KR20170098165A - A method for producing fusion protein-polysaccharide complex and use thereof - Google Patents

Abstract

The present invention relates to a method for producing a fusion protein-polysaccharide complex, which comprises a step of reacting a fusion protein including a sugar transferase and a target protein with carbohydrate; a fusion protein-polysaccharide composite produced through the method; and a method for refining and fixing a target protein by using the fusion protein-polysaccharide composite. When the fusion protein-polysaccharide composite of the present invention is used, a target protein can be very simply refined and fixed compared to a conventional protein refining and fixing method. Thus, the present invention can be widely utilized to produce a target protein and to produce a useful product by using the target protein.

Description

TECHNICAL FIELD [0001] The present invention relates to a fusion protein-polysaccharide complex and use thereof,

The present invention relates to a fusion protein comprising a glycosyltransferase and a target protein, an expression cassette thereof, and a vector for expressing a fusion protein comprising the expression cassette. The present invention also relates to a method for producing a fusion protein-polysaccharide complex, which comprises reacting the fusion protein with a carbohydrate; A fusion protein-polysaccharide complex prepared by the above production method; And a method for purification and immobilization of a target protein using the fusion protein-polysaccharide complex.

The first step in separating proteins from the biosystem is to break down the cells using physical techniques or surfactants to solubilize the desired enzyme. The desired protein is separated and purified stepwise from the thus-extracted cell solution using various physicochemical properties of the desired protein. Currently, most of the production proteins are separated and purified by chromatography. Chromatography methods include ion chromatography using the difference in electrostatic interaction with the carrier, Separation by molecular sieve filtration, adsorption chromatography using an affinity tag that is widely used in the production of recombinant proteins, and the like. However, this chromatographic protein purification method has a disadvantage that scale-up is difficult because it requires the use of an expensive resin, and it takes several steps.

Immobilized enzymes are slightly less active than natural enzymes, but generally have a high resistance to heat and chemicals, and can easily be recovered and reused in a reaction system. Therefore, the production of organic substances, removal of environmental pollutants, The application of this catalyst has been expanding as a catalyst that can be used repeatedly and stably for a long period of time. In particular, using a separation tube filled with immobilized enzyme, continuous production is possible because the reaction product is continuously injected from above and the product is obtained from below. Immobilization techniques include physical and chemical methods depending on the type of enzyme conjugation. Chemical methods include adsorption, ionic bonding, and covalent bonding, and physical methods include entrapment and encapsulation.

Under these circumstances, the present inventors have made intensive efforts to develop a novel method for purifying and immobilizing a target protein. As a result, they have developed a fusion protein-polysaccharide complex using a fusion protein containing a glycosyltransferase and a target protein, A protein purification method, a separation method, and an immobilization method, thereby completing the present invention.

It is an object of the present invention to provide a method for producing a fusion protein-polysaccharide complex comprising the step of reacting a fusion protein containing a glycosyltransferase and a target protein with a carbohydrate.

Another object of the present invention is to provide a process for producing a reaction solution containing a fusion protein-polysaccharide complex by reacting (a) a lysate or a culture of a transformant expressing a fusion protein containing a glycosyltransferase and a target protein with a carbohydrate step; (b) separating the fusion protein-polysaccharide complex from the reaction solution; And (c) separating the target protein from the fusion protein-polysaccharide complex.

Still another object of the present invention is to provide a method for producing a reaction solution containing a fusion protein-polysaccharide complex by reacting (a) a lysate or a culture of a transformant expressing a fusion protein containing a glycosyltransferase and a target protein with a carbohydrate step; And (b) separating the fusion protein-polysaccharide complex from the reaction solution.

Still another object of the present invention is to provide a fusion protein comprising a glycosyltransferase and a target protein; And a fusion protein-polysaccharide complex comprising a glycosyltransferase, which comprises a polysaccharide.

It is still another object of the present invention to provide a fusion protein comprising a glycosyltransferase and a target protein.

Still another object of the present invention is to provide a fusion protein expression cassette comprising a promoter and a polynucleotide encoding a glycosyltransferase and a target protein operably linked to the promoter.

It is still another object of the present invention to provide a vector for expressing a fusion protein comprising the fusion protein expression cassette.

One aspect of the present invention provides a method for preparing a fusion protein-polysaccharide complex, which comprises reacting a fusion protein containing a glycosyltransferase and a target protein with a carbohydrate.

Since the fusion protein of the present invention contains a sugar-transferase, it has a sugar-exchange activity and produces a carbohydrate, specifically, a polysaccharide derived from sucrose. In addition, the produced polysaccharide forms a natural bead, and the fusion protein is immobilized between the beads to produce a fusion protein-polysaccharide complex. Unlike the conventional protein purification method and immobilization method, the fusion protein-polysaccharide complex can purify and immobilize the target protein very simply by the same method as centrifugation. Can be widely used to produce useful products.

The term " glycosyltransferase "of the present invention means an enzyme capable of producing a glycosidic bond to various substrates, and capable of producing a polysaccharide containing a carbohydrate. The glycosyltransferase is included in the present invention without limitation as long as it is an enzyme capable of producing a polysaccharide, but specifically includes hexosyltransferase, pentosyltransferase, and sialyltransferase ), More specifically, a hexosyl transferase, and more specifically, amylosucrase, but is not limited thereto. Further, the sugar transferase of the present invention has no limitation on the origin of the enzyme if it is an enzyme having an activity capable of producing a polysaccharide.

In addition, the amylose sucrase is not limited as long as it can produce amylose due to its glycosylation activity. Specifically, it is derived from Deinococcus , more specifically, Deinococcus geothermalis .

Specifically, the amylose sucrase may be a protein having the amino acid sequence of SEQ ID NO: 1 or a protein encoded by the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

The term "target protein" of the present invention means a protein or peptide to be produced, and includes any kind of protein or peptide without limitation, but specifically includes antigens, antibodies, cell receptors, enzymes, structural proteins, Can be selected from the group consisting of. In one embodiment of the present invention, a fusion protein-polysaccharide complex was prepared using a green fluorescent protein (EGFP) or a beta-glucosidase (DGBG) as a luminescent protein.

The term "fusion protein" of the present invention refers to a protein artificially synthesized so that the glycosyltransferase binds to another protein or peptide, specifically, the glycosyltransferase and the target protein. In addition, the fusion protein may be a fusion protein in which the target protein is an N-terminal of a glycosyltransferase; C-terminal; Or may be directly connected to the N-terminus and the C-terminus, or may be linked via a linker. Also, the fusion protein may be directly connected to the N-terminal and C-terminal of the target protein, or may be connected through a linker.

The linker is not particularly limited as long as the glycosyltransferase of the fusion protein and the target protein are active. Specific examples of the linker include glycine (Gly, G), alanine (Alanine, Ala, A), leucine Leu, L), isoleucine (Ile, I), proline, Pro, P, serine, Ser, S, threonine, Thr, T, asparagine, Asn, N Aspartic acid, Asp, D, cysteine, Cys, C, glutamine, Gln, Q, glutamic acid, Glu, E, lysine, Lys, K, Arginine, arginine, arginine, arginine, arginine, arginine, arginine, arginine, arginine, arginine, arginine, arginine, arginine and arginine. More concretely, it can be connected by using several valine, leucine, aspartic acid, glycine, alanine, proline and the like. Considering the ease of gene manipulation, amino acids such as glycine, valine, leucine and aspartic acid, Five can be connected to each other. For example, in the present invention, the C-terminus of amylose sucrose and the N-terminus of beta-glucosidase are replaced with a linker (GS linker) or DKTKYTAS (SEQ ID NO: 29) consisting of the amino acid sequence of GGGGSGGGGS And then ligated through a linker composed of an amino acid sequence (EstO linker) to prepare a fusion protein.

The fusion protein may include a polypeptide having a sequence having a different amino acid sequence from the wild-type amino acid sequence of each domain contained in the fusion protein. Amino acid exchange in proteins and polypeptides that do not globally alter the activity of the molecule is known in the art. The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In addition, the protein may include a protein having increased structural stability or increased protein activity due to mutation or modification of the amino acid sequence, such as heat, pH and the like.

The fusion protein or the polypeptide constituting the fusion protein may be prepared by a chemical peptide synthesis method known in the art, or may be prepared by amplifying a gene encoding the fusion protein by PCR (polymerase chain reaction) or by a known method Followed by expression in a post-expression vector.

The protein of the present invention has not only the amino acid sequence shown in each of the above SEQ ID NOs, but also a homology of not less than 80%, specifically not less than 90%, more specifically not less than 95%, more specifically not less than 97% Lt; / RTI > As the sequence having such homology, any amino acid sequence which represents a protein exhibiting substantially the same or equivalent activity as the respective proteins is included without limitation. It is also apparent that amino acid sequences in which some sequences are deleted, modified, substituted or added are also included within the scope of the present invention, provided that they have such homology.

In addition, the gene encoding the protein of the present invention may contain not only the nucleotide sequence encoding the amino acid sequence shown in the above SEQ ID NOs, but also the nucleotide sequence having 80% or more, specifically 90% or more, more specifically 95% More specifically 98% or more, and most particularly 99% or more as a base sequence, which substantially encodes a protein exhibiting the same or corresponding efficacy as the respective proteins. Also, it is obvious that base sequences having deletion, modification, substitution or addition of some sequences are also included within the scope of the present invention if they are base sequences having such homology.

As used herein, the term "homology" means a base sequence or a similar degree of amino acid sequence of a gene encoding a protein. If the homology is sufficiently high, the expression product of the gene may have the same or similar activity have.

In one production example of the present invention, expression of a gene encoding a fusion protein fused with amylose sucrose (DGAS) derived from Deinococcus geothermalis and Green Fluorescent Protein (EGFP) Vector pHCXHD -egfp-dgas was prepared (Figure 2).

In addition, in one production example of the present invention, an expression vector having a gene encoding a fusion protein fused with DGAS and beta-glucosidase (DGBG) pET- dgas - dgbg was prepared using the overlap PCR method (Fig. 3).

In one production example of the present invention, an expression vector pET- dgas - gs - dgbg in which a gene encoding a fusion protein linked between DGAS and DGBG was linked by GS-linker was prepared (Fig.

In addition, in one production example of the present invention, an expression vector pET- dgas-esto - dgbg in which a gene encoding a fusion protein in which DGAS and DGBG are linked by an Esterase-O-linker (EstO linker) 5).

In addition, in one production example of the present invention, an expression vector pET- malE - dqas- ( lacZ alpha ) into which a gene encoding a fusion protein linking maltose binding protein (MalE) and DGAS with a linker is inserted (Fig. 6).

In one production example of the present invention, an expression vector pET- malE-dgas in which a gene coding for a fusion protein in which lacZ alpha fusion protein has been removed was ligated with a linker between MalE and DGAS (FIG. 7 ).

In addition, in a preparation example of the present invention, the DGAS-linker-MCS gene in which DGAS and MCS are linked by a linker was prepared using pMaL-cx2, and the DGAS-linker-MCS gene was inserted into pET21a (+ -dgas -linker-MCS (pET- dgas- M linker) was prepared (Fig. 8).

In one embodiment of the invention the prepared pHCXHD- egfp - was in the dgas vector is inserted into E. coli (E. coli) MC1061 incubated at 37 ℃ for 15 hours to express the fusion protein, the fusion protein containing the pET vector (DE3), in which the gene coding for the gene coding for IFN-γ was inserted, was cultured in the presence of 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) at 18 ° C. for 18 hours to induce expression of the fusion protein. Respectively. The supernatant obtained by centrifuging the pulverized liquid was injected into Ni-NTA affinity chromatography to purify the fusion protein. The purified fusion protein was found to have a fluorescence color and the target protein had EGFP activity, or it was confirmed that the target protein had a beta glucosidase activity due to the yellow of p- nitrophenol.

Therefore, in the present invention, the fusion protein has a glycoprotein-polysaccharide complex, purification and immobilization of the target protein, since the target protein is bound to the glycoconjugate, amylose sucrose, Can be used.

The term "carbohydrate" of the present invention means a compound used as a substrate of a glycosyltransferase, and specifically includes glucose, fructose, mannose, galactose, ribose, Maltose, sucrose, cellobiose, gentiobiose, melibiose, lactose, turanose, sophorose, amylose, but are not limited to, amylose, amylopectin, glycogen, and soluble starch.

Specifically, when a monosaccharide or a disaccharide and a polysaccharide are used together as a substrate to react with a fusion protein comprising a glycosyltransferase, the production rate of amylose or its analogous polysaccharide can be increased and the production of a fusion protein-polysaccharide complex The speed can be increased. When amylose is prepared using only a monosaccharide or a disaccharide as a substrate, the number of α-1,4 bonds in which the glycosyltransferase can extend glucose is small, but when amylopectin, glycogen, soluble starch is added, glucose The number of? -1,4 bond branches capable of elongating the?

In one embodiment of the present invention, when the monosaccharide or the disaccharide and the polysaccharide are added to the substrate reacting with the fusion protein containing the glycosyltransferase, the production rate of the fusion protein-polysaccharide complex increases, A 500 μl sugar solution and 500 μl of 100 mM Tris-HCl (pH 8.0) were mixed with 5 μl of amylopectin, glycogen or soluble starch (200 μl), and the substrate was reacted with a fusion protein containing a glycosyltransferase (Example 2 -2). Fusion protein - polysaccharide complexes were able to be produced in all three types of polysaccharides. When soluble starch and amylopectin were added, most of the fusion protein - polysaccharide complexes were produced in a short time and enzyme activity was strong. Thus, when monosaccharide or disaccharide and polysaccharide were added together to the substrate reacting with the fusion protein, the production rate of the fusion protein-polysaccharide complex was increased.

The term "fusion protein-polysaccharide complex" of the present invention refers to a complex in which a fusion protein having the above-mentioned glycosyltransferase activity is reacted with a carbohydrate and a fusion protein is immobilized between beads in which the resulting polysaccharide is tangled with each other . In addition, the fusion protein-polysaccharide complex maintains the original glycosyltransferase activity and the activity of the target protein. In addition, since the fusion protein-polysaccharide complex submerged by centrifugation, the reaction mixture containing the fusion protein-polysaccharide complex prepared by reacting the fusion protein with the carbohydrate can be centrifuged to obtain the complex. The fusion protein-polysaccharide complex is used in combination with a fusion protein-polysaccharide complex or a fusion protein-polysaccharide complex.

In one embodiment of the present invention, the reaction solution obtained by reacting the fusion protein and the sugar was centrifuged to obtain a precipitated white precipitate, which confirmed that the polysaccharide was synthesized. Through this, it was found that amylose sucrose (DGAS) of the fusion protein was active, and a fusion protein-polysaccharide complex was produced. Further, it was confirmed by fluorescence microscopy that EGFP was active in a fusion protein-polysaccharide complex comprising an EGFP-DGAS fusion protein and a polysaccharide (FIG. 10). Also, 40 μl of 10 mM p- nitrophenyl-β-D-glucopyranoside and 50 μl of 100 mM Tris-HCl (pH 8.0) were added to the resulting fusion protein-polysaccharide complex and reacted at 40 ° C. for 10 minutes. (DGBG) of the fusion protein-polysaccharide complex was confirmed by confirming the yellow of p- nitrophenol liberated by the fusion protein-polysaccharide complex (FIG. 11). As a result, using the fusion protein of the present invention, it was found that a fusion protein-polysaccharide complex was produced, and that the resulting fusion protein-polysaccharide complex had all the activities of the two fusion proteins.

In another aspect of the present invention, there is provided a method for producing a fusion protein comprising: (a) reacting a lysate or a culture of a transformant expressing a fusion protein containing a glycosyltransferase and a target protein with a carbohydrate, ; (b) separating the fusion protein-polysaccharide complex from the reaction solution; And (c) separating the target protein from the fusion protein-polysaccharide complex.

Since the fusion protein of the present invention is made of a carbohydrate-derived polysaccharide and the fusion protein is immobilized between the beads to form a fusion protein-polysaccharide complex, when the target protein is isolated from the separated fusion protein-polysaccharide complex, The target protein can be purified more simply than the protein purification method using a column or the like.

The "sugar transferase", "target protein", "fusion protein", "carbohydrate", and "fusion protein-polysaccharide complex" of the present invention are as described above.

In the present invention, the term "transformant" refers to a DNA that is introduced into a host and DNA can be cloned as a chromosome factor or by chromosomal integration, thereby introducing an external DNA into a cell, .

By such transformation, an expression vector comprising a polynucleotide encoding the fusion protein of the present invention or a part of the expression vector can be introduced into the host cell. Herein, a part of the expression vector refers to an activity of the fusion protein in the host cell Quot; means a portion of an expression vector that comprises a polynucleotide portion that encodes a fusion protein so as to confer expression of the fusion protein. For example, but not by way of limitation, T-DNA of a Ti plasmid transferred into a host cell in an Agrobacterium-mediated transformation method.

Any transformation method of the present invention can be used, and can be easily carried out according to a conventional method in the art. Generally, the transformation methods include the CaCl ₂ precipitation method, the Hanahan method which uses a reducing material called DMSO (dimethyl sulfoxide) for the CaCl ₂ method, the electroporation method, the calcium phosphate precipitation method, the protoplasm fusion method, Agrobacterium-mediated transformation, transformation with PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods. The method for transforming a vector comprising a polynucleotide encoding a fusion protein of the present invention is not limited to the above examples, and transformation or transfection methods commonly used in the art can be used without limitation.

The kind of the host cell that can be used in the production of the transformant in the present invention is not particularly limited as long as it is capable of expressing the polynucleotide of the present invention. Specific examples of the host which can be used in the present invention include bacteria belonging to the genus Escherichia such as E. coli ; Bacteria of the genus Bacillus such as Bacillus subtilis ; Bacteria of the genus Pseudomonas such as Pseudomonas putida ; Saccharomyces S. cerevisiae , yeast such as Schizosaccharomyces pombe for skiing; Animal cells, plant cells, and insect cells. Specific examples of the Escherichia coli strain that can be used in the present invention include C41 (DE3), BL21 or HB101, and specific examples of the Bacillus subtilis strain include WB700 or LKS87.

The transformant into which the expression vector containing the polynucleotide of the present invention is introduced may be in the form of a plant. Examples of the transformant include, but are not limited to, tobacco, Arabidopsis, potato, ginseng, sesame, citron and daisy.

The promoter contained in the vector for producing the transformant of the present invention may be any promoter so long as the polynucleotide of the fusion protein is expressed in the host. For example, Escherichia coli or phage-derived promoters such as trp promoter, lac promoter, PL promoter or PR promoter; Escherichia coli infectious phage-derived promoter such as T7 promoter, CaMV35S, MAS or histone promoter may be used. Artificially modified promoters such as the tac promoter may also be used.

The term "lysate " of the present invention means a pulverized liquid of a transformant or a supernatant obtained by centrifuging the pulverized liquid containing the fusion protein of the present invention.

The term "culture" of the present invention means a product containing the fusion protein of the present invention obtained after culturing the transformant. The culture includes both a form containing a transformant and a form in which a transformant is removed by centrifugation or the like in a culture solution containing the transformant.

 Since the lysate and the culture contain the fusion protein of the present invention, they have activity to produce a carbohydrate-derived polysaccharide, so that a fusion protein-polysaccharide complex can be produced.

The term "fusion protein-polysaccharide complex separation" of the present invention means to separate the complex from the reaction solution containing the fusion protein-polysaccharide complex prepared using the fusion protein, specifically, Separation can be carried out by precipitating the fusion protein-polysaccharide complex.

In one embodiment of the present invention, Escherichia coli MC1061 and BL21 (DE3) inserted with the recombinant vectors prepared in Production Examples 1 to 7 were cultured in the presence of continuous expression and IPTG (Isopropyl? -D-1-thiogalactopyranoside) And the cells were recovered. Then, the suspension system in Lai buffer (Lysis buffer) [50 mM NaH 2 PO 4, pH 7.0, 300 mM NaCl, 10 mM imidazole, pH 7.0], was pulverized ultrasound. Thereafter, the pulverized liquid was centrifuged to obtain a supernatant. Further, the fusion protein was purified from the supernatant using Ni-NTA affinity chromatography, and the activity of the amylose sucrose was confirmed by the reaction of the fusion protein with sugar and the coloring of DNS and reducing sugar. In addition, the activity of EGFP used as a target protein was confirmed by fluorescence microscopy. The activity of β-glucosidase was also confirmed by the yellow color of p- nitrophenol liberated by enzyme reaction using p- nitrophenyl-β-D-glucopyranoside.

The term "separation of a target protein" of the present invention refers to separation of a target protein from the fusion protein-polysaccharide complex. Specifically, the target protein can be isolated by treating the fusion protein-polysaccharide complex with a protease.

In one embodiment of the present invention, protease (Factor Xa) was treated with a fusion protein (DGAS-linker-DGBG) -saturated polysaccharide complex, and as a result, it was confirmed that DGAS and DGBG were separated by decomposing the linker. It was confirmed that it could be obtained in the supernatant portion (Fig. 12).

In another aspect of the present invention, there is provided a method for producing a fusion protein comprising: (a) reacting a lysate or a culture of a transformant expressing a fusion protein containing a glycosyltransferase and a target protein with a carbohydrate, ; And (b) separating the fusion protein-polysaccharide complex from the reaction solution.

The fusion protein of the present invention produces a bead made of a carbohydrate-derived polysaccharide, and a fusion protein is immobilized between the beads to form a fusion protein-polysaccharide complex. Since the complex thus formed exhibits the activity of the target protein, the complex can be separated and reacted with the substrate of the target protein, and then the complex can be separated and reused. Therefore, when the fusion protein- polysaccharide complex of the present invention is used, Can be immobilized.

The terms "glycoprotein", "target protein", "fusion protein", "transformant", "lysate", "culture" "carbohydrate", "fusion protein- polysaccharide complex" - separation of polysaccharide complex "is as described above.

Another embodiment of the present invention is a fusion protein comprising a glycosyltransferase and a target protein; ≪ / RTI > and a polysaccharide.

The fusion protein-polysaccharide complex of the present invention can purify and immobilize a desired protein in a very simple manner, unlike the existing methods for protein purification and immobilization, and produce useful products using target proteins and target proteins Can be widely used.

The "sugar transferase", "target protein", "fusion protein", and "fusion protein-polysaccharide complex" of the present invention are as described above.

Further, another embodiment of the present invention provides a fusion protein comprising a glycosyltransferase and a target protein.

The "sugar transferase", "target protein" and "fusion protein" of the present invention are as described above.

Yet another aspect of the present invention provides a fusion protein expression cassette comprising a promoter and a polynucleotide encoding a glycosyltransferase and a protein of interest operably linked to the promoter.

The "sugar transferase", "target protein" and "fusion protein" of the present invention are as described above.

The term "promoter" in the present invention refers to a polynucleotide sequence that is upstream of the coding region and contains a binding site for an RNA polymerase and has a transcription initiation activity to the mRNA of a downstream gene . In the expression cassette of the present invention, the promoter may be any promoter capable of initiating the expression of the fusion protein.

The term "operably linked" in the present invention means that when one polynucleotide fragment is combined with another polynucleotide fragment, each of these functions or expression is typically affected by other polynucleotide fragments, Means a combination in which each fragment in the combination combination has no detectable effect in performing its function. In addition, the expression cassette of the present invention may further comprise any transcription initiation regulatory sequence and transcription termination regulatory sequence for regulating transcription. Operable linkages can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.

In the present invention, the expression cassette may include, but is not limited to, a polynucleotide encoding a glycosyltransferase and a polynucleotide encoding a target protein. The linker is as described above.

Further, another embodiment of the present invention provides a vector for expressing a fusion protein comprising the expression cassette.

The "fusion protein" of the present invention is as described above.

The expression cassette may be in a form that is contained within a carrier that enables efficient introduction into the cell. The carrier is specifically a vector, and the vector can be used as both a viral vector and a non-viral vector. As viral vectors there may be mentioned, for example, lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus or avium fox, Avipox virus vectors, and the like, but the present invention is not limited thereto. Non-viral vectors include, for example, plasmid forms.

In addition, the expression vector may further comprise a selection marker. The "selection marker" is intended to facilitate screening of transformed cells into which the expression cassette of the present invention has been introduced. The selection marker that can be used in the expression vector of the present invention is not particularly limited as long as it is a gene that can easily detect or measure the introduction of the vector. However, the marker may be exemplified by a drug resistance, a nutritional requirement, Such as EGFP (Green Fluorescent Protein), puromycin, Neomycin (Neo), hygromycin (Hyg), Heath, etc., which confer a selectable phenotype, Histidinol dehydrogenase gene, hisD) or guanine phosphosribosyltransferase (Gpt).

The conjugated protein of the present invention and a fusion protein made of various target proteins can produce a fusion protein-polysaccharide complex from a carbohydrate. When the fusion protein-polysaccharide complex is used, unlike the conventional protein purification method and immobilization method, The target protein can be purified and immobilized. Thus, the present invention can be widely used for production of a target protein and production of a useful product using the target protein.

1 shows the structures of the fusion proteins prepared in Preparation Examples 1 to 7.
Fig. 2 is a graphical representation of the distribution of < RTI ID = 0.0 > Deinococcus & an illustrative dgas) - in geothermalis) derived from amyl sucrase dehydratase (DGAS) and green fluorescent protein (Green Fluorescent Protein, EGFP) fusion in which the fusion protein (an expression vector capable of expressing the EGFP-DGAS) (pHCXHD - egfp .
FIG. 3 is a diagram showing an expression vector (pET- dgas - dgbg ) capable of expressing a fusion protein (DGAS-DGBG) obtained by fusion of DGAS and beta-glucosidase ( DGBG ).
FIG. 4 is a diagram showing an expression vector (pET- dgas - gs - dgbg ) capable of expressing a fusion fusion protein (DGAS-GS-DGBG) in which a DGAS and a DGBG are linked by a GS-linker.
5 is a diagram showing an expression vector (pET- dgas - esto - dgbg ) capable of expressing a fusion protein (DGAS-EstO-DGBG) in which the DGAS and DGBG are linked by an Esterase-O-linker .
Fig. 6 is a diagram showing an expression vector ( pET- malE - dgas- ( lacZ a ) ) capable of expressing a fusion protein (MalE-DGAS) fused with DGAS and a maltose binding protein (MalE ) .
Fig. 7 shows an expression vector (pET- malE - dgas ) capable of expressing a fusion protein (MalE-DGAS) fused with MalE and DGAS.
FIG. 8 is a diagram showing an expression vector (pET- dgas- linker) capable of producing a variety of fusion proteins in which a target protein is introduced into an MCS portion and expressed with DGAS in front.
FIG. 9 is a photograph showing the polysaccharide synthesis activity of the fusion proteins (DGAS-DGBG, DGAS-GS-DGBG and DGAS-EstO-DGBG) containing DGAS.
FIG. 10 shows that EGFP is active in a fusion protein-polysaccharide complex comprising an EGFP-DGAS fusion protein and a polysaccharide.
FIG. 11 is a view showing beta glucosidase (DGBG) activity of a fusion protein (DGAS-DGBG, DGAS-GS-DGBG, DGAS-EstO-DGBG) -polysaccharide complex containing DGAS and DGBG.
FIG. 12 is a diagram for confirming that a target protein, DGBG, can be isolated from a fusion protein (DGAS-linker-DGBG) -polysaccharide complex. Here, kDA is a protein molecular weight marker. Lane 1 is a fusion protein-polysaccharide complex in which the reaction with sugar is completed; Lanes 2 to 5 are supernatants each of which has been subjected to one to four washing operations; Lane 6 is a fusion protein-polysaccharide complex in which four washing operations have been completed; Lane 7 represents the target protein DGBG separated from the fusion protein after the protease (Factor Xa) reaction and present in the supernatant; And lane 8 show the DGAS present in the polysaccharide pellet portion separated from the fusion protein after the protease reaction.
FIG. 13 shows the activity of DGBG immobilized through a fusion protein (DGAS-EstO-DGBG) -polysaccharide complex according to the number of times of reuse.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. The embodiments of the present invention are provided by way of example to facilitate a specific understanding of the present invention.

Manufacturing example  One. Amylosucrase < / RTI > and < RTI ID = Abstract Fluorescent Protein (Green Fluorescent Protein, EGFP ) Was fused Of the fusion protein  Produce

Deinococcus geothermalis) to produce the sucrase dehydratase (DGAS, SEQ ID NO: 1) and a green fluorescent protein (a fusion protein of an enzyme fused to Green Fluorescent Protein, EGFP) as derived from amyl, Day furnace the chromosome of Lactococcus fifth sseoreu Marlies as template DNA The forward primer F_N_DGAS and the reverse primer R_X_DGAS having the sequences shown in Table 1 were designed. The forward primer F_N_EGFP and the reverse primer R_N_EGFP were designed using the pET- egfp vector as a template DNA, and PCR was carried out by the method described in Table 2, respectively.

Primers used in PCR Name of the primer SEQ ID NO: Gene sequence F_N_DGAS Forward 3 5'-CAT ATG CTG AAA GAC GTG CTC ACT-3 ' R_X_DGAS Reverse 4 5'-CTC GAG TGC TGG AGC CTC CCC GGC-3 ' F_N_EGFP Forward 5 5'-CAT ATG GTG AGC AAG GGC GAG-3 ' R_N_EGFP Reverse 6 5'-CAT ATG CTT GTA CAG CTC GTC CAT GC-3 '

PCR conditions denaturalization
(denaturation) Pre-denaturation 94 ° C / 5 min Amplification
lt; / RTI > Denaturation 94 DEG C / 0.5 min 25 cycles Annealing 63 DEG C / 0.5 min Extension 72 ° C / 2 min Final Expansion
(final extension) Final extension 72 ° C / 7 min

The DGAS and pHCXHD vectors (also referred to as 'pHC vectors') obtained by the above PCR were treated with restriction enzymes Nde I and Xho I and ligation together to obtain pHCXHD-dgas Vector. The pHCXHD- dgas After again a vector with a restriction enzyme Nde I treatment, to connect the egfp gene the gene dgas front portion, pHCXHD - dgas vector was prepared (FIG. 2) - egfp. In addition, the pHCXHD - egfp - using dgas vector was prepared DGAS and EGFP fusion fusion proteins (DGAS-EGFP).

Manufacturing example  2. Amylose sucrase and  Beta-glucosidase (beta-glucosidase) glucosidase ) Was fused Of the fusion protein  Produce

In order to prepare an enzyme fused with amylose sucrose (DGAS) derived from Deinococcus zephullis malaria and beta-glucosidase (DGBG) derived from Deinococcus zephar males by the overlap PCR method, As a template DNA, a forward primer F_N_DGAS and a reverse primer R_ASBG having the sequences shown in Table 3 were designed. Also, the forward primer F_DGBG and the reverse primer R_DGBG_X were designed using the chromosome of Deinococcus zeussalmalis as a template DNA, and PCR was carried out by the method described in Table 4, respectively.

Primers used in PCR Name of the primer SEQ ID NO: Gene sequence F_N_DGAS Forward 7 5'-CAT ATG CTG AAA GAC GTG CTC ACT TCT GAA CT-3 ' R_ASBG Reverse 8 5'-AGT CTG GGT CAT TGC TGG AGC CTC CCC-3 ' F_ASBG Forward 9 5'-GAG GCT CCA GCA ATG ACC CAG ACT CGA-3 ' R_DGBG_X Reverse 10 5'-CTC GAG TCT CAG AAA TTG ACG GTA-3 '

PCR conditions denaturalization
(denaturation) Pre-denaturation 94 ° C / 5 min Amplification
lt; / RTI > Denaturation 94 DEG C / 0.5 min 25 cycles Annealing 63 DEG C / 0.5 min Extension 72 ° C / 4 min Final Expansion
(final extension) Final extension 72 ° C / 7 min

Each of the PCR products obtained through the above PCR was mixed and PCR was performed using the forward primer F_N_DGAS and the reverse primer R_DGBG_X using the template DNA as described in Table 4 above. The final product of approximately 3.3 kb of PCR product was ligated with pGEM-T Easy vector to obtain pGEM-T- dgas - dgbg Vector. Afterwards, it was confirmed that the nucleotide sequence was amplified properly by sequencing. The pGEM-T- dgas-dgbg vector and the expression vector pET21a (+) were treated with restriction enzymes Nde I and Xho I, and ligated together to obtain pET- dgas - dgbg (Fig. 3). Also, a fusion protein (DGAS-DGBG) fused with DGAS and DGBG was prepared using the above pET- dgas - dgbg vector.

Manufacturing example  3. DGAS and Of DGBG  Between GS - Linked by a linker Of the fusion protein  Produce

In order to prepare a fusion protein in which a GS-linker ( gs ) is connected between amylose sucrose (DGAS) and beta-glucosidase (DGBG) derived from Deinococcus zephar monkeys, The forward primer F_N_DGAS and the reverse primer R_DGAS_GS_BX having the sequences shown in Table 5 below were designed using the chromosome as the template DNA and PCR was performed according to the method described in Table 2 to obtain a DGAS-GS linker in which DGAS and GS- ≪ / RTI > Here, the amino acid sequence of the GS linker contained in the reverse primer is GGGGSGGGGS (SEQ ID NO: 28), the amino acid GS at the terminal portion of the linker corresponds to BamH I site, and the amino acid LE corresponds to Xho I site. Further, DGBG having BamH I site at the N-terminus and Xho I site at the C-terminus was obtained using the forward primer F_B_DGBG and the reverse primer R_DGBG_X under the PCR conditions shown in Table 2.

Primers used in PCR Name of the primer SEQ ID NO: Gene sequence F_N_DGAS Forward 11 5'-CAT ATG CTG AAA GAC GTG CTC ACT TCT GAA CT-3 ' R_DGAS_GS_BX Reverse 12 5'-CTC GAG GGA TCC ACC ACC GCC CGA GCC ACC GCC ACC TGC TGG AGC CTC CCC GGC GG -3 ' F_B_DGBG Forward 13 5'-GGA TCC ATG ACC CAG ACT CGA CCC GC-3 ' R_DGBG_X Reverse 14 5'-CTC GAG TCT CAG AAA TTG ACG GTA-3 '

The pET21a (+) vector and the DGAS-GS linker thus obtained were treated with restriction enzymes Nde I and Xho I, and ligated together to obtain pET- dgas - gs Vector. The pET- dgas-gs vector was further treated with restriction enzymes BamHI and XhoI to cut off the GS linker end portion and insert the downstream enzyme DGBG. As a result, the Xho I (amino acid LE) portion was removed and a pET- dgas - gs - dgbg vector in which a linker having the sequence GGGGSGGGGS (SEQ ID NO: 28) was completely bound between the two enzymes was obtained (FIG. In addition, the pET- dgas - gs - dgbg (DGAS-GS-DGBG) was prepared by linking DGAS and DGBG with a GS linker using a vector.

Manufacturing example  4. DGAS and Of DGBG  Linked by an Esterase-O-linker Of the fusion protein  Produce

In order to prepare a fusion protein in which the distance between amylose sucrose (DGAS) derived from Deinococcus zephyralis and the beta glucosidase (DGBG) is linked with an Esterase-O-linker (EstO linker, esto ) A forward primer F_N_DGAS and a reverse primer R1_DGAS_EstO having the sequence as shown in Table 6 were designed using the chromosome of Deinococcus zepharthalis as a template DNA. In this case, only a part of the Esterase O linker is synthesized in the reverse primer in order to prevent the annealing efficiency between the primer and the template DNA from deteriorating due to the presence of the template DNA and the non-complementary portion in the primer. Thus, the resultant PCR product was used again as template DNA, and the forward primer F_N_DGAS and the reverse primer R2_DGAS_EstO were used to obtain a DGAS-EstO linker gene to which a complete EstO linker was linked under the PCR conditions shown in Table 2. [ At this time, all the rest of the EstO linker is synthesized in the reverse primer. The amino acid sequence of the entire EstO linker is DKTKYTAS (SEQ ID NO: 29), the amino acid AS at the end of the linker is Nhe I site, and the amino acid LE corresponds to Xho I site do.

Using forward primer F_Nh_DGBG and reverse primer R_DGBG_X, DGBG having Nhe I site at the N-terminus and Xho I site at the C-terminus was obtained under the PCR conditions shown in Table 2.

Primers used in PCR Name of the primer SEQ ID NO: Gene sequence F_N_DGAS Forward 15 5'-CAT ATG CTG AAA GAC GTG CTC ACT TCT GAA CT-3 ' R1_DGAS_EstO Reverse 16 5'-CGT GTA TTT AGT TTT GTC TGC TGG AGC CTC CCC GGC-3 ' R2_DGAS_EstO Reverse 17 5'-CTC GAG GCT AGC CGT GTA TTT AGT TTT-3 ' F_Nh_DGBG Forward 18 5'-GGA TCC ATG ACC CAG ACT CGA CCC GC-3 ' R_DGBG_X Reverse 19 5'-CTC GAG TCT CAG AAA TTG ACG GTA-3 '

The pET21a (+) vector and the DGAS-Esto linker thus obtained were treated with restriction enzymes Nde I and Xho I, and ligated together to obtain pET- dgas - esto Vector. The pET- dgas-esto vector was further treated with restriction enzymes Nhe I and Xho I to cut the terminal portion of the EstO linker and insert the downstream enzyme DGBG. As a result, the Xho I (amino acid LE) portion was removed between the two enzymes, and a linker having the sequence of DKTKYTAS (SEQ ID NO: 29) was ligated to pET- dgas - esto - dgbg Vector was obtained (Fig. 5). Also, a fusion protein (DGAS-EstO-DGBG) in which DGAS and DGBG were linked by an EstO linker was prepared using the above pET- dgas-gs - dgbg vector.

Manufacturing example  5. Maltose  Binding protein (maltose binding protein, MalE )and DGAS  Linked by a linker MalE - DGAS Of the fusion protein  Produce

MalE-DGAS fusion protein with maltose binding protein (MalE) and DGAS linked by linker For the preparation, the forward primer F_N_malE and the reverse primer R_malE_X having the sequence shown in Table 7 were designed using the gene expression vector pMaL-cx2 (New England Biolabs) as the template DNA, and PCR was carried out by the method described in Table 2 To obtain the gene for MalE-lacZ alpha fusion protein (ACCESSION: AFE02922). PCR was performed using the forward primer F_E_DGAS and the reverse primer R_DGAS_H using the chromosome of Deinococcus zeussalmalis as the template DNA and the method described in Table 2. As a result, a 2 kb equivalent dgas gene was obtained.

Primers used in PCR Name of the primer SEQ ID NO: Gene sequence F_N_malE Forward 20 5'-CAT ATG AAA ATC GAA GAA GGT AAA CTG GTA-3 ' R_malE_X Reverse 21 5'-CTC GAG TCC GCC AAA ACA GCC AAG CTG CCA-3 ' F_E_DGAS Forward 22 5'-GAA TTC ATG CTG AAA GAC GTG CTC ACT TCT-3 ' R_DGAS_H Reverse 23 5'-AAG CTT TGC TGG AGC CTC CCC GGC GGT CAG-3 '

Also, pET21a (+) vector and the above-obtained handle-lacZ alpha fusion protein gene to MalE restriction enzymes Nde I and Xho I, then, with ligation pET- malE - was prepared (lacZα) vector. The pET- malE - (lacZα) processes the vector back to a restriction enzyme EcoR I and Hind III, MalE lacZ-alpha fusion protein gene was cut out of the MCS section therein, ligated with the above-obtained gene dgas pET- malE -dgas- ( lacZ? ) vector was prepared (Fig. 6). The MalE-lacZ fusion protein is composed of MalE-linker-MCS-lacZ alpha protein, and the MalE-DGAS fusion protein prepared using the pET- malE - dqas- ( lacZα ) vector is composed of MalE and DGAS as linkers It is connected.

Manufacturing example  6. MalE - DGAS Fusion protein lacZ alpha fusion protein Removed Jung Preparation of the sum protein

In order to prepare a fusion protein from which the lacZ alpha fusion protein was removed from the MalE-DGAS fusion protein, the forward primer F_N_malE having the sequence shown in Table 7 was used as the template DNA, and pMaL-cx2 (New England Biolabs) After designing the reverse primer R_malE_X2 having the sequence shown in Table 8, PCR was carried out as shown in Table 2 to obtain malE ( lacZ? ) Gene malE Only genes were obtained.

PCR was carried out by using the forward primer F_E_DGAS and the reverse primer R_DGAS_H having the sequence shown in Table 7 as a template DNA and using the chromosome of Deinococcus zeussalmalis as a template to obtain a 2 kb equivalent dgas gene .

Primers used in PCR Name of the primer SEQ ID NO: Gene sequence R_malE_X2 Reverse 24 5'-CTC GAG AAG CTT GCC TGC AGG TCG-3 '

Further, the pET21a (+) vector and the obtained malE The gene was treated with restriction enzymes Nde I and Xho I and ligated together to prepare a pET-MalE vector. Processing the vector pET-MalE back to the restriction enzyme EcoR I and Hind III, the cut part was MCS within the MalE gene, ligated with the above-obtained gene dgas pET- malE - dgas Vector (Fig. 7). The structure of malE gene is composed of malE- linker-MCS gene. As a result of inserting dgas gene instead of MCS, MalE-DGAS protein with lacZ alpha fusion protein was produced.

Manufacturing example  7. For the expression of various target proteins pET - DGAS - Preparation of Linker-MCS

PCR was carried out using the forward primer F_SacI_DGAS and the reverse primer R_DGAS_SacI_Mutant having the sequence shown in Table 9 as the template DNA, and a dgas gene equivalent to 2 kb was obtained as a result of the chromosome of Deinococcus zeosharmalis as the template DNA. Then, the ligation was cut to one side of pMaL-cx2 with restriction enzyme Sac I was inserted to a portion between the MalE gene dgas and linker in pMal-cx2. Using this plasmid as the template DNA, the forward primer F_N_DGAS having the sequence shown in Table 6 and the reverse primer R_malE_X3 having the sequence shown in Table 9 were subjected to PCR to obtain the DGAS-linker-MCS gene.

Primers used in PCR Name of the primer SEQ ID NO: Gene sequence F_SacI_DGAS Forward 25 5'-GAG CTC ATG CTG AAA GAC GTG CTC-3 ' R_DGAS_SacI_Mutant Reverse 26 5'-CGA GCT CGC TGG AGC CTC CCC GGC-3 ' R_malE_X3 Reverse 27 5'-CTC GAG GTC GAC TCT AGA GGA-3 '

The pET21a (+) vector and the obtained DGAS-linker-MCS gene were treated with restriction enzymes Nde I and Xho I and ligated together to prepare a pET- dgas -linker-MCS (pET- dgas- M linker) vector (Fig. 8). When the gene of the desired protein is inserted into the MCS portion of the pET- dgas -linker-MCS, various fusion proteins having the DGAS protein attached thereto can be produced.

The target protein may be any protein desired by a person skilled in the art, and may be a biological protein selected from the group consisting of medical, research and industrial proteins such as antigen, antibody, cell receptor, enzyme, structural protein, serum, Can be various proteins having activity.

Example  One. Of the fusion protein  Identification of expression and activity

To obtain a fusion protein of Figure 1 in large quantities, and that the pHCXHD- egfp -dgas vector of Preparative Example 1, Escherichia coli (E. coli) MC1061 inserted incubated at 37 ℃ for 15 hours was expressed a fusion protein, Preparation 2 E. coli BL21 (DE3) into which the pET vector was inserted was cultured at 18 DEG C for 18 hours in the presence of 0.5 mM IPTG (Isopropyl beta-D-1-thiogalactopyranoside) to induce the expression of the fusion protein. After the incubation, the cells were recovered by performing centrifugation at 4 ° C and 4,000 rpm for 30 minutes. Then, the suspension in the fusion buffer (Lysis buffer) [50 mM NaH 2 PO 4, pH 7.0, 300 mM NaCl, 10 mM imidazole, pH 7.0], was pulverized ultrasound. Thereafter, the pulverized liquid was subjected to centrifugation at 4 ° C and 12,000 rpm for 10 minutes, and a supernatant was obtained. The supernatant was injected into Ni-NTA affinity chromatography, and the recombinant protein was purified to confirm its expression.

Further, in order to measure the amylose sucrose (DGAS) activity of the fusion protein, the fusion protein expressed from the recombinant vector was reacted with sugar (sucrose). Amylose sucrase (DGAS) can synthesize or elongate amylose of alpha-1,4 linkages from sugar. First, 40 μl of a 1 M sugar solution and 50 μl of 100 mM Tris-HCl (pH 8.0) were mixed, and 10 μl of the purified fusion protein was added. After reacting at 30 ° C for 3 hours, 300 μl of DNS was added And the activity of amylose sucrase was confirmed by coloring DNS and reducing sugar in boiling water for 5 minutes.

Expression of the green fluorescent protein (EGFP) used as the target protein was confirmed by fluorescence microscopy.

In order to measure the activity of beta-glucosidase (DGBG), 40 μl of 10 mM p- nitrophenyl-β-D-glucopyranoside and 50 μl of 100 mM Tris-HCl (pH 8.0) were mixed, 10 μl of protein was added and reacted at 40 ° C for 10 minutes to confirm the yellow color of p- nitrophenol liberated by the enzyme reaction.

As a result, a fusion protein including a glycosyltransferase and a target protein was expressed, and the fusion protein had all the activities of the two fused enzymes.

Example  2. Fusion protein - Preparation of polysaccharide complexes and methods for increasing the production rate

Example  2-1. Fusion protein - Preparation of polysaccharide complex

In order to prepare a fusion protein-polysaccharide complex using the fusion protein containing the sugar-transferase, the fusion protein according to the above-mentioned preparation example was first reacted with sugar. The reaction solution was then centrifuged at 13,000 rpm to obtain a white precipitate (Fig. 9). As a result, it was found that DGAS of the fusion protein was active, and a fusion protein-polysaccharide complex was produced.

In addition, fluorescence microscopy confirmed that EGFP was active in the fusion protein-polysaccharide complex of EGFP-DGAS fusion protein and polysaccharide (FIG. 10).

Also, 40 μl of 10 mM p- nitrophenyl-β-D-glucopyranoside and 50 μl of 100 mM Tris-HCl (pH 8.0) were added to the resulting fusion protein-polysaccharide complex and reacted at 40 ° C. for 10 minutes. The yellow of p- nitrophenol liberated by the fusion protein-polysaccharide complex was found to be active (FIG. 11).

As a result, using the fusion protein of the present invention, it was found that a fusion protein-polysaccharide complex was produced, and that the resulting fusion protein-polysaccharide complex had all the activities of the two fusion proteins.

Example  2-2. Fusion protein - Methods for increasing production rate of polysaccharide complex

In order to confirm that the production rate of the fusion protein-polysaccharide complex increases when the monosaccharide or the disaccharide and the polysaccharide are added to the substrate reacting with the fusion protein, 200 μl of 1 M sugar solution and 100 mM Tris-HCl (pH 8.0) 500 [mu] l was mixed with 5% amylopectin, glycogen, or soluble starch (200 [mu] l) to prepare a substrate. Then, when 100 μl of the fusion protein was added to the substrate and the reaction was carried out at 45 ° C for 2 hours or more, when the amylopectin and the soluble starch were added, the fusion protein-polysaccharide complex was produced most in a short time and the enzyme activity was strong. This is because amylopectin and soluble starch have a high specificity as a substrate for DGAS, so sugar transfer for growing the branch has occurred rapidly.

Thus, the addition of a monosaccharide or a disaccharide and a polysaccharide to a substrate that reacts with the fusion protein increases the production rate of the fusion protein-polysaccharide complex.

Example  3. Fusion protein - Purification method of target protein using polysaccharide complex

Example  3-1. Fusion protein - Isolation of polysaccharide complex

E. coli expressing the fusion protein was pulverized by the method described in Example 1 and then centrifuged to prepare a supernatant containing the fusion protein. Then, the supernatant was reacted with sugar to prepare a fusion protein-polysaccharide complex.

Since the fusion protein-polysaccharide complex can be obtained by centrifugation, the desired protein can be easily purified from the cell lysate by one step by centrifugation. This indicates that the supernatant, which is a conventional purification method, is a very simple one-step purification method as compared with the method of injecting the supernatant into Ni-NTA affinity chromatography, and thus can be very useful for producing a target protein.

Example  3-2. Fusion protein - through the protease from the polysaccharide complex Of the target protein  detach

The commercially available pMaL-cx2 vector has a site inserted therein that is cleaved by a protease (Factor Xa). In addition, various pMAL vectors have protease cleavage sites such as Enterokinase and Genenase. The vectors of Preparative Examples 5 to 7 have a linker and a cleavage site between the fused proteins. When the protease is treated, the enzyme attached to the C-terminus of the linker is released. Therefore, when the protease is treated in the fusion protein-polysaccharide complex, the target protein at the C-terminus is cleaved, so that only the desired protein can be secondary purified again from the fusion protein.

Specifically, in order to confirm that the target protein can be isolated through the fusion protein-polysaccharide complex, the DGBG gene was inserted into the MCS portion of the pET- dgas -linker-MCS vector prepared in Preparation Example 7 as a target protein and expressed , E. coli having a fusion protein of DGAS-linker-DGBG was obtained. Escherichia coli expressing the fusion protein was pulverized by the method described in Example 3-1 and then centrifuged to obtain a supernatant containing the fusion protein. Then, the supernatant was reacted with sugar to prepare a fusion protein-polysaccharide complex. The fusion protein-polysaccharide complex was obtained by centrifugation, suspended in an equal amount of 100 mM Tris-HCl (pH 8.0) as the initial supernatant, and washed four times to obtain a fusion protein-polysaccharide complex by centrifugation , A fusion protein-polysaccharide complex in which another E. coli protein was washed was obtained. Then, 1 mg of the washed fusion protein-polysaccharide complex was reacted with 0.02 mg of Factor Xa, which is a protease, in a buffer of 20 mM Tris-HCl (pH 8.0) at 23 캜 for 6 hours or longer and centrifuged.

As a result, it was confirmed that the DGAS and DGBG were separated by decomposing the linker containing the protease (Factor Xa) sequence in the fusion protein of DGAS-linker-DGBG, and the separated DGBG could be obtained in the supernatant portion, (Fig. 12). ≪ tb > < TABLE >

Thus, it was found that the target protein can be easily purified using the fusion protein-polysaccharide complex of the present invention.

Example  4. Fusion protein - Immobilization method of target protein using polysaccharide complex

To confirm that the target protein can be immobilized through the fusion protein-polysaccharide complex, 50 mM Tris-HCl (pH 8.0) was added to the fusion protein-polysaccharide complex produced by the amylopectin and the soluble starch prepared in Example 2, The reaction mixture was reacted with 10 mM p- nitrophenyl-β-D-glucopyranoside as a substrate of protein (DGBG) The fusion protein-polysaccharide complex was again obtained. Then, the fusion protein-polysaccharide complex obtained again was reacted with p- nitrophenyl-β-D-glucopyranoside in the same manner as above and reused.

As a result, it was confirmed that the fusion protein-polysaccharide complex could be easily separated from the reaction mixture by centrifugation, and that the DGBG of the complex was maintained at 100% activity for up to 5 cycles, and the fusion protein- polysaccharide complex was reused (Fig. 13).

Thus, it can be seen that the fusion protein-polysaccharide complex of the present invention can easily immobilize a target protein and can be widely used for producing a useful product using a target protein.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> University-Industry Cooperation Group of Kyung Hee University <120> A method for producing fusion protein-polysaccharide complex and          use thereof <130> KPA141233-KR-P1 <150> KR 10-2016-0019296 <151> 2016-02-18 <160> 29 <170> Kopatentin 2.0 <210> 1 <211> 650 <212> PRT <213> Deinococcus geothermalis <400> 1 Met Leu Lys Asp Val Leu Thr Ser Glu Leu Ala Ala Gln Val Arg Asp   1 5 10 15 Ala Phe Asp Asp Asp Arg Asp Ala Glu Thr Phe Leu Leu Arg Leu Glu              20 25 30 Arg Tyr Gly Glu Asp Leu Trp Glu Ser Leu Arg Ala Val Tyr Gly Asp          35 40 45 Gln Val Arg Ala Leu Pro Gly Arg Leu Leu Glu Val Met Leu His Ala      50 55 60 Tyr His Ala Arg Pro Ala Glu Leu Arg Arg Leu Asp Glu Ala Arg Leu  65 70 75 80 Leu Arg Pro Asp Trp Leu Gln Arg Pro Glu Met Val Gly Tyr Val Ala                  85 90 95 Tyr Thr Asp Arg Phe Ala Gly Thr Leu Lys Gly Val Glu Glu Arg Leu             100 105 110 Asp Tyr Leu Glu Gly Leu Gly Val Lys Tyr Leu His Leu Met Pro Leu         115 120 125 Leu Arg Pro Arg Glu Gly Glu Asn Asp Gly Gly Tyr Ala Val Gln Asp     130 135 140 Tyr Arg Ala Val Arg Pro Asp Leu Gly Thr Met Asp Asp Leu Ser Ala 145 150 155 160 Leu Ala Arg Ala Leu Arg Gly Arg Gly Ile Ser Leu Val Leu Asp Leu                 165 170 175 Val Leu Asn His Val Ala Arg Glu His Ala Trp Ala Gln Lys Ala Arg             180 185 190 Ala Gly Asp Pro Lys Tyr Arg Ala Tyr Phe His Leu Phe Pro Asp Arg         195 200 205 Arg Gly Pro Asp Ala Phe Glu Ala Thr Leu Pro Glu Ile Phe Pro Asp     210 215 220 Phe Ala Pro Gly Asn Phe Ser Trp Asp Glu Glu Ile Gly Glu Gly Glu 225 230 235 240 Gly Gly Trp Val Trp Thr Thr Phe Asn Ser Tyr Gln Trp Asp Leu Asn                 245 250 255 Trp Ala Asn Pro Asp Val Phe Leu Glu Phe Val Asp Ile Ile Leu Tyr             260 265 270 Leu Ala Asn Arg Gly Val Glu Val Phe Arg Leu Asp Ala Ile Ala Phe         275 280 285 Ile Trp Lys Arg Leu Gly Thr Asp Cys Gln Asn Gln Pro Glu Val His     290 295 300 His Leu Thr Arg Ala Leu Arg Ala Ala Ala Arg Ile Val Ala Pro Ala 305 310 315 320 Val Ala Phe Lys Ala Glu Ala Ile Val Ala Pro Ala Asp Leu Ile His                 325 330 335 Tyr Leu Gly Thr Arg Ala His His Gly Lys Val Ser Asp Met Ala Tyr             340 345 350 His Asn Ser Leu Met Val Gln Leu Trp Ser Ser Leu Ala Ser Arg Asn         355 360 365 Thr Arg Leu Phe Glu Glu Ala Leu Arg Ala Phe Pro Pro Lys Pro Thr     370 375 380 Ser Thr Thr Trp Gly Leu Tyr Val Arg Cys His Asp Asp Ile Gly Trp 385 390 395 400 Ala Ile Ser Asp Glu Asp Ala Ala Arg Ala Gly Leu Asn Gly Ala Ala                 405 410 415 His Arg His Phe Leu Ser Asp Phe Tyr Ser Gly Gln Phe Pro Gly Ser             420 425 430 Phe Ala Arg Gly Leu Val Phe Gln Tyr Asn Pro Val Asn Gly Asp Arg         435 440 445 Arg Ile Ser Gly Ser Ala Ala Leu     450 455 460 Glu Thr Gly Asp Pro Gly Arg Ile Glu Asp Ala Val Arg Arg Leu Leu 465 470 475 480 Leu Leu His Thr Val Ile Leu Gly Phe Gly Gly Val Pro Leu Leu Tyr                 485 490 495 Met Gly Asp Glu Leu Ala Leu Leu Asn Asp Tyr Ala Phe Glu Asp Val             500 505 510 Pro Glu His Ala Pro Asp Asn Arg Trp Val His Arg Pro Gln Met Asp         515 520 525 Trp Ala Leu Ala Glu Arg Val Glu Glu Pro Ser Ser Pro Ala Gly     530 535 540 Arg Val Asn Thr Gly Leu Arg His Leu Leu Arg Val Arg Arg Asp Thr 545 550 555 560 Pro Gln Leu His Ala Ser Ile Glu Ser Gln Val Leu Pro Ser Pro Asp                 565 570 575 Ser Arg Ala Leu Leu Leu Arg Arg Asp His Pro Leu Gly Gly Met Val             580 585 590 Gln Val Tyr Asn Phe Ser Glu Glu Thr Val Met Leu Pro Ser Ser Val         595 600 605 Leu Arg Asp Val Leu Gly Asp His Val Gln Asp Arg Leu Ser Gly Ser     610 615 620 Ala Phe Arg Leu Asp Arg Pro Thr Val Arg Leu Glu Gly Tyr Arg Ala 625 630 635 640 Leu Trp Leu Thr Ala Gly Glu Ala Pro Ala                 645 650 <210> 2 <211> 1953 <212> DNA <213> Deinococcus geothermalis <400> 2 atgctgaaag acgtgctcac ttctgaactg gcggcgcagg tacgagacgc cttcgatgat 60 gaccgtgacg ccgagacgtt cctgctgcgg ctggaacgct acggcgagga cctctgggag 120 agcctgcgcg cggtgtatgg cgaccaggtg agggccttgc cagggcgact gctggaagtc 180 atgctccacg cctatcacgc ccgccccgcg gagctgcggc gtttggacga ggcccggctg 240 ctgcggcccg actggctgca acgtcccgag atggtgggct acgtcgccta caccgaccgt 300 tttgccggaa cgctgaaggg ggtagaggag cgcttggact acctggaggg cctgggtgtg 360 aagtacctgc acctgatgcc ccttctcagg ccgcgcgagg gcgaaaatga cggtggctac 420 gcggtgcagg attaccgagc ggtgcgtccc gacctgggca cgatggatga cctctcggcc 480 ctcgcgcggg cgctgcgggg ccgcggcatc agcctggtgc tggatctcgt gctgaaccac 540 gtggcgcgcg aacatgcgtg ggcccagaag gcgcgggcgg gtgatcccaa gtaccgggcc 600 tactttcatc tcttccccga ccgcaggggg ccggacgctt ttgaagccac ccttcctgag 660 atctttcccg acttcgcgcc gggcaacttc tcgtgggacg aggagatcgg tgaaggcgag 720 gggggctggg tctggaccac cttcaacagc taccagtggg acctgaactg ggccaacccc 780 gacgtgtttc tggagtttgt ggacatcatc ctctacctcg ccaaccgggg cgtggaggtg 840 ttccggctgg atgcgatcgc cttcatctgg aagcggctgg gaaccgactg ccaaaaccag 900 ccggaagttc accacctcac gcgggcgctg cgggcagccg cgcgcatcgt cgcgcccgca 960 gtcgccttta aggccgaggc gatcgtggcg cccgccgacc tgatccacta cctgggcacc 1020 cgtgcgcacc acggcaaggt gagcgacatg gcctaccaca acagcctgat ggtgcagctg 1080 tggagtagcc tcgccagccg gaatacccgt ctctttgagg aggcactgcg ggcgtttccc 1140 cccaagccca cgagcacgac ctgggggctg tacgtccgct gtcacgacga catcggctgg 1200 gccatcagcg acgaggacgc ggcccgggcc ggattgaacg gcgcggcgca ccggcacttt 1260 ctctcggact tctacagcgg tcagtttccc ggctcctttg cgcgggggct ggtgtttcag 1320 tacaacccgg tgaacggcga ccggcgcatc agtggctcgg cggccagcct cgctgggctg 1380 gaggcagcgc tggaaaccgg ggacccgggc cgcatcgagg acgcggtgcg tcgcctgctg 1440 ctcctccaca cggtcattct cggcttcggc ggggtgccgc tgctgtacat gggcgacgaa 1500 ctcgccctgc tgaatgacta cgccttcgag gacgtgcccg aacacgcgcc ggacaaccgc 1560 tgggtgcatc gcccgcagat ggattgggcc ctcgcggagc gggtgcggca ggagccttcc 1620 tcgcccgccg gacgggtgaa cacgggcctg cgccacctcc tgcgggtgcg ccgcgatacc 1680 ccgcagctgc acgccagcat cgagagccag gtgctgccca gccccgattc gcgtgcgctt 1740 ctgctgcgcc gcgaccatcc cctcggcggg atggtgcagg tgtacaactt cagcgaggag 1800 acggtgatgc tgcccagcca tgttctgcgg gacgtgctgg gggaccacgt ccaggaccgg 1860 ctgagcggga gtgcctttcg cctagatcgg cccaccgttc gcctggaggg ctaccgggca 1920 ctgtggctga ccgccgggga ggctccagca taa 1953 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> F_N_DGAS primer <400> 3 catatgctga aagacgtgct cact 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> R_X_DGAS primer <400> 4 ctcgagtgct ggagcctccc cggc 24 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> F_N_EGFP primer <400> 5 catatggtga gcaagggcga g 21 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> R_N_EGFP primer <400> 6 catatgcttg tacagctcgt ccatgc 26 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F_N_DGAS primer <400> 7 catatgctga aagacgtgct cacttctgaa ct 32 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R_ASBG primer <400> 8 agtctgggtc attgctggag cctcccc 27 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F_ASBG primer <400> 9 gaggctccag caatgaccca gactcga 27 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> R_DGBG_X primer <400> 10 ctcgagtctc agaaattgac ggta 24 <210> 11 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F_N_DGAS primer <400> 11 catatgctga aagacgtgct cacttctgaa ct 32 <210> 12 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> R_DGAS_GS_BX primer <400> 12 ctcgagggat ccaccaccgc ccgagccacc gccacctgct ggagcctccc cggcgg 56 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> F_B_DGBG primer <400> 13 ggatccatga cccagactcg acccgc 26 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> R_DGBG_X primer <400> 14 ctcgagtctc agaaattgac ggta 24 <210> 15 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F_N_DGAS primer <400> 15 catatgctga aagacgtgct cacttctgaa ct 32 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> R1_DGAS_EstO primer <400> 16 cgtgtattta gttttgtctg ctggagcctc cccggc 36 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R2_DGAS_EstO primer <400> 17 ctcgaggcta gccgtgtatt tagtttt 27 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> F_Nh_DGBG primer <400> 18 ggatccatga cccagactcg acccgc 26 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> R_DGBG_X primer <400> 19 ctcgagtctc agaaattgac ggta 24 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F_N_malE primer <400> 20 catatgaaaa tcgaagaagg taaactggta 30 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R_malE_X primer <400> 21 ctcgagtccg ccaaaacagc caagctgcca 30 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F_E_DGAS primer <400> 22 gaattcatgc tgaaagacgt gctcacttct 30 <210> 23 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R_DGAS_H primer <400> 23 aagctttgct ggagcctccc cggcggtcag 30 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> R_malE_X2 primer <400> 24 ctcgagaagc ttgcctgcag gtcg 24 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> F_SacI_DGAS primer <400> 25 gagctcatgc tgaaagacgt gctc 24 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> R_DGAS_SacI_Mutant primer <400> 26 cgagctcgct ggagcctccc cggc 24 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> R_malE_X3 primer <400> 27 ctcgaggtcg actctagagg a 21 <210> 28 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> GS-linker <400> 28 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser   1 5 10 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Esterase-O-linker <400> 29 Asp Lys Thr Lys Tyr Thr Ala Ser   1 5

Claims (20)

A method for producing a fusion protein-polysaccharide complex, comprising the step of reacting a fusion protein containing a glycosyltransferase and a target protein with a carbohydrate. The method for producing a fusion protein-polysaccharide complex according to claim 1, wherein the sugar transferase is at least one selected from the group consisting of hexosyltransferase, pentosyltransferase, and sialyltransferase. The method of producing a fusion protein-polysaccharide complex according to claim 1, wherein the sugar transferase is amylose sucrose. The method of claim 1, wherein the carbohydrate is selected from the group consisting of glucose, fructose, mannose, galactose, ribose, maltose, sucrose, cellobiose, gentiobiose, melibiose, lactose, turanose, Soluble starch, and soluble starch. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; The fusion protein according to claim 1, wherein the fusion protein comprises the N-terminus of the glycosyltransferase; C-terminal; Or the N-terminus and the C-terminus of the fusion protein-polysaccharide complex. 2. The method according to claim 1, wherein the fusion protein is a fusion protein-polysaccharide complex wherein the sugar transferase is bound to the N-terminus and the C-terminus of the target protein. The method for producing a fusion protein-polysaccharide complex according to claim 1, wherein the fusion protein is obtained by linking a glycosyltransferase and a target protein via a linker. The method for producing a fusion protein-polysaccharide complex according to claim 1, wherein the target protein is selected from the group consisting of an antigen, an antibody, a cell receptor, an enzyme, a structural protein, a serum and a cell protein. (a) producing a reaction solution containing a fusion protein-polysaccharide complex by reacting a lysate or a culture of a transformant expressing a fusion protein containing a glycosyltransferase and a target protein with a carbohydrate;
(b) separating the fusion protein-polysaccharide complex from the reaction solution; And
(c) separating the target protein from the fusion protein-polysaccharide complex. [10] The method of claim 9, wherein the step (b) comprises centrifuging the reaction mixture to separate the fusion protein-polysaccharide complex. 10. The method of claim 9, wherein the sugar transferase is at least one selected from the group consisting of hexosyltransferase, pentosyltransferase, and sialyltransferase. 10. The method for purifying a target protein according to claim 9, wherein the glycosyltransferase is amylose sucrose. 10. The method of claim 9, wherein the carbohydrate is selected from the group consisting of glucose, fructose, mannose, galactose, ribose, maltose, sucrose, cellobiose, gentiobiose, melibiose, lactose, turanose, sophorose, amylose, amylopectin, glycogen and Soluble starch, and soluble starch. [10] The method of claim 9, wherein the step (c) comprises cleaving a linker of the fusion protein in the fusion protein-polysaccharide complex to separate the target protein. (a) producing a reaction solution containing a fusion protein-polysaccharide complex by reacting a lysate or a culture of a transformant expressing a fusion protein containing a glycosyltransferase and a target protein with a carbohydrate; And
(b) separating the fusion protein-polysaccharide complex from the reaction solution. A fusion protein including a glycosyltransferase and a target protein; And a polysaccharide. A fusion protein comprising a glycosyltransferase and a target protein. A fusion protein expression cassette comprising a promoter and a polynucleotide encoding a glycosyltransferase and a protein of interest operably linked to the promoter. 19. The fusion protein expression cassette of claim 18, wherein the polynucleotide encoding the glycosyltransferase and the polynucleotide encoding the desired protein are linked by a linker. 19. A vector for expressing a fusion protein comprising the fusion protein expression cassette of claim 18 or 19.

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