Recombinant protein production using rice cultivar baromi-based transformant

Abstract

The present invention relates to a technique for producing a recombinant protein by using a transformant derived from the rice cultivar Baromi. The Baromi cultivar has a high regeneration efficiency and a short period until seeds are obtained, and has a floury rice characteristic, and thus can be pulverized for a short time at room temperature without pretreatment, thereby minimizing the denaturation and decomposition of proteins. Therefore, high-purity recombinant proteins can be efficiently recovered, and thus the present invention can be provided as a protein production platform suitable for industrial mass production.

Description

Production of recombinant protein using transgenic plants based on the Baromi rice variety

[0001] The present invention relates to the production of recombinant proteins using a transgenic plant based on the Baromi rice variety, and more specifically, to a technology for producing recombinant proteins using a transgenic rice plant into which a gene encoding a target protein has been introduced and seeds derived therefrom, and for efficiently recovering the target protein by crushing, extracting, and purifying the seeds.

[0002] Rice is a plant species for which transformation technology has been established relatively stably, and various vectors for plant expression with high transformation efficiency have already been widely developed. Based on this technological foundation, the introduction of genes and control of expression are relatively easy, and accordingly, rice has been widely used as a host plant for producing recombinant proteins.

[0003] In addition, rice has the advantage of having systematically established cultivation techniques as a representative crop. Since the agricultural technology and equipment required for the entire process from sowing to cultivation and harvesting are already well-equipped, economical large-scale cultivation and stable harvesting are possible. In this regard, rice can be evaluated as one of the plant species suitable for the production of recombinant proteins on an industrial scale, beyond the laboratory level.

[0004] In particular, the method of accumulating target proteins in rice seeds offers significant advantages in terms of storage and management. Since seeds can be stored stably for a long period, recombinant proteins can be stored relatively easily and retrieved when necessary. However, due to the nature of rice seeds as viable tissues, there is a problem in that the domestic or cross-border movement of transgenic rice seeds is subject to strict regulations under GMO-related laws. Consequently, significant restrictions may arise on movement and handling during actual industrial applications.

[0005] In contrast, if genetically modified rice seeds are processed into a viable powder form, they can be exempt from these GMO regulations, allowing for much more flexible responses in terms of transportation and handling. However, most existing rice varieties have limitations in that their hard seed tissues make processing into powder difficult. In particular, the frictional heat generated during the grinding process can cause denaturation of target proteins accumulated within the seeds, acting as an obstacle to ensuring protein stability (Aimutis and Shirwaiker, 2025).

[0006] To mitigate these issues, methods such as grinding seeds while soaked in water or grinding them while maintaining low temperatures have been proposed. However, in the case of water-based grinding methods, additional management processes—such as maintaining low temperatures to prevent protein degradation or microbial growth—are required when protein separation and purification cannot be performed immediately after grinding and the seeds must be transported to another location for processing. Consequently, this leads to limitations, such as increased process complexity and higher equipment and management costs.

[0007] Accordingly, in the present invention, when using the “Baromi 2” rice variety, the harvested seeds are ground in a short time without a pretreatment process of soaking in water or maintaining a low-temperature environment, thereby completing the invention by confirming the target protein.

[0008] Aimutis, W. R., & Shirwaiker, R. A. (2025). The challenges of co-extraction of animal and plant proteins from transgenic plants for use in food and feed. Frontiers in Plant Science, 16, 1626856.

[0009] The present invention aims to provide a transformant for efficiently producing a target protein and a method for producing a target protein using said transformant.

[0010] To achieve the above objective, the present invention provides a transformant derived from the Baromi variety into which a gene encoding a target protein, an expression construct comprising said gene, or a recombinant expression vector comprising said gene has been introduced.

[0011] In the present invention, the transformant may be characterized by comprising a transformed rice, a transformed plant cell, a transformed callus, a transformed seed, or a tissue or fraction derived from any one of these.

[0012] In the present invention, the transformant may be characterized by comprising a powder prepared from the transformant seed.

[0013] In the present invention, the powder may be characterized as being prepared by grinding the transformed seeds at room temperature for 10 seconds to 5 minutes.

[0014] In the present invention, the target protein may be characterized as being one or more selected from the group consisting of thaumatin, lactoferrin, transferrin, interleukin, transcription factor, membrane protein, insulin, cytokinin, growth factor, toxin protein, hormone, hormone analog, cytokine, movement protein, lysozyme, vaccine, enzyme, enzyme inhibitor, transport protein, structural protein, receptor, receptor fragment, biological defense inducer, storage protein, exploitative protein, reporter protein, artificially designed protein, hydrophobin, antigen, antibody, and antibody fragment.

[0015] The present invention also provides a method for producing a target protein comprising the following steps:

[0016] (a) a step of crushing a transgenic seed into which a gene encoding a target protein, an expression construct containing said gene, or a recombinant expression vector containing said gene has been introduced; and

[0017] (b) A step of recovering the target protein from the ground seed powder.

[0018] In the present invention, the above step (a) may be characterized by being powdered by mechanical grinding without a pretreatment process.

[0019] In the present invention, the pretreatment process may be characterized as being a moisture treatment or a cooling treatment.

[0020] In the present invention, the mechanical grinding may be characterized by being performed at room temperature for 10 seconds to 5 minutes.

[0021] In the present invention, the mechanical grinding may be characterized by being performed at a frequency of 10 to 50 Hz.

[0022] In the present invention, step (b) may be characterized by extracting the target protein from the crushed seed powder and then purifying and recovering it.

[0023] In the present invention, the purification may be characterized by being performed by chromatography, precipitation, membrane separation, filtration, or a combination thereof.

[0024] In the present invention, the transgenic seed may be characterized by being manufactured by including the following steps:

[0025] (i) a step of redifferentiating a transformed callus into transformed rice into which a recombinant expression construct or recombinant expression vector containing a gene encoding a target protein has been introduced; and

[0026] (ii) A step of harvesting transgenic seeds from the redifferentiated transgenic rice.

[0027] In the present invention, the regeneration efficiency of step (i) may be characterized as being 60% or higher.

[0028] In the present invention, the redifferentiation step may be characterized by inducing a stem within 10 to 18 days after culturing the transformed callus in a redifferentiation medium.

[0029] In the present invention, the step of harvesting the transformed seeds may be characterized by being performed within 45 to 65 days after the completion of Agrobacterium-mediated transformation.

[0030] When recombinant proteins are produced using the Baromi variety as in the present invention, the regeneration efficiency of the transformed callus is high and the time to flowering is short, allowing for the rapid and efficient acquisition of transformed seeds. Furthermore, due to the unique histological characteristics of the Baromi variety seeds, they can be processed into fine powder by grinding them briefly at room temperature without pretreatment processes such as moisture treatment or low-temperature maintenance. Consequently, the denaturation of the target protein caused by frictional heat generated during the grinding process is suppressed, thereby effectively maintaining the stability and yield of the recombinant protein. Moreover, by processing the transformed seeds into a viable powder state, the burden of GMO regulations related to seed movement and handling can be alleviated, thereby increasing operational flexibility and allowing the seeds to be utilized as a seed-based expression platform suitable for industrial applications for large-scale production.

[0031] Figure 1 is a schematic diagram showing the structure of a GFP expression vector for rice transformation.

[0032] Figure 2 shows the process of producing a transgenic rice plant expressing GFP through Agrobacterium-mediated transformation.

[0033] Figure 3 shows the results of comparing and analyzing the regeneration and growth processes of the transformed plants step by step after transforming Baromi and Dongjin variety seeds with Agrobacterium.

[0034] Figure 4A shows the results of observing GFP expression in the transformed callus and the seeds of the regenerated transformed plantlets using a fluorescence microscope after performing Agrobacterium-mediated transformation on Baromi and Dongjin variety seeds.

[0035] Figure 4B shows the results of western blot analysis of GFP expression in eight independent transformed calluses.

[0036] Figure 4C shows the results of western blot analysis of GFP expression in the leaves of 10 independent regenerated transgenic plants.

[0037] Figure 4D shows the results of western blot analysis of GFP expression in 4 to 5 T1 seeds harvested from two independent T0 transgenic plants.

[0038] Figure 5A shows the results of comparing the glutelin A protein band intensity through SDS-PAGE after extracting protein from Dongjin and Baromi variety seeds after crushing them for a certain period of time.

[0039] Figure 5B shows a graph comparing the relative protein extraction efficiency according to crushing time by quantifying the glutelin A protein band based on the SDS-PAGE results of Figure 5A.

[0040] Figure 6A shows a schematic diagram of a vector for Fc-InA fusion protein expression and the results of analyzing the Fc-InA fusion protein expressed by transforming the vector into the Baromi variety, purifying it based on Protein A, and then analyzing it by western blot. In Figure 6A, M represents the protein size marker, T represents the total soluble protein, P represents the pellet, E represents the eluted protein, and WT represents the wild-type total soluble protein.

[0041] Figure 6B shows a schematic diagram of a vector for the expression of pZipDB-InA and pZipDB-InB fusion proteins, and the results of analyzing the fusion proteins expressed by transforming the vector into the Baromi variety using His tag and purifying them by western blot. In Figure 6B, M represents the protein size marker, T represents the total soluble protein, Ft represents the flow-through fraction, W represents the washing fraction, B represents the resin binding fraction, E represents the eluted protein, and WT represents the wild-type total protein.

[0042] Figure 7A is a schematic diagram showing the structure of a dual expression vector pDIn(A+B)-f for the co-expression of human insulin A chain and B chain.

[0043] Figures 7B and 7C show the results of comparing and analyzing the expression and purification of recombinant proteins from seeds harvested by transforming the above vector into the Baromi and Dongjin varieties, respectively.

[0044] In FIGS. 7B and 7C, the WT lane represents protein extracted from non-transgenic wild-type rice seeds, the TSP lane represents total soluble protein extracted from transgenic rice seeds, and the isolation and purification lane represents recombinant protein purified from the Baromi and Dongjin varieties, respectively.

[0045]

[0046] The present invention will be described in more detail below.

[0047]

[0048] In the numerical ranges described in this specification, “to” is used to mean including both threshold ranges (greater than or equal to and less than), and when not including both threshold ranges, the numerical range is described as “greater than” and “less than.” In this specification, the term “about” used for numerical values ​​is used to mean a range that is expected to produce an effect substantially equivalent to the stated numerical value by a person skilled in the art; for example, it may be ±20%, ±10%, ±5%, etc. of the stated numerical value, but is not limited thereto.

[0049]

[0050] In this invention, it was confirmed that significant effects were achieved throughout the entire production of the target protein by utilizing a transgenic organism based on the Baromi variety. Specifically, the Baromi variety exhibited high regeneration efficiency of the transformed callus and shortened the time to regeneration and flowering, allowing for the rapid and efficient acquisition of transformed seeds. Furthermore, due to the characteristic of the endosperm of the Baromi variety having a powder-like texture, it was possible to process the seeds into a fine powder by grinding them briefly at room temperature without pretreatment processes such as moisture treatment or low-temperature maintenance. Consequently, it was confirmed that the denaturation of the target protein caused by heat generated during the grinding process was effectively suppressed. Moreover, it was confirmed that processing the transformed seeds into a viable powder state could alleviate the burden of GMO regulations regarding transportation and handling, and that stable recovery and purification were possible during the process of extracting and purifying the target protein from the ground seed powder. From these results, it was proven that this invention provides the effect of simplifying the process while maintaining the stability and yield of the recombinant protein, and simultaneously improving efficiency and practicality in terms of production and utilization.

[0051]

[0052] Accordingly, in one aspect, the present invention relates to a transformant derived from the Baromi variety into which a gene encoding a target protein, an expression construct comprising said gene, or a recombinant expression vector comprising said gene has been introduced.

[0053] The term “transformer” refers to a biological entity that has been genetically modified by introducing a gene encoding a target protein, an expression construct containing said gene, or a recombinant expression vector, and includes transformed rice, transformed plant cells, transformed calluses, transformed seeds, or tissues or fractions derived therefrom. The transformer in the present invention is characterized by comprising a plant body or a product derived therefrom of the Baromi variety.

[0054] In a specific embodiment, the transformant may comprise a powder prepared from the transformed seed, and the powder may be a powder prepared by mechanically grinding the transformed seed. For example, the powder may be prepared by mechanically grinding the transformed seed at room temperature for about 10 seconds to about 5 minutes.

[0055] The term “target protein” refers to a protein intended to be produced according to the present invention and may include all types of proteins capable of being expressed as recombinant proteins. The target protein may be an intracellular protein or a foreign protein, and the target gene encoding it may be included in an expression construct or recombinant expression vector for expressing the target protein.

[0056] In some embodiments, the target protein may be one or more selected from the group consisting of thaumatin, lactoferrin, transferrin, interleukin, transcription factor, membrane protein, insulin, cytokinin, growth factor, toxin protein, hormone, hormone analog, cytokine, movement protein, lysozyme, vaccine, enzyme, enzyme inhibitor, transport protein, structural protein, receptor, receptor fragment, biological defense inducer, storage protein, exploitative protein, reporter protein, artificially designed protein, hydrophobin, antigen, antibody, and antibody fragment.

[0057] The term “expression construct” refers to a nucleic acid structure in which one or more regulatory sequences required to express a target protein and a target gene are functionally combined. The expression construct may include a promoter, a gene encoding the target protein, and a terminator sequence as basic components, and may further include, as necessary, a 5′ uncoding region (5′ UTR), an intron, a leader sequence, a signal peptide sequence, a 3′ uncoding region (3′ UTR), and other expression regulatory sequences.

[0058] The term “recombinant” refers to a state in which a cell contains heterogeneous nucleic acids and replicates or expresses said nucleic acids to produce heterogeneous peptides or proteins. Recombinant cells can express genes or gene fragments that do not exist in cells in a natural state in a sense or antisense form, and also include cells that express genes that exist in a natural state but have been modified or reintroduced by artificial means.

[0059] A recombinant expression vector for expressing a target gene in a transformant according to the present invention may be used without limitation as long as it is a vector that can be stably maintained or expressed in plant cells. Such recombinant expression vectors may include plasmids that can be expressed in plants, and may include, for example, vectors in which a target gene expression cassette, CRT1 expression cassette, p38 expression cassette, etc. are introduced using the pCAMBIA1300 series, pRTVn, pRI101, pGreenII series, pTM series, pBI series, pBIN19, pPZP series, pEAQ series, pH7WG2, pK7WG2, Geminiviral vector, etc. as a basic backbone, but are not limited thereto.

[0060] In a preferred embodiment, the recombinant expression vector may be a Ti-plasmid-based vector capable of stably transferring a target gene into the genome of a plant cell through a T-DNA region when maintained in a host such as Agrobacterium tumefaciens or Agrobacterium rhizogenes. In particular, such a Ti-plasmid-based vector may be a binary vector system as described in EP 0120 516 B1 and U.S. Patent No. 4,940,838, which is advantageous in terms of gene introduction efficiency into plant cells and transformation stability. In one embodiment, the binary vector system may include a pCAMBIA series, pBI series, pGreen series, pPZP series, or a plant expression vector functionally equivalent thereto.

[0061] In addition, as other vectors for introducing the target gene according to the present invention into a plant host, viral vectors derived from double-stranded plant viruses including Cauliflower mosaic virus (CaMV), single-stranded viruses, or Gemini viruses may be used, and such vectors may be applied complementarily when the transformation efficiency in a specific plant host is relatively low.

[0062] More specifically, the recombinant expression vector according to the present invention may include an expression construct in which one or more expression regulatory sequences for regulating the expression of a target protein and a gene encoding the target protein are operably linked, using a conventional vector used for protein expression as a basic framework. The expression construct may include, for example, a promoter, a modified 5′ untranslated region (5′ UTR) that improves the translation efficiency of the target gene, and a terminator that improves the expression stability and level of the target gene, but is not limited thereto.

[0063] The recombinant expression vector may also include a ribosome binding site for translation initiation, a terminator for transcription termination, and one or more selection marker genes for selecting transformants. The selection marker genes may include, but are not limited to, herbicide resistance genes (e.g., glyphosate or phosphinothricin resistance genes), antibiotic resistance genes (e.g., kanamycin, G418, bleomycin, hygromycin, chloramphenicol resistance genes), aadA genes, etc.

[0064] Meanwhile, any host cell known in the art may be used as a prokaryotic host cell for the construction, amplification, or cloning of the above-mentioned recombinant expression vector, such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, Salmonella typhimurium, Serratia marcescens, or various Pseudomonas species.

[0065] The term “recombinant expression vector” refers to a vector containing an expression construct for expressing a target protein, and may include a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector capable of replicating and maintaining within a host cell. The recombinant expression vector according to the present invention comprises one or more expression regulatory sequences and a gene encoding a target protein, thereby enabling efficient and stable expression of a recombinant protein within plant cells, particularly rice (Oryza sativa) cells or tissues.

[0066] The recombinant expression vector may include a promoter suitable for expression in rice, for example, a rice-derived promoter or a constitutive promoter universally usable in plants, a tissue-specific promoter or a seed-specific promoter, and may further include appropriate terminator sequences, translation regulatory sequences, and selection marker genes for stable expression and accumulation of the target protein. The specific vector structure or composition of the recombinant expression vector is not limited as long as it can induce efficient and stable expression of the target protein within plant host cells, including rice.

[0067] A promoter refers to a DNA sequence for initiating and regulating the transcription of a target gene. It is located upstream of a structural gene and functions to initiate transcription by binding to RNA polymerase. In this specification, "plant promoter" refers to a promoter capable of initiating transcription in plant cells. In the present invention, any promoter capable of functioning in plant cells, particularly rice (Oryza sativa) cells, may be used without limitation. The promoter may include a constitutive promoter, a tissue-specific promoter, or a seed-specific promoter, and performs the function of regulating the expression level, timing of expression, or tissue of the target protein. Among these, a constitutive promoter is a promoter that exhibits continuous activity under most environmental conditions, developmental stages, or cell differentiation states. It can be preferably used in the present invention in that the selection and maintenance of transformants can be carried out at various tissues and stages. Therefore, the use of a constitutive promoter does not limit the selectability of transformants.

[0068] In one embodiment, the recombinant expression vector of the present invention may include, but is not limited to, an Fmm promoter, a double enhancer CaMV promoter, a CSVMV promoter, a 35MUFO promoter, a MacT promoter, an RbcS promoter, a Glu13a promoter, a GluB1 promoter, a GluB4 promoter, a Prolamin promoter, an FM'M-UD promoter, a CaMV 35S promoter, a 35S promoter, a de35S promoter, an actin promoter, a ubiquitin promoter, a pEMU promoter, an amylase promoter, or a Clp promoter that can be used. Functionally equivalent variants or similar promoters are also included within the scope of the present invention, as long as they can induce efficient expression of a target gene in plant cells, including rice.

[0069] The 5′ untranslated region (5′ UTR) is an untranslated nucleotide sequence located between the promoter and the target gene, situated upstream of the translation start codon. Although the 5′ UTR is not translated into a protein, it can influence the expression level of the target protein by regulating mRNA stability, ribosome binding efficiency, and translation initiation efficiency.

[0070] gBiP refers to a protein or sequence containing a signal sequence for inducing a target protein into the endoplasmic reticulum (ER). The gBiP signal sequence can play a role in improving the folding, stability, and accumulation of the protein by causing the target protein to move into the ER. The gBiP is merely an exemplary configuration of an ER targeting signal and can be replaced with other ER signal sequences that perform the same or similar functions.

[0071] The EK (enterokinase) cleavage sequence used in the present invention refers to a cleavage sequence positioned between a target protein and a purification tag or auxiliary sequence, which allows the purification tag to be selectively removed by enzymatic treatment after purification. Through the introduction of the EK cleavage sequence, the target protein can be separated from the tag after the purification process to obtain a protein having its original structure or function. The present invention is not limited to the use of the EK cleavage sequence and may include other functionally equivalent protease recognition sequences as long as they can remove the purification tag or auxiliary sequence from the target protein. For example, a cleavage sequence recognized by Factor Xa, Thrombin, TEV protease, PreScission protease, or HRV 3C protease may be used. Furthermore, the present invention is not limited to a cleavage sequence-based system, and other protein cleavage systems capable of selectively removing the tag from the target protein after purification, such as a cleavage system using a SUMO tag and SUMO protease, are also included within the scope of the present invention.

[0072] HDEL refers to a C-terminal retention signal sequence for retaining proteins in the endoplasmic reticulum (ER) of plant cells. The HDEL sequence inhibits the movement of target proteins from the ER to the extracellular space or other organelles, thereby providing the effect of increasing stability and accumulation within the ER. The present invention is not limited to HDEL sequences and may also include functionally equivalent ER retention signal sequences such as KDEL, SDEL, and KDEI, or variations thereof.

[0073] Each expression cassette according to the present invention may additionally include a sequence encoding a tag peptide depending on the purpose. The tag peptide is intended to facilitate the isolation and purification of recombinant proteins, and any tag peptide known in the art may be used without limitation. Specifically, the tag peptide may be one or more selected from the group consisting of Avi tag, Calmodulin tag, polyglutamate tag, E tag, FLAG tag, HA tag, His tag, Myc tag, CBM3 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, but is not limited thereto.

[0074] In one embodiment, the tag peptide may be a purification tag. A purification tag refers to a peptide or protein sequence fused to a target protein to facilitate the isolation and purification of the target protein. The purification tag functions to enable the selective isolation of the target protein through a purification process, such as affinity chromatography, without substantially affecting the original function of the target protein. In one embodiment, the purification tag may include affinity tags such as His tags and IgG-Fc tags in the embodiments of the present invention, but is not limited thereto, and various purification tags may be selectively used depending on the characteristics of the target protein or the purification method applied.

[0075] A terminator refers to a regulatory sequence that induces the precise termination and stabilization of transcribed mRNA. The terminator prevents unnecessary transcriptional extension and increases the stability of the generated mRNA, thereby performing the function of maintaining stable expression of the target protein. If necessary, a composite terminator consisting of two or more terminator sequences may be used. The terminators available for use in the present invention may be used without limitation as long as they are functionally operable in rice (Oryza sativa) cells, and may include, for example, nopaline synthase (Nos) terminator, octopine synthase (OCS) terminator, Cauliflower mosaic virus (CaMV) 35S terminator, tobacco RB7 terminator, Arabidopsis thaliana-derived HSP18.2 terminator, rbcS terminator, actin terminator, tRNA-Glu terminator, rice-derived glutelin gene terminator, prolamin gene terminator, ubiquitin gene terminator, and terminators functionally equivalent to these or combinations thereof (e.g., #9 terminator), but are not limited thereto.

[0076] In the present invention, the linker sequence may be a peptide linker for connecting protein sequences encoded by a target gene, an auxiliary gene, a purification tag, or other functional sequences, and may be optionally included as needed. The peptide linker provides flexibility to allow each component to operate independently without spatial or functional interference and may play a role in assisting the structural stability, proper folding, and maintenance of function of the fusion protein. In one embodiment, the peptide linker may include or be composed of a sequence consisting of about 1 to 20 amino acids.

[0077] The term “operably linked” means a state in which a gene or nucleic acid sequence is functionally linked to an expression control sequence and arranged so that the transcription and / or translation of the said gene can be performed by said expression control sequence. An “expression control sequence” means a DNA sequence that regulates the expression of an operably linked nucleic acid sequence in a specific host cell and may include a promoter, transcription initiation and control sequences, ribosome binding sites, transcription and translation termination sequences, etc. In the present invention, each component included in an expression construct or expression cassette should be understood to be operably linked to one another in a manner obvious to those skilled in the art, unless otherwise noted.

[0078] In the embodiments of the present invention, specific configurations, sequences, or examples are described for each component; however, these are for the convenience of explanation and should not be interpreted as limiting the scope of the present invention. Each component according to the present invention may include various modifications, substitutions, or combinations that are functionally identical or substantially equivalent to the configurations described herein.

[0079] Furthermore, the amino acid sequences and base sequences described herein are not limited to the provided sequences and may be interpreted to be extended to include sequences having sequence homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with said sequences. Sequences having such homology are included within the scope of the present invention insofar as they perform the same or similar functions as the sequences described in the present invention.

[0080] The “% of sequence homology” used in this specification refers to a value calculated based on the ratio of matching bases or amino acid residues in a comparison region after optimally aligning two sequences. The sequences in the comparison region may include some additions or deletions of bases or amino acids compared to a reference sequence, i.e., gaps, for optimal alignment, and the homology calculated with such gaps included is also interpreted as being included in the sequence homology of the present invention.

[0081]

[0082] In another aspect, the present invention relates to a method for producing a target protein comprising the following steps:

[0083] (a) a step of crushing a transgenic seed into which a gene encoding a target protein, an expression construct containing said gene, or a recombinant expression vector containing said gene has been introduced; and

[0084] (b) A step of recovering the target protein from the ground seed powder.

[0085] In the present invention, step (a) may involve pulverizing by mechanical grinding without a pretreatment process by utilizing the physical characteristics of the transgenic seed.

[0086] In one embodiment, the pretreatment process may be a moisture treatment or a cooling treatment. In the present invention, by not essentially including such a pretreatment process, it is possible to simplify the process and reduce process costs.

[0087] In another embodiment, the mechanical grinding may be performed at room temperature. In yet another embodiment, the mechanical grinding may be performed for about 10 seconds to about 5 minutes, or for any time selected within the above range. For example, the above mechanical grinding is about 10 seconds to about 5 minutes, about 10 seconds to about 4 minutes 30 seconds, about 10 seconds to about 4 minutes, about 10 seconds to about 3 minutes 30 seconds, about 10 seconds to about 3 minutes, about 10 seconds to about 2 minutes 30 seconds, about 10 seconds to about 2 minutes, about 10 seconds to about 1 minute 30 seconds, about 10 seconds to about 1 minute, about 20 seconds to about 5 minutes, about 20 seconds to about 4 minutes 30 seconds, about 20 seconds to about 4 minutes, about 20 seconds to about 3 minutes 30 seconds, about 20 seconds to about 3 minutes, about 20 seconds to about 2 minutes 30 seconds, about 20 seconds to about 2 minutes, about 20 seconds to about 2 minutes, about 20 seconds to about 1 minute 30 seconds, about 20 seconds to about 1 minute, also about 30 seconds to about It may be performed for 5 minutes, about 30 seconds to about 4 minutes 30 seconds, about 30 seconds to about 4 minutes, about 30 seconds to about 3 minutes 30 seconds, about 30 seconds to about 3 minutes, about 30 seconds to about 2 minutes 30 seconds, about 30 seconds to about 2 minutes, about 30 seconds to about 1 minute 30 seconds, about 30 seconds to about 1 minute, about 40 seconds to about 5 minutes, about 40 seconds to about 4 minutes 30 seconds, about 40 seconds to about 4 minutes 4 minutes, about 40 seconds to about 3 minutes 30 seconds, about 40 seconds to about 3 minutes, about 40 seconds to about 2 minutes 30 seconds, about 40 seconds to about 2 minutes, about 40 seconds to about 1 minute 30 seconds, or about 40 seconds to about 1 minute.

[0088] In another embodiment, the mechanical grinding may be performed at a frequency of about 10 Hz to about 50 Hz, or at any frequency selected within the above range. For example, the mechanical grinding is about 10 Hz to about 50 Hz, about 10 Hz to about 45 Hz, about 10 Hz to about 40 Hz, about 10 Hz to about 35 Hz, about 10 Hz to about 30 Hz, about 10 Hz to about 25 Hz, about 10 Hz to about 20 Hz, about 10 Hz to about 15 Hz, about 15 Hz to about 50 Hz, about 15 Hz to about 45 Hz, about 15 Hz to about 40 Hz, about 15 Hz to about 35 Hz, about 15 Hz to about 30 Hz, about 15 Hz to about 25 Hz, about 15 Hz to about 20 Hz, about 15 Hz to about 15 Hz, also about 20 Hz to about 50 Hz, about It can be performed at a frequency of 20 Hz to about 45 Hz, about 20 Hz to about 40 Hz, about 20 Hz to about 35 Hz, about 20 Hz to about 30 Hz, about 20 Hz to about 25 Hz, about 20 Hz to about 20 Hz, also about 25 Hz to about 50 Hz, about 25 Hz to about 45 Hz, about 25 Hz to about 40 Hz, about 25 Hz to about 35 Hz, about 25 Hz to about 30 Hz, about 25 Hz to about 25 Hz, about 30 Hz to about 50 Hz, about 30 Hz to about 45 Hz, about 30 Hz to about 40 Hz, about 30 Hz to about 35 Hz, or about 30 Hz to about 30 Hz.

[0089] In particular, the transgenic seed according to the present invention is derived from the Baromi variety and can be processed into a fine powder state with only short-term mechanical grinding, thereby effectively suppressing the frictional heat generated during the grinding process and, accordingly, minimizing thermal denaturation or degradation of the target protein.

[0090] In the present invention, step (b) may involve extracting a target protein from ground seed powder and then purifying and recovering it. In the present invention, the purification may be performed by chromatography, precipitation, membrane separation, filtration, or a combination thereof.

[0091] The transformed seeds obtained in this manner can be secured in a form in which the target protein is stably accumulated, and the transformed seeds, transformed plants, transformed calluses, or transformed plant cells are all included in the transformed bodies of the present invention. Subsequently, a recombinant protein can be produced by extracting and purifying the target protein from the transformed bodies. Conditions and processes regarding transformation, culture, regeneration, growth of plants and seed formation, and extraction and purification of the target protein that are not specifically specified in the present invention may be carried out according to conventional methods known in the art.

[0092] In the method for producing a target protein according to the present invention, the recovery of the target protein may be carried out through various separation and purification methods known in the art. Generally, a centrifugation process may be performed to remove cell debris and insoluble components from seed pulverized material or cell lysate, and subsequently, a precipitation process may be applied for the concentration and separation of the target protein. The precipitation process may include precipitation by salting out, such as salt precipitation methods like ammonium sulfate precipitation or sodium phosphate precipitation, and solvent precipitation methods using acetone or ethanol.

[0093] In addition, dialysis, electrophoresis, or various chromatography processes may be applied after or in parallel with the precipitation process to improve the degree of purification of the target protein. The chromatography processes may include ion exchange chromatography, gel-infiltration chromatography, high-performance liquid chromatography (HPLC), reverse-phase high-performance liquid chromatography (RP-HPLC), affinity column chromatography, etc., and ultrafiltration or membrane separation processes may also be applied alone or in combination. In a preferred embodiment, the purification process may be affinity chromatography utilizing a tag introduced to the target protein or specific binding characteristics of the target protein, but is not limited thereto.

[0094] According to an embodiment of the present invention, when recombinant protein is recovered from a pulverized material prepared using transgenic seeds derived from the Baromi variety, it was confirmed that the incorporation of unnecessary intrinsic proteins or degradation products is significantly reduced compared to samples derived from general rice varieties, allowing the target protein to be purified to a high purity using only relatively simple separation and purification processes. In particular, as the generation of frictional heat during the grinding process is suppressed, the denaturation or degradation of the target protein is minimized, resulting in a reduction or near absence of extra bands corresponding to impurities observed during the purification stage. Accordingly, the present invention provides a production method advantageous for the mass production and industrial application of recombinant protein by simultaneously improving the stability and purification efficiency of the recombinant protein and reducing the complexity of downstream processes.

[0095]

[0096] In the present invention, the transgenic seed may be produced by a method comprising the following steps:

[0097] (i) a step of redifferentiating a transformed callus into transformed rice into which a recombinant expression construct or recombinant expression vector containing a gene encoding a target protein has been introduced; and

[0098] (ii) A step of harvesting transgenic seeds from the redifferentiated transgenic rice.

[0099] In the present invention, the regeneration efficiency of step (i) may be about 60% or more, and in one embodiment, may be about 60% or more, about 65% or more, about 70% or more, or about 75% or more. Preferably, the regeneration efficiency may be significantly higher than the regeneration efficiency observed in general Japonica rice varieties, because stem induction and survival rates are stably maintained during the process of regenerating the transformed callus into a plant. In particular, when using the transformed callus derived from the Baromi variety according to the present invention, the variation in regeneration efficiency is relatively small and reproducibility is excellent, so stable regeneration results can be provided even in repeated transformation experiments. Such high regeneration efficiency enables the early acquisition of transformed plants and seeds, contributing to the shortening of the overall recombinant protein production process and the improvement of production efficiency.

[0100] In the present invention, the redifferentiation step may include inducing a stem within about 10 to about 18 days after culturing the transformed callus in a redifferentiation medium, and in one embodiment, the stem may be induced within about 10 to about 17 days, about 10 to about 16 days, about 12 to about 18 days, or about 12 to about 16 days. Preferably, the stem induction may be observed within about 2 weeks after transfer to the redifferentiation medium, which suggests that the viability and differentiation ability of the transformed callus are stably maintained. Such a shortened redifferentiation period enables the early acquisition of the transformed plant, thereby effectively shortening the time required for the entire transformation and seed production process.

[0101] In the present invention, the step of harvesting the transgenic seeds may be performed within about 45 to about 65 days after the completion of Agrobacterium-mediated transgenic transformation, and in one embodiment, within about 45 to about 60 days, about 45 to about 55 days, about 50 to about 65 days, or about 50 to about 60 days. Preferably, the transgenic seeds may be obtained within about 2 months under conditions where the flowering and seed maturation of the transgenic plant proceed normally, which means that the period from transgenic transformation to seed harvesting is relatively short. This possibility of early seed harvesting enables the rapid progress of subsequent generation (T1) analysis and recombinant protein production processes, thereby providing advantageous effects in terms of research and industrial application.

[0102] The introduction of the above-mentioned recombinant expression vector into plant cells, calluses, or plant bodies may be carried out by any one selected from the Agrobacterium sp.-mediated transformation method, particle gun bombardment, sonication, electroporation, or PEG (polyethylene glycol)-mediated transformation method. However, the present invention is not limited to these methods, and cases in which the recombinant expression vector is introduced into plant cells, calluses, or plant bodies using other transformation methods known in the art are also included within the scope of the present invention.

[0103] The transformant according to the present invention can be produced by a stable transformation method using plant material derived from the Baromi variety. In one embodiment, transformation can be performed by introducing a gene encoding a target protein, an expression construct containing said gene, or a recombinant expression vector into plant cells derived from the Baromi variety, e.g., a callus. The transformation can be performed through conventional methods known in the art, such as Agrobacterium-mediated transformation, and the transformed callus can be selected and proliferated on a selective medium.

[0104] Selected transgenic calluses can be cultured under appropriate regeneration conditions to induce stems and roots, thereby regenerating into transgenic plants. The Baromi variety according to the present invention has the advantage of high regeneration efficiency and a short regeneration period, allowing for the rapid acquisition of transgenic plants. The regenerated transgenic plants can form transgenic seeds by undergoing growth and flowering processes under greenhouse or field conditions.

[0105]

[0106] The present invention provides significant process and quality effects throughout the entire process of recombinant protein production by utilizing the physical and biological characteristics of the rice variety 'Baromi'.

[0107] First, the present invention demonstrates excellent effects in terms of the construction and production efficiency of transgenic plants. According to the examples, the Baromi variety showed higher regeneration (regeneration) efficiency during the transformation process compared to a general rice variety (Dongjin) (52% compared to 76%), and the time required for stem induction was shortened by about 5 days (19 days compared to 14 days). In addition, the Baromi variety produced a relatively large number of tillers, resulting in good establishment after soil transplantation, and consequently showed a tendency for increased seed yield at the flowering stage.

[0108] Second, the present invention has advantages in terms of ensuring protein stability and the efficiency of the grinding process. Since Baromi seeds have endosperms with floury characteristics that allow them to be easily powdered, unlike general rice varieties (Dongjin) which require a grinding time of about 20 to 25 minutes, they could be processed into fine powder with only a short grinding time of about 1 to 2 minutes. This short grinding time minimizes frictional heat generated during the grinding process, thereby contributing to suppressing the denaturation or decomposition of heat-sensitive target proteins. In fact, the protein extract obtained from a Baromi seed sample ground for about 1 minute was confirmed to be equivalent to that of a general rice seed sample ground for about 20 minutes, demonstrating that process time and costs can be reduced.

[0109] Third, compared to samples derived from ordinary rice seeds, the recombinant protein purified from Baromi seeds was isolated with no or significantly reduced extra bands corresponding to impurities, allowing for the acquisition of higher-purity recombinant proteins. This improvement in purification quality is attributed to the combined effect of reduced incorporation of endogenous proteins due to the histological characteristics of Baromi seeds and the suppression of frictional heat generation during the grinding process, which minimized denaturation or degradation of the target protein and surrounding proteins.

[0110]

[0111] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0112]

[0113] Example 1. Construction of an expression vector for rice transformation

[0114] To accumulate recombinant protein in the endoplasmic reticulum (ER) of rice seeds, a vector was constructed as follows.

[0115] First, Green Fluorescent Protein (GFP) was used as the target protein, and an ER-specific GFP recombinant gene was designed by fusing a BiP leader sequence for ER targeting to the N-terminus of GFP and an HDEL amino acid sequence for ER retention to the C-terminus. A 5'UTR sequence was added upstream of BiP, and an enterokinase cleavage site was additionally introduced between BiP and GFP. The GFP recombinant gene expression construct was designed to include an XbaI restriction enzyme recognition sequence at the 5' end and an XhoI restriction enzyme recognition sequence at the 3' end, and was then obtained through chemical synthesis (GenScript, USA).

[0116] Next, the above-mentioned GFP recombinant gene expression construct was inserted into a binary vector for plant expression. As the backbone of the expression vector, the pCAMBIA1300 vector (CAMBIA, Australia), which contains a hygromycin resistance gene as a selection marker, was used. The above-mentioned GFP recombinant gene expression construct and the pCAMBIA1300 vector were cleaved with XbaI and XhoI restriction enzymes (New England Biolabs, USA), respectively, and the target fragments were isolated and purified through agarose gel electrophoresis. The purified GFP recombinant gene expression construct (insert) and the pCAMBIA1300 backbone were ligated using T4 DNA ligase (New England Biolabs, USA), and as a result, the expression vector p1300-GFP-3PR, into which the GFP recombinant gene expression construct was inserted, was constructed.

[0117] Next, a composite terminator was additionally introduced to improve the transcription termination efficiency and expression stability of the GFP recombinant gene. The composite terminator was designed as a structure (3PR) by sequentially linking a 35S terminator derived from Cauliflower mosaic virus, a PIN2 terminator derived from Arabidopsis thaliana, and a matrix attachment site (MAS) derived from tobacco RB7. The terminator sequence was designed to include an XhoI restriction enzyme recognition sequence at the 5′ end and an EcoRI restriction enzyme recognition sequence at the 3′ end, and then obtained by chemical synthesis (GenScript, USA). The synthesized composite terminator fragment and the constructed p1300::GFP vector were cleaved with XhoI and EcoRI restriction enzymes, respectively, and then ligated using T4 DNA ligase (New England Biolabs, USA) to construct the final expression vector p1300::GFP::3PR (see Fig. 1), in which the composite terminator is linked downstream of the GFP gene.

[0118] The constructed vector was transformed into E. coli DH5α and selected for hygromycin resistance, and then confirmed to be properly cloned through restriction enzyme analysis and nucleotide sequence analysis.

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136] Example 2. Production of rice transgenic calluses and plantlets by Agrobacterium-mediated transformation

[0137]

[0138] 2-1. Sterilization of Plant Materials and Seeds

[0139] The Baromi and Dongjin rice varieties, which belong to Oryza sativa L. ssp. Japonica, were obtained from the National Institute of Food Science and Technology of the Rural Development Administration and used, respectively. 200 seeds of each variety were selected and surface sterilized by treating them in a 10% (v / v) sodium hypochlorite aqueous solution for 30 minutes. After sterilization, the seeds were washed three times with purified water to remove residual disinfectant, separated onto sterilized filter paper, and dried.

[0140]

[0141] 2-2. Callus Induction

[0142] Ten sterilized seeds of each variety were placed on a medium plate containing CI (callus induction) medium. Culture was performed in a 30°C incubator maintaining a photocycle of 16 hours of light and 8 hours of darkness. After approximately 3 weeks of culture, calluses were induced from the seeds (Fig. 2A). The induced calluses were lightly washed with sterile purified water, transferred to a new CI medium plate, and cultured for an additional 3 days to stabilize the callus condition before being used for Agrobacterium-mediated transformation (Fig. 2B).

[0143]

[0144] 2-3. Preparation of Agrobacterium Strains

[0145] For Agrobacterium-mediated transformation, the Agrobacterium tumefaciens EHA strain (BioApplications, Inc, Pohang) was used. The expression vector constructed in Example 1 was introduced into the Agrobacterium strain using electroporation. The transformed Agrobacterium was cultured at 28°C for 2 days in LB solid medium containing kanamycin and rifampicin.

[0146] Colony PCR was performed to confirm transformation, and single colonies showing a positive reaction were selected. The selected positive colonies were inoculated into new LB plates containing kanamycin and rifampicin and cultured for an additional 48 hours at 28°C. Subsequently, this culture was used for rice callus transformation.

[0147]

[0148] 2-4. Preparation of Agrobacterium Suspension and Co-culture

[0149] Agrobacterium into which the expression vector was introduced was recovered as independent colonies and suspended in CC (co-cultivation) medium to prepare an Agrobacterium suspension. The optical density (OD) of the suspension was adjusted to a range of approximately 0.05 to 0.2 at 600 nm. 40 ml of this Agrobacterium suspension was poured into a sterile plate, and the calluses induced in 2-2 were added and incubated for 1 minute and 30 seconds. Subsequently, the calluses were recovered, and co-culturing was initiated under dark conditions. For co-culturing, three sterile filter papers were placed in a new plate, and 3.2 ml of CC medium solution was poured onto them to sufficiently wet the filter papers. The calluses incubated in the Agrobacterium suspension for 1 minute and 30 seconds were transferred onto the wet filter papers of the prepared plate, and co-culturing was performed under dark conditions at 24°C (Fig. 2C).

[0150]

[0151] 2-5. Selection of Transgenic Calluses

[0152] After 3 days of co-culture, the calluses cultured with Agrobacterium were transferred to SI (selection I) medium containing antibiotics and cultured for 10 days (Fig. 2D). Subsequently, the calluses were transferred to S-II (selection II) medium and cultured for an additional week. Approximately 20 days after the start of co-culture, transformed calluses exhibiting resistance to hygromycin were selected (Fig. 2E).

[0153]

[0154] 2-6. Analysis of Plant Regeneration Efficiency

[0155] Selected transgenic calluses were transferred to R (regeneration) medium at approximately 20-25 days after co-culture to induce stem formation (Fig. 2F). During this process, the number of independent redifferentiated stems and the number of non-redifferentiated calluses were recorded, and the number of days required for redifferentiation was recorded for each variety. Redifferentiation was defined as the phenomenon in which a stem is formed from a callus.

[0156] The regeneration efficiency was calculated using the following formula.

[0157] Redifferentiation efficiency (%) = (Number of redifferentiated calluses / Number of transformed calluses) × 100

[0158] The regeneration efficiency and regeneration time required for each variety were compared using the above indicators.

[0159]

[0160] 2-7. Root Induction and Acclimation

[0161] The redifferentiated stems were transferred to hormone-free F2 medium and cultured to induce root formation. The medium was maintained in a cultivation room for 1-2 weeks under conditions of 30°C and a 16-hour light period. After sufficient root formation was confirmed, the plants underwent a hardening process to adapt to the external environment.

[0162] The curing process was performed by removing the lid of the plate, immersing the plant in sterile water, and maintaining it at room temperature for about 5 days (Fig. 2H).

[0163]

[0164] 2-8. Soil Transplantation and Analysis of Transgenic Plants

[0165] Plants that had completed hardening were transplanted into a soil bed in a greenhouse maintained at 30°C (Fig. 2I). After transplanting into the soil, leaf samples were collected and analyzed to check for GFP expression, and the number of days elapsed until flowering for each transgenic individual was recorded (Fig. 2J).

[0166]

[0167] The composition of the media used in each step is as shown in Table 1, and the reagents and media components used to prepare each medium were purchased from Sigma-Aldrich (Merck KGaA, St. Louis, MO, USA) unless otherwise noted.

[0168]

[0169] The results of comparing the efficiency of callus induction, transformation, and redifferentiation (stem formation) of the Baromi and Dongjin varieties are shown in Table 2.

[0170]

[0171] Callus induction efficiency = Number of induced calluses / Number of seeds x 100 Callus transformation efficiency = Number of transformed calluses / Number of co-cultured calluses x 100

[0172] Redifferentiation efficiency = Number of redifferentiated calluses / Number of transformed calluses x 100

[0173]

[0174] As confirmed in Table 2, when the Agrobacterium-mediated transformation method of the present invention was applied to two rice varieties belonging to the Japonica lineage, Dongjin and Baromi, it was possible to stably secure transformed plants with hygromycin resistance in both varieties, confirming that the transformation method of the present invention is applicable regardless of variety.

[0175] However, significant differences were observed between varieties during the regeneration process after transformation. In the case of the Baromi variety, the number of transformed calluses was 102, which was higher than the 89 of the Dongjin variety, and the number of redifferentiated (i.e., stem-formed) calluses was 78, which was significantly higher than the 52 of the Dongjin variety. In other words, the redifferentiation efficiency was 76% for the Baromi variety, which was significantly higher than the 58% of the Dongjin variety, confirming that the transformed calluses are highly efficient at producing stem-bearing plants. Additionally, the time required for stem induction averaged 14 days for the Baromi variety, which was shorter than the 19 days for the Dongjin variety, and the time required for flowering was also significantly shortened for the Baromi variety at 51 days compared to the 69 days for the Dongjin variety.

[0176] These results demonstrate that the transformation system of the present invention operates stably in both varieties, while showing that the Baromi variety is superior to the Dongjin variety in terms of regeneration efficiency and growth rate. This suggests that the Baromi variety is a more suitable rice variety for industrial applications requiring mass transformation and time efficiency for the production of recombinant proteins.

[0177]

[0178] Example 3. Analysis of Morphological Differences in Transformation and Regeneration Processes by Rice Variety

[0179] In the process of applying the Agrobacterium-mediated transformation method of the present invention to the Japonica strain varieties Baromi and Dongjin, morphological differences observed at each developmental stage of transformation and regeneration were compared and analyzed.

[0180] During the callus induction and antibiotic resistance screening process in the early stages of transformation, calluses derived from the Baromi variety tended to be relatively brown and darker overall compared to calluses derived from the Dongjin variety (Fig. 3A). These morphological differences were determined to reflect differences in physiological responses by variety under callus induction and antibiotic treatment conditions.

[0181] After proceeding to the regeneration stage, the formation of numerous green spots was observed in both varieties at approximately 2 weeks of culture in the regeneration medium. However, only some of the observed green spots were found to have the potential to actually differentiate into shoots, and at approximately 3 weeks, some of these green spots formed shoots and subsequently developed into fully regenerated stems. At this stage, the number of regenerated shoots observed in the Baromi variety was significantly higher than that of the Dongjin variety, suggesting that the Baromi variety possessed a relatively high regeneration potential during the regeneration stage after transformation (Fig. 3B).

[0182] Afterward, the regenerated stems were transplanted into a root-forming medium and subjected to the root development stage. As a result, seedlings derived from the Baromi variety showed a tendency to form relatively more tillers compared to seedlings derived from the Dongjin variety (Fig. 3C). This increase in the number of tillers observed in the Baromi variety was maintained even after transplanting into soil, and it was determined that this contributed to increasing the viability and resistance of the plants during the process of establishing in the soil environment.

[0183] In addition, after growth progressed and reached the flowering stage, plants derived from the Baromi variety tended to exhibit relatively higher seed productivity compared to plants derived from the Dongjin variety (Fig. 3D). In particular, compared to the seeds of the Dongjin variety, the seeds harvested from the Baromi variety were white in appearance and had a relatively soft texture and a mealy texture (Fig. 3E). These seed characteristics suggest that when the transformation and regeneration process of the present invention is combined with the growth characteristics of the Baromi variety, additional advantages can be provided in terms of growth stability after soil transplantation and final seed yield.

[0184]

[0185] Example 4. Confirmation of GFP expression in transformed calluses, regenerated plant stems, and seeds by rice variety

[0186] To confirm GFP expression in seeds harvested from transformed calluses and transformed plants, fluorescence images were taken of independent transformed calluses and seeds. GFP fluorescence images were acquired using a LAS-3000 imaging system (Fujifilm, Tokyo, Japan) and captured using filters suitable for the excitation and emission wavelength conditions of GFP. As a result, GFP-specific fluorescence signals were observed in the transformed calluses and seeds derived from transformed plants, confirming that the introduced GFP gene was properly expressed in the corresponding tissues (Fig. 4A).

[0187] To more quantitatively confirm the expression of GFP protein, proteins were extracted from the transformed callus, leaves, and seeds of the transformed plant, respectively, and western blot analysis was performed.

[0188] Specifically, after obtaining the transformed callus and the leaves of the transformed plant as independent individuals, each sample was placed in an E-tube and frozen with liquid nitrogen, then ground for 1 minute using a Retsch MM301 mixer mill equipped with a metal grinding container and metal balls, under conditions of 25 impacts per second. Meanwhile, the seed samples were ground under room temperature conditions, and the grinding time was adjusted according to the variety under the same conditions (Baromi: approximately 1 minute, Dongjin: approximately 25 minutes).

[0189] Protein extraction buffer (0.5 M NaCl, 100 mM Tris-HCl (pH 6.8), 0.5% Tween-20, Triton X-100, 400 mM sucrose, and 5% glycerol) was added to the prepared sample powder and vortexed for approximately 5 minutes. Protein was then extracted by stirring for approximately 4 hours at room temperature or 4°C. Subsequently, 2× phosphate buffer (PBS) was added, and the mixture was incubated overnight at 4°C with stirring. To remove cell debris and insoluble substances, the mixture was centrifuged at 14,000 × g for 15 minutes, and the supernatant was collected. The extracted protein sample was mixed with SDS sample buffer, heated for 5 minutes to denature it, and then subjected to electrophoresis using 10% SDS-PAGE.

[0190] The proteins separated after electrophoresis were transferred to a PVDF membrane, and then western blot analysis was performed using a GFP-specific primary antibody (anti-GFP antibody (Living Colors® GFP monoclonal antibody, Takara Bio Inc; HRP-conjugated mouse anti-goat IgG (Santa Cruz Biotechnology, USA) as a secondary antibody). As a result, specific bands corresponding to GFP were detected in the transformed callus, leaves, and seeds of the transformed plants, indicating that the GFP gene is stably expressed in various tissues and developmental stages of rice (Figs. 4B to 4D).

[0191]

[0192] Example 5. Comparison of Crushing Efficiency by Rice Variety

[0193] Seeds of the Baromi and Dongjin varieties obtained by transformation were harvested. The harvested seeds were dried at 37°C for 5 days to sufficiently remove moisture content, and then used in crushing experiments.

[0194] Among the dried seeds, 12 independent seeds were selected for each variety, and each seed was ground at a frequency of 20 revolutions per second using a Retsch MM301 mixer mill equipped with a metal grinding container and metal balls. During the crushing process, samples were collected for each variety at 1, 2, 3, 6, 8, 10, 12, 14, 16, 20, and 25 minutes to analyze the progress of crushing.

[0195] As a result, it was observed that the transgenic seeds of the Baromi variety reached a visually sufficiently crushed state within 1 minute of starting the crushing process, whereas the seeds of the Dongjin variety were relatively delayed in crushing under the same crushing conditions, requiring up to 25 minutes of continued crushing to reach a similar level of crushing.

[0196] To extract total protein from lysed samples collected at each time point, a non-reducing protein extraction buffer (Tris-HCl (pH 8), 250 mM NaCl, 400 mM sucrose, 100 mM EDTA, 1 mM PMSF, and 0.05% Tween) was added and vortexed for approximately 5 minutes, followed by stirring for approximately 4 hours at room temperature or 4°C. Subsequently, 2× phosphate buffer (PBS) was added, and the mixture was incubated overnight at 4°C with stirring. To remove cell debris and insoluble matter, the mixture was centrifuged at 14,000 × g for 15 minutes, and the supernatant was collected. The extracted protein samples were mixed with SDS sample buffer, heated for 5 minutes to denature them, and then subjected to electrophoresis using 10% SDS-PAGE.

[0197] Subsequently, protein bands were detected using Coomassie Brilliant Blue (CBB) staining. In addition, to quantitatively compare the degree of rice seed fragmentation, the signal intensity of gluten precursor protein bands observed on SDS-PAGE was used as an indicator of fragmentation efficiency. The signal intensity of gluten precursor bands detected in each sample from CBB-stained SDS-PAGE gel images was measured using ImageJ software and plotted as a graph (Fig. 5).

[0198] As a result, the total protein extracted from the transgenic seeds of the Baromi variety after crushing for 1 minute was almost the same as the total protein extracted from the Dongjin variety seeds after crushing for 20 minutes. This means that under the same crushing conditions, the Baromi variety seeds exhibit significantly higher crushing efficiency compared to the Dongjin variety seeds.

[0199]

[0200] Example 6. Isolation and purification of target protein from transformed Baromi seeds

[0201] 6-1. Objective: Protein Design and Vector Construction

[0202] The first target protein is a protein fused with the Fc region of human IgG, designed to enable separation and purification using its affinity for Protein A, and the second target protein is designed to include a His tag in the middle of the protein sequence, designed to enable separation and purification using its metal affinity with Ni2+-NTA beads.

[0203] To construct an expression vector expressing Fc-A:hIn-A, the uH7LH:GluB4:hFc-A:L1:EK:hIn-A DNA fragment was chemically synthesized (Gene Universal (USA)). The DNA fragment was cleaved with XbaI and XhoI restriction enzymes, and then p1300::Fc-A:hIn-A was constructed by ligating it to p1300-GFP-3PR, which had been cleaved with the same restriction enzymes. Subsequently, BiP:GFP:HDEL. was used to replace the promoter with the FMM-UD promoter. The promoter was obtained by cleaving Construct (yoon et al., 2023; Design of an artificial transcriptional system for production of high levels of recombinant proteins in tobacco (Nicotiana benthamiana), Front. Plant Sci., Sec. Plant Biotechnology 14. https: / doi.org / 10.3389 / fpls.2023.1138089) with PstI and XbaI restriction enzymes, and pFMM::Fc-A:hIn-A was constructed by ligating it to p1300::Fc-A:hIn-A treated with the same restriction enzymes. Subsequently, BiP:GFP:HDEL was used to replace the terminator with 3PR-t. Construct (yoon et al., 2023) was cleaved with XhoI and EcoRI restriction enzymes to obtain 3PR-t, and pFc-A:hIn-A was constructed by ligating it to pFMM::Fc-A:hIn-A treated with the same restriction enzymes (Fig. 6A).

[0204] To construct pZipDB-A:hIn-A and pZipDB-B:hIn-B, XbaI:Rubi:UTR:GluB4:GB1:ZipDB-A:His:2L:EK:In-A:XhoI and XbaI:Rubi:UTR:GluB4:GB1:ZipDB-B:2L:EK:In-B:XhoI DNA fragments were synthesized chemically (Gene Universal, USA). The two DNA fragments were cleaved with XbaI and XhoI restriction enzymes, and then ligated into pFc-A:hIn-A, which had been cleaved with the same restriction enzymes, to construct pZipDB-A:hIn-A and pZipDB-B:hIn-B (Fig. 6B).

[0205] Transgenic plants were produced by transforming Baromi seeds with the vectors prepared to express each target protein in the same manner as in Example 2 above, and then seeds were harvested from them and used for protein purification experiments.

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235] 6-2. Seed Grinding and Protein Extraction

[0236] A total of 10 g of Baromi seeds harvested from each transformant were taken and ground into a fine powder using a grinder for approximately 2 minutes. For the obtained seed powder, 10 mL of extraction buffer was added per 1 g of powder. The extraction buffer was prepared with a composition containing 0.5 M NaCl, 100 mM Tris-HCl (pH 6.8), 0.5% Tween-20, Triton X-100, 400 mM sucrose, and 5% glycerol. The mixture was vortexed for approximately 5 minutes, followed by stirring for approximately 4 hours at room temperature or 4°C to sufficiently extract proteins. Subsequently, 2× phosphate buffer (PBS) was added, and the mixture was incubated overnight at 4°C with stirring. To remove cell debris and insoluble substances, the mixture was centrifuged at 14,000 × g for 15 minutes, and the supernatant was collected. After performing three additional centrifugations under the same conditions, the final supernatant was used for protein purification.

[0237]

[0238] 6-3. Isolation and Purification of Fc-Fusion Human Insulin A Chain Protein Using Protein A Affinity Chromatography

[0239] Protein A agarose beads (Protein A Sepharose™, Cytiva) were used to purify the target protein containing the Fc region. The Protein A beads were washed three times with PBS buffer before use. The washed beads (50 μL slurry) were mixed with the protein supernatant (1 mL) prepared in Example 6-2 and incubated by gentle rotation at 4°C for approximately 2 hours to induce binding of the target protein. After binding, the beads were loaded into a gravity flow column to remove unbound proteins, and washed three times with PBS buffer to remove non-specifically bound proteins. Subsequently, the target protein (Fc-A:L1:EK:hIn-A) was eluted using IgG elution buffer (0.1 M glycine-HCl) at pH 2.0, and immediately after elution, 1 M Tris buffer at pH 8.8 was added to neutralize the solution. As a result, it was confirmed that the target protein was effectively eluted (Fig. 6A).

[0240]

[0241] 6-4. Isolation and Purification of His-Tagged ZipDB-A:hIn-A:ZipDB-B:hIn-B Proteins Using Ni2+-NTA Resin

[0242] Ni2+-NTA agarose beads (Qiagen, Hilden, Germany) were used to purify the target protein containing the His tag (ZipDB-A:hIn-A:ZipDB-B:hIn-B). The Ni2+-NTA beads were washed three times with PBS buffer (pH 7.4) before use. To inhibit non-specific protein binding, 3 mM imidazole was added to the protein supernatant (1 mL) prepared in Example 6-2, and then mixed with the washed Ni2+-NTA beads (50 μL slurry). The mixture was incubated at 4°C for approximately 2 hours using an end-over-end rotation method to induce binding of the target protein containing the His tag. After binding, the beads were loaded into a gravity flow column to remove unbound proteins, and non-specifically bound proteins were removed by washing three times with PBS washing solution (pH 7.4) containing 8 mM imidazole. Finally, the target protein (ZipDB-A:hIn-A:ZipDB-B:hIn-B) was eluted from the Ni2+-NTA beads using a PBS elution solution (pH 7.4) containing 400 mM imidazole, and as a result, it was confirmed that the target protein was effectively purified and eluted (Fig. 6B).

[0243]

[0244] Example 7. Comparison of Expression and Purification of Recombinant Heterodimeric Fc Fusion Human Insulin A+B Proteins in Baromi and Dongjin Variety Seeds

[0245] 7-1. Construction of a single expression vector co-expressing heterodimeric Fc fusion human insulin A and B chains

[0246] To simultaneously express human insulin A and B chains fused to heterodimeric Fc in rice, a single dual expression vector containing two independent expression cassettes in one vector was designed and constructed.

[0247] Transcription terminator 9 (Sequence No. 43) is a complex terminator comprising an Hsp18.2-derived termination sequence, a linker sequence, and a tRNA-Glu sequence, and was prepared by chemically synthesizing a DNA fragment designed to have an XhoI restriction enzyme recognition sequence at the 5′ end and an EcoRI restriction enzyme recognition sequence at the 3′ end (Gene Universal). After cleaving the DNA fragment with XhoI and EcoRI restriction enzymes, the DNA fragment was ligated into the p1300-GFP-3PR vector of Example 1 treated with the same restriction enzymes to remove the existing 3PR terminator and insert transcription terminator 9 in its place, thereby constructing a recombinant vector.

[0248] Next, the above vector was cleaved with MluI and ApaI restriction enzymes, and a DNA fragment was chemically synthesized and prepared in which the artificially reconstructed 35MUFO promoter (Sequence No. 44) had a MluI restriction enzyme recognition sequence at the 5′ end and an ApaI restriction enzyme recognition sequence at the 3′ end (Gene Universal, USA). The above DNA fragment was cleaved with MluI and ApaI restriction enzymes and then inserted into a vector treated with the same restriction enzymes to construct a promoter-substituted vector.

[0249] To introduce two expression cassettes into a single vector, PCR amplification was performed using transcription terminator 9 (SEQN 43) as a template. At this time, forward primer 9-Stu (SEQN 45) and reverse primer 9-Eco (SEQN 46), which contain a StuI restriction enzyme recognition sequence at the 5′ end, were used. By inserting the PCR amplification product into the corresponding restriction enzyme site of the vector, a pDouble vector capable of accommodating two independent expression cassettes was constructed.

[0250] The existing GFP expression cassette was removed by cleaving the constructed pDouble vector with XbaI and XhoI restriction enzymes. Subsequently, the XbaI:gBiP:Fc-B:hIn-B:XhoI expression cassette containing the BiP leader sequence (gBiP), Fc domain, and human insulin B chain sequence was chemically synthesized (Gene Universal). The pDgBiP:Fc:hIn-B vector was constructed by treating the expression cassette with the same restriction enzymes and inserting it into the pDouble vector.

[0251] Subsequently, a human insulin A chain expression cassette composed of ApaI:gBiP:Fc-A:2L:EK:hIn-A:StuI was chemically synthesized (Gene Universal). After cleaving the expression cassette with ApaI and StuI restriction enzymes, the vector pDhIn(A+B), capable of simultaneously expressing insulin A and B chains, was constructed by inserting it into a pDgBiP:Fc:hIn-B vector cleaved with the same restriction enzymes.

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260] 7-2. Protein Extraction from Transgenic Seeds

[0261] The vector prepared above was transformed into the Baromi and Dongjin varieties, respectively, using the same method as in Example 2, and seeds were harvested. 0.5 g of each harvested seed was taken and ground into a fine powder under the same conditions as in Example 5. Seeds of wild rice (WT) were also included as a control.

[0262] Baromi and Dongjin transgenic seed powders were placed in a protein extraction buffer containing 300 mM NaCl, 100 mM Tris, 2% glycerol, 0.2% Tween-20, 1 mM PMSF, and 3% PVP, and thoroughly homogenized. Subsequently, to increase protein extraction efficiency, the mixture was incubated with stirring for approximately 12-16 hours (overnight) at 4°C.

[0263] The next day, an equal volume of PBS buffer was added to the extract and sonicated for 30 seconds to promote protein elution. The extract was centrifuged twice in succession at 12,000 rpm for 15 minutes to remove insoluble cell residues, and the supernatant was recovered and filtered to be used as a total soluble protein (TSP) sample.

[0264]

[0265] 7-3. Purification of Fc-A:hIn-A / Fc-B:hIn-B Recombinant Proteins Using Protein A Affinity Chromatography

[0266] Total soluble protein samples from transgenic seeds of Baromi and Dongjin varieties were mixed with Protein A conjugated agarose beads (Protein A Sepharose™, Cytiva, Uppsala, Sweden) to induce binding of recombinant proteins containing the Fc region. For affinity binding, 200 μL of Protein A beads (based on slurry) was used per 100 mL of extract, and the mixture was incubated for about 1 hour at 4°C with gentle stirring in an end-over-end manner. After the binding reaction, the mixture was applied to a gravity flow column packed with Protein A beads to remove unbound proteins. Subsequently, to remove non-specifically bound proteins, the column was washed sequentially with (1) PBS, (2) PBS containing 400 mM NaCl, and (3) PBS containing 600 mM NaCl. After washing was completed, the recombinant protein Fc-A:hIn-A / Fc-B:hIn-B bound to Protein A beads was eluted using an elution buffer containing 200 mM glycine (pH 2.2), and the eluted fraction was immediately neutralized by adding 1 M Tris buffer (pH 8.8).

[0267]

[0268] 7-4. Analysis of Purified Recombinant Proteins (Fc-A:hIn-A and Fc-B-hIn-B)

[0269] The Fc-A:hIn-A / Fc-B:hIn-B proteins isolated and purified from the transformed seeds of the Baromi and Dongjin varieties were analyzed via SDS-PAGE. Total soluble protein (TSP) from wild-type (WT) and transformed Baromi seeds was used as the negative and positive controls in the western blot analysis. Following SDS-PAGE, the presence of recombinant proteins (Fc-A:hIn-A and Fc-B:hIn-B) was confirmed through Coomassie Brilliant Blue staining and western blot analysis using anti-human IgG antibodies derived from HRP-conjugated goats (Bethyl Laboratories, USA).

[0270] SDS-PAGE analysis results showed that the purified sample from the Baromi variety exhibited high purification purity with relatively few non-specific protein bands and a distinct single band in the target molecular weight region, whereas the purified sample from the Dongjin variety showed additional protein bands in the upper molecular weight region along with the target band, suggesting that some non-specific proteins were co-eluted.

[0271] This trend was confirmed more clearly through Western blot analysis. Specifically, while recombinant proteins containing the Fc region (i.e., double-stranded insulin recombinant protein) were detected by human anti-IgG antibodies in both the Baromi and Dongjin varieties, the band of the target protein was detected more clearly in samples derived from the Baromi variety compared to non-specific bands, indicating that the Baromi variety exhibited significantly more stable characteristics in terms of recombinant protein expression and purification efficiency.

[0272] These results suggest that seeds of the Baromi variety are a more suitable plant host for the production and subsequent purification processes of recombinant proteins, supporting their potential as a seed-based recombinant protein production platform.

[0273]

[0274] Although the present invention has been described above with specific details such as specific components, limited embodiments, and drawings, this is provided merely to aid in a more comprehensive understanding of the invention and the invention is not limited to the above embodiments. A person skilled in the art to which the invention pertains can make various modifications and variations from this description. Accordingly, the scope of the present invention should not be limited to the embodiments described above, and all things equivalent to or equivalently modified from the claims set forth below, as well as the claims themselves, shall be considered to fall within the scope of the concept of the present invention.

[0275]

[0276] [National R&D projects that supported this invention]

[0277] [Project ID] 2710015548

[0278] [Sub-project No.] 2022H1D3A2A02093527

[0279] [Ministry Name] Ministry of Science and ICT

[0280] [Specialized Research Management Agency] National Research Foundation of Korea

[0281] [Research Project Name] Support for the Expansion of Talent Utilization

[0282] [Research Project Title] Development of Production Technology for Protein Materials Essential to the Plant-Based Cultured Meat Industry

[0283] [Name of Project Performing Organization] Pohang University of Science and Technology

[0284] [Research Period] 2024-01-01 ~ 2024-12-31

Claims

1. A gene encoding a target protein, an expression construct comprising said gene, or a recombinant expression vector comprising said gene introduced therein, Transgenic organism derived from the Baromi variety.

2. In Paragraph 1, The above-mentioned transformant comprises transformed rice, transformed plant cells, transformed callus, transformed seeds, or tissues or fractions derived from any one of these. Transgenic organism.

3. In Paragraph 2, The above-mentioned transformant comprises a powder prepared from the above-mentioned transformant seed, Transgenic organism.

4. In Paragraph 3, The above powder is prepared by grinding genetically modified seeds at room temperature for 10 seconds to 5 minutes. Transgenic organism.

5. In Paragraph 1, The above-mentioned target protein is one or more selected from the group consisting of thaumatin, lactoferrin, transferrin, interleukin, transcription factor, membrane protein, insulin, cytokinin, growth factor, toxin protein, hormone, hormone analog, cytokine, movement protein, lysozyme, vaccine, enzyme, enzyme inhibitor, transport protein, structural protein, receptor, receptor fragment, biological defense inducer, storage protein, exploitative protein, reporter protein, artificially designed protein, hydrophobin, antigen, antibody, and antibody fragment. Transgenic organism.

6. A method for producing a target protein comprising the following steps: (a) a step of crushing a transgenic seed into which a gene encoding a target protein, an expression construct containing said gene, or a recombinant expression vector containing said gene has been introduced; and (b) A step of recovering the target protein from the ground seed powder.

7. In Paragraph 6, The above step (a) is powdered by mechanical grinding without a pretreatment process, Purpose: Method for producing protein.

8. In Paragraph 7, The above pretreatment process is a moisture treatment or a cooling treatment, Purpose: Method for producing protein.

9. In Paragraph 7, The above mechanical grinding is performed at room temperature for 10 seconds to 5 minutes, Purpose: Method for producing protein.

10. In Paragraph 7, The above mechanical grinding is performed at a frequency of 10 to 50 Hz, Purpose: Method for producing protein.

11. In Paragraph 6, The above step (b) is to extract the target protein from the crushed seed powder and then purify and recover it. Purpose: Method for producing protein.

12. In Paragraph 11, The above purification is performed by chromatography, precipitation, membrane separation, filtration, or a combination thereof. Purpose: Method for producing protein.

13. In Paragraph 6, A method for producing a target protein, wherein the above-mentioned transgenic seeds are manufactured by including the following steps: (i) a step of redifferentiating a transformed callus into transformed rice into which a recombinant expression construct or recombinant expression vector containing a gene encoding a target protein has been introduced; and (ii) A step of harvesting transgenic seeds from the redifferentiated transgenic rice.

14. In Paragraph 13, The regeneration efficiency of the above step (i) is 60% or higher, Purpose: Method for producing protein.

15. In Paragraph 13, The above redifferentiation step is in which a stem is induced within 10 to 18 days after culturing the transformed callus in a redifferentiation medium. Purpose: Method for producing protein.

16. In Paragraph 13, A method for producing a target protein, wherein the step of harvesting the transformed seeds is performed within 45 to 65 days after the completion of Agrobacterium-mediated transformation.

Images