KR20100077665A - A preparation method of solubility recombinant protein by use of peptidyl-prolyl cis-trans isomerase b as a fusion expression partner - Google Patents

Abstract

PURPOSE: A method for preparing a recombinant protein using peptidyl-proline cis-trans isomerase B(PpiB) as a fusion expression partner is provided to improve solubility and folding ability. CONSTITUTION: A method for preparing a recombinant protein comprises: a step of preparing an expression vector having peptidyl-proline cis-trans isomerase B(PpiB) gene and foreign protein gene; a step of introducing the expression vector to host cells to make a transformant; a step of culturing the transformant; and a step of inducing the expression of recombinant protein. The PpiB is denoted by sequence number 1. The expression vector additionally contains protein restriction enzyme recognition site.

Description

A preparation method of solubility recombinant protein by use of Peptidyl-prolyl cis-trans isomerase B as a fusion expression partner}

The present invention relates to a method for producing a recombinant protein using Peptidyl-prolyl cis-trans isomerase B (PpiB) as a fusion expression partner, and more specifically, 1) fusion expression partner (PpiB, which is a fusion expression partner), and preparing an expression vector connecting genes of a foreign protein, 2) introducing the expression vector into a host cell to prepare a transformant, and 3) converting the transformant It relates to a method for producing a recombinant protein comprising the step of culturing to induce expression of the recombinant protein, and to a recombinant protein produced by the method.

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, the method of cloning hG-CSF gene and expressing 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 soluble expression rate, effective tertiary structure formation, and high yield of its own protein. 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, it is possible to lower the protein expression rate of Escherichia coli to perform low temperature expression (Hammarstrom et al., Protein Sci. 11: 313-321, 2002), or to induce conditions using various promoters. (Qing et al., Nat Biotechnol . 22: 877-882, 2004), co-expression of molecular chaperone or protein folding regulators and target proteins (de Marco and De Marco, J Biotechnol. 109: 45-52, 2004 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 the soluble protein known to be stably produced in E. coli as a fusion expression partner to the N-terminus of the target protein 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 trying to develop a new concept of a fusion expression partner for expressing a high value-added medical or industrial recombinant protein in the soluble state in E. coli, 2-HEDS (2-hydroxyethyldisulfide), etc. Peptidyl-prolyl cis-trans isomerase B (PpiB), which can maintain its structural stability even under stress, was identified and used as a fusion expression partner of hG-CSF, a target protein. As a result, it expressed high soluble hG-CSF and hG-CSF remained soluble even after removal of PpiB. 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 peptidyl-proline cis-trans isomerase B, a protein in Escherichia coli that maintains its structural stability even in a strong stress environment that inhibits the folding of the protein as a universal 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 as the native form in a soluble state.

In order to achieve the above object, the present invention

1) As a fusion expression partner, a gene of peptidyl-prolys cis-trans isomerase B (PpiB) and an expression vector linking a gene of a foreign protein are prepared. step;

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 preparing a soluble foreign protein from which PpiB has been removed, comprising the step of treating the protein restriction enzyme with an expression vector further comprising a protein restriction enzyme recognition site between the PpiB gene and the foreign protein gene. It provides a manufacturing method.

The present invention also provides a gene construct comprising a gene of PpiB 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 PpiB is prepared by further treating a protein restriction enzyme in addition to the recombinant fusion protein.

The present invention produces a recombinant protein using a peptidyl-prolyl cis-trans isomerase B (PpiB), which maintains structural stability and increases expression even in a strong stress environment, as a fusion expression partner. By overcoming the limitations on the solubility and folding of existing fusion partners, it can be widely used in producing 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) A gene for the peptidyl-prolyl cis-trans isomerase B (PpiB) as a fusion expression partner, and an expression vector connecting the gene of a foreign protein are prepared. step;

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, PpiB 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 PpiB gene and the foreign protein gene of the expression vector of step 1) It provides a method for producing a soluble foreign protein containing PpiB is 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, PpiB, a protein whose expression level was increased by 2.7 times or more in a soluble state in a stress environment that inhibited the formation of a correct structure, was identified (see FIG. 1). As a result of the opposite increase in soluble expression of PpiB protein under conditions in which most proteins form insoluble aggregates or decrease in expression level, PpiB 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 PpiB 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 PpiB protein as a fusion expression partner, the present inventors fused PpiB 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, transfected 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 alone in E. coli forms mostly insoluble aggregates as shown by 4-5% of soluble expression (see FIG. 3A), whereas recombinant hG expressed using PpiB as a fusion expression partner. -CSF was found to significantly increase the amount of soluble expression to 80% (see Fig. 3B and 3C). Therefore, when PpiB 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, which is easy to form insoluble aggregates.

Based on the fact that the hG-CSF used for the medical treatment should be separated from the fusion expression partner to avoid the antigen antibody response, the remaining hG-CSF can remain soluble even when PpiB is separated from the hG-CSF. In order to determine whether PPIB 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 without PpiB was still present in a soluble state (see FIG. 4).

In order to determine whether the production method of hG-CSF using PpiB protein as a fusion expression partner is induced and expressed to maintain the structure of hG-CSF, hG-CSF purified commercially and hG commercially available 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 PpiB of the present invention as a fusion expression partner can express a medical or industrial expression recombinant protein with a high ratio of soluble protein, and can form the same protein structure as that of a natural type. 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 PpiB and a gene of a foreign protein as a fusion expression partner, and an expression vector comprising the gene construct.

The expression vector is included as a fusion partner of the gene of PpiB and the foreign protein 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.

The expression vector is a polynucleotide encoding a protein cleavage enzyme recognition site may be linked between the gene of PpiB and the gene of the foreign protein to be operatively linked. 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 separation and purification of the recombinant protein in order to operably link the gene of PpiB and the gene of 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 PpiB and a gene of a foreign protein as a fusion expression partner. Preferably, the PpiB includes an amino acid sequence as set forth in SEQ ID NO: 1, but is not limited thereto. 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 PpiB has been prepared by adding enterokinase to the recombinant fusion protein.

Recombinant protein prepared by the manufacturing method using the PpiB of the present invention as a fusion expression partner is expressed in a high proportion 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 electrophoresed using a 12.5% polyacrylamide gel, and then protein spots were detected by silver staining according to the Rabilloud method (Rabilloud T., Methods Mol Biol , 1999). The stained gels were scanned using a GS-710 densitometer and the protein spots were identified using PDQuest software version 6.1. The volume of the protein spot was measured and compared with the increase and decrease in the expression level of the protein separated under 2-HEDS (10 mM) based on the protein separated at 37 ° C under normal conditions. Was selected as the spot of protein increased by more than 2.7 times (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 with a 2.7-fold increase in expression under 2-HEDS stress was identified as Peptidyl-prolyl cis-trans isomerase B (PpiB) (Table 1).

Identification of Proteins with 2.7-fold Increase in Expression Under 2-HEDS Stress Gene name Gene approach
Number ^a Protein name Isoelectric point (pI) / molecular weight (kDa) order
Similarity (%) Theoretical value ^b Experimental value ^c
ppiB P23869 Peptidyl-prolyl
cis-trans
isomerase B 5.51 / 18.15 5.43 / 18.34 21

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

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

c: Experimental values were calculated from two-dimensional electrophoretic gel images.

Example 3 Preparation of a single expression vector (pET-hGCSF) and a PpiB fusion expression vector (pET-PpiB-hGCSF) of hG-CSF

The present inventors prepared an expression vector comprising Escherichia coli protein PpiB 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 PpiB, 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 PpiB consisting of 164 amino acids (SEQ ID NO: 1) excluding codons was amplified by PCR. 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 GCT GAA ATT ACC GCA TCC-3 (SEQ ID NO: 4) 'and 2) 5'-CTC GAG AGA CTG CTT GGA CAT CGC-3 (SEQ ID NO: Number: 5) '. Were inserted into each process a gene of PpiB 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- PpiB-hGCSF 'was named.

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-PpiB-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 vector were used to produce hG-CSF alone and hG-CSF fused with PpiB. A portion of the spawn culture cultured for at least 8 hours was inoculated (1%) in the main culture LB medium (+100 mg / L kanamycin), and then cultured at 130 rpm at 37 ° C. 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 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. 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 PpiB, the degree of solubility was only 4-5%, whereas when PpiB and hG-CSF were fused, it was significantly increased to 80% (FIG. 3).

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

The present inventors produced and purified the recombinant protein using E. coli BL21 (DE3) transformed with the fusion expression vector (pET-PpiB-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) PpiB- (entokinase site) hGCSF] fused with PpiB 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) PpiB- (entokinase site) hGCSF attached to agarose beads 500 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 hG-CSF remained soluble even after PpiB was removed, and the purified hG-CSF was a mouse anti-human G-CSF monoclonal antibody (mouse anti-human). 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 diagram showing the image of two-dimensional gel electrophoresis of E. coli protein body under normal environment.

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

(C) is a graph comparing the relative spot intensities of PpiB in a 2-HEDS stress environment.

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

Figure 3 shows the results of analysis of SG-CSF alone expression and fusion expression using PipB 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 diagram showing the result of SDS-PAGE analysis of hG-CSF and PipB fusion expression.

(C) is a graph showing the expression of hG-CSF alone and the solubility (%) of fusion-expressing cells of PipB 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 PipB was removed by enterokinase treatment.

(B) shows the result of Western blot analysis of purified recombinant hG-CSF after PipB 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 PipB :: 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          Peptidyl-prolyl cis-trans isomerase B as a fusion expression          partner <130> 8p-09-08 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 164 <212> PRT <213> Escherichia coli <400> 1 Met Val Thr Phe His Thr Asn His Gly Asp Ile Val Ile Lys Thr Phe   1 5 10 15 Asp Asp Lys Ala Pro Glu Thr Val Lys Asn Phe Leu Asp Tyr Cys Arg              20 25 30 Glu Gly Phe Tyr Asn Asn Thr Ile Phe His Arg Val Ile Asn Gly Phe          35 40 45 Met Ile Gln Gly Gly Gly Phe Glu Pro Gly Met Lys Gln Lys Ala Thr      50 55 60 Lys Glu Pro Ile Lys Asn Glu Ala Asn Asn Gly Leu Lys Asn Thr Arg  65 70 75 80 Gly Thr Leu Ala Met Ala Arg Thr Gln Ala Pro His Ser Ala Thr Ala                  85 90 95 Gln Phe Phe Ile Asn Val Val Asp Asn Asp Phe Leu Asn Phe Ser Gly             100 105 110 Glu Ser Leu Gln Gly Trp Gly Tyr Cys Val Phe Ala Glu Val Val Asp         115 120 125 Gly Met Asp Val Val Asp Lys Ile Lys Gly Val Ala Thr Gly Arg Ser     130 135 140 Gly Met His Gln Asp Val Pro Lys Glu Asp Val Ile Glu Ser Val 145 150 155 160 Thr Val Ser Glu                 <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> PpiB primer I <400> 4 catatggctg aaattaccgc atcc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PpiB primer II <400> 5 ctcgagagac tgcttggaca tcgc 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 for the peptidyl-prolyl cis-trans isomerase B (PpiB) as a fusion expression partner, and an expression vector connecting the gene of a foreign protein are prepared. step; 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 the method of producing a recombinant protein consisting of obtaining it. 2. The method of claim 1, wherein PpiB is set forth in SEQ ID NO: 1. The method of claim 1, wherein the foreign protein has a biological activity selected from the group consisting of antigen, antibody, cell receptor, enzyme, structural protein, serum and cellular protein. 4. The method of claim 3, wherein the foreign protein is 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 PpiB gene 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. Method for producing a recombinant protein, characterized in that. The recombinant protein of claim 1, wherein the expression vector uses any one selected from the group consisting of pT7, pET / Rb, pGEX, pET28a (+), pET-22b (+) and pGEX as a backbone vector. Manufacturing method. The method of claim 1, wherein the host cell is E. coli ( E. coli ) method for producing a recombinant protein. The method of claim 1, wherein the culturing is a method for producing a recombinant protein, characterized in that the culture under stress environmental conditions that interfere with the formation of the tertiary structure of the protein. 10. The method of claim 9, wherein the stress environment is a condition for producing a recombinant protein, characterized in that the addition of 2-HEDS (2-hydroxyethyldisulfide). A method for preparing soluble foreign protein from which PpiB has been removed, comprising adding an enterokinase to the recombinant protein prepared by the method of claim 5. A gene construct comprising a gene of PpiB and a gene of foreign protein as a fusion expression partner. The gene construct of claim 12, wherein the gene construct further comprises a protein restriction enzyme recognition site between the PpiB gene and the foreign protein gene. The method of claim 13, 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. Gene construct characterized in that. 13. The gene construct according to claim 12, wherein the gene construct is represented by Fig. 2 (B). An expression vector comprising the gene construct of claim 12. The expression vector of claim 16, wherein the expression vector uses any one selected from the group consisting of pT7, pET / Rb, pGEX, pET28a (+), pET-22b (+), and pGEX as a skeletal vector. A transformant transduced with the expression vector of claim 16. A recombinant fusion protein prepared by the method of claim 1. A soluble foreign protein from which PpiB prepared by the method of claim 5 is removed.

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