KR101077613B1 - A preparation method of solubility recombinant protein by use of Putative HTH―type transcriptional regulator yjdC as a fusion expression partner - Google Patents

Abstract

The present invention relates to a method for preparing a recombinant protein using a putative HTH-type transcriptional regulator yjdC (YjdC) as a fusion expression partner, and more specifically, 1) a fusion expression partner YjdC, and the step of preparing an expression vector connecting the gene of the foreign protein, 2) introducing the expression vector into the host cell to prepare a transformant, and 3) culturing the transformant to recombinant protein It relates to a method for producing a recombinant protein comprising the step of inducing expression and obtaining thereof, and to a recombinant protein produced by the method. YjdC of the present invention is a protein in E. coli that can maintain its structural stability even under stress environment, and a method for producing a recombinant protein using the gene of the protein as a fusion expression partner is a soluble and folded ( It can be usefully used for the production of medical and industrial proteins because it can maintain the structural stability of the protein of interest and express it with a high production yield by improving the folding ability.

 Putative HTH-type transcriptional regulator yjdC (YjdC), fusion expression partner, stress environment

Description

A preparation method of solubility recombinant protein by use of Putative HTH-type transcriptional regulator yjdC as a fusion expression partner}

The present invention relates to a method for preparing a recombinant protein using a putative HTH-type transcriptional regulator yjdC (YjdC) as a fusion expression partner, and more specifically, 1) a fusion expression partner YjdC, and the step of preparing an expression vector connecting the gene of the foreign protein, 2) introducing the expression vector into the host cell to prepare a transformant, and 3) culturing the transformant to recombinant protein It relates to a method for producing a recombinant protein comprising the step of inducing expression and obtaining thereof, and to a recombinant protein produced by the method.

The formation of neutrophil granulocytes or monocytic macrophages in the human body has been known from myeloid differentiation and proliferation experiments, and it is known that there are certain factors that stimulate the formation of these colonies in living plants (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 collectively referred to as Colony Stimulating Factor (such as interleukin, EPO, M-CSF, GM-CSF and G-CSF) regulate the proliferation and differentiation of blood cells from progenitor stem cells. (Nicola, NA, Annu. Rev. Biochem. 58: 45-77, 1989), depending on their specificity, can be classified as follows: 1) M-CSF (monophage macrophage stimulating factor) 2) GM-CSF (granulocytes and monocytic macrophage stimulating factor) proliferate / differentiate hepatocytes of granulocytes or monocytic macrophages to stimulate colony formation, 3 multi-CSF (pluripotent cell stimulator) acts on hepatocytes of undifferentiated pluripotent cells to form pluripotent cells and 4) G-CSF (granulocyte colony stimulator) forms granulocytes Burgss 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).

Human granulocyte colony-stimulating factor (hG-CSF) is a glycoprotein of about 20 KDa and the gene is present on chromosome 17. Produced mainly by macrophage, activated T cells, fibroblasts or endothelial cells, it is known to play a role in proliferation of neutrophil colonies, differentiation from progenitor cells to neutrophil cells, and active stimulation of mature neutrophil cells (Nagata et al., EMBO J. 5 (3): 575-581, 1986). Indeed hG-CSF stimulates neutrophil colonization in vitro, works with IL-3 to stimulate colonization of blasts, eukaryotic cells and macrophages, and some leukemia myeloma lines are hG- It is known to mature by CSF.

Clinical uses of hG-CSF include: 1) increasing the number of neutrophils dose-dependently against neutropenia in patients with hematologic malignancies, 2) malignant, lymphoma, lung cancer, ovarian cancer, testicular tumor, urethral epithelial cancer, and In chemotherapy for various cancers such as acute leukemia, neutropenia due to chemotherapy can be quickly recovered, 3) neutropenia due to myelodysplastic syndrome, and 4) acute non-lymphatic leukemia and chronic bone marrow. Restoring neutropenia in bone marrow transplantation of leukemia, 5) restoring neutropenia due to congenital or idiopathic neutropenia and aplastic anemia, and 6) preventing the development of mucositis or febrile neutropenia caused by anti-tumor chemotherapy. (Drug Evaluations Annual 1993, American Medical Associations p. 2232-2333).

As described above, several genetic engineering attempts have been made to obtain large amounts of hG-CSF, which is very useful for medical purposes. Among them, a method of cloning the hG-CSF gene and expressing it in E. coli has been mainly studied (Misawa and Kumagai 1999, Biopolymers. 51 (4): 297-307, 1999).

To be used as a fusion expression partner, it has to be characterized by high protein soluble expression rate, effective tertiary structure formation and high yield. Research into the structural stability and production of proteins has been conducted in various ways. The structural stability is reported by the method of confirming the retention time of the purified protein under environmental conditions (high temperature, low pH, high osmotic pressure, and protein structure inhibitors) difficult to form a tertiary structure protein (Park et al., Appl. Microl. Biotech. 75 (2): 347-355, 2007) It is the application of several stresses to a purified protein that analyzes the structural stability of the protein, while the inventors The first attempt was made to directly analyze the structural stability of proteins expressed in response to cells.

Escherichia coli has a high growth rate compared to other host cells, and thus high concentration culture is possible, and since the entire genetic information is revealed, it is widely used as a strain for producing a large amount of recombinant protein. In general, the formation of an active tertiary structure requires the assistance of various proteins (molecular chaperone) to assist in the formation of the structure and undergo a number of modifications. However, because E. coli is not able to perform the complete protein tertiary structure formation process in eukaryotic cells, it is highly likely to form insoluble aggregates by directly producing relatively complex structures such as human-derived medical proteins or industrial enzymes. Ding et al., Acta.Crystallogr.D . Biol.Crystallogr . 58: 2102-2108, 2002). Therefore, in order to increase the availability of the target protein in Escherichia coli, the expression rate of Escherichia coli is lowered to perform low temperature expression (Hammarstrom et al., Protein Sci. 11: 313-321, 2002), or induced by using various promoters. Conditions (Qing et al., Nat Biotechnol . 22: 877-882, 2004), co-expression of molecular chaperone or protein folding regulators with the desired protein (de Marco and De Marco, J Biotechnol. 109: 45-52, Although many methods have been tried, the most commonly used method is the use of a fusion expression system that simultaneously expresses a fusion expression partner and a target protein. Indeed, several fusion expression partners that induce high soluble expression of foreign proteins in E. coli have been studied and reported over the years (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 the recombinant protein is expressed using Escherichia coli, most of the expressed proteins fail to form an accurate tertiary structure and form insoluble aggregates. In addition, post-translational processes such as refloding, which are dissolved and diluted with a denaturing agent such as guanidine hychloride or urea to make the inactive insoluble aggregate of the recombinant protein thus produced active ( Since post-translational processes are required, additional processes are required after protein expression to provide a factor of economic inefficiency (Marston, FA, Biochem. J. 240 (1): 1-12, 1986).

In order to overcome this problem, methods for producing a desired polypeptide by fusing at the N-terminus of the target protein using a soluble expression partner known to be stably produced in Escherichia coli have been studied. Representative fusion expression partners studied so far as stable soluble proteins include maltose binding protein (MBP), beta-galactosidase, thioredoxin (Trx) and glutathione- Glutathione-S-transferase (GST). This fusion expression partner has the advantage of easily purifying the desired protein by using affinity chromatography (Bach et al., J. Mol. Biol. 312 (1): 79-93, 2001; Smith and Johnson, Gene. 67 (1): 31-40, 1988; La Vallie et al., Biotechnology (NY). 11 (2): 187-193, 1993). However, these fusion-expressing partners are already patented in many countries, and their use is subject to many restrictions. Therefore, it is necessary to find a new fusion expression partner that can function as a fusion expression partner and to build a new concept of fusion expression partner library.

Accordingly, the present inventors are working on developing a new concept of fusion expression partner for expressing a high value-added medical or industrial recombinant protein in an soluble state in E. coli, and stress such as 2-HEDS (2-hydroxyethyldisulfide), which is difficult to form correctly. As a result of finding a putative HTH-type transcriptional regulator yjdC (YjdC) that can maintain its structural stability even in the environment, it was expressed in Escherichia coli as a fusion expression partner of hG-CSF, a target protein. Highly soluble hG-CSF was expressed and hG-CSF remained soluble even after removal of YjdC. As a result, the present invention was completed by confirming that it is possible to increase the expression level of soluble hG-CSF and maintain structural safety.

An object of the present invention is to use a putative HTH-type transcriptional regulator YjdC, an E. coli protein that maintains its structural stability even in a strong stress environment that inhibits protein folding, by using a natural fusion expression partner that can be used in various target proteins. It is to provide a method for producing a recombinant protein of the same structure in a soluble state.

In order to achieve the above object, the present invention

1) preparing an expression vector linking a gene of Putative HTH-type transcriptional regulator yjdC (YjdC) and a foreign protein gene as a fusion expression partner;

2) preparing a transformant by introducing the expression vector into a host cell; And

3) culturing the transformant to induce the expression of the recombinant protein and to provide a method for producing a recombinant protein consisting of obtaining the same.

In addition, the present invention provides a method for producing a soluble foreign protein of YjdC removed, comprising the step of treating the protein restriction enzyme in the expression vector further comprising a protein restriction enzyme recognition site between the gene of the YjdC and the foreign protein gene It provides a manufacturing method.

The present invention also provides a gene construct comprising a gene of YjdC and a gene of foreign protein as a fusion expression partner.

In addition, the present invention provides an expression vector comprising the gene construct.

The present invention also provides a transformant to which the expression vector is transduced.

The present invention also provides a recombinant fusion protein prepared by the above method.

In addition, the present invention provides a soluble foreign protein from which YjdC is removed, which is prepared by further processing a protein restriction enzyme in addition to the recombinant fusion protein.

The present invention is a method of producing a recombinant protein using a putative HTH-type transcriptional regulator YjdC (YjdC), which maintains structural stability and increases expression even in a strong stress environment, as a fusion expression partner. Because of its ability to overcome the solubility and folding limitations of fusion-expressing partners, it can be widely used in the production of high value-added medical or industrial recombinant proteins.

Hereinafter, the term used by this invention is demonstrated.

In the present invention, the term "heterologous protein" or "target protein" is a protein that a person skilled in the art intends to produce in large quantities, and transforms by inserting a polynucleotide encoding the protein into a recombinant expression vector. It means any protein that can be expressed in the body.

In the present invention, the term “recombinant protein” or “fusion protien” refers to another protein linked to the N-terminus or C-terminus of the sequence of the original foreign protein or to which another amino acid sequence is added. Means protein. The recombinant protein of the target protein in which the fusion expression partner is removed from the recombinant fusion protein by a recombinant fusion protein form or protein cleavage enzyme connected to the target protein of the fusion expression partner of the present invention.

In the present invention, the term "expression vector" is a linear or circular DNA molecule consisting of fragments encoding a polypeptide of interest operably linked to additional fragments provided for transcription of the expression vector. Such additional fragments include promoter and termination code sequences. Expression vectors also include one or more replication initiation points, one or more selection markers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or contain elements of both.

Hereinafter, the present invention will be described in detail.

The present invention

1) preparing an expression vector linking a gene of Putative HTH-type transcriptional regulator yjdC (YjdC) and a gene of a foreign protein as a fusion expression partner;

2) preparing a transformant by introducing the expression vector into a host cell; And

3) culturing the transformant to induce the expression of the recombinant protein and to provide a method for producing a recombinant protein consisting of obtaining the same.

In the above production method, YjdC of step 1) is preferably used to include an amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto, any sequence having a high homology of 90% or more with the sequence can be used Do.

In the above production method, the foreign protein of step 1) is preferably a protein having a biological activity selected from the group consisting of antigen, antibody, cell receptor, enzyme, structural protein, serum and cellular protein, human granulocyte colony More preferably, but not limited to, using a granulocyte colony-stimulating factor (hG-CSF).

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

In addition, the present invention, the step of treating the protein restriction enzyme to the recombinant fusion protein prepared by additionally comprising a protein restriction enzyme recognition site between the YjdC gene and the foreign protein gene of the expression vector of step 1) It provides a method for producing a soluble foreign protein containing YjdC removed.

In the above method, the protein restriction enzyme recognition site is any one selected from the group consisting of Xa factor recognition site, enterokinase cleavage site, Genenase I recognition site, and Furin recognition site. It is preferably, but more preferably, it is an enterokinase recognition site, but is not limited thereto.

The present inventors present a protein with high solubility expressed in high solubility against 2-HEDS (2-hydroxyethyldisulfide), which is an oxidizing agent in E. coli protein, and the protein has excellent self-folding ability, which ultimately improves the availability of the target protein. Under the assumption that it could be used as a fusion-expressing partner capable of increasing and maintaining an active form, proteins in the general environment of E. coli and proteins in 2-HEDS stress state were analyzed using secondary electrophoresis and MALDI-TOF. As a result, YjdC was identified as a protein whose expression level increased more than three times in a soluble state in a stress environment that inhibited the formation of a correct structure (see FIG. 1). As a result of the opposite increase in the soluble expression level of YjdC protein under conditions where most proteins form insoluble aggregates or decrease in expression level, YjdC protein is not only structurally stable but also its intrinsic function is involved in a mechanism against stress. Able to know. Therefore, it can be seen that YjdC obtained through the analysis of E. coli protein bodies under stress can be used as a fusion expression partner.

In order to verify the efficiency of the YjdC protein as a fusion expression partner, the present inventors fused YjdC to the amino terminus of hG-CSF and inserted it into an expression vector for E. coli to prepare an expression vector (see FIG. 2), and then, transformed into E. coli strains. A transformant was prepared to obtain a product containing the desired proteins, and the product was analyzed using SDS-PAGE. As a result, hG-CSF expressed solely in E. coli forms mostly insoluble aggregates as shown by less than 4% of the soluble expression, whereas recombinant hG-CSF expressed using YjdC as a fusion expression partner was expressed in soluble expression. It was found to increase significantly to 95% (see Figure 3). Therefore, when YjdC is used as a fusion expression partner, it can be seen that there is an excellent effect in inducing hG-CSF to be expressed in a soluble state that is easy to form insoluble aggregates.

The inventors have found that hG-CSF, which is used for medical treatment, should be separated from the fusion expression partner to avoid antigen-antibody reactions, so that the remaining hG-CSF remains soluble even when YjdC is separated from hG-CSF. In order to determine whether the YjdC was removed from recombinant hG-CSF using enterokinase, the recombinant hG-CSF was purified using metal chromatography, followed by SDS-PAGE and Western blot. And analyzed. hG-CSF is first made into a precursor in the human body, and the precursor is made into hG-CSF having the N-terminal amino acid sequence of Thr-Pro-Leu through a process of change by protease. However, when expressed in E. coli, methionine corresponding to the start codon is present at the N-terminus according to the amino peptidase enzyme titer. The mixture is very difficult to remove during the purification process, and when expressed as an inactive insoluble protein, it has to go through the process of making a soluble form having activity, the yield is disadvantageous. The present invention overcomes these disadvantages by preparing hG-CSF, which is cleaved with enterokinase to remove methionine. As a result of the analysis, it was confirmed that recombinant hG-CSF from which YjdC was removed was still present in a soluble state (see FIG. 4).

In order to find out whether the production method of hG-CSF using YjdC protein as a fusion expression partner is induced and expressed to maintain the structure of hG-CSF, hG-CSF which is commercially available with the purified recombinant hG-CSF The modification of protein secondary structure of -CSF was confirmed using circular dichroism spectroscopy (CD) analysis. As a result, it was confirmed that the purified recombinant hG-CSF of the present invention has the same protein secondary structure as hG-CSF used for treatment (see FIG. 5).

Therefore, the method for producing a recombinant protein using the YjdC of the present invention as a fusion-expressing partner can express the recombinant protein for medical or industrial use as a high ratio of soluble protein, and can form the same protein structure as that of the natural form. It is possible to omit the refolding process by the denaturing agent or the reducing agent which has been required, thereby establishing an efficient production process.

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

The expression vector is a fusion partner of the YjdC gene and a foreign protein gene is operably linked to the skeletal vector, the skeletal vector is pT7, pET / Rb, pGEX, pET28a (+), pET-22b (+) And it is preferable to use a variety of vectors capable of transforming E. coli selected from the group consisting of pGEX, it is more preferred to use pET28a (+), but is not limited thereto.

In order for the expression vector to be operably linked to the gene of the YjdC and the foreign protein, a polynucleotide encoding a protein cleavage enzyme recognition site may be connected between the gene of the YjdC and the foreign protein. When the fusion expression partner and the target protein are industrial proteins, the fusion expression partner may decrease the function of the target protein, and in the case of medical proteins, the fusion expression partner may be removed because it may cause an antigen antibody response. In this case, the protein cleavage enzyme recognition site may be used as the Xa factor recognition site, the enterokinase recognition site, the Genenase I recognition site, or the Furin recognition site, or any two or more of them are sequentially connected. have.

The expression vector may be linked to a polynucleotide encoding a tag for separation and purification to facilitate the separation and purification of the recombinant protein in order to operably link the gene of the YjdC and the foreign protein. In this case, the separation and purification tag may be used alone or selected from the group consisting of GST, poly-Arg, FLAG, poly-His and c-myc, or any two or more of them in sequence.

The present invention also provides a transformant in which the expression vector is transduced into a host cell.

Preferably, the expression vector is a vector including a gene construct including a gene of YjdC and a gene of a foreign protein as a fusion expression partner. It is preferable to use the YjdC including an amino acid sequence represented by SEQ ID NO: 1, but not limited thereto, and any sequence having a high homology of 90% or more with the sequence may be used. The foreign protein is preferably a protein having a biological activity selected from the group consisting of antigen, antibody, cell receptor, enzyme, structural protein, serum and cellular protein, more preferably using hG-CSF, but is not limited thereto. It doesn't work. The host cell is preferably E. coli ( E. coli ) is not limited to this.

Also provided is a recombinant fusion protein produced by the method of the invention.

In addition, the present invention provides a soluble foreign protein from which YjdC is removed prepared by adding enterokinase to the recombinant fusion protein.

Recombinant protein prepared by the production method using the YjdC of the present invention as a fusion expression partner is expressed in a high ratio of soluble and forms the same protein structure as that of the natural type, and thus refolding by a denaturing agent or reducing agent required for insoluble aggregates. ) Process can be omitted. High value-added medical or industrial recombinant proteins can be produced as target proteins.

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 E. coli protein sieve analysis

<1-1> Recovery of Soluble Protein from Escherichia Coli

The present inventors selected the E. coli BL21 (DE3) strain for proteomic analysis. A portion of the spawn cultures incubated for 8 hours or more was inoculated in LB medium, and then cultured at 130 rpm at 37 ° C. (control) in the incubator (control group), and a protein body under stress that inhibited proper folding of the protein to form its own tertiary structure was removed. In order to obtain a part of the seed culture was inoculated in LB medium containing 2-HEDS (10 mM) and cultured under the same conditions (experimental group). E. coli were all induced by IPTG (1 mM) when the OD ₆₀₀ reached 0.5. The cell culture was centrifuged at 6,000 rpm at 4 ℃ to recover the cell precipitate, and washed twice with 40 mM Tris buffer (pH 8.0). The washed E. coli precipitate was washed with 500 µl of lysis buffer; 8M urea, 4% (w / v) chaps, 40 mM Tris, protease inhibitor cocktail] and suspended using an ultrasonic shredder. After cell breakage, the cells were centrifuged at 12,000 rpm and 4 ° C for 60 minutes to remove cell breakage, and the supernatant was separated. Protein concentration of the separated supernatant was measured using a Bio-Rad protein assay kit. The supernatant containing 30 mg of soluble protein was fractionated and rehydration solution [rehydration solution; Suspended in 2M thiourea, 8M urea, 4% w / v chaps, 1% w / v DTT, 1% w / v mobile ampholyte, pH 4.7]. Samples for 2-Dimensional polyacrylamide gel electrophoresis (2D-PAGE) were cryopreserved at -80 ° C.

<1-2> Analysis of Escherichia Coli Proteins by Two-Dimensional Gel Electrophoresis

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

The one-dimensional isoelectric point separation process for separating the cryopreserved protein by pI was performed using a Bio-Rad Protein IEF cell electrophoresis system, and an IPG gel strip having a linear pH range of 4-7. (17 cm, ReadyStrip) was used to rehydrate the protein contained in the rehydration solution overnight. 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 contain an equilibration solution containing 1% DTT; 50 mM Tris, pH 8.6, 6 M urea, 30% v / v glycerol, 2% SDS, a little bromophenol blue] for 15 minutes followed by 2.5% The reaction was carried out for 15 minutes in an equilibration solution containing iodoacetamide. The equilibrated gel strip was then subjected to a second electrophoresis using the PROTEAN II Xi cell system (Bio-Rad) to separate proteins by molecular weight. Protein spots were detected by silver staining according to the Rabilloud method (Rabilloud T., Methods Mol Biol , 1999). Stained gels were scanned with a UMAX powerlook 1100 scanner and image density software per area of each protein spot shown on the gel was measured and analyzed using Image Master software v 4.01 (Amersham Biosciences). After comparing and analyzing the expression level of the protein under the condition of HEDS stress, the spot of the protein in which the spot volume was increased by three times or more than the general culture condition was selected (FIG. 1).

Example 2 MALDI-TOF-MS Analysis and Protein Identification

The present inventors extracted the protein spot selected by Example 1 and deposited it in Korea Basic Science Institute, and identified it using MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization-Time Of Flight) -MS analysis. .

Specifically, the protein spots selected by Example 1 were extracted from silver-stained gels for MALDI-TOF-MS analysis (Gharahdaghi F, et al. , Electrophoresis , 20: 601-605, 1999). . The protein spot was extracted and trypsin (trypsin, 10.15 mg / ml) digestion process in 25 mM ammonium bicarbonate (pH 8.0) solution at 37 ℃ overnight. The digested peptide was extracted using 5% v / v TFA, 50% v / v ACN solution, this process was repeated three times and dried using a vacuum centrifuge. The dried peptide was dissolved in a 50% ACN / 0.1% TFA solution, and deposited in the Korea Institute of Basic Science, the molecular weight was measured using a MALDI-TOF-MS system (Voyager DE-STR instrument; Biosystems, USA). Peptide mass fingerprints of peptides measured were performed using MS-FIT (http://prospector.ucsf.edu/prospector/4.0.8/html/msfit.htm) on the Prospector website. Swiss-Prot was used as the MS-FIT database. As a result of protein identification, the protein whose expression level increased more than three times under 2-HEDS stress was identified as Putative HTH-type transcriptional regulator yjdC (YjdC) (Table 1).

Identification of Proteins with More Than Three-fold Increase in Expression Under 2-HEDS Stress Conditions Gene name Gene approach
Number ^a Protein name Isoelectric point (pI) / molecular weight (kDa) Sequence coverage ^b Folding change yjdC P0ACU7 Putative HTH-type transcriptional regulator yjdC 4.95 / 21.93 29 3.03 ± 0.16

a: Gene accession number is an identification number for genetic information retrieval from ExPASy Proteomics Server (http://www.expasy.org/).

b: Sequence coverage was collected using "ALDENTE: PEPTIDE MASS FINGERPRINTING TOOL" (http://au.expasy.org/tools/aldente/).

Example 3 Preparation of hG-CSF Expression Vector (pET-hGCSF) and YjdC Fusion Expression Vector (pET-YjdC-hGCSF)

The present inventors prepared an expression vector comprising E. coli protein YjdC having an increased amount of soluble expression under environmental stress selected by the method of <Example 1> and <Example 2> as a fusion expression partner.

A cDNA sequence of human-derived G-CSF was constructed using a vector having a cDNA sequence of hG-CSF as a template so as to include Xho I at the 5'-end of the hG-CSF and a Hind III restriction enzyme recognition site at the 3'-end. Two types of synthetic DNA [primer I: 5'-CTCGAGGACGATGACGA TAAAACCCCCCTGGGCCCTGCC-3 (SEQ ID NO: 2) 'and primer II: 5'-AAGCTTTCAGGGCTGGGCAA GGTGGCG-3 (SEQ ID NO: 5) No. 3) '] was used to amplify the gene encoding the recombinant hG-CSF by PCR. hG-CSF amplified a gene encoding a total of 175 amino acid sequences with start codons removed and stop codons. The N-terminus of hG-CSF was cleaved by enterokinase immediately following the Xho I restriction site. It was designed to have a site (DDDDK; SEQ ID NO: 6). PCR was performed for the DNA polymerase buffer [0.25 mM dNTPs, 50 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 20 mM Tris-HCl (pH 8.8), 2 mM MgSO ₄ , 0.1% Triton X-100]. 100 ng of template DNA, 50 pmol of each primer was added thereto, followed by Taq DNA polymerase. The reaction conditions were 30 seconds at 95 ° C. (denaturation), 30 seconds at 52 ° C. (annealing), 72 A total of 30 runs were carried out at 60 ° C. (elongation) (hereinafter all PCRs described were according to the above conditions unless otherwise noted). After the PCR was completed, the amplified DNA was separated using a 1% agarose gel, and both ends of the amplified DNA were cut with Xho I and Hind III to express pET-28a (+) (Novagen) expression vector. Restriction enzymes were inserted into the Xho I and Hind III sites, and the resulting plasmid was named 'pET-hGCSF'.

Further, in order to obtain a gene of the E. coli-derived YjdC, 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 YjdC consisting of 191 amino acids (SEQ ID NO: 1) excluding codons was amplified by PCR method. At this time, Nde I restriction enzyme recognition sequence at the 5 'end and Xho I restriction enzyme recognition sequence at the 3' end are subjected to PCR, where Nde I at the 5 'end of the amplified DNA fragment and Xho I restriction enzyme at the 3' end. To have. Primers for the PCR are as follows: 1) 5'-CAT ATG CAA CGT GAA GAT GTA GTA CTG-3 (SEQ ID NO: 4) 'and 2) 5'-CTC GAG GGT CAA TGC ACC ACC CTG-3 (SEQ ID NO: Number: 5) '. Were inserted into each process a gene of YjdC 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- YjdC-hGCSF '.

Example 4 Soluble Expression of Recombinant Protein and Identification Using SDS-PAGE

<4-1> Preparation of E. coli transformants

The present inventors are the expression vector prepared in Example 3 by the method described by Hanahan (Hanahan D, DNA Cloning vol. 1 109-135, IRS press 1985). pET-hGCSF and pET-YjdC-hGCSF were transformed into E. coli BL21 (DE3) (Studier and Moffatt, J. Mol. Biol. 189 (1): 113-130, 1986) by heat shock method. Next, colonies showing kanamycin resistance were selected by culturing in LB medium containing kanamycin (+100 mg / L kanamycin).

<4-2> Culture of E. coli transformants and gene expression

E. coli transformants transformed with the recombinant expression vectors were used to produce hG-CSF alone and hG-CSF fused with YjdC. A portion of the spawn culture cultured for 8 hours or more was inoculated (1%) in the main culture LB medium (+100 mg / L kanamycin) and then incubated at 37 ° C. at 250 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, the cells were further incubated for 3-4 hours under the same conditions, and the cell cultures were centrifuged at 4 ° C. at 6,000 rpm for 5 minutes to recover the cell precipitates. The recovered cell precipitates were collected in a 5 ml percolation solution [10 mM Tris-HCl buffer solution. (pH 7.5), 10 mM EDTA], and then disrupted using an ultrasonic crusher (Branson Sonifier). After crushing, the supernatant and the protein aggregates were separated by centrifugation (13,000 rpm × 10 minutes), and the separated supernatants and the insoluble aggregates were separated into 5 × SDS (0.156 M Tris-HCl, pH 6.8, 2.5% SDS, 37.5% glycerol). , 37.5 mM DTT) was mixed with 1: 4 and boiled at 100 ° C for 10 minutes. Boiled samples were loaded on a 12% SDS-PAGE gel (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (12% Tris-Glycine gel), developed at 120 V for 3 hours, and then stained by Coomassie staining. By doing so, the expression level of each recombinant protein was confirmed with a densitometer (Densitometer, Bio-Rad, USA), and the solubility (%) was calculated according to Equation 1.

As a result, when hG-CSF was expressed alone without YjdC, the degree of solubility was only less than 4%, whereas when fusion expression of YjdC and hG-CSF was significantly increased to 95% (FIG. 3).

<Example 5> Purification and analysis of recombinant hG-CSF from which YjdC is isolated

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

Specifically, the cells were disrupted and centrifuged at 13,000 rpm for 10 minutes, and the separated supernatants were combined with 500 mL of Ni-NTA agarose beads (QIAGEN) for purification using metal affinity. Wash with binding buffer [pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole] prior to reaction with Ni-NTA agarose beads It was. The binding of the supernatant containing the fusion protein [(His × 6) YjdC- (entokinase site) hGCSF] fused with YjdC and Ni-NTA agarose beads proceeded at 4 ° C. and after 2 hours of sufficient binding, Wash twice with 8 mL of washing buffer (pH 8.0, 50 mM sodium phosphate, 300 mM sodium chloride (NaCl), 50 mM imidazole). Next step, PBS buffer [PBS buffer; 137 mM sodium chloride (NaCl), 2.7 mM potassium chloride (KCl), 10 mM sodium monophosphate (Na ₂ HPO ₄₎ , 2 mM potassium monophosphate (KH ₂ PO ₄ ), pH 7.4] It was further washed. Enterokinase solution (5 μl enterokinase, 10 μl enterokinase buffer, 50 μl PBS, 445 μl PBS (Invitrogen)) in (His × 6) YjdC- (entokinase site) hGCSF attached to agarose beads. Reaction was carried out at 22 ° C. for 12 hours, the elution fraction was received, and the soluble and insoluble solutions were separated by centrifugation at 13,000 rpm for 15 minutes. SDS-PAGE analysis confirmed that the hG-CSF remained soluble even after YjdC was removed. Western blot analysis using G-CSF monoclonal antibody (Clone 3D1, Santa Cruz Biotechnology Inc, CA) was confirmed (Fig. 4).

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

The inventors used EK-Away ^™ resin (Invitrogen, Germany, Cat. No. R180-01) to remove enterokinase used during the purification, and the solution containing enterokinase was removed from the microcon YM-10 (Millipore, Ireland). ) To prepare the sample for analysis by concentrating (0.4167 μg / μl) as needed for CD analysis. The prepared recombinant hG-CSF sample was commissioned by Korea Basic Science Institute (Ochang) to analyze the circular dichroism spectroscopy (CD) by the JASCO J-710 spectropolarimeter (spectropolarimeter). Comparative analysis of the protein secondary structure of recombinant hG-CSF purified by the above method and hG-CSF [Grasin Prefilled-Syringe (Filgrastim), Kirin, Japan)] It was confirmed to have (Fig. 5).

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

(A) is a picture showing two-dimensional gel electrophoresis of E. coli protein under normal and 2-HEDS stress environment.

(B) is a graph comparing the relative spot intensity of YjdC in normal environment and 2-HEDS stress environment.

FIG. 2 is a diagram showing a schematic expression of a single expression vector of human granulocyte colony stimulating factor (hG-CSF) and a fusion expression vector using E. coli YjdC gene as a fusion expression partner.

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

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

3 is a diagram showing the results of analysis of SG-CSF alone expression and fusion expression using YjdC as a fusion expression partner by SDS-PAGE (M: molecular marker; S: soluble fraction of recombinant E. coli derived cell lysates; And IS: insoluble fraction of recombinant E. coli derived cell lysate).

(A) is a figure showing the result of SDS-PAGE analysis of hG-CSF expression alone.

(B) is a figure showing the result of SDS-PAGE analysis of the expression of fusion with hG-CSF and YjdC.

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

4 is a diagram showing the results of SDS-PAGE analysis of the purified hG-CSF and Western blot analysis thereof (M: molecular marker; 1: soluble fraction of recombinant cell lysate; 2: purified hG-CSF) Soluble fraction; and 3: insoluble fraction of purified hG-CSF) (arrows indicate purified hG-CSF).

(A) is a diagram showing the result of SDS-PAGE analysis of the availability of purified recombinant hG-CSF after YjdC was removed by enterokinase treatment.

(B) is a figure showing the result of Western blot analysis of purified recombinant hG-CSF after YjdC was removed by enterokinase treatment.

5 is a graph showing the results of circular dichroism spectroscopy (CD) analysis for confirming whether the secondary structure of the purified hG-CSF and the natural hG-CSF is modified.

(A) is a diagram showing the results of analysis of purified recombinant hG-CSF isolated from YjdC :: hG-CSF by circular dichroism spectroscopy (CD).

(B) is a figure showing the result of analyzing the commercial standard hG-CSF by circular dichroism spectroscopy (CD).

<110> Korea University Industrial & Academic Collaboration Foundation <120> A preparation method of solubility recombinant protein by use of          Putative HTH-type transcriptional regulator yjdC as a fusion          expression partner <130> 8p-09-11 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 191 <212> PRT <213> Escherichia coli <400> 1 Met Gln Arg Glu Asp Val Leu Gly Glu Ala Leu Lys Leu Leu Glu Leu   1 5 10 15 Gln Gly Ile Ala Asn Thr Thr Leu Glu Met Val Ala Glu Arg Val Asp              20 25 30 Tyr Pro Leu Asp Glu Leu Arg Arg Phe Trp Pro Asp Lys Glu Ala Ile          35 40 45 Leu Tyr Asp Ala Leu Arg Tyr Leu Ser Gln Gln Ile Asp Val Trp Arg      50 55 60 Arg Gln Leu Met Leu Asp Glu Thr Gln Thr Ala Glu Gln Lys Leu Leu  65 70 75 80 Ala Arg Tyr Gln Ala Leu Ser Glu Cys Val Lys Asn Asn Arg Tyr Pro                  85 90 95 Gly Cys Leu Phe Ile Ala Ala Cys Thr Phe Tyr Pro Asp Pro Gly His             100 105 110 Pro Ile His Gln Leu Ala Asp Gln Gln Lys Ser Ala Ala Tyr Asp Phe         115 120 125 Thr His Glu Leu Leu Thr Thr Leu Glu Val Asp Asp Pro Ala Met Val     130 135 140 Ala Lys Gln Met Glu Leu Val Leu Glu Gly Cys Leu Ser Arg Met Leu 145 150 155 160 Val Asn Arg Ser Gln Ala Asp Val Asp Thr Ala His Arg Leu Ala Glu                 165 170 175 Asp Ile Leu Arg Phe Ala Arg Cys Arg Gln Gly Gly Ala Leu Thr             180 185 190 <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> YjdC primer I <400> 4 catatgcaac gtgaagatgt actg 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> YjdC primer II <400> 5 ctcgagggtc aatgcaccac cctg 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) A gene of the putative HTH-type transcriptional regulator yjdC (YjdC) described in SEQ ID NO: 1 as a fusion expression partner, and a gene of a recombinant protein to which a gene of a foreign protein is linked. Preparing an expression vector including a construct; 2) preparing a transformant by introducing the expression vector of step 1) into a host cell; 3) culturing the transformant of step 2) to induce expression of and obtain the recombinant protein; And 4) A method of improving the soluble expression and structural stability of the foreign protein, comprising the step of separating the foreign protein from the recombinant protein obtained in step 3). delete The method of claim 1, wherein the foreign protein has a biological activity selected from the group consisting of antigens, antibodies, cell receptors, enzymes, structural proteins, serum and cellular proteins, soluble expression and structural stability of the foreign protein How to improve. 4. The method of claim 3, wherein said foreign protein is a human granulocyte colony-stimulating factor (hG-CSF). The method of claim 1, wherein the expression vector further comprises a protein restriction enzyme recognition site between the gene of YjdC and the foreign protein gene. The method of claim 5, wherein the protein restriction enzyme recognition site is any one selected from the group consisting of Xa factor recognition site, enterokinase cleavage site, Genenase I recognition site, and Furin recognition site. Characterized in that one, the method of improving the soluble expression and structural stability of the foreign protein. According to claim 1, The expression vector is a skeleton vector, characterized in that any one selected from the group consisting of pT7, pET / Rb, pGEX, pET28a (+), pET-22b (+) and pGEX A method of improving soluble expression and structural stability of a protein. 2. The method of claim 1, for the host cell to improve availability and structural stability of the expression, foreign proteins, characterized in that Escherichia coli (E.coli). The method of claim 1, wherein the culturing is performed under stress environmental conditions that interfere with the formation of tertiary structure of the protein. 10. The method of claim 9, wherein the stress environmental conditions are conditions in which 2-HEDS (2-hydroxyethyldisulfide) is present. The method of claim 1, wherein the separation of step 4) is performed by treating enterokinase with the recombinant protein obtained in step 3). . A gene construct for improving soluble expression and structural stability of a foreign protein, comprising a gene of the YjdC and a foreign protein as described in SEQ ID NO: 1 as a fusion expression partner. 13. The gene construct of claim 12, wherein the gene construct further comprises a protein restriction enzyme recognition site between the gene of YjdC and the gene of the foreign protein. The method of claim 13, wherein the protein restriction enzyme recognition site is any one selected from the group consisting of factor Xa, enterokinase recognition site (enterokinase cleavage site), genenase (Genenase I) recognition site and Furin recognition site. Gene construct for improving the soluble expression and structural stability of foreign proteins, characterized in that one. An expression vector for improving soluble expression and structural stability of a foreign protein, comprising the gene construct of claim 12. The expression vector of claim 12, wherein the expression vector is represented by FIG. 2 (B). 16. The method according to claim 15, wherein the expression vector is any one selected from the group consisting of pT7, pET / Rb, pGEX, pET28a (+), pET-22b (+) and pGEX as a skeletal vector. Expression vector for improving the soluble expression and structural stability of the protein. A transformant transduced with the expression vector of claim 15. delete delete

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