WO2014088255A1

WO2014088255A1 - Base sequence for enhancing translation efficiency of target proteins in plants

Info

Publication number: WO2014088255A1
Application number: PCT/KR2013/010856
Authority: WO
Inventors: 황인환; 김영현; 박민희; 조규형; 이용직; 손은주
Original assignee: 주식회사 바이오앱
Priority date: 2012-12-06
Filing date: 2013-11-27
Publication date: 2014-06-12
Also published as: KR101449155B1; KR20140073710A

Abstract

The present invention relates to a DNA fragment, which is synthesized for the enhancement of translation efficiency, and to a recombinant vector comprising same. More particularly, the present invention relates to a DNA fragment and to a recombinant vector comprising the DNA fragment, wherein the DNA fragment is composed of 21 base sequences expressed by SEQ ID No: 1, and can enhance the translation efficiency of a target protein. The DNA fragment according to the present invention can be inserted upstream of a target protein, thereby being constructed as a recombinant vector. As a result, a plant which is transformed with the vector can improve the translation of a target protein and allow the target protein to be stably accumulated by inducing the targeting of the target protein to a particular location in the plant. Thus, the present invention has the effect of producing target proteins from a plant in large amounts.

Description

Sequences for Enhancing Translation Efficiency of Target Proteins in Plants

The present invention relates to a novel base sequence that can enhance the translation efficiency of genes in order to produce a large amount of the protein of interest in plants.

Many biopharmaceuticals used to date have been mostly produced using animal cells, bacteria and fungi (Granz PR et al., 1996; Ma JK et al., 1999; Pen J, 1996). However, the production of recombinant protein using a microbial system has the advantage that mass production is easy, while glycoproteins have problems of proper folding of proteins and post-translational modification such as glycosylation. It is not suitable for the production of. The problem has been solved by using animal cells, but it has emerged as another problem to be solved that animal cells can be infected with human infectious viruses and that a large cost is required to expand equipment for mass production.

Plant cells, unlike animal cells, are unlikely to be infected with a virus that can infect the human body, and have well-established experiences and infrastructure related to cultivation and harvesting from the past to the present, so that only the cultivated area for mass production is secured. Since mass production is always possible, the cost equipment investment for mass production is relatively inexpensive.

However, even with these advantages, one of the obstacles to protein production in plants is that protein expression in plants or plant cells is not well utilized because the level of protein expression that can be produced in plants has not been achieved on a commercial scale.

The protein is produced through a transcriptional step and a translational step that generate mRNA. There are two methods of regulating the expression of proteins, a method of greatly increasing the amount of transcription and a method of increasing translation efficiency. However, even if the production of mRNA is improved by improving the efficiency of transcription step to effectively express the useful protein in the plant, the produced mRNA is not all used for translation, and in some cases inhibits the production of mRNA. (Napoli C et al., 1990; van der Krol AR et al., 1990).

In addition, as the amount of protein finally synthesized from a gene is found to be more important in the translational stage than in the transcriptional stage, the expression of protein at the translational stage, not at the transcriptional stage, is not required for mass production of effective useful proteins. The development of adjustable technology is needed.

In order to overcome the problems of the prior art, the inventors of the present invention, while studying the method to promote the synthesis of the protein in the translation step by mass production of useful proteins from the plant, the base of the 5 'UTR in the adjacent upper portion of the translation start codon The present invention was completed by paying attention to the sequence and artificially synthesizing the 5 'UTR sequence.

It is therefore an object of the present invention to provide a DNA fragment capable of enhancing the translational efficiency of a protein and / or a recombinant vector comprising said DNA fragment.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention is a DNA fragment consisting of the nucleotide sequence of SEQ ID NO: 1, the DNA fragment is to improve the translation (translation) of the target protein located downstream of the plant cell Provided is a DNA fragment for improving translation efficiency.

The present invention also provides a recombinant vector comprising the DNA fragment for improving the translation efficiency.

In one embodiment of the present invention, the recombinant vector may be a promoter, a DNA fragment for enhancing translation efficiency, and a gene encoding a target protein to be operably linked sequentially.

In another embodiment of the present invention, the recombinant vector may be one operably linked to a base sequence that can target (target) and retain the target protein (ER) in plant cells.

In another embodiment of the present invention, the base sequence that can target the target protein to the endoplasmic reticulum (ER) in the plant cell is the nucleotide sequence represented by SEQ ID NO: 3 encoding the BiP (chaperone binding protein) protein The base sequence capable of retaining the target protein may be a base sequence encoding a HDEL (His-Asp-Glu-Leu) or KDEL (Lys-Asp-Glu-Leu) peptide.

In another embodiment of the present invention, the recombinant vector may be one operably linked to the nucleotide sequence of SEQ ID NO: 4 encoding the cellulose-binding domain (CBD).

In addition, the present invention provides a method for mass-producing a protein of interest from a plant comprising introducing a recombinant vector into the plant.

In one embodiment of the present invention, the step of introducing the recombinant vector to the plant is PEG precipitation method (Polyethylen glycol), Agrobacterium (Agrobacterium sp.)-Mediated method, particle gun bombardment (particle gun bombardment) It may be made using any one method selected from the group consisting of silicon carbide whiskers, sonication, and electroporation.

In another embodiment of the invention, the plant is Arabidopsis, soybean, tobacco, eggplant, pepper, potato, tomato, cabbage, radish, cabbage, lettuce, peach, pear, strawberry, watermelon, melon, cucumber, carrot, and Dicotyledonous plants selected from the group consisting of celery; Or a monocotyledonous plant selected from the group consisting of rice, barley, wheat, rye, corn, sugar cane, oats, and onions.

The DNA fragment according to the present invention is added upstream of the target protein to be produced as a recombinant vector, so that the plant transformed with the recombinant vector can improve the translation of the target protein and induce targeting to a specific place in the plant to target the target protein. This can be accumulated stably.

1 shows a schematic diagram of recombinant vector to which a) M17 and b) R21 are added for expression of fusion protein GFP in Arabidopsis protoplasts.

Figure 2 confirms the expression of GFP from Arabidopsis protoplasts transformed with the recombinant vector of Figure 1 by Western analysis. lane 1: Marker, lane 2: M17-GFP, lane 3: R21 GFP, lane 4: Negative control

3 is a recombinantly operably linked signal sequence (gBiP), target protein (GFP) sequence, cellulose-binding domain (CBD), and HDEL sequence that can be targeted to DNA fragments and endoplasmic reticulum for enhancing translation efficiency according to the present invention. The schematic of the vector is shown.

4 is confirmed by Western blot whether the target protein GFP is properly targeted to the endoplasmic reticulum from the Arabidopsis protoplasts transformed with the recombinant vector of FIG. 3.

The present inventors pay attention to the development of 5'untranslated regions (5'UTRs) that can enhance the translation of genes in order to find a way to mass-produce a target protein in plants, and Arabidopsis thaliana ) synthesized an arbitrary 5 'UTR sequence based on the 5' UTR sequence of the small subunit (RbcS) gene of ribulose bisphosphate carboxylase / oxygenase (RuBisCo), which is known to have excellent translation efficiency. .

In general, 5 'untranslated region (5' UTR) of mRNA performs a number of functions in the gene expression process, the biggest feature of these functions is involved in the regulation of mRNA translation efficiency. The nucleotide sequence of the 5 'UTR in the immediate upper portion of the translation initiation codon has been reported to influence the efficiency of the translation step (Xiong W et al., 2001; Rogozin IB et al., 2001; Gallie DR et al., 1989; Mignone F et al. , 2002; Kawaguchi R et al., 2002; Kawaguchi R et al., 2005), a representative example is the 5 ′ UTR of the small subunit (RbcS) gene of ribulose bisphosphate carboxylase / oxygenase (RuBisCo). This 5 'UTR has a back base or more of nucleotides, and the 3' UTR has a few kilo bases longer. In addition, a sequence belonging to 5'UTR, which is not a predetermined position such as a Shine-Dalgarno sequence, which is known as a ribosome binding site sequence located at 5 'UTR in prokaryotes, is a ribosome binding site sequence which is also called a ribosome binding site sequence in eukaryotes. Have been reported (Kozak M, 1987; Hamilton et al., 1987; Yamauchi et al., 1991; Joshi et al., 1997).

In this regard, the present inventors use a small subunit (RbcS) of ribulose bisphosphate carboxylase / oxygenase (RuBisCo), which is known to have excellent translation efficiency among the sequences of 5 'UTR of Arabidopsis in a way to mass-produce a target protein. The 5 'UTR nucleotide sequence represented by SEQ ID NO: 1 was synthesized based on the 5' UTR sequence of the gene, and it was confirmed that the sequence had excellent translation efficiency (see Example 1).

Therefore, the present invention is a DNA fragment consisting of the nucleotide sequence of SEQ ID NO: 1, the DNA fragment is a DNA fragment for enhancing the translation efficiency, characterized in that to improve the translation (translation) of the target protein located downstream of the plant cell to provide.

In addition, the present invention is a DNA fragment consisting of the nucleotide sequence of SEQ ID NO: 1, wherein the DNA fragment is a DNA fragment for enhancing the translation efficiency, characterized in that to enhance the translation (translation) of the target protein located downstream of the plant cell It provides a recombinant vector comprising a.

More specifically, the recombinant vector is a promoter based on an existing vector used for protein expression, promoter, the DNA fragment according to the present invention to enhance the translation efficiency of the protein, and the gene encoding the target protein sequentially It may be a recombinant expression vector operably linked.

In the present invention, the term "expression vector" refers to a plasmid, virus or other medium known in the art in which the polynucleotide sequence encoding the DNA fragment and the target protein according to the present invention can be inserted or introduced. The DNA fragment and the polynucleotide sequence encoding the target protein according to the present invention may be operably linked to an expression control sequence, wherein the operably linked gene sequence and the expression control sequence are selected markers and replication origin. It may be included in one expression vector containing together. “Operably linked” can be genes and expression control sequences linked in such a way as to enable gene expression when the appropriate molecule is bound to the expression control sequences. An "expression control sequence" refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a particular host cell. Such regulatory sequences include promoters for carrying out transcription, any operator sequence for controlling transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control termination of transcription and translation.

The promoter is not particularly limited as long as it can express an insertion gene in a plant, but is not limited thereto, and the 35S RNA and 19S RNA promoters of CaMV; Full-length transcriptional promoters derived from Peak Water Mosaic Virus (FMV) and coat protein promoters of TMV. Also suitable vectors for introducing the DNA fragments according to the invention and polynucleotide sequences encoding the desired proteins into plant cells are Ti plasmids and plant viral vectors. Examples of suitable vectors are, but are not limited to, p35S. Those skilled in the art can select a vector suitable for introducing the DNA fragment and the polynucleotide sequence encoding the protein of interest according to the present invention, in the present invention plant cell polynucleotide sequence encoding the DNA fragment and the protein of interest according to the invention Any vector that can be introduced into can be used.

In addition, the present inventors use the DNA fragments of the present invention to enhance the translational efficiency of the protein of interest, thereby enhancing protein expression as well as inducing and retaining the targeting of the target protein to specific places in plant cells. Recombinant expression vector was constructed to see if it can be.

In general, in the case of overexpressing a target protein in a transgenic plant or cell, the phenomenon of proteolytic degradation often occurs. However, when foreign proteins are targeted to various intracellular organelles, these proteins can be stored more stably. In particular, if the target protein is targeted to the endoplasmic reticulum, the degradation of the protein can be minimized than that present in the cytoplasm. For example, when the human epidermal growth factor is targeted to the endoplasmic reticulum in tobacco, the yield of the protein is about 10 ⁴ times higher. It is known (Wirth S et al., 2004). In addition, the use of ER retention signal peptides such as KDEL or HDEL keeps the target protein in the endoplasmic reticulum, thereby increasing folding and assembly by molecular chaperones, resulting in protein degradation. Can be minimized (Nuttall J et al., 2002). As such an example, when the target protein is retained in the endoplasmic reticulum than when it is sent to the secretory pathway, the yield of the target protein has been known to increase by about 10 to 100 times (Hellwig S et al., 2004). In addition, the chloroplasts of plants are not only huge protein reservoirs containing more than 40% of the total water soluble protein but also a large number, making them a good target for the storage of target proteins. The plant's chloroplasts contain only the minimal protease necessary for protein processing, which means that the protein of interest can be stored in high concentrations in the chloroplasts by preventing degradation by the protease.

Therefore, the present inventors have found that the DNA fragment of the present invention consists of a promoter, a nucleotide sequence of SEQ ID NO: 1 which enhances the translation efficiency of a protein, and a plant to a recombinant expression vector in which a gene encoding a target protein is operably linked. Recombinant vectors were additionally operably linked to specific locations of the vesicles or chloroplasts to target the target protein.

At this time, when targeting the target protein to the endoplasmic reticulum (ER) in the plant cell, a base sequence encoding a BiP (chaperone binding protein) protein may be used, and preferably a cDNA of BiP to enhance the translation efficiency. Instead, the nucleotide sequence represented by SEQ ID NO: 3, which is a genomic DNA of BiP containing an intron, may be used. A base sequence for retaining the target protein is not limited thereto. Preferably, the base sequence encoding the HDEL (His-Asp-Glu-Leu) or KDEL (Lys-Asp-Glu-Leu) peptide is preferred. Can be used. The N-terminus of the BiP protein contains a signal sequence that determines targeting to the endoplasmic reticulum, which may serve to target the target proteins to the endoplasmic reticulum. In addition, when targeting the target protein to the chloroplast in the plant cells (nucleotide chlorophyll a / b binding protein) can be used to coat the base sequence. Cab (chlorophyll a / b binding protein) has a transit peptide necessary for targeting to chloroplasts at the N-terminus, and when the peptide is used, target proteins can be normally targeted to chloroplasts (kavanagh TA). Et al., 1988).

Therefore, when using an expression vector using such BiP signal sequence, vesicle maintenance signal sequence, or CAB transit peptide, the target protein can be targeted to the endoplasmic reticulum or chloroplast, and the target protein can be accumulated at a high level for mass production. It has an effect.

The present invention further provides a plant transformed by introducing the recombinant vector according to the present invention into a plant using methods known in the art.

Method for introducing the recombinant vector of the present invention into the plant is useful for but not limited to precipitation by PEG (Polyethylenglycol), Agrobacterium (Agrobacterium sp.) - method according to the parameters, the particle gun bombardment method (particle gun bombardment ), Silicon carbide whiskers, sonication, or electroporation. In one embodiment of the present invention, Arabidopsis was transformed with the recombinant vector of the present invention using the precipitation method by polyethylene glycol (PEG).

In one embodiment of the present invention, the Arabidopsis was used as the plant, but this is only a preferred example, and the plant may be a dicotyledonous plant or a monocotyledonous plant, but the dicotyledonous plant is not limited thereto. Tobacco, eggplant, pepper, potato, tomato, cabbage, radish, cabbage, lettuce, peach, pear, strawberry, watermelon, melon, cucumber, carrot or celery and the like, and the monocotyledonous plant, but is not limited thereto, rice, Barley, wheat, rye, corn, sugar cane, oats, onions, and the like.

In addition, the present invention can provide a method for mass-producing a target protein from a plant by introducing a recombinant vector containing a DNA fragment comprising SEQ ID NO: 1 of the present invention into a plant.

In the above, the target protein is a term meaning a protein to be produced, and is not limited to a specific protein. In addition, reporter proteins may be included as the target protein, wherein the reporter protein is a protein expressed by a reporter gene and refers to a labeling protein indicating its activity in a cell by its presence. In one embodiment of the present invention, green fluorescent protein (GFP) was used as the target protein.

The method for producing a target protein from the transformed plant comprises introducing a recombinant vector according to the present invention into a plant or a protein translation enhancing DNA fragment according to the present invention and a nucleotide sequence encoding the target protein operably connected to the plant or the The cells may be transformed with the recombinant expression vector, then incubated for a suitable time to express the protein of interest, and then obtained from the transformed plant or cell. At this time, the method for expressing the target protein can be used as long as it is known in the art. In addition, the recovery of the target protein highly expressed in the transformed plant or cell can be carried out through various separation and purification methods known in the art, and the cell lysate is usually removed to remove cell debris. After centrifugation, precipitation, for example, salting out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.) may be performed, and dialysis, electrophoresis and various columns are performed. Chromatography may be performed. As the chromatography, a target protein of the present invention can be purified by applying techniques such as ion exchange chromatography, gel-penetration chromatography, HPLC, reverse phase-HPLC, affinity column chromatography, or ultrafiltration alone or in combination. (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989) Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182. Academic Press. Inc., San Diego, CA (1990)).

In addition, the present invention is to encode a cellulose-binding domain (CBD) of the cellulase in the recombinant vector of the present invention to more easily and quickly separate and purify the target protein from the transformed plant A recombinant vector was prepared, in which the nucleotide sequence represented by SEQ ID NO: 4 is further operably linked.

Therefore, in the present invention, the target protein expressed by using a recombinant vector including a cellulose-binding domain may be in a fused form with a cellulose-binding domain, and can be easily separated and purified through chromatography using a cellulose carrier as a column. have. The produced protein of the fused form may be cleaved of the cellulose binding domain using an appropriate enzyme and the like, followed by additional separation and purification to produce the non-fused form of the protein of interest.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[실시예 1]Example 1

번역효율을 증가시키는 5' UTR의 개발Development of 5 'UTR to increase translation efficiency

In order to develop a 5 'UTR that can enhance the translation of genes to increase the production of the protein of interest, a small amount of ribulose bisphosphate carboxylase / oxygenase (RuBisCo), which is known to have excellent translation efficiency in Arabidopsis An arbitrary 5 'UTR sequence was developed and synthesized based on the 5' UTR sequence of the subunit (RbcS) gene. The synthesized 5 'UTR was named M17, the 5' UTR of the RbcS gene was named R21, and each base sequence is shown in Table 1 below.

Table 1

designation	Sequence	SEQ ID NO:
M17 5 'UTR	5'- GGC GTG TGT GTG TGT TAA AGA -3 '	One
R21 5 'UTR	5'- CAC AAA GAG TAA AGA AGA ACA -3 '	2

[실시예 2]Example 2

개발된 5' UTR의 식물세포 내에서 발현량 확인Expression level of the developed 5 'UTR in plant cells

In order to confirm that M17 translation efficiency was increased during protein expression, a green fluorescent protein (GFP) was used as a reporter gene, and as shown in FIG. 1, M17 was placed on the start codon of the GFP. As a control, R21, a 5 'UTR derived from RbcS, was installed on top of the start codon of GFP. In addition, PEG (Polyethylen glycol) precipitation method was introduced to protoplasts of Arabidopsis to determine whether M17 is effective in improving translation efficiency.

As a result, as shown in Fig. 2, the expression effect of the group in which M17 was installed was better than that of R21, a 5 ′ untranslated sequence derived from RbcS, which is known to be involved in relatively high expression in Arabidopsis.

From the above, it can be seen that M17 has an effect of improving the expression rate by enhancing the translation efficiency of the target protein.

[실시예 3]Example 3

식물 세포 내의 특정 장소에 목적 단백질을 수송 시 개발된 5' UTR 번역 효율 분석Analysis of the developed 5 'UTR translation efficiency when transporting the target protein to a specific place within plant cells

The inventors investigated whether M17, the 5 'UTR developed by the present inventors, shows excellent translation efficiency even in a vector designed to transport a target protein to a specific place for stable storage and increased yield of the protein. To this end, as shown in Figure 3, M17 5 'UTR and control group R21 5' UTR were added to the vector transporting the target protein to the endoplasmic reticulum. At this time, the vector produced to transport the target protein to the endoplasmic reticulum (ER) is a base sequence encoding the BiP (chaperone binding protein) involved in the endoplasmic reticulum transport, retaining the target protein in the endoplasmic reticulum It consists of a base sequence encoding the HDEL (His-Asp-Glu-Leu) for, and a base sequence encoding the cellulose-binding domain (cellulose-binding domain) used for the purification of proteins. In the vector, the target protein, Green fluorescent protein (GFP), which is a reporter gene, was used, and these vectors were introduced into Arabidopsis protoplasts using PEG precipitation and compared with expression efficiency.

As a result, as shown in FIG. 4, the expression of the 5 'untranslated region M17 was superior to that of the control group R21, confirming that the 5' untranslated region M17 was more effective in transporting the target protein GFP into the endoplasmic reticulum. It was.

From the above, it can be seen that the 5 'untranslated region M17 of the present invention having excellent translation efficiency can be usefully used for targeting and retention of a target protein to a specific place.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

Plants transformed with a recombinant vector comprising a DNA fragment of the present invention can be usefully used to produce a large amount of the protein of interest.

<210> 1

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> M17 5 'UTR

<400> 1

ggcgtgtgtg tgtgttaaag a

<210> 2

<211> 21

<212> DNA

<213> Arabidopsis thaliana-RbcS 5 'UTR

<400> 2

cacaaagagt aaagaagaac a

<210> 3

<211> 272

<212> DNA

<213> Arabidopsis thaliana-BiP

<400> 3

atggctcgct cgtttggagc taacagtacc gttgtgttgg cgatcatctt cttcggtgag

tgattttccg atcttcttct ccgatttaga tctcctctac attgttgctt aatctcagaa

ccttttttcg ttgttcctgg atctgaatgt gtttgtttgc aatttcacga tcttaaaagg

ttagatctcg attggtattg acgattggaa tctttacgat ttcaggatgt ttatttgcgt

tgtcctctgc aatagaagag gctacgaagt ta

<210> 4

<211> 502

<212> DNA

<213> Clostridium stercorarium-CBD

<400> 4

ttacacatgg catggatgaa ctatacaaat taggaggtgg aggtggaccc cgggcacacc

accaccacca ccactttcga agttcaccag tgcctgcacc tggtgataac acaagagacg

catattctat cattcaggcc gaggattatg acagcagtta tggtcccaac cttcaaatct

ttagcttacc aggtggtggc agcgccattg gctatattga aaatggttat tccactacct

ataaaaatat tgattttggt gacggcgcaa cgtccgtaac agcaagagta gctacccaga

atgctactac cattcaggta agattgggaa gtccatcggg tacattactt ggaacaattt

acgtggggtc cacaggaagc tttgatactt atagggatgt atccgctacc attagtaata

ctgcgggtgt aaaagatatt gttcttgtat tctcaggtcc tgttaatgtt gactggtttg

tattctcaaa tcaagaactt ag

Claims

DNA fragment consisting of the nucleotide sequence of SEQ ID NO: 1, wherein the DNA fragment is a DNA fragment for enhancing translation efficiency, characterized in that to promote the translation (translation) of the target protein located downstream of the plant cell.
Recombinant vector comprising a DNA fragment for improving the translation efficiency of claim 1.
The method of claim 2,

The recombinant vector is a recombinant vector, characterized in that the promoter, the DNA fragment for enhancing the translation efficiency, and the gene encoding the target protein is operably linked in sequence.
The method of claim 3,

The recombinant vector is a recombinant vector, characterized in that the nucleotide sequence capable of targeting and retention of the target protein (ER) in the plant cell additionally operably linked.
The method of claim 4, wherein

The base sequence capable of targeting the target protein to the ER (ER) in the plant cell is a nucleotide sequence represented by SEQ ID NO: 3 encoding a BiP (chaperone binding protein) protein, and retains the target protein. A nucleotide sequence which can be a nucleotide sequence encoding HDEL (His-Asp-Glu-Leu) or KDEL (Lys-Asp-Glu-Leu) peptide, recombinant vector.
The method of claim 4, wherein

The recombinant vector is a recombinant vector, characterized in that the nucleotide sequence of SEQ ID NO: 4 encoding the cellulose-binding domain (CBD) is additionally operably linked.
A method for mass-producing a protein of interest from a plant comprising the step of introducing the recombinant vector of any one of claims 2 to 6 into a plant.
The method of claim 7, wherein

The step of introducing the recombinant vector into the plant is PEG precipitation (Polyethylen glycol), Agrobacterium ( Agrobacterium sp. )- Mediated method, particle gun bombardment, silicon carbide whiskers (Silicon carbide whiskers) ), Sonication, and electroporation, the method comprising any one selected from the group consisting of.
The method of claim 7, wherein

The plant is a dicotyledonous plant selected from the group consisting of Arabidopsis, soybean, tobacco, eggplant, pepper, potato, tomato, cabbage, radish, cabbage, lettuce, peach, pear, strawberry, watermelon, melon, cucumber, carrot, and celery ; Or a monocotyledonous plant selected from the group consisting of rice, barley, wheat, rye, corn, sugar cane, oats, and onions.