KR101292240B1 - DNA fragment for targeting to chloroplast and recombinant vectors containing the same - Google Patents

Abstract

The present invention relates to a DNA fragment for chloroplast targeting and a recombinant vector comprising the DNA fragment. More specifically, the present invention relates to a chloroplast comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO. The present invention relates to a DNA fragment for targeting, a recombinant vector comprising the DNA fragment, and a method for mass-producing a target protein from a plant, the method comprising introducing the recombinant vector into a plant. Chloroplast targeting DNA fragments and recombinant vectors comprising the same according to the present invention has an efficient effect of targeting the target protein expressed in the transformed plant to chloroplasts, which are organelles of plants having excellent storage and stability, and translation of proteins. When operably additionally linking the gene that promotes efficiency to the recombinant vector has the effect of producing a large amount of the target protein from the plant.

Chloroplast, targeting, plant, protein, translation, recombinant vector, transformation

Description

DNA fragment for targeting to chloroplast and recombinant vectors containing the same}

The present invention relates to a DNA fragment for chloroplast targeting and a recombinant vector comprising the DNA fragment, which can efficiently transfer a target protein to the chloroplast of a plant so as to mass-produce a target protein from a plant.

In general, plants have a great potential for the production of biopharmceutical proteins and peptides that can be used as biopharmaceuticals because they are easy to transform and economically inexpensive as protein materials. On the other hand, to date, most biopharmaceuticals have typically been produced from transformed cultured mammalian cells, bacteria, fungi and the like (Ganz PR et al., 1996; Ma JK et al., 1999; Pen J, 1996). However, the production of therapeutic proteins in plants compared to these mammalian cells, bacteria, and fungi can lead to a reduction in the risk of contamination by pathogens and higher yields, along with economics such as production in seeds or other storage organs. There are several advantages in terms of quality. In addition, plants can be potentially inexpensive targets to produce recombinant products, cultivate, harvest, store, and process transformed grains, and can also use current infrastructure and require relatively little capital investment. This gives a great prospect in the commercial production of biologics.

Therefore, the development of plant expression systems for transforming plant cells to obtain the desired useful protein with high efficiency has been in the spotlight, and these plant expression systems are selected for the expression level of recombinant protein used by plants to target host proteins to organs. It is attractive in that it can be improved by using classification and targeting mechanisms, and has the advantage of easily producing large quantities of plant-derived biopharmaceuticals.

However, the production of target proteins using plants can be an important factor in determining plant-based production potential, including plant species selection, tissue selection, expression and recovery strategies, and post-translational processing.

Recently, many methods for transforming the nucleus have been studied in the method of producing useful proteins using plant cells. However, since genes introduced into the nucleus are usually introduced with one or a few genes, there is a limit to mass production of useful proteins. In addition, when a foreign gene is inserted into a heterochromatin site on a chromosome, even if it is transformed, there is a problem in that the degree of transformation varies depending on the site to be introduced without expression. Therefore, in recent years, many efforts have been made to induce mass expression by introducing genes into mitochondria, chloroplasts, or pigments.

In particular, the use of chromosomes such as chloroplasts to produce useful proteins has the following advantages: First, due to the ploidy of the plastid genome system, which has thousands of genomic copies per cell, Foreign proteins can accumulate in the chloroplasts. Therefore, the transformed chloroplast is an ideal expression plant for the high yield of protein production. Second, the chloroplast is maternally inherited to the next generation, so it is much more ecologically stable than traditional transformation technology. It contains minimal proteolytic enzymes, which are essential for protein quality control and protein processing, which minimizes the degradation of foreign proteins. In addition, gene silencing and location effects (side effect) has the advantage of less side effects.

Therefore, recently, studies to produce useful proteins in the chloroplasts of plants have been conducted. Looking at the related technologies, Korean Patent No. 542,053 describes the transfer of the protein from the nucleotide sequence of sweet potato-derived ADP-glucose kinase cDNA to chloroplasts. Disclosed is a vector for targeting a target gene to chloroplast using the required DNA sequence, and Korean Patent No. 362320 discloses a gene targeting a protein to chloroplast using a rbcS (ribulose bisphosphate carboxylase oxygenase) promoter. Disclosed is a method for expression of.

However, the process of producing a useful protein through transformation into chloroplasts according to the prior art has a disadvantage in that the efficiency of migration to chloroplasts is low, and thus there is a problem in that the desired protein cannot be produced as desired.

Therefore, there is a need for the development of a new technology that can efficiently introduce foreign proteins into the chloroplast to mass-produce the target protein in a stable form.

Accordingly, an object of the present invention is to provide a DNA fragment for chloroplast targeting, which can efficiently move and accumulate a target protein expressed in plant cells into chloroplasts.

Another object of the present invention is to provide a recombinant vector comprising the DNA fragment for chloroplast targeting.

Another object of the present invention is to provide a method for mass-producing a target protein from a plant, comprising the step of introducing the recombinant vector into the plant.

In order to achieve the object of the present invention as described above, the present invention provides a DNA fragment for chloroplast targeting comprising a base sequence encoding the amino acid sequence of SEQ ID NO: 16 in the base sequence consisting of SEQ ID NO: 1.

In one embodiment of the present invention, the DNA fragment may be composed of a base sequence encoding the amino acid sequence of SEQ ID NO: 14.

The present invention also provides a recombinant vector comprising the DNA fragment for chloroplast targeting according to the present invention.

In one embodiment of the present invention, the recombinant vector may be operably linked to the promoter, the chloroplast targeting DNA fragment according to the present invention and the gene encoding the target protein sequentially.

 In one embodiment of the present invention, the recombinant vector is a nucleotide sequence for enhancing the protein translation efficiency of any one selected from the group consisting of SEQ ID NO: 20 to 31 between the promoter and the chloroplast targeting DNA fragment according to the invention Possibly connected.

In one embodiment of the present invention, the target protein may be green fluorescent protein (GFP).

The present invention also provides a method for mass-producing a target protein from a plant by transferring and accumulating the target protein including the step of introducing the recombinant vector according to the present invention into a plant chloroplast.

In one embodiment of the invention, the introduction of the recombinant vector into the plant Agrobacterium ( Agrobacterium sp . ) -Mediated method, particle gun bombardment, silicon carbide whiskers, sonication, electroporation and PEG precipitation (Polyethylen glycol) Any one selected from can be used.

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

In one embodiment of the present invention, the target protein may be green fluorescent protein (GFP).

Chloroplast targeting DNA fragments and recombinant vectors comprising the same according to the present invention have a highly efficient effect of targeting the target protein expressed in the transformed plant to chloroplasts, which are organelles of plants having excellent storage and stability, and translation of the protein. When operably additionally linking the gene that promotes efficiency to the recombinant vector has the effect of producing a large amount of the target protein from the plant.

The present invention provides a DNA fragment for chloroplast targeting that can safely accumulate and transport useful proteins to chloroplasts, which are plant specific organelles, as a method for maximizing the production of useful proteins in plant cells. In one embodiment, there is provided a DNA fragment for chloroplast targeting comprising a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 16 among nucleotide sequences consisting of SEQ ID NO: 1.

The inventors used Cab protein, a chloroplast a / b binding protein, to find DNA fragments that can transfer useful proteins expressed in plant cells to chloroplasts.

Plant chloroplasts are organs responsible for photosynthesis and are excellent targets for foreign protein storage because they have excellent protein storage capacity such that over 40% of the plant proteins are chloroplast proteins. In addition, chloroplasts have their own genome, which is capable of producing proteins by electronically translating genetic information. However, most chloroplast proteins are known to migrate to chloroplasts after they are synthesized from genetic information maintained in the cell nucleus. Therefore, among the proteins in the plant, the proteins to be transferred to the chloroplasts are systemized to move the protein to the chloroplast by using a precursor sequence called transit peptide at the amino terminus of the protein. have. However, these transit peptides do not have a specially conserved sequence among the various chloroplast proteins.

On the other hand, Cab protein, which has a property of binding to chlorophyll, is known to function as an aid of antenna pigment in photosystems (PS) I and II that perform photosynthesis in chloroplasts, and Cab moves proteins to chloroplasts. It has been shown that it acts in the same way (Santini C. et. Al., 1994; Kavanagh TA. Et. Al., 1998).

Therefore, the inventors of the present invention can more efficiently transfer proteins to chloroplasts and safely accumulate them, and at the same time, establish DNA fragments of the Cab proteins to establish optimal conditions for mass production of proteins from plants. The best DNA fragments were identified first.

That is, in one embodiment of the present invention, the present inventors PCR to obtain Cab fragments having 87, 67, 47, 27 and 7 peptide sequences, respectively, of the nucleotide sequence of the Cab protein represented by SEQ ID NO: 1 DNA fragments encoding the peptide fragments were prepared by the method. Then, recombinant vectors including each of the DNA fragments amplified by PCR were prepared and transformed into Arabidopsis, and then each Cab was subjected to fluorescence microscopy and Western blot. The effect of transfer of the target protein to chloroplasts by the fragments was investigated.

As a result, transfer of the green fluorescent protein used as the target protein to the chloroplast was not observed when the DNA fragments encoding the Cab 7, 17 and 27 peptides were used, but the DNA fragments encoding the peptides of Cab 87, 67 and 47. In the case of, the green fluorescent protein shifted to the chloroplast, and especially when using the DNA fragment encoding the peptide of Cab 87, almost 90% of the protein shifted to the chloroplast (see FIGS. 2 and 3). .

Therefore, through the above results, the present inventors have an activity in which a Cab fragment including at least 47 amino acids in the peptide sequence of Cab protein transfers a target protein to chloroplast, and in particular, the Cab fragment consisting of 87 amino acids is 90% or more protein chloroplast. It can be seen that there is an action to move the protein, and using the Cab 87 fragment can transfer the protein to the chloroplast with better efficiency than conventional.

In the present invention, the amino acid sequences of Cab fragments consisting of the 87, 67, 47, 27 and 7 peptides are shown in SEQ ID NOs: 14 to 19, respectively.

Therefore, the present invention can provide a DNA fragment for chloroplast targeting comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 in the base sequence consisting of SEQ ID NO: 1 in transferring the protein expressed in the plant to the chloroplast. Preferably, the DNA fragment may be a DNA fragment consisting of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14 which can target the protein to the chloroplast with a high efficiency of 90% or more.

In addition, the present invention may provide a recombinant vector comprising the DNA fragment for chloroplast targeting according to the present invention.

More specifically, the recombinant vector according to the present invention has a conventional vector used for protein expression as a backbone, the promoter, the chloroplast targeting DNA fragment of the present invention and the gene encoding the target protein are sequentially operably linked It may be a recombinant vector.

In the present invention, the term "expression vector" refers to a plasmid, virus or other medium known in the art, into which the DNA fragment for chloroplast targeting and the polynucleotide sequence encoding the target protein according to the present invention can be inserted or introduced. do. 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. In addition, "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 regulating 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. Examples of the promoter include, but are not limited to, 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 include, but are not limited to, binary vectors such as pCHF3, pPZP, pGA and pCAMBIA series. 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 Any vector can be used as long as it can be introduced into it.

In addition, the recombinant vector according to the present invention may be operably linked between any one of the promoter and the chloroplast targeting DNA fragments of any one selected from the group consisting of SEQ ID NO: 20 to 31 for enhancing protein translation efficiency.

In the present invention, the "nucleotide sequence for improving protein translation efficiency" is the base sequence of the 5 'untranslated region (5'UTR: 5'untranslated region) described in the present patent application No. 2009-0081403 As a nucleotide sequence having an activity capable of translating the target protein with high efficiency.

   In general, the 5 'untranslated region (UTR) of mRNA is known to play an important role in post-transcriptional regulation of gene expression, regulation of mRNA transport out of the nucleus, efficiency of protein translation and regulation of mRNA stability. have. In particular, the 5 'UTR is also known as the leader sequence and contains a ribosome binding site (RBS), known in bacteria as the Shine-Delarno sequence (AGGAGGU). This 5 'UTR is one hundred or more nucleotides in length, and the 3' UTR is several kilobases 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 sequence belonging to 5'UTR, which is also 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 addition, translation of mRNA may change protein production depending on its efficiency, which is very important for gene regulation. The 5'UTR is mainly used to regulate mRNA translation, and the context of the sequence around the translation initiation codon, which is part of the 5'UTR, is used to regulate translation efficiency (Xiong W et al., 2001; Rogozin IB et al., 2001; Gallie). DR et al., 1989; et al Mignone F, 2002; Kawaguchi R et al., 2002,2005), and typical examples are the small subunit of ribul Los bisphosphate carboxy la kinase / octanoic cyclooxygenase (RUBISCO) (subunit; RbcS) 5 of the gene 'UTR is being used.

Therefore, in the present invention, in the patent application No. 2009-0081403 as a base sequence for enhancing the protein translation efficiency to mass-produce the target protein through the effect of the translation of the protein to the chloroplast of the target protein by the Cab 87 fragment 5'UTR sequences that have been identified can be used, preferably 5'UTR sequences represented by SEQ ID NOs: 20 to 31 described in the table below. In addition, in the present invention, the entirety of the patent application No. 2009-0081403 was referred to by reference in the present invention, Cab 87 fragments according to the present invention produced in one embodiment of the present invention and as a base sequence for enhancing protein translation efficiency A cleavage map of the recombinant vector comprising the sequence of UTR 35 (SEQ ID NO: 25) is shown in FIGS. 1C and 1D, respectively.

5'UTR number Base sequence SEQ ID NO: UTR 1 AGAGAAGACGAAACACAAAAG 20 UTR 2 GAGAGAAGAAAGAAGAAGACG 21 UTR 6 AAAACTTTGGATCAATCAACA 22 UTR 7 CTCTAATCACCAGGAGTAAAA 23 UTR 24 AGAAAAGCTTTGAGCAGAAAC 24 UTR 35 AACACTAAAAGTAGAAGAAAA 25 U1 AAA AGAGAAGACGAAACACAAAAA 26 U1 CCC AGAGAAGACGAAACACAACCC 27 U1 GGG AGAGAAGACGAAACACAAGGG 28    U1 (-4,5G) AGAGAAGACGAAACACGGAAG 29    U1 (-4,5C) AGAGAAGACGAAACACCCAAG 30      UAAG AAGAAGAAGAAGAAGAAGAAG 31

On the other hand, in the case of overexpressing a target protein in a transgenic plant or cell, the phenomenon of proteolytic degradation often occurs. However, when targeting foreign proteins with various intracellular organelles, these proteins can be stored more stably. In particular, when target proteins are targeted to chloroplasts, the plant's chloroplasts contain only the minimum amount of protease required for protein processing. This means that the protein of interest can be stored at a high concentration in the chloroplast by preventing the protein of interest from being degraded by the protease.

Therefore, when using the recombinant vector of the present invention comprising a nucleotide sequence for enhancing the translation efficiency of the chloroplast targeting DNA and protein according to the present invention, as well as the effect of targeting the target protein linked to the vector to the chloroplast with high efficiency There is an effect that can be mass produced by accumulating the target protein to a high level.

In one embodiment of the present invention, a recombinant vector and UTR 35 sequence operably linked to a UTR 35 sequence (nucleotide sequence of SEQ ID NO: 25) and a target protein to enhance protein translation efficiency and a green fluorescent protein (GFP) as a target protein, Cab After transforming Arabidopsis with a recombinant vector operably linked to the nucleotide sequence of the 87 fragment and the nucleotide sequence encoding the green fluorescent protein (GFP), the amount of GFP protein expressed in the Arabidopsis pole was Western blot As a result, when the vector containing the Cab 87 fragment was used, the expression level of the protein was increased by about 6 times or more compared to the vector containing the Cab 87 fragment (see Fig. 4).

Further, in another embodiment of the present invention, as a result of quantitatively measuring the expression level of the target protein expressed in the plant transformed with the recombinant vector according to the present invention, the amount of the target protein expressed is transformed. It was found that the amount corresponds to about 0.5% of the total protein present in the plant, and this expression amount is 0.13% of oleosin, which is known to increase the protein accumulation rate by targeting the protein with chloroplast oil bodies. It was shown to be superior to the efficiency (see FIG. 5).

Therefore, through the above results, the inventors of the present invention can effectively move the target protein to the chloroplast targeting DNA fragments, thereby effectively inhibiting the degradation of the target protein from various proteolytic enzymes present in the cytoplasm. It was found that a large amount of the target protein can be produced. Furthermore, the present invention can provide a transformed plant by introducing the recombinant vector according to the present invention into the plant using a method known in the art.

A method of introducing into the plant the recombinant vector of the present invention is useful for but not limited to Agrobacterium (Agrobacterium sp.) - method according to the parameters, the particle gun bombardment method (particle gun bombardment), silicon carbide whisker methods (Silicon carbide whiskers), sonication, electroporation and precipitation by polyethylene glycol (PEG) may be used. In an embodiment of the present invention Agrobacterium (Agrobacterium sp.) - using the method mediated by the Arabidopsis thaliana was transformed with the recombinant vector of the present invention.

In addition, a plant transformed with the recombinant vector of the present invention, that is, a plant capable of producing the target protein with high efficiency because the target protein is moved and accumulated in the chloroplast in the plant, is a conventional breeding method in the art. Or through an asexual propagation method. More specifically, the plant of the present invention may be obtained through oily breeding, which is a process of producing seeds from the pollination process of flowers and breeding from the seeds. In addition, after transforming a plant with a recombinant vector according to the present invention, it can be obtained through an asexual propagation method which is a process of induction, rooting and soil purification of callus according to a conventional method. That is, the fragments of the plant transformed with the recombinant vector according to the present invention are inoculated into a suitable medium known in the art, and then cultured under appropriate conditions to induce the formation of callus, and when the shoots are formed, transfer to a hormone-free medium to culture. do. After about 2 weeks, the shoots are transferred to rooting medium to induce roots. The plant according to the invention can be obtained by root derivation, which is then transplanted into soil and purified. Plants transformed in the present invention may include whole plants as well as tissues, cells or seeds obtainable therefrom.

In the present invention, the plant may be a dicotyledonous plant or a monocotyledonous plant, and the dicotyledonous plant is not limited thereto, but the Arabidopsis, soybean, tobacco, eggplant, pepper, potato, tomato, Chinese cabbage, radish, cabbage, lettuce, peach , Pears, strawberries, watermelons, melons, cucumbers, carrots and celery. The monocotyledonous plants may include, but are not limited to, rice, barley, wheat, rye, corn, sugarcane, oats and onions.

Furthermore, the present invention can provide a method for mass-producing a target protein from a plant transformed with the recombinant vector according to the present invention.

The target protein that can be used in the present invention refers to a protein to be produced by the genetic engineering method according to the present invention, is not particularly limited. Preferably for commercial use, i.e., agriculturally or medically useful, may include proteins that need to be produced in large quantities, such as, but not limited to, serum proteins (e.g. Factor VII , Blood factors including VIII and IX), immunoglobulins, cytokines (eg interleukin), α-, β- and γ-interferons, colony stimulating factors (including G-CSF and GM-CSF), platelet induced growth factors (PDGF), phospholipase-activated protein (PLAP), insulin, tumor necrosis factor (TNF), growth factor (e.g. tissue growth factor and endothelial growth factor such as TCFα or TGFβ), hormones (e.g. vesicle-stimulating hormone , Thyroid-stimulating hormone, antidiuretic hormone, pigment and parathyroid hormone, progesterone-releasing hormone and derivatives thereof, calcitonin, calcitonin gene related peptide (CGPR), enke Enkephalin, somatomedin, erythropoietin, hypothalamic secretion factor, prolactin, chronic gonadotropin, tissue plasminogen activator, growth hormone releasing peptide (GHPR), thymic humoral factor (thymic) humoral factor (THF) and the like. In addition, the protein of interest of the present invention may include enzymes, and examples thereof may include carbohydrate-specific enzymes, proteolytic enzymes, redox enzymes, transferases, hydrolases, lyases, isomerases and ligases. have. In addition, reporter proteins may be included as the target protein, which is a protein expressed by a reporter gene, and indicates the activity of its activity in the 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 in which a chloroplast targeting DNA fragment according to the present invention and a nucleotide sequence encoding the target protein are operably linked to a promoter or The cells can 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 of expressing the target protein may be any method known in the art. In addition, recovery of the target protein highly expressed in the transformed plant or cell can be carried out through a variety of separation and purification methods known in the art, usually for removing the cell debris, etc. After centrifugation of the seafood, precipitation, for example, salting out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.) can be performed, and dialysis, electrophoresis and various Column chromatography, and the like. As the chromatography, a target protein of the present invention may be purified by applying techniques such as ion exchange chromatography, gel-penetration chromatography, HPLC, reverse phase-HPLC, affinity column chromatography, and 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 the cellulose-binding domain (CBD) of the cellulase in the recombinant vector of the present invention described above to more easily and quickly separate and purify the target protein from the transformed plant A recombinant vector was additionally operably linked to a nucleotide sequence, preferably the nucleotide sequence represented by SEQ ID NO: 32.

 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 the cellulose-binding domain, and thus, it may be easily separated and purified through chromatography using a cellulose carrier as a column. Can be.

In addition, the produced protein of the fused form can be used to cut the cellulose binding domain using an appropriate enzyme, etc., and then to further separate and purify to produce a non-fused form of the target protein.

In one embodiment of the present invention, a cellulose-binding domain (cellulose-binding) in a vector including a promoter, a nucleotide sequence for enhancing protein translation, a chloroplast targeting DNA sequence, and a nucleotide sequence encoding a target protein (GFP) When a recombinant vector operably linked to a domain (CBD) is introduced into a plant, a highly expressed target protein from the transformed plant can be isolated and purified in almost 90% yield using a cellulose-binding domain. Confirmed. In addition, when using a vector in which the TEV protease site was additionally linked to the recombinant vector linked to the CBD sequence, it was confirmed that only the target protein in which the CBD was cleaved by the TEV protease was purified.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

< Example  1>

With chloroplasts Targeting  for Cab Transit Sequence of  making

The present inventors produced various fragments as follows using the sequence of Cab (chlorophyll a / b binding protein) in order to target the protein of interest as chloroplasts and store and produce it in a stable state. That is, fragments of Cab7, Cab17, Cab27, Cab47, Cab67 and Cab87 described in Table 2 below, namely, by PCR using the DNA sequence of Cab described in SEQ ID NO: 1 as a template and the primers described in Table 1 below, Cab transit peptide fragments were prepared respectively.

[Table 1]

Primer Sequences Used for the Construction of Cab Fragments

Cab fragment Primer sequence SEQ ID NO: Cab 87 F  5'-ATGGCGTCGAACTCGCTTATG-3 ' 2 Cab 87 R  5'-CTCTGACTCTTTGTATCTCTCA-3 ' 3 Cab 67 F  5'-CGTATCAGAATGGCTGCTCACATGAGTAAAGGAGAAGAACTT-3 ' 4 Cab 67 R  5 'AAGTTCTTCTCCTTTACTCATCCCAAAGTCACCAGGAGCAGA-3' 5 Cab 47 F  5'-CGTATCAGAATGGCTGCTCACATGAGTAAAGGAGAAGAACTT-3 ' 6 Cab 47 R  5'-AAGTTCTTCTCCTTTACTCATGTGAGCAGCCATTCTGATACG-3 ' 7 Cab 27 F  5'-CTTCCAAGTCTAAATTCGTAATGAGTAAAGGAGAAGAACT-3 ' 8 Cab 27 R  5'-AGTTCTTCTCCTTTACTCATTACGAATTTAGACTTGGAAG-3 ' 9 Cab 17 F  5'-TAGCCGCCGTGTACCCTTCGATGAGTAAAGGAGAAGAACT-3 ' 10 Cab 17 R  5'-AGTTCTTCTCCTTTACTCATCGAAGGGTACACGGCGGCTA-3 ' 11 Cab 7 F  5'-ATGGCGTCGAACTCGCTTATGATGAGTAAAGGAGAAGAACT-3 ' 12 Cab 7 R  5'-AGTTCTTCTCCTTTACTCATCATAAGCGAGTTCGACGCCAT-3 ' 13

[Table 2]

Amino acid sequence of the Cab fragment

SEQ ID NO: Amino acid sequence SEQ ID NO: 14 (Cab 87) MASNSLMSCGIAAVYPSLLSSSKSKFVSAGVPLPNAGNVGRIRMAAHWMPGEPRPAYLDGSAPGDFG FDPLGLGEVPANLERYKESE SEQ ID NO: 15 (Cab 67) MASNSLMSCGIAAVYPSLLSSSKSKFVSAGVPLPNAGNVGRIRMAAHWMPGEPRPAYLDGSAPGDFG SEQ ID NO: 16 (Cab 47) MASNSLMSCGIAAVYPSLLSSSKSKFVSAGVPLPNAGNVGRIRMAAH SEQ ID NO: 17 (Cab 27) MASNSLMSCGIAAVYPSLLSSSKSKFV SEQ ID NO: 18 (Cab 17) MASNSLMSCGIAAVYPS SEQ ID NO: 19 (Cab 7) MASNSLM

< Example  2>

Cab Transit Sequence  Chloroplasts containing For targeting  Construction of Recombinant Vectors

For the transient peptide fragments of Cab7, Cab17, Cab27, Cab47, Cab67, and Cab87 prepared in Example 1, these fragments were included to identify the fragments having the highest targeting effect to the chloroplasts. Recombinant vectors were constructed in the following manner. In other words, as a DNA fragment, that is, a 5'UTR (5 'untranslated region) sequence, which enhances the translation efficiency of the color flower mosaic virus 35S promoter and protein, the inventors have developed and applied for a patent (No. 2009-0081403) 326, a vector which typically uses a region of the sequence comprising the nucleotide sequence consisting of SEQ ID NO: 25, a gene sequence encoding a green fluorescent protein (GFP), and a stop codon containing a 35S terminator (NOS) A 326-35S-UTR35: Cab fragment (Cab7, 17, 27, 47, 67 or 87): GFP: NOS, which was a chloroplast targeting recombinant vector according to the present invention, was prepared by connecting to a vector, and FIG. 1A was prepared according to the present invention. Sequential linkage of 35Sp-UTR35: Cab fragments: GFP: NOS in one recombinant vector, 326-35S-UTR35: Cab87: GFP: NOS, a recombinant expression vector containing Cab 87 fragments, was produced A cleavage map of the vector is shown in Fig. 1C. .

SEQ ID NO: 25 UTR Sequence: 5'-AACACTAAAAGTAGAAGAAAA-3

< Example  3>

Selection of Optimal Cab Transient Sequences for Chloroplast Targeting by Fluorescence Microscopy and Western Blot

Recombinant vectors comprising each of the six Cab fragments prepared in Example 2 (Cab7, 17, 27, 47, 67 or 87) were PEG-mediated transformation methods well known in the art ( Jin et al., Plant Cell 13: 1511-1526, 2001) were used to transform Arabidopsis vulgaris. The transformed Arabidopsis plants were subjected to fluorescence microscopy and Western blotting analysis. Expression of GFP protein and migration to chloroplasts were observed.

In the analysis by fluorescence microscopy, the recombinant vectors were transformed into the Arabidopsis and 24 hours later, the degree of fluorescence of GFP, a fluorescent protein linked to Cab fragments, was visually observed through a microscope. The Arabidopsis leaves transformed with the recombinant vectors according to the present invention were stored in an artificial environment room at 23 ° C. for 24 hours, and then W5 solution (154 mM NaCl, 125 mM CaCl ₂ , 5 mM KCl, 5 mM Glucose, 1.5 mM MES, and storage solution). pH 5.6), remove the denatured buffer (10% SDS, β-mercaptoethanol) for protein extraction, break the protoplasts, and then bathe in boiling water and centrifuge for 10 minutes at 4 ℃, 13,000 rpm. And supernatant were separated. The supernatant was then subjected to SDS-PAGE electrophoresis, followed by immunoblotting using monoclonal anti-GFP antibody (Clontech cat no. 632381). Migration and GFP protein expression levels were analyzed.

As a result, as shown in Figure 2, using a recombinant vector containing Cab 7, 17 and 27 fragments GFP protein with green fluorescence shifted from protoplasts to chloroplasts could not be observed at all, in the case of Cab 47 Some green fluorescent GFP proteins migrated to chloroplasts that were never seen in Cab 27 were observed. In addition, Cab 47 confirms that a significant amount of green fluorescence is observed in the cytoplasm, while only a few are observed in the chloroplast, though 47 nucleotides in the Cab's sequence can shift the green fluorescence protein to chloroplast, although the chloroplast targeting efficiency is lower. It can be seen that it is the minimum unit of the transient peptide. In particular, since the red color shown in the drawing is a self-fluorescence color coming from chlorophyll, it can be determined that the GFP protein is transferred to chloroplast by Cab for the cases where green fluorescence is seen on the red color. In addition, in the case of Cab 67, some were targeted to chloroplasts, but some were still able to observe the green fluorescence remaining in the cytoplasm, and compared to Cab 47, Cab 67 confirmed that more green fluorescence was observed in the chloroplast. It was found that the targeting efficiency to chloroplast was higher than that of 47. In the case of Cab 87, most of the green fluorescence was observed in the chloroplast under the fluorescence microscope, while almost no in the cytoplasm.

Therefore, it can be seen from the above results that Cab 47 is the smallest unit that can move the protein to chloroplasts for Cab fragments prepared in the present invention, and in the case of Cab 87, more than 90% of the target protein can be moved to chloroplasts. Chloroplast targeting efficiency was found to be very good.

 In addition, in the analysis of Western blot results, after the Cab fragment, ie, Cab Transit Peptide, was transferred to the chloroplast, cleavage was caused by a transit peptidase that specifically cleaves the Transit Peptide. The size of the band in the phase will be different. Therefore, the green fluorescent protein, which cannot move to chloroplasts and remains in the cytoplasm, appears as a large size in which the transgenic peptides are connected as it is, whereas when moved to chloroplasts, the protein is cleaved by enzymes and thus appears as a small size.

As shown in FIG. 3, the western blot according to the present invention showed that in the case of Cab 87, almost all proteins were cut in size, and thus, most of the GFP protein was moved into the chloroplast. Unsized protein was detected in very small amounts. On the other hand, in Cab 67, more than 50% of the protein was found to be truncated protein, which also showed the effect of chloroplast targeting, but its efficiency was observed to be somewhat lower than that of Cab 87. The case was observed in the size of the whole or intermediate form including the transient peptides. In the case of Cab 47, a distinct double band was observed, with the larger band representing the protein prior to migration to the chloroplast, and the smaller band representing the protein transferred to the chloroplast, indicating that the migration effect was less than 50%. . In addition, in the case of Cab 27, 17, and 7, the result of the microscopic analysis showed that the green fluorescence transferred to the chloroplast was not observed at all, and the protein band of the truncated form could not be observed. Only full-sized proteins that could not migrate and remain in the cytoplasm were observed.

Therefore, the present inventors found that the minimum unit for chloroplast movement among the cab transition sequences produced by the present invention was a cab 47 sequence, and in particular, in the case of Cab 87. It was found that more than 90% of the proteins could be transferred to chloroplasts.

< Example  4>

Cab  87 Transit In sequence  Protein expression and stability analysis

Of Cab Transition Sequence Fragments Made In The Present Invention Through Example 3

As Cab 87 was identified as the optimal transition sequence to transfer the target protein to chloroplasts, it was investigated whether Cab 87 also affected the expression efficiency and stability of the target protein. To this end, protoplasts of plant cells were obtained from a Arabidopsis transformed with a recombinant vector comprising Cab 87 in Example 3 by a method known in the art, followed by the monoclonal anti-GFP method described in Example 3. The expression level of GFP protein was analyzed by immunoblotting using an antibody (monoclonal anti-GFP antibody, Clontech cat no. 632381). At this time, the control group was used as a protoplast of a plant transformed with a vector not including a Cab sequence, that is, a vector containing only Cab fragments in a vector including Cab fragments prepared in the present invention.

As a result, as shown in Figure 4, despite using the same UTR 35 sequence, which is a DNA fragment that enhances the expression efficiency of the protein using Cab 87, the expression of GFP protein is weak compared to the case without using Cab 87 It increased more than 6 times. These results suggest that if proteins expressed in the cytoplasm fail to migrate to chloroplasts and are present in the cytoplasm, they are more likely to be degraded by intracellular enzymes, such as proteolytic enzymes, but they can be safely protected from these proteolytic actions when transferred to chloroplasts. It can be expected that a large amount of protein can be obtained.

Therefore, according to the above results, the Cab 87 transit sequence according to the present invention not only has an excellent effect of targeting the expressed target protein to chloroplast, but also accumulates proteins stably because the chloroplasts of the organelles of plant cells have a very good protein storage capacity. I could see that you can.

< Example  5>

Production of Transgenic Plants

A transgenic plant capable of mass-producing a target protein in the chloroplast was prepared using a Cab transit sequence, a sequence capable of targeting the target protein as a chloroplast. Plant transformation plasmid was cut from the region containing 35Sp-UTR35: Cab 87: GFP in the recombinant vector prepared in Example 2 using pstI and EcoRI restriction enzymes at the multicloning site of pCAMBIA3300 vector using pCAMBIA3300-35Sp-UTR35 A recombinant vector for transforming: Cab 87: GFP: HA: NOS was prepared, and a cleavage map of the prepared vector is shown in FIG. 1D. Thereafter, the transformed recombinant vector was prepared using the Agrobacterium-mediated transformation method. At this time, pCABIA3300, a basic vector of the prepared vector for transformation, was selected for plants transformed by the method of the present invention through a Vasta resistance test. In addition, the plants selected by the resistance test obtained a first generation of transformation through the Western blot to find a line that expresses the protein well, and in the second generation of transformation, a death marker: the surviving subject is 1: 3 using a selection marker. The clean lines were selected as one copy, and these lines were then designated as the third generation of transformation, and in the third generation of transformation, homo lines were selected to express all the proteins for all surviving individuals. This secured a transformed plant line.

< Example  6>

Determination of the expression level of the protein of interest in plants using the vector according to the present invention

The inventors examined the degree of expression efficiency in plants with respect to the Cab 87 sequence, which was found to be the most efficient among the chloroplast targeting sequences prepared in the present invention. To this end, the entire protein was first extracted from Arabidopsis transformed with a recombinant vector for transformation of a plant comprising a sequence encoding Cab 87 and GFP protein prepared in Example 5. At this time, the extraction of the protein is carried out in a denatured buffer (denaturation buffer; 10% SDS, β-mercaptoethanol) for extracting the transformed Arabidopsis leaves, the protoplasts are broken, and then heated in boiling water and heated at 4 ℃, 13,000 rpm 10 The supernatant was obtained by centrifugation for a minute. Subsequently, the supernatant isolated was subjected to SDS-PAGE electrophoresis, and then transformed into a recombinant vector according to the present invention by Western blotting using a monoclonal anti-GFP antibody (Clontech cat no. 632381). The expression level of the GFP protein in the plants was measured. At this time, the control group was used to isolate and purify the GFP protein expressed in bacteria using a vector linking GFP to an expression vector having a His label known in the art and to quantify the amount of purified protein.

As a result, as shown in FIG. 5, it was found that the amount of GFP protein expressed from the plant transformed by the vector including Cab 87 reached about 0.5% of the total protein of the plant, and this amount was the amount of oil in the chloroplast. By targeting the protein with the body, it is much higher than the efficiency of 0.13% of oleosin, which is known to increase the accumulation of protein.

< Example  7>

Isolation and Purification of Proteins from Arabidopsis Transformed with Recombinant Vectors According to the Present Invention

The inventors have isolated and purified proteins accumulated and expressed in chloroplasts of plants using the vectors prepared in the present invention. A recombinant protein for transformation of pCAMBIA3300-35Sp-UTR35: Cab 87: GFP: HA: NOS, a vector prepared in the above-described example, as a protein pure purification label, and a cellulose binding domain (CBD) and a TEV protease for removing the same. A vector was obtained by further linking the cleavage site of ie, ie, the substrate site of the TEV protease and the cellulose at the C-terminus of the HA label using the pCAMBIA3300-35Sp-UTR35: Cab 87: GFP: HA: NOS as a base vector. The binding domain (CBD) was fused to produce pCAMBIA3300-35Sp-UTR35: Cab 87: GFP: HA: TEV: CBD vector. At this time, the production of the vector is based on the nucleotide sequence encoding the substrate site of the TEV protease known in the art and the cellulose binding domain of SEQ ID NO: 32, using a primer pair of SEQ ID NO: 33 and SEQ ID NO: 34 After PCR amplification, pCAMBIA3300-35Sp-UTR35: Cab 87: GFP: HA: TEV: CBD vector was prepared by cloning at the C-terminus of the HA label of the pCAMBIA3300-35Sp-UTR35: Cab 87: GFP: HA plasmid. The connection structure of 35Sp-UTR35: Cab 87: GFP: HA: TEV: CBD in the vector is shown in FIG. 1B.

SEQ ID NO: 33 (CBD F Primer): TTACACATGGCATGGATGAACT

SEQ ID NO: 34 (CBD R primer): CTAAGTTCTTGATTTGAGAATAC

The pCAMBIA3300-35Sp-UTR35: Cab 87: GFP: HA: TEV: CBD vector was then introduced into the Arabidopsis protoplasts by PEG-mediated transformation (Jin et al., Plant Cell 13: 1511-1526,2001), a fusion protein expressed in protoplasts of Arabidopsis (Cab 87: GFP: HA: TEV: CBD fused protein) was isolated and subjected to Western blotting, and the purified fusion protein It was analyzed whether the fusion protein and the CBD were separated through the purification of and without TEV protease treatment. Specifically, protein extraction transformed into Arabidopsis protoplasts was performed with protein extract solution (10 mM HEPES pH 7.5, 10 mM NaCl, 3 mM MgCl ₂ , 5 mM DTT, 5 mM EDTA, 5 mM EGTA 0.2% Triton X-100, protease inhibitor The extract was centrifuged at 4 ° C. and 13,000 rpm for 10 minutes to separate the precipitate and the supernatant. 0.3-0.5% cellulose beads (cellulose bead, Sigma S3504 Cellulose Type 20) were added to the supernatant separated therefrom, incubated at 4 ° C for 3-4 hours, and then passed through a Poly-Prep chromatography column (Bio-Rad, USA). Cellulose beads were packed and passed through 5 ml of the extract used for protein extraction to remove nonspecifically bound proteins. Cellulose beads were transferred to a new tube and elution buffer containing 1.6% of cellobios. ), Incubated for 10-30 minutes, and then precipitated the cellulose beads by centrifugation at 4 ° C and 13,000 rpm, and taking only the supernatant containing the protein in the form of fused pure Cab 87: GFP: HA: TEV: CBD. It was used for the next experiment. Since the fusion protein contained an amino acid sequence that was cut by the TEV protease, it was analyzed whether it was actually cut by the TEV protease. The assay was performed by treating the pure purified fusion protein with TEV protease for 6 hours at 4 ° C., followed by Western blotting using monoclonal anti-GFP antibody (monoclonal anti-GFP antibody, Clontech cat no. 632381) after SDS-PAGE. It was confirmed through.

As a result, as shown in Figure 6, it can be seen that the fusion protein expressed in the chloroplast of the plant stored (Cab 87: GFP: HA: TEV: CBD fused form protein) is easily purified by the method of the present invention Also, when the TEV protease was treated, it was confirmed that the fusion protein was cut by the TEV protease. On the other hand, when the TEV protease was not treated, a large sized protein was detected, and the protein band was detected in the small size of the CBD-removed form in the TEV protease-treated group. .

Therefore, through the embodiments of the present invention described above, the inventors have identified chloroplasts in which the DNA fragment comprising 47 amino acid sequences of Cab, ie, the amino acid sequence of SEQ ID NO: 16, is expressed in a plant. And DNA fragments consisting of 87 amino acid sequences of Cab, ie, the amino acid sequence of SEQ ID NO: 14, are capable of mobilizing and accumulating chloroplasts of the target protein with high efficiency. Therefore, when using the chloroplast targeting DNA fragments according to the present invention, it was found that the target protein can be produced in large quantities from the plant very effectively.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Figure 1a is a schematic diagram showing the connection form of the 35Sp-UTR35: Cab fragment (Cab 7, 17, 27, 47, 67 or 87): GFP: NOS portion in the recombinant vector for targeting the chloroplast of the target protein according to the present invention Figure 1b is a schematic diagram showing the connection form of 35Sp-UTR35: Cab 87: GFP: HA: TEV: CBD in the recombinant vector prepared in one embodiment of the present invention, Figure 1c is an embodiment of the present invention 1 shows a cleavage map of a recombinant vector for plant transformation comprising Cab 87 prepared in one embodiment of the present invention.

Figure 2 is a chloroplast of GFP protein expressed in Arabidopsis after transforming Arabidopsis using a recombinant vector for transformation of a plant comprising Cab 7, 17, 27, 47, 67 or 87 prepared in the present invention The pattern of the targeting of the furnace was observed through a fluorescence microscope.

Figure 3 is transformed Arabidopsis using a recombinant vector for transformation of plants comprising Cab 7, 17, 27, 47, 67 or 87 prepared in the present invention, the expression of GFP protein expressed in Arabidopsis The pattern shows the result of Western blotting using an antibody against GFP, and the upper arrow in Cab 87 lane is not cut by the enzyme that specifically cleaves the transient peptide because the protein is not transferred to the chloroplast. The protein is shown, and the down arrow shows the protein in the form that has been moved to the chloroplast and cut by the enzyme.

4 is a transgenic Arabidopsis transformed using a recombinant recombination vector of a plant comprising UTR35: GFP and UTR35: Cab 87: GFP, the amount of GFP protein expressed in the Arabidopsis is the antibody to GFP The comparison result is shown by using western blotting.

5 is expressed from Arabidopsis transformed with a recombinant vector for transformation of a plant comprising UTR35: Cab 87: GFP to confirm the expression and accumulation efficiency of the target protein by the Cab 87 transition sequence according to the present invention The amount of GFP protein was expressed in bacteria and compared with the quantified amount of purified GFP via Western blot.

Figure 6 is isolated from the Arabidopsis transformed with pCAMBIA3300-35Sp-UTR35: Cab 87: GFP: HA: TEV: CBD recombinant vector prepared in one embodiment of the present invention using CBD, The protein isolated and purified by the treatment of TEV protease was shown by Western blot using a GFP antibody.

<110> Helix.co.ltd. <120> DNA fragment for targeting to chloroplast and recombinant vectors          containing the same <160> 34 <170> Kopatentin 1.71 <210> 1 <211> 261 <212> DNA <213> Artificial Sequence <220> <223> cab transit DNA seq. <400> 1 atggcgtcga actcgcttat gagctgtggc atagccgccg tgtacccttc gcttctctct 60 tcttccaagt ctaaattcgt atccgccgga gttccactcc caaacgccgg gaatgttggt 120 cgtatcagaa tggctgctca ctggatgcct ggcgagccac gaccagctta ccttgacggt 180 tctgctcctg gtgactttgg gtttgaccca cttggacttg gagaagttcc agcgaacctt 240 gagagataca aagagtcaga g 261 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> cab 87 F primer <400> 2 atggcgtcga actcgcttat g 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> cab 87 R primer <400> 3 ctctgactct ttgtatctct ca 22 <210> 4 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> cab 67 F primer <400> 4 cgtatcagaa tggctgctca catgagtaaa ggagaagaac tt 42 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> cab 67 R primer <400> 5 aagttcttct cctttactca tcccaaagtc accaggagca ga 42 <210> 6 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> cab 47 F primer <400> 6 cgtatcagaa tggctgctca catgagtaaa ggagaagaac tt 42 <210> 7 <211> 42 <212> DNA <213> Artificial Sequence <220> 223 47 cab primer <400> 7 aagttcttct cctttactca tgtgagcagc cattctgata cg 42 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> cab 27 F primer <400> 8 cttccaagtc taaattcgta atgagtaaag gagaagaact 40 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> 223 cab 27 R primer <400> 9 agttcttctc ctttactcat tacgaattta gacttggaag 40 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> cab 17 F primer <400> 10 tagccgccgt gtacccttcg atgagtaaag gagaagaact 40 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> 223 cab 17 R primer <400> 11 agttcttctc ctttactcat cgaagggtac acggcggcta 40 <210> 12 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> cab 7 F primer <400> 12 atggcgtcga actcgcttat gatgagtaaa ggagaagaac t 41 <210> 13 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> cab 7 R primer <400> 13 agttcttctc ctttactcat cataagcgag ttcgacgcca t 41 <210> 14 <211> 87 <212> PRT <213> Artificial Sequence <220> <223> cab 87 peptide <400> 14 Met Ala Ser Asn Ser Leu Met Ser Cys Gly Ile Ala Ala Val Tyr Pro 144 148 153 158 Ser Leu Leu Ser Ser Ser Lys Ser Lys Phe Val Ser Ala Gly Val Pro             163 168 173 Leu Pro Asn Ala Gly Asn Val Gly Arg Ile Arg Met Ala Ala His Trp         178 183 188 Met Pro Gly Glu Pro Arg Pro Ala Tyr Leu Asp Gly Ser Ala Pro Gly     193 198 203 Asp Phe Gly Phe Asp Pro Leu Gly Leu Gly Glu Val Pro Ala Asn Leu 208 213 218 223 Glu Arg Tyr Lys Glu Ser Glu                 228 <210> 15 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> cab 67 peptide <400> 15 Met Ala Ser Asn Ser Leu Met Ser Cys Gly Ile Ala Ala Val Tyr Pro 155 159 164 169 Ser Leu Leu Ser Ser Ser Lys Ser Lys Phe Val Ser Ala Gly Val Pro             174 179 184 Leu Pro Asn Ala Gly Asn Val Gly Arg Ile Arg Met Ala Ala His Trp         189 194 199 Met Pro Gly Glu Pro Arg Pro Ala Tyr Leu Asp Gly Ser Ala Pro Gly     204 209 214 Asp Phe Gly 219 <210> 16 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> cab 47 peptide <400> 16 Met Ala Ser Asn Ser Leu Met Ser Cys Gly Ile Ala Ala Val Tyr Pro 166 170 175 180 Ser Leu Leu Ser Ser Ser Lys Ser Lys Phe Val Ser Ala Gly Val Pro             185 190 195 Leu Pro Asn Ala Gly Asn Val Gly Arg Ile Arg Met Ala Ala His         200 205 210 <210> 17 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> cab 27 peptide <400> 17 Met Ala Ser Asn Ser Leu Met Ser Cys Gly Ile Ala Ala Val Tyr Pro 177 181 186 191 Ser Leu Leu Ser Ser Ser Lys Ser Lys Phe Val             196 201 <210> 18 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> cab 17 peptide <400> 18 Met Ala Ser Asn Ser Leu Met Ser Cys Gly Ile Ala Ala Val Tyr Pro 188 192 197 202 Ser     <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> cab 7 peptide <400> 19 Met Ala Ser Asn Ser Leu Met 199 203 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> utr 1 <400> 20 agagaagacg aaacacaaaa g 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> utr 2 <400> 21 gagagaagaa agaagaagac g 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> utr 6 <400> 22 aaaactttgg atcaatcaac a 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> utr 7 <400> 23 ctctaatcac caggagtaaa a 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> utr 24 <400> 24 agaaaagctt tgagcagaaa c 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> utr 35 <400> 25 aacactaaaa gtagaagaaa a 21 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> u1 aaa <400> 26 agagaagacg aaacacaaaa a 21 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> u1 ccc <400> 27 agagaagacg aaacacaacc c 21 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> u1 ggg <400> 28 agagaagacg aaacacaagg g 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> u1 (-4,5G) <400> 29 agagaagacg aaacacggaa g 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> U1 (-4,5C) <400> 30 agagaagacg aaacacccaa g 21 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> uaag <400> 31 aagaagaaga agaagaagaa g 21 <210> 32 <211> 502 <212> DNA <213> Artificial Sequence <220> <223> CBD DNA seq <400> 32 ttacacatgg catggatgaa ctatacaaat taggaggtgg aggtggaccc cgggcacacc 60 accaccacca ccactttcga agttcaccag tgcctgcacc tggtgataac acaagagacg 120 catattctat cattcaggcc gaggattatg acagcagtta tggtcccaac cttcaaatct 180 ttagcttacc aggtggtggc agcgccattg gctatattga aaatggttat tccactacct 240 ataaaaatat tgattttggt gacggcgcaa cgtccgtaac agcaagagta gctacccaga 300 atgctactac cattcaggta agattgggaa gtccatcggg tacattactt ggaacaattt 360 acgtggggtc cacaggaagc tttgatactt atagggatgt atccgctacc attagtaata 420 ctgcgggtgt aaaagatatt gttcttgtat tctcaggtcc tgttaatgtt gactggtttg 480 tattctcaaa tcaagaactt ag 502 <210> 33 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CBD F primer <400> 33 ttacacatgg catggatgaa ct 22 <210> 34 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CBD R primer <400> 34 ctaagttctt gatttgagaa tac 23

Claims (12)

delete delete delete delete Promoter, DNA fragment for enhancing protein translation efficiency consisting of SEQ ID NO: 20, Chloroplast targeting DNA fragment comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 of the base sequence consisting of SEQ ID NO: 1 and gene encoding the target protein Recombinant vectors comprising operably linked in sequence. The method of claim 5, The chloroplast targeting DNA fragment is a recombinant vector comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 14. The method of claim 5, The DNA fragment for enhancing the protein translation efficiency in the DNA base sequence consisting of SEQ ID NO: 20, A DNA fragment consisting of SEQ ID NO: 26 in which three bases at the 3 'end are substituted with adenine (A); A DNA fragment consisting of SEQ ID NO: 27 in which 3 bases at the 3 'end are substituted with cytosine (C); A DNA fragment consisting of SEQ ID NO: 28 in which three bases at the 3 'end are substituted with guanine (G); A DNA fragment consisting of SEQ ID NO: 29, wherein the fourth and fifth bases from the 3 'end are substituted with guanine (G); A DNA fragment consisting of SEQ ID NO: 30 in which the fourth and fifth bases from the 3 'end are replaced with cytosine (C); Recombinant vector, characterized in that it is selected from the group consisting of; DNA fragment consisting of SEQ ID NO: 31 in which the three bases AAG is repeated in the DNA base sequence consisting of SEQ ID NO: 20. The method of claim 5, The target protein is a recombinant vector, characterized in that the green fluorescent protein (GFP). A method of mass-producing a target protein from a plant by transferring and accumulating the target protein into the chloroplast of the plant, the method comprising introducing the recombinant vector of any one of claims 5 to 8 into the plant. 10. The method of claim 9, Introduction into a plant of the recombinant vector is Agrobacterium (Agrobacterium sp.) - method according to the parameters, the particle gun bombardment method (particle gun bombardment), silicon carbide whisker methods (Silicon carbide whiskers), ultrasound treatment (sonication), electrical A method comprising using any one selected from the group consisting of electroporation and PEG precipitation (Polyethylen glycol). 10. The method of claim 9, 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. 10. The method of claim 9, The target protein is characterized in that the green fluorescent protein (GFP).

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