KR101342974B1 - Novel peptide tag and uses thereof - Google Patents

Abstract

The present invention relates to a peptide tag derived from Bacteriophytochrome, BphP which is photoreceptor protein of Deinococcus radiodurans and antibodies which can specifically recognize the peptide tags or hybridoma cell lines which can produce the same, to polynucleotide which codes the peptide tag or a vector or a transformant including the same, and to fusion protein which includes the peptide tag or methods for manufacturing, detecting, and purifying the same. The novel peptide tag according to the present invention has a short length and completely removes non-specific responses in which conventional c-myc tags and FLAG tags have. Also, fusion protein expressed in a recombinant cell can be efficiently detected or purified if the novel peptide tag and the antibody for the same according to the present invention are used. Therefore the novel peptide tag and an epitope tagging system including the antibody for the same can be applied in various fields such as confirming locations of specific proteins inside cells, investigation of functions, detection, purification, protein-protein interaction, and the likes.

Description

New peptide tag and uses thereof

The present invention relates to novel peptide tags that can be used in epitope tagging systems for the detection or purification of proteins of interest, antibodies to them and their use, and more particularly to light receiving proteins of Deinococcus radiodurans. It relates to a peptide tag derived from phosphorus phytochrome (BphP) and an antibody capable of specifically recognizing the peptide tags, to a hybridoma cell line capable of producing the same. The present invention also relates to a polynucleotide encoding the peptide tag or a vector or transformant containing the same, a fusion protein comprising the peptide tag, and a method or kit for producing, detecting or purifying the fusion protein .

Useful proteins or polypeptides can be produced by synthesis or isolated from natural sources. However, such a method is disadvantageous in terms of cost, time, and production volume. Therefore, it is preferable to produce a target protein and a target polypeptide through culturing a transformant produced by recombination to over-express the target protein or the target polypeptide. However, the polypeptides produced in the cell environment (particularly short polypeptides) may be sensitive to degradation due to the action of the proteases present in the cells, and the antibodies to all the target proteins may not be present, It may not be easy to purify a target protein in which no antibody exists.

Protein tagging or epitope tagging has been used to overcome this problem. Protein tagging or epitope tagging is performed by preparing a recombinant nucleic acid molecule in which a coding sequence of an epitope tag is linked to a coding sequence of a target protein and expressing it in a suitable host cell and then using an antibody against an epitope tag , Which is a recombinant DNA method used for detecting, quantifying, purifying a target protein, locating a target protein in a cell, or identifying a function. There are a number of commercially available epitope tags and ligand antibodies thereon, and when selecting the appropriate epitope tag and antibody thereto, Western blot analysis, immunoprecipitation, immunofluorescence, The target protein can be detected or purified by immunocytochemistry, immunoaffinity purification, or the like, thereby eliminating the need for antibody production to the target protein.

Currently, epitope tags used for protein tagging or epitope tagging include proteins with as little as 6 amino acid residues (for example, 6xHis tag) to 40kDa (MBP) Many unique tags are available (see Stevens, RC, (2000) Structure Fold Des 8: R177-85) and typically a peptide tag consisting of 3 to 30 amino acids is used. His-tagged proteins are specifically trapped on Ni-NTA (nickel-nitrilotriacetic acid) resin, which can be eluted by EDTA or imidazole. In addition, maltose binding protein (MBP, 396 amino acids, 40 kDa), staphylococcus protein A, calmodulin-binding peptide (CBP, 26 amino acids, 2.96 kDa), GFP (238 amino acids, 27 kDa) And glutathione-S-transferase (GST, 211 amino acids, 26 kDa) can also be used for both prokaryotic and eukaryotic proteins. In general, the most frequently used epitope tags include c-myc tags, HA tags, and FLAG tags. The c-myc tag is an epitope tag of 10 amino acids in length derived from human c-myc protein (Evans et al., (1985) Mol. Cell. Biol., 12: 3610-3616), HA tagged influenza hemoglobin Is an epitope tag of nine amino acid lengths derived from the influenza hemagglutinin HA-1 protein (Field et al., (1988) Mol. Cell. Biol., 8: 2159-2165). The FLAG tag is an eight amino acid length epitope tag derived from bacteriophage T7 (Hopp et al., (1988) Bio / Technology, 6: 1204-1210).

On the other hand, the epitope tag used in epitope tagging preferably minimally affects the three-dimensional structure and biological activity of the target protein when fused with the target protein. Generally, a long epitope tag (for example, a GST tag or MBP tag) have problems such as changing the function of a target protein. On the other hand, relatively short epitope tags such as the FLAG tag and the c-myc tag have very little effect on the characteristics of the target protein fused with it, In some cases it is currently used primarily because it does not need to be removed from the fusion protein. However, since the amino acid sequence of the c-myc tag and the FLAG tag contains the same sequence in many proteins of known proteins in living cells, it induces a nonspecific reaction of the tag-recognizing antibody, and this non- (Ksenija Gasic et al., (2005) Plant molecular biology reporter 23: 9-16). In order to overcome this nonspecific reaction problem, an affinity purification system using two different tag fusions in succession to the N-terminus of the protein has been developed (Rigaut, G., et al. (1999) Nat Biotechnol 17: 1030 -2).

Therefore, it is possible to detect a novel peptide tag having a short amino acid sequence and a fusion protein expressed therein in a recombinant host cell at the same time while eliminating nonspecific reaction which is a problem in an epitope tagging system using a conventional epitope tag and an antibody therefor It is necessary to develop an antibody that can be used for purification and purification.

It is an object of the present invention to provide a novel peptide tag useful for the detection or purification of a target protein, its use, and the like.

Another object of the present invention is to provide a novel antibody capable of specifically recognizing a novel epitope tag or selectively binding to a novel epitope tag, and its use, and the like.

The inventors of the present invention conducted research to develop a peptide tag and an antibody therefor useful for the detection or purification of a target protein. The inventors of the present invention have found that a bacteriophycochrome, a photoreceptor protein of Deinococcus radiodurans, A hybridoma producing a monoclonal antibody is obtained by using Bacteriophytochrome (BphP) or a fragment thereof as an immunogen, and a Bacteriophytochrome (BphP) of Deinococcus radiodurans is administered to a subject To complete the present invention by performing an epitope mapping to find a peptide tag having 8 to 9 amino acids.

 The present invention provides a peptide tag comprising the amino acid sequence of SEQ ID NO: 2 to solve the above object.

The present invention also provides a polynucleotide encoding a peptide tag comprising the amino acid sequence of SEQ ID NO: 2.

The present invention also provides primer pairs for synthesizing polynucleotides encoding peptide tags comprising the amino acid sequence of SEQ ID NO: 2.

The present invention also provides a recombinant vector comprising a polynucleotide encoding the peptide tag.

The present invention also provides a fusion protein comprising a target protein and a peptide tag linked thereto and comprising the amino acid sequence of SEQ ID NO: 2.

The present invention also provides a polynucleotide encoding the fusion protein.

The present invention also provides a recombinant vector comprising a polynucleotide encoding the fusion protein.

The present invention also provides a polynucleotide encoding a peptide tag comprising the amino acid sequence of SEQ ID NO: 2, a recombinant vector comprising the polynucleotide encoding the peptide tag, a target protein and an amino acid sequence of SEQ ID NO: 2 linked thereto It provides a transformant, characterized in that any one selected from the group consisting of a polynucleotide encoding a fusion protein comprising a peptide tag and a recombinant vector comprising the polynucleotide encoding the fusion protein.

The present invention also provides a method for producing a fusion protein comprising culturing the transformant to express the fusion protein.

In addition, the present invention comprises the steps of binding a fusion protein comprising a target protein and a peptide tag linked thereto and comprising an amino acid sequence of SEQ ID NO: 2 in contact with an antibody against a peptide tag comprising the amino acid sequence of SEQ ID NO: 2; And it provides a method for detecting a fusion protein comprising the step of confirming the presence or analysis of the amount of the fusion protein bound to the antibody.

In addition, the present invention comprises the steps of binding a fusion protein comprising a target protein and a peptide tag linked thereto and comprising an amino acid sequence of SEQ ID NO: 2 in contact with an antibody against a peptide tag comprising the amino acid sequence of SEQ ID NO: 2; And it provides a method for purifying the fusion protein comprising the step of recovering the fusion protein bound to the antibody.

In addition, the present invention provides a polynucleotide encoding a peptide tag comprising an amino acid sequence of SEQ ID NO: 2, a primer pair for synthesizing a polynucleotide encoding a peptide tag comprising an amino acid sequence of SEQ ID NO: 2, coding the peptide tag A recombinant vector comprising a polynucleotide, a polynucleotide encoding a fusion protein comprising a target protein and a peptide tag linked thereto and comprising an amino acid sequence of SEQ ID NO: 2 and a recombinant vector comprising a polynucleotide encoding the fusion protein It provides a kit for fusion protein expression comprising any one selected from the group consisting of.

In addition, the present invention provides a polynucleotide encoding a peptide tag comprising an amino acid sequence of SEQ ID NO: 2, a primer pair for synthesizing a polynucleotide encoding a peptide tag comprising an amino acid sequence of SEQ ID NO: 2, coding the peptide tag A recombinant vector comprising a polynucleotide, a polynucleotide encoding a fusion protein comprising a target protein and a peptide tag linked thereto and comprising an amino acid sequence of SEQ ID NO: 2 and a recombinant vector comprising a polynucleotide encoding the fusion protein Any one selected from the group consisting of; And an antibody to a peptide tag comprising the amino acid sequence of SEQ ID NO: 2 or a hybrid protein detection or purification kit comprising any one selected from the hybridoma cell lines producing the antibody.

The novel peptide tag according to the present invention has the advantages of completely eliminating the nonspecific reaction of conventional c-myc tag and FLAG tag while having a short length. In addition, when the novel peptide tag and the antibody thereof are used, the fusion protein expressed in the recombinant cell can be detected or purified very efficiently. Therefore, the epitope tagging system including the novel peptide tag and the antibody according to the present invention can be applied to a variety of fields such as the intracellular localization, functional identification, detection, purification and protein interactions of a specific protein.

FIG. 1 shows the results of purifying DrBphP and DrBphN using His tag column chromatography. FIG. 1 (A) is a schematic diagram showing a gene construct used for expressing BphP protein and BphN protein of Deinococcus radiodurans. FL, full length (1-755 amino acids, BphP); ? PHY / HKD (1-321 amino acids, BphN); Figure 1 (B) shows the results of SDS-PAGE after purification of BphP apoprotein and BphN apoprotein of Deinococcus radiodurans using Ni ⁺ -NTA affinity chromatography and incubation with BV for 20 minutes , The result of detection of BV binding by zinc-induced fluorescence (right) and the result of staining with Coomassie Blue (left). 1: BphP crude extract, 2: purified BphP, 3: BphN crude extract, 4: purified BphN. Purification conditions - Binding & Washing: pH 8.0, 100 mM Tris, 200 mM NaCl, 10 mM imidazole / elution: pH 8.0, 100 mM Tris, 200 mM NaCl,
FIG. 2 is a graph showing the result of Western blot analysis of a monoclonal antibody selected using purified BphP and BphN. SDS-PAGE was performed on the purified BphP and BphN and western blotting was performed using the purified monoclonal antibodies (2B8, 2C11, 3B2, 3D2, 3H7) of Bphp. P; BphP, N; BphN. Western blot confirmed that the 2B8 antibody in the monoclonal antibody recognizes an epitope present at the N-terminus of the BphP protein.
FIG. 3 shows the result of confirming whether the PCD structure constituting the N-terminal region reacts with 5 kinds of monoclonal antibodies of BphP, Oat PhyA, which is an otopicochrome protein similar to DrBphP.
Figure 4 shows the results of the first-order epitope mapping for antibody 2B8 through Western blot, and Figure 5 shows the results of second epitope mapping for antibody 2B8 via Western blot. In Figs. 4 and 5, "a-GST" represents an anti-GST antibody.
Fig. 6 is a Western blot result showing that a fusion protein in which the BRT tag is attached to the N-terminus of the GST protein is normally expressed on E. coli and the fusion protein can be detected by the 2B8 antibody. 5, "a-myc" represents an anti-myc antibody and "a-GST" represents an anti-GST antibody.
FIG. 7 is a Western blot result showing that a fusion protein to which a BRT tag is attached to the N-terminus of GFP is normally expressed in plant cells and that a fusion protein can be detected by 2B8 antibody. 7, "Myc" represents an anti-myc antibody.

One aspect of the present invention relates to a novel peptide tag that can be used in epitope tagging for purification or detection of a target protein and the like. The novel peptide tag according to the present invention comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a peptide comprising the amino acid sequence of SEQ ID NO: 2. In the context of the present invention, peptide tags can be used interchangeably with protein tags or epitope tags. The term "peptide tag" in the present invention means a peptide that can be fused to a target protein and used as a tag. The term "epitope" in the present invention means an antigen binding site recognized by a specific antibody, or a certain site of an antigen that reacts with B cells or T cells. In the present invention, when the peptide tag includes the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, the length of the peptide tag is not particularly limited. For example, when considering the number of amino acid residues of a commonly used epitope tag, Preferably a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide consisting of the amino acid sequence of SEQ ID NO: 2. A peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide consisting of the amino acid sequence of SEQ ID NO: 2 is derived from Bacteriophytochrome (BphP), a light-receiving protein of Deinococcus radiodurans. Specifically, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 has the amino acid sequence of SEQ ID NO: 3 of Bacteriophytochrome (BphP), the light-receiving protein of Deinococcus radiodurans, And the amino acid residues corresponding to the 11th position from the 3 < rd > In addition, the peptide consisting of the amino acid sequence of SEQ ID NO: 2 has the amino acid sequence of SEQ ID NO: 3 of Bacteriophytochrome (BphP), the light-receiving protein of Deinococcus radiodurans, And a phenylalanine residue which is an amino acid residue corresponding to the eighth position or the ninth position in the amino acid residues corresponding to the 11th position from the third position.

The peptide tag consisting of the amino acid sequence of SEQ ID NO: 1 or the peptide tag consisting of the amino acid sequence of SEQ ID NO: 2 according to the present invention can be prepared by a conventional method using a bacteriophychrome of Deinococcus radiodurans as an immunogen, , And obtaining purified antibodies from the hybridoma cell line and then performing epitope mapping using the antibodies. Hereinafter, the bacteriophycomic of Deinococcus radiodurans is denoted as "DrBphP ". Deinococcus Radio Duchenne is an extreme microorganism that can grow in cryogenic, dry, hypoxic, and strong acid environments. Phytochrome is a photoreceptor protein capable of absorbing external light signals and transmitting a signal to the lower part. In the conventional theory, it is known that it exists only in higher plants. Recently, phytochrome, proteobacteria, actinobacteria, fungi and the like Several phytochrome-like photoreceptors have been found. Among them, the phytochrome-like photoreceptors found in Deinococcus radiodustans and Pseudomonas aeruginosa were named BphP, of which DrBphP was composed of 755 amino acids represented by the amino acid sequence of SEQ ID NO: 3 (Davis SJ, et al., (1999) Science 286, 2517-2520). BphP is composed of an N-terminal region structure similar to that of phytochrome, but the histidine kinase domain (HKD), which is different from phytochrome, exists at the C-terminal region [Karniol R, et al., (2005) Photosynth Res 392, 103-116; Giraud E et al., (2008) Photosynth Res 97, 141-153]. And DrBphP combines with the bilirubidin (BV) to form a reversible Pr / Pfr isoform without the aid of other elements [Vierstra RD et al., (2000) Semin Cell Dev Biol 11, 511-521 ; Bhoo SH, et al., (2001) Nature 414, 776-779; Rockwell NC, et al., (2006) Annu Rev Plant Biol 57, 837-858].

Hereinafter, the steps of epitope mapping for determining a novel peptide tag according to the present invention will be described in detail. First, a gene (SEQ ID NO: 4) corresponding to the entire BphP of Deinococcus radiodurans and a polypeptide consisting of amino acids 1 to 321 (hereinafter referred to as BphN) based on the N-terminal of DrBphP (SEQ ID NO: 5) was prepared (see Fig. 1 (A)), inserted into the pET28a (+) vector, and then expressed using BL21 competent cells. Protein purification was then performed with Ni ⁺ -NTA affinity chromatography (QIAGEN) using the His tag contained in the pET28a (+) vector. Since the expressed BphP protein (SEQ ID NO: 3) and BphN protein (SEQ ID NO: 6) contain both PAS and GAF domains and Cys-24 residues which play an important role in the binding of BV, This is possible. Therefore, in order to confirm the expression and purification of BphP and BphN proteins, BV was added to the purified BphP protein and BphN protein, and the mixture was incubated at room temperature for 10 minutes. SDS-PAGE was then performed, followed by coomassie staining and zinc blotting As a result, the expression and purification of BphP protein and BphN protein were confirmed (see FIG. 1 (B)). Next, in order to prepare a monoclonal antibody against BphP, BphP protein or BphN protein of Deinococcus radiodurans was mixed with Complete Freund's adjuvant as an antigen in peritoneal cavity of 6-week old female BALB / c mice After intraperitoneal injection, mice were sacrificed to obtain B lymphocytes. Then, B lymphocytes and myeloma cells were mixed and cultured by cell fusion. After that, HAT medium and HT medium were changed, antibody production of fusion cells was confirmed by ELISA, and a total of 24 hybridoma cell line rear clones were selected. A total of 24 candidate hybridoma cell clones were tested for Fusion plate ELISA with BphP protein and BphN protein injected as antigen (Table 3). As a result, the 2B8 clone and 2C11 clone were found to recognize the N-terminal region of the BphP protein because the BphP protein and the BphN protein of Deinococcus radiodurans were both highly expressed. However, 3B2 and 3D2 clones were found to recognize the C-terminal region of the BphP protein, indicating that the BphP protein has a high level and the BphN protein has a value close to zero. In contrast, the 3H7 clone exhibited a high level of BphP protein and a low level of BphN protein, suggesting that the N-terminal region of the BphP protein was also recognized to some extent. Then, the 5 clones of the hybridoma cell line were subjected to a primary cloning plate ELISA test and a secondary cloning plate ELISA test. Ultimately, ascites was purified to purify the antibody. As a result, 5 monoclonal antibodies, 2B8 antibody, 2C11 antibody, 3B2 antibody, 3D2 antibody and 3H7 antibody were obtained. Next, the purified antibodies were subjected to Western blotting to confirm whether the purified and purified BphP protein and BphN protein had the same results. Each 5 μg of purified BphP protein and BphN protein was used as a primary antibody at a ratio of 1: 1000 to 2% skim milk. HRP-mouse antibody (SIGMA) was added at a ratio of 1: 10000 And subjected to Western blotting. As a result, 2B8 antibody and 2C11 antibody resulted in binding to BphP protein and BphN protein as a result of ELISA test on hybridoma cell line clone (FIG. 2). Therefore, all of the two antibodies could be considered to bind to the N-terminal region of BphP. The 3B2 antibody and the 3D2 antibody showed a band for the BphP protein but no band for the BphN protein, indicating that the antibody binds to the C-terminal region of the BphP protein. However, in the case of the 3H7 antibody, only the BphP protein was recognized, unlike the results in the ELISA test. In addition, the degree of binding of each antibody was most strongly bound to 2B8 antibody and 2C11 antibody binding at the N-terminal site, and the 3D2 antibody thought to recognize the C-terminal site exhibited a weaker recognition band. Followed by 3H7 antibody and 3B2 antibody in that order. On the other hand, DrBphP has a structure similar to that of phytochrome. Especially, PCDs constituting N-terminal region have very similar structures including PAS, GAF and PHY domains. Therefore, And whether or not they responded. However, all five monoclonal antibodies against the BphP protein of Deinococcus radiodurans did not bind to the Ophtocrome protein (Oat PhyA) (FIG. 3). Therefore, it was confirmed that the monoclonal antibodies against the BphP protein of Deinococcus radiodurans are new BphP-specific antibodies recognizing epitopes different from those of the existing Oat phytochrome proteins. By isolating an epitope to be recognized, it has been found that it can be used as a specific peptide tag which is not contained in any protein of a known organism. Specifically, a DrBphP recombinant partial peptide was prepared to identify the epitope of 2B8 antibody most strongly bound to the BphP protein of Deinococcus radiodurans, and epitope mapping was performed Respectively. Specifically, the amino acid sequence of the 3-12 position, the amino acid sequence of the 3-12 position (SEQ ID NO: 1), the amino acid sequence of the position 3-10, 4- of the entire amino acid sequence (SEQ ID NO: 3) of DrBphP Peptide fragments each comprising an amino acid sequence at position 12 were prepared and confirmed for binding by Western blot. As a result, it was confirmed that the 2B8 antibody accurately recognizes and binds to 9 amino acids (SEQ ID NO: 1) corresponding to 3-11 positions based on the N-terminal of the entire amino acid sequence of DrBphP (SEQ ID NO: 3) . From these results, it was confirmed that the epitope of antibody 2B8 was a peptide having the amino acid sequence of SEQ ID NO: 1, and it could be used as a peptide tag. In addition, peptide fragments containing an amino acid sequence in which one amino acid residue of the amino acid sequence of SEQ ID NO: 1 was deleted were prepared, and epitope mapping was performed using 2B8 antibody. As a result, it was confirmed that another epitope of the antibody 2B8 was a peptide having the amino acid sequence of SEQ ID NO: 2.

In addition, the peptide tag of the present invention may be provided in a form combined with other known epitope tags to secure various functions in addition to the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2. For example, the peptide tag of the present invention may have HA tag, FLAG tag, His tag, BCCP (biotin) in the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 in order to improve the detection ability, detection ability, availability, etc. of the target protein. It may be provided in the form of a double tag in which a carboxyl carrier protein (MBP) or a maltose binding protein (MBP) is bound (see US Patent Publication No. 20050221308, US Publication No. 20060099710, US Patent Publication No. 6462254). In addition, the peptide tag of the present invention may be a triple tag in which two or more tags selected from the HA tag, the 6 × His tag, the c-myc tag and the V5 tag are attached to the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: Tag (see US-A-20100184612). In addition, the peptide tag of the present invention may be provided in a form in which the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 is repeated or a form in which some amino acid residues in the repeated amino acid sequence are substituted or deleted, (See U.S. Patent No. 7,135,624).

When the peptide tag according to the present invention includes the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, the corresponding antibody can be used. For example, as an antibody against a peptide tag comprising the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, there is the above-mentioned 2B8 antibody. Meanwhile, in the peptide tag of the present invention, some amino acids constituting the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 are individually substituted, deleted, added or modified, and at the same time, Such as variants that retain at least 75% or more based on the binding activity, and the like. Substitution of the amino acid is preferably accomplished by conservative amino acid replacement, in which the nature of the peptide is not altered. Further, the modification of the amino acid may be carried out by glycosylation, acetylation, phosphorylation, and the like. In addition, the peptide tag according to the present invention may contain a labeled amino acid. In addition, the peptide tag according to the present invention may include a peptide having increased structural stability against heat, pH, or the like due to mutation or modification of the amino acid sequence or increased activity against the antibody. Table 1 below shows amino acids that can replace amino acids in the peptide by conservative amino acid substitutions.

Amino acid residues in peptides Conservative substituent Ala Ser Arg Lys Asn Gln, His Asp Glu Gln Asn Cys Ser Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln Met Leu, Ile Phe Met, Leu, Tyr Ser Thr, Gly Thr Ser, Val Trp Tyr Tyr Trp, Phe Val Ile, Leu

Another aspect of the present invention relates to a polynucleotide encoding the novel peptide tag described above and a recombinant vector comprising the polynucleotide. The term "polynucleotide" as used herein means any non-modified or modified polyribonucleotide (RNA) or polydeoxyribonucleotide (DNA). The polynucleotides can be single- or double-stranded DNA, DNA which is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA which is a mixture of single- and double- Or a hybrid molecule comprising DNA and RNA which may be a mixture of single- and double-stranded regions. In addition, the polynucleotide of the present invention includes a primer used for synthesizing the above-mentioned novel peptide tag as single-stranded RNA or DNA. The polynucleotides of the present invention can also be used interchangeably with nucleic acid molecules or oligonucleotides.

Polynucleotides encoding the peptides may or may not contain untranslated sequences (e. G., Introns) (e. G., CDNA). The information on which the peptide is encoded is specified using codons. Typically, the amino acid sequence is encoded by a polynucleotide using a universal genetic code. "Codon" refers to a triplet of nucleotides that determine the amino acid sequence in a polypeptide chain. Most organisms use 20 or 21 amino acids to produce their polypeptides, which are protein or protein precursors. Because there are four possible nucleotides, adenine (A), guanine (G), cytosine (C) and thymine (T) in DNA, there are 64 possible triplets capable of coding 20 amino acids and terminal signals. Because of this redundancy, most amino acids are coded by one or more triplets. This allows a change in the nucleotide sequence without affecting the amino acid sequence of the encoded polypeptide, which is referred to as "silent variation" by "codon degeneracy ". All silent variations of the polynucleotides encoding the novel peptide tags of the present invention are within the scope of the present invention. On the other hand, codons that embody a single amino acid can not be used at the same frequency, and each organism often exhibits a particular " codon-bias "for one of several codons encoding the same given amino acid. If the coding region contains a large number of clusters of rare codons or rare codons, the level of expression can be increased by eliminating the rare codons using gene rearrangement or mutagenesis. [J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001), at 15.12]. Thus, "codon selection" can be used to optimize expression in a selected host. The most preferred codons are codons found primarily in highly expressed genes. In " codon preference "in E. coli, Konigsberg, et al., Proc. Nat'l. Acad. Sci. U.S.A. 80: 687-91 (1983).

When the nucleotide sequence is prepared by chemically synthesizing, it is possible to use a method well known in the art, for example, a method described in Engels and Uhlmann, Angew Chem IntEd Engl., 37: 73-127, 1988 , Triesters, phosphites, phosphoramidites and H-phosphate methods, PCR and other auto primer methods, and oligonucleotide synthesis on solid supports.

Thus, the polynucleotide encoding the novel peptide tag of the present invention can be composed of a variety of base sequences, for example a polynucleotide encoding a peptide tag consisting of the amino acid sequence of SEQ ID NO: 1 is preferably a base of SEQ ID NO: 7 And the polynucleotide encoding the peptide tag consisting of the amino acid sequence of SEQ ID NO: 2 may preferably be composed of the nucleotide sequence of SEQ ID NO:

The invention also provides a recombinant vector comprising a polynucleotide encoding a novel peptide tag. For example, the recombinant vector according to the present invention may comprise a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7 or a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8. The recombinant vector may be provided in the form of a polynucleotide encoding a novel peptide tag inserted into a cloning vector or an expression vector using known standard methods. And can be provided in the form of a recombinant vector. As used herein, the term "vector" refers to any medium for cloning and / or transfer of a base into a host cell. The vector may be a replica, in which other DNA fragments can bind to result in replication of the bound fragment. Refers to any genetic unit (e.g., a plasmid, phage, cosmid, chromosome, or virus) that functions in vivo as an autologous unit of DNA replication, that is, . The term "vector" includes viral and non-viral mediators for introducing a base into a host cell in vitro, in vitro or in vivo. The term "vector" may also include mini-spherical DNA. For example, the vector may be a plasmid without a bacterial DNA sequence. Removal of abundant bacterial DNA sequences in the CpG region has been done to reduce transgene expression silencing and to bring about a more sustained expression from the plasmid DNA vector. The term “vector” may also include transposons (Szping, et al. J. MoI. Biol. 302: 93-102 (2000)), such as Sleeping Beauty, or artificial chromosomes. The term "cloning vector" in the present invention is defined as a substance capable of carrying a DNA fragment into a host cell and regenerating it. In the present invention, the cloning vector may further include a polyadenylation signal, a transcription termination sequence, and a multiple cloning site. Wherein the multiple cloning site comprises at least one restriction site of an endonuclease restriction enzyme. In addition, the cloning vector may further include a promoter. For example, in the present invention, the polynucleotide encoding the novel peptide tag may be located upstream of a polyadenylation signal and a transcription termination sequence, and may comprise at least one endonuclease an endonuclease restriction site may be located upstream of a polyadenylation signal and a transcription termination sequence. In addition, the term "expression vector" in the present invention is defined as a DNA sequence necessary for transcription and translation of the cloned DNA in an appropriate host. In addition, the term "expression vector" in the present invention means a gene construct comprising an essential regulatory element operably linked to an insert such that the insert is expressed when present in the cell of the individual. The expression vector can be prepared and purified using standard recombinant DNA techniques. The expression vector is not particularly limited as long as it expresses a desired gene in various host cells of prokaryotic and eukaryotic cells and produces a desired protein. However, the expression vector has a promoter that exhibits strong activity, A vector capable of producing a large amount of exogenous protein in a form similar to that of the wild type. The expression vector preferably contains at least a promoter, an initiation codon, a gene encoding a desired protein, and a termination codon terminator. In addition, DNA encoding the signal peptide, additional expression control sequences, non-translation regions on the 5 'side and the 3' side of the desired gene, a selectable marker region, or a replicable unit may be suitably contained. A "promoter" means a minimal sequence sufficient to direct transcription. Also, a promoter construct sufficient to allow expression of a regulatable, promoter-dependent gene that is induced by a cell type-specific or external signal or agent may be included, and such constructs may be located at the 5 ' or 3 ' . Both conservative and inducible promoters are included. Promoter sequences may be derived from prokaryotes, eukaryotes or viruses. The term "operably linked" means that polynucleotide sequence associations on a single polynucleotide are modulated by one function. For example, if the promoter is capable of controlling the expression of the coding sequence (i. E., The coding sequence is under transcriptional control of the promoter), the promoter may be operatively linked to the coding sequence or the ribosomal binding site If present, the ribosomal binding site is operatively linked to the coding sequence. The coding sequence may be operatively linked to the regulatory sequence in the sense or antisense direction. The expression vector according to the present invention is preferably a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 7 as a polynucleotide encoding a novel peptide tag or a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 8 and a polynucleotide encoding a target protein .

Another aspect of the invention relates to a fusion protein comprising the novel peptide tag described above. The term "fusion protein" in the present invention means a polymer in which two or more peptides, oligopeptides, polypeptides or proteins having different functions are linked to each other. In the present invention, the fusion protein can be used interchangeably with a fusion polypeptide, a fusion oligopeptide, a recombinant polypeptide, a recombinant oligopeptide or a recombinant protein. The first portion of the fusion protein according to the invention comprises at least one of the novel peptide tags described above and the second portion of the fusion protein comprises at least one of the target proteins. In the present invention, a target protein can be used interchangeably with a target peptide, a target oligopeptide or a target polypeptide. The fusion protein in which the novel peptide tag according to the present invention is linked to the codon region of the target protein can be efficiently detected or purified using an antibody against the novel peptide tag. The peptide tag according to the present invention can be inserted anywhere within the protein, as long as it does not substantially affect the C-terminus of the target protein as well as the N-terminus or the functionality of the protein. Here, the fact that the protein does not substantially affect the functionality thereof means that the activity of the protein is retained by 80% or more, preferably 95% or more, before fusion of the peptide tag. As the target protein used in the present invention, any protein can be used. For example, a short-chain FV (ScFv) of humanized antibody AKA / HzK to TAG-72 which is a cancer-associated antigen, thrombopoietin (TPO), B - lymphocyte stimulator (B-lymphocyte stimulator), and the like. A fusion protein according to the present invention comprises a combination of two or more peptides, for example, a peptide tag and a combination of a target protein, wherein the two or more peptides may be linked by covalent bonds or noncovalent bonds. At this time, the peptide tag and the target protein may be directly connected to each other without any mediator, or may be indirectly connected by a mediator such as a peptide linker. In a preferred embodiment, the fusion protein comprises at least one cleavable peptide linker, which can then be designed such that the cleavable linker is chemically and / or enzymatically cleaved so that the desired protein can be recovered from the fusion protein. Thus, a fusion protein according to the invention can preferably be designed to include one or more tags, a cleavable peptide linker and a target protein. Linkers generally do not have specific biological activity other than to associate proteins or to maintain a minimum spacing or other spatial relationship between these proteins, but the constituent amino acids of the peptide linker are not affected by the properties of the molecule, such as folding, net charge, or hydrophobicity May be selected to have some effect. The peptide linker may optionally include a site for the separation of the fused constituent polypeptide, for example, a site that can be cleaved by a protease. The cleavable peptide linker may be from 1 to about 50 amino acids in length, preferably from 1 to about 20 amino acids. As an example of an enzyme-cleavable peptide linker, it may include an aspartic acid-proline dipeptide (D-P) moiety which may contain a caspase-3 truncation sequence and which is cleavable by an acid. A cleavable peptide linker can be incorporated into the fusion protein using a number of techniques well known in the art. In addition, a flexible peptide linker can be used, and peptide linkers having a GGSGGT amino acid sequence, a GGGGS amino acid sequence, and a GGGGSGGGGS amino acid sequence can be used, but are not limited thereto. A peptide linker can be operably linked in-frame between polynucleotides encoding each peptide of the fusion protein and the polynucleotide comprising the peptide linker and expressed through an expression vector.

Means for preparing peptides (peptide tags, cleavable peptide linkers, target peptides, and / or fusion peptides) in the present invention are well known in the art (see, for example, Stewart et al., Solid Phase Peptide Synthesis, Peone Synthesis Protocols, Humana Press, Totowa, NJ, 1994); and [Bodanszky, Principles of Peptide Synthesis, Springer-Verlag, New York, Reference). The various components of the fusion peptide described herein (tag peptide, target peptide, and truncable linker / truncation sequence) can be prepared by known chemical synthetic methods (see, for example, Hermanson, Greg T. , Bioconjugate Techniques, Academic Press, New York (1996)), chemical synthesis is often limited to peptides that are less than about 50 amino acids in length due to cost and / or impurities. The peptides described herein can preferably be prepared using standard recombinant DNA and molecular cloning techniques [Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And Silhavy, TJ, et al., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Cold Spring Harbor, NY (1984); And Ausubel, FM et. al., < / RTI > Short Protocols in Molecular Biology, 5th Ed. Current Protocols and John Wiley and Sons, Inc., NY, 2002)

Another aspect of the invention relates to a transformant comprising the above-described expression vector. In the present invention, the term "transformant" means a cell transformed by introduction of an expression vector having a polynucleotide encoding at least one target protein into a host cell. Methods for preparing the transformant by introducing the expression vector into a host cell include transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome mediated transfection liposemmediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, electroinjection, PEG, and the like A chemical treatment method, a method using a gene gun or the like, but the present invention is not limited thereto. When the transformant expressing the expression vector is cultured in a nutrient medium, a large amount of the fusion protein can be produced, and the fusion protein can be isolated. The culture medium and culture conditions can be appropriately selected and used depending on the host cell. The conditions such as temperature, medium pH and incubation time should be appropriately adjusted so as to be suitable for cell growth and mass production of the protein during culturing. The host cell that can be transformed with the expression vector according to the present invention is not limited in its kind as long as it is known in the art such as prokaryotic cells, plant cells, insect cells, animal cells, etc. Preferably, A host having high expression efficiency of introduced DNA is usually used. For example, as host cells, well-known prokaryotic hosts such as Escherichia, Pseudomonas, Bacillus, Streptomyces can be used, and Escherichia coli can be preferably used. Expression of the protein by the host cell can be induced using IPTG (isopropyl-1-thio-β-D-galactopyranoside), and the induction time can be controlled to maximize the amount of protein. In the present invention, the recombinantly produced protein can be recovered from the medium or cell lysate. If membrane bound, it can be liberated from the membrane by using a suitable surfactant solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells used for protein expression can be disrupted by various physical or chemical means such as freeze-thaw cycles, sonication, mechanical disruption or cell disruption, and can be isolated or purified by conventional biochemical separation techniques (Sambrook Inc., San Diego, Calif., USA), < RTI ID = 0.0 > Molecular Cloning: A laborarory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989. Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 1990). For example, a method of separating or purifying a protein expressed by a host cell includes electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion-exchange chromatography, affinity chromatography, immuno- adsorption affinity chromatography, , Gel permeation HPLC), isoelectric focusing, and various variations or combinations thereof. Meanwhile, the fusion protein comprising the peptide tag according to the present invention can be separated and purified using immunoadsorption affinity chromatography using an antibody against the peptide tag.

Another aspect of the present invention relates to an antibody against the novel peptide tag described above and a hybridoma cell line capable of producing the antibody. An antibody according to the present invention is an antibody that specifically recognizes or specifically or selectively binds to a peptide tag consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide tag consisting of the amino acid sequence of SEQ ID NO: 2. The term "antibody" in the present invention refers to a substance produced by stimulation of a specific antigen in the immune system, which can specifically bind to the specific antigen to cause an antigen-antibody reaction, , And functional fragments of antibody molecules having an activity equivalent to that of the native form. The natural type antibody generally has a structure having two light chains and two heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. A functional fragment of an antibody molecule refers to a fragment having an antigen binding function and includes Fab, F (ab ') 2, F (ab') 2, Fv and the like. Fabs in the antibody fragment have one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. F (ab ') 2 is produced by disulfide bonding of cysteine residues in the hinge region of Fab'. Recombinant techniques for generating Fv fragments with minimal antibody fragments having only a heavy chain variable region and a light chain variable region are disclosed in WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88 / 09344. The double-stranded Fv (dsFv) is linked to a light chain variable region by a disulfide bond and a light chain variable region. A short chain Fv (scFv) is generally linked to a variable region of a heavy chain and a variable region of a light chain through a peptide linker by a covalent bond . Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, an F (ab ') 2 fragment can be obtained by cutting a whole antibody into papain and obtaining a Fab and digesting with pepsin) Can be produced through recombinant DNA technology. For the purpose of the present invention, the antibody may be an antibody capable of specifically binding to the peptide tag of the present invention, preferably in the form of a Fab or whole antibody, more preferably in the form of a monoclonal antibody . The present invention also provides a hybridoma cell line capable of producing a monoclonal antibody to the above-mentioned novel peptide tag. The hybridoma cell line was deposited with KCTC (Korea Research Institute of Bioscience and Biotechnology) on September 24, 2012, and the deposit number is KCTC 12283BP.

The monoclonal antibody having specific binding to the peptide tag of the present invention can be prepared by using the peptide tag or the polypeptide comprising the peptide tag as an immunogen. More specifically, the peptide tag is first injected into the subcutaneous, muscular, intravenous, or abdominal cavity of a mammal other than a human by one or more injections as an immunogen. At this time, the peptide tag is used in a mixed form with a Freund adjutant as necessary. The mammals other than humans are preferably selected from the group consisting of mice, rats, hamsters, marmots, rabbits, cats, dogs, pigs, goats, sheep, donkeys, horses or cattle (transgenic mice producing human antibodies) A transgenic animal engineered to produce an antibody derived from another animal, and more preferably a mouse, rat, hamster, guinea pig or rabbit. Immunization may be performed one to four times every about 1 to 21 days from the first immunization to obtain cells that produce antibodies from immunosensitive mammals about 1 to 10 days after the final immunization. The number of times of immunization and the time interval may be appropriately changed depending on the characteristics of the immunogen used. Hybridomas secreting monoclonal antibodies can be prepared according to the methods of Keira and Mirtstein et al. (Nature, 1975, Vol. 256, p. 495-497) and the equivalent method. The mammal is preferably selected from the group consisting of spleen, lymph node, bone marrow, or tonsil collected from an animal other than the above-mentioned immunosensitive human, preferably a mammalian animal that does not have an ability to produce an antibody, Hybridoma cells can be prepared by cell fusion of myeloma cells of the present invention. The myeloma cells used for the cell fusion include mouse-derived myeloma P3 / X63-AG8. 653 (653), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63-Ag8. U1 (P3U1), SP2 / 0-Ag14 (Sp2 / O, Sp2), PAI, F0 or BW5147; Rat-derived myeloma 210 RCY3-Ag. 2.3; Or human derived myeloma U-266 AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15. The cell fusion is carried out, for example, by a fusion promoter such as polyethylene glycol or Sendai virus or a method using an electric pulse. For example, in a fusion medium containing a fusion promoter, an antibody-producing cell and a mammalian- Is suspended at a ratio of about 1: 1 to 1:10, and in this state, it is cultured at about 30 to 40 DEG C for about 1 to 5 minutes. As the fusion medium, for example, MEM medium, RPMI1640 medium, and Iscove's modified Dulbecco's medium may be used, and serum components such as bovine serum are preferably excluded. A method for screening a hybridoma cell clone producing a monoclonal antibody is characterized in that the obtained fusion cell is transferred to a selection medium such as HAT medium and cultured at about 30 to 40 DEG C for about 3 to 3 weeks to prepare a hybridoma cell Of cells. Subsequently, the hybridoma cells are cultured on a microtiter plate or the like. Then, the reactivity between the immunogen and the culture supernatant used for the immunological reaction of the animals other than human, described above, is measured by radioactive substance-marked immuno-antibody (RIA) It is confirmed through immunoassay such as ELISA (Enzyme-Linked Immunosorbent Assay), and hybridoma cells with increased reactivity are searched. Hybridoma cell clones producing the monoclonal antibodies found above exhibit specific binding ability to the immunogen. The monoclonal antibody of the present invention can be obtained by culturing a hybridoma cell line in vitro or in vivo. For culturing, a conventional method for culturing cells derived from mammals is used, and in order to collect a monoclonal antibody from a culture or the like, an ordinary method in this field for purifying an antibody in general is used. Methods for purifying antibodies include, for example, salting out, dialysis, filtration, concentration, centrifugation, fractional precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography, Gel electrophoresis and isoelectric point electrophoresis, and they are applied in combination as needed. The purified monoclonal antibody is then concentrated, dried, and stored in a liquid or solid phase depending on the application. In addition, the monoclonal antibody of the present invention can be obtained by amplifying the DNA encoding the heavy chain and the light chain variable region with known DNA encoding the normal region of the heavy chain and the light chain (for example, Japanese Patent Application Laid-Open No. 2007-252372) The genes thus ligated are synthesized by PCR or chemical synthesis, and the synthesized gene is introduced into a known expression vector (for example, pcDNA 3.1 (marketed by Invitrogen) or the like, and the transformant is prepared using the expression vector , A host cell such as transformed CHO cells or Escherichia coli can be cultured and expressed. From such a culture solution, purified antibody can be obtained by using Protein A column or the like.

Yet another aspect of the present invention relates to the novel peptide tag described above, the polynucleotide encoding the peptide tag, or the use of the antibody for the peptide tag.

For example, one example of the present invention provides a method for producing the aforementioned fusion protein. A method for producing a fusion protein according to an embodiment of the present invention includes culturing a transformant into which a polynucleotide encoding a fusion protein has been introduced to express a fusion protein. At this time, the polynucleotide encoding the fusion protein is preferably introduced into the transformant in a form contained in the recombinant vector. In addition, the method for producing a fusion protein according to the present invention may further comprise the step of recovering the expressed fusion protein. In addition, the recombinant vector includes a polynucleotide encoding a peptide tag and a polynucleotide encoding a target protein linked thereto, wherein the two polynucleotides preferably include a site that can be cleaved by a protease or the like Lt; RTI ID = 0.0 > linker. ≪ / RTI >

In addition, one example of the present invention provides a method for detecting the aforementioned fusion protein. A method of detecting a fusion protein according to an exemplary embodiment of the present invention includes: binding a fusion protein to which a peptide tag and a target protein are linked by contacting the antibody against the peptide tag; And identifying or quantitating the presence of the fusion protein bound to the antibody. Herein, the method of detecting a fusion protein according to an exemplary embodiment of the present invention may be performed by Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunofluorescence, immunocytochemistry, An array or the like can be used. Wherein the protein microarray is immobilized with a specific antibody to the peptide tag of the present invention on a glass or silicon substrate and can be used for the analysis of large samples.

In addition, one example of the present invention provides a method for purifying the fusion protein described above. A method for purifying a fusion protein according to an exemplary embodiment of the present invention includes: binding a fusion protein to which a peptide tag and a target protein are linked by contacting the antibody against the peptide tag; And recovering the fusion protein bound to the antibody. At this time, the antibody is immobilized on a support, preferably a solid support. Examples of the support for immobilizing the antibody include a column, a bead, an adsorbent, a nitrocellulose paper and the like, and the antibody can be immobilized on the support through various known immobilization methods. A specific example of a method for purifying a fusion protein according to the present invention includes contacting a sample containing a tagged protein with a support on which an antibody is immobilized, wherein the tagged protein is a protein covalently linked to a peptide tag , Wherein the peptide tag has a specific binding activity to the antibody and the contacting is carried out under conditions that the antibody binds to the peptide tag and comprises removing the unbound components and separating the tagged protein from the support . At this time, the supporter and the antibody immobilized thereto can be used as a medium for immunoaffinity chromatography, and the immunoaffinity column chromatography can be carried out using a column packed with the medium. By the purification method of the present invention, a fused protein purified with high purity can be obtained, and further, the function of the target protein is not affected at all.

In addition, one example of the present invention provides a kit for expression, detection, or purification of a fusion protein. The kit for expression, detection or purification of the fusion protein according to the present invention comprises the above-mentioned novel peptide tag, a polynucleotide encoding the peptide tag, a primer pair for synthesizing the polynucleotide, a recombinant vector containing the polynucleotide, Or a transformant transformed with a nucleotide or a recombinant vector. In addition, the kit for expression, detection or purification of the fusion protein according to the present invention may include any one selected from the above-mentioned antibody against the novel peptide tag or hybridoma cell line producing the antibody. In addition, the kit for expressing the fusion protein according to the present invention preferably comprises a recombinant vector comprising a polynucleotide encoding the novel peptide tag of the present invention, and the kit for detecting or purifying the fusion protein according to the present invention is preferably Lt; RTI ID = 0.0 > of the < / RTI > novel peptide of the present invention. Here, the recombinant vector constituting the kit is preferably provided in such a form that the user can produce an expression vector containing the polynucleotide encoding the fusion protein. The fusion protein is a polypeptide to which a peptide tag and a target protein are linked. In addition, the antibody constituting the kit is preferably provided in a fixed form on a suitable support. In addition, the kit for detecting a fusion protein preferably further comprises means for detecting an antibody against a peptide tag. Here, the means capable of detecting the antibody of the present invention is preferably a secondary antibody. In addition, the antibody or secondary antibody of the present invention constituting the kit for detecting a fusion protein may be labeled with a labeling substance such as enzyme, radioactive substance, fluorescent substance, etc., and the labeling method is preferably conjugation . In addition, the fusion protein purification kit may further comprise a protease for separation of the peptide tag. In addition, the kit according to the present invention may further include instructions, restriction enzymes, ligases, buffer solutions and the like.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to clearly illustrate the present invention, and do not limit the scope of protection of the present invention.

Example  One: Deinococus Of the radioactive deurans (Deinococcus radiodurans) BphP  Gene and BphN  Gene Cloning

(SEQ ID NO: 4) coding for the entire length of Deanococcus radiodurans bacteriophytechrome monophrotein (BphP) and a polypeptide (BphN) consisting of amino acids 1 to 321 based on the N-terminus of DrBphP The coding gene (SEQ ID NO: 5) was cloned using PCR. Specifically, a primer for cloning the BphP gene or a primer for cloning the BphN gene is used as a template for a gene encoding the full length of Deinococcus radiodurans bacteriophytochrome protein (BphP) (SEQ ID NO: 4) PCR was performed with Ex-Taq (TAKARA) polymerase for about 30 cycles to obtain amplified BphP DNA product and BphN DNA product. Table 2 below shows the primers used to obtain amplified BphP DNA products and BphN DNA products. The primers shown in Table 2 and all the primers described below were manufactured by Bioneer co., KR.

SEQ ID NO: Type of primer The base sequence (5 '- > 3') The restriction enzyme recognition site contained in the primer 9 Forward primer for BphP cloning GCCATATGATGAGCCGGGACCCGTTGCCC Nde I 10 Reverse primer for BphP cloning GCCTCGAGTCAGGCATCGGCGGCTCCCGG Xho I 11 Forward primer for BphN cloning GCCATATGATGAGCCGGGACCCGTTGCCC Nde I 12 Reverse primer for BphN cloning GCCTCGAGCGCTTCCTTGACCTGAACTTG Xho I

The amplified PCR product was run on 1% agarose gel (QA-Agarose gel, Q-BIO, CAT # AGAH0250) and the size of amplified DNA was confirmed by ultraviolet irradiation. After removing only the desired DNA from the amplified products, the desired DNA was isolated using gel elution kit (FavorPrep GEL / PCR purification kit, FAVORGEN, CAT # FAGCK001-1) , And the nucleotide sequence was determined by commissioning with Bioneer co., KR. As a result, it was confirmed that the DNA product generated through PCR had BphP DNA and BphN DNA as desired target sequences.

Then, BphP DNA product and BphN DNA product amplified using restriction enzymes Nde I and Xho I were inserted into pET28a (+) vector (manufacturer: Novagen), and BL21 competent cells (manufactured by RBC, Taipei, Taiwan) was transformed by heat shock at 42 ° C for 1 minute and then cultured on LB medium containing kanamycin to express BphP protein and BphN protein.

Example  2 : Deinococus Of the radioactive deurans (Deinococcus radiodurans) BphP  Protein and BphN  Expression of protein

Colony colonies transformed into BL21 competent cells and grown on LB medium were inoculated on LB medium containing kanamycin and seed culture was carried out at 37 ° C for about 8 hours or more. The seeded cells were inoculated on LB medium containing kanamycin, grown to a concentration of 0.6 at OD ₆₀₀ , IPTG was added to give a total concentration of 0.5 mM, and protein expression was measured at 25 ° C for 6 hours . The cells were then centrifuged to discard the supernatant and the pellet resuspended in binding buffer (100 mM Tris-Cl, 150 mM NaCl, and 10 mM imidazole, pH 8.0). Next, the pellet resuspension was sonicated, the cells were disrupted, and the supernatant was separated by centrifugation again. The protein was purified using a Ni ⁺ -NTA column (QIAGEN). The purified protein was then stored at -70 < 0 > C.

The BphP protein and the BphN protein are capable of binding to BV alone, as they contain both the PAS, the GAF domain, and the Cys-24 residue, which play an important role in BV binding. Virilverdin (BV, 2 μg / ml, Frontier, Scientific, Carnforth, UK) was added to purified Apo-BphP protein and Apo-BphN protein to obtain holo-BphP protein and reacted for 10 minutes at room temperature. Subsequently, the reaction samples were subjected to SDS-PAGE, followed by reaction with zinc acetate solution (20 mM zinc acetate, 150 mM Tris-Cl, pH 7.0) for 20 minutes. In addition, the reaction samples were subjected to SDS-PAGE followed by coomassie staining. The left side of FIG. 1 (B) shows the result of coomassie staining, and the right side of FIG. 1 (B) shows the result of the zinc blot. As shown in FIG. 1 (B), it was confirmed that the expression and purification of BphP protein and BphN protein were smooth.

Example  3: Deinococus Of the radioactive deurans (Deinococcus radiodurans) BphP  Protein and BphN  Preparation of monoclonal antibodies specifically binding to proteins

3-1: Immunization

The purified BphP protein or BphN protein was used as an antigen, and complete Freund's adjuvant was mixed therein, and injected into the abdominal cavity of female BALB / c mice at 6 weeks of age. Immersion was mixed with vortexer for 1 hour and then injected intraperitoneally with Incomplete Freund's adjuvant and antigen in the same manner. After 2 weeks, blood was drawn from the tail of the mouse and used for cell fusion when the antibody titer after 1/1000 to 1.0 was measured after the ELISA test. At 4 weeks after the second immunization for cell fusion, the same amount of antigen was diluted in PBS and injected intraperitoneally. Three days later, mice were sacrificed and cell fusion was performed.

3-2: Cell fusion for monoclonal antibody production

As a B lymphocyte cell source, the abdominal incision was made to remove the fat and other organs as much as possible, and the spleen was aseptically removed to prevent excessive bleeding. The cells were homogenized with a cell strainer, diluted with the basic medium, and centrifuged twice. B lymphocytes and myeloma cells were mixed at an appropriate ratio, centrifuged for 5 minutes, the supernatant was discarded, and the remaining cell mixture was gently shaken. 10 ml of prewarmed RBC lysis buffer was added and suspended, followed by incubation at 37 ° C for 20 minutes FBS was added and centrifuged. Cell fusion was performed as follows. First, PEG (polyethlyene glycerol) 1500 solution was slowly added to the centrifuged cell mixture while maintaining the temperature at 37 ° C, and then the reaction was stopped by adding the basic medium again. Then, the cells were quickly centrifuged, the supernatant was discarded, and the remaining cell mixture was carefully suspended in a macrophage culture medium, and the cells were placed in a 96-well tissue culture plate and cultured in a carbon dioxide incubator at 37 ° C.

3-3: Selection of fusion cell

Five days after the cell fusion, HAT medium, a selective medium for fused cells, was added to each well. Then, the HAT medium was added at the intervals of 3 days in the same manner. After 11 days of cell fusion, HT medium supplemented with HT supplement was added to the complete medium and observed with a microscope every day for confirming the growth of the fusion cells. The supernatant of the wells in which the growth has been confirmed is taken and the antibody production is confirmed by ELISA. The fusion cell line once determined to secrete the desired antibody is subjected to 1, 2 and 3 cloning by limiting dilution, ≪ / RTI >

3-4: Selection of fusion cells by ELISA

Immunostimulation of immunity and monoclonal antibody production were carried out at 4 ° C in a 96 well tissue culture plate by adding antigen protein solution diluted with ELISA coating buffer solution. Subsequently, the antigen protein solution was inhaled and removed, and the blocking solution was added to the remaining surface of each well, followed by reaction at 37 ° C for 1 hour. The blocking solution was then removed and washed three times with PBST solution. The fusion cell culture supernatant was added to each well, reacted at 37 ° C, and the solution was removed and washed three times with PBST solution. A solution of Horseradish peroxidase (HRP) conjugated anti-mouse immunoglobulin (anti-mouse IgG) diluted in PBS was added to each well. OPD (orthophenylenediamine dihydrochloride) substrate solution was added to the wells, reacted at room temperature for 10 minutes, and the reaction was stopped with stop solution. At this time, the absorbance was measured with an ELISA reader at 492 nm in order to confirm the degree of the reaction, and a total of 24 candidate hybridoma cell clones were selected based on the measured results.

3-5: Fusion plate ELISA test to confirm the binding with the antigen and purification of the antibody

A total of 24 candidate hybridoma cell clones were tested by Fusion plate ELISA with BphP protein and BphN protein injected as antigen, and the degree of binding was confirmed. The results are shown in Table 3 below. As shown in the following Table 3, 2B8 clones and 2C11 clones were found to be highly expressed in both BphP protein and BphN protein of Deinococcus radiodurans, and it was presumed to recognize the N-terminal region of BphP protein . However, 3B2 and 3D2 clones were found to recognize the C-terminal region of the BphP protein, indicating that the BphP protein has a high level and the BphN protein has a value close to zero. In contrast, the 3H7 clone exhibited a high level of BphP protein and a low level of BphN protein, suggesting that the N-terminal region of the BphP protein was also recognized to some extent.

Hybridoma cell line clone The degree of binding with the antigen protein (absorbance at 492 nm) BphN BphP (full) 1B6 0.0660 1.3190 1B11 0.1560 2.3700 1D6 0.0550 2.3180 2B8 2.3400 2.4200 2 C11 2.3480 2.4040 2D9 0.1430 2.2730 2E3 2.1720 1.8020 2F7 0.1220 2.4430 2H8 0.0650 1.3440 3A4 1.9180 2.3770 3B2 0.0980 2.2900 3B3 0.0700 2.1650 3C11 1.9730 2.2590 3 D2 0.0640 2.2830 3D10 0.1180 2.4280 3D11 1.6850 2.3130 3F10 0.0730 2.3570 3G2 0.0880 2.0510 3G9 0.0800 1.5580 3H1 1.7840 2.0950 3 H7 0.4640 2.3490 4B7 1.9730 2.2620 4E2 0.2050 2.3770 4E3 0.0550 2.2650

Then, the 5 clones of the hybridoma cell line were subjected to a primary cloning plate ELISA test and a secondary cloning plate ELISA test. Ultimately, ascites was purified to purify the antibody. As a result, 5 monoclonal antibodies, 2B8 antibody, 2C11 antibody, 3B2 antibody, 3D2 antibody and 3H7 antibody were obtained. Purification of the antibody was carried out in the following manner. After 0.5 ml of pristane was injected intraperitoneally into BALB / c mice over 8 weeks, hybridoma cells were cultured after 1 to 10 days, washed three times with PBS, and then washed with PBS (5 × 10 ⁶ cells / And injected into the abdominal cavity using a syringe. After 1 to 10 days of ascites, the mice were sacrificed by cervical dislocations and a small hole was made in the abdomen. The ascites was collected using a serum separation tube while avoiding the flow of ascites. The mixture was allowed to stand at room temperature for about 1 hour and centrifuged to remove RBC. An appropriate amount of PBS was added to the collected samples, and the mixture was slowly mixed with ammonium sulfate at 4 ° C. After centrifugation at 4 ° C, the solution was passed through a filter. An appropriate amount of Protein A and DFMF mixed beads was added, and then Tris buffer solution was washed with 10 times of the bed volume. After adding the antibody and mixing well with the protein A / G, the fraction was collected, washed with Tris buffer, and then eluted several times with glycine. The purified antibody was dialyzed in PBS buffer for 3 hours, replaced with PBS buffer overnight, dialyzed overnight, and dialyzed.

Example  4: Specificity analysis of monoclonal antibodies

Western blotting was performed to confirm whether the obtained antibodies exhibited the same results for the pre-expressed and purified BphP protein and BphN protein. Each of the monoclonal antibodies having a concentration of 1 mg / ml as the primary antibody was used at a ratio of 1: 1000 to 2% skim milk, and HRP-mouse antibody ( SIGMA) at a ratio of 1: 10000 to perform Western blotting. As a result, 2B8 antibody and 2C11 antibody resulted in binding to BphP protein and BphN protein as a result of ELISA test on hybridoma cell line clone (FIG. 2). Therefore, all of the two antibodies could be considered to bind to the N-terminal region of BphP. The 3B2 antibody and the 3D2 antibody showed a band for the BphP protein but no band for the BphN protein, indicating that the antibody binds to the C-terminal region of the BphP protein. However, in the case of the 3H7 antibody, only the BphP protein was recognized, unlike the results in the ELISA test. In addition, the degree of binding of each antibody was most strongly bound to 2B8 antibody and 2C11 antibody binding at the N-terminal site, and the 3D2 antibody thought to recognize the C-terminal site exhibited a weaker recognition band. Followed by 3H7 antibody and 3B2 antibody in that order.

On the other hand, DrBphP has a structure similar to that of phytochrome. Especially, PCDs constituting N-terminal region have very similar structures including PAS, GAF and PHY domains. Therefore, And whether or not they responded. However, all five monoclonal antibodies against the BphP protein of Deinococcus radiodurans did not bind to the Ophtocrome protein (Oat PhyA) (FIG. 3). Therefore, it was confirmed that monoclonal antibodies against the BphP protein of Deinococcus radiodurans are new BphP-specific antibodies recognizing epitopes different from the antibodies against the existing Oat phytochrome protein.

The present inventors have developed a hybridoma cell line 2B8 of the 2B8 antibody most strongly bound to the BphP protein of Deinococcus radiodurans on September 24, 2012 by KCTC ), And the accession number of hybridoma cell line 2B8 is KCTC 12283BP. The isotype of 2B8 antibody was determined by ELISA using a mouse antibody. The heavy chain isotype of the 2B8 antibody was IgG 2a and the light chain isotype was kappa.

Example  5: 2B8 monoclonal antibody Epitope  For confirmation Epitope Mapping

5-1: Primary epitope mapping

A recombinant partial peptide of DrBphP was constructed and epitope mapping was performed to identify the epitope of 2B8 antibody which is the most strongly bound monoclonal antibody against BphP protein of Deinococcus radiodurans.

First, a DNA encoding an amino acid sequence at 3-12 positions (SEQ ID NO: 13), an amino acid sequence at 3-11 positions (SEQ ID NO: 1) based on N-terminal of the entire amino acid sequence of DrBphP (SEQ ID NO: A DNA encoding the amino acid sequence at positions 3-10, and a DNA encoding the amino acid sequence at positions 4-12 were prepared. PCR was performed using primers and PrimeSTAR HS (TAKARA, R040) polymerase that were prepared for about 30 cycles to obtain amplified DNA products. The PCR reaction for DNA cloning does not require a separate template, and in one cycle of PCR, a template for amplification can be obtained by combining a forward primer and a reverse primer. Table 4 below shows the primers used in PCR to obtain DNA amplification products encoding the four DrBphP recombinant partial peptides.

SEQ ID NO: Type of primer The base sequence (5 '- > 3') The restriction enzyme recognition site contained in the primer 14 BphP 3-12 a.a. Forward primer for cloning GC CTCGAG GGATCC ATG CGGGACCCGTTGCCCTTTTTT BamH I 15 BphP 3-12 a.a. Reverse primer for cloning GC GAGCTC GAATTC TCA AAGCGGTGGAAAAAAGGGCAA EcoR I 16 BphP 3-11 a.a. Forward primer for cloning GC CTCGAG GGATCC ATG CGGGACCCGTTGCCCTTTTTT BamH I 17 BphP 3-11 a.a. Reverse primer for cloning GC GAGCTC GAATTC TCA CGGTGGAAAAAAGGGCAACGG EcoR I 18 BphP 3-10 a.a. Forward primer for cloning GC CTCGAG GGATCC ATG CGGGACCCGTTGCCCTTTTTT BamH I 19 BphP 3-10 a.a. Reverse primer for cloning GC GAGCTC GAATTC TCA TGGAAAAAAGGGCAACGGGTC EcoR I 20 BphP 4-12 a.a. Forward primer for cloning GC CTCGAG GGATCC ATG GACCCGTTGCCCTTTTTTCCA BamH I 21 BphP 4-12 a.a. Reverse primer for cloning GC GAGCTC GAATTC TCA AAGCGGTGGAAAAAAGGGCAA EcoR I

The amplified PCR product was run on 1% agarose gel (QA-Agarose gel, Q-BIO, CAT # AGAH0250) and the size of amplified DNA was confirmed by ultraviolet irradiation. Then, only the DNA band corresponding to the DNA amplification product was cut out, and only the desired DNA was isolated using a gel elution kit (FavorPrep GEL / PCR purification kit, FAVORGEN, CAT # FAGCK001-1). Then, pJET (CloneJET PCR cloning kit, Fermentas, # K1232) vector. DH5? Competent cells (manufactured by Invitrogen), about 30 to 50 占 퐇 of Escherichia coli, were added to a binding vector of about 10 to 20 占 퐇, incubated on ice for 20 minutes and then heat shocked for 1 minute at 42 占 폚 shock, and then resuspended on ice for 20 minutes, spread on an LB plate containing ampicillin, and incubated overnight at 37 ° C. for transformation. Then, when the colonies grew, the cells were inoculated on LB medium containing ampicillin and cultured overnight at 37 ° C. Then, plasmids were grown using the FavorPrep plasmid extraction kit (FAVORGEN, cat # FAPDE300) And extracted. Then, the extracted plasmid was consigned to Bioneer co., KR and the nucleotide sequence thereof was measured. As a result, it was confirmed that the DNA product generated through PCR had a desired base sequence.

After cleavage of the plasmid and pGEX4T1 vector (GE Healthcare, Piscataway, NJ, USA) using restriction enzymes BamH I and EcoR I, the N-terminus of the entire amino acid sequence (SEQ ID NO: 3) of the target DNA DrBphP DNA encoding the amino acid sequence at position 3-12 (SEQ ID NO: 13), DNA encoding the amino acid sequence at position 3-11 (SEQ ID NO: 1), DNA encoding amino acid sequence at position 3-10 The DNA encoding the amino acid sequence at position -12 was ligated to the pGEX4T1 vector, which is a protein expression vector, respectively. Then, BL21 competent cells (manufactured by RBC, Taipei, Taiwan), which is Escherichia coli, And then cultured on LB medium containing ampicillin to induce protein expression. Then, the cultured cells were disrupted using sonication, and the supernatant obtained by centrifugation was separately separated, and then GST resin (Glutathion S Epharose ™ High Performance, GE Healthcare). Western blotting was performed on the purified protein and the BphP protein of Deinococcus radiodurans. 2B8 monoclonal antibody and anti-GST antibody were used as primary antibodies and Horseradish peroxidase (HRP) conjugated anti-mouse immunoglobulin (anti-mouse IgG; Sigma) was used as the secondary antibody. Further, in order to develop a sample transferred to the cellulose membrane after SDS-PGAE, Amersham ™ ECL ™ Western Blotting Detection Reagents (GE Healthcare, RPN2106OL / AF) were used. Figure 4 shows Western blot results of primary epitope mapping for 2B8 antibody. As shown in FIG. 4, the epitope of the antibody 2B8 is 9 amino acids (SEQ ID NO: 1) corresponding to 3-11 positions based on the N-terminal of the entire amino acid sequence of DrBphP (SEQ ID NO: 3) and is used as a peptide tag Respectively. In addition, the peptide tag consisting of the amino acid sequence of SEQ ID NO: 1 was named "BRT tag".

5-2: Secondary epitope mapping

Through the primary epitope mapping, it was confirmed that the BRT tag consisting of the amino acid sequence of SEQ ID NO: 1 was an epitope of 2B8 antibody. The present inventors conducted a secondary epitope mapping to see whether the 2B8 antibody recognizes the DrBphP modified partial peptide, which is a peptide in which one amino acid of the amino acid sequence of SEQ ID NO: 1 constituting the BRT tag is deleted.

First, a peptide (denoted by 3-11-Δ4D) containing an amino acid sequence of 3-11 positions based on the N-terminus of the entire amino acid sequence of DrBphP (SEQ ID NO: 3) and simultaneously deletion of aspartic acid which is a 4th amino acid residue, (SEQ ID NO: 3) of DrBphP, a peptide containing the amino acid sequence at the 3-11 position based on the N-terminus of the DrBphP, and the 5th amino acid residue proline deleted (3-11-Δ5P (SEQ ID NO: 3) of DrBphP, and a peptide (3-11 (SEQ ID NO: 3) which contains an amino acid sequence of 3-11 positions based on the N-terminus of the DrBphP and at the same time a leucine- -Δ6L), a peptide containing the amino acid sequence of 3-11 positions based on the N-terminus of the entire amino acid sequence of DrBphP (SEQ ID NO: 3) and at the same time the 7th amino acid residue proline deleted 3-11-Δ7P) (SEQ ID NO: 3) of DrBphP, a peptide (3-11-Δ8F) containing an amino acid sequence of 3-11 positions based on the N-terminus of the DrBphP and having a deletion of phenylalanine, which is an eighth amino acid residue Primers were prepared in order to clone the DNA encoding the gene encoding the gene coding for each gene. PCR was performed using primers and PrimeSTAR HS (TAKARA, R040) polymerase that were prepared for about 30 cycles to obtain amplified DNA products. The PCR reaction for DNA cloning does not require a separate template, and in one cycle of PCR, a template for amplification can be obtained by combining a forward primer and a reverse primer. Table 5 below shows the primers used in PCR to obtain DNA amplification products coding for the five DrBphP modified partial peptides. The procedure after obtaining the DNA amplification product by PCR is the same as the first-order epitope mapping, so the explanation is omitted.

SEQ ID NO: Type of primer The base sequence (5 '- > 3') The restriction enzyme recognition site contained in the primer 22 3-11-forward primer for Δ4D cloning GCGGATCCATGCGGCCGTTGCCCTTTTTTCCACCG BamH I 23 Reverse primer for 3-11-Δ4D cloning GCGAATTCTCACGGTGGAAAAAAGGGCAACGGCCG EcoR I 24 3-11-Δ5P forward primer for cloning GCGGATCCATGCGGGACTTGCCCTTTTTTCCACCG BamH I 25 Reverse primer for 3-11-Δ5P cloning GCGAATTCTCACGGTGGAAAAAAGGGCAAGTCCCG EcoR I 26 3-11-Δ6L forward primer for cloning GCGGATCCATGCGGGACCCGCCCTTTTTTCCACCG BamH I 27 Reverse primer for 3-11-Δ6L cloning GCGAATTCTCACGGTGGAAAAAAGGGCGGGTCCCG EcoR I 28 3-11-forward primer for Δ7P cloning GCGGATCCATGCGGGACCCGTTGTTTTTTCCACCG BamH I 29 Reverse primer for 3-11-Δ7P cloning GCGAATTCTCACGGTGGAAAAAACAACGGGTCCCG EcoR I 30 3-11-Δ8F forward primer for cloning GCGGATCCATGCGGGACCCGTTGCCCTTTCCACCG BamH I 31 Reverse primer for 3-11-Δ8F cloning GCGAATTCTCACGGTGGAAAGGGCAACGGGTCCCG EcoR I

Figure 5 shows Western blot results of secondary epitope mapping for 2B8 antibody. As shown in FIG. 5, the other epitope of the antibody 2B8 is located at the eighth position or the ninth position from the amino acid residues corresponding to the 11th position from the 3'th position of the entire amino acid sequence of DrBphP (SEQ ID NO: 3) (SEQ ID NO: 2), and another epitope of the 2B8 antibody can also be used as a peptide tag. The peptide tag consisting of the amino acid sequence of SEQ ID NO: 2 was named "BRT-ΔF tag".

Example  6: BRT  Preparation of fusion protein using tag BRT  Detection of Fusion Protein Using Antibody to Tag

A fusion protein was prepared using the BRT tag of the present invention, and the fusion protein was detected using a B28 antibody as a monoclonal antibody against the BRT tag.

6-1: Expression and detection of fusion protein in E. coli

In order to confirm whether the fusion protein containing the BRT tag of the present invention is normally expressed on E. coli and to determine whether the expressed fusion protein can be detected as a 2B8 monoclonal antibody, GST (Glutathion-S-Transferase ) ≪ / RTI > protein, a DNA construct coding for a fusion protein to which the BRT tag is linked. Primers were prepared for cloning the DNA encoding the BRT tag of the present invention and the DNA encoding the c-myc tag, respectively. PCR was performed using primers and PrimeSTAR HS (TAKARA, R040) polymerase that were prepared for about 30 cycles to obtain amplified DNA products. The PCR reaction for DNA cloning does not require a separate template, and in one cycle of PCR, a template for amplification can be obtained by combining a forward primer and a reverse primer. Table 6 below shows the primers used in the PCR for obtaining the above two DNA amplification products. The desired DNA was isolated from the amplified PCR product and ligated to pJET (CloneJET PCR cloning kit, Fermentas, # K1232) vector. Then, the above-mentioned binding vector was consigned to Bioneer co., KR to measure the nucleotide sequence. As a result, it was confirmed that the DNA product generated through PCR had a desired base sequence.

SEQ ID NO: Type of primer The base sequence (5 '- > 3') The restriction enzyme recognition site contained in the primer 32 Forward primer for BRT tag cloning GCCTCGAGGGATCCATGCGGGACCCGTTGCCCTTTTTT BamH I 33 Reverse primer for BRT tag cloning GCGAGCTCGAATTCTCACGGTGGAAAAAAGGGCAACGG EcoR I 34 forward primer for c-myc tag cloning GCGGATCCATGGAACAAAAATTAATTTCTGAAGAAGATTTA BamH I 35 reverse primer for c-myc tag cloning GCGAATTCTCATAAATCTTCTTCAGAAATTAATTTTTGTTC EcoR I

The isolated DNA product was then ligated to a pGEX4T1 vector (GE Healthcare, Piscataway, NJ, USA) containing the DNA coding for the GST (Glutathion-S-Transferase) protein as well as an expression vector. The DNA product and pGEX4T1 vector (GE Healthcare, Piscataway, NJ, USA) were digested with restriction enzymes BamH I and EcoR I and ligated using ligase. Then, the pGEX4T1 vector containing the desired DNA The cells were transformed by heat shock at 42 ° C for 1 minute in BL21 competent cells (manufacturer: RBC, Taipei, Taiwan), cultured on LB medium containing ampicillin, and fused with BRT- (Expressed as 3 < 11 > :: GST) and a fusion protein (designated Myc :: GST) to which c-myc tag was linked to the GST protein were specifically expressed. Specifically, BL21 competent cells were transformed into LB medium Cole grown on (colony) of cultured ampicillin containing the LB inoculated to the medium and at least about 8 hours at 37 ℃ species inoculated species cultured cells in LB medium with ampicillin containing and until the value at OD ₆₀₀ become 0.6 IPTG was added so that the total concentration was 0.5 mM and protein expression was induced for 6 hours at 25 ° C. After that, a commercially available GST resin (Glutathion Sepharose ™ High Performance, GE Healthcare). Western blotting was performed on the purified protein and the BphP protein of Deinococcus radiodurans. 2B8 monoclonal antibody and anti-myc antibody were used as primary antibodies and Horseradish peroxidase (HRP) conjugated anti-mouse immunoglobulin (anti-mouse IgG; Sigma) was used as the secondary antibody. Further, in order to develop a sample transferred to the cellulose membrane after SDS-PGAE, Amersham ™ ECL ™ Western Blotting Detection Reagents (GE Healthcare, RPN2106OL / AF) were used. Fig. 6 is a Western blot result showing that a fusion protein in which the BRT tag is attached to the N-terminus of the GST protein is normally expressed on E. coli and the fusion protein can be detected by the 2B8 antibody. As shown in FIG. 6, the fusion protein tagged with the N-terminus of the GST protein was smoothly expressed in E. coli , and the expressed protein was well detected without nonspecific binding by the 2B8 antibody. In addition, the epitope tagging system using the BRT tag and 2B8 antibody showed almost the same level of effect as compared with the epitope tagging system using the c-myc tag and antibody against the commonly used tag.

6-2: Expression and detection of fusion proteins in plant cells

In order to confirm whether the fusion protein including the BRT tag of the present invention is normally expressed on plant cells and to confirm whether the expressed fusion protein can be detected as a 2B8 monoclonal antibody, a BRT tag is connected before GFP (Green Fluorescent Protein) A DNA construct encoding the fusion protein was prepared. DNA encoding the fusion protein (represented by BRT :: GFP) to which the BRT tag of the present invention is linked is cloned into GFP and a DNA encoding a fusion protein (represented by Myc :: GFP) to which c-myc tag is linked to GFP Primers for each were prepared. PCR was performed for about 30 cycles using primer and PrimeSTAR HS (TAKARA, R040) polymerase, and DNA encoding GFP (SEQ ID NO: 36) was used as a template for PCR. Table 7 below shows the primers used in the PCR for obtaining the above two DNA amplification products. The desired DNA was isolated from the amplified PCR product and ligated to pJET (CloneJET PCR cloning kit, Fermentas, # K1232) vector. Then, the above-mentioned binding vector was consigned to Bioneer co., KR to measure the nucleotide sequence. As a result, it was confirmed that the DNA product generated through PCR had a desired base sequence.

SEQ ID NO: Type of primer The base sequence (5 '- > 3') The restriction enzyme recognition site contained in the primer 37 Forward primer for BRT :: GFP cloning GCCTCGAGATGAGGGATCCACTTCCATTCTTCCCTCCAATGGTGAGCAAGGGCGAG Xho I 38 Reverse primer for BRT :: GFP cloning GCGAGCTCTTACTTGTACAGCTCGTCCAT Sheet I 39 Forward primer for Myc :: GFP cloning GGAGAGGACCTCGAGATGGAACAAAAGCTTATTTCTGAAGAAGATCTTATGGTGAGCAAGGGCGAG Xho I 40 Reverse primer for Myc :: GFP cloning GCGAGCTCTTACTTGTACAGCTCGTCCAT Sheet I

The isolated DNA product was then ligated into the expression vector pBY030.2R vector, and the vector was used to transform Agrobacterium LBA4404 cells. Since then, Nicotiana benthamiana (tobacco) Agrobacterium LBA4404 was syringe infiltrated into the lower surface of the leaf, and a fusion protein (designated BRT :: GFP) to which a BRT tag was linked and a fusion protein to which c-myc tag was linked to GFP (Myc :: GFP) were transiently expressed in tobacco plants, respectively. For detailed experiments, Imogen et < RTI ID = 0.0 > al. (NATURE PROTOCOL, 2006, Vol. 1, No. 4, 2019-2025). Agrobacterium was cultivated at a concentration of up to 0.7 at OD ₆₀₀ and then injected into the plant. After that, the plant was maintained in the culture room for 5 days and the sample was harvested. The presence or absence of the transfection was confirmed by plants treated with ultraviolet light (UV) to show GFP fluorescence. The harvested samples were rapidly frozen in liquid nitrogen and stored at -80 ° C. Tobacco leaf samples were then pulverized with a TissueLyser (QIAGEN), and an extraction buffer (125 mM Tris-HCL pH 8.8, 1% SDS, 10% Glycerol, and 50 mM Na ₂ S ₂ O ₅ ) Only the supernatant was isolated and used for Western blot analysis. FIG. 7 is a Western blot result showing that a fusion protein to which a BRT tag is attached to the N-terminus of GFP is normally expressed in plant cells and that a fusion protein can be detected by 2B8 antibody. As shown in FIG. 7, the fusion protein tagged with BRT at the N-terminal of GFP was expressed smoothly regardless of leaf growth in plant cells, and the expressed protein was well detected without any specific binding by 2B8 antibody. In addition, the epitope tagging system using the BRT tag and 2B8 antibody showed almost the same level of effect as compared with the epitope tagging system using the c-myc tag and antibody against the commonly used tag.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Therefore, the protection scope of the present invention should be construed as including all embodiments falling within the scope of the claims appended to the present invention.

Korea Biotechnology Research Institute KCTC12283BP 20120924

Attach an electronic file to a sequence list

Claims (22)

Peptide tag consisting of the amino acid sequence of SEQ ID NO: 2.
A polynucleotide encoding a peptide tag consisting of the amino acid sequence of SEQ ID NO.
The polynucleotide of claim 2, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8.
A primer pair for synthesizing a polynucleotide encoding a peptide tag consisting of the amino acid sequence of SEQ ID NO.
The primer pair of claim 4, wherein the primer pair comprises a forward primer having a nucleotide sequence of SEQ ID NO: 30 and a reverse primer having a nucleotide sequence of SEQ ID NO: 31. 6.
A recombinant vector comprising the polynucleotide of claim 2.
7. The recombinant vector of claim 6, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8.
The recombinant vector of claim 6, wherein the recombinant vector is a cloning vector or an expression vector.
The method of claim 8, wherein the expression vector further comprises a target polynucleotide encoding the target protein,
The target polynucleotide is a recombinant vector, characterized in that linked to a polynucleotide encoding a peptide tag consisting of the amino acid sequence of SEQ ID NO: 2.
A fusion protein comprising a peptide of interest and a peptide tag linked thereto and consisting of the amino acid sequence of SEQ ID NO: 2.
A polynucleotide encoding the fusion protein of claim 10.
A recombinant vector comprising the polynucleotide of claim 11.
Introducing any one selected from the group consisting of the polynucleotide of any one of claims 2 or 3, the recombinant vector of any one of claims 6 to 9, the polynucleotide of claim 11 and the recombinant vector of claim 12 Characterized by a transformant.
A method for producing a fusion protein comprising culturing the transformant of claim 13 to express the fusion protein.
15. The method of claim 14, wherein the method for producing a fusion protein further comprises the step of recovering the expressed fusion protein.
Contacting the fusion protein of claim 10 with an antibody against a peptide tag consisting of the amino acid sequence of SEQ ID NO: 2 to bind; And
Confirming the presence or analysis of the amount of the fusion protein bound to the antibody.
The method of claim 16, wherein the antibody is a monoclonal antibody produced by a hybridoma cell line having an accession number of KCTC 12283BP.
Contacting the fusion protein of claim 10 with an antibody against a peptide tag consisting of the amino acid sequence of SEQ ID NO: 2 to bind; And
A method for purifying a fusion protein comprising recovering a fusion protein bound to the antibody.
19. The method of claim 18, wherein said antibody is a monoclonal antibody produced by a hybridoma cell line with an accession number of KCTC 12283BP.
The polynucleotide of any one of claims 2 or 3, the primer pair of any one of claims 4 or 5, the recombinant vector of any one of claims 6 to 9, the polynucleotide of claim 11 and 12 A fusion protein expression kit comprising any one selected from the group consisting of the recombinant vector of claim.
The polynucleotide of any one of claims 2 or 3, the primer pair of any one of claims 4 or 5, the recombinant vector of any one of claims 6 to 9, the polynucleotide of claim 11 and 12 Any one selected from the group consisting of the recombinant vector of claim; And
A kit for detecting or purifying a fusion protein comprising any one selected from an antibody against a peptide tag comprising the amino acid sequence of SEQ ID NO: 2 or a hybridoma cell line producing the antibody.
22. The kit for detecting or purifying a fusion protein according to claim 21, wherein the antibody is a monoclonal antibody produced by a hybridoma cell line having an accession number of KCTC 12283BP.

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