KR101023518B1 - A preparation method of solubility recombinant protein by use of Aspartate carbamoyltransferase catalytic chain as a fusion expression partner - Google Patents

Abstract

The present invention relates to a method for producing a recombinant protein using an aspartate carbamoyltransferase catalytic chain (PyrB) as a fusion expression partner, and more particularly, to a method for producing a recombinant protein using a fusion expression partner PyrB, and an exogenous protein; 2) introducing the expression vector into a host cell to produce a transformant; and 3) culturing the transformant to express the recombinant protein And obtaining the recombinant protein, and a recombinant protein produced by the method. PyrB of the present invention is a protein in E. coli that can maintain its own structural stability even under a stress environment. As a method for preparing a recombinant protein using the gene of this protein as a fusion expression partner, the availability and folding folding ability, thereby maintaining the structural stability of the target protein and expressing it at a high production yield. Therefore, the protein can be usefully used for production of medical and industrial proteins.

 Aspartate carbamoyltransferase catalytic chain (PyrB), fusion expression partner, stress environment

Description

Technical Field [0001] The present invention relates to a preparation method of a soluble recombinant protein using aspartate carbamoyltransferase catalytic chain as a fusion expression partner,

The present invention relates to a method for producing a recombinant protein using an aspartate carbamoyltransferase catalytic chain (PyrB) as a fusion expression partner, and more particularly, to a method for producing a recombinant protein using a fusion expression partner PyrB, and an exogenous protein; 2) introducing the expression vector into a host cell to produce a transformant; and 3) culturing the transformant to express the recombinant protein And obtaining the recombinant protein, and a recombinant protein produced by the method.

The fact that neutrophilic granulocytes or monocyte macrophages are formed in the body has already been known through bone marrow differentiation proliferation experiments and it is known that there are certain factors that stimulate the formation of these colonies in living beings (Pluznik and Sachs, J ... Cell Physiol 66 (3 ):..... 319-324, 1965; Bradley and Metcalf, Aust J. Exp Bio Med Sci 44 (3): 287-299, 1996). Factors commonly referred to as colony stimulating factors (interleukins, EPO, M-CSF, GM-CSF, and G-CSF) regulate the proliferation and differentiation of hematopoietic cells from progenitor stem cells plays a role (Nicola, NA, Annu Rev. Biochem 58:.. 45-77, 1989), depending on the specificity can be classified as follows: 1) M-CSF (macrophage-stimulating factor-stage configuration), mainly single 2) GM-CSF (granulocyte and monocyte macrophage stimulating factor) stimulates formation of colony by proliferating / differentiating granulocyte or monocyte macrophage hepatocytes, and 3) ) Multi-CSF (pluripotent cell stimulating factor) acts on hepatocytes of undifferentiated pluripotent cells to form pluripotent cell colonies, and 4) G-CSF (granulocyte colony stimulating factor) forms granular white blood cell colonies (Burgess et al., J. Biol. Chem. 252 (6): 1998-2003, 1977; Burgess et al. 1980, Biochem. J. 185 (2): 301-314 , 1980; Nicola et al., J. Biol. Chem. 258 (14): 9017-9023, 1983).

The human granulocyte colony-stimulating factor (hG-CSF) is a glycoprotein of about 20 KDa and the gene is present on chromosome 17. It is mainly produced by macrophages, activated T cells, fibroblasts or endothelial cells and is known to play a role in proliferation of neutrophil colonies, differentiation into progenitor cells from progenitor cells, and stimulation of mature neutrophil cells (Nagata et al., EMBO J. 5 (3): 575-581, 1986). Actually, hG-CSF stimulates the formation of neutrophils in vitro and works together with IL-3 to stimulate the formation of colonies of cells, nucleoli and macrophages, and some leukemic myeloid leukemic lines express hG- It is known to be mature by CSF.

Clinical uses of hG-CSF are as follows: 1) increase the number of neutrophils in a dose-dependent manner for neutropenia in hematologic malignancies; 2) increase the number of neutrophils in malignant, lymphoma, lung cancer, ovarian cancer, testicular tumors, Acute myelogenous leukemia, and acute leukemia, and 3) to rapidly recover neutropenia caused by bone marrow dysplasia syndrome, and 4) to prevent acute non-lymphocytic leukemia and chronic bone marrow 5) to restore neutropenia due to congenital or idiopathic neutropenia and aplastic anemia, 6) to prevent the occurrence of mucositis or febrile neutropenia by antitumor chemotherapy, (Drug Evaluations Annual 1993, American Medical Associations, p. 2232-2333).

As described above, various genetic engineering attempts have been made to obtain a large amount of hG-CSF which is very useful for medical use. Among them, hG-CSF gene is cloned and expressed in Escherichia coli (Misawa and Kumagai 1999, Biopolymers. 51 (4): 297-307, 1999).

In order to be used as a fusion expression partner, it should have characteristics such as high solubility expression rate of its own protein, effective tertiary structure formation and high yield. Studies on the structural stability and production of protein have been carried out in various fields. The structural stability is confirmed by the function maintenance time of the purified protein in an environmental condition (high temperature, low pH, high osmotic pressure, and an environment containing a protein structure inhibitor) in which the protein is difficult to form a tertiary structure (Park et al., Appl. Microl. Biotech. 75 (2): 347-355, 2007). While this analyzes the structural stability of proteins by applying multiple stresses to the purified protein, The first attempt was made to directly analyze the structural stability of intracellular proteins expressed by the reaction.

E. coli has a higher growth rate than other host cells and can be cultured at a high concentration. Since the entire genetic information is known, it is widely used as a strain to produce a large amount of recombinant protein. Generally, in order to form the tertiary structure of the active form, various proteins (molecular chaperone) that help in the formation of the structure are needed and various transformation processes are required. However, since Escherichia coli does not completely perform the process of forming a tertiary structure of proteins in eukaryotic cells, it is highly likely to form insoluble aggregates when directly producing proteins having relatively complex structures such as human-derived medical proteins and industrial enzymes Ding et al., Acta . Crystallogr. D. Biol . Crystallogr . 58: 2102-2108, 2002). Therefore, in order to increase the solubility of the target protein in E. coli, the expression rate of E. coli is lowered to perform low-temperature expression (Hammarstrom et al., Protein Sci. 11: 313-321, 2002) Simultaneous expression of a molecular chaperone or protein folding regulator and a protein of interest (de Marco and De Marco, J Biotechnol . 109: 45-52, 2004), optimization of conditions (Qing et al., Nat Biotechnol . 22: 877-882, 2004). However, the most commonly used method is the use of a fusion expression system that simultaneously expresses fusion partner and target protein. Indeed, several fusion expression partners have been studied and reported for many years in Escherichia coli to induce high expression of exogenous proteins (Esposito and Chatterjee, Curr. Opin. Biotechnol. 17: 353-358, 2006; Kapust and Waugh, Protein Sci. 8: 1668-1674, 1999; Sachdev and Chirgwin, Biochem. Biophys. Res. Commun. 244: 933-937, 1998).

As described above, when a recombinant protein is expressed using E. coli, an accurate tertiary structure is not formed and most of the expressed protein forms an insoluble aggregate. In addition, in order to make an inert insoluble aggregate of the recombinant protein thus produced active in a post-translational process such as refluxing in which it is dissolved by using a denaturant such as guanidine-hydrochloride (urea) or urea (diluted) (Marston, FA, Biochem., J. 240 (1): 1-12, 1986), which requires an additional process after protein expression, thereby providing a factor of economic inefficiency.

In order to overcome this problem, a method of producing a desired polypeptide by using a soluble protein known to be stably produced in E. coli as a fusion expression partner and fusing it to the N-terminal of a target protein has been studied. Maltose binding protein (MBP), beta-galactosidase, Thioredoxin (Trx), and glutathione-binding protein have been widely studied as stable fusion proteins. And Glutathione-S-transferase (GST). Such fusion expression partners have the advantage that they can easily purify the desired protein using an affinity chromatography method (Bach et al., J. Mol. Biol. 312 (1): 79-93, 2001; Smith and Johnson, Gene 67 (1): 31-40, 1988; LaVallie et al, Biotechnology (NY) 11 (2):... 187-193, 1993). However, these fusion partners are already registered in various countries as patents, and their use is subject to many restrictions. Therefore, it is necessary to find a new fusion expression partner capable of functioning as a fusion expression partner, and it is required to construct a new fusion fusion expression partner library.

Accordingly, the present inventors have made efforts to develop new fusion fusion expression partners for expressing high-value-added medical or industrial recombinant proteins in E. coli in a soluble state, and have found that stresses such as 2-hydroxyethyldisulfide (2-HEDS) Aspartate carbamoyltransferase catalytic chain (PyrB), which is able to maintain its own structural stability in the environment, was identified and expressed as a fusion partner of hG-CSF, a target protein, in Escherichia coli. As a result, Of hG-CSF and hG-CSF remained soluble after removal of PyrB. As a result, it has been confirmed that the expression of soluble hG-CSF can be increased and structural safety can be maintained, thereby completing the present invention.

It is an object of the present invention to provide an asparagine carbamoyltransferase catalytic chain, which is a protein in E. coli that maintains its own structural stability even under a strong stress environment that inhibits protein folding, as a universal fusion expression partner that can be utilized in various target proteins, And a method for producing a recombinant protein having the same structure as that of the recombinant protein in a soluble state.

In order to achieve the above object,

1) preparing an expression vector comprising a gene of an aspartate carbamoyltransferase catalytic chain (PyrB) and a gene of an exogenous protein as a fusion expression partner;

2) introducing the expression vector into a host cell to produce a transformant; And

3) culturing the transformant to induce expression of the recombinant protein and obtaining the recombinant protein.

The present invention also provides a method for producing a PyrB-deleted soluble foreign protein, which comprises the step of treating a protein restriction enzyme in an expression vector additionally containing a protein restriction enzyme recognition site between a PyrB gene and a foreign protein gene And a manufacturing method thereof.

In addition, the present invention provides a gene construct comprising a gene of PyrB as a fusion expression partner and a gene of a foreign protein.

The present invention also provides an expression vector comprising the gene construct.

In addition, the present invention provides a transformant into which the expression vector is transduced.

In addition, the present invention provides a recombinant fusion protein produced by the above production method.

In addition, the present invention provides a PyrB-deleted soluble foreign protein prepared by treating the recombinant fusion protein with a protein restriction enzyme.

The present invention relates to a method for producing a recombinant protein by using an aspartate carbamoyltransferase catalytic chain (PyrB) as a fusion expression partner, which maintains structural stability and exhibits increased expression even in a strong stress environment, It can be used extensively in producing high value added medical or industrial recombinant proteins because it can overcome the limitations of solubility and folding that partners have.

Hereinafter, terms used in the present invention will be described.

In the present invention, the term " heterologous protein "or" target protein "refers to a protein that a person skilled in the art intends to produce in large quantities. In the present invention, a polynucleotide encoding the protein is inserted into a recombinant expression vector, ≪ / RTI > refers to all proteins capable of expression in the body.

In the present invention, the term "recombinant protein" or "fusion protein" refers to a protein that is linked to another protein at the N-terminus or C-terminus of the sequence of the original foreign protein, Protein. Means a recombinant fusion protein in which the fusion expression partner of the present invention and the target protein are linked or a recombinant protein of the target protein in which the fusion fusion expression partner has been removed from the recombinant fusion protein by the protein cleavage enzyme.

In the present invention, the term "expression vector" is a linear or circular DNA molecule consisting of a fragment encoding a polypeptide of interest operably linked to an additional fragment provided in the transcription of the expression vector. Such additional fragments include promoter and termination coding sequences. The expression vector also includes at least one replication origin, at least one selection marker, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or contain both elements.

Hereinafter, the present invention will be described in detail.

The present invention

1) preparing an expression vector comprising a gene of an aspartate carbamoyltransferase catalytic chain (PyrB) and a gene of an exogenous protein as a fusion expression partner;

2) introducing the expression vector into a host cell to produce a transformant; And

3) culturing the transformant to induce expression of the recombinant protein and obtaining the recombinant protein.

In the above-mentioned preparation method, it is preferable to use the one containing the amino acid sequence of SEQ ID NO: 1 in the step 1), but not limited thereto, and the sequence having high homology of 90% Do.

In the above production method, the exogenous protein of step 1) preferably uses a biologically active protein selected from the group consisting of an antigen, an antibody, a cell receptor, an enzyme, a structural protein, a serum and a cell protein, It is more preferable to use a granulocyte colony-stimulating factor (hG-CSF), but it is not limited thereto.

In the above production method, the host cell of step 2) is preferably, but not limited to, E. coli.

The present invention also provides a method for producing a recombinant fusion protein comprising the step of treating a recombinant fusion protein prepared by further comprising a protein restriction enzyme recognition site between a PyrB gene and an exogenous protein gene of the expression vector of step 1) Lt; RTI ID = 0.0 > PyrB < / RTI >

In the above production method, the protein restriction enzyme recognition site may be any one selected from the group consisting of Xa factor recognition site, enterokinase cleavage site, Genenase I recognition site and purine recognition site More preferably an enterokinase recognition site, but is not limited thereto.

The present inventors have found that a protein having high solubility and high yield against 2-HEDS (2-hydroxyethyldisulfide), which is an oxidizing agent of E. coli proteome, exists and the protein has excellent self-folding ability, The proteome in the normal environment of Escherichia coli and the protease in the 2-HEDS stress state were analyzed using secondary electrophoresis and MALDI-TOF, assuming that it could be used as a fusion expression partner capable of maintaining high and active type. As a result, PyrB, which is a protein whose expression level increased more than 7-fold in a soluble state in a stress environment inhibiting proper structure formation, was identified (see FIG. 1). As a result of the reverse increase in the soluble expression level of PyrB protein in an environment in which most of the protein forms insoluble aggregates or decreases the expression level, PyrB protein is not only very stable in structure but also is related to the mechanism of the original function against stress Able to know. Therefore, it can be seen that PyrB obtained through analysis of E. coli proteome according to stress can be used as a fusion expression partner.

In order to verify the efficiency of the PyrB protein as a fusion expression partner, the present inventors synthesized an expression vector (see FIG. 2) by inserting PyrB into the amino terminal of hG-CSF and inserting it into an expression vector for Escherichia coli To prepare a transformant, to obtain a product containing the desired proteins, and then analyzing the product using SDS-PAGE. As a result, while recombinant hG-CSF expressed by using PyrB as a fusion expression partner formed soluble insoluble aggregates in hG-CSF alone expressed in Escherichia coli (Fig. 3A), the soluble expression amount was remarkably increased to 20 times or more (See Figs. 3B and 3C). Therefore, when PyrB is used as a fusion expression partner, it can be seen that there is an excellent effect in inducing the expression of hG-CSF in a soluble state which is easy to form an insoluble aggregate.

Based on the fact that the hG-CSF used for medical treatment should be separated from the fusion expression partner in order to avoid the antigen-antibody reaction, even when PyrB is separated from the hG-CSF, the remaining hG-CSF alone can remain soluble , Recombinant hG-CSF was purified using metalchromatography after removal of PyrB from recombinant hG-CSF using enterokinase, followed by SDS-PAGE and Western blotting Respectively. hG-CSF is first made into a precursor in the human body, and this precursor is made into hG-CSF having an N-terminal amino acid sequence of Thr-Pro-Leu through a protease-altering process. However, when expressed in Escherichia coli, methionine corresponding to the start codon is present at the N-terminal according to the amino-peptidase enzyme activity, and in some cases, a protein in which N-terminal methionine is not removed It is very difficult to remove it during the purification process, and when it is expressed as an insoluble insoluble protein, it is required to be made into a soluble form having activity. In this case, the yield is low. In the present invention, this disadvantage is overcome by producing methionine-deleted hG-CSF by cleaving with an enterokinase. As a result of the above analysis, it was confirmed that the recombinant hG-CSF from which PyrB was removed remained in a soluble state (see FIG. 4).

In order to investigate whether the hG-CSF production method using the PyrB protein as a fusion expression partner induces and expresses the hG-CSF inherent structure, the present inventors used the purified recombinant hG-CSF and commercially available hG- Degradation of the protein secondary structure of CSF was confirmed by using a circular dichroism spectroscopy (CD) analysis. As a result, it was confirmed that the purified recombinant hG-CSF of the present invention had the same protein secondary structure as hG-CSF used for therapeutic (see Fig. 5).

Therefore, the method of producing recombinant protein using PyrB of the present invention as a fusion expression partner is capable of expressing a high-rate soluble protein in a medical or industrial expression form and can form the same protein structure as the native type, It is possible to omit the process of refolding by the required denaturant or reducing agent, thereby making it possible to construct an efficient production process.

The present invention also provides a gene construct comprising a gene of PyrB and a gene of a foreign protein as a fusion expression partner, and an expression vector comprising the gene construct.

The expression vector includes pT7, pET / Rb, pGEX, pET28a (+), pET-22b (+), and pET-22b And pGEX, and it is more preferable to use pET28a (+), but it is not limited thereto.

The expression vector may be linked with a polynucleotide encoding a protein cleavage enzyme recognition site between the PyrB gene and the foreign protein gene so that the PyrB gene and the foreign protein gene are operably linked. When the fusion expression partner and the target protein are industrial proteins, the fusion expression partner may degrade the function of the target protein. In the case of a medical protein, the fusion expression partner is preferably removed because 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 have.

In order to operably link the gene of PyrB with the gene of the foreign protein, the expression vector may be linked with a polynucleotide encoding a tag for separation and purification so that the recombinant protein can be easily separated and purified. At this time, the tag for separation and purification can be selected from the group consisting of GST, poly-Arg, FLAG, poly-His, and c-myc, or may be used by sequentially connecting two or more of them.

In addition, the present invention provides a transformant wherein the expression vector is transfected into a host cell.

The expression vector is preferably a vector including a gene construct containing a PyrB gene and a gene of a foreign protein as a fusion expression partner, but is not limited thereto. The PyrB preferably includes the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. Any sequence having high homology of 90% or more with the above sequence may be used. Preferably, the exogenous protein uses a biologically active protein selected from the group consisting of an antigen, an antibody, a cell receptor, an enzyme, a structural protein, a serum and a cell protein, more preferably hG-CSF It does not. Preferably, the host cell uses E. coli but is not limited thereto.

In addition, a recombinant fusion protein produced by the method of the present invention is provided.

In addition, the present invention provides soluble PyrB-derived exogenous proteins prepared by addition of enterokinase to the recombinant fusion protein.

The recombinant protein produced by the production method using the PyrB of the present invention as a fusion expression partner is expressed at a high rate of solubility and forms the same protein structure as that of the native form. Therefore, refolding by the denaturant or reducing agent required for the insoluble aggregate ) Process can be omitted. A high value-added medical or industrial recombinant protein can be used as a target protein to produce it.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1 Analysis of Escherichia coli Protein

<1-1> Recovery of soluble protein in Escherichia coli

The present inventors selected E. coli BL21 (DE3) strains and analyzed the protein bodies. A portion of the seed culture that had been cultured for more than 8 hours was inoculated into the LB medium and cultured at 37 ° C at 200 rpm in the incubator (control group), and the protein body under stress which inhibited the proper folding of the protein, To obtain LB medium containing 2-HEDS (10 mM), a part of the seed culture was inoculated and cultured under the same conditions (experimental group). All E. coli were induced by IPTG (1 mM) when OD ₆₀₀ reached 0.5. The cell culture was centrifuged at 6,000 rpm at 4 ° C, and the cell lysate was recovered and washed twice with 40 mM Tris buffer (pH 8.0). The washed E. coli precipitate was suspended in 500 [mu] l of disruption buffer [lysis buffer; 8 mM urea, 4% (w / v) CHAPS, 40 mM Tris, protease inhibitor cocktail) and disrupted using an ultrasonic disrupter. After disruption of the cells, the cells were centrifuged at 12,000 rpm at 4 ° C for 60 minutes, and the supernatant was separated. The protein concentration of the supernatant was measured using a Bio-Rad protein assay kit. The supernatant containing 30 mg of soluble protein was fractionated to obtain a rehydration solution. And suspended in 2M thiourea, 8M urea, 4% w / v CHAPS, 1% w / v DTT, 1% w / v mobile ampholyte, pH 4.7] 2-Dimensional polyacrylamide gel electrophoresis (2D-PAGE) samples were stored frozen at -80 ° C.

<1-2> Analysis of Escherichia coli Protein by 2-D Gel Electrophoresis

The protein bodies of the control and experimental groups obtained by the method of Example <1-1> were analyzed using 2-dimensional gel electrophoresis.

One-dimensional isoelectric point separation for separating the cryopreserved proteins by pI was performed using a Bio-Rad Protein IEF cell electrophoresis system and a linear pH 4-7 IPG gel strip (17 cm, ReadyStrip) was used to rehydrate the proteins contained in the rehydrated solution overnight. For 2 hours at 500 V, 30 minutes at 1000 V, 30 minutes at 2000 V, 30 minutes at 4000 V, and 70000 VHr (Volt-hours) at 8000 V. The rehydrated IPG gel strips were washed with an epilating solution containing 1% DTT. After reacting for 15 minutes in 50 mM Tris, pH 8.6, 6 M urea, 30% v / v glycerol, 2% SDS, some bromophenol blue) And reacted in an equilibration solution containing iodoacetamide for 15 minutes. The equilibrated gel strips were electrophoresed using 12.5% polyacrylamide gels and then protein spots were detected by silver staining. The stained gels were scanned using a GS-710 densitometer and protein spots were confirmed using PDQuest software version 6.1. The volume of the protein spot was measured and compared with the increase or decrease in the expression level of the protein isolated under 2-HEDS (10 mM) based on the protein isolated under the normal condition at 37 ° C., (Fig. 1).

&Lt; Example 2 > MALDI-TOF-MS analysis and protein identification

The present inventors extracted protein spots selected according to Example 1 and deposited them with the Korean Basic Science Institute and identified them using MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization-Time Of Flight) -MS analysis .

Specifically, protein spots selected by <Example 1> were extracted from silver-stained gel for MALDI-TOF-MS analysis (Gharahdaghi F, et al. , Electrophoresis , 20: 601-605, 1999) . Protein spots of extracted protein spots were digested with trypsin (10.15 mg / ml) in a 25 mM ammonium bicarbonate (pH 8.0) solution overnight at 37 ° C. The digested peptides were extracted with 5% v / v TFA and 50% v / v ACN solution. This procedure was repeated three times and dried using a vacuum centrifuge. The dried peptide was dissolved in 50% ACN / 0.1% TFA solution, and the molecular weight was measured using a MALDI-TOF-MS system (Voyager DE-STR instrument; Biosystems, USA) Peptide mass fingerprints of the peptides were measured using the MS-FIT (http://prospector.ucsf.edu/prospector/4.0.8/html/msfit.htm) on the Prospector website and the protein identifications Swiss-Prot was used as the MS-FIT database. As a result of protein identification, a protein having an expression level of at least 7-fold increased under 2-HEDS stress was confirmed to be an aspartate carbamoyltransferase catalytic chain (PyrB) as an Escherichia coli aspartate.

Identification of proteins with expression levels over 7-fold increased under 2-HEDS stress conditions Gene name Gene access number ^a Protein name Isoelectric point (pI) / molecular weight (kDa) Sequence similarity (%) Theoretical value ^b Experimental value ^c pyrB P0A786 Aspartate Carbamoyltransferase catalytic chain 6.13 / 34.28 6.25 / 35.40 23%

a: Gene access number: This is an identification number for searching the gene information in ExPASy Proteomics Server (http://www.expasy.org/).

b: Theoretical values were collected using Compute pI / Mw tool (http://www.expasy.org/tools/pi_tool.html).

c: Experimental value was calculated from two-dimensional electrophoresis gel image.

Example 3 Production of hG-CSF alone expression vector (pET-hGCSF) and PyrB fusion expression vector (pET-PyrB-hGCSF)

The present inventors prepared an expression vector containing the E. coli protein PyrB as a fusion expression partner with increased soluble expression under environmental stresses selected by the methods of Example 1 and Example 2 above.

(SEQ ID NO: 2) having the base sequence of hG-CSF gene (primer I: 5'-CTCGAGGACGATGACGA TAAAACCCCCCTGGGCCCTGCC-3 (SEQ ID NO: 2)) having the nucleotide sequence of hG-CSF gene and the primer II: 5'-AAGCTTTCAGGGCTGGGCAA GGTGGCG-3 (SEQ ID NO: 3) '] was used to amplify a gene encoding recombinant hG-CSF by PCR. The hG-CSF amplified a gene coding for a total of 175 amino acid sequences with the start codon removed and the stop codon. The hG-CSF was amplified by the enterokinase immediately following the Xho I restriction site at the N- (DDDDK; SEQ ID NO: 6). PCR reaction buffer for the DNA polymerase [0.25 mM dNTPs, 50 mM KCl , 10 mM (NH 4) 2 SO 4, 20 mM Tris-HCl (pH8.8), 2 mM MgSO 4, 0.1% Triton X-100] 100 ng of template DNA and 50 pmol of each primer were added and the reaction was carried out using Taq DNA polymerase. The reaction conditions were 95 ° C for 30 seconds (denaturation), 52 ° C for 30 seconds (annealing), 72 (Elongation) for a total of 30 runs (all PCRs described below are subject to the above conditions unless otherwise noted). After completion of the PCR, the amplified DNA was separated by using 1% agarose gel, and the both ends of the amplified DNA were cut into Xho I and Hind III to obtain pET-28a (+) (Novagen) expression vector The plasmid was inserted into the restriction enzymes Xho I and Hind III, and the plasmid thus constructed was named 'pET-hGCSF'.

Further, in order to obtain a gene of the E. coli-derived PyrB, E. coli strain BL21 (DE3 ) [F - omp T hsd S B (rB - mB -)] to into the genome (genomic) DNA extract is suspended in this mold PyrB consisting of 311 amino acids (SEQ ID NO: 1) except the codon was amplified by PCR method. At this time, PCR was carried out by inserting Nde I restriction enzyme recognition sequence at the 5 'end and Xho I restriction enzyme recognition sequence at the 3' end so that Nde I at the 5 'end of the amplified DNA fragment and Xho I restriction enzyme site at the 3' Respectively. The primers for the PCR were as follows: 1) 5'-CAT ATG GCT AAT CCG CTA TAT CAG-3 (SEQ ID NO: 4) 'and 2'5'-CTC GAG CAG TAC CAG ATC GCG ATT- Number: 5) '. Were inserted into each process a gene of PyrB amplified by PCR in the Nde I and Xho I place the vector by processing the above-prepared plasmid pET-hGCSF with Nde I and Xho I to Nde I and Xho I, this' pET- PyrB-hGCSF &quot;.

Example 4 Solubility Expression of Recombinant Protein and Confirmation Using SDS-PAGE

<4-1> Preparation of Escherichia coli Transformants

The present inventors used the expression vector prepared in Example 3 by the method described by Hanahan (Hanahan D, DNA Cloning vol.19-135, IRS press 1985) pET-hGCSF and pET-PyrB-hGCSF were transformed into Escherichia coli BL21 (DE3) (Studier and Moffatt, J. Mol. Biol. 189 (1): 113-130, 1986) by thermal shock method Next, colonies showing resistance to kanamycin were selected by culturing in LB medium (+100 mg / L kanamycin) containing kanamycin.

<4-2> Culture and Gene Expression of Escherichia coli Transformants

Using the E. coli transformant transformed with the recombinant expression vector, hG-CSF fused with hG-CSF and PyrB alone was produced. A portion of the seed culture which had been cultured for 8 hours or longer was inoculated (1%) in the cultured LB medium (+100 mg / L kanamycin) and cultured at 37 ° C at 200 rpm. When the OD ₆₀₀ of the culture reached 0.5, 1 mM IPTG (isopropylthio-β-D-galactoside) was added to induce the expression of the recombinant gene. After addition of IPTG, cells were further cultured for 3 to 4 hours under the same conditions. The cell culture was centrifuged at 6,000 rpm for 10 minutes at 4 ° C to collect the cell precipitate. The collected cell precipitate was suspended in 10 mM Tris-HCl buffer (pH 7.5) Suspended, and then disrupted using an ultrasonic disrupter (Branson Sonifier). After the disruption, the supernatant and the protein aggregate were separated by centrifugation (13,000 rpm × 10 minutes), and the separated supernatant and insoluble aggregate were suspended in 5 × SDS (0.156 M Tris-HCl, pH 6.8, 2.5% SDS, 37.5% glycerol , 37.5 mM DTT) and boiled at 100 ° C for 10 minutes. The boiled samples were loaded on a 12% SDS-PAGE gel (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (12% Tris-Glycine gel) and developed for 3 hours at 120 V. The samples were then stained with Coomassie staining, (Densitometer, Bio-Rad, USA), and the solubility (%) was calculated according to the following equation (1).

As a result, when hG-CSF alone was expressed without PyrB, the degree of solubility was less than 4%, whereas when PyrB and hG-CSF were fusion-expressed, the degree of solubility was significantly increased to 80% (FIG.

Example 5 Purification and Analysis of Recombinant hG-CSF from which PyrB Is Detached

The present inventors produced and purified recombinant proteins using E. coli BL21 (DE3) transformed with the fusion expression vector (pET-PyrB-hGCSF) prepared in Example 4 above. Since the transformant used pET-28a (+), six histidine residues were located at the N-terminal region of the expressed fusion protein, and purification through Ni ²⁺ metal affinity chromatography due to the affinity of histidine possible by Ni-NTA agarose beads using (agarose bead) (QIAGEN) was performed and purification of Ni ²⁺ metal affinity chromatography.

Specifically, the cells were disrupted, centrifuged at 13,000 rpm for 10 minutes, and the separated supernatant was combined with 500 mL of Ni-NTA agarose beads (QIAGEN) for purification using metal affinity. Before reaction with the Ni-NTA agarose beads, washing was performed using a binding buffer (pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole) Respectively. The binding of the supernatant containing Ni-NTA agarose beads with the supernatant containing the fusion protein [(x6) PyrB- (enterokinase moiety) hGCSF fusion protein fused to PyrB proceeded at 4 &lt; 0 &gt; And then washed twice with 8 mL of washing buffer [pH 8.0, 50 mM sodium phosphate, 300 mM sodium chloride (NaCl), 50 mM imidazole]. In the next step, PBS buffer solution [PBS buffer; The cells were treated once with 137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM potassium phosphate monobasic (KH ₂ PO ₄ ), pH 7.4 Further washing was carried out. Enterokinase solution (5 占 퐇 of enterokinase, 50 占 퐇 of 10 占 ¢ enterokinase buffer, 445 占 퐇 of PBS (Invitrogen)) 500 (PEGylation inhibitor) into hGCSF (HisX6) PyrB- (enterokinase site) attached to agarose beads The reaction was carried out at 22 ° C for 12 hours. The elution fraction was taken and centrifuged at 13,000 rpm for 15 minutes to separate the soluble and insoluble solution. The separated samples showed that hG-CSF remained soluble even after PyrB was removed through SDS-PAGE analysis. This purified hG-CSF was detected by mouse anti-human G-CSF monoclonal antibody Western blot analysis using G-CSF monoclonal antibody (Clone 3D1, Santa Cruz Biotechnology Inc, CA) (FIG. 4).

Example 6 Identification of Protein Secondary Structure by Analysis of Circular Dichroism Spectroscopy (CD)

The enterokinase-free solution was removed using the EK-Away ^TM resin (Invitrogen, Germany, Cat. No. R180-01), and the solution with the enterokinase removed was diluted with microcon YM-10 (Millipore, Ireland ) (0.4167 [mu] g / [mu] l) by the amount necessary for CD analysis to prepare a sample for analysis. The prepared recombinant hG-CSF sample was analyzed by a JASCO J-710 spectropolarimeter with a circularly polarized dichroism spectroscopy (CD) by Korea Basic Science Institute (Ochang). CSF (Grasin Prefilled-Syringe (Filgrastim), Kirin, Japan), which is currently being marketed for treatment with purified recombinant hG-CSF, (Fig. 5).

Figure 1 is a graph showing the results of comparative analysis of E. coli BL21 (DE3) proteome under normal environment and 2-HEDS stress environment.

(A) is an image showing an image of two-dimensional gel electrophoresis of Escherichia coli protein under normal circumstances.

(B) is an image showing an image of a two-dimensional gel electrophoresis of an E. coli protein body in a 2-HEDS stress environment.

(C) is a graph comparing relative spot intensities of PyrB in a normal environment and a 2-HEDS stress environment.

FIG. 2 is a diagram showing a vector expression for expression of PyrB fusion with a single expression vector of human granulocyte colony stimulating factor (hG-CSF).

(A) is a figure showing a single expression vector of hG-CSF.

(B) is a figure showing a plasmid vector for fusion expression of hG-CSF including a His ₆ tag and an enterokinase cleavage site.

FIG. 3 is a graph showing the results of SDS-PAGE analysis of hG-CSF alone and PyrB fusion expression (M: molecular marker; S: soluble fraction of recombinant E. coli-derived cell lysate; and IS: recombinant Escherichia coli Insoluble fraction of the resulting cell lysate).

(A) shows the result of SDS-PAGE analysis of hG-CSF alone expression.

(B) shows SDS-PAGE analysis results of hG-CSF and PyrB fusion expression.

(C) is a graph showing solely the expression of hG-CSF and the solubility (%) of cells expressing fusion of PyrB and hG-CSF.

Figure 4 is an image showing the results of SDS-PAGE analysis and Western blot analysis of purified hG-CSF (M: molecular marker; S: soluble fraction of purified hG-CSF; and IS: purified hG - insoluble fraction of CSF) (arrow indicates purified hG-CSF).

(A) shows the results of SDS-PAGE analysis of the availability of purified recombinant hG-CSF after removal of PyrB by treatment with enterokinase.

(B) is a graph showing the result of western blot analysis of purified recombinant hG-CSF after removal of PyrB by treatment with enterokinase.

5 is a graph showing the results of the circularly dichroism spectroscopy (CD) analysis of the purified hG-CSF.

(A) is a figure showing the result of analysis of purified recombinant hG-CSF separated from PyrB :: hG-CSF by circular dichroism spectroscopy (CD).

(B) is a graph showing the result of analysis of a commercial standard hG-CSF by a circularly polarized dichroism spectroscopy (CD).

<110> Korea University Industrial & Academic Collaboration Foundation <120> A preparation method of solubility recombinant protein by use of          Aspartate carbamoyltransferase catalytic chain as a fusion          expression partner <130> 8p-09-05 <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 311 <212> PRT <213> Escherichia coli <400> 1 Met Ala Asn Pro Leu Tyr Gln Lys His Ile Ile Ser Ile Asn Asp Leu   1 5 10 15 Ser Arg Asp Asp Leu Asn Leu Val Leu Ala Thr Ala Ala Lys Leu Lys              20 25 30 Ala Asn Pro Gln Pro Glu Leu Leu Lys His Lys Val Ile Ala Ser Cys          35 40 45 Phe Phe Glu Ala Ser Thr Arg Thr Arg Leu Ser Phe Glu Thr Ser Met      50 55 60 His Arg Leu Gly Ala Ser Val Val Gly Phe Ser Asp Ser Ala Asn Thr  65 70 75 80 Ser Leu Gly Lys Lys Gly Glu Thr Leu Ala Asp Thr Ile Ser Val Ile                  85 90 95 Ser Thr Tyr Val Asp Ala Ile Val Met Arg His Pro Gln Glu Gly Ala             100 105 110 Ala Arg Leu Ala Thr Glu Phe Ser Gly Asn Val         115 120 125 Gly Asp Gly Ser Asn Gln His Pro Thr Gln Thr Leu Leu Asp Leu Phe     130 135 140 Thr Ile Gln Glu Thr Gln Gly Arg Leu Asp Asn Leu His Val Ala Met 145 150 155 160 Val Gly Asp Leu Lys Tyr Gly Arg Thr Val His Ser Leu Thr Gln Ala                 165 170 175 Leu Ala Lys Phe Asp Gly Asn Arg Phe Tyr Phe Ile Ala Pro Asp Ala             180 185 190 Leu Ala Met Pro Gln Tyr Ile Leu Asp Met Leu Asp Glu Lys Gly Ile         195 200 205 Ala Trp Ser Leu His Ser Ser Ile Glu Glu Val Met Ala Glu Val Asp     210 215 220 Ile Leu Tyr Met Thr Arg Val Gln Lys Glu Arg Leu Asp Pro Ser Glu 225 230 235 240 Tyr Ala Asn Val Lys Ala Gln Phe Val Leu Arg Ala Ser Asp Leu His                 245 250 255 Asn Ala Lys Ala Asn Met Lys Val Leu His Pro Leu Pro Arg Val Asp             260 265 270 Glu Ile Ala Thr Asp Val Asp Lys Thr Pro His Ala Trp Tyr Phe Gln         275 280 285 Gln Ala Gly Asn Gly Ile Phe Ala Arg Gln Ala Leu Leu Ala Leu Val     290 295 300 Leu Asn Arg Asp Leu Val Leu 305 310 <210> 2 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> hG-CSF primer I <400> 2 ctcgaggacg atgacgataa aacccccctg ggccctgcc 39 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> hG-CSF primer II <400> 3 aagctttcag ggctgggcaa ggtggcg 27 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PyrB primer I <400> 4 catatggcta atccgctata tcag 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PyrB primer II <400> 5 ctcgagcagt accagatcgc gatt 24 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> enterokinase cleavage site <400> 6 Asp Asp Asp Asp Lys   1 5  

Claims (20)

1) preparing an expression vector comprising a gene of an aspartate carbamoyltransferase catalytic chain (PyrB) and a gene of an exogenous protein as a fusion expression partner; 2) introducing the expression vector into a host cell to produce a transformant; And 3) culturing the transformant to induce expression of the recombinant protein and obtaining the recombinant protein. 2. The method for producing a recombinant protein according to claim 1, wherein PyrB is represented by SEQ. 2. The method according to claim 1, wherein the exogenous protein has a biological activity selected from the group consisting of an antigen, an antibody, a cell receptor, an enzyme, a structural protein, a serum and a cell protein. 4. The method according to claim 3, wherein the exogenous protein is a human granulocyte colony-stimulating factor (hG-CSF). 2. The method according to claim 1, wherein the expression vector further comprises a protein restriction enzyme recognition site between the PyrB gene and the foreign protein gene. 6. The protein restriction enzyme recognition site according to claim 5, wherein the protein restriction enzyme recognition site is selected from the group consisting of an Xa factor recognition site, an enterokinase cleavage site, a Genenase I recognition site and a purine recognition site &Lt; / RTI &gt; The recombinant protein according to claim 1, wherein the expression vector is selected from the group consisting of pT7, pET / Rb, pGEX, pET28a (+), pET-22b (+ Gt; 2. The method according to claim 1, wherein the host cell is E. coli . The method for producing a recombinant protein according to claim 1, wherein the culture is cultured under a stress environmental condition that prevents formation of a tertiary structure of the protein. [Claim 11] The method according to claim 9, wherein the stress environmental condition is a condition in which 2-HEDS (2-hydroxyethyldisulfide) is added. A method for preparing a soluble PyrB-derived foreign protein, which comprises treating a recombinant protein produced by the method of claim 5 with a protein restriction enzyme. A gene construct comprising an aspartate carbamoyltransferase catalytic chain (PyrB) gene and a gene for an exogenous protein as a fusion expression partner. 13. The genetic construct according to claim 12, wherein the gene construct further comprises a protein restriction enzyme recognition site between the PyrB gene and the foreign protein gene. 14. The protein restriction enzyme recognition site according to claim 13, wherein the protein restriction enzyme recognition site is selected from the group consisting of an Xa factor recognition site, an enterokinase cleavage site, a Genenase I recognition site and a purine recognition site Wherein the genetic construct is characterized by being a genetic construct. 13. The genetic construct of claim 12, wherein the genetic construct is represented by Figure 2 (B). 12. An expression vector comprising the genetic construct of claim 12. The expression vector according to claim 16, wherein the expression vector comprises any one selected from the group consisting of pT7, pET / Rb, pGEX, pET28a (+), pET-22b (+) and pGEX as a skeletal vector. A transformant transformed with the expression vector of claim 16. A recombinant fusion protein produced by the method of claim 1. A soluble foreign protein produced by the method of claim 5, wherein the PyrB is removed.

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