KR100368073B1 - Preparation of Peptides by Use of Human Glucagon Sequence as a Fusion Expression Partner - Google Patents

Abstract

The present invention relates to a method for effectively preparing a recombinant protein or peptide using a fusion protein of the X _G -LY type comprising a strong self-binding peptide as a fusion partner.

X _G : Fusion partner (polypeptide comprising amino acid sequence of all or part of human glucagon protein or analogue thereof)

L : linker (sequence for codon alignment or recognition sequence for peptide cleavage)

Y : recombinant protein or peptide

More specifically, a gene encoding a fusion protein comprising a polypeptide comprising an amino acid sequence of all or part of a human glucagon protein or an analog thereof as a fusion partner, a vector comprising the gene, a microorganism transformed with the vector, and The present invention relates to a method for preparing a fusion protein, and according to the present invention, it is possible to maximize the intracellular production yield and purity of the aggregate of the recombinant protein, and the separated aggregate can be easily water-soluble without using denaturing agent or reducing agent.

Description

Preparation method of recombinant protein using fusion partner {Preparation of Peptides by Use of Human Glucagon Sequence as a Fusion Expression Partner}

The present invention relates to a method for effectively preparing a recombinant protein or peptide using a fusion protein of the X _G -LY type comprising a strong self-binding peptide as a fusion partner.

X _G : Fusion partner (polypeptide comprising amino acid sequence of all or part of human glucagon protein or analogue thereof)

L : linker (sequence for codon alignment or recognition sequence for peptide cleavage enzyme)

Y : recombinant protein or peptide

More specifically, the present invention provides a gene encoding a fusion protein comprising a polypeptide comprising an amino acid sequence of all or part of a human glucagon protein or an analog thereof as a fusion partner, a vector comprising the gene, and a vector transformed with the vector. It relates to a microorganism and a method for producing a fusion protein.

In order to produce a variety of important proteins that are difficult to obtain in human or animal organs by genetic recombination method in microorganisms, the gene of the target protein is cloned into an expression vector to obtain a recombinant expression vector and transformed into appropriate host cells and cultured. Escherichia coli has the most known information about its genes and metabolism compared to other organisms, and is used as a host for producing useful foreign proteins by genetic recombination technology. However, when the target protein is directly expressed in E. coli, it is necessary to make a form in which methionine is added to the N-terminus and convert it to a native protein, which is added to the N-terminus of the target protein. When applied to humans or other animals, the immune response (immunogenecity) may be induced or the structure may be unstable to perform the original protein function. In addition, when the expressed protein is highly toxic such as an antibacterial peptide, it may inhibit the growth of the host, and in the case of a polypeptide having a molecular weight of 10,000 or less, it is degraded by protease present in the host cell. As a result, stable mass production becomes difficult.

As a method for solving this phenomenon, a method of fusion expressing an appropriate fusion partner at the N-terminus of the target protein is used. Specifically, one method is to produce a desired polypeptide using a water-soluble protein known to be stably produced in a selected host as a fusion partner. Soluble proteins known to be stably mass-produced in Escherichia coli up to now include beta-galactosidase (beta-galactosidase), maltose binding protein (45 kDa), and glutathione-S-transferase (glutathione-S). -transferase, 26 kDa). However, when using the protein as a fusion partner, these fusion proteins have an advantage that can be easily purified by affinity chromatography, etc., while the fusion partner is relatively large in size compared to the polypeptide to be prepared by the fusion partner According to the size of the peptide has a disadvantage in that the yield is significantly reduced. In addition, the expressed fusion protein can not be used as an effective production method when the cytotoxicity is maintained as it is.

Alternatively, a fusion partner can be used to express the desired polypeptide in the form of insoluble aggregates in cells (Ulman, A. 1984, Gene 29, 27-31; Nilsson, B.). et al. 1985, EMBO J. 4, 1075-1080; Di Guan et al. 1988, Gene 67, 21-30; La Vallie, ER et al. 1993, Bio / Technology 11, 187-193). This method has the advantage that the recombinant protein of interest can be easily separated from the cell culture during the initial separation process, but the intermediates of the folding of the polypeptide expressed during the formation of aggregates are intermolecular. Purity in aggregates of the desired polypeptide by non-selective binding with other protein impurities (chaperon, ribosome, early factor, etc.) of the host cell by disulfide bond or hydrophobic interaction. There is a downside (Anna Mituraki et al., 1989, Bio / Technol. 7 , 690-697).

When the protein is produced as insoluble aggregates, in order to purely separate the active protein therefrom, it is generally dissolved using a denaturant such as guanidine hydrochloride or urea, followed by dilution through dilution. You have to go through the refolding process. In the case of aggregates produced by disulfide bonds, not only must they be treated together with a reducing agent for dissolution, but there are still many problems in the refolding process, which may be a major cause of reduced production yield (Marston FAO 1986, Biochem.J . 240, 1-12; Mitraki, A. and King, J. 1989, Bio / Technology 7, 690-697).

Human glucagon is a hormone secreted from the pancreatic Langerhans alpha cells (α-cells of the islets of Langerhans) that regulates glucose levels in the blood by antagonism with insulin, a hypoglycemic drug or endoscopy and X-ray diagnosis As a supplement, it 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. 1998, Appl. Microbiol. Biotechnol. 49, 364-370). Although glucagon is a small peptide (3.5 kDa), X-ray analysis shows alpha helix and forms oligomers by strong hydrophobic bonds between molecules in order to maintain a stable state. (Sasaki, K. et al. 1975 Nature 257, 751-757). Therefore, glucagon, a small-sized peptide with strong hydrophobic self-association, can be effectively used as a fusion partner to produce aggregates containing high purity of target protein while improving intracellular production yield. .

Accordingly, the present inventors recombined a polypeptide containing an amino acid sequence of human glucagon or an analog of strong self-combination as a fusion partner of N-terminal to stably mass-produce foreign proteins from recombinant E. coli in the form of insoluble aggregates. The present invention was completed by developing a method for maximizing intracellular production yield and purity of aggregates as well as finding that the aggregates are easily soluble in an alkaline solution without using any denaturing agent or reducing agent.

The present invention is to produce the target protein from the recombinant E. coli in the form of insoluble aggregates, to improve the production yield in the culture medium and the purity of the target protein in the aggregate and to separate the resulting insoluble aggregates into active proteins without using denaturing or reducing agents, It is an object of the present invention to provide a method for preparing a recombinant protein in the form of a fusion protein.

Figure 1 shows the manufacturing process of the expression vector pT7IL-2,

Figure 2 shows the manufacturing process of the expression vector pT1GIL-2,

Figure 3 shows the manufacturing process of the expression vector pT2GIL-2,

Figure 4 shows the manufacturing process of the expression vector pG1IL-2,

Figure 5 shows the manufacturing process of the expression vector pG2IL-2,

Figure 6 shows the manufacturing process of the expression vector pG3IL-2,

Figure 7 shows the expression level (%) of the intracellular expression of recombinant protein produced in E. coli transformed with each expression vector,

8 shows the content (purity,%) of the recombinant protein contained in the insoluble precipitate recovered from the culture medium of each E. coli transformant,

9 shows the solubility (%) in the alkaline solution of the fusion protein precipitate recovered from the culture medium of each E. coli transformant,

Figure 10 shows the result of performing Western blot after separating the fusion protein precipitate recovered from the culture medium of each E. coli transformant by non-reducing SDS-PAGE,

A) lane 1: molecular weight size marker

Lane 2: aggregate produced by Escherichia coli transformed with the expression vector pT7IL-2

B) lane 1: molecular weight size marker

Lane 2: aggregate produced by Escherichia coli transformed with the expression vector pT1GIL-2

Lane 3: aggregate produced by Escherichia coli transformed with the expression vector pT2GIL-2

Lane 4: aggregate produced by E. coli transformed with the expression vector pG1IL-2

Lane 5: aggregate produced by Escherichia coli transformed with the expression vector pG2IL-2

Lane 6: aggregate produced by Escherichia coli transformed with the expression vector pG3IL-2

11 is a ratio of the conjugates between the same recombinant protein molecules from the Western blot analysis performed after separating the fusion protein precipitate recovered from the culture medium of each E. coli transformant by non-reducing SDS-PAGE It is shown.

In order to achieve the above object, the present invention provides a method for producing a target protein from Escherichia coli with high purity and high yield using a polypeptide comprising an amino acid sequence of human glucagon or its analog having strong self-binding ability as a fusion partner. . Specifically, the present invention uses a hybrid polypeptide consisting of a heterologous peptide derived from human glucagon and human tumor necrosis factor as a fusion partner, or repeats only one to three amino acid sequences of human glucagon. Provided is a method for mass-producing a recombinant protein in the form of a high purity insoluble aggregate (inclusion body) using the polypeptide as a fusion partner.

The glucagon constituting the fusion partner may use all or part of an amino acid sequence, and furthermore, the same effect may be achieved by using an analog in which a part is modified.

Human tumor necrosis factor or its mutein forming a fusion partner with glucagon may include the entire amino acid sequence or a portion thereof, and in addition, a polyhistidine sequence may be included in the fusion partner to facilitate separation of the recombinant protein.

In addition, a peptide containing an enterokinase cleavage site may be used as a linker in the fusion partner protein.

Meanwhile, the present invention provides a method for obtaining a human protein or an analog thereof from a microorganism using a recombinant plasmid including the fusion partner. In particular, the present invention provides a method of simply reactivating and purifying a recombinant protein by dissolving insoluble aggregates of the recombinant protein mass-produced in the above manner in an alkaline solution and then restoring the pH to neutral again.

Hereinafter, the present invention will be described in detail.

The present invention produces a recombinant protein in the form of a fusion protein using human glucagon, which is a peptide hormone having a molecular weight of 3.5 kDa and stably expressed as a soluble protein in E. coli.

Glucagon is a peptide with an alpha-helix structure consisting of 29 amino acids and is a peptide hormone stably expressed in Escherichia coli. Furthermore, glucagon can be effectively used as a fusion partner because it has a strong self-bonding tendency to form oligomers by hydrophobic bonds in order to maintain a unique alpha-helix structure. Specifically, the present invention produces a recombinant protein using a polypeptide including all or part of human glucagon having the amino acid sequence of SEQ ID NO: 1 as a fusion partner.

More specifically, the present invention provides a method for producing a recombinant protein using various peptides / polypeptides including the glucagon amino acid sequence (G peptide) of SEQ ID NO: 1 as a fusion partner as follows.

1) From N-terminus [peptide (T1 peptide) consisting of amino acids 1-57 of the N-terminus of human tumor necrosis factor (T1 peptide)]-[G-peptide]-[polyhistidine sequence (His6)] Polypeptides consisting of ( fusion partner T1G )

2) From N-terminus of [peptide (T2 peptide) consisting of the 8th to 12th amino acids of N-terminus among amino acid sequences of human tumor necrosis factor]]-[polyhistidine sequence (His10)]-[G peptide] Peptide Consisting in Sequence ( Fusion Partner T2G )

3) Peptide ( fusion partner G1 ) consisting of [G peptide]-[polyhistidine sequence (His6)] from N-terminus

4) Peptide ( fusion partner G2 ) consisting of [peptide G peptide repeated twice (G2 peptide)]-[polyhistidine sequence (His6)] from N-terminus

5) Peptide ( fusion partner G3 ) consisting of [peptide G repeats three times in a row (G3 peptide)]-[polyhistidine sequence (His6)] from the N-terminus

The polyhistidine sequence allows the fusion protein to be easily purified by using a metal chelating resin, a cation exchange resin, etc. by using a positive charge of histidine at a metal affinity and low pH.

In the present invention, human interleukin-2 is used as a model protein to express human interleukin-2 in the form of a fusion protein by using the above five fusion partners and produces high purity protein aggregates. Looking at the method of producing a recombinant expression vector to produce a fusion protein in detail,

1) First, the gene encoding human interleukin-2 is amplified by polymerase chain reaction (PCR),

2) preparing the expression vector pT7IL-2 capable of producing the interleukin-2 by inserting the prepared DNA into the pT7-7 vector including the T7 promoter,

3) Next, Asp Asp Asp Asp Lys (DDDDK, hereinafter referred to as D4K), which is an enterokinase cleavage site, is added to the C-terminal portion of each of the five fusion partners (T1G, T2G, G1, G2, and G3). PCR amplifies the nucleotide sequence encoding the polypeptide, and then

4) The amplified DNA fragment was inserted into the 5'-terminal portion of the interleukin-2 gene in the expression vector pT7IL-2, whereby interleukin-2 was expressed as a fusion protein containing the D4K sequence, followed by enterokinase in the purification process. Cleavage by means of a vector produces a vector in which only interleukin-2 can be isolated and produced (pT1GIL-2, pT2GIL-2, pGIL-2, pG2IL-2, pG3IL-2).

In addition, DNA encoding the polypeptide gene having the enterokinase cleavage site added to the C-terminus of each of the fusion partners is lac, trp, tac, pL, T3, T7, SP6, SV40, λ (pL / pR). The vector system can be constructed by directly inserting into a vector having a suitable promoter, or by combining DNAs having these promoters with ribosome binding sites with the polypeptide (fusion partner-D4K) DNA, and then inserting them into various plasmids. .

After constructing the expression vector system by inserting the DNA encoding the desired protein or peptide into the 3 'end of the vector DNA, the fusion protein can be produced by culturing the transformant prepared by introducing the expression vector into a suitable host.

In the present invention, transformants were prepared by introducing the expression vectors producing the interleukin-2 fusion protein into a suitable host (Hanahan, D. 1985, DNA Cloning 1, 109-135, IRS press).

Specifically, the E. coli transformant obtained by transforming the expression vectors pT1GIL-2, pT2GIL-2, pG1IL-2, pG2IL-2, pG3IL-2 and the like into Escherichia coli BL21 (DE3) strains Deposited to GenBank on November 13, 1998 (Accession No .: KCTC 0555BP, KCTC 0556BP, KCTC 0552BP, KCTC 0553BP, KCTC 0554BP).

The transformed recombinant E. coli can be cultured under suitable culture conditions to produce a fusion protein comprising interleukin-2. The cultured cells are treated with lysozyme digestion, lyolysis, sonication, or French press, and then centrifuged or filtered to obtain extracts containing the fusion protein. The fusion protein can be separated by common purification methods such as dissolution of the extract, ultrafiltration, dialysis, ion exchange chromatography, gel filtration, electrophoresis and affinity chromatography. Treatment of the isolated fusion protein with an appropriate amount of enterokinase yields only interleukin-2 therefrom.

When expressing interleukin-2 using a fusion partner containing human glucagon of the present invention, the expression rate is significantly improved (50 to 60% of the total protein in Escherichia coli), compared to the direct expression of only interleukin-2. (In about 80% of the total amount of aggregate constituent protein) can be obtained. Unlike insoluble aggregates, the insoluble aggregates are easily dissolved in a large amount in an alkaline solution.However, in the case of a general insoluble aggregate, the fused recombinant protein of the present invention has to be dissolved in a denaturant or reducing agent and then refolded. Alkaline dissolution alone completes the process and overcomes the difficulties of refolding.

This unique phenomenon is due to the role of the N-terminal fusion partner, most of the high-purity recombinant protein is aggregated by the hydrophobic interaction between the same molecules.

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 contents of the present invention are not limited by the examples.

Example 1 Preparation of pT7IL-2 Plasmid Directly Expressing Interleukin-2

The interleukin-2 producing strain (KCTC 8258P) obtained from the GenBank was used to mass express the genes of the interleukin-2 gene and the interleukin-2 fusion protein in E. coli using the T7 promoter expression system. Plasmid pNKM21 in this strain contains a modified interleukin-2 gene in which the cysteine, the 125th amino acid of native interleukin-2, is substituted with serine, which is regulated by the PL promoter. . Using the interleukin-2 gene contained in this plasmid as a template, the interleukin-2 gene encoding 133 amino acid sequences was amplified by PCR using the primers described below.

1) GCA CAT ATG GCA CCT ACT TCA AGT TCT (pNIL)

2) GCA AAG CTT CTA TTA AGT TAG TGT TGA GAT GAT (pHIL)

PCR was performed on DNA DNA in 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). 100ng, 50 pmol of each primer was added and then performed using Taq DNA polymerase. The reaction conditions were performed 30 times in total at 95 ° C./30 sec (denaturation), 52 ° C./30 sec (annealing), and 72 ° C./60 sec (elongation) (all PCR described below was carried out according to the above conditions unless otherwise specified). do). After completion of PCR, the amplified DNA was analyzed using a 1% agarose gel. The amplified DNA was purified using Qiagen Gel extraction kit and treated with restriction enzymes NdeI and HindIII to have NdeI restriction enzyme sites at the 5 'end and HindIII restriction enzyme sites at the 3' end. The expression plasmid pT7-7 was inserted into the NdeI and HindIII sites. The resulting plasmid was named pT7IL-2 (see FIG. 1 ).

Example 2. Preparation of Interleukin-2 Expression Vector Using Human Glucagon Gene as Fusion Partner

end. Preparation of pT1GIL-2 Plasmid

DNA of T1 peptide (peptide consisting of 1st to 57th amino acids of N-terminus among amino acid sequences of human tumor necrosis factor) by PCR using human mononuclear cDNA library (purchased from Clontech) made from U937 cell line as a template After amplification was purified using a Qiagen Gel extraction kit (Qiagen Gel extraction kit). The purified DNA was treated with BamHI and NdeI, respectively, to have an NdeI restriction enzyme site at the 5 'end and a BamHI restriction site at the 3' end, and inserted into the NdeI and BamHI sites of the pT7-7 vector. The resulting plasmid was named pT7-T1.

Using a glucagon gene prepared by a DNA synthesizer as a template, fragment 1 (fragment-1) was amplified by PCR using the following primer.

1) GCA GGA TCC GAC GAC GAC GAC AAA CAC TCT CAG GGT (BGD4K)

2) TTT GTC GTC GTC GTC CTC GAG AGT GTT CAT CAG CCA (XGD4K)

After completion of PCR, the amplified DNA was analyzed using 1% agarose gel, and the amplified DNA was purified using a Qiagen Gel extraction kit.

In addition, fragment 2 (fragment-2) was amplified by PCR using the following primer, using the interleukin-2 gene contained in the plasmid pT7IL-2 as a template.

1) GAC GAC GAC GAC AAA GCA CCT ACT TCA AGT TCT (XID4K)

2) GCA AAG CTT CTA TTA AGT TAG TGT TGA GAT GAT (HIL)

After completion of PCR, the amplified DNA was analyzed using 1% agarose gel, and the amplified DNA was purified using a Qiagen Gel extraction kit.

Fragment 1 and fragment 2 were used as templates, respectively, and a second PCR was performed using the primers described below.

1) GCA GGA TCC GAC GAC GAC GAC AAA CAC TCT CAG GGT (BGD4K)

2) GCA AAG CTT CTA TTA AGT TAG TGT TGA GAT GAT (HIL)

After PCR, the amplified DNA was analyzed using 1% agarose gel, and the amplified DNA was purified using a gel extraction kit. The purified DNA was treated with BamHI and HindIII, respectively, and inserted into BamHI and HindIII sites of the pT7-T1 vector to have a BamHI restriction site at the 5 'end and a HindIII restriction site at the 3' end. The resulting plasmid was named pT7-T1GIL-2.

The plasmid pT7-T1GIL-2 as a template was used to amplify the XH6IL-2 fragment by PCR using the following primers.

1) GCA CTC GAG CAT CAT CAT CAT CAT AGC AGC GAC GAC GAC GAC AAA GCA (XH6)

2) GCA AAG CTT CTA TTA AGT TAG TGT TGA GAT GAT (HIL)

After PCR, the amplified DNA was analyzed using 1% agarose gel, and the amplified DNA was purified using a gel extraction kit. The purified DNA was treated with XhoI and HindIII, respectively, and inserted at the XhoI and HindIII sites of the pT7-T1GIL-2 vector with XhoI restriction enzyme sites at the 5 'end and HindIII restriction enzyme sites at the 3' end. The resulting plasmid was named pT1GIL-2 (see FIG. 2 ).

I. Preparation of pT2GIL-2 Plasmid

NdeI restriction enzyme site at the 5 'end of the amino acid sequence Pro-Ser-Asp-Lys-Pro (peptide consisting of the 8th to 12th amino acids of the N-terminal end of the amino acid sequence of human tumor necrosis factor) and poly at the 3' end The histidine sequence (His10) and the BamHI restriction site were added and inserted into the NdeI and BamHI sites of the pT7-7 vector. The resulting plasmid was named pT7-T2.

The GIL-2 (BH) fragment was amplified by PCR using the following primers as a template of the Glucagon-fused interleukin-2 fusion protein contained in the plasmid pT7-T1GIL-2.

1) GCA GGA TCC CAC TCT CAG GGT ACT TTC (GBAM)

2) GCA AAG CTT CTA TTA AGT TAG TGT TGA GAT GAT (HIL)

After PCR, the amplified DNA was analyzed using 1% agarose gel, and the amplified DNA was purified using a gel extraction kit. The purified DNA was treated with BamHI and HindIII, respectively, to have a BamHI restriction site at the 5 'end and a HindIII restriction site at the 3' end, and then inserted into BamHI and HindIII sites of the pT7-T2 vector. The resulting plasmid was named pT2GIL-2 (see FIG. 3 ).

All. Preparation of pG1IL-2 Plasmid

The GH6IL-2 (NH) fragment was amplified by PCR using the following primers as a template of the Glucagon-fused interleukin-2 fusion protein contained in the plasmid pT1GIL-2.

1) GCA CAT ATG CAC TCT CAG GGT ACT (GNDE)

2) GCA AAG CTT CTA TTA AGT TAG TGT TGA GAT GAT (HIL)

After PCR, the amplified DNA was analyzed using 1% agarose gel, and the amplified DNA was purified using a gel extraction kit. The purified DNA was treated with 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 inserted into NdeI and HindIII sites of the expression vector pT7-7. The resulting plasmid was named pG1IL-2 (see FIG. 4 ).

la. Preparation of pG2IL-2 Plasmid

The GH6IL-2 (BH) fragment was amplified by PCR using the following primers as a template using the Glucagon-fused interleukin-2 fusion protein gene in the plasmid pGIL-2.

1) GCA GGA TCC CAC TCT CAG GGT ACT TTC (GBAM)

2) GCA AAG CTT CTA TTA AGT TAG TGT TGA GAT GAT (HIL)

After PCR, the amplified DNA was analyzed using 1% agarose gel, and the amplified DNA was purified using a gel extraction kit. The purified DNA was treated with BamHI and HindIII, respectively, to have the BamHI restriction site at the 5 'end and the HindIII restriction site at the 3' end, and inserted into the BamHI and HindIII sites of the expression vector pT7-7. The resulting plasmid was named pGIL-2 (BH).

G (NB) fragments were amplified by PCR using a glucagon gene prepared with a DNA synthesizer as a template and the following primers.

1) GCA CAT ATG CAC TCT CAG GGT ACT (GNDE)

2) GCA GGA TCC AGT GTT CAT CAG CCA (GBAM-3)

After completion of PCR, the amplified DNA was analyzed using 1.5% agarose gel and the amplified DNA was purified using a gel extraction kit. The purified DNA was treated with NdeI and BamHI, respectively, to have an NdeI restriction enzyme site at the 5 'end and a BamHI restriction site at the 3' end, and inserted into the NdeI and BamHI sites of the pGIL-2 (BH) vector. The resulting plasmid was named pG2IL-2 (see FIG. 5 ).

hemp. Preparation of pG3IL-2 Plasmid

The G (EB) fragment was amplified by PCR using the glucagon gene contained in the plasmid pGIL-2 as a template.

1) GCA GAA TTC CAC TCT CAG GGT ACT (GECO)

2) GCA GGA TCC AGT GTT CAT CAG CCA (GBAM-3)

After completion of PCR, the amplified DNA was analyzed using 1.5% agarose gel and the amplified DNA was purified using a gel extraction kit. The purified DNA was treated with EcoRI and BamHI, respectively, to have an EcoRI restriction enzyme site at the 5 'end and a BamHI restriction site at the 3' end, and then inserted into the EcoRI and BamHI sites of the pGIL-2 (BH) vector. The resulting plasmid was named pG2IL-2 (EH).

In addition, G (NE) fragments were amplified by PCR using the glucagon gene contained in the plasmid pGIL-2 as a template.

1) GCA CAT ATG CAC TCT CAG GGT ACT (GNDE)

2) GCA GAA TTC AGT GTT CAT CAG CCA (GECO-3)

After completion of PCR, the amplified DNA was analyzed using 1.5% agarose gel and the amplified DNA was purified using a gel extraction kit. The purified DNA was treated with NdeI and EcoRI, respectively, to have an NdeI restriction enzyme site at the 5 'end and an EcoRI restriction enzyme site at the 3' end, and then inserted into the NdeI and EcoRI sites of the pG2IL-2 (EH) vector. The resulting plasmid was named pG3IL-2 (see FIG. 6 ).

Example 3. Preparation of E. coli transformants

By the method described by Hanahan (Hanahan D. 1985, DNA Cloning 1, 109-135, IRS press) the expression vectors pT7IL-2, pT1GIL-2, pT2GIL-2, pG1IL-2, pG2IL-2, pG3IL- E. coli BL21 (DE3) (Studier, FA and Moffatt, BA 1986, 189, 113-130) were transformed with 2, and then colonies resistant to ampicillin were selected.

The selected recombinant E. coli was deposited with the Korea Institute of Science and Technology Biotechnology Research Institute Gene Bank on November 23, 1998. (Accession No .: pT7IL-2 is KCTC 0555BP, pT2GIL-2 is KCTC 0556BP, pG1IL-2 is KCTC 0552BP, pG2IL-2 is KCTC 0553BP, pG3IL is KCTC 0554BP).

Example 4 Culture of Escherichia Coli Transformant and Production of Fusion Protein

A part of the spawn culture cultured all day was inoculated (1%) in this culture medium (LB medium, +100 mg / L ampicillin) and then incubated at 37 ° C. at 200 rpm. When OD ₆₀₀ of the culture reached 0.4, IPTG (isopropylthio-β-D-galactoside) was added (0.5 mM) to induce the expression of recombinant genes. After addition of IPTG, the cells were further incubated for 3-4 hours under the same conditions.

Example 5 Selection of Highly Producible E. Coli Transformants

E. coli BL21 (DE3) was transformed with each of the expression vectors, and 10 colonies were selected from each plate. Selected colonies were seeded and some of them were inoculated (1%) in 2 ml of LB medium (+100 mg / L ampicillin) and then incubated at 37 ° C. at 200 rpm. When the OD ₆₀₀ of the culture reached 0.4, IPTG was added (0.5 mM) to induce the expression of the recombinant gene, followed by further incubation for 3-4 hours under the same conditions. The cell culture solution was centrifuged at 6000 rpm to recover the cell precipitate, and the recovered cell precipitate was suspended in distilled water and then crushed using an ultrasonic crusher (Branson Sonifier). Proteins in the lysate were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (14% Tris-Glycine gel), and then the expression rate of each recombinant protein was analyzed using a densitometer (Bio-Rad). Colonies showing the maximum expression rate were inoculated in LB medium and cultured, and then stored at -70 ° C with 15% glycerol. As a result of analyzing the expression rate of each protein, the expression rate of all fusion proteins was much higher than that of direct expression of interleukin-2, especially in the case of using fusion partners T2G and G3. Exceeded 60% of (see FIG. 7 ).

Example 6 Isolation of Insoluble Aggregates from Cell Cultures

The cell culture solution was centrifuged at 6000 rpm to recover the cell precipitate, and the coliform precipitate was suspended in distilled water and then crushed using an ultrasonic crusher (Branson Sonifier). After centrifugation of the crushed solution at 5000g for 10 minutes, the supernatant and precipitates were analyzed by SDS-PAGE (14% Tris-Glycine gel) to confirm their recovery. Protein aggregates were washed twice with 0.5% TritonX-100 and the necessary assays were performed.

Example 7 Preparation of Interleukin-2 Expression Vector Using Human Glucagon-Like Gene as Fusion Partner and Expression of Fusion Protein by Transformant

The effect of glucagon analogues on IL-2 expression was examined by modifying a portion of the glucagon sequence ( ⁶ Phe-> ⁶ Lys; ¹⁰ Tyr-> ¹⁰ Lys; ¹³ Tyr-> ¹³ Lys). The preparation method of IL-2 recombinant (GK3IL-2) fused with glucagon analogue is as follows. Oligonucleotides GK3-1 and GK3-2 having base sequences of SEQ ID NO: 2 and SEQ ID NO: 3 , respectively, were amplified by PCR using primers GNDE and GXHO as templates.

1) GCA CAT ATG CAC TCT CAG GGT ACT (GNDE)

2) GAC CTC GAG AGT GTT CAT CAG CCA (GXHO)

After completion of PCR, the amplified DNA was analyzed using 2% agarose gel and the amplified DNA was purified using the Qiagen gel extraction kit. The purified DNA was treated with NdeI and XhoI, respectively, to have an NdeI restriction enzyme site at the 5 'end and an XhoI restriction enzyme site at the 3' end, and inserted into the NdeI and XhoI sites of the expression vector pIL-2 (NX). The resulting plasmid was named pGK3IL-2. The pIL-2 (NX) vector is a vector obtained by removing the G3 sequence by treating the pG3IL-2 vector with NdeI and XhoI.

The expression level of the pseudo-glucagon fused IL-2 recombinant (GK3IL-2) was investigated using the same method as in the above example, and the expression level thereof was compared with that of G1IL-2. As a result, as shown in Table 1 , even when a certain similar glucagon is fused, it shows a high expression level.

GIL-2 GK3IL-2 Expression level (%) 25.02 32.89

Experimental Example 1. Purity of Recombinant Protein Expressed

The highly productive E. coli strains selected in Example 5 were each cultured in 100 ml of LB medium (+100 mg / l ampicillin) after spawning. When the OD ₆₀₀ of the culture reached 0.4, IPTG was added (0.5 mM) to induce the expression of the recombinant gene, followed by further incubation for 3-4 hours under the same conditions. The insoluble protein aggregate produced from the cell culture was recovered by the method of Example 6. Each recovered aggregate was washed twice with 0.5% TritonX-100, analyzed by SDS-PAGE (14% Tris-Glycine gel), and the recombinant protein in each aggregate using a bio-rad. The purity of was analyzed.

As a result, when the interleukin-2 was directly expressed without the fusion partner, the purity in the aggregate was only about 40%, but when expressed with the five fusion partners, the purity of the recombinant protein in the aggregate was significantly increased (see FIG. 8 ). . In particular, when using the fusion partner G3 was confirmed that the purity increased by about 80%.

Experimental Example 2 Solubilization Characteristics of Aggregates

The insoluble protein obtained from each recombinant strain was dried by lyophilization method, and each insoluble protein was suspended in an alkaline solution of pH 12 at a concentration of 50 mg / ml, and then diluted twice in sequence using the same pH 12 solution. The solubility of insoluble protein was investigated. After each step, the sample was separated into a supernatant and a precipitate by centrifugation (5000 g, 10 minutes), and each was quantified using a Bradford quantitative method, and the solubility was determined by the following formula. The precipitate was dissolved again in a large amount of pH 12 solution for quantification and then quantified.

As shown in Figure 9 it can be seen that the solubility of the protein aggregate obtained by expressing with the fusion partner compared to the protein aggregate obtained by directly expressing the interleukin-2. In particular, when using the fusion partner G3 showed solubility close to 100% even in the aggregate concentration of 8 g / L or more. As such, it is possible to dissolve a large amount of insoluble protein aggregates simply by changing the pH without using a denaturing agent or a reducing agent, which is a unique phenomenon identified in the present invention, and may be usefully used in the separation and purification process. In addition, recombinant proteins readily dissolved in alkaline solutions at pH 12 can be reactivated by lowering the pH of the solution back to about 8.

Experimental Example 3 Binding Properties Between Protein Molecules Constituting Aggregates

The following Western blot analysis was performed to determine the binding characteristics (between the same recombinant protein molecules or between recombinant proteins and other host protein molecules) between the constituent proteins in the resulting aggregates. First, proteins in each aggregate were separated by non-reducing SDS-PAGE (14% Tris-Glycine gel), followed by Western blot. The primary antibody was a mouse-derived anti-interleukin-2 monoantibody IgG1, and the secondary antibody was a goat-derived anti-mouse antibody IgG attached with an alkaline phosphatase enzyme. As shown in FIG. 10 , in protein aggregates produced by direct expression of interleukin-2, interleukin-2 forms multimers of various sizes mainly by disulfide bonds with other host proteins. However, in the case of interleukin-2 fusion protein, it can be seen that most of them are bound by hydrophobic interaction between the same molecules of recombinant protein.

Bio-Rad was used to analyze the proportion of the same recombinant protein molecular complex (hydrophobically-bound homologous multimer) in each protein aggregate, and is shown in FIG. 11 . As a result, when only interleukin was produced, only about 32% of the conjugates between the same recombinant proteins were produced. However, when produced in the form of the fusion protein of the present invention, the hydrophobic bonds between the same proteins range from 60% to as much as 90% or more. Could. As such, aggregates produced by hydrophobic bonds between the same proteins can be easily dissociated in the purification process as described above, which is very advantageous.

As described above, according to the present invention, by using a polypeptide comprising the amino acid sequence of human glucagon as a fusion partner, the recombinant protein can be expressed in high yield in Escherichia coli, and at the same time, the purity of the recombinant protein in the generated insoluble aggregates. Can greatly improve.

In addition, aggregates produced with the expression of the fused recombinant protein are easily dissolved in large amounts in alkaline solutions, and in general, unlike insoluble aggregates, which must be dissolved in a denaturant or reducing agent and then refolded, the process is completed by alkaline dissolution alone. You can overcome the difficulties of folding.

Claims (13)

A method for producing a recombinant protein or peptide using an X _G -LY type fusion protein comprising a peptide having strong self-binding as a fusion partner in culturing a polypeptide by culturing the transformed microorganism. X _G : fusion partner (human glucagon peptide, or human glucagon analog peptide of SEQ ID NO: 1) L : linker (recognition sequence of cleavage enzyme) Y : recombinant protein or peptide delete The method of claim 1, wherein the human glucagon analogue ⁶ Phe → ⁶ Lys, ¹⁰ Tyr → ¹⁰ Lys, ¹³ Tyr → ¹³ Lys is modified. The method of claim 1, wherein the amino acid sequence of the human tumor necrosis factor or its mutein is further included in the fusion partner. The method of claim 1, wherein the linker has an Asp Asp Asp Asp Lys (DDDDK, D4K) amino acid sequence that can be recognized by enterokinase. The method of claim 1, wherein the polyhistidine sequence is included in the fusion partner. The method of claim 1, wherein the recombinant protein is interleukin-2 or an analog thereof. 8. The method of claim 7, wherein the recombinant E. coli transformant, which produces human Interleukin-2 or its analogs, in combination with the fusion partner is cultured to disrupt cells, obtain protein precipitates, dissolve in alkaline solution, and restore the pH to neutral conditions. Method for producing a recombinant protein or peptide, characterized in that by reactivating and purifying the protein. The method of claim 8, wherein the fusion protein isolated during purification is cleaved with enterokinase to purify human interleukin-2 or an analog thereof. The expression vector of claim 1 is prepared by inserting a gene of a fusion partner, a linker, a recombinant protein or a peptide into a pT7-7 vector. The expression vector of claim 10, wherein the recombinant protein is interleukin-2 or an analog thereof. Polypeptide consisting of peptides (T1 peptide)-[G-peptide]-[polyhistidine sequence (His6)] consisting of the N-terminal 1st to 57th amino acids in the amino acid sequence of human tumor necrosis factor Expression vector pT1GIL-2 (microbial accession number KCTC 0555BP.), Which is T1G and recombinant protein is IL-2, T2G, a peptide consisting of peptides consisting of the 8th to 12th amino acids at the N-terminus (T2 peptide)-[polyhistidine sequence (His10)]-[G peptide] in the amino acid sequence of human tumor necrosis factor Expression vector pT2GIL-2 (microbial accession number KCTC 0556BP.), Wherein the recombinant protein is IL-2; Expression vector pG1IL-2 (microbial accession number KCTC 0552BP), wherein the fusion partner is G1, which is a peptide consisting of glucagon [G peptide]-[polyhistidine sequence (His6)] of SEQ ID NO: 1, and the recombinant protein is IL-2, Expression vector wherein the fusion partner is G2, and the recombinant protein is IL-2, which is a peptide consisting of [G peptide is repeated twice (G2 peptide)]-[polyhistidine sequence (His6)] in which glucagon is repeated twice pG2IL-2 (Microbial Accession No. KCTC 0553BP), Expression vector in which the fusion partner is G3 and the recombinant protein is IL-2, which is a peptide consisting of [G peptide is repeated three times (G3 peptide)]-[polyhistidine sequence (His6)] in which glucagon is repeated three times pG3IL-2 (Microbial Accession Number KCTC 0554BP), and An expression vector selected from the group consisting of an expression vector pGK3IL-2 (microbial accession number KCTC 0582BP) wherein the fusion partner is a GK3 glucagon analogue and the recombinant protein is IL-2. delete