KR20180023099A - Interferon-beta signal peptide variant and use thereof - Google Patents

Abstract

The present invention relates to an interferon-beta signal peptide variant and a method for producing a target protein using the same. According to the present invention, the interferon-beta signal peptide variant ensures an enhanced alpha-helix structure and hydrophobicity on a hydrophobic portion, and an expression level of recombinant proteins remarkably increases in a host cell compared to prior arts when the interferon-beta signal peptide variant is applied to recombinant vectors or host cell systems. Accordingly, it is possible to effectively improve efficiency in producing target proteins using the same.

Description

Interferon-beta signal peptide variants and uses thereof < RTI ID = 0.0 >

The present invention relates to an interferon-beta signal peptide mutant and a method for producing a target protein using the mutant.

The ultimate goal in the production of recombinant proteins is to develop recombinant protein production technology with high production efficiency with high specificity. To achieve this goal, studies have been conducted on recombinant vectors and host cell systems, including, for example, in expressing the desired protein in host cells, such as Bacillus subtilis and E. coli , Various expression vectors have been developed. However, in the expression of the target protein, the recombinant protein is converted into an insoluble inclusion body in the host cell, degraded by proteolytic enzymes, and fails to be chemically modified in the expression process. Defective factors still exist.

In order to solve such a conventional problem, introduction of a vector system which promotes the secretion of recombinant proteins reduces the formation of insoluble inclusion bodies and the like, so that the expression amount of the recombinant protein, There is a plan to promote. In general, the host cell distinguishes between the protein to be present in the cytoplasm and the protein to be separated out of the cell by a signal peptide present in the precursor protein. In this connection, it is known that the precursor protein contains no signal peptide It has been reported that the protein is not expressed externally because it is not secreted and that the expression pattern of the protein can be changed according to the nature of the amino acid constituting the signal peptide. And their properties have an important influence on the production of recombinant proteins.

On the other hand, human interferon with antiviral activity is an important cytokine protein group that inhibits cell proliferation and regulates the natural immune response. Among them, interferon-beta (IFN-beta) It has a size of 25 kD and a sugar chain of 20 kD. Currently, studies on the clinical application of interferon-beta have been actively conducted. Among them, interferon-beta has been widely used as a therapeutic agent for alleviating and alleviating multiple sclerosis. In addition, therapeutic effect on hepatitis and melanoma It has been reported. Regarding the production of such interferon-beta, glycerol or a low-temperature culture method or a microporous microcarrier may be used as a method for reducing the aggregation phenomenon occurring in the production process of human interferon-beta using CHO cells, (Korean Patent Laid-Open No. 10-2009-0123622). However, there is no report on a method using a signal peptide of interferon-beta.

Under these circumstances, research on new technologies for improving the production efficiency of interferon-beta, which exhibits clinically significant activity, and for reducing the production cost therefrom has been actively conducted, but it is not yet very successful.

DISCLOSURE OF THE INVENTION The present invention has been conceived to solve the problems as described above. The present inventors have made intensive efforts to improve the production efficiency of recombinant proteins, and as a result, they have found that, by using an interferon-beta signal peptide or its gene having a mutated hydrophobic region, The amount of recombinant protein expressed in the cells can be remarkably improved. Based on this finding, the present invention has been completed.

Accordingly, an object of the present invention is to provide a mutant interferon-beta signal peptide mutant having a hydrophobic amino acid or a gene encoding the same.

It is another object of the present invention to provide a recombinant vector comprising the interferon-beta signal peptide mutant for enhancing the expression of a target protein, and a transformant therefor.

It is still another object of the present invention to provide a method for producing a target protein, which comprises introducing the recombinant vector into a host cell to obtain a transformant.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides an interferon-beta signal in which the 8th to 10th Gln-Ile-Ala in the amino acid sequence of SEQ ID NO: 1 are substituted with Leu- A peptide mutant or a gene encoding the peptide mutant.

In one embodiment of the present invention, the gene may consist of the nucleotide sequence of SEQ ID NO: 6.

The present invention provides a recombinant vector comprising a gene encoding the signal peptide mutant and a gene encoding a target protein, and a transformant into which the recombinant vector is introduced into a host cell.

In one embodiment of the present invention, the vector may be pcDNA3.1 / Zeo, and the host cell may be a CHO cell (Chinese Hamster Ovary cell) or a HEK 293 cell (Human Embryonic Kidney 293 cell).

In another embodiment of the present invention, the target protein may be interferon-beta.

The present invention provides a method for producing a recombinant vector, comprising: introducing the recombinant vector into a host cell to obtain a transformant; And culturing the transformant, and recovering the desired protein from the transformant.

In one embodiment of the present invention, the vector may be pcDNA3.1 / Zeo, the host cell may be a CHO cell (Chinese Hamster Ovary cell) or HEK 293 cell (Human Embryonic Kidney 293 cell) Beta.

The interferon-beta signal peptide mutant according to the present invention further enhances the hydrophobic property and the alpha -helical structure of the hydrophobic region and is applied to a recombinant vector and a host cell system. As a result, And the production efficiency of the target protein can be effectively enhanced by using the present invention.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a schematic diagram showing a mutation site and a specific amino acid sequence of an interferon-beta signal peptide mutant according to the present embodiment. FIG.
FIG. 2 is a result of checking the interferon-beta signal peptide mutant gene, interferon-beta gene and (b) ligation of (a) according to the present embodiment by Overlap PCR.
3 is a cleavage map of a recombinant vector containing the interferon-beta signal peptide mutant gene according to the present embodiment.
FIG. 4 shows the result of checking the interferon-beta gene without signal peptide through Colony PCR.
FIG. 5 shows results obtained by introducing a recombinant vector containing the interferon-beta signal peptide mutant gene according to the present example, followed by Colony PCR.
FIG. 6 shows the result of comparing the expression level of interferon-beta in HEK293A cells according to the signal peptide mutation using a sandwich ELISA.

Hereinafter, the present invention will be described in detail.

The present invention provides an interferon-beta signal peptide mutant in which the 8th to 10th Gln-Ile-Ala in the amino acid sequence of SEQ ID NO: 1 is substituted with Leu-Leu-Leu, or a gene encoding the same.

 As used herein, the term "signal peptide" is a peptide consisting of about 15 to 30 amino acid residues and includes a basic amino acid region at the N-terminus, a hydrophobic region, (Cleavage region). Among these, the hydrophobic region generally contains 6 to 15 amino acid residues and is known to be involved in targeting and immobilizing proteins with an endoplasmic reticulum membrane. In the present invention, based on the fact that the hydrophobic nature and the enhancement of the? -Helical structure for the hydrophobic region in the signal peptide can effectively enhance the secretion and expression of the target protein, the interferon-beta signal peptide mutant Respectively.

As used herein, the term "signal peptide variant" means that at least one of the amino acid residues of the interferon-beta signal peptide of SEQ ID NO: 1 is within the range capable of carrying out the function of the signal peptide that secretes or expresses the protein outside the cell Deletion, addition, insertion, or substitution of a peptide.

  The interferon-beta signal peptide mutant according to the present invention is characterized in that the amino acid sequence of SEQ ID NO: 1 has an 8th to 10th sequence selected from the group consisting of phenylalanine (Phe), tryptophan (Trp), isoleucine (Ile) (Leu), Proline (Pro), Methionine (Met), Valine (Val) or Alanine (Ala), preferably Leu-Leu- 2, which has at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 70%, of the amino acid sequence shown in SEQ ID NO: 2, Or an amino acid sequence having 95% or more sequence homology.

 Also, the gene encoding the signal peptide mutant may preferably include all kinds of base sequences capable of coding the amino acid sequence shown in SEQ ID NO: 2, and most preferably, consists of the nucleotide sequence shown in SEQ ID NO: 6 And may comprise a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NO: 6 have.

In another aspect of the present invention, the present invention provides a recombinant vector for promoting expression of a target protein, which comprises a gene encoding the signal peptide mutant and a gene encoding a target protein, and a transformant into which the recombinant vector is introduced into a host cell to provide.

 As used herein, the term "vector" refers to a DNA construct comprising an exogenous DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. More specifically, the recombinant vector according to the present invention is an expression vector capable of expressing a target protein or transcribing a target RNA in a host cell, and includes an essential regulatory element operatively linked to express the gene insert . An example of such a vector may be a plasmid vector, a bacteriophage vector, a cosmid vector, a yeast artificial chromosome (YAC) vector, etc., and may be a plasmid vector, more specifically pcDNA3.1 / Zeo for the purpose of the present invention , But is not limited thereto.

The recombinant vector may be constructed so that a recombinant peptide in the host cell, that is, a signal peptide mutant gene is inserted at the 5 'end of the target protein gene to enhance the expression of the target protein, and (a) (B) an antibiotic resistance gene that allows the host cell transformed with the plasmid vector to be selected, and (c) a foreign DNA fragment can be inserted into the plasmid vector. The restriction enzyme cleavage site, and the like, which are known to those skilled in the art. As an example, the gene encoding the signal peptide mutant of the present invention and / or the gene encoding the desired protein may be operably linked to a conventional gene expression control sequence such as a promoter, transcription termination sequence, and the like. Operatively linked "means a functional linkage between a gene expression control sequence and another nucleotide sequence, wherein the gene expression control sequence is operably linked to transcription and / or translation of another nucleotide sequence Can be adjusted.

As used herein, the term " transformation " as used in the present invention means that a DNA strand or plasmid having a foreign gene other than that of the original cell has been introduced into the host cell, Refers to a molecular biological technique that changes the genetic character of a cell by binding it to DNA. For the purpose of the present invention, the transfection means that the gene encoding the signal peptide mutant and the gene encoding the target protein are inserted into the host cell to express the desired protein. Preferably, the host cell is a CHO cell (Chinese Hamster Ovary cell) or HEK 293 cell (Human Embryonic Kidney 293 cell).

In the present invention, the term "target protein" is a protein to be ultimately expressed through the above recombinant vector and the host system, preferably insulin, cytokine (interleukin, tumor necrosis factor, interferon, colony stimulating factor, Erythropoietin, and the like, and more preferably, it may be interferon-beta, but is not limited thereto.

In one embodiment of the present invention, a recombinant vector containing a mutant in which the hydrophobic region of the interferon-beta signal peptide was mutated was transformed (see Examples 1 and 2), and as a result, the existing interferon-beta The expression level of interferon-beta in the transformant into which the recombinant vector containing the nucleotide sequence of SEQ ID NO: 6 was introduced was remarkably increased compared with the case of using the signal peptide or other mutant (see Example 3).

Thus, as another embodiment of the present invention, the present invention provides a method for producing a recombinant vector, comprising: introducing the recombinant vector into a host cell to obtain a transformant; And culturing the transformant, and recovering the desired protein from the transformant.

The transformant can be produced by introducing the recombinant vector into a host cell as described above, and the introduction method can be introduced by a known technique, that is, a chemical method, a thermal shock method, an electroporation method or the like .

In addition, the target protein of the present invention can be isolated or recovered from the culture of the transformant by methods known in the art. For example, the target protein may be isolated from the culture by conventional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. In addition, the target protein may be purified by a variety of methods known in the art including chromatography (e.g., ion exchange, affinity, hydrophobicity and size exclusion), electrophoresis, fractional solubility (e.g. ammonium sulfate precipitation) ≪ / RTI >

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

Example  1. Preparation of recombinant vector for promoting interferon-beta expression

1-1. Interferon-beta signal Peptides  Mutation of gene

First, PCR was performed using an oligonucleotide (63 bp) encoding human interferon-beta signal peptide as a template. Specifically, 50 μl of PCR reaction solution [template (1 μl), primer F (100 pmole, 1 μl), primer R (100 pmole, 1 μl), polymerase (1 μl), dNTP (2.5 mM, buffer (10 μl), distilled water (31 μl)] at 95 ° C for 5 minutes, and then 1) denaturation at 95 ° C for 1 minute; 2) annealing at 58 ° C for 1 min; 3) Reaction was carried out at 72 ° C for 15 minutes, followed by reaction at 72 ° C for 7 minutes. This procedure was repeated 18 times (18 cycles).

Then, using a conventional site direct mutation method including the step of treating Dpn I in the PCR product (30.5 l) obtained from the above, the hydrophobic region of the human interferon-beta signal peptide is mutated, Interferon-beta signal peptide mutant genes were prepared and their sequence information is shown in Table 1 below.

1-2. Preparation of recombinant vector for promoting interferon-beta expression

The plasmid vector was linearized by treating the pcDNA3.1 / Zeo plasmid vector with restriction enzymes HindIII and XbaI overnight at 37 ° C. Then, overlap PCR was performed to subclone the human interferon-beta gene except the gene of the mutant obtained in Example 1-1 and the original signal peptide into the pcDNA3.1 / Zeo plasmid vector. Specifically, 50 μl of PCR reaction solution [template (1 μl), primer F (100 pmole, 1 μl), primer R (100 pmole, 1 μl), polymerase (1 μl), dNTP (2.5 mM, buffer (10 μl), distilled water (31 μl)] at 95 ° C for 5 minutes, and then 1) denaturation at 95 ° C for 1 minute; 2) annealing at 55 ° C for 1 min; 3) Reaction was performed at 72 ° C for 90 seconds, followed by reaction at 72 ° C for 7 minutes. This procedure was repeated 25 times (25 cycles). Thereafter, they were cultured overnight at 37 ° C, and each interferon-beta signal peptide mutant gene and Human Interferon-beta gene were ligation (INFB3 (Q → L), INFB4 (QI → LL) INFO5 (QIA? LLL)) were confirmed, and pcDNA3.1 / Zeo-IFNB (Q? L), pcDNA3.1 / Zeo-IFNB (QI? LL) LLL) recombinant vector. In order to compare the expression level of interferon-beta with signal peptide, pcDNA3.1 / Zeo-IFNB (wt) with wild type signal peptide and pcDNA3.1 / Zeo-IFNB without signal peptide signal peptide w / o).

As a result, as shown in FIG. 4 and FIG. 5, a band of about 600 bp size was confirmed by comparison with a 100 bp size marker. As a result, the recombinant vector was introduced into the competent cell and transfected with the recombinant vector. there was. In addition, a colony with a band of 600 bp in size was selected and cultured in LB amp liquid medium in an amount of 3 ml. Cells were grown overnight at 37 ° C and 200 rpm, and the plasmid vector was purified by mini-prep using a plasmid mini kit , And their sequencing was performed again (not shown).

Example  2. Transformation using a recombinant vector for increasing interferon-beta expression

HEK293A cells and CHO-DG44 cells were transfected by introducing the recombinant vectors prepared in Example 1 under the same conditions. HEK293A cells are fetal bovine serum 10% was added with IMDM (Iscove's Modified Dulbecco's Medium , GIBCO) culture medium and 37 ℃, 5% CO ₂ (DMEM-F12, Dulbecco's Modified Eagle Medium F-12, GIBCO) supplemented with 10% fetal bovine serum.

Specifically, cells were seeded and cultured at a concentration of 1 × 10 ⁶ cells / dish in a 10-ml culture medium in a 100 × 20 mm cell culture dish in a third passage, followed by replacing 8 ml of the dish medium with 70% cell confluence After one hour, the cells were transfected with lipofectamine 2000 (Invitrogen). Midi-prep DNA (7.5 μg) was added to serum free IMDM / DMEM-F12 (300 μl) and Lipofectamine 2000 (12 μl) was also added to serum free IMDM / DMEM-F12 (300 μl) After incubation at room temperature, DNA and lipofectamine 2000 were mixed and incubated at room temperature for 20 minutes to form DNA-Lipofectamine 2000 complex. After that, DNA-lipofectamine 2000 complex was added to a dish containing cells in IMDM / DMEM-F12 medium and incubated at 37 ° C in 5% CO ₂ After incubation for 24 hours in an incubator, the dish medium was replaced by 8 ml each, and after 48 hours, the supernatant in the dish was obtained.

Example  3. Interferon-beta expression level comparison

In this example, the amount of expression of interferon-beta according to the signal peptide mutation was quantitatively analyzed by sandwich ELISA. Specifically, the standard recombinant protein was used as the pBR assay, Human Interferon Beta 1a, and mammalian (Catalog No: 11410-2). To confirm and correct the interferon-beta recombinant protein concentration, VeriKine ™ Human IFN Beta ELISA Kit (Catalog No. 41410) was used according to the manufacturer's instructions. Incubation was carried out at room temperature (RT), 22 to 25 ° C, and wash buffer was used with 300 μl of wash buffer per well.

As a result, as shown in Tables 2, 3 and 6, in the case of pcDNA3.1 / Zeo-IFNB (signal peptide w / o) without signal peptide, no interferon-beta was expressed at all, When the HEK293A cells (see Table 2 and FIG. 6) and the CHO-DG44 cells (see Table 3) were transformed with the pcDNA3.1 / Zeo-IFNB (QIA? LLL) recombinant vector, the expression level of interferon- type signal peptide, and the expression level of interferon-beta in HEK293A cells was about 6 times higher than that of pcDNA3.1 / Zeo-IFNB (wt).

However, in the case of pcDNA3.1 / Zeo-IFNB (Q → L) and pcDNA3.1 / Zeo-IFNB (QI → LL), although the hydrophobic region in the signal peptide was mutated, In contrast, the expression level of interferon-beta did not show a significant difference or decreased, and that the mutation of the signal peptide had a significant effect on the expression level of the target protein such as Interferon-beta. Interferon-beta (QIA? LLL) level (SEQ ID NO: 6).

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Sejong Industry-Academy Cooperation Foundation <120> Interferon-beta signal peptide variant and use thereof <130> IP-2016-166-KR_P16U10C1397 <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 21 <212> PRT <213> Interferon beta signal peptide <400> 1 Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser   1 5 10 15 Thr Ala Leu Ser              20 <210> 2 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Interferon beta signal peptide variant 5 protein <400> 2 Met Thr Asn Lys Cys Leu Leu Leu Leu Leu Leu Leu Leu Cys Phe Ser   1 5 10 15 Thr Ala Leu Ser              20 <210> 3 <211> 63 <212> DNA <213> Interferon beta signal peptide nucleotide <400> 3 atgaccaaca agtgtctcct ccaaattgct ctcctgttgt gcttctccac tacagctctt 60 tcc 63 <210> 4 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Interferon beta signal peptide variant 3 nucleotide <400> 4 atgaccaaca agtgtctcct cctgattgct ctcctgttgt gcttctccac tacagctctt 60 tcc 63 <210> 5 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Interferon beta signal peptide variant 4 nucleotide <400> 5 atgaccaaca agtgtctcct cctgctggct ctcctgttgt gcttctccac tacagctctt 60 tcc 63 <210> 6 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Interferon beta signal peptide variant 5 nucleotide <400> 6 atgaccaaca agtgtctcct cctgctgctg ctcctgttgt gcttctccac tacagctctt 60 tcc 63

Claims (8)

An interferon-beta signal peptide mutant in which the 8th to 10th Gln-Ile-Ala in the amino acid sequence of SEQ ID NO: 1 are substituted with Leu-Leu-Leu.
A gene encoding the signal peptide mutant of claim 1.
3. The gene according to claim 2, wherein the gene comprises the nucleotide sequence of SEQ ID NO: 6.
A recombinant vector comprising a gene encoding the signal peptide mutant of claim 2 and a gene encoding a target protein.
5. The recombinant vector according to claim 4, wherein the target protein is interferon-beta.
5. The recombinant vector according to claim 4, wherein the vector is pcDNA3.1 / Zeo.
A transformant, wherein the recombinant vector of any one of claims 4 to 6 is introduced into a host cell.
8. The transformant according to claim 7, wherein the host cell is a CHO cell (Chinese Hamster Ovary cell) or HEK 293 cell (Human Embryonic Kidney 293 cell).

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