JP2007037498A - Method for producing protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To establish a method for removing amino-terminal methionine corresponding to initiation codon. <P>SOLUTION: The method for producing a protein free from amino-terminal methionine by a recombinant comprises the introduction of a plasmid having a gene encoding the object protein and a plasmid having a gene encoding methionine aminopeptidase into a cell and the culture of the cell under a condition to effect the expression of the protein gene to a level lower than that of the methionine aminopeptidase gene. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing a protein containing no methionine at the amino terminus by a gene recombination technique.

When a protein is biosynthesized in a cell, its amino terminus begins with a methionine (formyl methionine in prokaryotes) that corresponds to the start codon of mRNA (generally AUG). It is removed by enzymatic reaction in protein species. Thus, many actual protein molecules do not contain methionine at the amino terminus. On the other hand, advances in gene recombination technology have made it possible to produce useful proteins using microorganisms and animal cells. In particular, E. coli is most widely used as a host for production of recombinant proteins from the viewpoint of cost, but when recombinant proteins are expressed using E. coli and other microorganisms as hosts, they are expressed in animal cells. May not be as accurate as you would see it. Addition of an amino-terminal methionine derived from the start codon is one such inaccurate processing. Removal of methionine added to the amino terminus of the recombinant protein is usually performed by methionine aminopeptidase derived from the host. However, when the expression level of the recombinant protein is high, most of the expressed recombinant protein Often remain methionine at the amino terminus. For example, in human growth hormone expressed in Escherichia coli, the methionine addition rate reaches almost 100% [Non-patent document 1: Nature, 293 , 408 (1981)], and 50% [non-patented in interferon-α]. Reference 2: Journal of Interferon Research (J. Interferon Res.), 1 , 381 (1981)], non-glycosylated human interleukin-2, molecular species starting with alanine, similar to natural human interleukin-2 In addition to (rIL-2), the presence of a molecular species (Met-rIL-2) having methionine added to the amino terminus (having a methionine residue at the amino terminus) has been observed.

  In some cases, it is possible to separate a recombinant protein with methionine added at the amino terminus and a recombinant protein without methionine added at the amino terminus by purification. Separation is often difficult and disadvantageous in terms of purification yield. Therefore, reducing the proportion of recombinant protein with methionine added to the amino terminus is expected to be effective in terms of purification yield. In addition, a recombinant protein in which methionine is added to the amino terminus may not have the original activity, or antigenicity may be a problem when the recombinant protein is used as a pharmaceutical product. For example, as seen in recombinant human growth hormone and the like, an increase in antigenicity has been pointed out as compared to mature type. Therefore, in order to use a precursor protein produced by gene recombination technology for a pharmaceutical or the like, it is necessary to selectively remove this amino terminal methionine to obtain a mature protein.

As a method for chemically removing amino-terminal amino acids, Dixon in 1964 caused DL-alanylglycine to react with glyoxylic acid, pyridine, and copper acetate, resulting in an amino group rearrangement reaction to produce pyruvoylglycine. [Non-Patent Document 3: Biochemistry Journal (Biochem. J.), 92 , 661 (1964)], and further, when a compound is reacted with thiosemicarbazide, amide bond cleavage occurs to produce glycine. Non-patent document 4: Biochemistry Journal (Biochem. J.), 90 , 2C (1964)]. Subsequently, this reaction was applied to cytochrome c-551 (Pseudomonas cytochrome c-551), and it was reported that amino-terminal glutamic acid was removed [Non-Patent Document 5: Biochem. J., 94 , 463 (1965)]. However, these methods have problems in terms of reaction efficiency and reaction specificity, and have a lack of practicality because they may affect the target protein. Therefore, as a method for selectively removing amino-terminal methionine, a method in which methionine aminopeptidase is allowed to act is most desirable. Examples of methionine aminopeptidase that can be used in this method include Escherichia coli [Patent Document 1: JP-A-62-115281 and Non-Patent Document 6: Journal of Bacteriol., 169 , 751 (1987). ] Salmonella typhymurium [Patent Document 2: Japanese Patent Publication No. Hei 3-502400 and Non-Patent Document 7: European Journal of Biochemistry (Eur. J. Biochem.), 180 , 23 (1989)], methionine aminopeptidase derived from yeast (Patent Document 3: JP-A-4-500155) and Bacillus subtilis (Patent Document 4: JP-A-3-285684) have been proposed. Yes. These methionine aminopeptidases have been isolated as proteins and their genes have also been revealed. Other methods for removing the amino terminal methionine or producing a recombinant protein without the methionine added include secretory expression [Non-Patent Document 8: Gene, 55 , 189 (1987)], A method of expressing as a fusion protein [Non-patent Document 9: Science, 198 , 1056 (1977)] is known. However, the secretory expression method has a disadvantage that the production amount is lower than that of normal expression. In addition, regarding the method of expressing as a fusion protein and the method of removing methionine using an aminopeptidase after expression, it is necessary to perform the operation of cleaving the fusion protein or removing methionine after the expression. This is inconvenient because it leads to a decrease in final yield and purity.
JP-A-62-115281 Japanese Patent Publication No. Hei 3-502400 Japanese National Patent Publication No. 4-500155 JP-A-3-285684 Nature, 293, 408 (1981) Journal of Interferon Research, 1,381 (1981) Biochem. J., 92, 661 (1964) Biochem. J., 90, 2C (1964) Biochemistry Journal (Biochem. J.), 94, 463 (1965) Journal of Bacteriol, 169,751 (1987) European Journal of Biochemistry (Eur. J. Biochem.), 180, 23 (1989) Gene, 55, 189 (1987) Science, 198, 1056 (1977)

  Even in the same protein, molecular species with methionine added at the amino terminus and those with no methionine may differ from each other in protein conformation, biological activity, and stability. It is believed that the addition may result in increased antigenicity. Therefore, it is considered to be meaningful to establish a method for removing the amino terminal methionine corresponding to this initiation codon from the viewpoint of industrial use. In order to solve this problem, a method of subjecting a severe chemical reaction to a protein never gives satisfactory results. That is, it is considered difficult to find a chemical reaction that can remove the amino-terminal methionine residue under mild conditions without denaturing the final product protein. Also, it is considered that using other methods is inconvenient because the operation is complicated and the final yield and purity are lowered.

  As a result of earnest research on a method for producing a protein containing no methionine at the amino terminus by a gene recombination technique, the present inventors have found that a plasmid having DNA encoding the target protein and a plasmid having DNA encoding methionine aminopeptidase It was found that the amino terminal methionine can be removed unexpectedly and efficiently by culturing under the condition that the target protein gene is expressed at a lower level than the methionine aminopeptidase gene. More specifically, DNA encoding a target protein is inserted into a low copy number plasmid, and the resulting expression plasmid is introduced into a cell together with a high copy number plasmid having DNA encoding methionine aminopeptidase. By culturing under conditions in which both aminopeptidases are expressed, the methionine residue is removed from the protein having a methionine residue at the amino terminus, and there is an extra methionine residue at the amino terminus without reducing its activity. The inventors have found a method for obtaining an unexpectedly high protein in a high yield, and further advanced the research to complete the present invention.

That is, the present invention is as follows.
1. A plasmid having a gene encoding a target protein and a plasmid having a gene encoding a methionine aminopeptidase are introduced into a cell, and the protein gene is cultured under conditions such that the expression of the protein gene is lower than that of the methionine aminopeptidase gene. And a method for producing a protein from which amino terminal methionine has been removed in a recombinant form.
2. Item 2. A method for producing a protein from which the amino-terminal methionine has been removed according to Item 1, wherein the expression level of the methionine aminopeptidase gene is 1.5 times or more of the expression level of the protein gene.
3. Item 3. A method for producing a protein in which the amino-terminal methionine according to Item 1 or 2 is removed in a recombinant using Escherichia coli as a cell.
4). Item 4. The method for producing a protein from which the amino-terminal methionine has been removed according to any one of Items 1 to 3, wherein the methionine aminopeptidase is derived from Archia.
5. DNA encoding the protein of interest is inserted into a low copy number plasmid, and the resulting expression plasmid is introduced into a cell along with a high copy number plasmid having DNA encoding methionine aminopeptidase, both the protein and the methionine aminopeptidase A method for producing a protein from which an amino-terminal methionine has been removed, in a recombinant form, characterized by culturing under a condition that expresses.
6). The amino terminal methionine-removed protein according to Item 5, wherein the low copy number plasmid maintains 1 to 20 copies per cell and the high copy number plasmid maintains 50 to 500 copies per cell. Manufacturing method.
7). Item 7. A method for producing a protein in which the amino-terminal methionine of Item 5 or 6 is removed in a recombinant using Escherichia coli as cells.
8). Item 8. A method for producing a protein from which the amino-terminal methionine has been removed according to any one of Items 5 to 7, wherein the methionine aminopeptidase is derived from Archia.
9. A transformant into which a plasmid having DNA encoding a target protein and a plasmid having DNA encoding methionine aminopeptidase are introduced.
10. It has a low copy number plasmid in which DNA encoding the protein of interest can be expressed and a high copy number plasmid in which DNA encoding methionine aminopeptidase is expressed so that the copy number of the high copy number plasmid is low. Transformants that are more than 1.5 times the number of plasmids.
11. Item 11. Use of the transformant according to Item 9 or 10 for producing a recombinant protein from which amino terminal methionine has been removed.

  According to the present invention, a protein containing no methionine at the amino terminus can be produced by a gene recombination technique. Therefore, a protein having no extra methionine residue at the amino terminus can be obtained in high yield without reducing the activity of the target protein. According to the present invention, a peptide or protein having a natural amino acid sequence can be advantageously produced industrially.

  The target protein that is the subject of the present invention is not particularly limited in terms of type and molecular weight, and can be applied in a wide range from low molecular weight so-called peptides to high molecular weight proteins. Moreover, even if it is a protein which consists of a several subunit, this invention is applicable by introduce | transducing DNA into a cell on the same conditions. Preferable examples include soluble proteins such as hormones, growth factors, enzymes, nuclear receptors, and the like.

The methionine aminopeptidase that is the subject of the present invention is not particularly limited, but is preferably one that has been isolated as a protein and whose gene has been clarified. Examples of such methionine aminopeptidase include Escherichia coli, Salmonella typhimurium, yeast, Bacillus subtilis, and archeon-derived methionine aminopeptidase. In particular, methionine aminopeptidase derived from a hyperthermophilic archaea is desirable because of its excellent stability.
Examples of cells (transformants) that produce proteins include bacteria such as E. coli, yeast, cyanobacterium, mammalian cells, animal cells such as insect cells, plant cells, and the like, preferably E. coli.

As a method for changing the gene expression level of the present invention, a method of recombining each gene DNA into a plasmid having a different copy number and utilizing a gene quantitative effect associated with the copy number difference is most easily applied. Alternatively, the gene expression level can also be changed by controlling and regulating the expression of each gene under the control of different promoters. Alternatively, the expression level may be controlled by using regulatory elements such as enhancers and silencers.
In order to effectively carry out the present invention, the expression level of the methionine aminopeptidase gene is preferably 1.5 times or more the expression level of the target protein gene, and the expression level of the methionine aminopeptidase gene is More preferably, it is at least 2.5 times the expression level. The upper limit of the expression level of the methionine aminopeptidase gene is not particularly limited, but is preferably 100 times or less the expression level of the target protein gene.

  As the low copy number plasmid used in the present invention, one that maintains 1 to 20 copies per cell is preferably used. Examples of such plasmids include pETcoco-1 (1 copy / cell), pACYC (˜10 copies / cell), pSC101 (˜6 copies / cell), ColE1 (about 15 copies / cell), and the like.

As the high copy number plasmid used in the present invention, a plasmid that maintains 25 copies or more, preferably 50 to 500 copies or more per cell is suitably used. Such plasmids include pGEM (300-700 copies / cell) pUC18 (500-700 copies / cell), pUC19 (500-700 copies / cell), pBR322 (about 25 copies / cell), pET vector (-40 Copy / cell).
In order to carry out the present invention more efficiently, the medium composition and culture conditions may be examined in detail for each protein derived from the inserted gene. By conducting these studies and obtaining optimum conditions, it is expected that the methionine removal efficiency will be close to 100%.

The present invention will be described more specifically with reference to the following reference examples and examples, but the present invention is not limited thereto.
Reference Example 1 (Construction of target protein expression plasmid)
To obtain histone A (HAfA) derived from the hyperthermophilic archaeoglobus fulgidus, an oligonucleotide designed with a NdeI cleavage site adjacent to the upstream of the start codon of the structural gene and a ribosome binding site and PstI cleavage site upstream (SEQ ID NO: 1) ), An oligonucleotide having a BamHI cleavage site downstream of the stop codon (SEQ ID NO: 2) and an oligonucleotide having a structural gene internal sequence (SEQ ID NO: 3) were synthesized.
5'-AACTGCAGTAAGGAGGAATAGCATATGGCTGAGTTGCCGATGGCACCGGTTGACAGATTGATTAGGAAGGCTGGGGCTGAGAGAGTAAGT-3 '(SEQ ID NO: 1);
5'-TTCCACAGCTTTCTTAGCAACGGTAATTGCGTAGTCCTCAAGCACTTCGACCATCTTTTCCACTGCATCTGCACTTACTCTCTCAGCCCC-3 '(SEQ ID NO: 2);
5′-GGAATTCTTACATCGAGAGAGCGAGCTTAATGTCGTCCGCAGTGACGGTCTTTCTGCCAGAGTGCTTCGCAATTTCCACAGCTTTCTTAG-3 ′ (SEQ ID NO: 3).

The hafA gene was synthesized by overlapping PCR using these three oligonucleotides. The amplified fragment was ligated to pUC19 cut with HincII and introduced into E. coli DH10B. A transformant was selected using ampicillin resistance and β-galactosidase activity as indices, and a plasmid was recovered from the strain to obtain pUC-HAfA (HincII). pUC-HAfA (HincII) was cleaved with PstI and BamHI to recover a fragment of about 0.2 kbp, and then ligated to pUC19 cleaved with PstI and BamHI. This was introduced into Escherichia coli DH10B, a transformant was selected using ampicillin resistance as an index, and a plasmid was recovered from the strain to obtain an expression plasmid pUC-HAfA. Furthermore, pUC-HAfA was cleaved with PstI and BamHI, and the HAfA gene fragment was recovered and ligated to pSTV29 cleaved with PstI and BamHI. This was introduced into Escherichia coli DH10B, a transformant was selected using ampicillin resistance as an index, and a plasmid was recovered from the strain to obtain an expression plasmid pSTV-HAfA (500 to 700 copies / cell).
Reference Example 2 (Construction of methionine aminopeptidase expression plasmid)
In order to obtain the methionine aminopeptidase (AfMAP) gene derived from the hyperthermophilic archaeoglobus fulgidus, a primer having an NdeI cleavage site adjacent to the upstream of the start codon of the structural gene, a ribosome binding site, and a PstI cleavage site upstream (SEQ ID NO: 4) ), And a primer having a BamHI cleavage site adjacent to the downstream of the stop codon (SEQ ID NO: 5), PCR was performed using A. fulgidus chromosomal DNA as a template, and the amplified fragment was recovered.
5'-AACTGCAGTAAGGAGGAATAGCATATGGATGACGAGGTAAGGGAAAAGCTG-3 '(SEQ ID NO: 4);
5′-TTGGATCCTTATTTAGTGGTTATGGTTGCTCCTCCGTCC-3 ′ (SEQ ID NO: 5).

The amplified fragment was ligated to HSTII cleaved pSTV29 and introduced into E. coli DH10B. A transformant was selected using chloramphenicol resistance and β-galactosidase activity as indicators, and a plasmid was recovered from the strain to obtain pSTV-AfMAP (HincII). pSTV-AfMAP (HincII) was cleaved with PstI and BamHI to recover a fragment of about 0.9 kbp, and then ligated to pUC19 cleaved with PstI and BamHI. This was introduced into Escherichia coli DH10B, a transformant was selected using ampicillin resistance as an index, and a plasmid was recovered from the strain to obtain an expression plasmid pUC-AfMAP (500 to 700 copies / cell).
Reference Example 3 (Preparation of recombinant and production of target protein)
A pSTV-HAfA and pUC-AfMAP expression plasmid-bearing strain BL21 ((5) in Table 1 below) was added to a 2YT medium (1.6% bactotryptone, chloramphenicol (34 μg / ml) and ampicillin (50 μg / ml). The cells were cultured overnight at 37 ° C. in a test tube containing 5 ml of 1.0% yeast extract and 0.5% NaCl. 2 ml of the obtained culture solution was cultured with shaking at 37 ° C. in a 500 ml Sakaguchi flask containing 200 ml of 2YT medium supplemented with chloramphenicol (34 μg / ml) and ampicillin (50 μg / ml). When the turbidity of the culture reached about 0.4 at OD ₆₆₀ , isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the mixture was further cultured at 37 ° C. for 4 hours. The cells obtained by centrifuging the culture solution were stored frozen at -80 ° C.

  The pSTV-HAfA and pUC19 expression plasmid holding strain BL21 (Table 1 (4) below) and the pSTV-HAfA expression plasmid holding strain BL21 (Table 1 (3)) were treated in the same manner as described above.

In addition, pET-HAfA expression plasmid holding strain BL21 (DE3) CodonPlusRIL ((2) in Table 1 below) was added to 2YT medium (1.6% bactotryptone, 1.0% yeast extract, 0.5% NaCl) containing ampicillin (50 μg / ml). ) Cultivated overnight at 37 ° C in a test tube containing 5 ml. 2 ml of the obtained culture solution was cultured with shaking at 37 ° C. in a 500 ml Sakaguchi flask containing 200 ml of 2YT medium supplemented with ampicillin (50 μg / ml). When the turbidity of the culture reached about 0.4 at OD ₆₆₀ , isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the mixture was further cultured at 37 ° C. for 4 hours. The cells obtained by centrifuging the culture solution were stored frozen at -80 ° C.

(Example)
By co-expressing the MAP and the target protein in E. coli, the starting methionine of the target protein is removed.
MAP ・ ・ ・ ・ ・ ・ ・ ・ AfMAP (Archaeoglobus fulgidus-derived MAP)
Target protein: HAfA (Archaeoglobus fulgidus-derived histone A)
N-terminal amino acid sequence of target protein ... (M) AELPM ...
Manufacturing conditions of (2) and (3) in Table 1 below
pET-HAfA expression plasmid-carrying strain BL21 (DE3) CodonPlusRIL ((2) in Table 1 below) is added to 2YT medium (1.6% bactotryptone, 1.0% yeast extract, 0.5% NaCl) containing ampicillin (50 μg / ml) in 5 ml. The cells were cultured overnight at 37 ° C. in a test tube containing. 2 ml of the obtained culture solution was cultured with shaking at 37 ° C. in a 500 ml Sakaguchi flask containing 200 ml of 2YT medium supplemented with ampicillin (50 μg / ml). When the turbidity of the culture reached about 0.4 at OD ₆₆₀ , isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the mixture was further cultured at 37 ° C. for 4 hours. The cells obtained by centrifuging the culture solution were stored frozen at -80 ° C.

The pSTV-HAfA expression plasmid-bearing strain BL21 ((3) in Table 1 below) is mixed with 5 ml of 2YT medium (1.6% bactotryptone, 1.0% yeast extract, 0.5% NaCl) containing chloramphenicol (34 μg / ml). The cells were cultured overnight at 37 ° C in the contained test tube. 2 ml of the obtained culture solution was cultured with shaking at 37 ° C. in a 500 ml Sakaguchi flask containing 200 ml of 2YT medium supplemented with chloramphenicol (34 μg / ml). When the turbidity of the culture reached about 0.4 at OD ₆₆₀ , isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the mixture was further cultured at 37 ° C. for 4 hours. The cells obtained by centrifuging the culture solution were stored frozen at -80 ° C.

Manufacturing conditions of (4) and (5) in Table 1 below
The pSTV-HAfA and pUC-AfMAP both expression plasmid-bearing strain BL21 or pSTV-HAfA and pUC19 both expression plasmid-bearing strain BL21 were mixed with 2YT medium (1.6% bacterial) containing chloramphenicol (34 μg / ml) and ampicillin (50 μg / ml). The cells were cultured overnight at 37 ° C. in a test tube containing 5 ml of tryptone, 1.0% yeast extract, 0.5% NaCl). 2 ml of the obtained culture solution was cultured with shaking at 37 ° C. in a 500 ml Sakaguchi flask containing 200 ml of 2YT medium supplemented with chloramphenicol (34 μg / ml) and ampicillin (50 μg / ml). When the turbidity of the culture reached about 0.4 at OD ₆₆₀ , isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the mixture was further cultured at 37 ° C. for 4 hours. The cells obtained by centrifuging the culture solution were stored frozen at -80 ° C.

From the results of Table 1 above, the following became clear.
○ In the original strain, the starting methionine of HAfA has been completely removed ((1)).
○ By reducing the copy number of E. coli expression plasmid (reducing the expression level), we succeeded in increasing the proportion of HAfA from which the starting methionine was removed ((2), (3)).
○ Further, by co-expression with MAP, we succeeded in increasing the HAfA ratio (~ 92%) ((5) and (4) are control experiments).

Claims (11)

A plasmid having a gene encoding a target protein and a plasmid having a gene encoding a methionine aminopeptidase are introduced into a cell, and the protein gene is cultured under conditions such that the expression of the protein gene is lower than that of the methionine aminopeptidase gene. And a method for producing a protein from which amino terminal methionine has been removed in a recombinant form. The method for producing a protein from which amino-terminal methionine has been removed according to claim 1, wherein the expression level of the methionine aminopeptidase gene is 1.5 times or more of the expression level of the protein gene. A method for producing a protein from which amino terminal methionine has been removed according to claim 1 or 2, using Escherichia coli as a cell, in a recombinant form. The method for producing a protein from which an amino-terminal methionine has been removed according to any one of claims 1 to 3, wherein the methionine aminopeptidase is derived from archaea. DNA encoding the protein of interest is inserted into a low copy number plasmid, and the resulting expression plasmid is introduced into a cell along with a high copy number plasmid having DNA encoding methionine aminopeptidase, both the protein and the methionine aminopeptidase A method for producing a protein from which an amino-terminal methionine has been removed, in a recombinant form, characterized by culturing under a condition that expresses. 6. The amino terminal methionine-depleted protein of claim 5, wherein the low copy number plasmid maintains 1-20 copies per cell and the high copy number plasmid maintains 50-500 copies per cell. The manufacturing method. The method for producing a protein from which amino terminal methionine has been removed according to claim 5 or 6, using Escherichia coli as cells. The method for producing a protein from which amino-terminal methionine has been removed according to any one of claims 5 to 7, wherein the methionine aminopeptidase is derived from archaea. A transformant into which a plasmid having DNA encoding a target protein and a plasmid having DNA encoding methionine aminopeptidase are introduced. It has a low copy number plasmid in which DNA encoding the protein of interest can be expressed and a high copy number plasmid in which DNA encoding methionine aminopeptidase is expressed so that the copy number of the high copy number plasmid is low. Transformants that are more than 1.5 times the number of plasmids. Use of the transformant according to claim 9 or 10 for producing a recombinant protein from which amino terminal methionine has been removed.