JP2008054673A - Method for producing recombinant protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for forcedly forming insoluble granules of a recombinant protein in host cells without producing the recombinant protein as a fused protein. <P>SOLUTION: The method for producing the insoluble granules of the recombinant protein using host cells is provided, comprising the step of transforming the host cells using a DNA encoding a protein differing from the above recombinant protein and forming insoluble granules when produced singly in the host cells and a second DNA encoding the recombinant protein, and the step of culturing the transformed host cells to produce the recombinant protein and the latter protein differing from the recombinant protein, respectively. Using this method, protein with low stability in host cells or protein affecting the proliferation or survival of host cells can be stably mass-produced. Furthermore, this method enables a recombinant protein of higher purity to be prepared more easily because of no need of expressing a desired protein as a fused protein with another protein, therefore because of no need of chemically and/or enzymatically cleaving a fused protein in the step of retrieving the aimed recombinant protein. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a method for producing an insoluble granule of a recombinant protein in a host cell, and a method for producing a recombinant protein partially including the method.

When a heterologous protein is expressed as a recombinant protein in a host cell, particularly when a heterologous protein derived from a higher eukaryotic cell is produced as a recombinant protein using a host cell that is a microbial cell, the recombinant protein is expressed in the host cell. It is empirically well known to form inactive and insoluble aggregates within. Such insoluble aggregates of proteins are generally called insoluble granules, inclusion bodies, inclusion bodies, or the like. In addition, since these terms are synonymous with each other, they are hereinafter referred to as insoluble granules in the present invention.

When the recombinant protein forms an insoluble granule in the host cell, in order to recover the active recombinant protein, the insoluble granule is removed from the host cell, and the insoluble granule is solubilized and the protein is refolded. Thus, it must be converted into an active recombinant protein. In this solubilization and refolding, insoluble granules are solubilized in a solution containing a denaturant typified by guanidine hydrochloride, and then the denaturant is gradually removed from the solution to allow the protein to take an active three-dimensional structure. This requires complicated operations and a long time. Therefore, various methods for avoiding the formation of insoluble granules have been studied in the production of recombinant proteins.

On the other hand, when insoluble granules of the recombinant protein are formed in the host cell, the degradation and modification of the recombinant protein by a modification enzyme such as a protease possessed by the host cell is suppressed, and as a result, the recovery rate of the recombinant protein is improved. The effect can be expected. It is also possible to avoid the influence of the protein on the growth of the host cell by producing a protein that may affect the growth of the host cell in the form of insoluble granules. Furthermore, since the isolation and purification of insoluble granules is relatively easy, it can be advantageous to produce recombinant proteins with insoluble granules in large-scale recombinant protein production, particularly industrial production. For these reasons, a method of actively forming insoluble granules of the recombinant protein in the host cell is also particularly important in industrial production of the recombinant protein.

However, whether or not the recombinant protein forms insoluble granules in the host cell
Depending on the intrinsic properties of the protein, it is generally difficult to predict whether a protein will form insoluble granules in a host cell. Therefore, a method for forming an insoluble granule of a recombinant protein artificially or forcibly in a host cell is expected and has been developed.

A representative example of the method is a method in which the protein to be produced is expressed as a fusion protein with a protein that is known to form insoluble granules in the host cell, and insoluble granules of the fusion protein are formed. (For example, Patent Document 1 and Patent Document 2). There is also a method for promoting the formation of insoluble granules of recombinant protein using mutant host cells that have lost the ability to produce certain proteins inherent in the host cell, which are involved in the correct folding of the protein in the host cell. It has been reported (Patent Document 3).

However, the method of forming insoluble granules of the fusion protein involves DNA encoding the fusion protein,
Designing a DNA that encodes a fusion protein via a linker peptide having a specific amino acid sequence that can be cleaved chemically or enzymatically, preparing it, and introducing it into a host cell Recombinant operation is required.

Furthermore, in order to isolate and purify the recombinant protein from the insoluble granules, in addition to solubilization of the insoluble granules and refolding of the fusion protein, the peptide bond of the fusion protein is chemically or enzymatically cleaved at a predetermined position. Thus, an operation for recovering and purifying the recombinant protein is required.

This chemical or enzymatic cleavage of the peptide bond itself is a complicated operation, which is disadvantageous in terms of workability and production cost when producing a recombinant protein on an industrial scale.

In addition, this method has a problem that if the specificity of the peptide bond cleavage site is low, peptide bonds other than the predetermined position in the fusion protein are cleaved, and the recovery rate of the recombinant protein is lowered. When such non-specific peptide bond cleavage occurs, the resulting peptide fragment becomes an impurity, making purification of the recombinant protein more difficult.

In addition, the method of using a mutant host cell that has lost the ability to produce certain proteins inherent in the host cell involved in the proper protein folding is limited to such a mutant host cell. In addition, it does not apply to all recombinant proteins.
JP-A-5-328992 JP-A-8-187094 Special table 2006-513710

The present invention provides a new method for forcibly forming insoluble granules of a recombinant protein in a host cell without producing the recombinant protein as a fusion protein.

As a result of numerous previous studies on the production of various recombinant proteins in host cells, a considerable number of proteins that form insoluble granules when produced alone in host cells have been reported. The present inventors, together with a desired protein, particularly a protein that does not form an insoluble granule when produced alone in the host cell, produces a protein that forms an insoluble granule when produced alone in the host cell. It was found that a protein that originally does not form or only slightly forms insoluble granules when produced in the host cell, forms insoluble granules, and the following inventions have been completed.

(1) A method for producing an insoluble granule of a recombinant protein using a host cell, wherein the protein is different from the recombinant protein and forms a insoluble granule when produced alone in the host cell. A step of transforming the host cell with a DNA encoding and a DNA encoding the recombinant protein, and culturing the transformed host cell to produce a protein different from the recombinant protein and the recombinant protein. A method for producing an insoluble granule of the recombinant protein, comprising a step of producing a protein that forms an insoluble granule when produced alone in the host cell.

(2) The method according to (1), wherein the host cell is a microbial cell.

(3) The method according to (2), wherein the microbial cell is an Escherichia bacterium.

(4) An expression vector incorporating a DNA encoding a recombinant protein and a DNA encoding a protein that is different from the recombinant protein and forms an insoluble granule when produced alone in the host cell. The production method according to (1), wherein a host cell is transformed using the expressed expression vector.

(5) DNA encoding a recombinant protein and a DN different from the recombinant protein and encoding a protein that forms insoluble granules when produced alone in the host cell
The production method according to (1), wherein a host cell is transformed with an expression vector incorporating A.

(6) DNA encoding a recombinant protein and a DN different from the recombinant protein and encoding a protein that forms insoluble granules when produced alone in the host cell
The method according to (4) or (5), wherein A is linked to different expression promoters.

(7) DNA encoding a recombinant protein and a DN that is different from the recombinant protein and encodes a protein that forms insoluble granules when produced alone in the host cell
The method according to (4) or (5), wherein A is linked to the same type of expression promoter.

(8) A transformed host cell, a DNA encoding a recombinant protein, and a DNA encoding a protein that is different from the recombinant protein and forms an insoluble granule when produced alone in the host cell A transformed host cell transformed by

(9) A method for producing a recombinant protein, comprising the following steps.

1) A DNA that encodes the recombinant protein and a protein that is different from the recombinant protein and that forms an insoluble granule when produced alone in the host cell
Preparing a host cell transformed with DNA;
2) causing the transformed host cell to produce insoluble granules of the recombinant cell;
3) recovering insoluble granules of the recombinant protein from the host cell,
4) a step of solubilizing the recovered insoluble granules of the recombinant protein in a solution containing a denaturing agent, and 5) a step of removing the denaturing agent from the solution and refolding the recombinant protein.

(10) The method according to (9), wherein the host cell is a microbial cell.

(11) The method according to (10), wherein the microbial cell is an Escherichia bacterium.

(12) An expression vector incorporating a DNA encoding a recombinant protein and a DNA encoding a protein that is different from the recombinant protein and forms an insoluble granule when produced alone in the host cell The production method according to (9), wherein a host cell is transformed with the expressed expression vector.

(13) DNA encoding a recombinant protein and a protein that is different from the recombinant protein and that forms an insoluble granule when produced alone in the host cell
The production method according to (9), wherein a host cell is transformed with an expression vector into which DNA is incorporated.

(14) DNA encoding a recombinant protein and a protein that is different from the recombinant protein and forms an insoluble granule when produced alone in the host cell
The method according to (12) or (13), wherein the DNAs are linked to different expression promoters.

(15) DNA encoding a recombinant protein and a protein different from the recombinant protein, which encodes a protein that forms insoluble granules when produced alone in the host cell
The method according to (12) or (13), wherein the DNA is linked to the same type of expression promoter.

According to the method of the present invention, a protein having low stability in a host cell, a protein capable of affecting the growth and survival of a host cell, and the like can be produced in large quantities and stably. In addition, it is not necessary to express the desired protein as a fusion protein with another protein, and therefore, it is not necessary to cleave the fusion protein chemically and / or enzymatically in the recovery process of the recombinant protein. Proteins can be prepared more easily.

One aspect of the present invention is a method for producing an insoluble granule of a recombinant protein using a host cell, wherein the insoluble granule is a protein that is different from the recombinant protein and is produced alone in the host cell. What is the step of transforming the host cell with DNA encoding the protein forming the DNA and the DNA encoding the recombinant protein, and the recombinant protein and the recombinant protein by culturing the transformed host cell A method for producing an insoluble granule of the recombinant protein comprising the steps of producing different proteins, each of which produces an insoluble granule when produced alone in the host cell.

The method of the present invention is a method suitable for producing a protein that does not form an insoluble granule or only a small amount when it is expressed alone in a host cell as a recombinant protein. In particular, the amount of expression in the host cell is affected by the effects of feedback control, etc., because the protein is prone to degradation and / or modification by the protease etc. possessed by the host cell by not forming insoluble granules. This method is particularly suitable for producing recombinant proteins that are difficult to enhance, or that are produced as active forms in the host cell, and that adversely affect the growth or viability of the host cell. is there.

Some of these proteins include ABF-2 (AntiBacterial Factor-2), ASABF (Ascaris
Antibacterial proteins such as Suum AntiBacterial Factor, FMBP-1 (Fibroin modulator-binding p
rotein-1) and the like. However, any protein other than those described above can be used as an object of the present invention as long as it is determined that production as an insoluble granule is advantageous.

The type of host cell used in the method of the present invention is not particularly limited, but it is preferable to use a host cell, particularly a microbial cell, which is suitable for recombinant production of proteins, for which various expression systems have been established. For example, Escherichia bacteria, Bacillus bacteria, Pseudomonas bacteria, Sacch
Examples of suitable host cells include aromyces yeast, Pichia yeast, and Aspergillus filamentous fungi. In particular, the use of Escherichia bacteria, particularly Escherichia coli, is particularly preferred.

The protein that forms insoluble granules when produced alone in the host cell used in the method of the present invention is a protein different from the recombinant protein to be produced by the method of the present invention,
Proteins that form insoluble granules when produced alone in any of the host cells described above, most of which are heterologous proteins to the host cell. Hereinafter, the above-mentioned protein used in the present invention is referred to as “insoluble granule-inducing protein”.

In studies on the recombinant production of proteins so far, many proteins having the property of forming insoluble granules in host cells have been reported (for example, review by Misawa et al., Biopol
ymers, Refolding of therapeutic proteins produced in Echerichia coli as inclusio
n bodies, 1999, Vol. 51, No. 4, pp. 297-307). In the present invention, any of these can be used as an insoluble granule-inducing protein in the host cell. In addition, a protein newly confirmed to have the property of forming insoluble granules when produced alone in a host cell can also be used as the insoluble granule-inducing protein referred to in the present invention. Furthermore, for example, a fusion protein designed to form an insoluble granule as described in Patent Document 1 or 2, which is a protein that forms an insoluble granule when produced alone in a host cell. Can be used as an insoluble granule-inducing protein.

Insoluble granule-inducing proteins that can be used when Eschericia bacteria are selected as host cells include α-lactalbumin, lysozyme, EGF, growth hormone, and interleukin-2.
In addition, interleukin 6, fusion proteins prepared by the methods described in Patent Documents 1 and 2, and other proteins specifically described in the review of Misawa et al. Can be specifically exemplified. Also
Examples of insoluble granule-inducing proteins when microbial cells other than Eschericia are selected as the host include proinsulin (Cousens L. et al., Ge
ne, 1987, Vol. 61, No. 3, pp. 265-275), prochymosin in the case of Bacillus bacteria (
Parente D. et al., FEMS Microbial Lett., 1991, Vol. 15, 61 (2-3): 243-9). In this case, referring to whether the recombinant protein to be produced by the method of the present invention has a positive or negative charge in the environment in the host cell, a protein having a charge opposite to the charge is used as an insoluble granule-inducing protein. It is preferable to select.

The method of the present invention is a method comprising the step of transforming the host cell using DNA encoding an insoluble granule-inducing protein and DNA encoding a recombinant protein.

The DNA used in this step is a DNA having an ORF sequence encoding each amino acid sequence of an insoluble granule-inducing protein or a recombinant protein and a functional promoter sequence that can be produced in a host cell, and other terminators A base sequence suitable for expression in a host cell such as a sequence may be included. Each protein may have a tag sequence or the like useful for purification of a protein such as His-tag.

Such DNA can be easily prepared by a person skilled in the art using a general genetic recombination technique used in producing a protein recombinantly. For example, an expression vector having an appropriate functional promoter sequence capable of expressing a protein in a host cell is incorporated with an ORF encoding an insoluble granule-inducing protein or a recombinant protein, and a recombinant vector D
NA can be created, but of course not limited to this method.

The DNA encoding the insoluble granule-inducing protein of the present invention and the DNA encoding the recombinant protein may be separate expression vector DNAs, or insoluble granules unless the fusion protein consisting of both proteins is encoded. It may be DNA having an ORF encoding the inducing protein and an ORF encoding the recombinant protein in one expression vector.

In addition, DNA encoding insoluble granule-inducing protein and DNA encoding recombinant protein are:
Each protein may be expressed under the control of an expression promoter unique to each protein, but an ORF encoding an insoluble granule-inducing protein under the control of a functional promoter capable of artificially inducing expression
It is preferable that the ORF encoding the recombinant protein is linked and expressed by a functional promoter capable of induction. In this case, the ORF encoding the insoluble granule-inducing protein and the ORF encoding the recombinant protein may be expressed by different functional promoters or by the same functional promoter. It is preferably expressed by the same type of functional promoter in that it can be performed by operation. When expressed by the same type of functional promoter, the same type of functional promoter may be placed upstream of the ORF encoding the insoluble granule-inducing protein and the ORF encoding the recombinant protein. An ORF encoding an insoluble granule-inducing protein and an ORF encoding a recombinant protein may be expressed in series downstream of the promoter using so-called read-through of RNA polymerase.

The above-mentioned functional promoter capable of inducing artificial expression is, for example, a functional promoter that can be induced by adding IPTG, lactose or other compounds to the medium, medium composition, culture temperature, medium pH and other culture conditions. It may be a functional promoter that can be induced by changing, a functional promoter that can be induced by applying a physical stimulus such as light or electricity, or any other functional promoter.

Non-limiting examples of such functional promoters include T7 available in Escherichia bacteria.
Promoter, lac promoter, trp promoter, λPL promoter, etc. Alkaline protease-derived promoter, neutral protease-derived promoter, α-amylase-derived promoter, GAL1 promoter, GAL10 promoter, Pichia genus available in Saccharomyces yeast Examples include, but are not limited to, AOX1 promoter that can be used in yeast.

The method for transforming host cells using the above DNA is a suitable method that can be used for each host cell from the calcium phosphate method, electroporation method, PEG method, and other methods without requiring any particular limitation or ingenuity. Can be selected. A host cell transformed in this way is also an embodiment of the present invention.

Another step of the present invention is a step of culturing transformed host cells to produce a recombinant protein and an insoluble granule-inducing protein, respectively.

The transformed host cells may be cultured by setting various culture conditions such as medium composition, temperature, pH, oxygen concentration and the like to conditions suitable for the growth of the host cells. Such conditions are known in each host cell and do not require trial and error for those skilled in the art. In addition, for transformed host cells grown under such culture conditions, the expression of DNA encoding the recombinant protein and the insoluble granule-inducing protein under the control of the functional promoter is expressed for each functional promoter. Recombinant protein and insoluble granule-inducing protein can be produced by induction by a predetermined method.

By the above method, insoluble granules of the recombinant protein can be produced in the transformed host cell.

Another embodiment of the present invention is a method for producing a recombinant protein comprising the following steps: 1) preparing a host cell transformed with a DNA encoding the recombinant protein and a DNA encoding an insoluble granule-inducing protein. Process,
2) causing the transformed host cell to produce insoluble granules of the recombinant cell;
3) recovering insoluble granules of the recombinant protein from the host cell,
4) a step of solubilizing the recovered insoluble granules of the recombinant protein in a solution containing a denaturing agent, and 5) a step of removing the denaturing agent from the solution and refolding the recombinant protein.

The above steps 1) and 2) correspond to the method for producing the insoluble granules of the recombinant protein of the present invention described in detail above. Steps 3) to 5) excluding these may be carried out by using a general method until the insoluble granules of the protein are recovered from the host cells to obtain the active protein, and no special operation is required.

In step 3), insoluble granules are recovered by destroying host cells containing insoluble granules using techniques such as sonication, crushing, freezing and thawing, and lytic enzyme treatment. This is a step of separating and recovering from the components. The method for disrupting the cells and the conditions for centrifugation can be performed by employing methods or conditions widely known by those skilled in the art.

Step 4) is a step of solubilizing the insoluble granules by placing the insoluble granules in a suitable solution containing a denaturant capable of dissolving the insoluble granules. In the present invention, any conventionally known modifier can be used. Non-limiting examples include guanidine hydrochloride, potassium thiocyanate, urea, sodium dodecyl sulfate (SDS), Triton X-100, and the like. In general, a buffer aqueous solution containing these denaturants at a high concentration (eg, about 4 to 7 M) is prepared
The insoluble granules of the recovered recombinant protein may be added to this aqueous solution, and the insoluble granules may be solubilized while incubating at low or room temperature for several hours to several days.

Step 5) is a step of refolding the recombinant protein by removing the denaturant from the buffered aqueous solution containing the denaturant and the solubilized recombinant protein. Detailed conditions in this step may be in accordance with general conditions for refolding the protein solubilized by the denaturant. Usually, the recombinant protein can be refolded by gradually decreasing the concentration of the denaturing agent in the buffer solution by dialysis for several hours to several days.

The active recombinant protein recovered and purified through the above operation is a protein that is not in the form of a fusion protein with another protein, particularly an insoluble granule-inducing protein. Therefore, the present invention does not require the operations of breaking the peptide bond at a predetermined position of the fusion protein and further recovering the recombinant protein, which are required in the conventional method of forming insoluble granules in the form of the fusion protein. In addition, a recombinant protein can be produced with high purity without contamination of partial fragments of the fusion protein caused by cleavage of the peptide bond and contamination of peptide fragments caused by non-specific cleavage of the peptide bond.

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.

<Example 1>
1) Antimicrobial peptide ABF-2 (AntiBacterial Factor-2, GenBank En derived from C. elegans)
try: AB029810) and the antibacterial peptide ASABF derived from the nematode Ascaris.suum (Ascaris Suum An
tiBacterial Factor, GenBank Entry: D83529), designed and synthesized so that the recognition sequences of restriction enzymes NdeI and XhoI are introduced into the 5 'and 3' ends of the amplified DNA fragment, respectively, for each ORF excluding the signal sequence PCR reaction was performed using the primer DNA to prepare an amplified DNA fragment. In addition, silkworm (Bombix mori) DNA-binding protein FMBP-1 (Fibroin modulator-binding pro
tein-1, GenBank Entry: AB163434) and the 5 'end of the amplified DNA fragment and 3
'An amplified DNA fragment was prepared by performing a PCR reaction using a primer DNA designed and synthesized so that the recognition sequences of restriction enzymes AseI and XhoI were introduced at the ends.

Each of the amplified DNA fragments obtained as described above was recombined into a commercially available expression vector pET-22b opened with restriction enzymes NdeI and XhoI, and pET / ABF-2 / HT, pET / ASABF / HT and pET / FMBP / HT was obtained. The primer DNA used in the above operation is a histidine tag derived from pET-22b (His-tag, H
T) is designed to be added to the C-terminus of the recombinant protein located upstream, so pET / ABF-2 / HT, pET / ASABF / HT and pET / FMBP / HT are derived from pET-22b ABF-2 / HT, ASABF / HT and FMBP / HT, which are recombinant proteins with HT added to the C-terminus, are expressed.

2) For ORF excluding the signal sequence of human lactalbumin (HLA, Human α-lactalbmin, GenBank Entry: HSLACTG), the restriction enzyme NdeI is present at the 5 ′ end and 3 ′ end of the amplified DNA fragment, respectively.
A PCR reaction was carried out using primer DNA designed and synthesized so that the recognition sequence of XhoI was introduced, and an amplified DNA fragment was prepared. This was recombined with a commercially available expression vector pCOLADuet-1 (Novagen) that was opened with restriction enzymes NdeI and XhoI to obtain an expression vector pCOLA / HLA.

3) Transformants (TF / ABF-2 / HT, TF / ASABF) expressing the respective antibacterial peptides and HLA independently by introducing the vectors prepared in 1) and 2) above into E. coli BL21 (DE3).
/ HT, TF / FMBP / HT). Moreover, pCOLA / HLA prepared in 2) is introduced into Escherichia coli BL21 (DE3) to obtain a transformant. Further, the vector prepared in 1) is further introduced into this transformant, and HLA and antibacterial peptide or DNA are introduced. Transformant capable of expressing binding protein (TF / ABF-2)
/ HT / HLA, TF / ASABF / HT / HLA, TF / FMBP / HT / HLA). E. coli transformed with pET-22b alone (Control 1) and E. coli transformed with pCOLADuet-1 alone (Control 2)
Prepared.

4) For each transformant obtained in 3), TF / ABF-2 / HT, TF / ASABF / HT, TF / FMBP / HT and Contro
l For LB medium containing ampicillin, TF / ABF-2 / HT / HLA, TF / ASABF / HT / HLA, TF / FMBP
For HT / HLA and LB medium containing ampicillin and kanamycin for HT / HLA, and LB medium containing kanamycin for Control2, each was cultured at 37 ° C until OD = 0.8 (600 nm), and then IPTG was brought to a final concentration of 1 mM. In addition, after further culturing for 4 hours, the cells were collected by centrifugation.

5) The cells recovered in 4) were suspended in 0.6 ml of 20 mM Tris-HCL buffer, subjected to ultrasonic treatment and centrifugation for 10 seconds at an output of 10 W, and insoluble granules were collected as a precipitate. Precipitate was run in SDS-containing electrophoresis sample buffer (100 mM Tris-HCl pH6.8, 25% glycerol, 1% SDS, 0.02% Coomasieie G
-250, 2% β-mercaptoethanol) and heated at 98 ° C. for 10 minutes. For heated samples, Tricine-PAGE using 16.5% polyacrylamide gel (Schagger H et al., Anal. Bioch
em., 1987, volume 166, number 2, pages 368-379).

When the gel after electrophoresis was stained with CBB, TF / ABF-2 / HT, TF / ASABF / HT, TF / FMBP
In HT / HT, there was almost no characteristic band compared to control1 and Control2, but in TF / ABF-2 / HT / HLA, TF / ASABF / HT / HLA, TF / FMBP / HT / HLA, Control1 As a characteristic band for Control2 and Control2, respectively, protein bands corresponding to each antibacterial peptide and
Bands corresponding to HLA were observed (FIGS. 1a-1c).

In addition, the gel after electrophoresis is stained using a kit (Invirtogen) that can stain only the protein having His-tag instead of CBB, and using a UV transilluminator.
In addition to selective fluorescence detection of proteins with His-tags (Figures 1a to 1c), images of the gel after staining were captured using a CCD camera device, and protein analysis was performed using analysis software (CS Analyzer 2.0). Quantification was performed. As a result, TF / ABF-2 / HT / HLA, TF / ASABF / HT / HLA and TF / ABF-2 / HT, TF / ASABF / HT, and TF / ASABF / HT / HT TF / FMBP / HT / H
In LA, at least 8-fold, 6-fold and 3-fold increase in the expression level was confirmed.

<Example 2>
1) For the ORF excluding the signal sequence of the antimicrobial peptide ABF-2 derived from the nematode C. elegans used in Example 1, the restriction enzymes NdeI and XhoI were respectively present at the 5 ′ end and 3 ′ end of the amplified DNA fragment. A PCR reaction was performed using a primer DNA designed and synthesized so that a recognition sequence was introduced, and an amplified DNA fragment was prepared. This amplified DNA fragment was recombined with a commercially available expression vector pET-22b opened with restriction enzymes NdeI and XhoI to obtain vector pET / ABF-2 that produces ABF-2 without His-tag.
The primer DNA used in the above operation is designed so that a stop codon is inserted upstream of the HT derived from pET-22b. Therefore, pET / ABF-2 is an ABF-2 that has no HT at the C-terminus. To express.

2) For ORF excluding the signal sequence of human lysozyme (HLZ, Human lysozyme, GenBank Entry: HUMLSZA), recognition sequences of restriction enzymes NdeI and XhoI are introduced at the 5 'end and 3' end of the amplified DNA fragment, respectively. PCR reaction is performed using primer DNA designed and synthesized as described above, and amplified DN
A fragment was prepared. This is a commercial expression vector pCOLADu that has been opened with restriction enzymes NdeI and XhoI.
The expression vector pCOLA / HLZ was obtained by recombination with et-1 (Novagen).

3) The vectors prepared in 1) and 2) above and pCOLA / HLA prepared in 2) of Example 1 are introduced into Escherichia coli BL21 (DE3) to express ABF-2, HLA and HLZ, respectively. Transformants (TF / ABF-2, TF / HLA, TF / HLZ) were obtained. Moreover, pCOLA / HLA and pCOLA / HLZ prepared in 2) are introduced into E. coli BL21 (DE3) to obtain transformants, and pE is further added to the transformants.
Transformant capable of expressing both HLA or HLZ and ABF-2 by introducing T / ABF-2 (TF / ABF-2 / HLA,
TF / ABF-2 / HLZ) was obtained. E. coli transformed with pET-22b alone (Control1) and p
E. coli transformed with COLADuet-1 alone (Control 2) was prepared. The same operation as 4) 5) of Example 1 was performed, and the gel after Tricine-PAGE was stained with CBB (FIG. 2).

As a result, in TF / ABF-2 / HLA and TF / ABF-2 / HLZ, in addition to the bands corresponding to HLA and HLZ, characteristic bands corresponding to ABF-2 were observed. In addition, the expression level of ABF-2 in TF / ABF-2 / HLA and TF / ABF-2 / HLZ was significantly increased compared to the expression level of ABF-2 in TF / ABF-2.

<Example 3>
Transformant TF / ABF-2 / HLA prepared in Example 2 (host cell expressing both HLA and ABF-2)
OD = 0.8 (600 nm) at 37 ° C with 250 ml of LB medium containing ampicillin and kanamycin
Then, IPTG was added to a final concentration of 1 mM, and the culture was further continued for 4 hours, and then the cells were collected by centrifugation.

The collected cells were suspended in 25 ml of 20 mM Tris-HCL buffer, subjected to ultrasonic treatment and centrifugation for 180 minutes at an output of 180 W, and insoluble granules were collected as a precipitate. The precipitate was washed twice with 10 ml of an aqueous solution containing 0.5% Triton-X100, then suspended in 20 mM Tris-HCl pH 8.0 containing 8 M urea, 3 mM EDTA, and 200 mM β-mercaptoethanol, and incubated at 37 ° C. for 3 hours.

After incubation, the supernatant collected after centrifugation at 9400 xg for 30 minutes was added to the equilibration buffer (20 mM Tris-HCl p containing 6 M urea, 3 mM EDTA, 3 mM β-mercaptoethanol).
The column was packed with a cation exchange resin (SP-sepharose) equilibrated with H8.0). After washing the column with the equilibration buffer, linear gradient elution of 0 to 1 M NaCl based on the equilibration buffer was performed to recover ABF-2 from the column.

Dialyze the collected ABF-2 fraction using dialysis buffer (2 mM reduced glutathione, 0.2 mM).
This was repeated using 20 mM Tris-HCl pH 8.0) containing oxidized glutathione. Each time the urea content was reduced, a part of the sample was collected and subjected to Tricine-PAGE (FIG. 3). By this operation, ABF-2 can be produced in large quantities.

<Example 4>
1) Recognition sequences of restriction enzymes NdeI and XhoI at the 5 'and 3' ends of the amplified DNA fragment against ORF encoding PHS001 (hypothetical protein, Uniprot entry: O74079), a protein of unknown function derived from P. horikoshii A PCR reaction was performed using primer DNA designed and synthesized so that was introduced, and an amplified DNA fragment was prepared.

  The amplified DNA fragment obtained as described above was recombined with a commercially available expression vector pET-22b opened with restriction enzymes NdeI and XhoI to obtain pET / PHS001 / HT. The primer DNA used in the above operation is designed so that a histidine tag (His-tag, HT) derived from pET-22b is added to the C-terminus of the recombinant protein located upstream thereof. PHS001 / HT expresses PHS001 / HT, which is a recombinant protein in which HT derived from pET-22b is added to the C-terminus.

  2) Transformants that individually introduce PHS001 / HT and HLZ by introducing the vector prepared in 1) above and pCOLA / HLZ prepared in 2) of Example 2 into E. coli BL21 (DE3), respectively. TF / PHS001 / HT, TF / HLZ) were obtained. In addition, transformants can be obtained by introducing pCOLA / HLZ prepared in 2) into E. coli BL21 (DE3), and pET / PHS001 / HT can be further introduced into this transformant to express both HLZ and PHS001 / HT. A transformant (TF / PHS001 / HT / HLZ) was obtained. In addition, E. coli transformed with only pET-22b (Control 1) and E. coli transformed with only pCOLADuet-1 (Control 2) were prepared. The gel after Tricine-PAGE obtained by performing the same operation as 4) 5) of Example 1 was stained with CBB (upper figure 4).

  As a result, a characteristic band (arrow →) corresponding to PHS001 was observed in addition to the band corresponding to HLZ from TF / PHS001 / HT / HLZ (upper right lane in FIG. 4). In addition, the expression level of PHS001 in TF / PHS001 / HT / HLZ was significantly increased compared to the expression level of PHS001 in TF / PHS001 / HT (upper left in FIG. 4).

  In addition, the gel after electrophoresis is stained with a kit (Invitrogen) that can stain only a protein having a His-tag instead of CBB, and a protein having a His-tag using a UV transilluminator. In addition to selective fluorescence detection (bottom of FIG. 4), an image of the gel after staining was taken using a CCD camera device, and protein was quantified using analysis software (CS Analyzer 2.0). As a result, even when the background noise in TF / PHS001 / HT was taken into account, an increase in the expression level of TF / PHS001 / HT / HLZ was confirmed by at least 3-fold.

CBB staining of SDS-PAGE performed on cell extracts of transformants TF / ABF-2 / HT / HLA (lane right) and TF / ABF-2 / HT (lane left) prepared in Example 1 (top) The results of HT staining (bottom) are also shown. The arrow represents ABF-2. SDS-PAGE CBB staining (upper) and HT staining (upper) of cell extracts of transformants TF / ASABF / HT / HLA (lane right) and TF / ASABF / HT (lane left) prepared in Example 1 ( The results of (lower) are shown. The arrow represents ASABF. SDS-PAGE CBB staining (upper) and HT staining (upper) of cell extracts of transformants TF / FMBP / HT / HLA (lane right) and TF / FMBP / HT (lane left) prepared in Example 1 ( The results of (lower) are shown. The arrow represents FMBP-1. CBB staining of SDS-PAGE performed on cell extracts of transformants TF / ABF-2 (lane left), TF / ABF-2 / HLZ (lane center), and TF / ABF-2 / HLA (lane right) Results are shown. The purification result of ABF-2 co-expressed with human-α-lactalbumin is shown. From each lane, molecular weight markers, insoluble granules, supernatant obtained by solubilizing insoluble granules, cation exchange resin column passage, ABF-2 recovered from cation exchange resin (before dialysis), dialysis Each ABF-2 is shown below. SDS-PAGE CBB staining (upper) and HT staining (upper) of the cell extracts of the transformants TF / PHS001 / HT / HLZ (lane right) and TF / PHS001 / HT (lane left) prepared in Example 4 The results of (lower) are shown. The arrow represents PHS001.

Claims (15)

A method for producing an insoluble granule of a recombinant protein using a host cell, wherein the DNA encodes a protein that is different from the recombinant protein and forms an insoluble granule when produced alone in the host cell And a step of transforming the host cell using the DNA encoding the recombinant protein, and culturing the transformed host cell to produce the recombinant protein and a protein different from the recombinant protein, wherein the host cell A method for producing an insoluble granule of the recombinant protein comprising a step of producing a protein that forms an insoluble granule when produced alone in the protein. The method of claim 1, wherein the host cell is a microbial cell. The method according to claim 2, wherein the microbial cell is an Escherichia bacterium. An expression vector incorporating a DNA encoding a recombinant protein, and an expression incorporating a DNA encoding a protein that is different from the recombinant protein and forms an insoluble granule when produced alone in the host cell Transforming host cells with vectors,
The manufacturing method according to claim 1. Host cells using DNA encoding a recombinant protein and an expression vector incorporating a protein that is different from the recombinant protein and that forms a protein that forms an insoluble granule when produced alone in the host cell The production method according to claim 1, wherein DNA encoding a recombinant protein and DNA encoding a protein that is different from the recombinant protein and forms an insoluble granule when produced alone in the host cell,
The method according to claim 4 or 5, wherein each is linked to a different expression promoter. DNA encoding a recombinant protein and DNA encoding a protein that is different from the recombinant protein and forms an insoluble granule when produced alone in the host cell,
The method according to claim 4 or 5, wherein the method is linked to a homologous expression promoter. A transformed host cell transformed with DNA encoding a recombinant protein and DNA encoding a protein that is different from the recombinant protein and forms an insoluble granule when produced alone in the host cell Transformed host cell. A method for producing a recombinant protein, comprising the following steps.
1) A DNA that encodes the recombinant protein and a protein that is different from the recombinant protein and that forms an insoluble granule when produced alone in the host cell
Preparing a host cell transformed with DNA;
2) causing the transformed host cell to produce insoluble granules of the recombinant cell;
3) recovering insoluble granules of the recombinant protein from the host cell,
4) a step of solubilizing the recovered insoluble granules of the recombinant protein in a solution containing a denaturing agent, and 5) a step of removing the denaturing agent from the solution and refolding the recombinant protein. The method of claim 9, wherein the host cell is a microbial cell. The method according to claim 10, wherein the microbial cell is an Escherichia bacterium. An expression vector incorporating a DNA encoding a recombinant protein, and an expression incorporating a DNA encoding a protein that is different from the recombinant protein and forms an insoluble granule when produced alone in the host cell Transforming host cells with vectors,
The manufacturing method according to claim 9. Host cells using DNA encoding a recombinant protein and an expression vector incorporating a protein that is different from the recombinant protein and that forms a protein that forms an insoluble granule when produced alone in the host cell The production method according to claim 9, wherein DNA encoding a recombinant protein and DNA encoding a protein that is different from the recombinant protein and forms an insoluble granule when produced alone in the host cell,
The method according to claim 12 or 13, wherein each is linked to a different expression promoter. DNA encoding a recombinant protein and DNA encoding a protein that is different from the recombinant protein and forms an insoluble granule when produced alone in the host cell,
The method according to claim 12 or 13, wherein the method is linked to a homologous expression promoter.