KR20140073710A - Nucleotide sequence to promote translation efficiency in plants - Google Patents

Abstract

The present invention relates to a DNA fragment, which is synthesized for the enhancement of translation efficiency, and to a recombinant vector comprising the 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 the sequence number 1, and can enhance the translation efficiency of target protein. The DNA fragment according to the present invention can be inserted to an upstream of the target protein, thereby being constructed as the recombinant vector. As a result, a plant which is transformed using the recombinant vector can improve the translation of the 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 an effect of mass-producing the target protein from the plant.

Description

[0001] The present invention relates to a nucleotide sequence for promoting translation efficiency of a target protein in plants,

The present invention relates to a novel nucleotide sequence capable of enhancing the translation efficiency of a gene to produce a target protein in a large amount in a plant.

Many of the live medicines currently used have been produced using animal cells, bacteria, fungi, etc. (Granz PR et al., 1996; Ma JK et al., 1999; Pen J, 1996). However, production of recombinant protein using microorganism system has advantages in that it is easy to mass-produce, but there is a problem that proper folding of protein and post-translational modification after protein synthesis such as glycosylation do not occur, . ≪ / RTI > The above problem has been solved by using animal cells, but the problem that animal cells can be infected with viruses capable of infecting with human beasts and the cost for expanding the facilities for mass production has become another problem to be solved.

In the case of plant cells, unlike animal cells, there is no possibility of being infected with a virus capable of infecting the human body, and the experience and infrastructure related to cultivation and harvest from the past to the present are well established and only the cultivation area for mass production is secured Since mass production is possible at all times, it is advantageous that the cost of equipment investment for mass production is relatively low.

However, one of the obstacles to the production of proteins in plants by these advantages is that, when proteins are expressed in plants or plant cells, the level of protein expression that can be produced in plants is not as high as that of commercial scale.

Protein production generates proteins through the transcription and translation stages of mRNA production. Methods for controlling the expression of proteins include a method of increasing the transfer amount and a method of increasing translation efficiency. However, in order to efficiently express the useful protein in the plant, the efficiency of the transcription step is improved to increase the production of mRNA. In addition, not only the mRNA produced is not used for translation but occasionally inhibits mRNA production (Napoli C et al., 1990; van der Krol et al., 1990).

In addition, it has been found that the amount of the finally synthesized protein from the gene plays a more important role in the translation step than in the transcription step. Therefore, in order to mass-produce an effective protein efficiently, It is necessary to develop a controllable technology.

Therefore, in order to overcome the problems of the prior art, the inventors of the present invention have been studying a method for mass-producing a useful protein from a plant by promoting the synthesis of the protein in the translation step, The present inventors completed the present invention by artificially synthesizing the 5 ' UTR base sequence.

Accordingly, an object of the present invention is to provide a DNA fragment capable of promoting translation efficiency of a protein and / or a recombinant vector comprising the DNA fragment.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the object of the present invention as described above, the present invention provides a DNA fragment comprising the nucleotide sequence of SEQ ID NO: 1, wherein the DNA fragment promotes translation of the target protein located downstream in the plant cell And a DNA fragment for promoting translation efficiency.

In addition, the present invention provides a recombinant vector comprising the DNA fragments for promoting translation efficiency.

In one embodiment of the present invention, the recombinant vector may be one in which a promoter, a DNA fragment for promoting translation efficiency, and a gene encoding a target protein are sequentially operatively linked.

In another embodiment of the present invention, the recombinant vector may further be operably linked to a base sequence capable of targeting and retention of a target protein to an endoplasmic reticulum (ER) in a plant cell.

In another embodiment of the present invention, the base sequence capable of targeting a target protein from the plant cell to the endoplasmic reticulum (ER) comprises a base sequence (SEQ ID NO: 3) encoding a BiP (chaperone binding protein) , And the base sequence capable of retention of the target protein may be a nucleotide sequence encoding an HDEL (His-Asp-Glu-Leu) or KDEL (Lys-Asp-Glu-Leu) peptide.

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

In addition, the present invention provides a method for mass-producing a target protein from a plant, which comprises introducing a recombinant vector into a plant.

In one embodiment of the present invention, the step of introducing the recombinant vector into a plant may be carried out by a method such as PEG precipitation method (Polyethylen glycol), Agrobacterium sp. -Mediated method, particle gun bombardment method, , Silicon carbide whiskers, sonication, and electroporation. [0034] The present invention is not limited to the above-described embodiments.

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

Since the DNA fragment according to the present invention is added to the upstream of the target protein to produce a recombinant vector, the plant transformed with the recombinant vector can improve translation of the target protein and induce targeting to a specific site in the plant, So that the target protein can be produced in large quantities from the plant.

Brief Description of the Drawings Fig. 1 shows a recombinant vector schematic diagram in which a) M17 and b) R21 are respectively added for the expression of a fusion protein GFP in Arabidopsis protoplasts.
FIG. 2 shows the expression of GFP from Arabidopsis protoplasts transformed with the recombinant vector of FIG. 1 through Western analysis. lane 1: Marker, lane 2: M17-GFP, lane 3: R21 GFP, lane 4: Negative control
FIG. 3 is a graph showing the relationship between the DNA fragment for promoting translation efficiency according to the present invention and the recombinant signal sequence (gBiP), target protein (GFP) sequence, cellulosic-binding domain (CBD) Fig.
FIG. 4 shows the expression of target protein GFP as an endoplasmic reticulum from the Arabidopsis protoplast transformed with the recombinant vector of FIG. 3 and confirmed by Western blotting.

The present inventors have to find a way to mass produce the desired protein in plants, the 5 'untranslated region can promote translation of the gene: paying attention to (5'UTR 5'untranslated region) development, Arabidopsis thaliana (Arabidopsis an optional 5 'UTR base sequence was synthesized based on the base sequence of the 5' UTR of the small subunit (RbcS) gene of ribulose bisphosphate carboxylazase / oxygenase (RuBisCo) .

In general, the 5 'untranslated region (5' UTR) of mRNA performs various functions during gene expression, but the most important feature of these functions is the regulation of mRNA translation efficiency. It has been reported that the nucleotide sequence of the 5 'UTR present in the immediate vicinity of the translation initiation codon affects 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 of which is the 5 'UTR of the small subunit (RbcS) gene of ribulose bisphosphate carboxylazase / oxigenase (RuBisCo). The length of this 5 'UTR is a hundred bases or more of nucleotides, and the length of the 3' UTR is a few kilobases longer. In addition, a sequence belonging to the 5'UTR, which is not a predetermined site such as a Shine-Dalgarno sequence known as a ribosome binding site sequence located in the 5 'UTR in a prokaryote but can also be referred to as a ribosome binding site sequence in eukaryotes, (Kozak M, 1987; Hamilton et al., 1987; Yamauchi et al., 1991; Joshi et al., 1997) have been reported.

Accordingly, the present inventors have found that a small subunit (RbcS) of ribulose bisphosphate carboxylazase / oxygenase (RuBisCo), which is known to have excellent translation efficiency among sequences of 5 'UTR of Arabidopsis thaliana, The 5 'UTR base sequence shown in SEQ ID NO: 1 was synthesized based on the 5' UTR sequence of the gene, and it was confirmed that the above sequence had excellent translation efficiency (see Example 1).

Accordingly, the present invention provides a DNA fragment comprising the nucleotide sequence of SEQ ID NO: 1, wherein the DNA fragment promotes translation of a target protein located downstream in the plant cell, to provide.

The present invention also provides a DNA fragment comprising the nucleotide sequence of SEQ ID NO: 1, wherein the DNA fragment promotes translation of a target protein located downstream in the plant cell. ≪ / RTI >

More specifically, the recombinant vector comprises a DNA fragment according to the present invention, which promotes translation efficiency of a promoter and a protein, with a conventional framework used for protein expression as a basic framework, and a gene encoding a target protein are sequentially Lt; RTI ID = 0.0 > operably linked < / RTI >

The term "expression vector" in the present invention means plasmids, viruses or other vectors known in the art into which the DNA fragment according to the present invention and a polynucleotide sequence encoding the target protein can be inserted or introduced. The polynucleotide sequence encoding the DNA fragment and the protein of interest according to the present invention may be operably linked to an expression control sequence, wherein the operably linked gene sequence and expression control sequence comprise a selection marker and a replication origin, May also be included in an expression vector containing the same. Said "operably linked" may be a gene and expression control sequence linked in such a way as to enable gene expression when a suitable molecule is coupled to an expression control sequence. "Expression control sequence" means a DNA sequence that regulates the expression of a polynucleotide sequence operably linked to a particular host cell. Such regulatory sequences include promoters for conducting transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling transcription and translation termination.

Examples of the promoter include, but are not limited to, the 35S RNA and 19S RNA promoters of CaMV, as long as they are capable of expressing the inserted gene in plants. May be a full-length transcriptional promoter derived from Peakwort mosaic virus (FMV) and a coat protein promoter of TMV. Suitable vectors for introducing the polynucleotide sequence encoding the DNA fragment and the target protein according to the present invention into a plant cell include a Ti plasmid and a plant virus vector. Examples of suitable vectors include, but are not limited to, p35S. Those skilled in the art can select the DNA fragment according to the present invention and a vector suitable for introducing the polynucleotide sequence encoding the target protein. In the present invention, the DNA fragment according to the present invention and the polynucleotide sequence encoding the target protein are expressed in plant cells Any vector that can be introduced into the vector is available.

The present inventors have also found that the use of the DNA fragment of the present invention capable of enhancing the translation efficiency of a target protein not only promotes protein expression but also induces targeting of a target protein to a specific site in a plant cell, ). The recombinant expression vector was constructed.

In general, when over-expressing a target protein in a transgenic plant or a cell, proteolytic degradation often occurs. However, when targeting foreign proteins with various intracellular organelles, these proteins can be stored more stably. In particular, when the target protein is targeted to the endoplasmic reticulum, degradation of the protein can be minimized as compared with that in the cytoplasm. For example, when the human epithelial growth factor is targeted to the endoplasmic reticulum in the tobacco, the yield of the protein is increased by about 10 ⁴ times (Wirth S et al., 2004). In addition, using an ER retention signal peptide such as KDEL or HDEL allows the target protein to remain in the endoplasmic reticulum, thereby increasing folding and assembly by the molecular chaperone, resulting in degradation of the protein Can be minimized (Nuttall J et al., 2002). As an example, it is known that when the target protein is retentioned in the endoplasmic reticulum rather than the secretory pathway, the yield of the target protein is increased by 10 to 100 times (Hellwig S et al., 2004). In addition, chloroplasts in plants are not only a huge protein reservoir containing more than 40% of the total water soluble protein, but also have a large number of them. In the chloroplast of a plant, only the minimum protease necessary for protein processing is contained. This means that the target protein can be stored at a high concentration in the chloroplast by preventing it from being decomposed by the protease.

Therefore, the present inventors have found that a DNA fragment of the present invention comprising the nucleotide sequence of SEQ ID NO: 1 promoting the translation efficiency of a promoter and a protein, and a recombinant expression vector in which a gene encoding the target protein is operably linked in sequence, A recombinant vector was additionally operably linked to a specific site of the target protein, e. G., A marker sequence capable of targeting the target protein to the endoplasmic reticulum or chloroplast.

In this case, when targeting the target protein to the endoplasmic reticulum (ER) in the plant cell, a nucleotide sequence encoding a chaperone binding protein (BiP) protein may be used. Preferably, Instead, the nucleotide sequence shown in SEQ ID NO: 3, which is a genomic DNA of BiP containing an intron, can be used. Although the base sequence for retention of the target protein is not limited thereto, it is preferable that the base sequence encoding HDEL (His-Asp-Glu-Leu) or KDEL (Lys-Asp-Glu-Leu) Can be used. The N-terminus of the BiP protein contains a signal sequence that determines targeting to the endoplasmic reticulum and can serve to target the target proteins to the endoplasmic reticulum. In addition, when targeting the target protein to the chloroplast in the plant cell, a base sequence coating with Cab (chlorophyll a / b binding protein) can be used. Cab (a chlorophyll a / b binding protein) has a transit peptide necessary for targeting to the chloroplast at the N-terminus, so that when the peptide is used, the target protein can be normally targeted to the chloroplast (kavanagh TA , 1988).

Therefore, when an expression vector using such a BiP signal sequence, an endoplasmic reticulum retention signal sequence, or a trans peptide of Cab is used, target protein can be targeted to an endoplasmic reticulum or chloroplast, and a target protein can be accumulated at a high level There is an effect.

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

The method of introducing the recombinant vector of the present invention into a plant is not limited thereto. However, the method of precipitation by PEG (polyethylenglycol), Agrobacterium sp. -Mediated method, particle gun bombardment , Silicon carbide whiskers, sonication, electroporation, or the like can be used. In one embodiment of the present invention, Arabidopsis thaliana was transformed with the recombinant vector of the present invention using a precipitation method using PEG (Polyethylenglycol).

The plant may be a dicotyledonous plant or a monocotyledonous plant. Examples of the dicotyledonous plant include, but are not limited to, Arabidopsis thaliana, soybean, Such as tobacco, eggplant, pepper, potato, tomato, cabbage, radish, cabbage, lettuce, peach, pear, strawberry, watermelon, melon, cucumber, carrot or celery, Barley, wheat, rye, corn, sorghum, oats, or onions.

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

The term "target protein" used herein means a protein to be produced, and is not limited to a specific protein. 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, a green fluorescent protein (GFP) was used as the target protein.

The method for producing the desired protein from the transformed plant can be carried out by introducing into the plant a recombinant vector according to the present invention in which the protein translation enhancing DNA fragment according to the present invention and the nucleotide sequence encoding the target protein are operably linked to the promoter, Can be obtained from transformed plants or cells after transforming the cells with a recombinant expression vector and culturing them for a suitable time to express the desired protein. At this time, the method of expressing the target protein may be any method 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. Usually, in order to remove cell debris and the like, And then precipitating (for example, ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol and the like), and the like can be carried out, and dialysis, electrophoresis, Chromatography, and the like. As the chromatography, the target protein of the present invention can be purified by applying techniques such as ion exchange chromatography, gel-permeation chromatography, HPLC, reversed phase-HPLC, affinity column chromatography, ultrafiltration or the like, alone or in combination Cold Spring Harbor Laboratory Press (1989), Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, ; Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182. Academic Press, Inc., San Diego, Calif. (1990)).

In addition, in the present invention, in order to more easily and rapidly isolate and purify the target protein from the transformed plant, the above-described recombinant vector of the present invention encodes a cellulase-binding domain (CBD) A recombinant vector was additionally operably linked to the nucleotide sequence, preferably the nucleotide sequence shown in SEQ ID NO: 4.

Therefore, in the present invention, the expressed target protein may be fused with a cellulose binding domain by using a recombinant vector comprising a cellulose-binding domain, and the cell can be easily separated and purified by chromatography using a cellulose carrier as a column have. The fused form of the target protein thus produced can be cleaved to the cellulose-binding domain using an appropriate enzyme and then subjected to further separation and purification to produce a target protein in a non-fusion form.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided to further understand the present invention, but the present invention is not limited by the following examples.

The 5 ' UTR Development of

In order to develop a 5 'UTR capable of promoting translation of a gene so as to increase the production of a target protein, a small amount of ribulose bisphosphate carboxylazase / oxygenase (RuBisCo), which is known to have excellent translation efficiency in Arabidopsis thaliana An arbitrary 5 'UTR base sequence was developed and synthesized based on the base sequence of the 5' UTR of the subunit (RbcS) gene. The synthesized 5 'UTR was named M17, and the 5' UTR of RbcS gene was named R21. The nucleotide sequences of the 5 'UTRs are shown in Table 1 below.

designation Base 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

The developed 5 ' UTR Identification of expression levels in plant cells

Green fluorescence protein (GFP) was used as a reporter gene in order to confirm whether M17 translation efficiency was increased during protein expression. As shown in Fig. 1, M17 And R21, a 5 'UTR derived from RbcS, was set as a control on the start codon of GFP. And we investigated whether M17 is effective in improving translation efficiency by introducing them into Arabidopsis protoplasts by PEG (polyethylene glycol) precipitation method.

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

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

When the target protein is transported to a specific place in a plant cell, UTR  Translation efficiency analysis

In order to achieve stable storage and increase yield of the protein, M17, a 5 'UTR developed by the inventors of the present invention, examined whether the vector exhibited excellent translation efficiency even in a vector prepared to transport the target protein to a specific site. To this end, as shown in Fig. 3, M17 5 'UTR and control R21 5' UTR were added to the vector that transports the target protein into the endoplasmic reticulum. At this time, the vector prepared for transporting the target protein to the endoplasmic reticulum (ER) includes a nucleotide sequence coding for chaperone binding protein (BiP) involved in the transport of the endoplasmic reticulum, retention of the target protein in the endoplasmic reticulum , A nucleotide sequence encoding HDEL (His-Asp-Glu-Leu), and a nucleotide sequence encoding a cellulose-binding domain used for purification of proteins. Green fluorescent protein (GFP), a reporter gene, was used as a target protein in the above vector. These vectors were introduced into Arabidopsis protoplasts using PEG precipitation method and the expression efficiency was compared.

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, and it was confirmed that the 5'-untranslated region M17 was more effective in transporting the target protein GFP into the endoplasmic reticulum Respectively.

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

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> POSTECH Academy-industry Foundation <120> Nucleotide sequence to promote translation efficiency in plants <130> PB12-10864 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> M17 5 'UTR <400> 1 ggcgtgtgtg tgtgttaaag a 21 <210> 2 <211> 21 <212> DNA <213> Arabidopsis thaliana - RbcS 5 'UTR <400> 2 cacaaagagt aaagaagaac a 21 <210> 3 <211> 272 <212> DNA <213> Arabidopsis thaliana - BiP <400> 3 atggctcgct cgtttggagc taacagtacc gttgtgttgg cgatcatctt cttcggtgag 60 tgattttccg atcttcttct ccgatttaga tctcctctac attgttgctt aatctcagaa 120 ccttttttcg ttgttcctgg atctgaatgt gtttgtttgc aatttcacga tcttaaaagg 180 ttagatctcg attggtattg acgattggaa tctttacgat ttcaggatgt ttatttgcgt 240 tgtcctctgc aatagaagag gctacgaagt ta 272 <210> 4 <211> 502 <212> DNA <213> Clostridium stercorarium - CBD <400> 4 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

Claims (9)

1. A DNA fragment comprising the nucleotide sequence of SEQ ID NO: 1, wherein the DNA fragment promotes translation of a target protein located downstream in the plant cell. A recombinant vector comprising the DNA fragments for promoting translation efficiency according to claim 1. 3. The method of claim 2,
Wherein the recombinant vector is operatively linked in sequence with a promoter, a DNA fragment for promoting translation efficiency, and a gene encoding the target protein. The method of claim 3,
Wherein said recombinant vector is further operably linked to a base sequence capable of targeting and retention of the desired protein from the plant cell to the endoplasmic reticulum (ER). 5. The method of claim 4,
A nucleotide sequence capable of targeting a target protein to the ER in the plant cell is a nucleotide sequence of SEQ ID NO: 3 encoding a BiP (chaperone binding protein) Wherein the base sequence is a nucleotide sequence encoding an HDEL (His-Asp-Glu-Leu) or KDEL (Lys-Asp-Glu-Leu) peptide. 5. The method of claim 4,
Wherein the recombinant vector is further operatively linked to a nucleotide sequence of SEQ ID NO: 4 encoding a cellulose-binding domain (CBD). A method for mass-producing a target protein from a plant comprising the step of introducing the recombinant vector of any one of claims 2 to 6 into the plant. 8. The method of claim 7,
The step of introducing the recombinant vector into a plant may be carried out by a method such as PEG precipitation method (Polyethylen glycol), Agrobacterium sp . A method selected from the group consisting of an atomic method, a particle gun bombardment method, a silicon carbide whisker method, a sonication method, and an electroporation method &Lt; / RTI &gt; 8. The method of claim 7,
The plant is a dicot 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, ; Or a terminal plant selected from the group consisting of rice, barley, wheat, rye, maize, sorghum, oats, and onions.

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