KR20190086908A - Method of producing recombinant protein by combining multimerization moiety and solubility controlling technique and use thereof - Google Patents

Abstract

The present invention relates to a method of producing recombinant proteins through combination of a multiplexing element and a solubility controlling technique, and a use thereof and, more specifically, to a method of producing recombinant fusion proteins, in which a domain capable of forming multimers through a disulfide bond is included, and a solubility controlling technique is grafted in and out of the domain and a target protein, and then fused and expressed, thereby producing recombinant proteins having enhanced solubility and folding properties, and to a use thereof for medical or industrial production. According to the present invention, the method of producing recombinant proteins by using a multiplexing element as a fusion partner and grafting a translation control peptide is capable of overcoming limits regarding solubility and folding properties that the existing fusion partner has and exhibits excellent in vivo stability, thereby being applied widely to prepare proteins for industrial purposes as well as protein drugs.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing a recombinant protein by combining a multiplexing factor and a solubility control technique,

The present invention relates to a recombinant protein production method and a use thereof by grafting a multiplexing element and a solubility control technology, and more particularly, to a recombinant protein production method comprising a domain capable of forming a complex through a disulfide bond, To a method for producing a recombinant fusion protein having an improved solubility and folding by grafting with an internal or external part of a target protein and its use for pharmaceutical production or industrial production.

Recombinant proteins (including peptides) are proteins that encode high-value-added genetic products derived from animals, plants, yeast, bacteria, etc., and transgenic proteins that are genetically engineered to produce large quantities of products Quot; Particularly, when producing useful recombinant proteins using recombinant microorganism technology, Escherichia coli, which has a universal technology for genetic engineering and is easy to mass-grow, is widely used.

A variety of expression systems have been developed to produce recombinant proteins in E. coli. However, when E. coli is used as a host cell, the recombinant protein is degraded by proteolytic enzymes in E. coli, and the yield of the recombinant protein is often lowered. In particular, when the peptide having a molecular weight of 10 kDa or less is expressed, have. In addition, overexpression of recombinant proteins in E. coli often results in the formation of insoluble structures known as inclusion bodies, leading to the need to return proteins to the active form (Marston FA, Biochem J 240 (1): 1 -12, 1986).

In order to obtain recombinant proteins of high activity in E. coli in a high yield, various methods of optimizing genetic engineering techniques and expression conditions have been attempted, and the expression of fusion with fusiom partner, which helps folding of the target proteins, It is generally used.

The fusion of the proteins or polypeptides having the ability to improve expression at the amino terminus leads to overexpression of the protein. Examples of the fusion partner include maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (Trx), N-utilizing substance protein A (NusA), and the like. These fusion partners are known to be capable of easily purifying the expressed fusion protein using affinity chromatography and assist folding of the target protein by using different molecular biological mechanisms. However, since the fusion partner has a relatively large size as compared with the target protein, the yield of the target protein is remarkably deteriorated depending on the structure and size of the fusion target. This is because the mass ratio of the target protein or the peptide to the fusion protein is relatively small. In addition, in the case of peptide-based drugs, maintenance of effective blood concentration is particularly difficult as compared with other low-molecular-weight drugs, and solubility is generally low, making formulation difficult. Short half-life, low in-vivo stability and low bioavailability (BA) are important improvements in the development of drugs for the prevention and treatment of peptides. Also, when fused with a polypeptide or protein having no target function or enzyme activity, the recombinant protein in the form of a monomer has a possibility of lowering the target ability or losing its activity, so that the above method can be applied to a protein useful for medical or industrial purposes It is difficult to apply the method to such a problem.

Korean Patent Publication No. 2014-0112888 (Apr. 24, 2014).

In order to solve the above-mentioned problems, the present invention provides a method for producing oligonucleotides, which comprises a cartilage oligomeric matrix protein (COMP) for inducing oligomerization in order to produce small peptides or proteins at a high yield, And to provide a method for producing an active recombinant fusion protein in which a specific sequence is grafted inside and outside of a fusion protein and a target protein (peptide), thereby remarkably improving the stability and targeting ability in the body.

One aspect of the present invention is

a) a gene encoding an autogenous multimeric protein; And

  Wherein the nucleic acid sequence encoding the peptide for controlling the translation rate is selected from the group consisting of a gene encoding a target protein or a nucleic acid sequence encoding a fusion protein of the self- Preparing a vector comprising the sequence;

b) transforming the vector into a host cell to produce a transformant; And

c) culturing the transformant to obtain a recombinant protein;

And a method for producing the recombinant fusion protein.

Further, another aspect of the present invention relates to a recombinant fusion protein produced by the above-mentioned method for producing a recombinant fusion protein.

The method of producing the recombinant protein by using the multiplexing element according to the present invention as a fusion partner and grafting the translation modulating peptide not only overcomes the limitations on the water solubility and foldability of the existing fusion partner, It is expected to be widely used for industrial applications including protein drugs.

1 is a result of SDS-PAGE of cartilage oligomer substrate protein (COMP) expression, confirming self oligomerization of a cartilage oligomer substrate protein according to reducing agent oil (A) no (B) added to a sample. (lane T, total protein; S, soluble protein; U, unbound protein: W, Washing step; Number 1-10, refined protein).
FIG. 2 shows the result of SDS-PAGE of the labeled protein fused with the cartilage oligomer substrate protein. As a result, the oligomer of the cartilage oligomer substrate protein-labeled protein with or without the reducing agent added to the fluorescent protein mCherry (A) and Luciferase Rluc8 It was confirmed. (lane T, total protein; S, soluble protein; U, unbound protein: W, Washing step; Number 1-9, purified protein).
FIG. 3 shows the results of confirming the activity of the fluorescent proteins mCherry (A) and rufferase Rluc8 (B), which are fusion-expressed with cartilage oligomer substrate proteins. (T, total protein; S, soluble protein).
FIG. 4 shows the result of SDS-PAGE in which insulin was fused to glutathione-S-transferase (GST) (A) and maltose binding protein (MBP) (B) (Total, total protein, Soluble, soluble protein, insoluble, insoluble protein).
Figure 5 shows SDS-PAGE results of expression of insulin fused with cartilage oligomeric protein containing translation modulating peptide. Expression rates were calculated at the expressed protein / total protein ratio. COMP-proinsulin (R2-C-In); D, pET-R3-COMP-proinsulin (R3- C-In); E, pET_R4-COMP-proinsulin (R4-C-In).
FIG. 6 shows SDS-PAGE results after removal of trypsin-treated fusion partner GST (A) fused with insulin and cartilage oligomer substrate protein (B).
FIG. 7 shows the result of confirming residual insulin in the mouse blood. (Insulin (R4-c), insulin protein obtained by trypsin treatment of R4-C-In protein).
FIG. 8 shows the result of checking glucose concentration in mouse blood by insulin.
Figure 9 shows SDS-PAGE results of expression of human growth hormone fused with cartilage oligomeric protein containing translation modulating peptide. The oligomerization of the cartilage oligomeric substrate protein-human growth hormone fusions according to the reducing agent oil (A) (B) added to the sample was confirmed. (lane T, total protein; S, soluble protein; U, unbound protein: W, Washing step; Number 1-10, refined protein).
FIG. 10 shows the result of confirming residual human growth hormone in mouse blood. (CR-hGH, human growth hormone fused with cartilage oligomeric substrate protein).
Figure 11 shows the results of SDS-PAGE expression of granulocyte stimulating factor fused with cartilage oligomeric protein containing translation modulating peptide. The oligomerization of the cartilage oligomeric substrate protein-human growth hormone fusions according to the reducing agent oil (1) (2) added to the sample was confirmed.
FIG. 12 shows the result of confirming residual granulocyte stimulating factor in mouse blood. (RC-G_CSF, granulocyte stimulating factor fused with cartilage oligomeric matrix protein).
FIG. 13 shows SDS-PAGE (A) and Gel filtration (B) expression of epidermal growth factor receptor (EGFR) binding domain fused with a cartilage oligomer substrate protein containing a translation modulating peptide. The oligomerization of the cartilage oligomeric substrate protein-human growth hormone fusions with or without reducing agent added to the sample was confirmed.
FIG. 14 shows the results of flow cytometry (FACS) using HT29 cells (A) and A549 cells (B) to confirm the binding ability of a cartilage oligomer substrate protein containing a translation modulating peptide to a epidermal growth factor receptor binding domain fused.
FIG. 15 is a result of FITC labeling of the epidermal growth factor receptor binding domain fused with the cartilage oligomer substrate protein containing the translational control peptide, and confirming the binding ability by HT-29 cells by enzyme-linked immunosorbent assay.
FIG. 16 shows SDS-PAGE results of expression of an RGD binding peptide and a fluorescent protein mCherry fusion (A) fused with COMP and an RGD binding peptide (B) fused with a cartilage oligomer substrate protein. Oligomerization of COMP-RGD binding peptide fusions with or without reducing agent added to the sample.
Figure 17 shows the results of SDS-PAGE (A) and Western bolt (B) expression of the RGD binding peptide fused with the cartilage oligomeric protein per time.
Figure 18 shows SDS-PAGE results of expression of an RGD binding peptide fused with a cartilage oligomeric protein containing a translation modulating peptide. (1, pET_T-COMP without derivative 2, pET_T-COMP with derivative 3, pET_T-R-COMP without derivative 4, pET_T-R-COMP with derivative).
Figure 19 shows the results of SDS-PAGE of RGD binding peptide and fluorescent protein mCherry fusion expression fused with cartilage oligomer substrate protein containing translation modulating peptide. (A, pET_T-COMP-R1-mCherry; B. pET_T-R2-COMP-mCherry; C, pET_T-COMP-mCherry-R3).
FIG. 20 shows the binding potency of the RGD binding peptide and the fluorescent protein mCherry fused with the cartilage oligomer substrate protein containing the translational control peptide by fluorescence intensity (A) and enzyme-linked immunosorbent assay (B). (TC-R1-M, pET_T-COMP-R1-mCherry; T-R2-CM, pET_T-R2-COMP-mCherry; TCM-R3, pET_T-COMP-mCherry-R3; TM, pET_T-mCherry).

The present invention will now be described in detail with reference to the accompanying tables or drawings.

Where a drawing is described, it is provided as an example to enable those skilled in the art to fully understand the spirit of the invention. Therefore, the present invention is not limited to the illustrated drawings, but may be embodied in other forms, and the drawings may be exaggerated in order to clarify the spirit of the present invention.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

In the present invention, the term "target protein" refers to any protein that a person skilled in the art intends to produce in large quantities, and which can be expressed in a transformant by inserting a polynucleotide encoding the protein into a recombinant expression vector.

Also, a "recombinant protein" or "fusion protein" means a protein to which another protein is linked or another amino acid sequence is added to the N-terminal or C-terminal of the original target protein sequence. Refers to a recombinant fusion protein in which the fusion partner of the present invention and the target protein are linked or a recombinant protein of the target protein from which the fusion partner has been removed by the protein cleavage enzyme.

An " expression vector "is a linear or circular DNA molecule composed of elements that are provided for gene transcription and translation, and fragments encoding operably linked polypeptides composed of additional fragments. Additional fragments include promoter and transcription termination sequences. The expression vector includes one or more replication start points and one or more selection markers and the like. Expression vectors are generally derived from plasmid or viral DNA, or comprise elements of both.

"Self-assembling multimeric protein" or "coiled coil domain" refers to a protein, protein subunit or peptide that forms a self-organizing structure or pattern when multiple proteins are assembled and forms an oligomerization Subunit or peptide.

Hereinafter, the present invention will be described in detail.

The present invention

a) a gene encoding an autogenous multimeric protein; And

  Wherein the nucleic acid sequence encoding the peptide for controlling the translation rate is selected from the group consisting of a gene encoding a target protein or a nucleic acid sequence encoding a fusion protein of the self- Preparing a vector comprising the sequence;

b) transforming the vector into a host cell to produce a transformant; And

c) culturing the transformant to obtain a recombinant protein;

And a method for producing the recombinant fusion protein.

In the present invention, the self-assembling multispecific protein is not limited to a source or a structure, but may be a matrix protein family, a matrix protein family, or the like, as long as it does not interfere with the object of the present invention as a fusion partner. May include a domain belonging to a transcription protein family, a growth factor family or a secretory protein family, more preferably a cartilage matrix protein or cartilage matrix protein And may be a domain present in the cartilage oligomeric matrix protein (COMP).

The cartilage oligomer substrate protein (COMP) is an oligomeric glycoprotein having a molecular weight of 524 kDa, and is mainly present in the upper part of articular cartilage. In all types of cartilage, cartilage is synthesized by chondrocytes, the cartilage oligomeric matrix protein consists of five identical sulfide-linked subunits, the apparent Mr of each subunit is composed of about 100 kDa, the content of cysteine (Hedbom E, J Biol Chem 25: 267 (9): 6132-6, 1992). The nucleotide sequence, amino acid sequence and structural model of such a cartilage oligomeric substrate protein have been disclosed (Oldberg A, J Biol Chem 5: 267 (31): 22346-50, 1992; M ㆆ rgelin M, J Biol Chem 25: 267 (9): 6137-41,1992). By oligomerization through introduction of a multiplexing induction region of the protein (oligopeptide domain associated with multiplexing in the entire COMP protein), it protects against protease and improves the folding effect of protein In addition, from the results obtained in one embodiment of the present invention, even if the target protein expressed by fusion with the protein has the form of an oligomer, there is an effect of increasing the expression amount while maintaining the activity, (See FIG. 3).

In the present invention, the nucleic acid sequence encoding the peptide for controlling the translation rate is not particularly limited as long as it does not interfere with the object of the present invention, but for example, by collecting a rare codon of the host cell And preferably one or two or more nucleic acid sequences selected from SEQ ID NO: 23 to SEQ ID NO: 34. In order to improve the inhibition of translational efficiency due to bias of the codon usage of the host cell, it is necessary to further introduce a peptide capable of rapidly supplying the amino acid (aa) -tRNA, or to change the codon in the recombinant protein Thereby inducing an increase in protein expression. More specifically, in one embodiment of the present invention, rare codons were collected by analyzing the codon usage of host strains for the preparation of peptides for controlling the translation rate.

In the present invention, the concept of the "rare codon" In general, the rate of translation is most influenced by the rapid supply of aa-tRNA, which is an amino acid-linked state, when the codon is decoded. Therefore, the rate of translation of a codon in which aa-tRNA is sufficiently present in the cell is rapid, and the lack of aa-tRNA in the cell leads to a decrease in translation speed. That is, the gene copy of tRNA can be used as the first measure of the concentration of the corresponding tRNA in the cell. The typical codon optimization method reflects the codon frequency, which is due to the fact that the codon frequency is known to be proportional to the number of tRNA copies. In the present invention, a codon frequency corresponding to each amino acid (aa) of E. coli was collected using a gene copy of tRNA on the basis of 100% as a whole (http://gtrnadb.ucsc.edu/) With reference to the method disclosed in Korean Patent Publication No. 1446054, a codon having a codon frequency of 3% or less, preferably 0.1 to 1.0% in each amino acid was defined as a rare codon, and up to 3 amino acids were collected for one amino acid. In the method using the tRNA gene copy, 0 to 2 isoacceptor tRNAs may be selectively used in combination, but the scope of the present invention is not limited thereto.

A skeleton vector includes a peptide for controlling the translation rate and a gene for a COMP protein and a gene for a foreign protein as a fusion partner and includes a promoter or a selection marker capable of promoting skeletal vector transcription that can be used in the method of the present invention can do. The vector of the present invention may be a selection marker and may include an antibiotic resistance gene commonly used in the art. Examples thereof include ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

The recombinant vector of the present invention comprises a nucleic acid sequence enabling proliferation in a prokaryotic host cell, particularly E. coli , and includes, for example, a cloning origin of a colE1 or p15A bacterial origin or a cloning origin of a bacteriophage such as f1 origin can do. However, the vectors that can be used in the present invention are not limited to those shown above.

The translation rate-controlling peptide and the fusion partner of the present invention may be included in the recombinant vector as described above, but may be inserted directly into the chromosome of the host cell. For such chromosome insertion, conventional homologous recombination methods, transposon-mediated gene insertion or Cre-LoxP recombination system can be used.

Furthermore, a nucleic acid sequence encoding a protein cleavage enzyme recognition site may be linked between the COMP protein and the exogenous protein operably in the gene of the fusion partner of the vector of the present invention. When the objective protein to be fused with the fusion partner is an industrial protein, the fusion partner may degrade the function of the target protein, and in the case of a medical protein, the fusion partner is preferably removed since it may cause an antigen-antibody reaction. Herein, the protein cleavage enzyme recognition site may be a Xa factor recognition site, an enterokinase recognition site, a Genenase I recognition site, or a purine recognition site, or two or more of them may be used in sequence However, it does not limit the kind of protein-cleaving enzyme or the method of connection.

In the expression of the recombinant protein, the translation rate-controlling peptide can be freely changed in amino acid length and introduction position according to the degree of bias of the codon usage of the fusion partner and the foreign protein, and redesigned to enable soluble and insoluble expression depending on the purpose of expression . If it is difficult to introduce additional amino acids into the fusion partner or foreign protein of the present invention or codon usage analysis is required to introduce the translation rate controlling peptide into the fusion partner or foreign protein, internal base sequences may be changed.

In the present invention, the peptide for controlling the translation rate is not particularly limited in its sequence and length insofar as it does not impair the object of the present invention, but it may be composed preferably in the form of 10 amino acid sequences or less, The nucleotide sequence encoding the amino acid can be selected from the self-assembling multispec protein or the gene encoding the recombinant protein of the present invention Or it is also possible to apply the codon in the gene by changing it.

As can be seen from the results of one embodiment of the present invention, by applying the peptide for controlling the translation rate through the above-described method, the self-assembling multispecific protein (for example, recombinant fusion protein, COMP) -recombinant protein was increased (see FIGS. 5, 9, 12, 15 and 20, etc.). Particularly, by controlling the translation speed through the above-mentioned method, the peptide for controlling the translation speed is not applied, and the effect of making the amount of phage and the solubility remarkably improved in comparison with merely fusion of the self-assembly multimeric protein and the target protein can be obtained .

According to another aspect of the present invention, in order to facilitate post-purification treatment in the pharmaceutical and industrial fields, the recombinant protein may be added to an insoluble aggregate (inclusion body) The recombinant fusion protein of the example of the present invention was remarkably improved in the expression level compared to the wild type protein and was expressed as an insoluble aggregate for ease of treatment after purification (see FIG. 5).

From the above, it was confirmed through the production method of the recombinant fusion protein of the present invention that a recombinant fusion protein selectively showing solubility and insolubility can be produced, while remarkably improving the expression level, if necessary.

In addition, according to one embodiment of the present invention, in order to verify the activity of the recombinant protein produced by the method according to the present invention, it was confirmed that the recombinant fusion protein exhibits its original activity even if the fusion partner is not separately removed 8, 11, 14, 16).

In the present invention, the nucleic acid sequence grafted to the inside or the outside of the gene coding for the target protein is not limited unless it interferes with the object of the present invention, but may include a nucleic acid sequence encoding a leader sequence or a tag for separation and purification .

The leader sequence is not particularly limited. For example, nucleic acid sequences of any one or two or more genes selected from the group consisting of p elB, gene Ⅲ, ompA, ompB, ompC, ompD, ompE, ompF, ompT, phoA and phoE And the position of the protein or peptide expressed through it may be a form that is internal to the bacteria or exposed to the cell membrane, and the site showing the activity may be expressed alone or in a form located in the fused protein.

In the present invention, the nucleic acid sequence encoding the tag for separation and purification is not limited unless it interferes with the object of the present invention. However, any nucleic acid sequence selected from the group consisting of GST, poly-Arg, FLAG, poly- Or sequentially linking nucleic acid sequences encoding one or more tags. According to one embodiment of the present invention, the vector of the present invention comprises a gene coding for the above-mentioned translation rate controlling peptide, a fusion partner protein, a gene encoding a target protein and a region for inserting a tag for separation and purification into a pET24a framework vector In the form of a recombinant protein comprising the fusion partner, the target protein and the tag for separation and purification.

The gene coding for the target protein can be cloned through a restriction enzyme site, for example, when a nucleic acid sequence encoding a protein cleavage site is used, ), So that the target protein of the original form can be purified when the protein is cleaved with the protein cleaving enzyme after secretion of the target protein, but the scope of the present invention is not limited thereto.

In another aspect of the present invention, the present invention relates to a host cell transformed with a vector produced in the above-described form.

In the present invention, the host cell is a not particularly limited it does not inhibit the object of the present invention, Escherichia genus (Escehreichia sp.), Salmonella in (Salmonellae sp.), Yersinia genus (Yersinia sp.), Bacteria of the genus Shigella sp., Enterobacter sp., Pseudomonas sp., Proteus sp. Or Klebsiella sp., And the like. It is preferably, but not exclusively, Salmonella spp., Escherichia spp., Yersinia spp., More preferably Escherichia bacteria.

In the present invention, the method of delivering the recombinant vector of the present invention into a host cell can be carried out by a CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. (1973)), Hanahan, D., J. Mol. Biol., ≪ RTI ID = 0.0 > 166: 557-580 (1983)) and electroporation method (Dower, WJ et al., Nucleic Acids Res., 16: 6127-6145 (1988)). It is not.

In the present invention, the culturing of the host cell may be carried out by appropriately introducing a cell culture method known in the art or a modified method thereof, as long as the object of the present invention is not impaired. For example, when the host cell is E. coli , the culture medium for culturing the transformed host cell may be a natural medium or a synthetic medium containing a carbon source, a nitrogen source, an inorganic salt and the like which can be efficiently used by E. coli. have.

Such carbon sources include, but are not limited to, carbohydrates such as glucose, fructose, sucrose; Starch, hydrolyzate of starch; Organic acids such as acetic acid and propionic acid; Alcohols such as ethanol, propanol, and glycerol, and the nitrogen source may include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate; Peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean extract, soy hydrolyzate; Various fermented cells and their degradation products, and the like. The inorganic salt may include, but is not limited to, potassium dihydrogen phosphate, dihydroxy hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, manganese sulfate, copper sulfate, calcium carbonate and the like.

In the present invention, the cultivation of the host cells can be usually carried out under aerobic conditions such as shaking culture or rotating machine. When culturing, the culturing temperature is preferably in the range of 10 to 40 ° C, and the culturing time may be 5 to 7 days, but is not limited thereto. In the present invention, a suitable culture cell concentration can be selected and cultured in order to obtain a recombinant protein at a high yield. The pH of the culture medium during culturing can preferably be kept in the range of 3.0 to 9.0, which is an inorganic or organic acid, , Urea, calcium carbonate, ammonia, and the like. When necessary during culturing, antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin, and tetracycline may be appropriately selected for maintenance and expression of the recombinant vector.

In addition, the produced recombinant protein can be separated and purified from the cultured cells or culture medium according to a method well known to those skilled in the art. For example, the microbial cells may be separated by centrifugation or the like and then subjected to purification by salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium pressure liquid chromatography, reverse phase chromatography, Chromatography or the like, or a combination thereof. Proteins secreted in the surface layer of the microorganism according to the present invention may be isolated and purified in the same manner as in the case of expression in microbial cells after a method well known to those skilled in the art, for example, by increasing the salt concentration or using a surfactant . In some cases, the protein secreted into the surface layer of the microorganism may not be solubilized, but may be used, for example, as an immobilized enzyme.

In the present invention, the objective protein is a physiologically active protein including hormones and receptors thereof, biological response modifiers and receptors thereof, cytokines and their receptors, enzymes, antisera, antibody fragments, Such as, for example, human growth hormone (hGH), insulin, follicle-stimulating hormone (FSH), human chorionic gonadotropin, parathyroid hormone (PTH) (GPO), granulocyte macrophage colony stimulating factor (GM-CSF), interferon alpha, interferon beta, interferon gamma, interleukin, marophage ) Activation factor, tumor necrosis factor, tissue plasminogen activator, blood coagulation factor VII / VIIa, human bone morphogenic protein 2 (hBMP2), KGF nocyte growth factor (PDGF), platelet-derived growth factor (PDGF), alpha -galactosidase A, iduronate-2-sulfatase, lactase, But are not limited to, adenosine deaminase, butyrylcholinesterase, chitinase, glutamate decarboxylase, imiglucerase, lipase, uricase, platelet-activating factor acetylhydrolase, neutral endopeptidase, urokinase, streptokinase, myeloperoxidase, superoxide dismutase, L-asparaginase, monoclonal antibody, polyclonal antibody, scFv, Fab, Fab ', F (ab') 2 And Fd. Or more, but is not limited thereto.

Also, the target protein may be a physiologically active peptide, such as a glucagon-like peptide-1 (GLP- 1) and its analogues, somatostatin and its analogues, LHRH luteinizing hormone-releasing hormone agonists and antagonists, adrenocorticotropic hormone, growth hormone-releasing hormone, oxytocin, thymosin alpha-1, A group consisting of fragments of calcitonin, vasopressin analogues, somatostatins, bombesin, cholecystokinin, gastrin analogue, epidermal growth factor receptor (EGFR), human fibronectin extradomain B (EDB) , But the present invention is not limited thereto.

Further, another example of peptide application for molecular imaging, which is capable of early diagnosis of cancer, includes αVβ3 integrin binding RGD peptide, gastric cancer endothelium binding peptide, vascular cell adhesion molecule-1 targeting peptide (VCAM-1) peptide, matrix metalloproteinase Targeting peptide, urokinase plasminogen activator (uPA) binding peptide, urokinase-type plasminogen activator receptor (uPAR) homing peptide, ErbB-2 targeting peptide, plectin-1 pancreatic ductal adenocarcinoma targeting peptide, galectin- (LA Landon and SL Deutscher, J. Cell Biochem, 90: 509, 2003).

Further, another aspect of the present invention relates to a recombinant fusion protein produced through the above-mentioned method for producing a recombinant fusion protein.

In the present invention, the recombinant fusion protein is not particularly limited, but a multiplex region element of the COMP protein translated from the nucleic acid sequence of SEQ ID NO: 1 is most ideal.

The recombinant protein or polypeptide fused product according to the present invention exhibits increased expression and solubility as compared with the wild type protein and has a multimeric structure by binding a physiologically active protein or a physiologically active peptide to a COMP protein to maintain persistence in the body, To increase the physiological activity by the structure of the oligomer form as compared with the wild type monomer

A protein or a low-molecular weight peptide having a short in vivo half-life and low stability can not have effective drug delivery in the blood, so that it is possible to have desirable pharmacokinetic properties as a method for designing the recombinant protein fusion of the present invention in which the safety of the body is appropriately modified Do. 10 and 13, the serum half-life (t _1/2 ) of the recombinant human growth hormone protein fusion (CR-hGH) of the present invention is 11.4 hours, The concentration reaching time (T _max ) was 4 hours. On the other hand, the serum half-life (t _1/2 ) of human growth hormone (hGH) is 3.6 hours and the peak reaching time of blood (T _max ) is 2.4 hours. In addition, the plasma half-life (t1 / 2) of the recombinant granulocyte stimulating factor protein fusant (RC-G_CSF) is 18.4 hours and the maximum blood concentration reaching time (Tmax) is 8.1 hours. On the other hand, the plasma half-life (t _1/2 ) of the granulocyte stimulating factor (G_CSF) is 9.4 hours and the maximum blood concentration reaching time (T _max ) is 4.3 hours.

From these results, it can be confirmed that the recombinant human growth hormone protein fusions and the recombinant granulocyte stimulating factor protein fusions according to the present invention have significantly increased body stability as compared with the wild type. As a result of confirming the effects of recombinant proteins and peptides on the in vivo activity of the recombinant protein fusions, it was found that the human growth hormone treated in the body increased in body weight, leukocyte counts in the granulocyte stimulating factor, luciferase activity and EGFR binding domain And RGD binding peptides were found to be effective in both wild type and recombinant protein fusions, and improved activity was observed in the recombinant protein fusants. Therefore, it can be seen that the recombinant protein fusions of the present invention exhibit effective in vivo activity. However, the physiologically active protein used in the present invention has been selected to confirm the stability and activity of the recombinant protein fusants in the body, and the application range is not limited thereto. In addition, the diagnostically effective agent used for imaging the in vivo structure or fluid may include, but is not limited to, beta -galactosidase, green fluorescent protein, blue fluorescent protein, luciferase, A marker gene that encodes an easily detectable protein.

The recombinant fusion protein prepared by the method of the present invention has a small size, so that the yield of the target protein can be greatly improved as compared with the case where the conventional fusion partner is used. In addition, from the results according to one embodiment of the present invention, not only can the method of the present invention be universally applied to proteins useful for medical purposes or industrially, as shown in Figs. 8 to 11, Can induce the water-soluble expression of the target protein, and are suitable for inducing expression in an active form that performs a unique function.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. It is to be understood that the embodiments are illustrative of the present invention in more detail, and the scope of the present invention is not limited thereto.

[Reagents, Materials and Protocols]

Bacterial strains, medium and culture conditions

LacIqA (lacZ) M15] hsdR17 (rK-mK < + >)) for E. coli XL1-Blue (endA1 gyrA96 (nalR) thi-1 recA1 relA1 lacglnV44 F '

(RB-mB-)? (DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB +] K-12 (lambda S) for protein expression: E. coli BL21 (DE3) (ompT gal dcm lon hsdSB

Plasmids for cloning and expression: pET24a (Novagen, USA)

Cultivation conditions: E. coli strains were cultured at 37 ° C using Luria-Bertani (LB) medium (Difco Labaratories, USA) containing 1% NaCl and 50 μg / ml kanamycin was added to all media.

Production and transformation of competent cells

The competent cells of E. coli XL1-Blue used for cloning were inoculated into LB liquid medium, pre-cultured and inoculated with 1% of the total volume of SOB medium supplemented with 20 mM MgCl2 (super-optimized broth) Incubated for 24 hours and washed in Inoue TB buffer.

E. coli BL21 (DE3) used for protein expression and purification was inoculated in 100 ml LB medium, cultured at 37 ° C, and harvested at OD ₆₀₀ of 0.4, washed in 0.1 M CaCl ₂ , and absorbance was measured. The prepared competent cells were stored at -80 ° C after rapid freezing in liquid nitrogen.

Transformation was carried out by mixing the recombinant plasmid and E. coli competent cells, leaving them on ice for 10 minutes, treating them at 42 ° C for 90 seconds, and for 2 minutes on ice. The transformants were plated on LB solid medium containing antibiotics and cultured at 37 ° C.

[Example 1] Preparation of recombinant protein expression vector

Recombinant plasmids were prepared by homologous recombination method using Ez-Fusion Cloning kit (Enzynomics, Korea) after digesting pET24a with Nde I or using a sequence containing restriction enzyme recognition sites in pET24a and primers The amplified gene was treated with restriction enzymes and then constructed using T4 DNA ligase (Enzynomics, Korea).

In order to insert the COMP gene into the skeletal vector pET24a, the template synthetic DNA (SEQ ID NO: 1) was subjected to PCR (Professional Basic Thermocycler (SEQ ID NO: 1)) using the primers COMP1 and COMP2 and the speed-pfu polymerase (Nanohelix, Biometra, Germany) and cloned. COMP2 in the primers included a linker to maximize flexibility to maintain the activity of the fused protein.

The nucleic acid sequence of SEQ ID NO: 1 is a synthetic sequence in which a histidine tag is attached to the multiplexing region of the COMP protein.

The vector into which the COMP gene was inserted was named pET_COMP and a vector (pET_COMP-his) was prepared by cloning using primers COMP1 and COMP3 in order to add a His tag sequence at the C-terminus for purification.

Then, the primers corresponding to the respective genes (Rluc8, mCherry, proinsulin, RGD, EGFR, hGH and G-CSF) (see Table 1) were cloned after PCR amplification to fuse the target protein into pET_COMP. The amplified nucleotides were inserted into the above pET_COMP to prepare an expression vector.

Table 1. Primers for recombinant protein cloning

[Example 2] Design and application of peptide for controlling translation rate

A translation rate-regulating peptide was designed to induce the solubility of the recombinant protein or, if necessary, to aggregate, to bind the COMP-target protein fusions to each of the target proteins of Example 1.

Analysis of E. coli codon usage (codon usage) and rare codon collection

In order to apply a translation rate-controlling peptide to a recombinant fusion protein composed of a COMP-target protein, the codon utilization of E. coli was analyzed by an open method so that the number of rare codons with a frequency of less than 1% was less than 3 per amino acid, and the number of tRNAs were collectively taken together (Table 2).

Table 2. Collected E. coli strains Rare codons

Based on the analyzed rare codons, a pair of primers was prepared as shown in Table 3 so as to be fused to the COMP-protein fusions by combining the translation rate-regulating peptides, followed by cloning. The peptide to be fused is a peptide sequence in which the primers of SEQ ID NOs: 23 to 34 are introduced into the target gene and translated

Table 3. Primers for Translation Rate Modulating Peptides

The amplified nucleotides were inserted into the vector for introduction of each COMP-target protein and named according to the inserted position (denoted by R) and the sequence (denoted by numerals).

[Example 3] Expression of multimeric protein (COMP) as a fusion partner and confirmation of activity

1: COMP only expression and purification

The recombinant vector pET-COMP-his was transformed through the method described above.

Single colonies were selected from the solid medium and inoculated into 20 ml LB liquid medium supplemented with 50 占 퐂 / ml kanamycin and pre-cultured at 37 占 폚 for 4 hours. When the OD ₆₀₀ was 2.0 at the time of absorbance measurement, the cells were inoculated 1% in 500 ml LB liquid medium supplemented with 50 μg / ml kanamycin and cultured at 37 ° C and 200 rpm.

When the OD ₆₀₀ reached 0.4-0.5, 0.2 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to induce overexpression of the protein. When the OD _{600 was} 2.0, the cultured cells were treated with 4 Lt; 0 > C for 10 minutes at 6,000 rpm. The harvested cells were rapidly frozen in liquid nitrogen and stored at -80 ° C.

Cells were stored at -80 ° C in ice water and liquid nitrogen for 3 times. Cells were washed with 100 ml of binding buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.5), then 100 μg / ml lysozyme was added and reacted on ice for 20 minutes. Thereafter, the suspension was subjected to a 5-second shaking procedure and a 10-second stabbing procedure five times using an ultrasonic disintegrator (Vibra-cellTM; Sonic & materials), and the cells were disrupted for 30 minutes in one cycle.

The disrupted cells were centrifuged at 10,000 x g for 60 minutes at 4 < 0 > C to remove insoluble precipitates, and the supernatant containing soluble proteins was recovered. The recovered supernatant was completely removed using a 0.45 μm syringe filter and purified using Fast Protein Liquid chromatography (Biologic duoFlow System, Bio-Rad). Since the protein used in this experiment contains his-tag, the Ni-NTA column (BIO-RAD) was used. After reaching the equilibrium with the binding buffer, the filtered supernatant was added to 2 ml / min, and proteins with weak binding ability to nickel were isolated using wash buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM immidazole, pH 7.5).

Subsequently, the immidazole concentration was gradually increased at a rate of 5 ml / min in an elution buffer (50 mM Tris-HCl, 300 mM NaCl, 300 mM immidazole, pH 7.5) Respectively. The recovered protein was dialyzed 3 to 5 times with 1 L of phosphate-buffered saline (PBS) or the salt was removed using a desalting column (GE healthcare). The bradford assay The concentration of protein was quantified.

SDS-PAGE was performed by conventional methods to confirm the expression of the recombinant protein, and the presence or absence of a reducing agent was determined in the SDS-PAGE sample dye to confirm oligomerization of the protein due to disulfide bond.

As a result of the above experiment, the COMP protein was confirmed to be soluble in overexpression under the above conditions and oligomerized according to disulfide bond (FIG. 1).

2: Fusion expression and purification of COMP-labeled proteins

In order to confirm expression changes when the target protein and the COMP protein were fused, the labeled protein was fused to the COMP protein.

The recombinant vector pET_COMP-mCherry (C-mCherry), pET_COMP-Rluc8 (C-rluc8) was pre-cultured through the above-described transformation method and then cultured in the same manner as in Example 3-1, And purified using a Ni-NTA column (BIO-RAD).

Thereafter, over-expression of soluble proteins was confirmed by SDS-PAGE, and it was confirmed that the fusion protein of the recombinant COMP-labeled protein was oligomerized in the sample without the reducing agent (FIG. 2).

3: Confirmation of activity of COMP-labeled protein fusants

To confirm the activity change upon fusion of the target protein and the COMP protein, the fibrin activity of the COMP-protein purified from the above Example 3-2 was measured.

To measure the fluorescence of C-mCherry, fluorescence wavelength (Ex 587 nm / Em 610 nm) was examined using a Tecan Infinite M200 Plate Reader. Coelenterazine (BIOTIUM) was used as a substrate to measure the activity of C- And the luminescence was measured.

As a result, it was confirmed that the activity of the labeled protein fused to COMP was maintained, and it was confirmed that the function or structure was maintained even when the target protein was fused to the COMP protein (FIG. 3).

 [Example 4] Expression of COMP-insulin fusions and confirmation of activity

 1: COMP-insulin fusion expression and insulin yield

The insulin gene was cloned into the expression vector of the present invention to confirm its expression.

In addition, for comparison of the degree of expression, a known fusion partner, maltose binding protein (MBP) and glutathione-S-transferase (GST) were fused.

COMP-proinsulin (R2-C-In), pET_R3-COMP-proinsulin (R3-C-In), pET_R4-COMP- (MBP-In) and pMAL_proinsulin (MBP-In) were transformed by the above-mentioned method, respectively. After pre-culture, the cells were cultured in the same manner as in Example 3, Respectively.

Expression was then confirmed by SDS-PAGE.

As a result, the strain into which the vector containing only the insulin gene alone did not express the insulin protein, but the vector containing the insulin gene fused with the fusion partner overexpressed the insulin (FIG. 4).

Image J image program was used to compare the amount of target protein expressed relative to the total protein amount of the strain.

As a result, it was confirmed that MBP-insulin fusions were expressed in a similar range of 32% and recombinant insulin protein fusions were in a range of 25.3 to 36.6% (FIG. 5).

Then, trypsin was treated to remove the fusion partner of the expressed insulin protein and make it active. The expressed proinsulin fusions were dissolved in 8 M urea (pH 10.8) and 3 mM cysteine was added to the clarified proinsulin fusion solution. Thereafter, the mixture was stirred at 4 ° C for 24 hours, then fixed at pH 8.5 with hydrochloric acid, and treated so that the ratio of proinsulin to trypsin was 100: 1. Thereafter, the reaction was carried out at 37 ° C and the sample was sampled over time and analyzed by SDS-PAGE.

As a result, as shown in FIG. 6, it can be seen that the amount of the protein lost is smaller than that of the existing fusion protein, and the yield of insulin as the target protein is improved. This is because the COMP protein used in the present invention has a relatively small size .

2: Pharmacokinetic studies of insulin prepared from recombinant insulin protein fusions

6-week-old C57BL / 6 mice were divided into experimental groups consisting of 4 mice per group.

In one experimental group, C57BL / 6 mice were subcutaneously injected with insulin prepared by the above procedure subcutaneously at a dose of 50 μg per g, and injected at 0, 10, 20, 30, 40, 60, 120, 180, And after 360 minutes blood was drawn into the heparin treated tubes to obtain serum after centrifugation.

Each sample was analyzed by the following enzyme immunoassay.

First, a monoclonal antibody against insulin (ab9569, Abcam) was diluted to 10 μg / ml in phosphate buffer and 100 μl was dispensed into a 96-well plate and left at room temperature for 15 to 18 hours .

After removing the unattached antibody, 200 μl of phosphate buffer solution in which 1% bovine serum albumin was dissolved was dispensed, left at room temperature for 2 hours, washed three times with washing solution (phosphate buffer, 0.05% tween 20) . Samples were diluted with 1% bovine serum albumin dissolved phosphate buffer, added to 96 well plate and reacted at room temperature for 2 hours. After washing the plate 5 times with washing solution, the insulin monoclonal antibody-biotin conjugate conjugated with sulfo-NHS-biotin (pierec biotechnology, USA) was diluted in diluted solution and dispensed 100 μl into a 96-well plate. The plate was reacted at room temperature for 2 hours, washed with washing solution 5 times, and then treated with streptavidin-HRP solution at room temperature for 30 minutes. After washing 5 times with washing solution, 100 μl of TMB (3,3 ', 5,5'-tetramethylbenzidine) and hydrogen peroxide solution were added to each well and reacted for 15 minutes in a dark room.

Then, 100 μl of 1 M sulfuric acid was added to each well to terminate the reaction, and the absorbance at 450 nm was measured with an analyzer (Tecan Infinite M200). Quantitative values of each sample were calculated by regression analysis after creating a standard curve for the standard material.

The results are shown in Fig. From this, the serum half-life of insulin produced in a recombinant insulin fusion protein of this invention (t _1/2) is 42 minutes and the maximum density arrival time (T _max) in blood was 13 minutes, and serum half-life of insulin on the market (t _{1 / 2} ) was 47 minutes and the maximum time to reach blood concentration (T _max ) was 12 minutes.

From these results, it was confirmed that the insulin prepared from the recombinant insulin protein fusions according to the present invention had the same or better in vivo stability than the products sold on the market.

3: In vivo activity test of recombinant insulin protein fusants

Diabetic model mice, 9-week-old db / db mice (Harlan Laboratories), were divided into experimental groups consisting of 5 mice per group. In the absence of dietary restriction, insulin and commercially available insulin prepared by the above procedure were injected subcutaneously into mice of each experimental group at a dose of 150 μg / g, and the concentrations of 0, 1, 3, 6, 12, After 24, 48, and 52 hours, blood glucose was measured using a glucose meter.

The results are shown in Fig. From these results, each insulin was kept low in blood glucose until 24 hours and then showed similar value to the control group. From this, it was confirmed that insulin prepared by the manufacturing method according to the present invention had activity equal to or higher than that of a product sold on the market, and thus it could be used as a medical protein.

 [Example 5] Expression of recombinant human growth hormone protein fusion and confirmation of activity

1: expression and purification of recombinant human growth hormone protein fusions

Human growth hormone was fused to the COMP protein to determine recombinant human growth hormone protein fusion expression.

The recombinant vector pET_COMP-R-hGH (CR-hGH) was transformed in the same manner as described above, and pre-cultured, and then cultured in the same manner as in Example 4 to induce protein expression, and the Ni-NTA column ).

Thereafter, over-expression of the soluble protein was confirmed by SDS-PAGE, and it was confirmed that the fusion protein of the recombinant COMP-human growth hormone protein was oligomerized in the sample to which the reducing agent was not added.

2: Pharmacokinetics of recombinant human growth hormone protein fusions

6-week-old C57BL / 6 mice were divided into experimental groups consisting of 4 mice per group.

One experimental group of C57BL / 6 mice was subcutaneously injected with a recombinant human growth hormone protein fusion prepared by the above procedure subcutaneously at a dose of 100 μg per mouse and diluted with phosphate buffer solution. Blood was collected at 0, 1, 2, 4, 8, 12, 16, 24, and 48 hours, and centrifuged to obtain serum. As a control, human growth hormone Eutropin (LG Life Science) was injected subcutaneously at a dose of 50 μg per mouse.

Each sample was analyzed by the following enzyme immunoassay.

First, the anti-growth hormone antibody (ab74324, Abcam) against human growth hormone was coated and blocked according to the method of Example 5 at a concentration of 5 μg / ml. After dilution, And reacted for 2 hours. Human growth hormone monoclonal antibody-biotin conjugate was used, and the subsequent procedure was performed according to the method of Example 5 above.

The results are shown in Fig.

As a result, the blood half-life (t _1/2 ) of the recombinant human growth hormone protein fusion (CR-hGH) of the present invention was 11.4 hours and the maximum blood concentration reaching time (T _max ) was 4 hours. (t _1/2 ) of the hGH (hGH) is 3.6 hours, and the maximum blood concentration reaching time (T _max ) is 2.4 hours.

From the above results, it was confirmed that the recombinant human growth hormone protein fusions of the present invention have improved body stability compared to human growth hormone.

 [Example 6] Expression of recombinant granulocyte stimulating factor protein fusants and confirmation of activity

1: Recombinant granulocyte stimulating factor protein fusion expression and purification

The recombinant granulocyte stimulating factor protein fusion protein was fused to the COMP protein in the presence of granulocyte stimulating factor.

The recombinant vector pET_R-COMP-G_CSF (RC-G_CSF) and pET_COMP-R_GCSF (CR-G_CSF) were transformed as described above and pre-cultured, followed by culturing as in Example 4 to induce protein expression And purified using a Ni-NTA column (BIO-RAD).

 Thereafter, over-expression of soluble proteins was confirmed by SDS-PAGE and oligomerization of the recombinant protein fusions was confirmed in the sample to which no reducing agent was added (FIG. 11).

2: Pharmacokinetics of recombinant granulocyte stimulating factor protein fusants

6-week-old C57BL / 6 mice were divided into experimental groups consisting of 4 mice per group.

Recombinant granulocyte stimulating factor protein fusions (R-C-G_CSF) prepared by the above procedure were subcutaneously injected into C57BL / 6 mice in one experimental group at a dosage of 200 ug / g, and diluted with a phosphate buffer solution. Blood was collected at 0, 1, 2, 4, 8, 12, 16, 24, 30, and 48 hours, and centrifuged to obtain serum. As a control, granulocyte stimulating factor Filgrastim (Gracin, Jeil Pharm) was subcutaneously injected at a dose of 100 ug per mouse.

Each sample was analyzed by the following enzyme immunoassay.

First, the anti-G-CSF antibody (ab9691, Abcam) against the granulocyte stimulating factor was coated and blocked at a concentration of 5 μg / ml according to the method of Example 5, For 2 hours. The granulocyte stimulating factor monoclonal antibody-biotin conjugate was used, and the subsequent procedure was carried out according to the method of Example 5 above.

The results are shown in Fig.

As a result, the plasma half-life (t _1/2 ) of the recombinant granulocyte stimulating factor protein fusion (RC-G_CSF) of the present invention was 18.4 hours and the maximum blood concentration reaching time (T _max ) was 8.1 hours. (T _1/2 ) is 9.4 hours and the maximum blood concentration reaching time (T _max ) is 4.3 hours.

From the above results, it was confirmed that the recombinant granulocyte stimulating factor protein fusions according to the present invention have relatively increased body stability as compared with the control group.

 [Example 7] Recombinant target-directed domain fusions and activity confirmation

1: Recombinant target-directed domain fusion expression and purification

The EGFR binding protein was fused to the N-terminus of the COMP protein to reveal recombinant target-directed domain fusion expression.

The recombinant vector pET_EGFR-R-COMP (EGFR-RC) was transformed as described above and pre-cultured, and then cultured as in Example 4 to induce expression of the protein. Using Ni-NTA column (BIO-RAD) Lt; / RTI >

Thereafter, over-expression of soluble proteins was confirmed by SDS-PAGE, and it was confirmed that the recombinant target-directed domain fusions were oligomerized in a sample to which no reducing agent was added. Also, gel filtration showed that the multimeric protein Homogeneity was confirmed (Fig. 13).

2: Identification of recombinant target-directed domain fusant activity

To confirm the activity of purified recombinant target-directed domain fusions (EGFR-R-C), FACS was performed using cell lines overexpressing EGFR on the cell surface.

First, dialyzed with 0.1 M sodium carbonate buffer (pH 9.0) for fluorescence labeling of recombinant target-directed domain fusions, 20 μg of FITC (Fluorescein isothiocyanate) was added per mg of fusion substance, Lt; / RTI >

Subsequently, the cells were dialyzed against phosphate buffered saline (PBS) to remove free dye, buffered and stored at -20 ° C.

HT29 cells and A549 cells were cultured in DMEM (Dulbecco's modified Eagle's medium) at 5% CO ₂ and 37 ° C using a medium containing 10% FBS (Fetal Bovine Serum).

After 24 hours of incubation, the cells were washed once with 1 x DPBS (Dulbecco's Phosphate Buffered Saline). Then, the cells were prepared so as to have a cell number of 2 x 10 < ⁵ > / ml, and the FITC-labeled recombinant target-oriented domain fusions were reacted by concentration.

Subsequently, recombinant target-directed domain fusions conjugated with cell lines were measured at the fluorescence wavelength (Ex 490 nm / Em 525 nm) using FACS.

As a result, as shown in FIG. 14, it was found that the cell line treated with the FITC-labeled recombinant target-oriented domain fusion had a higher fluorescence value than that of the control, and the binding ability was confirmed through this.

In addition, since the COMP protein can be self-assembled oligomerization, the COMP fusions having binding strength to the complex can have higher avidity even when they have the same binding affinity when applied to binding proteins , And an enzyme-linked immunosorbent assay was performed using a cell line to confirm this.

In each divider and 5% CO _2, 37 ℃ conditions the HT29 cell culture in the same way the number of cells in the 96 well plate so that the ^{2 x 10 4/100 μl /} well and the cells were cultured for 18 hours. After 4% p-formaldehyde treatment for cell fixation, FITC-labeled recombinant target-directed domain fusions and EGFR binding protein were reacted per well for 30 minutes per concentration.

After washing three times with PBS, the fluorescence was measured at a fluorescence wavelength (Ex 490 nm / Em 525 nm) using an analyzer (Tecan Infinite M200 Plate Reader).

As a result, as shown in FIG. 15, since the recombinant target-directed domain fusion (EGFR-RC) exhibits a fluorescent value higher than that of the EGFR-binding single protein at a concentration of 10 nmol, the binding activity is enhanced by multiplexing by the COMP protein Respectively.

 [Example 8] Expression of recombinant target-directed peptide fusion and confirmation of activity

1: Recombinant target-directed peptide fusion expression and purification

Recombinant Targeted Peptide Fusion of the targeting peptide to the COMP protein to confirm fusion expression.

The target peptide is a recombinant vector pET_T-mCherry (TM), pET_T-COMP (TC), pET_T-COMP-mCherry (TCM), pET_T-R-COMP (TRC), pET_T (TC-R1-M), pET_T-R2-COMP-mCherry (T-R2-CM) and pET_T-COMP-mCherry-R3 After the pre-culture, the protein was cultured as in Example 4 to induce the expression of the protein and purified using a Ni-NTA column (BIO-RAD).

As a result of confirming the expression of the recombinant protein by SDS-PAGE, overexpression of the clone (pET_T-COMP, pET_T-COMP-mCherry) fused with the target protein alone to the COMP protein was confirmed but the soluble ratio was small 16). When COMP-peptide fusions were analyzed by western blotting, peptide portions were degraded with the lapse of incubation time, and two sizes of proteins were identified (Fig. 17).

These results suggest that the low-molecular peptide, which is unstable by the protease in the host, is degraded. In contrast, in the case of a recombinant protein fused clone containing the translation rate-regulating peptide expressed by the present invention, a fusion protein (pET_T-R-COMP, FIG. 18) or a fused fusion protein (pET_T-COMP-R1-mCherry , pET_T-R2-COMP-mCherry, pET_T-COMP-mCherry-R3, FIG. 19) were all over soluble and did not show degradation by protein hydrolytic enzymes even after the lapse of incubation time.

In general, even when a multimeric protein is used as a fusion partner to express a low-molecular peptide, there is a limit to decompose the low-molecular peptide. However, the COMP-peptide fusion comprising the translation rate- Etc., were found to have a remarkable effect.

2: Confirmation of activity of recombinant target-oriented peptide fusants

In order to confirm the activity of the purified recombinant target-directed peptide fusions of the present invention, binding capacity was analyzed using animal cell lines.

U87MG cells were cultured in a medium containing 5% CO ₂ and MDA-MB-435 cells and 10% FBS (Fetal Bovine Serum) in RPMI 1640 medium at 37 ° C in α-MEM medium as a positive control for the target- Lt; / RTI >

As a control, M21L cells were used in high glucose DMEM medium.

After 24 hours of incubation, the cells were washed once with 1 x DPBS. Then, the cells were cultured in a 24-well plate at a density of 3 × 10 ⁵ / ml. After incubation for 16 hours, the cells were washed with DPBS and blocked with DPBS containing 10 mg / ml BSA (bovine serum albumin). Then, 500 nM of purified protein was added to each well for reaction.

The fusants used were pET_T-COMP-R1-mCherry (TC-R1-M), pET_T-R2-COMP-mCherry PET_T-mCherry (TM) was used.

After 30 minutes, the cells were washed thoroughly with DPBS and measured at fluorescence wavelength (Ex 587 nm / Em 610 nm) using an analyzer (Tecan Infinite M200 Plate Reader) and COMP fusions conjugated with the cell line.

As a result, it was confirmed that the cells were bound to the fusions in the positive control, and the fluorescence intensities of the TCM fusions were higher than the binding fluorescence intensities of the COMP protein and the control fusion fused with the translation rate controlling peptide, .

Then, ELISA using his-tag was performed.

Cells were prepared as above and 8% p-formaldehyde of the same volume per well was added and allowed to react for 15 minutes. After washing with DPBS and blocking with DPBS containing 10 mg / ml BSA, 500 nM of purified protein was added to each well for reaction.

After washing with DPBS and adding anti-His antibody (ab18184, Abcam), rabbit anti-Fab antibody (ab87422, Abcam) was added and reacted.

After sufficiently washing with DPBS and adding TMB (3,3 ', 5,5'-tetramethylbenzidine), the reaction was stopped by mixing 1 M sulfuric acid and measured by absorption at 450 nm.

As a result, it was possible to confirm the fused cells bound to the cells in the positive test group. As in the case of the fluorescence measurement, the fluorescence protein exhibited a higher binding force in the recombinant target-oriented peptide fusions compared to the binding force of the fused target- The present inventors completed the present invention by confirming that COMP fusions have higher binding activity as in Example 8 (Fig. 20).

<110> University Industry Liaison Office of Chonnam National University <120> ethod of producing recombinant protein by combining          multimerization moiety and solubility controlling technique and          use thereof <130> P17120761474 <160> 35 <170> KoPatentin 3.0 <210> 1 <211> 234 <212> DNA <213> Artificial Sequence <220> <223> COMP protein multimerization moiety + histidine tag <400> 1 atgccgccta ctccgccaac tccgtctccg tctactccgc caactccgtc tccgggatcc 60 ggagacctgg gcccgcagat gctgcgtgaa ctgcaggaaa ccaacgctgc tctgcaggac 120 gttcgtgact acctgcgtca gctggttcgt gaaatcacct tcctgaaaaa caccgttatg 180 gaatgcgacg cttgcggtat gcagcagact agtcatcacc atcaccatca ctaa 234 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> COMP1 <400> 2 atacatatgc cgcctactcc gccaactccg 30 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> COMP2 <400> 3 gccgcaagct ttcgtgatcc tccctgctgc ataccg 36 <210> 4 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> COMP3 <400> 4 gccgcaagct tcaccatcac catcaccatt cgtgatcctc cctgctgcat accg 54 <210> 5 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> MCH1 <400> 5 cagcagggag gatcacgagt gagcaagggc gaggaggata aca 43 <210> 6 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> MCH2 <400> 6 gtgctcgagt gcggccgcct acttgtacag ctcgtccatg c 41 <210> 7 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> RLU1 <400> 7 cagcagggag gatcacgagc ttccaaggtg tacgaccc 38 <210> 8 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> RLU2 <400> 8 gtgctcgagt gcggccgctc aatgatgatg atgatgatgg tcga 44 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> INSU1 <400> 9 atatcatatg tttgtgaacc aacacctgtg c 31 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> INSU2 <400> 10 atataagctt ctatccgcag tagttctcca gctgg 35 <210> 11 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> COINSU1 <400> 11 cagcagggag gatcacgatt tgtgaaccaa cacctgtgc 39 <210> 12 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> COINSU2 <400> 12 gtgctcgagt gcggccgcct atccgcagta gttctccagc tgg 43 <210> 13 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> COHGH1 <400> 13 cagcagggag gatcacgaga attcaggact gaatcgtgct caca 44 <210> 14 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> COHGH2 <400> 14 gtgctcgagt gcggccgcga attcaacagg catctactga gtgg 44 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> COCSF1 <400> 15 cagcagggag gatcacgagg acctgccacc cagagc 36 <210> 16 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> COCSF2 <400> 16 gtgctcgagt gcggccgctc agggctgggc aagg 34 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> EGFCO1 <400> 17 gaaggagata tacatatgga aaccattacc gtgag 35 <210> 18 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> EGFCO2 <400> 18 agttggcgga gtaggcgggg atcctccacc tcctgatcca ccacctccag aaccgcctcc 60 accggtcggg caaataatg 79 <210> 19 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> RGDM1 <400> 19 atatcatatg gcttgtgact gcaggggaga ctgcttctgc ggtgtgagca agggcga 57 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> RGDM2 <400> 20 gactcgaagc ttctacttgt acagctcgtc catgcc 36 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> RGDCO1 <400> 21 gaaggagata tacatatggc ttgtgactgc agggga 36 <210> 22 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> RGDCO2 <400> 22 agttggcgga gtaggcggac cccagaagca gtctcccct 39 <210> 23 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> R1COMPIN <400> 23 atatacatat gtgccactgc caccgatctc gatctccgcc tactccgcca a 51 <210> 24 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> R2COMPIN <400> 24 atatacatat gcgatgtcga tgttgccact gccacaggtc tccgcctact ccgccaa 57 <210> 25 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> R3COMPIN <400> 25 atatacatat gcgatctcga tctaggcaca ggccgcctac tccgccaa 48 <210> 26 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> R4COMPIN <400> 26 atatacatat gcgatgtcga tgtaggcaca ggcactgccc gcctactccg ccaa 54 <210> 27 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> COMRHGH <400> 27 cttgcggtat gcagcagagg tgtaggtgtc gatctgaatt caggactgaa tcgtg 55 <210> 28 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> RCOMCSF <400> 28 tatacatatg cgacaccgac acaggtgtag gtgttgctct tgctctccgc ctactccgcc 60 aact 64 <210> 29 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> COMRCSF <400> 29 tgcggtatgc agcagcgaca ccgacactgc tgttgctgta gg 42 <210> 30 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> EGFRRCOM <400> 30 cgatgtcgat gttgctcttg cccgcctact ccgccaact 39 <210> 31 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> TRCOM <400> 31 aggcacaggc actgctgttg ctgtccgcct actccgccaa ct 42 <210> 32 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> TCOMR1M <400> 32 tgcggtatgc agcagtgcca ctgccaccga tgtcgatgta gg 42 <210> 33 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> TR2COMM <400> 33 caccgacaca ggtgtaggcc gcctactccg ccaact 36 <210> 34 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> TCOMMR3 <400> 34 cgacaccgac actgctcttg ctctagg 27 <210> 35 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> COMP protein multimerization moiety + histidine tag: protein          sequence <400> 35 Met Pro Pro Thr Pro Pro Thr Pro Ser Ser Thr Pro Pro Thr Pro   1 5 10 15 Ser Pro Gly Ser Gly Asp Leu Gly Pro Gln Met Leu Arg Glu Leu Gln              20 25 30 Glu Thr Asn Ala Leu Gln Asp Val Arg Asp Tyr Leu Arg Gln Leu          35 40 45 Val Arg Glu Ile Thr Phe Leu Lys Asn Thr Val Met Glu Cys Asp Ala      50 55 60 Cys Gly Met Gln Gln Thr Ser His His His His His  65 70 75

Claims (18)

a) a gene encoding an autogenous multimeric protein; And
Wherein the nucleic acid sequence encoding the peptide for controlling the translation rate is selected from the group consisting of a gene encoding a target protein or a nucleic acid sequence encoding a fusion protein of the self- Preparing a vector comprising the sequence;
b) transforming the vector into a host cell to produce a transformant; And
c) culturing the transformant to obtain a recombinant protein;
&Lt; / RTI &gt; The method according to claim 1,
The self-assembling multispecific protein includes a domain contained in a matrix protein family, a transcription protein family, a growth factor family, or a secretory protein family. Wherein the recombinant fusion protein is a recombinant fusion protein. 3. The method of claim 2,
Wherein the domain belonging to the substrate protein group is a domain contained in a cartilage matrix protein or a cartilage oligomeric matrix protein (COMP). The method of claim 3,
Wherein the cartilage oligomer substrate protein is composed of five identical sulfide binding subunits. The method according to claim 1,
Wherein the nucleic acid sequence encoding the peptide for controlling the translation rate is obtained by collecting a rare codon of the host cell. 6. The method of claim 5,
Wherein the nucleic acid sequence encoding the peptide for the translation rate regulation is selected from SEQ ID NO: 23 to SEQ ID NO: 34. 6. The method of claim 5,
Wherein the collection of the rare codons is performed by analyzing the frequency of codons and the number of isoacceptor tRNA genes. 8. The method of claim 7,
Wherein the frequency of the rare codon is 0.1 to 1%. 8. The method of claim 7,
Wherein the number of genes of the isoceptor tRNA is 0 to 2. The method according to claim 1,
Wherein the nucleic acid sequence grafted to the inside or the outside of the gene encoding the target protein is a nucleic acid sequence encoding a leader sequence or a tag for separation and purification. 11. The method of claim 10,
Wherein the leader sequence is a nucleic acid sequence of any one or two or more genes selected from the group consisting of p elB, gene Ⅲ, ompA, ompB, ompC, ompD, ompE, ompF, ompT, phoA and phoE . &Lt; / RTI &gt; 11. The method of claim 10,
The nucleic acid sequence encoding the tag for separation and purification is sequentially linked with a nucleic acid sequence encoding any one or two or more tags selected from the group consisting of GST, poly-Arg, FLAG, poly-His and c-myc &Lt; / RTI &gt; The method according to claim 1,
The host cell may be selected from the group consisting of Escherichia sp., Salmonella sp., Yersinia sp., Shigella sp., Enterobacter sp. Wherein the recombinant fusion protein is a bacterium of the genus Pseudomonas sp., Proteus sp. Or Klebsiella sp. The method according to claim 1,
Wherein the target protein is a physiologically active protein including hormones and receptors thereof, biological response modifiers and receptors thereof, cytokines and receptors thereof, enzymes, antisera, and antibody fragments A method for producing a recombinant fusion protein. 15. The method of claim 14,
The target protein may be selected from the group consisting of human growth hormone (hGH), insulin, follicle-stimulating hormone (FSH), human chorionic gonadotropin, parathyroid hormone (PTH) EPO), platelet growth promoting factor (TPO), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), interferon alpha, interferon beta, interferon gamma, interleukin, Tumor necrosis factor, tissue plasminogen activator, blood clotting factor VII / VIIa, human bone morphogenic protein 2 (hBMP2), keratinocyte growth factor (KGF), platelet- derived growth factor, alpha -galactosidase A, iduronate-2-sulfatase, lactase, adenosine deaminase, Butyrylcholine &lt; / RTI &gt; a chitinase, a glutamate decarboxylase, an imiglucerase, a lipase, a uricase, a platelet-activating factor, a platelet-activating factor acetylhydrolase, neutral endopeptidase, urokinase, streptokinase, myeloperoxidase, superoxide dismutase, botulinum toxin, collagenase, hyaluronidase ), L-asparaginase, monoclonal antibody, polyclonal antibody, scFv, Fab, Fab ', F (ab') 2 and Fd A method for producing a recombinant fusion protein. 15. The method of claim 14,
The target protein may be selected from the group consisting of glucagon-like peptide-1 (GLP-1) and its analogues, somatostatin and its analogues, luteinizing hormone-releasing hormone (LHRH) agonists and antagonists, adrenocorticotropic Hormones such as adrenocorticotropic hormone, growth hormone-releasing hormone, oxytocin, thymosin alpha-1, calcitonin, vasopressin analogues, peptide hormone and fragments of the physiologically active protein of claim 15, which are selected from the group consisting of somatostatins, bombesin, cholecystokinin and gastrin analogues, epidermal growth factor receptor (EGFR), human fibronectin extradomain B (EDB) &Lt; / RTI &gt; A recombinant fusion protein produced by a method for producing a recombinant protein of any one of claims 1 to 16. 18. The method of claim 17,
Wherein said recombinant fusion protein is a recombinant fusion protein characterized by a COMP protein multiplexing region introduced and translated from SEQ ID NO: 1.

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