effect of improving the expression level of the target protein.

Example 4. Wild-type plants RbcS Gene Missing In plants RbcS Comparative analysis of protein expression levels due to fusion

In order to analyze the expression level of the RbcS fusion protein in the RbcS gene-deficient plant, the mutant Arabidopsis thaliana deficient in the RbcS gene was used as a host cell.

Specifically, protoplasts were isolated from the leaves of wild-type Arabidopsis and the mutant Arabidopsis deficient in the RbcS gene, and the PTH:HA gene in which the entire RbcS gene (SEQ ID NO: 1) was fused was added to the protoplast by a PEG-mediated transformation method. After incubation for 24 hours after the introduction, the protoplasts were collected, lysed, and quantified to perform Western blot analysis.

As a result, as shown in FIG. 3, it was found that the expression of PTH:HA in which the entire RbcS gene was fused was higher in the mutant in which the RbcS gene was deleted than in the wild type Arabidopsis thaliana. From this, it was found that the expression level of the target protein can be efficiently improved by utilizing the method of expressing the RbcS fusion protein in the mutant in which the RbcS gene is deleted.

Example 5. RbcS Construction of transgenic plants expressing fusion protein

Transgenic plants with improved expression of the target protein were prepared by RbcS fusion. The plasmid for plant transformation was constructed as follows. First, a plasmid DNA containing Leptin as a template is used as a forward primer consisting of'BamHI-leptin N-termianl sequence' (SEQ ID NO: 12) and a reverse primer consisting of'XhoI-stop codon-HA C-terminal sequence' (SEQ ID NO: 13). ) To perform a PCR reaction to amplify the leptin gene in which the HA-tag was fused. The amplified PCR product was digested with BamHI/XhoI, purified, and then cloned into RbcS:PTH:HA plasmid DNA digested with BamHI/XhoI to prepare RbcS:leptin:HA.

Gene construct in the form of a 35S promoter-RbcS:Leptin:HA:NOS terminator using XbaI and EcoRI restriction enzymes at the multi-cloning site of the pCAMBIA1300 vector by cutting the region containing RbcS:Leptin:HA from the prepared recombinant vector By cloning, a recombinant vector for transformation was prepared. Thereafter, the transformed plant was prepared using the Agrobacterium-mediated transformation method using the prepared recombinant vector for transformation. At this time, pCABIA1300, which is a basic vector of the prepared transformation vector, was selected for plants transformed by the method of the present invention through hygromycin resistance test. In addition, the plants selected by the resistance test were found to be well-expressed lines through Western blot to secure the first generation of transformation (Fig. 4), and in the second generation of transformation, the “dead individuals: Lines where "surviving individuals" come out cleanly at a ratio of 1:3 were selected, and among these lines, individuals surviving all three generations of transformation were selected as homos, and the transformed plant line was secured by maintaining the line. As shown in FIG. 4, several bands were observed in transformants 1 to 5, 8, and 11, confirming that the target protein (RbcS:Leptin:HA) was highly expressed.

Example 6. RbcS Pepsin resistance effect of fusion protein

As in Example 5, a transgenic plant of RbcS-FL:Leptin:HA was prepared, and resistance to pepsin was investigated in vitro using this.

Specifically, the transgenic plants were grown in a greenhouse for about 4 weeks, then the leaves were harvested, lyophilized, and finely ground to produce a powder. Transgenic plants in the form of powder were each 10 mg, mixed with a buffer of pH 2.0, and then treated with pepsin at a concentration of 0, 200,400 ng/μl, and reacted at 37° C. for 30 minutes. After that, in order to lower the activity of pepsin, the temperature was lowered by placing the sample on ice for about 10 minutes, and the activity of pepsin was once again lowered by increasing the pH by treating with a 1.5 M Tris buffer of pH 8.8. Thereafter, the cells were broken by sonication, mixed with a buffer and boiled for 10 minutes, and then the residue was removed at 14,000 rpm. Thereafter, a sample corresponding to 100 μg was quantified, SDS-PAGE was performed, and Western blot was performed. As a control for comparing the resistance of the fusion protein according to the pepsin treatment, a chapheron binding protein (BiP), an ER protein, and a chlorophyll a-b binding protein (Lhcb4) protein, which forms a complex protein were used. As a result, as shown in Figure 5, in the case of the Leptin protein (RFL-Leptin) in which the entire RbcS gene was fused, the reaction with pepsin treated at a high concentration showed higher degradation resistance than Bip, and another complex was formed. Decomposition resistance was similar to that of Lhcb4.

In the above, the resistance to degradation according to the pepsin treatment according to the concentration of the RbcS fusion protein was confirmed, and then the reaction time for 400 ng/μl pepsin was varied to 30 minutes, 1 hour, and 2 hours to confirm the degradation. As a result, as shown in FIG. 6, despite the prolonged reaction time, it still showed resistance to degradation unlike other proteins.

Example 7. RbcS - Leptin Confirmation of stability in the digestive tract

In order to measure the stability of the RbcS-FL:leptin:HA protein in the digestive tract, mice were forcibly fed, and food was obtained from the stomach and small intestine, followed by Western blot.

Specifically, the leaves of the plant transformed with RbcS-FL:Leptin:HA were freeze-dried under liquid nitrogen, and then finely ground to prepare a powder state. This was suspended in PBS, and 80 mg (based on dry leaf weight)/subject was directly administered to the stomach of C57BL/6 mice (n = 5) using zoned in order to accurately control the time in the digestive tract. After 30 minutes of administration, food was obtained from the stomach and small intestine, put into RIPA lysis buffer, and subjected to ultrasonic pulverization. Cell residues were removed by centrifugation (14,000 xg), and 20 μg of protein was loaded onto an SDS-PAGE gel by Bradford protein quantification to perform Western blotting. Using an HA antibody, it was attempted to confirm the presence of RbcS-FL:leptin:HA.

As a result, as shown in FIG. 7, when food was collected from the stomach, RbcS-FL:leptin:HA of the original molecular weight was detected in most individuals. At the same time, in the small intestine, digestion appeared to be more advanced than the stomach, but the original molecular weight of RbcS-FL:leptin:HA was detected. From this, it was confirmed that when the target protein in which RbcS was fused was administered orally, it was stably present in the digestive tract without being degraded by digestive enzymes.

Example 8. RbcS Of fusion proteins Rubisco Confirmation of complex formation

Blue-Native PAGE (polyacrylamide gel electrophoresis) was performed to confirm whether RbcS-FL:Leptin:HA prepared in the above example still forms a rubisco complex regardless of whether or not the Leptin gene is fused.

Blue-Native PAGE analysis is a method to identify a protein in the form of a polymer, and specifically, the following process was performed. Transgenic plants were grown in a greenhouse for about 4 weeks, then the leaves were harvested, lyophilized, and finely ground to produce a powder. Transgenic plants in the form of powder were each 10 mg and mixed in Bis-Tris buffer, cells were broken and quantified by ultrasonic grinding, followed by n-dodecyl beta-, a non-ionic detergent. It was dissolved by adding D-maltoside (n-Dodecyl β-D-maltoside). Thereafter, after mixing with CBB-G (coomassie brilliant blue-G250), electrophoresis on a Blue-Native gel having a concentration gradient of 4 to 16%, Western blot was performed in the same manner as in the above example.

As a result, it was confirmed that the fusion protein formed the Rubisco complex as shown in FIG. 8.

Example 9. Other purposes For protein RbcS Of fusion proteins Rubisco Confirmation of complex formation

As confirmed in Example 8, in order to make it clear that the RbcS-fused target protein forms a rubisco complex, other target proteins other than Leptin were identified.

Specifically, the Leptin:HA portion was removed from the RbcS-Leptin:HA recombinant vector, and a DNA fragment in which exendin-4 (SEQ ID NO: 14) and GM1 binding peptide (G15) (SEQ ID NO: 16) were fused was recombined (RFeEx4fG15). . The prepared recombinant vector was introduced into protoplasts isolated from Arabidopsis leaves by a PEG-mediated transformation method, and the amount of RbcS fusion protein expressed after 24 hours was confirmed by Western blot analysis. As a result, as shown in FIG. 9, it was confirmed that the target fusion protein (RFeEx4fG15) was overexpressed in the expected size (29 kD).

In addition, in order to check whether the target fusion protein (RFeEx4FG15) forms a Rubisco complex, a recombinant vector was introduced into the protoplast in the same manner as described above, and Blue native-PAGE was performed. As a result, as shown in FIG. 10, the original 29 kD fusion protein was identified as a larger protein complex than the band (rubisco complex) seen near 480 kD in CBB (coomassie brilliant blue), which means that the target fusion protein is normally ruby It means that the SCCO complex has been formed. From this, it was confirmed that irrespective of the type of protein of interest, when the RbcS fragment of the present invention was used as a fusion partner, the Rubisco complex could be stably formed.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> A method of high level expression of target proteins as RbcS fusion proteins and method of preparing composition for oral delivery of the RbcS-fused target proteins expressed in leaf tissues as protein drugs <130> 1065369 <150> KR 10-2016-0062409 <151> 2016-05-20 <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 782 <212> DNA <213> Artificial Sequence <220> <223> rbcS <400> 1 atggcttcct ctatgctctc ttccgctact atggttgcct ctccggctca ggccactatg 60 gtcgctcctt tcaacggact taagtcctcc gctgccttcc cagccacccg caaggctaac 120 aacgacatta cttccatcac aagcaacggc ggaagagtta actgcatgca ggtcatttat 180 atttcttctt tcactttttt attattccat atgatttttt tcggttcttt cttcgaatct 240 acataaacta atatcattgg aaaaatcgaa aaaataggtg tggcctccga ttggaaagaa 300 gaagtttgag actctctctt accttcctga ccttaccgat tccgaattgg ctaaggaagt 360 tgactacctt atccgcaaca agtggattcc ttgtgttgaa ttcgagttgg aggtaattaa 420 acaaaattta aacatctata taaactagct agatcttagg aaaatttggt ttaatatatt 480 aggatcttga tttatataaa catgttcaaa atgttatctg agtggtttgt aacatgtggt 540 ttgtatagca cggatttgtg taccgtgagc acggtaactc acccggatac tatgatggac 600 ggtactggac aatgtggaag cttcccttgt tcggttgcac cgactccgct caagtgttga 660 aggaagtgga agagtgcaag aaggagtacc ccaatgcctt cattaggatc atcggattcg 720 acaacacccg tcaagtccag tgcatcagtt tcattgccta caagccacca agcttcaccg 780 gt 782 <210> 2 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> rbcS <400> 2 Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala 1 5 10 15 Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala 20 25 30 Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser 35 40 45 Asn Gly Gly Arg Val Asn Cys Met Gln Met Gln Val Trp Pro Pro Ile 50 55 60 Gly Lys Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Asp Leu Thr Asp 65 70 75 80 Ser Glu Leu Ala Lys Glu Val Asp Tyr Leu Ile Arg Asn Lys Trp Ile 85 90 95 Pro Cys Val Glu Phe Glu Leu Glu His Gly Phe Val Tyr Arg Glu His 100 105 110 Gly Asn Ser Pro Gly Tyr Tyr Asp Gly Arg Tyr Trp Thr Met Trp Lys 115 120 125 Leu Pro Leu Phe Gly Cys Thr Asp Ser Ala Gln Val Leu Lys Glu Val 130 135 140 Glu Glu Cys Lys Lys Glu Tyr Pro Asn Ala Phe Ile Arg Ile Ile Gly 145 150 155 160 Phe Asp Asn Thr Arg Gln Val Gln Cys Ile Ser Phe Val Ala Tyr Lys 165 170 175 Pro Pro Ser Phe Thr Gly 180 <210> 3 <211> 835 <212> DNA <213> Artificial Sequence <220> <223> 35S promoter <400> 3 agattagcct tttcaatttc agaaagaatg ctaacccaca gatggttaga gaggcttacg 60 cagcaggtct catcaagacg atctacccga gcaataatct ccaggaaatc aaataccttc 120 ccaagaaggt taaagatgca gtcaaaagat tcaggactaa ctgcatcaag aacacagaga 180 aagatatatt tctcaagatc agaagtacta ttccagtatg gacgattcaa ggcttgcttc 240 acaaaccaag gcaagtaata gagattggag tctctaaaaa ggtagttccc actgaatcaa 300 aggccatgga gtcaaagatt caaatagagg acctaacaga actcgccgta aagactggcg 360 aacagttcat acagagtctc ttacgactca atgacaagaa gaaaatcttc gtcaacatgg 420 tggagcacga cacacttgtc tactccaaaa atatcaaaga tacagtctca gaagaccaaa 480 gggcaattga gacttttcaa caaagggtaa tatccggaaa cctcctcgga ttccattgcc 540 cagctatctg tcactttatt gtgaagatag tggaaaagga aggtggctcc tacaaatgcc 600 atcattgcga taaaggaaag gccatcgttg aagatgcctc tgccgacagt ggtcccaaag 660 atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa 720 agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgcacaa tcccactatc 780 cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagaga acacg 835 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> HA tag <400> 4 tacccatacg atgttccaga ttacgct 27 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HA tag <400> 5 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> XbaI-RbcS N-termianl sequence F primer <400> 6 tctagaatgg cttcctctat gctctcttc 29 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> BamHI-RbcS C-terminal sequence R primer <400> 7 ggatcctacc ggtgaagctt ggtgg 25 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> BamHI-PTH N-termianl sequence F primer <400> 8 ggatccaatc tgtgagtgaa atacagctta tgc 33 <210> 9 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> XhoI-stop codon-HA C-terminal sequence R primer <400> 9 ctcgagtcag gaagcgtaat ctggaacatc gtatgggtaa gcccggggct gggatttagc 60 tttagttaat acattc 76 <210> 10 <211> 240 <212> DNA <213> Artificial Sequence <220> <223> rbcS transit peptide <400> 10 atggcttcct ctatgctctc ttccgctact atggttgcct ctccggctca ggccactatg 60 gtcgctcctt tcaacggact taagtcctcc gctgccttcc cagccacccg caaggctaac 120 aacgacacta cttccatcac aagcaacggc ggaagagtta actgcatgca ggtgtggcct 180 ccgattggaa agaagaagtt tgagactctc tcttaccttc ctgaccttac cgattccggg 240 240 <210> 11 <211> 80 <212> PRT <213> Artificial Sequence <220> <223> rbcS transit peptide <400> 11 Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala 1 5 10 15 Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala 20 25 30 Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser 35 40 45 Asn Gly Gly Arg Val Asn Cys Met Gln Val Trp Pro Pro Ile Gly Lys 50 55 60 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Asp Leu Thr Asp Ser Gly 65 70 75 80 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BamHI-leptin N-termianl sequence F primer <400> 12 ggatccatgt gctggagacc cctg 24 <210> 13 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> XhoI-stop codon-HA C-terminal sequence R primer <400> 13 ctcgagtcag gaagcgtaat ctggaacatc gtatgggtaa gcccgggggc attcagggct 60 aacatccaac 70 <210> 14 <211> 117 <212> DNA <213> Artificial Sequence <220> <223> Exendin-4 <400> 14 catggtgaag gaacatttac cagtgacttg tcaaaacaga tggaagagga ggcagtgcgg 60 ttatttattg agtggcttaa gaacggagga ccaagtagcg gggcacctcc gccatcg 117 <210> 15 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Exendin-4 <400> 15 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> G15 <400> 16 aggtcttcta ctaagcctcc tctttctcct cttggt 36 <210> 17 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> G15 <400> 17 Arg Ser Ser Thr Lys Pro Pro Leu Ser Pro Leu Gly 1 5 10

Claims (7)

A fusion protein comprising a target protein and a RbcS peptide fragment bound to the 5 &apos; end of the target protein.
The method according to claim 1,
Wherein the RbcS peptide fragment is the amino acid sequence of SEQ ID NO: 2.
The method according to claim 1,
Wherein the fusion protein is a giant complex of 500 to 800 kD.
The method according to claim 1,
Wherein said fusion protein is resistant to digestive enzymes.
A pharmaceutical formulation composition for oral administration comprising a fusion protein comprising an RbcS peptide fragment at the 5 'end of a target protein as an active ingredient.
A pharmaceutical formulation composition for oral administration comprising a transgenic plant expressing a fusion protein in which an RbcS peptide fragment is bound to the 5 'terminus of a target protein as an active ingredient.
The method according to claim 5 or 6,
Wherein the RbcS peptide fragment is the amino acid sequence represented by SEQ ID NO: 2.

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