KR20190030263A - Recombinant vector for separating and purifying target protein in plant - Google Patents

Abstract

The present invention relates to a recombinant vector for separating and purifying a protein in plant cells and a method for separating and purifying a target protein using the same. A target protein expression system using plant cells according to the present invention solves problems of conventional plant cell culturing methods and mass-produces the target protein containing a biopharmaceutical protein, and thus makes it possible to commercialize biopharmaceuticals such as plant-derived protein products, thereby being very effective.

Description

The present invention relates to a recombinant vector for separating and purifying a protein from a plant,

The present invention relates to a recombinant vector for the separation and purification of a protein in plant cells and a method for separating and purifying a target protein using the recombinant vector.

The remarkable development of molecular biology and genetic engineering techniques has also been applied to the field of plants, and efforts to produce useful physiologically active substances from plants are steadily continuing. When a useful substance is produced in a plant, it is possible to dramatically reduce the production cost, and it is possible to produce various pollutants such as viruses, cancer genes, and enterotoxins that can be generated in a conventional method of isolating and purifying a protein synthesized in animal cells or microorganisms It can be excluded from the source, and unlike animal cells and microorganisms, it has an advantage that it can be stored and managed for a long time as a seed even in the commercialization stage. In addition, when the demand of the useful substance surges, it is advantageous compared to the existing animal cell system in terms of equipment technology and cost required for mass production, so that it is possible to supply according to the demand that is increased in the shortest period.

However, even these advantages have been the drawbacks of relatively low protein expression and difficulty in separating and purifying proteins from plant cells in comparison with other hosts including animal cells. Many studies have been carried out, and there have been attempts to increase the amount of protein expression in plant cells by various methods and to separate and purify proteins efficiently.

On the other hand, Cellulose-binding module 3 (CBM3) is a cellulose binding domain obtained from C. thermocellum and has very high binding power to cellulose. Therefore, it has several properties for use as an affinity tag for pure protein separation. It is known that CBM3 is specific for amorphous cellulose and has a high affinity. When CBM3 is fused to a target protein, it is less likely to interfere with the fusion target protein and also helps to fold the target protein (Wen Wan et al. 2011). In microorganisms, CBM3 has been reported to increase the yield of protein production. Cellulose to which CBM3 binds is environmentally friendly as an affinity resin, and has various advantages such as a very low price. cellulose is not chemically reactive and can be used in various buffer solutions and will not react with proteins. In addition, there are also advantages that various forms of cellulose can be easily obtained commercially. Therefore, it has various conditions that can be the most suitable friendly resin for pure protein separation. Due to its affinity with human body, it has already been applied to various medical and human body products. Small ubiquitin-related modifier (SUMO) is a small protein that modifies many of the proteins studied following ubiquitin in the ubiquitin-like molecules (Ubls) group of eukaryotes. SUMO is present in various target proteins through the C- It is linked to the covalent bond by forming an isopeptide with the ε-amino moiety of the lysine. To fuse this SUMO to the target protein, the SUMO-specific protease must remove the extra portion present at the C-terminal region from the SUMO precursor. At this time, the SUMO-specific protease recognizes glycine-glycine motif, which is specifically present at the C-terminal portion of the SUMO domain, and removes the extra portion at the C-terminus of the GG sequence. The SUMO protease recognizes the SUMO domain when it makes SUMO from the SUMO precursor and cleaves the C-terminal region of the two glycines, so the SUMO protease can show a very high specificity, and the type of amino acid following the two glycine residues , The di-glycine residues are followed by diverse proteins and the mature SUMO domain at the C-terminus of the precisely di-glycine is removed, so that a specific protein at the C-terminal region does not have an extra amino acid It has the characteristics to be able to. Therefore, target protein can be fused to SUMO to make fusion protein of SMO-target, and SUMO domain can be removed from this to make target protein having no extra amino acid residue at the desired N-terminus.

As a result of intensive efforts to facilitate separation and purification of target proteins in plant cells, the present inventors have found that it is easy to separate and purify proteins when CBM3 and SUMO are fused to a target protein. Thus completing the present invention.

The present invention relates to a process for producing SUMO domain; And CBM3 or sCBD are fused to the target protein to induce high expression of the recombinant protein. In the case of CBM3, the expression of the protein is further enhanced by using the Mp domain, and the recombinant protein is purified from the total protein extract of plant cells at low cost Purified recombinant protein CBM3 or sCBD used for the purification of the recombinant protein was completely immobilized using CBM3 in AMC to remove the affinity tag, And a method of producing the same.

Another object of the present invention is to provide a cell-binding module (CBM3) or a cellulose-binding module (sCBD); And (b) a small ubiquitin-related modifier (SUMO).

It is still another object of the present invention to provide a method for separating and purifying a target protein comprising the following steps.

(a) preparing the above-mentioned recombinant vector;

(b) introducing the recombinant vector into a plant to produce a transgenic plant;

(c) culturing the transgenic plant to express the target protein;

(d) binding the expressed target protein to cellulosic beads; And

(e) separating and purifying only the target protein by treating bdSEBNP1 with the target protein bound to the cellulose bead.

In order to accomplish the object of the present invention as described above, the present invention provides a method of manufacturing a cell-binding module (CBM3) or a cell binding module (CBM); And (b) SUMO (Small ubiquitin-related modifier).

The Cellulose-binding module 1 (CBM3) of the present invention is a gene encoding the amino acid sequence of SEQ ID NO: 2, sCBD (Cellulose-binding module 1) is a gene coding the amino acid sequence of SEQ ID NO: 1, SUMO (Small ubiquitin -related modifier) may be a gene encoding the amino acid sequence of SEQ ID NO: 3.

The recombinant vector of the present invention may further comprise Mp (mannosylated peptide region).

The Mp (mannosylated peptide region) of the present invention may be a gene encoding the amino acid sequence of SEQ ID NO: 8.

The recombinant vector of the present invention may further include one or more selected from the group consisting of BiP (chaperone binding protein) and HDEL (His-Asp-Glu-Leu) peptides.

The recombinant vector of the present invention comprises a 35S promoter derived from cauliflower mosaic virus, a 19S RNA promoter derived from cauliflower mosaic virus, a plant actin protein promoter and a ubiquitin protein promoter And a promoter selected from the group consisting of a promoter and a promoter.

The cleavage map of the recombinant vector of the present invention can be shown in Fig.

In order to accomplish still another object of the present invention, there is provided a method for separating and purifying a target protein comprising the following steps.

(a) preparing the above-mentioned recombinant vector;

(b) introducing the recombinant vector into a plant to produce a transgenic plant;

(c) culturing the transgenic plant to express the target protein;

(d) binding the expressed target protein to cellulosic beads; And

(e) separating and purifying only the target protein by treating bdSEBNP1 with the target protein bound to the cellulose bead.

The cellulosic beads of the present invention may be prepared by mixing and dissolving a mixture of carboxyl methyl cellulose (CMC), methyl cellulose (MC), amorphous cellulose (AMC), hydroxy ethyl cellulose (HEC) Hydroxypropylmethylcellulose (HPMC) may be used as the cellulose beads.

The bdSEBNP1 of the present invention may be a gene encoding the amino acid sequence of SEQ ID NO: 4.

The bdSEBNP1 of the present invention is expressed in E. coli.

The present invention relates to a method for purifying a recombinant protein, such as a medical protein, in a plant, purifying and separating purified cellulose using an inexpensive cellulose as an affinity resin, And then mass-producing only the target protein portion. Since the recombinant protein of the present invention includes the SUMO domain, it can induce the high expression of the recombinant protein in the plant, and the target protein is fused to SUMO to make the fusion protein of SUMO-target and the SUMO domain is removed therefrom It has a di-glycine motif, SUMO protease site, which is a protease that recognizes SUMO. Therefore, it is effective to purely isolate the target protein only through proteolytic cleavage. In addition, the inclusion of cellulose-binding module 3 (CBM3) of Clostridium thermocellum or sCBD (cellulose binding module 1) of Trichoderma reesei, which is an affinity tag for pure separation of recombinant proteins, The purified recombinant protein can be purified and separated, purified and then immobilized on the cellulose resin. Furthermore, the recombinant protein gene has the effect of inducing accumulation of high concentrations in ER (ER) by including BiP signal sequence and ER retention signal HDEL at N- and C-terminus of the gene, respectively. It also includes enhancing the expression level by including the MP domain in the front of the cellulose binding domain and in the back of the BiP. Therefore, the present invention can obtain a high-efficiency and high-expression target protein from a plant, and thus can be utilized for the mass production of industrial or medical proteins using plants.

1 is a schematic diagram of a method for pure separation of a target protein using CBM3-bdSUMO of the present invention.
Fig. 2 is a cleavage map of a recombinant expression vector for purifying and isolating a target protein of the present invention.
3 is a cleavage map of a recombinant expression vector for purifying and separating a target protein of the present invention.
4 is a schematic diagram of a recombinant expression vector expressed in plants or Escherichia coli according to the present invention.
FIG. 5 is a Western blot photograph showing the decomposition activity of SUMO fusion protein in vivo using the Arabidopsis protopalst expression system by bdSUMO-specific proteinase (bdSENP1).
6 is a Western blot photograph of bdSENP1 purified from Escherichia coli transformed with a recombinant expression vector containing a gene construct according to the present invention using a Ni2 + -NTA agarose column.
7 is a Western blot photograph showing the expression of Mp-CBM3-SUMO fusion hIL6 of the present invention in plants.
8 is a photograph showing Mp-CBM3-SUMO-IL6 separation and AMC immobilization using AMC of the present invention.
9 is a photograph showing the in vitro hydrolysis activity of Mp-CBM3-bdSUMO fusion hIL6 immobilized on AMC using AbdSENP1 of the present invention.
FIG. 10 is a photograph of SDS-page and Western blot showing hIL6 purely isolated from the Mp-CBM3-bdSUMO-IL6 fusion protein immobilized on AMC of the present invention by using bdSENP and removing the tag for purification and column chromatography .
FIG. 11 shows the results of STAT3 activity test in human LNCaP by hIL-6 produced by the plant of the present invention.

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

As described above, the present inventors used the SUMO domain to induce high expression of a recombinant protein in a plant, and the SUMO domain has a SUMO protease site, a di-glycine motif, which proteolytic cleavage And pure recombinant proteins can be purified and purified from total protein extracts of plant cells expressing recombinant proteins using CBM3 (Cellulose-binding module 3) or sCBD (cellulose binding module 1) at low cost And it was confirmed that the recombinant protein was immobilized by AMC (amorphous cellulose) using CBM3, and bdSENP1 was used to purify and purify only the target protein by pure separation without additional sequence. (See Fig. 1)

Accordingly, the present invention is directed to a method of producing a cellulosic material comprising: (a) Cellulose-binding module 3 (CBM3) or cellulose binding module 1 (sCBD); And (b) SUMO (Small ubiquitin-related modifier).

The recombination refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. Recombinant cells can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term " recombinant expression vector " means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element.

The recombinant expression vector and an expression vector comprising a suitable transcription / translation control signal can be constructed by methods known to those skilled in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. Suitable vectors for expressing the DNA fragment and the gene encoding the target protein in plant cells according to the present invention include pUC19-based plasmids or Ti plasmid vectors.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a so-called T-region to a plant cell when present in a suitable host such as Agrobacterium tubapassense. Other types of Ti-plasmid vectors are currently being used to transfer hybrid DNA sequences to plant cells, or to protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0120 516 B1 and US 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

Examples of suitable vectors include, but are not limited to, 326 GFP or binary vectors such as the pCAMBIA family. Those skilled in the art will appreciate that the DNA fragment and the gene encoding the desired protein according to the present invention may be expressed in plant cells Any vector that can be selected can be used.

More specifically, the recombinant vector can be produced by sequentially sequencing the DNA fragment according to the present invention and the gene encoding the target protein, wherein the DNA fragment according to the present invention, which promotes the expression level of the promoter and the target protein, Linked recombinant expression vector. In addition, the expression vector may include a ribosome binding site and a transcription terminator as a translation initiation site. The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, And aadA gene, but the present invention is not limited thereto.

In the recombinant vector of the present invention, the promoter is a 19S RNA promoter derived from cauliflower mosaic virus, a 35S promoter derived from cauliflower mosaic virus, a 19S RNA promoter derived from cauliflower mosaic virus, an actin protein promoter and a ubiquitin protein promoter The term " promoter " refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription . A " plant promoter " is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Because the selection of transformants can be made by various tissues at various stages, Thus, a constitutive promoter does not limit selectivity.

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

Any host cell known in the art may be used as the host cell capable of continuously cloning and expressing the vector of the present invention in a stable and prokaryotic cell, for example, E. coli JM109, E. coli BL21, E. coli RR1 , E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus tulifene, and Salmonella typhimurium, Serratia marcesensus and various Pseudomonas species And enterobacteria and strains such as the species.

When the vector of the present invention is transformed into eukaryotic cells, yeast (Saccharomyce cerevisiae), insect cells, human cells (for example, Chinese hamster ovary, W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells. The host cell is preferably a plant cell, wherein the plant is selected from the group consisting of food crops including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and millet; Vegetable crops including Arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Special crops including ginseng, tobacco, cotton, sesame seed, sugar cane, sugar beet, perilla, peanut and rapeseed; Apple trees, pears, jujubes, peaches, grapes, citrus fruits, persimmons, plums, apricots, and banana; Roses, carnations, chrysanthemums, lilies, and tulips.

The method of delivering the vector of the present invention into a host cell can be carried out by the CaCl2 method, Hanahan, D., J. MoI. Biol., 166: 557-580 (1983) An electric drilling method or the like. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

The term " operably linked " may be a gene and expression regulatory factor linked in such a way as to enable expression of the gene when the appropriate molecule is coupled to the expression regulatory element.

The Cellulose-binding module 1 (CBM3) is a gene coding for the amino acid sequence of SEQ ID NO: 2, sCBD (Cellulose-binding module 1) is a gene coding for the amino acid sequence of SEQ ID NO: 1, SUMO (Small ubiquitin- ) May be a gene coding for the amino acid sequence of SEQ ID NO: 3. More specifically, the gene is preferably at least 70%, more preferably at least 80%, even more preferably at least 90% May comprise a nucleotide sequence having at least 95% sequence homology. &Quot;% of sequence homology to polynucleotides " is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The SUMO (small ubiquitin-related modifier) may be derived from Brachypodium distachyon, CBM3 (Cellulose-binding module 3) may be derived from Clostridium thermocellum, and sCBD (cellulose binding module 1) may be derived from Trichoderma reesei.

The term "target protein" used herein means a protein to be produced, and is not limited to a specific protein. Specifically, the target protein may be an antigen, an antibody, an antibody fragment, a structural protein, a regulatory protein, a transcription factor, a toxin protein, a hormone, a hormone analog, a cytokine, an enzyme, an enzyme inhibitor, , A storage protein, a movement protein, an exploitive protein, and a reporter protein.

The recombinant vector may further include Mp for the purpose of increasing the level of protein expression. The Mp is a mannosylated peptide region and is a 60 amino acid fragment of alanine 231 to aspartic acid 290 of PTPRC (protein tyrosine phosphatase, receptor type, C). The term target protein refers to a protein to be produced, and is not limited to a specific protein. Specifically, the target protein may be an antigen, an antibody, an antibody fragment, a structural protein, a regulatory protein, a transcription factor, a toxin protein, a hormone, a hormone analog, a cytokine, an enzyme, an enzyme inhibitor, , A storage protein, a movement protein, an exploitive protein, and a reporter protein.

The Mp may be a gene coding for the amino acid sequence of SEQ ID NO: 8. More specifically, the gene may be at least 70%, more preferably at least 80%, even more preferably at least 90% May comprise a nucleotide sequence having at least 95% sequence homology. &Quot;% of sequence homology to polynucleotides " is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The recombinant vector may further include any one selected from the group consisting of BiP (chaperone binding protein) and HDEL (His-Asp-Glu-Leu) peptides, and the BiP (chaperone binding protein) And the HDEL (His-Asp-Glu-Leu) peptide may be a gene coding for the sequence of SEQ ID NO: 6.

The recombinant vector is selected from the group consisting of 35S promoter derived from cauliflower mosaic virus, 19S RNA promoter derived from cauliflower mosaic virus, actin protein promoter of plant, and ubiquitin protein promoter Any one promoter may be further included.

The cleavage map of the recombination vector can be represented by Fig. 2, and preferably can be represented by Fig.

In order to accomplish still another object of the present invention, the present invention provides a method for producing a recombinant vector, comprising: (a) preparing a recombinant vector according to claim 1; (b) introducing the recombinant vector into a plant to produce a transgenic plant; (c) culturing the transgenic plant to express the target protein; (d) binding the expressed target protein to cellulosic beads; And (e) separating and purifying only the target protein by treating bdSEBNP1 with the target protein bound to the cerulose bead. And a method for separating and purifying a target protein.

The method for producing a target protein from the transformed plant cells can be obtained from transformed cells after transforming plant cells with the recombinant vector according to the present invention and culturing the plant for a suitable time to express the target protein . At this time, the method of expressing the target protein may be any method known in the art.

In the above method, a transformant was prepared in Arabidopsis thaliana, but not limited thereto. Both protoplasts or protoplasts isolated from monocotyledonous plants can be used in the method of the present invention.

The cellulose beads may be selected from the group consisting of carboxyl methyl cellulose (CMC), methyl cellulose (MC), amorphous cellulose (AMC), hydroxy ethyl cellulose (HEC) Hydroxypropyl methyl cellulose (HPMC), and most preferably amorphous cellulose (AMC).

The bdSEBNP1 may be expressed in E. coli, and the bdSEBNP1 may preferably be a gene encoding the amino acid sequence of SEQ ID NO: 4. Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 4 . &Quot;% of sequence homology to polynucleotides " is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI > The bdSEBNP1 can be isolated and purified using Ni2 + -NTA agarose column.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Genetic information and method of securing

The cellulose-binding module family 3 (CBM3; SEQ ID NO: 2) was obtained from Clostridium thermocellum and the small ubiquitin-related modifier (bdSUMO; SEQ ID NO: 3) was obtained from Trichoderma reesei. ) Were obtained from Brachypodium distachyon (bd), and the nucleotide sequences of sCBD, CBM3 and bdSUMO genes were designed to optimize codon usage in Arabidopsis thaliana and were produced in biona.

SEQ ID NO: order The base sequence (or amino acid sequence) One sCBD TQSHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCL 2 CBM3 VSGNLKVEFYNSNPSDTTNSINPQFKVTNTGSSAIDLSKLTLRYYYTVDGQKDQTFWCDHAAIIGSNGSYNGITSNVKGTFVKMSSSTNNADTYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSFKSASQFVEWDQVTAYLNGVLVWGKEP
3 bdSUMO HINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMTAIAFLFDGRRLRAEQ TPDELEMEDGDEIDAMLHQTGG (bdSENPI amino acids 248) 4 bdSENP1 PFVPLTDEDEDNVRHALGGRKRSETLSVHEASNIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLKCHFFNTFFYKKLINGGYDYKSVRRWTTKRKLGYNLIDCDKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDVHAWKQEGVQNLPLQENGWDCGMFMLKYIDFYSRDMELVFGQKHMSYF RRRTAKEILDLKAG 5 HDEL HDEL 6 BiP MARSFGANSTVVLAIIFFGCLFALSSAIEEATKL 7 MP ANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPD

sCBD or CBM3; And plant expression vector containing bdSUMO and target protein

sCBD, CBM3 and bdSUMO (Bioneer Corp., Daejeon, South Korea) were introduced into E. coli (Top 10) and amplified. Plasmid DNA isolated from Escherichia coli was then digested with restriction enzymes to obtain necessary sites. GFP is a green fluorescent protein that is widely used as a marker protein for in vivo localization. Also, human interleukin-6 (IL-6) (SEQ ID NO: 5) was utilized as an example of a practical target protein.

construction of a fusion construct with sCBD, bdSUMO and GFP; These domains were obtained by PCR, followed by overlapping PCR products of each domain. The overlap PCR products were digested with restriction enzymes BamHI and XhoI and then introduced into a plant expression vector called 326-ER, which was digested with the same restriction enzymes. This 326-MELsCH vector has a leader sequence for targeting ERPs of BiP (chaperone binding protein), the 35S promoter of cauliflower mosaic virus, and the translational enhancer sequence xx 5'-UTR and HSP terminator. HDEL (His-Asp-Glu-Leu) was added to the ER retention signal, which was included in the reverse primer to be introduced during the PCR process. PCR products were identified by DNA sequencing. This plant expression vector was named 326-MELsCH-bdSUMO-GFP (SEQ ID NO: 10) (FIG. 4).

In order to produce human IL-6 in plants, the CBM3-bdSUMO-IL-6-HDEL construct (SEQ ID NO: 12) was prepared by the same method as used for GFP above. The MP domain was introduced between BiP and CBM3 to increase the expression level of this fusion recombinant protein. The Mp domain is a domain with amino acid positions 231 to 290 of the human protein tyrosine phosphatase, receptor type C (PTPRC), which has six N-glycosylation sites, and is known to greatly enhance protein expression in plants. Fusion constructs were also used to link Mp and CBM3 for the first time through overlap PCR, and overlap PCR for bdSUMO and hIL-6. These two fragments were introduced into a 326-ER vector to construct a fusion construct with BiP-Mp-CBM3-SUMO-IL-6-HDEL. Then, 326 vector was introduced into a binary vector pCAMBIA1300. pCAMBIA1300 has a promoter such as 326 vector, 5'-UTR and HSP terminator. This expression vector construct was named 1300-ME-MCBM3-bdSUMO-IL-6 (Fig. 2). 1300-ME-MCBM3-bdSUMO-IL-6 was introduced into the A. tumefaciens GV3101 strain by electroporation and the expression was confirmed by transient expression on the leaf of Nicotiana benthamiana.

Construction of bdSENP1 expression vector for expression of plant and Escherichia coli

bdSENP1 was obtained from Brachypodium distachyon (bd). The bdSENP1 gene was prepared by chemical synthesis from Bioneer (Korea). bdSENP1 was introduced into Escherichia coli (Top 10) and propagated, followed by restriction enzyme digestion to construct a plant and Escherichia coli expression vector. To construct a plant expression vector, the bdSENP1 plasmid was cut with BamHI and XhoI and introduced between the BiP of the 326-MELsCH vector and the C-terminal ER retention signal HDEL (His-Asp-Glu-Leu). This plant vector was named 326-MELsCH-bdSENP1 (Fig. 4).

A pRSET expression vector was used to construct the bdSENP1 vector for expression of E. coli. BdSENP1 was first amplified by PCR using BamH1 and EcoR1 primers, and BamH1 and EcoR1 were cut and introduced into pRSET cut with the same restriction enzymes. The obtained clones were sequenced by automated DNA sequencing. This expression vector for E. coli was named pRSET-bdSENP1 (Fig. 4).

Expression of fusion proteins with recombinant proteins sCBD-bdSUMO and CBM3-bdSUMO in plants

The expression of these fusion proteins with sCBD-bdSUMO and CBM3-bdSUMO in plants was confirmed by protoplasts obtained from the leaf cell of Arabidopsis through polyethyleneglycol (PEG) -mediated transformation, or by the addition of silencing suppressor (TCV-CP) and Nicotiana benthamiana And the expression of agrobacterium-infiltration-mediated transient expression was confirmed. To confirm the expression of recombinant proteins in Arabidopsis mesophyll protoplasts transfected with sCBD-bdSUMO-GFP and bdSENP1, protoplasts were first settled through centrifugation (500 rpm, 10 mins) and the protoplast pellet was resuspended in 130 l extraction buffer (10 sec, 30W) for 10 min at room temperature. The protoplasts were then incubated for 10 min at room temperature. The cell extracts thus obtained were centrifuged (3000 g, 10 min) to remove precipitates and only the supernatant was obtained and used for protein quantification. In order to obtain protein extracts from Nicotiana benthamiana leaf tissue, Mp-CBM3-bdSUMO-IL-6 was added to the leaf tissue volume (50 mM Tris-HCl, pH 7.5, 150 mM NaCl , 1 mM DTT, 0.1% [v / v], Triton X-100 and protease inhibitor. Leaf extracts were centrifuged (12000 g, 10 min) to obtain supernatant, which was used for the next analysis such as protein quantification.

Proteolytic cleavage of bdSUMO fusion protein by bdSENP1 in the endoplasmic reticulum of plant cells

First, we examined the expression of SUMO fusion protein and bdSENP1 in protoplasts obtained from the leaves of Arabidopsis thaliana. We also investigated whether the bdSENP1 cleaves the SUMO fusion protein. For this purpose, SUMO fusion expression vector 326-MELsCH-bdSUMO-GFP and bdSENP1 expression vector 326-MELsCH-bdSENP1 (Fig. 1) were introduced into the protoplast through PEG technique. These vectors were introduced into protoplasts either alone or in combination. 326-GFP was used as a control. Protein extracts were obtained from the transformed protoplasts and western blot analysis was performed using appropriate antibodies to confirm protein expression. The amount of total protein was also confirmed by CBB staining (Table 3). Western blot analysis using anti-His antibody revealed that bdSENP1 was 28 kDa in size. When western blot analysis was performed using GFP antibody, the fusion protein was found to be 42 kDa when only sCBD-SUMO-GFP was expressed. On the other hand, when bdSENP1 was expressed in the same manner as the sCBD-SUMO-GFP fusion protein, the GFP antibody recognized 29 kDa protein (Fig. 5B). On the other hand, after western blot analysis with GFP antibody and removal of the GFP antibody, western blot analysis of this same western blot with sCBD antibody confirmed the 13 kDa band corresponding to CBD-bdSUMO (Fig. 5C). These results suggest that bdSENP1 effectively cleaves SUMO-containing recombinant proteins in the endoplasmic reticulum.

Expression of bdSENP1 in E. coli and purification using Ni2 + -NTA agarose column

To produce bdSENP1 in E. coli, pRSET-bdSENP1 was introduced into BL21 (DE3) pLysS. Transformed E. coli was obtained using ampicillin resistance. For protein expression and pure purification, the following procedure was followed. One colony was selected and cultured in 5 ml SOB containing ampicillin (50 μg / ml). Shaking (225 rpm) was performed at 37 ° C. The next day, it was grown at 37 ° C until it reached 0.4-0.6 at OD600 after 200 ml SOB inoculation with ampicillin (50 μg / ml). IPTG was allowed to reach a final concentration of 1 mM and then grown at 37 ° C for an additional 3 hours. The E. coli was sedimented by centrifugation (12,000 rpm, 10 min) and the supernatant was removed. E. coli was dissolved by sonication in 20 ml cold lysis buffer (40 mM Tris, 300 mM NaCl, 2 mM DTT, 20 mM Na2 PO4, 1% Triton-X-100, 10 mM imidazole, PMSF, pH-7.5). At this time, sonication was performed for 2 s on ice and 3 s pulse was treated for 10 min. After the sonication, E. coli extract was centrifuged (15,000 g, 30 min, 4 ° C) to remove precipitate, and the supernatant was taken. Recombinant bdSENP1 was purified using Ni2 + -NTA agarose column (Qiagen, Valencia, Calif.) Using the method described below. 1 ml of Ni2 + -NTA agarose resin was filled in the empty column and the storage solution was removed. After washing the column with 10 ml binding buffer (40 mM Tris, 300 mM NaCl, 20 mM Na2PO4, 10 mM imidazole, pH-7.5), the supernatant of E. coli containing bdSENP1 was introduced into the column. The flow of the solution was adjusted to about 0.5 mL / min. Ni2 + -NTA agarose column was washed 4 times with 10 ml of washing buffer (40 mM Tris, 300 mM NaCl, 20 mM Na2PO4, 30 mM imidazole, pH-7.5). bdSENP1 was isolated from the column using 1 ml of 250 mM imidazole and 1xPBS buffer at pH 7.4. Protein concentrations were determined by Bradford assay. The exact quantification and purity of bdSENP1 was confirmed by Commassie-staining after SDS-PAGE (Fig. 6A) and bands of bdSENP1 were confirmed by Western blot analysis using anti-his antibody (Qiagen, Valencia, CA) 6B). According to SDS-PAGE analysis, the protein purity was estimated to be over 95%, and the yield of bdSENP1 in E. coli was 6 mg / L.

Nicotiana in benthamiana Mp - CBM3 -SUMO-IL-6 and the expression of this recombinant protein CBM3  Used AMC  Confirm binding

In the present invention, CBM3 was used as an affinity tag to purify recombinant proteins purely from whole plant protein extracts. CBM3 is known to tightly couple to AMC. For this purpose, a recombinant protein was designed so that CBM3 is located at the N-terminus of the SUMO domain. Expression of the constructed construct was induced by agrobacterium infiltration in leaf tissue of Nicotiana benthamiana. The leaves of Nicotiana benthamiana were homogenized and centrifuged at 13,000 rpm for 20 minutes to obtain a supernatant. It was isolated by SDS-PAGE and then separated by Commassie blue stain (Fig. 7A). The supernatant was subjected to SDS-PAGE and western blot analysis revealed the expression of Mp-CBM3-SUMO-IL-6 (FIG. 7). Western blot analysis of antibodies against IL-6 produced in mice confirmed proteins between 55-75 kDa (Figure 7B). In order to perform affinity purification using cellulose resin, the whole protein extract was bound to amorphous cellulose (AMC) microcrystalline powder (Sigma-Aldric). The cellulose affinity purification method is described below. Cellulose was hydrated by immersing it in distilled water in four times the volume. In this state, the microparticles suspended in the aqueous solution were removed. This procedure was repeated 5 times. Finally, cellulose beads were mixed with water at a ratio of 1: 1 (V / V), and sodium azide (0.1%) was added thereto and stored at 4 ° C. To induce the binding of recombinant proteins containing CBM3 to cellulose, 1 ml of Nicotiana bentamiana leaf extract was mixed with 500 l of cellulose beads and 2 ml of centrifuge tube and mixed at 4 ° C using an orbital shaker (60 rpm). It is known that CBM3 binds about 300 mg / g to AMC when producing yeast protein. After allowing the CBM3 fusion protein to bind to the cellulose for about one hour, the cellulose beads were sedimented by simple centrifugation and the supernatant was removed. Cellulose beads were washed with 40 mM Tris-HCl buffer (pH 7.5), which was 4 times the bead volume, to remove weakly nonspecific cellulose-bound proteins. The proteins bound to the cellulose beads were put in a 2x SDS-PAGE sample buffer and were favored by denaturation. We tried to liberate CBM3 fusion proteins expressed from plants under various conditions from cellulose beads, but recombinant proteins containing CBM3 were not released. Therefore, it can be said that CBM3 irreversibly binds strongly to cellulose. In addition, the CBM3 fusion protein bound to cellulose beads was obtained from cellulose beads by denaturation, developed by SDS-PAGE, and then found to have a purity of 95% or more when confirmed by Commassie-staining (FIG. 8). In this way, the CBM3 fusion protein can be used as an affinity column for pure protein separation.

Plant-derived recombinant proteins Mp - CBM3 -SUMO-IL- 6 to CBM3  In this study, we used bdSENP1 as a substrate for the selective cleavage of IL-6

Separation of proteins after production of recombinant protein The affinity tag used for purification can introduce unnecessary activity into a target protein to be produced. Especially in a medical protein, addition of additional amino acid residues is strictly restricted in addition to the sequence of the target protein. Therefore, it is very important to remove tags for separation and purification. The present invention provides a method for completely removing an affinity tag using SUMO. In particular, a recombinant protein is bound to a cellulose bead, and only a target protein is separated and purified from cellulose beads using bdSENP1, a SUMO-specific protease. Using this method, proteins can be separated and purified efficiently at 4 ° C for 1-2 hours. First, it was confirmed that bdSENP1 recognizes SUMO and cleavage reaction can be performed with the protein bound to the cellulose bead (outlined in Figure 1). Briefly, the Mp-CBM3-SUMO-IL-6 recombinant protein was bound to AMC as in Example 7. AMC beads were then added to the bdSENP1 reaction buffer (25 mM Tris-HCl buffer (pH 7.5), 0.1% NP- 250 mM NaCl, 1 mM DTT) and then suspended in bdSENP1 reaction buffer (500 ml). To this was added bdSENP1 protease (5 g) and the reaction was allowed to proceed for 1-2 hours at 4 ° C with gentle shaking (60 rpm). After the reaction, IL-6 is released from the cellulose bead and will be present in the incubation buffer (FIG. 9). On the other hand, CBM3 is still irreversibly bound to AMC like SUMO domain. Therefore, IL-6 and beSENP1 were present in the supernatant, and bdSENP1 had its tag at the N-terminus. Therefore, bdSENP1 was removed through a Ni2 + -NTA column to purify IL-6 purely. Using the present invention, a larger scale of IL-6 was produced in plants and purified and purified to confirm the contents of the present invention. Human IL-6-containing BiP-CBM3-SUMO-IL-6-HDEL constructs were expressed in Nicotiana benthamiana using transient expression technique. Total protein extracts were prepared from 25 g of Nicotiana benthamiana leaves expressing recombinant proteins using 25 ml protein extraction buffer. CBM3-SUMO-IL-6-HDEL recombinant protein with CBM3 domain was bound to AMC by adding 5 ml of AMC and gently shaking (60 rpm) in a 4 ° C orbital shaker for 1 hour . Followed by washing 4 times with 40 mM Tris-HCl buffer (pH 7.5) corresponding to 4 times the beads volume. IL-6 release was induced by SUMO protease in the presence of AMC-bound BiP-Mp-CBM3-SUMO-IL-6-HDEL recombinant protein bound to AMC. To this end, AMC bound to BiP-Mp-CBM3-SUMO-IL-6-HDEL was washed with bdSENP1 reaction buffer, suspended in 10 ml of bdSENP1 reaction buffer, and purified and purified bdSENPI protease (50 g) And incubated for 2 hours at 4 ° C with shaking gently (60 rpm). In this way, IL-6 fused to the C-terminal is present in the supernatant and Mp-CBM3-SUMO is still bound to AMC. Thus, AMC and Mp-CBM3-SUMO were removed by simple centrifugation (Fig. 10). After the reaction, bdSENP1 was also in the supernatant. Therefore, bdSENP1 was designed to bind specifically to his tag, so it was removed using Ni2 + -NTA agarose column. Finally, the extra proteins with a small amount of contamination were ultimately obtained with size-exclusion chromatography using Superdex 200 column. SDS-PAGE analysis and Western blot analysis showed that IL-6 was isolated and purified from Nicotiana benthamiana at> 95% purity. Bradford protein assay and SDS-PAGE analysis showed that 80 μg of purified IL-6 was obtained from 1 g of leaf tissue. Then, the activity of plant-produced IL-6 was examined.

Activation of human LNCaP cells by STAT3 phosphorylation using plant-produced IL-6

In order to verify the activity of hIL-6 produced by plants, we used the phenomenon of activation of STAT3 signaling pathway by hIL-6 using LNCaP cells (androgen-sensitive human prostate adenocarcinoma cells). hIL-6 binds first to the IL-6 receptor (gp80) and then to gp130. After this, gp130 forms a dimer, and this dimer type gp130 induces the activation of Janus kinases (Jaks) to phosphorylate tyrosine residues of gp130. This process activates the SHP2 / Erk / Map pathway and the transcription factors STAT1v and STAT3. LNCaP cells were treated with 150 ng / mL hIL-6 produced by the plant and the level of STAT3 phosphorylation (P-STAT3) was determined at 30 min. A time-course study was then performed to determine the degree of phosphorylation up to 1 hour (Figure 11 A and Figure 11 B). The activity of the plant-produced protein was compared with a commercially purchased hIL-6 produced in E. coli, and it was confirmed that the activity was similar to that of the protein produced in the same concentration of E. coli. It was confirmed that the plant-produced hIL-6 is a protein showing the same activity.

The experiment was carried out as follows. LNCaP (prostate cancer cell line) was cultured in RPMI 1640 medium (Welgene, LM-011-01) containing 10% FBS. Culture was started by seeding 0.8x10 ⁶ LNCaP cells in a 60 mm incubator. After one day, the cells were changed to RPMI 1640 medium without 10% FBS, and then grown in serum-free condition for 8 hours. After treatment of IL-6 from plants or Escherichia coli, phosphorylation was measured over time. Cells were treated with IL-6 and washed with PBS (phosphate-buffered saline) at 2, 15, 30 and 60 min. The harvested cells were pulverized with RIPA (1X protease inhibitor) and the proteins were extracted. Protein concentration was measured with the BCA assay kit (Thermo Pierce, 23225). Protein samples were mixed with 5X Lamaeli buffer and electrophoresed using 8% PAGE gel. Antibodies against STAT3 or anti-p-STAT3 antibodies (Santacruz sc-482, anti-p-STAT3, Santacruz sc-8052) And anti-beta actin antibody (Santacruz sc-47778) was used to quantify the loading. Clarity ECL substrate (Bio-Rad, 170-5061) was used for Western blot image generation and images were captured using Image Quant LAS500 (GE lifescience). In order to verify the activity of hIL-6 produced by plants, we used the phenomenon of activation of STAT3 signaling pathway by hIL-6 using LNCaP cells (androgen-sensitive human prostate adenocarcinoma cells). hIL-6 binds first to the IL-6 receptor (gp80) and then to gp130. After this, gp130 forms a dimer, and this dimer type gp130 induces the activation of Janus kinases (Jaks) to phosphorylate tyrosine residues of gp130. This process activates the SHP2 / Erk / Map pathway and the transcription factors STAT1 and STAT3. LNCaP cells were treated with 150 ng / mL hIL-6 produced by the plant and the level of STAT3 phosphorylation (P-STAT3) was confirmed at 30 minutes. A time-course study was then performed to determine the degree of phosphorylation up to 1 hour (Figure 11 A and Figure 11B). The activity of the plant-produced protein was compared with a commercially purchased hIL-6 produced in E. coli, and it was confirmed that the activity was similar to that of the protein produced in the same concentration of E. coli. It was confirmed that the plant-produced hIL-6 is a protein showing the same activity.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Recombinant vector for separating and purifying target protein in          plant <130> 1062619 <160> 11 <170> KoPatentin 3.0 <210> 1 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Cellulose-binding module 1 from Trichoderma reesei <400> 1 Thr Gln Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro   1 5 10 15 Thr Val Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr              20 25 30 Ser Gln Cys Leu          35 <210> 2 <211> 158 <212> PRT <213> Artificial Sequence <220> <223> Cellulose-binding module 1 from Clostridium thermocellum <400> 2 Val Ser Gly Asn Leu Lys Val Glu Phe Tyr Asn Ser Asn Pro Ser Asp   1 5 10 15 Thr Thr Asn Ser Ile Asn Pro Gln Phe Lys Val Thr Asn Thr Gly Ser              20 25 30 Ser Ala Ile Asp Leu Ser Lys Leu Thr Leu Arg Tyr Tyr Tyr Thr Val          35 40 45 Asp Gly Gln Lys Asp Gln Thr Phe Trp Cys Asp His Ala Ala Ile Ile      50 55 60 Gly Ser Asn Gly Ser Tyr Asn Gly Ile Thr Ser Asn Val Lys Gly Thr  65 70 75 80 Phe Val Lys Met Ser Ser Ser Thr Asn Asn Ala Asp Thr Tyr Leu Glu                  85 90 95 Ile Ser Phe Thr Gly Gly Thr Leu Glu Pro Gly Ala His Val Gln Ile             100 105 110 Gln Gly Arg Phe Ala Lys Asn Asp Trp Ser Asn Tyr Thr Gln Ser Asn         115 120 125 Asp Tyr Ser Phe Lys Ser Ala Ser Gln Phe Val Glu Trp Asp Gln Val     130 135 140 Thr Ala Tyr Leu Asn Gly Val Leu Val Trp Gly Lys Glu Pro 145 150 155 <210> 3 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> SUMO from Brachypodium distachyon <400> 3 His Ile Asn Leu Lys Val Lys Gly Gln Asp Gly Asn Glu Val Phe Phe   1 5 10 15 Arg Ile Lys Arg Ser Thr Gln Leu Lys Lys Leu Met Asn Ala Tyr Cys              20 25 30 Asp Arg Gln Ser Val Asp Met Thr Ala Ile Ala Phe Leu Phe Asp Gly          35 40 45 Arg Arg Leu Arg Ala Glu Gln Thr Pro Asp Glu Leu Glu Met Glu Asp      50 55 60 Gly Asp Glu Ile Asp Ala Met Leu His Gln Thr Gly Gly  65 70 75 <210> 4 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> bdSENP1 from Brachypodium distachyon <400> 4 Pro Phe Val Pro Leu Thr Asp Glu Asp Glu Asp Asn Val Arg His Ala   1 5 10 15 Leu Gly Gly Arg Lys Arg Ser Glu Thr Leu Ser Val His Glu Ala Ser              20 25 30 Asn Ile Val Ile Thr Arg Glu Ile Leu Gln Cys Leu Asn Asp Lys Glu          35 40 45 Trp Leu Asn Asp Glu Val Ile Asn Leu Tyr Leu Glu Leu Leu Lys Glu      50 55 60 Arg Glu Leu Arg Glu Pro Asn Lys Phe Leu Lys Cys His Phe Phe Asn  65 70 75 80 Thr Phe Phe Tyr Lys Lys Leu Ile Asn Gly Gly Tyr Asp Tyr Lys Ser                  85 90 95 Val Arg Arg Trp Thr Thr Lys Arg Lys Leu Gly Tyr Asn Leu Ile Asp             100 105 110 Cys Asp Lys Ile Phe Val Pro Ile His Lys Asp Val His Trp Cys Leu         115 120 125 Ala Val Ile Asn Ile Lys Glu Lys Lys Phe Gln Tyr Leu Asp Ser Leu     130 135 140 Gly Tyr Met Asp Met Lys Ala Leu Arg Ile Leu Ala Lys Tyr Leu Val 145 150 155 160 Asp Glu Val Lys Asp Lys Ser Gly Lys Gln Ile Asp Val His Ala Trp                 165 170 175 Lys Gln Glu Gly Val Gln Asn Leu Pro Leu Gln Glu Asn Gly Trp Asp             180 185 190 Cys Gly Met Phe Met Leu Lys Tyr Ile Asp Phe Tyr Ser Arg Asp Met         195 200 205 Glu Leu Val Phe Gly Gln Lys His Met Ser Tyr Phe Arg Arg Arg Thr     210 215 220 Ala Lys Glu Ile Leu Asp Leu Lys Ala Gly 225 230 <210> 5 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> BiP <400> 5 Met Ala Arg Ser Phe Gly Ala Asn Ser Thr Val Val Leu Ala Ile Ile   1 5 10 15 Phe Phe Gly Cys Leu Phe Ala Leu Ser Ser Ala Ile Glu Glu Ala Thr              20 25 30 Lys Leu         <210> 6 <211> 272 <212> DNA <213> Artificial Sequence <220> <223> BiP <400> 6 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> 7 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> HDEL <400> 7 His Asp Glu Leu   One <210> 8 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> MP <400> 8 Ala Asn Ile Thr Val Asp Tyr Leu Tyr Asn Lys Glu Thr Lys Leu Phe   1 5 10 15 Thr Ala Lys Leu Asn Val Asn Glu Asn Val Glu Cys Gly Asn Asn Thr              20 25 30 Cys Thr Asn Asn Glu Val His Asn Leu Thr Glu Cys Lys Asn Ala Ser          35 40 45 Val Ser Ile Ser His Asn Ser Cys Thr Ala Pro Asp      50 55 60 <210> 9 <211> 184 <212> PRT <213> Artificial Sequence <220> <223> human interleukin 6 <400> 9 Met Val Pro Pro Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His Arg   1 5 10 15 Gln Pro Leu Thr Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile              20 25 30 Leu Asp Gly Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn          35 40 45 Met Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu      50 55 60 Pro Lys Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu  65 70 75 80 Glu Thr Cys Leu Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val                  85 90 95 Tyr Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln Ala             100 105 110 Arg Ala Val Gln Met Ser Thr Lys Val Leu Ile Gln Phe Leu Gln Lys         115 120 125 Lys Ala Lys Asn Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr Thr Asn     130 135 140 Ala Ser Leu Leu Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp 145 150 155 160 Met Thr Thr His Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser                 165 170 175 Ser Leu Arg Ala Leu Arg Gln Met             180 <210> 10 <211> 376 <212> PRT <213> Artificial Sequence <220> <223> MELsCH-SUMO-GFP <400> 10 Met Ala Ser Ser Met Ala Ser Ser Ala Thr Met Val Ala Ser Ser Ala   1 5 10 15 Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala              20 25 30 Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser          35 40 45 Asn Gly Gly His Ile Asn Leu Lys Val Lys Gly Gln Asp Gly Asn Glu      50 55 60 Val Phe Phe Arg Ile Lys Arg Ser Thr Gln Leu Lys Lys Leu Met Asn  65 70 75 80 Ala Tyr Cys Asp Arg Gln Ser Val Asp Met Thr Ala Ile Ala Phe Leu                  85 90 95 Phe Asp Gly Arg Arg Leu Arg Ala Glu Gln Thr Pro Asp Glu Leu Glu             100 105 110 Met Glu Asp Gly Asp Glu Ile Asp Ala Met Leu His Gln Thr Gly Gly         115 120 125 Ala Gly Thr Pro Arg Gly Met Ser Lys Gly Glu Glu Leu Phe Thr Gly     130 135 140 Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys 145 150 155 160 Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu                 165 170 175 Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro             180 185 190 Thr Leu Val Thr Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr         195 200 205 Pro Asp His Met Lys Arg His Asp Phe Phe Lys Ser Ala Met Pro Glu     210 215 220 Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr 225 230 235 240 Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg                 245 250 255 Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly             260 265 270 His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala         275 280 285 Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Thr Arg His Asn     290 295 300 Ile Glu Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr 305 310 315 320 Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser                 325 330 335 Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met             340 345 350 Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr His Gly Met Asp         355 360 365 Glu Leu Tyr Lys His Asp Glu Leu     370 375 <210> 11 <211> 510 <212> PRT <213> Artificial Sequence <220> ME-MCBM3-bdSUMO-IL-6 <400> 11 Met Ala Asn Ile Thr Val Asp Tyr Leu Tyr Asn Lys Glu Thr Lys Leu   1 5 10 15 Phe Thr Ala Lys Leu Asn Val Asn Glu Asn Val Glu Cys Gly Asn Asn              20 25 30 Thr Cys Thr Asn Asn Glu Val His Asn Leu Thr Glu Cys Lys Asn Ala          35 40 45 Ser Val Ser Ser Ser His Asn Ser Cys Thr Ala Pro Asp Gly Gly Gly      50 55 60 Gly Ser Gly Gly Gly Ser Ser Ser Val Ser Gly Asn Leu Lys Val Glu  65 70 75 80 Phe Tyr Asn Ser Asn Pro Ser Asp Thr Thr Asn Ser Ile Asn Pro Gln                  85 90 95 Phe Lys Val Thr Asn Thr Gly Ser Ser Ala Ile Asp Leu Ser Lys Leu             100 105 110 Thr Leu Arg Tyr Tyr Tyr Thr Val Asp Gly Gln Lys Asp Gln Thr Phe         115 120 125 Trp Cys Asp His Ala Ala Ile Gly Ser Asn Gly Ser Tyr Asn Gly     130 135 140 Ile Thr Ser Asn Val Lys Gly Thr Phe Val Lys Met Ser Ser Ser Thr 145 150 155 160 Asn Asn Ala Asp Thr Tyr Leu Glu Ile Ser Phe Thr Gly Gly Thr Leu                 165 170 175 Glu Pro Gly Ala His Val Gln Ile Gln Gly Arg Phe Ala Lys Asn Asp             180 185 190 Trp Ser Asn Tyr Thr Gln Ser Asn Asp Tyr Ser Phe Lys Ser Ala Ser         195 200 205 Gln Phe Val Glu Trp Asp Gln Val Thr Ala Tyr Leu Asn Gly Val Leu     210 215 220 Val Trp Gly Lys Glu Pro Gly Pro Arg Gly Gly Gly Gly Gly Ser Gly 225 230 235 240 Gly Gly Gly Ser Met His Ile Asn Leu Lys Val Lys Gly Gln Asp Gly                 245 250 255 Asn Glu Val Phe Phe Arg Ile Lys Arg Ser Thr Gln Leu Lys Lys Leu             260 265 270 Met Asn Ala Tyr Cys Asp Arg Gln Ser Val Asp Met Thr Ala Ile Ala         275 280 285 Phe Leu Phe Asp Gly Arg Arg Leu Arg Ala Glu Gln Thr Pro Asp Glu     290 295 300 Leu Glu Met Glu Asp Gly Asp Glu Ile Asp Ala Met Leu His Gln Thr 305 310 315 320 Gly Gly Met Val Pro Pro Gly Glu Asp Ser Lys Asp Val Ala Ala Pro                 325 330 335 His Arg Gln Pro Leu Thr Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg             340 345 350 Tyr Ile Leu Asp Gly Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys         355 360 365 Ser Asn Met Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu     370 375 380 Asn Leu Pro Lys Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe 385 390 395 400 Asn Glu Glu Thr Cys Leu Val Lys Ile Ile Thr Gly Leu Leu Glu Phe                 405 410 415 Glu Val Tyr Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu             420 425 430 Gln Ala Arg Ala Val Gln Met Ser Thr Lys Val Leu Ile Gln Phe Leu         435 440 445 Gln Lys Lys Ala Lys Asn Leu Asp Ala Ile Thr Thr Pro Asp Pro Thr     450 455 460 Thr Asn Ala Ser Leu Leu Thr Lys Leu Gln Ala Gln Asn Gln Trp Leu 465 470 475 480 Gln Asp Met Thr Thr His Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu                 485 490 495 Gln Ser Ser Leu Arg Ala Leu Arg Gln Met His Asp Glu Leu             500 505 510

Claims (11)

(a) Cellulose-binding module 3 (CBM3) or Cellulose-binding module 1 (sCBD); And
(b) SUMO (Small ubiquitin-related modifier)
A recombinant vector for the isolation and purification of the target protein. 2. The method of claim 1, wherein the cellulo-binding module 3 (CBM3) is a gene encoding the amino acid sequence of SEQ ID NO: 2, the sCBD (Cellulose-binding module 1) is a gene encoding the amino acid sequence of SEQ ID NO: (Small ubiquitin-related modifier) is a gene encoding the amino acid sequence of SEQ ID NO: 3. 2. The recombinant vector of claim 1, further comprising a mannosylated peptide region (Mp) in the recombinant vector. 4. The recombinant vector according to claim 3, wherein the Mp (mannosylated peptide region) is a gene encoding the amino acid sequence of SEQ ID NO: 8. The recombinant vector according to claim 1, wherein the recombinant vector further comprises any one or more selected from the group consisting of BiP (chaperone binding protein) and HDEL (His-Asp-Glu-Leu) peptides. The recombinant vector according to claim 1, wherein the recombinant vector is selected from the group consisting of 35S promoter derived from cauliflower mosaic virus, 19S RNA promoter derived from cauliflower mosaic virus, actin protein promoter and ubiquitin protein promoter &Lt; / RTI &gt; further comprising a promoter selected from the group consisting of: &lt; RTI ID = 0.0 &gt; 2. The recombinant vector according to claim 1, wherein the cleavage map of the recombination vector is represented by Fig. (a) preparing a recombinant vector according to claim 1;
(b) introducing the recombinant vector into a plant to produce a transgenic plant;
(c) culturing the transgenic plant to express the target protein;
(d) binding the expressed target protein to cellulosic beads; And
(e) separating and purifying only the target protein by treating bdSEBNP1 with the target protein bound to the cerulose beads;
/ RTI &gt; of the target protein. The method of claim 8, wherein the cellulose bead is selected from the group consisting of carboxyl methyl cellulose (CMC), methyl cellulose (MC), amorphous cellulose (AMC), hydroxy ethyl cellulose HEC), and hydroxypropyl methyl cellulose (HPMC). The method for separating and purifying a target protein according to claim 1, 9. The method according to claim 8, wherein said bdSEBNP1 is a gene encoding the amino acid sequence of SEQ ID NO: 4. 9. The method according to claim 8, wherein the bdSEBNP1 is expressed in Escherichia coli.

Images