KR100407792B1 - Preparation method of recombinant protein by use of human glucagon-derived peptide analogue as a fusion expression partner - Google Patents

Abstract

The present invention relates to a method for effectively preparing a recombinant protein using a human glucagon-derived analog polypeptide as a fusion partner, specifically, a gene encoding a fusion protein using a polypeptide comprising an amino acid sequence of a human glucagon analog as a fusion partner. Obtaining a fragment; Preparing an expression vector comprising the gene fragment; Preparing a transformed microorganism transformed with the expression vector; Inducing expression of a target protein from the transformed microorganism and producing the recombinant insoluble aggregate; And dissolving the recombinant insoluble aggregate in an alkaline solution to solubilize to obtain an active recombinant protein. Aggregates produced from the transformed microorganism by the production method of the present invention can be easily soluble without using a denaturing agent and reducing agent, etc., and the recombinant protein can be obtained in the form of low molecular weight oligomer due to the interaction during the aggregation of the recombinant protein. .

Description

Preparation method of recombinant protein by use of human glucagon-derived peptide analogue as a fusion expression partner}

The present invention relates to a method for effectively preparing a recombinant protein using a human glucagon-derived analog polypeptide as a fusion partner, specifically, a gene encoding a fusion protein using a polypeptide comprising an amino acid sequence of a human glucagon analog as a fusion partner. Obtaining a fragment; Preparing an expression vector comprising the gene fragment; Preparing a transformed microorganism transformed with the expression vector; Inducing expression of a target protein from the transformed microorganism and producing the recombinant insoluble aggregate; And dissolving the recombinant insoluble aggregate in an alkaline solution to solubilize to obtain an active recombinant protein.

In order to produce a variety of important proteins that are difficult to obtain from human or animal organs from microorganisms using gene recombination methods, the gene of the target protein is cloned into an expression vector to obtain a recombinant expression vector and transformed into an appropriate host cell to transform. After selection of the cells, the transformed cells are cultured to obtain the desired protein.

E. coli, which is most used as a host cell for this, is known to have the most information about its genes and metabolic systems compared to other organisms, so it is very easy to produce useful foreign proteins by genetic recombination technology. However, since the target protein expressed directly from E. coli is produced in a form in which methionine is added at the N-terminus, a step of converting it into a native protein is necessary. The target protein in the form of methionine added to the N-terminus may cause an immune response when applied to humans or other animals, or the structure may become unstable and thus fail to function as an original protein. In addition, when the target protein to be expressed is highly toxic, such as an antibacterial peptide, it may inhibit the growth of the host cell, and in the case of a polypeptide having a molecular weight of 10,000 or less, the protein present in the host cell It is decomposed by the enzyme to make stable mass production difficult.

As a method for solving these problems, a method of fusion expressing an appropriate fusion partner at the N-terminus of the target protein has been used. The method uses a water-soluble protein known to be stably produced in a selected host as a fusion partner to produce the desired polypeptide. As is known, the water-soluble proteins stably produced in Escherichia coli include β-galactosidase, maltose binding protein (45 kDa), and glutathione-S-transferase (glutathione). -S-transferase, 26 kDa). However, in the case of using the proteins as N-terminal fusion partners, the fusion proteins produced therefrom have an advantage that can be easily purified using affinity chromatography, etc. Compared to the polypeptide, the size is relatively very large, and the yield of the polypeptide is significantly reduced according to the size of the fusion portion. In addition, the expressed fusion protein can not be used as an effective production method even if the cytotoxicity is maintained as it is.

Alternatively, a method of expressing a target polypeptide in the form of insoluble aggregates in a cell using a fusion partner may be used (Ulman, A., Gene, 29, 27-31, 1984; Nilsson, B). et al., EMBO J. , 4, 1075-1080, 1985; Di Guan et al., Gene , 67, 21-30, 1988; La Vallie, ER et al., Bio / Technology , 11, 187-193. , 1993). The method has the advantage that the recombinant protein of interest can be easily separated from the cell culture in the initial separation process, but the folding intermediate of the polypeptide expressed during the formation of insoluble aggregates can By selectively binding other impurities such as chaperon, ribosome, prefactor protein, etc. in the host cell by intermolecular disulfide bond or hydrophobic interaction, It has the disadvantage of lowering the purity in insoluble aggregates (Anna Mituraki et al., Bio / Technol., 7, 690-697, 1989).

In particular, when the activity of the desired recombinant protein is in the binding of the receptor, a large aggregate is formed due to the hydrophobic nature of the recombinant protein itself, and even though it is soluble in water, a large molecular weight multimer is formed so that the receptor binding activity per unit mass is increased. There is a problem that is greatly reduced. In general, in order to purely separate an active protein from an insoluble aggregate, a denaturant such as guanidine hydrochloride or urea is used to dissolve the insoluble aggregate, and then dilute and refold it. It is reactivated only after refolding. In addition, in the case of aggregates produced by disulfide bonds, not only the treatment with a reducing agent must be used together for dissolution, but also many problems have been raised in the refolding process. (Marston, FAO, Biochem. J., 240, 1-12, 1986; Mitraki, A. and King, J., Bio / Technology, 7, 690-697, 1989).

On the other hand, human glucagon is a hormone secreted from the pancreas of α-cells of the islets of Langerhans to regulate glucose concentration in the blood by antagonism with insulin. X-ray diagnostic adjuvant is a peptide hormone already commercialized in the United States and Japan. In addition, glucagon is a peptide consisting of 29 amino acids and is known to be stably produced in the form of a fusion protein in Escherichia coli (Shin et al., Appl. Microbiol. Biotechnol ., 49, 364-370, 1998). ). As such, although glucagon is a very small peptide with a size of 3.5 kDa, X-ray analysis shows an α-helix structure, and the oligomer itself is formed by strong hydrophobic bonds between molecules in order to maintain a stable state. ) (Sasaki, K. et al., Nature , 257, 751-757, 1975). Therefore, glucagon, a small sized peptide with strong hydrophobic self-association, is very useful as a fusion partner for producing aggregates containing high purity of target protein while improving yield of target protein from host cells. Can be effectively used.

Thus, the present inventors efficiently produce recombinant fusion proteins using a polypeptide comprising an amino acid sequence of human glucagon or an analog thereof as an N-terminal fusion partner in order to stably mass produce a target protein from a recombinant transformant. When a specific glucagon analogue is used, the desired recombinant protein is stably expressed, and aggregates isolated from the cell culture are easily soluble in alkaline solution without using denaturing and reducing agents, as well as remarkable multimer formation of the recombinant protein. The present invention was completed by confirming that it is possible to produce a recombinant fusion protein having significantly reduced specific activity by forming a low molecular weight multimer.

An object of the present invention is to produce a protein of interest in the form of an insoluble aggregate from a transforming microorganism, in which the insoluble aggregate is separated into an active protein without using a denaturing agent or a reducing agent, while at the same time inducing the formation of a low molecular weight recombinant protein multimer. It is to provide a method for producing a recombinant protein in the form of a protein.

Figure 1 shows the manufacturing process of the expression vector pT7IL-2 directly expressing human interleukin-2,

Figure 2 shows the manufacturing process of the expression vector pGIL-2 expressing human interleukin-2 fused with the natural human glucagon gene,

Figure 3 shows the manufacturing process of the expression vector pGβIL-2 expressing human interleukin-2 in the form of a fusion protein using a human glucagon-derived analog polypeptide as a fusion partner,

4 shows human interleukin-2 (IL-2) expressed directly from the expression vector of FIG. 1 and human interleukin-2 (Gβ · IL-2) expressed in the form of a fusion protein with a fusion partner from the expression vector of FIG. Is the result of comparing solubility,

A: protein aggregate (0.4 g / L) containing IL-2 (80 mg / L) produced by direct expression

B: protein aggregate (1.0 g / L) containing Gβ-IL-2 (300 mg / L) produced by fusion expression

FIG. 5 shows human interleukin-2 (IL-2) expressed directly from the expression vector of FIG. 1 , human interleukin-2 (G • IL-2) fused with a native human glucagon gene expressed from the expression vector of FIG. Alkali lysis process (pH 8->12-> 8) of human interleukin-2 (Gβ-IL-2) expressed in fusion protein form using a fusion partner containing human glucagon analogue expressed from the expression vector of FIG . Western blot analysis of dissolved aggregates is then shown.

Lane 1: IL-2

Lane 2: GIL-2

Lane 3: Gβ-IL-2

In order to achieve the above object, the present invention uses a polypeptide comprising a similar amino acid sequence of human glucagon with strong self-binding as a fusion partner to produce the target protein in the form of insoluble aggregates from the transforming microorganism and then alkaline solution The present invention provides a method for preparing a desired protein in a low molecular weight oligomer form by dissolving easily.

The present invention also provides a recombinant expression vector using a polypeptide comprising the amino acid sequence of the human glucagon analogue as a fusion partner.

In addition, the present invention provides a transformed microorganism transformed with the recombinant expression vector.

Hereinafter, the present invention will be described in detail.

The present invention is to produce a target protein in the form of an insoluble aggregate from the transforming microorganism using a polypeptide comprising a similar amino acid sequence of human glucagon with strong self-binding as a fusion partner and then easily dissolved in an alkaline solution to produce the target protein. Provided are methods for preparing low molecular weight multimeric forms.

The manufacturing method of the present invention

1) preparing a fusion protein in the form of an insoluble aggregate in which a glucagon-derived polypeptide is fused to a target protein;

2) dissolving the fusion protein in the form of an insoluble aggregate in an alkaline solution to prepare a water-soluble aggregate; And

3) restoring the pH of the solution to neutral to reactivate the water soluble aggregates.

The present inventors expressed human interleukin-2 using a fusion partner containing human glucagon analogues according to the preparation method as a preferred embodiment. In the above, the glucagon-derived polypeptide is preferably a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10 . The recombinant human interleukin-2 fusion protein thus prepared occupies 30-40% of the total protein produced by the transforming microorganism, and the expression rate is markedly improved compared to that when only human interleukin-2 is directly expressed without a fusion partner. Produced in the form of insoluble aggregates containing proteins. Since the insoluble aggregates are easily dissolved in a large amount in an alkaline solution, the insoluble aggregates produced when Interleukin-2 is expressed alone are dissolved in a denatured or reducing agent and then reactivated by refolding. Reprocessing recombinant proteins can be obtained by the process alone, thereby overcoming the problem of refolding. This characteristic is due to the role of the fusion partner linked to the N-terminus, the binding mode between the aggregated recombinant protein molecules is converted from disulfide bonds to hydrophobic bonds between the same molecules.

In addition, the water-soluble recombinant protein by the alkali dissolution process is obtained in the form of a low molecular-weight oligomer having a uniform molecular weight distribution of about 110 kDa, which is a hydrophobic amino acid residue (F22) of glucagon C-terminus. Glucagon-derived analogs, all of which are substituted with glycine (W25, L26), are used as fusion partners, which may be because the recombinant proteins readily stabilize at low molecular weight stages when aggregated.

The present invention also provides a recombinant expression vector using a polypeptide comprising an amino acid sequence of a human glucagon analogue as a fusion partner in order to produce a target protein in an insoluble aggregate form using a fusion partner.

The recombinant expression vector is

1) amplifying a gene encoding a protein of interest by a polymerase chain reaction (hereinafter, abbreviated as "PCR") to obtain a gene fragment (step 1);

2) amplifying a gene encoding a human glucagon-derived analog polypeptide by PCR to obtain a gene fragment (step 3);

3) preparing an expression vector comprising the gene fragment of the human glucagon derived analog of step 2;

4) preparing a recombinant vector expressing a target protein in the form of a fusion protein by inserting the gene fragment of step 1 into the 5'-terminal portion of the gene fragment of the human glucagon-derived analog contained in the expression vector of step 3 (step Prepared by 4).

The present inventors prepared a recombinant expression vector for producing human interleukin-2 as a target protein using a human glucagon-derived analog polypeptide as a fusion partner as a preferred embodiment.

Specifically, the gene encoding human interleukin-2 of step 1 was amplified by PCR from the plasmid DNA of the interleukin-2 producing strain using the primers described in SEQ ID NO: 1 and SEQ ID NO: 2 .

The expression vector of step 2 is a vector having suitable promoters such as lac, trp, tac, pL, T3, T7, SP6, SV40 and λ (pL / pR). -2 gene fragment was inserted directly.

Glucagon of step 3 is a peptide having a α-helix structure composed of 29 amino acids and is a peptide hormone having a molecular weight of 3.5 kDa that is stably expressed in E. coli. Furthermore, glucagon can be effectively used as a fusion partner because of its strong self-bonding tendency to form oligomers by hydrophobic bonds in order to maintain a unique α-helix structure stably. Specifically, the present inventors produced a recombinant protein of human interleukin-2 using a polypeptide comprising a human glucagon-derived analogue having the amino acid sequence of SEQ ID NO: 10 as a fusion partner.

The polypeptide comprising the human glucagon-derived analog is a fusion partner Gβ consisting of [Gβ peptide]-[polyhistidine sequence (His 6)] from the N-terminus, wherein the polyhistidine sequence exhibits a metal affinity and a low pH. In the histidine has a positive charge property, the fusion protein produced using the fusion partner can be easily separated and purified using a metal chelating resin and a cation ion exchange resin. Can be.

In addition, the polypeptide of the fusion partner may be used as a linker (polypeptide) containing a polypeptide or a recognition site of the cleavage enzyme for coordination. The inventors have linked a polypeptide comprising Asp Asp Asp Asp Lys (DDDDK, hereinafter abbreviated as "D4K"), which is described by SEQ ID NO: 11 at the C-terminal end of the fusion partner Gβ, as an enterokinase cleavage site. Used.

In addition, the DNA fragment encoding the polypeptide fusion partner Gβ-D4K to which the enterokinase cleavage site is added at the C-terminus of the fusion partner is lac, trp, tac, pL, T3, T7, SP6, SV40 and λ (pL / Vector systems can be constructed by directly inserting into a vector having a suitable promoter, such as pR), or by combining DNA promoters having ribosome binding sites with these promoters, and inserting them into various plasmids.

The recombinant expression vector of step 4 is a gene fragment of a fusion partner comprising the amino acid sequence of the human glucagon-derived analog and enterokinase cleavage site (D4K) at the 5'-end of the interleukin-2 gene in the expression vector of step 2 Interleukin-2 is expressed in the form of a fusion protein comprising the D4K sequence.

Human interleukin-2 fusion protein produced by the recombinant expression vector can be easily separated and purified by metal chelated resin or cation exchange resin by histidine sequence attached to the fusion partner. By treating the enzyme, only human interleukin-2 protein from which the fusion partner has been removed can be isolated, which simplifies the separation and purification process, thereby facilitating recovery of the target protein from the manufacturing process.

In addition, the present invention provides a transformed microorganism wherein the recombinant expression vector is transformed to produce a target protein in the form of a fusion protein.

As a preferred embodiment, the present inventors have used a recombinant expression vector expressing interleukin-2 using the human glucagon analog as a fusion partner (Hanahan, D., DNA Cloning, 1, 109-135, IRS press, 1985,). E. coli transformants were prepared by introducing into E. coli cells according to the method and deposited in July 13, 2000 with the Korea Institute of Biotechnology Gene Bank (Accession Number: KCTC 0830BP).

The transformed recombinant E. coli can be cultured under appropriate culture conditions to produce a fusion protein comprising interleukin-2, and the cultured cells are lysozyme digestion, lyolysis, ultrasonic crushing, or French press. After treatment with a cell or the like, the cell lysate containing the fusion protein can be obtained by centrifugation or filtration. The human interleukin-2 fusion protein can be separated through general purification methods such as cell lysate lysis, ultrafiltration, dialysis, ion exchange chromatography, gel filtration, electrophoresis and affinity chromatography. Only interleukin-2 protein from which the fusion partner was removed by an appropriate amount of enterokinase treatment can be obtained.

Hereinafter, the present invention will be described in detail with reference to Examples.

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

Example 1 Preparation of Recombinant Expression Vector Expressing Interleukin-2 Directly

The present inventors used the plasmid pNKM21 of the interleukin-2 producing strain (KCTC 8258P), which was distributed from the Biotechnology Research Institute Gene Bank, to produce an expression system stably mass producing interleukin-2 from the transforming microorganism. The interleukin-2 producing strain has a modified interleukin-2 gene in which the cysteine, which is the 125th amino acid of the native interleukin-2 gene, is substituted with serine, and the modified interleukin-2 gene is PL. It contains the plasmid pNKM21 whose expression is regulated by a promoter.

In order to secure the interleukin-2 gene, 133 amino acid sequences were isolated from the interleukin-2 producing strain by using plasmid DNA as a template and pNIL described in SEQ ID NO: 1 and a pHIL primer pair described in SEQ ID NO: 2 . The interleukin-2 gene encoding A was amplified by the PCR method.

PCR was performed by mixing 0.25 mM dNTPs, template DNA 100 ng, and 50 pmol of each primer to Taq DNA in a buffer for DNA polymerase reaction (50 mM KCl; 20 mM Tris-HCl (pH 8.0); 2 mM MgCl ₂ ). It was performed using a polymerase (Taq DNA polymerase, TaKara). PCR reaction conditions were denatured by heating at 95 ℃ for 5 minutes, repeat the reaction of 95 ℃, 30 seconds (denaturation), 55 ℃, 30 seconds (annealing), 72 ℃, 60 seconds (elongation) 30 times and finally Amplification at 72 ° C. for 10 minutes. After the PCR was completed, the amplified DNA was electrophoresed on a 1% agarose gel (agarose gel) to confirm that the band is detected at the 411 bp position. After the bands were cut from the gel, the DNA amplified from the gel was purified using a Qiagen Gel extraction kit. The DNA thus prepared was treated with restriction enzymes NdeI and HindIII, respectively, to have an NdeI restriction enzyme site at the 5 'end and a HindIII restriction enzyme site at the 3' end, and then the DNAs were restriction enzymes NdeI and HindIII positions of the expression vector pT7-7. It was inserted into the recombinant expression vector (direct expression) direct expression (interleukin-2) was prepared and named it pT7IL-2 ( Fig. 1 ).

Example 2 Preparation of Recombinant Expression Vector Expressing Interleukin-2 Using Gene of Human Glucagon or Analogue as Fusion Partner

<2-1> Preparation of pGIL-2 Plasmid

In order to ensure the human glucagon gene fragment, the reaction conditions of Example 1 using the GXXb1 primer pair described the glucagon gene produced by using a DNA synthesizer to GND1 and SEQ ID NO: 4 as the template described in SEQ ID NO: 3 PCR was performed to amplify the glucagon gene fragment.

After PCR, the amplified DNA was electrophoresed on a 2% low-melting agarose gel to confirm that the band was detected at 99 bp, and the band at the position was cut from the gel. DNA amplified from the gel was purified in the same manner as described above. The purified DNA was treated with restriction enzymes NdeI and XbaI, respectively, to have an NdeI restriction enzyme site at the 5 'end and an XbaI restriction enzyme site at the 3' end, and then inserted into the restriction enzymes NdeI and XbaI positions of the expression vector pT7-7. The expression vector was prepared and named pT-G.

In order to fuse the interleukin-2 gene to the glucagon gene secured as described above, PCR was performed using the interleukin-2 gene inserted into the expression vector pT7IL-2 prepared in Example 1 as a template. Amplified.

The first PCR reaction was carried out under the same reaction conditions as in Example 1 using primer HD4K described in SEQ ID NO: 5 and primer pHIL described in SEQ ID NO: 2 , and the second PCR reaction was carried out HDX1 described in SEQ ID NO: 6 And interleukin-2 gene was amplified using the DNA amplified by the first PCR reaction under the same reaction conditions using the primer pHIL described in SEQ ID NO: 2 as a template. The amplified DNA was subjected to electrophoresis on a 1% agarose gel to confirm that a band was detected at 411 bp, and the DNA amplified from the gel was purified in the same manner as in Example 1. The purified DNA was treated with restriction enzymes XhoI and HindIII, respectively, to have XhoI restriction enzyme sites at the 5 'end and HindIII restriction enzyme sites at the 3' end, and then restriction enzymes XhoI and HindIII of the expression vector pT7-G prepared above. Recombinant expression vectors were prepared by insertion into positions and named pGIL-2 ( FIG. 2 ).

<2-2> Preparation of pGβIL-2 Plasmid

In order to manufacture an expression system for producing human interleukin-2 using a human glucagon-derived analog polypeptide as a fusion partner, the expression vector pGIL-2 of <2-1>, wherein the interleukin-2 gene is fused to the glucagon gene, is templated. PCR was performed to amplify the GβIL-2 fragment containing the fusion partner.

The first PCR reaction was carried out under the same reaction conditions as in Example 1 using primer T7P described in SEQ ID NO: 7 and primer GG3-2 described in SEQ ID NO: 9 , and DNA fragment-1 amplified therefrom by 2%. After confirming by electrophoresis on the low-melting agarose gel was purified in the same manner as in Example 1. In the second PCR reaction, DNA fragment-2 was obtained by the same method as described above after performing a PCR reaction using primers GG3-1 described in SEQ ID NO: 8 and primers pHIL described in SEQ ID NO: 2 . The third PCR reaction amplifies the GβIL-2 DNA fragment containing the DNA fragment-1 and fragment-2 obtained as described above as a template and a human glucagon-derived analog polypeptide as a fusion partner using a T7P / pHIL primer pair. It was. The amplified GβIL-2 DNA fragment was electrophoresed on a 1% agarose gel to confirm that the band was detected at the 531 bp position. Was purified.

The GβIL-2 DNA fragment comprises as a fusion partner a human glucagon-derived analog polypeptide having the amino acid sequence set forth in SEQ ID NO: 10 . The polypeptide including the human glucagon-derived analog is a fusion partner Gβ composed of [Gβ peptide]-[polyhistidine sequence (His 6)] from the N-terminus, and has a metal affinity and a positive charge at low pH. Containing histidine having a fusion protein produced using the fusion partner can be easily separated and purified using a metal chelating resin (cation ion exchange resin) and the like. In addition, the protein of the fusion partner may be used as a linker (polypeptide) containing a polypeptide or a recognition site of a cleavage enzyme for coordination. The inventors have linked a polypeptide comprising Asp Asp Asp Asp Lys (DDDDK, hereinafter abbreviated as "D4K"), which is described by SEQ ID NO: 11 at the C-terminal end of the fusion partner Gβ, as an enterokinase cleavage site. Used.

The amplified GβIL-2 DNA fragment was treated with restriction enzymes NdeI and HindIII, respectively, to have an NdeI restriction enzyme site at the 5 'end and a HindIII restriction enzyme site at the 3' end, followed by restriction enzymes NdeI and HindIII of the expression vector pT7-7. Inserted at the position, using a human glucagon analogue as a fusion partner, a recombinant expression vector expressing Interleukin-2 was prepared and named pGβIL-2 ( FIG. 3 ).

Example 3 Preparation of E. Coli Transformant

In order to prepare E. coli transformants transformed with the recombinant expression vector pGβIL-2 of Example 2, this technique using the expression vectors pT7IL-2, pGIL-2 and pGβIL-2 prepared in Examples 1 to 2 Ampicillin after transformation into Escherichia coli BL21 (DE3) (Studier, FA and Moffatt, BA, 113-130,1986) according to one method (Hanahan D., DNA Cloning, 1, 109-135, IRS press, 1985). Colonies showing ampicillin resistance were selected by transforming the expression vector by culturing in a medium containing (amphicillin).

E. coli transformant BL21 (DE3) / pGβIL-2 transformed with the recombinant expression vector pGβIL-2 expressing interleukin-2 using human glucagon analogs selected as above as a fusion partner was transferred to the Biotechnology Research Institute Gene Bank 2000. It was deposited on July 13, 2014 (Accession No .: KCTC 0830BP).

Example 4 Isolation of Insoluble Aggregates from Cell Cultures

Cell culture solution obtained by culturing the high-productivity E. coli transformant selected from Example 3 in 100 mg / L ampicillin-added LB medium was centrifuged at 6,000 rpm to separate the culture medium and the cells, and only the cell precipitates were recovered. . The recovered E. coli precipitate was suspended in distilled water and crushed using an ultrasonic crusher. The lysate thus separated was centrifuged at 5,000 g for 10 minutes to separate the supernatant and precipitate, and then analyzed by SDS-PAGE (14% Tris-Glycine gel) to confirm the expression of recombinant proteins. Insoluble aggregates of the recombinant protein were separated by washing twice with 0.5% Triton X-100 and the required analysis was performed.

Experimental Example 1 Measurement of Alkaline Solubility of Insoluble Aggregates

The insoluble protein produced from the recombinant transformant of Example 3 was dried by lyophilization and suspended in an alkaline solution of pH 12 to a final concentration of 50 mg / ml. The solubilization of the insoluble protein produced from each recombinant transformant was examined by diluting the sample sequentially two-fold with the same pH 12 alkaline solution. To the amount of each of protein, each of each dilution step was centrifuged for 5,000 g, 10 minutes, remove the sample with the supernatant and precipitate by using the Bradford (Bradford) DETERMINATION supernatant present in the by Equation (1) Solubility was determined. The amount of protein in the precipitate was quantified after dissolving the precipitate again with a large amount of pH 12 alkaline solution.

<Equation 1>

As shown in FIG. 4 , the expression vector pT71L-2 expressing interleukin-2 directly expresses interleukin-2 with a fusion partner comprising a human glucagon analogue polypeptide as compared to protein aggregates produced from transformed recombinant transformants. It can be seen that the solubility of the protein aggregates produced from the recombinant transformant transformed with the recombinant expression vector pGβ1L-2 is much improved. In the case of using the fusion partner Gβ containing the human glucagon analogue, the solubility was close to 100% even at a protein aggregate concentration of 1 g / L. As such, protein aggregates produced from recombinant transformants using a fusion partner containing human glucagon analogues can be dissolved in large quantities by simply changing pH without using any denaturing or reducing agents at the time of mass production of insoluble protein aggregates. Separation and purification can be facilitated. In addition, recombinant insoluble protein aggregates that are readily dissolved in alkaline solutions at pH 12 can be reactivated by lowering the pH of the solution back to about 8, thereby overcoming the problems encountered in existing refolding.

Experimental Example 2 Multimer Formation Characteristics of Recombinant Proteins Following Alkali Dissolution

In order to investigate the degree of multimer formation of the water-soluble recombinant protein after the alkali dissolution process as in Experimental Example 1, the proteins in each aggregate were separated by native-PAGE (14% Tris-Glycine gel) and then Western blot ) Analysis was performed. As the primary antibody, anti-interlukin-2 monoclonal antibody (Biosource, USA) derived from mouse was used. The secondary antibody was derived from goat with alkaline phosphatase attached. Anti-mouse antibody IgG (Santa Cruz, USA) was used.

As a result, the recombinant protein aggregate produced by the recombinant vector expressing human interleukin-2 or the recombinant protein aggregate produced by the expression vector fused with native human glucagon is a multimer of large molecular weight. However, the recombinant human interleukin-2 generated by the recombinant expression vector fused with the human glucagon analogue Gβ peptide showed a significant decrease in molecular weight and a uniform molecular weight distribution ( FIG. 5 ). As a result of analyzing the molecular weight of the recombinant protein fused with Gβ peptide, which is a human glucagon analogue, by gel filtration chromatography, the molecular weight was determined to be about 110 kDa.

As described above, the present invention provides a method for stably expressing recombinant human interleukin-2 from a transformed microorganism by using a polypeptide comprising a similar amino acid sequence derived from human glucagon as a fusion partner. Recombinant human interleukin-2 fusion protein produced by the preparation method of the present invention is characterized in that a large amount is easily dissolved in an alkaline solution, unlike the insoluble aggregates generally produced after being dissolved in a denaturing agent or a reducing agent and then refolding. Indicates. In addition, the method of the present invention increases the specific activity (receptor binding activity per unit mass) of the fusion protein because the recombinant fusion protein is dissolved in the form of low molecular weight oligomer after the alkali dissolution step. It can be useful for development.

Claims (9)

1) preparing a fusion protein in the form of an insoluble aggregate in which a glucagon-derived polypeptide having an amino acid sequence set forth in SEQ ID NO: 10 is fused to a protein of interest; 2) dissolving the fusion protein in the form of an insoluble aggregate in an alkaline solution to prepare a water-soluble aggregate; And 3) restoring the pH of the solution to neutral to reactivate the water soluble aggregate, the method of producing a recombinant protein using a glucagon-derived polypeptide as a fusion partner. delete The method of claim 1, wherein the target protein of step 1 is human interleukin-2. The method of claim 1 wherein the alkaline solution of step 2 is pH 12. The method of claim 1, wherein the fusion protein of step 1 further comprises a polyhistidine sequence (His6) between the protein of interest and the glucagon-derived polypeptide. The method of claim 1, wherein the fusion protein of step 1 further comprises a peptide comprising an enterokinase cleavage site of SEQ ID NO: 11 between the protein of interest and the glucagon-derived polypeptide. A pGβIL-2 recombinant expression vector expressing human interleukin-2 in the form of a fusion protein using a glucagon-derived polypeptide having an amino acid sequence set forth in SEQ ID NO: 10 as a fusion partner. 8. The recombinant expression vector of claim 7, wherein the fusion partner comprises an enterokinase cleavage site and a histidine binding site. E. coli transformants transformed with the recombinant expression vector of claim 7 (Accession Number: KCTC 0830BP).