JPH0575760B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides dihydrofolate reductase (hereinafter also referred to as DHFR) expressed in an insolubilized state in Escherichia coli by genetic recombination technology, which is present at the amino terminal side. Methods for isolating and purifying fusion proteins, such as expression results in E. coli of a fusion gene created by ligating a heterologous gene to the 3' end of a modified E. coli-derived DHFR gene with the open reading frame of the genetic code aligned. Among the obtained recombinant fusion proteins, the present invention relates to a method for solubilizing a fusion protein that is expressed and accumulated as an insoluble protein in Escherichia coli cells and highly purifying the solubilized fusion protein. The method for separating and purifying a fusion protein of the present invention is suitable for use in the fields of microbial industry, fermentation industry, and pharmaceutical manufacturing.

[Conventional techniques and problems] When trying to produce polypeptides with small molecular weights or proteins that do not have a stable higher-order structure within E. coli cells, it is not enough to efficiently express the gene that encodes the protein itself. Degradation occurs at the same time as it is produced by the action of proteolytic enzymes existing in the body, making it impossible to accumulate and produce a large amount of the target polypeptide or protein within the bacterial body. To avoid this, proteins are expressed and produced as fusion proteins with proteins that are stably expressed and accumulated in E. coli.
The present inventors have already identified proteins derived from Bacillus subtilis and Escherichia coli as proteins used for stable production of unstable polypeptides or proteins.
Developing a method to utilize DHFR (Japanese Patent Application Laid-open No. 1983
-87981, JP-A-63-102696, JP-A-63-267276,
Japanese Patent Application Publication No. 63-245679, Japanese Patent Application Publication No. 63-245680, Patent Application No. 1986
-085406, Japanese Patent Application 1986-258597, Patent Application 1987-
302154, patent application 1983-302155, patent application 1982-302156, etc.). Proteins other than DHFR include β-galactosidase (K. Itakura, et al., Science,
vol.198, 1056 (1977)), tryptophan synthase (K.Nagahari, et al., Agric.Biol.Chem.,
vol.51, 845 (1987)), human growth hormone (M.
Ikehara, et al., Proc. Natl. Acad. Sci. USA,
vol. 83, 4695 (1986)) are known.

Stable production of heterologous polypeptides or proteins using DHFR is based on the fact that fusion proteins with DHFR are accumulated and produced within E. coli cells as soluble proteins with DHFR enzyme activity. This method was superior in terms of separation and purification of fusion proteins.

However, when the polypeptide or protein to be fused with DHFR is changed in various ways, certain polypeptides or proteins (herein, exemplified with respect to growth hormone-releasing factor derivatives and human prolactin) are present at the carboxy-terminal side. It became clear that the DHFR fusion protein accumulates as an insolubilized protein, and this was considered to be a major problem in the separation and purification of the target fusion protein.

Many examples are known regarding insolubilization when foreign proteins are expressed in E. coli (F.
A.O.Marston, Biochem.J.vol.240, 1 (1986)),
Regarding separation and purification of a target protein, a method is used in which an insolubilized protein is solubilized with a protein denaturing agent such as urea, and then purified in the presence of the denaturing agent. However, purification methods in the presence of denaturing agents are limited in their applicability, and in the presence of denaturing agents, the physiological activity of the target protein is inactivated, causing major problems in identifying and detecting the target protein during purification. ing.

[Problems to be Solved by the Invention] Under these circumstances, the present invention aims to improve DHFR expressed and accumulated in Escherichia coli as an insolubilized protein.
This was developed for the purpose of providing a method for solubilizing and highly purifying fusion proteins.

[Means for Solving the Problem] As a result of intensive research to develop a method for solubilizing and high-level purification of the DHFR fusion protein, the present inventors have discovered that insoluble proteins can be solubilized with acetic acid or a denaturing agent. Further, the inventors have discovered that the objective can be achieved by subjecting the compound to a specific chromatographic treatment after diluting it with a buffer as the case may be, and based on this finding, the present invention has been accomplished.

That is, the present invention provides a method for isolating and purifying a fusion protein accumulated as an insoluble protein in E. coli by expressing a gene encoding a fusion protein in which a heterologous protein is bound to the carboxy terminus of E. coli dihydrofolate reductase. After disrupting E. coli cells that expressed and produced insoluble proteins, the precipitated fraction obtained by centrifugation was solubilized with acetic acid, and the solubilized fusion protein was highly purified by reversed-phase high-performance liquid chromatography. The fusion protein is accumulated as an insoluble protein in the E. coli body by using a characteristic fusion protein separation and purification method and by expressing a gene encoding a fusion protein in which a heterologous protein is bound to the carboxy-terminal side of E. coli dihydrofolate reductase. In a protein separation and purification method, after disrupting E. coli cells that have expressed and produced an insoluble protein, the precipitated fraction obtained by centrifugation is solubilized with a protein denaturant, and the solubilized fusion protein is diluted with a buffer solution. The present invention provides a method for separating and purifying a fusion protein, which is characterized by activating dihydrofolate reductase and highly purifying the fusion protein by mesotrixate-linked affinity chromatography using dihydrofolate reductase activity as a guideline. As a bacterial cell that expresses and produces DHFR fusion protein as an insoluble protein,
Escherichia coli containing the recombinant plasmid pSG1-12, which the present inventors have already invented
Deposited as FERMBP-2149, patent application No. 63-293389
), Escherichia coli containing the recombinant plasmid pGRF44-22 (deposited as FERMBP-2152 to the FIKEN, described in patent application 1983-294203), E. coli containing the recombinant plasmid pGRFM44-6 (deposited at the FEI
Deposited as FERMBP-2151, patent application No. 63-294204
), Escherichia coli containing recombinant plasmid pPRLh4 (deposited with FIKEN as FERMBP-2153,
(described in Japanese Patent Application No. 63-296913), but the present invention is not limited to these bacterial cells.

The present invention comprises a method for culturing bacterial cells, a method for separating insolubilized proteins from bacterial cells, a method for solubilizing insolubilized proteins, and a method for highly purifying solubilized proteins. The configuration contents will be explained in order below.

Method for culturing bacterial cells When the DHFR fusion protein is expressed and accumulated as an insoluble protein, the ratio of the protein that accumulates in an insolubilized state and the protein that does not become insolubilized changes depending on the culture temperature. The rate of insolubilization increases as the culture temperature increases. Therefore, the culture temperature is preferably the highest temperature (usually 37°C to 42°C) at which the bacterial cells can grow. The percentage of insolubilized protein is 5000 after disrupting the cultured bacterial cells.
The precipitate and supernatant fractions were separated by centrifugation at 10,000 revolutions/min for 10 to 20 minutes, and after SDS-polyacrylamide electrophoresis (abbreviated as SDS-PAGE), this and the total cell protein were separated into Kumazi-Brilliant fractions. The degree of staining of the target protein band can be measured using a densitometer and compared based on the blue staining pattern.

Culture of bacterial cells that express and produce insolubilized protein of DHFR fusion protein (hereinafter abbreviated as insolubilized fusion protein) is carried out in YT+Ap medium (medium 11
It can be cultured in a liquid medium containing 5 g of NaCl, 5 g of yeast extract, 8 g of tryptone, and 50 mg of ampicillin sodium. Other media include ST+Ap medium (a liquid medium containing 2g of glucose, 1g of dipotassium phosphate, 5g of polypeptone, 5g of yeast extract, and 50mg of ampicillin sodium in medium 11). Any medium can be used as long as the body grows, but as far as we have investigated, YT+ is suitable for the production of DHFR fusion proteins.
Ap medium was optimal.

Escherichia coli containing the expression-producing insoluble protein is inoculated into a medium and cultured at 37°C until the late logarithmic growth phase or stationary phase. The cultured bacterial cells are collected by centrifugation at 5000 rpm. From the medium 11, bacterial cells with a wet weight of 2 to 5 g are obtained.

Bacterial collection and subsequent operations are performed at low temperatures (between 0 and 10°C, preferably 4°C) unless otherwise specified.

Isolation of insolubilized fusion protein from bacterial cells The bacterial cells obtained by culturing can be disrupted by any method that can crush the bacterial cells, such as a method using a French press, a sonication method, a method using glass beads, etc. It can be applied in various ways. Here, a method using a French press will be described, but the present invention is not limited to the method of crushing bacterial cells.

Suspend in buffer 1 (10 mM potassium phosphate buffer containing 0.1 mM sodium ethylenediaminetetraacetate, pH 7.0) twice the wet weight, and crush the cells using a French press. 5000 ml of bacterial cell disruption solution
Centrifuge at 10,000 rpm for 10 minutes to obtain a precipitate.
For the purpose of washing the obtained precipitate, it is suspended in Buffer 1 and centrifuged at 5,000 to 10,000 rpm for 10 minutes to obtain a precipitate (washing of the precipitate). This cleaning operation is carried out in 2 steps.
Repeat 3 times. The obtained protein fraction is called an insolubilized fraction.

Through this operation, the purity of the insolubilized fusion protein is approximately 50 to 90%.

Methods for solubilizing insolubilized fusion proteins Methods for solubilizing insolubilized proteins include:
() A method using acetic acid and () A method using a protein denaturant are effective.

() Method using acetic acid Dissolve the insolubilized fraction in an acetic acid aqueous solution of the same volume (ml) as the wet weight of the bacterial cells used in grams. Materials insoluble in acetic acid are removed by centrifugation. The obtained supernatant is referred to as the acetic acid insolubilized fraction. The concentration of the acetic acid aqueous solution used is effectively between 15 and 30%. This operation increases the purity of the target fusion protein to approximately
Increases to over 90%.

() Method using a protein denaturing agent As a protein denaturing agent, urea or chlorine guanidine is described, but it is a denaturing agent that can solubilize an insolubilized fusion protein and that has a side chain on the amino acid residue of the protein. Any protein denaturing agent can be used as long as it does not have an adverse effect such as modification of protein, and the present invention is not limited to the protein denaturing agent used.

The insolubilized fraction is dissolved in an aqueous urea solution or an aqueous guanidine hydrochloride solution in an amount equal to the wet weight in grams of the bacterial cells used. Substances insoluble in urea or guanidine hydrochloride are removed by centrifugation. The obtained supernatants are referred to as a urea solubilized fraction and a guanidine hydrochloride solubilized fraction, respectively. It is effective to use a concentration of urea of 4M or more, and a concentration of guanidine hydrochloride of 3M or more. This operation increases the purity of the target fusion protein to about 80% or more.

Advanced purification method for solubilized fusion protein () Fusion protein solubilized using acetic acid The acetic acid solubilized fraction was subjected to high performance liquid chromatography (hereinafter abbreviated as HPLC) using a reversed phase carrier.
Separate and purify. Silica gel carriers with octyl groups are effective as carriers for reversed phase systems.
Elution is performed by applying a concentration gradient of 15% to 50% acetonitrile in 0.1% trifluoroacetic acid (abbreviated as TFA), and the elution position can be determined by examining the absorption at 280 nm. Even under such conditions, the solubilized fusion protein partially has DHFR activity, and the target fusion protein can be confirmed in the elution fraction. The fusion protein of interest is eluted between 45-48% acetonitrile concentration. By this operation, the target fusion protein can be completely purified. Various HPLC devices are available for use in this operation. In the examples, a Shimadzu LC-4A type HPLC apparatus is used, but the present invention
It is not limited to devices. In addition, an Inertsil-ODS column manufactured by Gascro Kogyo is used as a reversed-phase carrier, but various silica gel carriers having octyl groups can be used, and therefore, the present invention is applicable to the carrier used. is not limited.

() Fusion protein solubilized using a protein denaturing agent Protein fraction solubilized using a denaturing agent
By diluting the protein 10 times or more with Buffer 1, the fusion protein solubilized in a denatured state can be reactivated. Buffer 1 is described as the buffer to be diluted, but buffers with buffering capacity within this range (phosphate buffer, Tris buffer, histidine buffer, Gud buffer) are recommended for pH 5 to 8. As far as we have investigated, effective reactivation has been achieved for liquids, etc.). Therefore, the present invention is not limited to the buffer used for dilution.

High-level purification of the target fusion protein reactivated by dilution of Buffer 1 is carried out using DHFR activity as a guideline.
This is achieved using infinity chromatography coupled with mesotrixate (hereinafter abbreviated as MTX). The MTX-bound agarose gel carrier used can be a commercially available product (for example, sold by Sigma).

A standard fusion protein solution reactivated by dilution of Buffer 1 is adsorbed onto an MTX-agarose affinity column equilibrated with Buffer 1 in advance. After adsorption, wash with buffer 1 containing 1M KCl. For washing, measure the absorbance of the eluate from the column at 280 nm, and continue to flow the same buffer until the absorbance becomes 0.1 or less. Enzyme elution was performed using 1M KCl and
10mM potassium phosphate buffer containing 3mM folic acid,
It is carried out using pH 9.0, and the eluate is fractionated in fixed amounts using a fraction collector. Measure the DHFR activity of the fractionated eluate, and collect the fractions containing enzyme activity. The obtained enzyme solution is dialyzed against buffer solution 1 three times. With this operation,
The fusion protein of interest can be completely purified.

Furthermore, if the dialysis is incomplete, the enzyme solution obtained by dialysis may contain folic acid, which may interfere with assays for protein content using absorbance at 280 nm. It will be done. To that end, here we describe a method of utilizing DEAE-Toyopearl column chromatography, but the use of this method does not limit the separation and high-level purification method of fusion proteins.

The dialyzed enzyme solution is adsorbed onto a DEAE-Toyopearl column equilibrated with buffer 1 in advance.
After adsorption, wash with buffer 1 containing 0.1M KCl. For washing, measure the absorbance of the eluate from the column at 280 nm, and continue to flow the same buffer until the absorbance becomes 0.01 or less. Elute the enzyme using buffer 1.
A linear concentration gradient of KC1 from 0.1M to 0.3M is used, and a fixed amount of the eluate is fractionated using a fraction collector. About the fractionated eluate
Measure absorbance at 280 nm and DHFR activity. Collect fractions with a constant value of enzyme activity/absorbance at 280 nm. By this operation, folic acid can be removed with good reproducibility.

DHFR enzyme activity was determined using the reaction solution (0.05mM dihydrofolic acid, 0.06mM NADPH, 12mM 2-mercaptoethanol, 50mM phosphate buffer (PH
7.0)) is carried out by taking a 1 ml cuvette, adding the enzyme solution to it, and measuring the change in absorbance at 340 nm over time at 30°C. One unit of enzyme is defined as the amount of enzyme required to reduce 1 micromole of dihydrofolate per minute under the above reaction conditions. This measurement can be easily performed using a spectrophotometer.

Reagents, devices, etc. used in the present invention may be commercially available products, except as specifically described. Furthermore, the various operations described herein can be easily and reproducibly performed by those skilled in the art. Note that the commercially available reagents used are required to be of special grade or higher quality.

Next, examples of the present invention will be shown.

Example 1 DHFR-Bovine Growth Hormone Releasing Factor Fragment Fusion Protein The DHFR-Bovine Growth Hormone Releasing Factor Fragment Fusion Protein was produced using the recombinant plasmid pSG1-
12 and is encrypted on the Microtechnology Institute deposit number
This is a fusion protein produced by E. coli FERMBP-2149 (hereinafter abbreviated as BP-2149 strain).

For the BP-2149 strain, when using YT+Ap medium,
At 37°C, 90% of the fusion protein becomes insoluble, and at 30°C, about 50% of the fusion protein becomes insoluble, but at 20°C, almost 100% becomes insoluble.
accumulates in the bacterial body as a soluble protein. Therefore, when culturing was performed at 37°C for 16 hours using YT+Ap medium 31, the culture was further performed at 42°C for 1 hour. After culturing, the bacterial cells were collected by centrifugation at 5000 rpm for 10 minutes, suspended in 300 ml of buffer 1, and centrifuged again at 5000 rpm for 10 minutes to collect the bacterial cells. As a result, bacterial cells with a wet weight of 11 g were obtained. Suspend the obtained bacterial cells in 22 ml of buffer 1, crush the bacterial cells using a French press, centrifuge the resulting bacterial cell suspension at 5000 rpm for 10 minutes, and collect the precipitate. Ta. The precipitate was white in color and was suspended in 30 ml of Buffer 1, and centrifuged again at 5000 rpm for 10 minutes to collect the precipitate.
This operation was repeated three times. The obtained precipitate was
Dissolve the insoluble part in 11 ml 15% acetic acid, 15000
The precipitate was removed by centrifugation at rotation/min for 15 minutes to obtain a supernatant (approximately 14 ml). The resulting supernatant was separated by reverse phase HPLC. HPLC 0.5ml of supernatant
using a device (Shimadzu LC-4A, inertsil-ODS column) from 15% to 50% in 0.1% trifluoroacetic acid.
% acetonitrile concentration gradient can be used for elution and separation. Eluates can be detected by measuring absorbance at 280 nm. A peak of the desired fusion protein was obtained 34 minutes after sample injection, and the peak fraction was separated. This peak fraction possessed DHFR activity, which was approximately 0.7 units/mg protein. After drying the separated eluate using an evaporator, a small amount of water was added and lyophilized to remove the solvent, yielding a fusion protein. Approximately 0.9 mg of the fusion protein was recovered by one HPLC operation (that is, by repeating this operation, 19.8 mg of the fusion protein could be separated). The obtained preparation was shown to be a homogeneous protein preparation by SDS-PAGE, and growth hormone-releasing factor peptide fragments were produced by bromcyan treatment, indicating that growth hormone-releasing factor peptide fragments were produced. It was useful as a raw material (patent pending).

Example 2 DHFR-Bovine Growth Hormone Releasing Factor Derivative Fusion Protein The DHFR-Bovine Growth Hormone Releasing Factor Derivative Fusion Protein was produced using recombinant plasmid pGRFM44-6.
It is encrypted on
This is a fusion protein produced by E. coli FERMBP-2151 (hereinafter abbreviated as BP-2151 strain).

For the BP-2151 strain, when using YT+Ap medium,
At 37°C, almost all of the fusion protein becomes insolubilized, but at 30°C, about 65% becomes insolubilized, and at 20°C, almost 100% accumulates in the bacterial body as soluble protein. Therefore, using YT+Ap medium 31,
Culture was performed at 37°C for 16 hours. After culturing, 5000 rotations/
Collect the bacterial cells by centrifugation for 10 minutes and remove the bacterial cells.
Suspend in 300 ml of buffer 1 and spin again at 5000 revolutions/min.
The cells were collected by centrifugation for 10 minutes. As a result, bacterial cells with a wet weight of 13 g were obtained. The obtained bacterial cells were suspended in 26 ml of buffer solution 1, and the bacterial cells were crushed using a French press. The resulting bacterial cell suspension was centrifuged at 5000 rpm for 10 minutes, and the precipitate was collected. . The precipitate was white, and was suspended in 30 ml of buffer 1, and centrifuged again at 5000 rpm for 10 minutes to collect the precipitate. This operation was repeated three times. The obtained precipitate was dissolved in 14 ml of buffer 1 containing 4M urea, and the insoluble portion was heated at 15,000 revolutions/min.
The precipitate was removed by centrifugation for 15 minutes to obtain a supernatant (approximately 14 ml). The supernatant was diluted by adding 10 times the volume (140 ml) of Buffer 1. In the diluted solution,
It contained 930 units of DHFR activity. Add to this 10 g of MTX pre-equilibrated with buffer 1.
- Agarose gel was added and left overnight with gentle stirring to allow the fusion protein to adsorb onto the MTX agarose gel. After filling the gel after this operation into a column and passing the supernatant through the column,
Washed with buffer 1 containing 1M KCl. For washing, the absorbance at 280 nm of the eluate from the column was measured, and the same buffer solution was continued to flow (approximately 150 ml) until the absorbance became 0.1 or less. Enzyme elution was performed in 10mM potassium phosphate buffer containing 1M KCl and 3mM folic acid, pH 9.0.
A fixed amount (approximately 5 ml) of the eluate was fractionated using a fraction collector. DHFR activity was measured for the fractionated eluate, and fractions containing enzyme activity were collected (approximately 25 ml). The obtained enzyme solution was dialyzed against buffer 1 three times. When the dialyzed sample was examined by SDS-PAGE, it was shown to be a homogeneous protein sample. This preparation has 502 units of DHFR activity (recovery rate 54%),
It also contained approximately 20 mg of fusion protein.

Example 3 DHFR-Prolactin Fusion Protein The DHFR-Prolactin fusion protein is encoded on the recombinant plasmid pPRLh4,
This is a fusion protein produced by Escherichia coli (hereinafter abbreviated as BP-2153 strain) with the FERMBP-2153 deposit number.

For the BP-2153 strain, when using YT+Ap medium,
Approximately 70% becomes insolubilized at 37°C, and at 30°C, approximately 90% or more accumulates in the bacterial body as soluble protein. Therefore, using YT+Ap medium 31, the cells were cultured at 37°C for 16 hours and then further cultured at 42°C for 2 hours. After culturing, the bacterial cells were collected by centrifugation at 5000 rpm for 10 minutes, suspended in 300 ml of buffer 1, and centrifuged again at 5000 rpm for 10 minutes to collect the bacterial cells. As a result, bacterial cells with a wet weight of 10 g were obtained. Suspend the obtained bacterial cells in 20 ml of buffer 1, disrupt the bacterial cells using a French press, centrifuge the resulting bacterial cell suspension at 5000 rpm for 10 minutes, and collect the precipitate. Ta. The precipitate is white. It is suspended in 30 ml of buffer solution 1, and the precipitate is rotated again at 5000 rpm.
Centrifugation was performed for 10 minutes and the precipitate was collected. This operation was repeated three times. 10ml of the obtained precipitate
Dissolved in buffer 1 containing 3M guanidine hydrochloride,
The insoluble portion was removed as a precipitate by centrifugation at 15,000 rpm for 15 minutes to obtain a supernatant (approx.
ml). The supernatant was diluted by adding 10 times the volume (100 ml) of Buffer 1. There are 680 units in the diluted solution.
DHFR activity was included. To this was added 10 g of MTX-agarose gel equilibrated with Buffer 1 in advance, and the mixture was allowed to stand overnight with gentle stirring to allow the fusion protein to be adsorbed onto the MTX agarose gel. The gel subjected to this operation was packed into a column, and the supernatant liquid was passed through the column, followed by washing with buffer 1 containing 1M KCl. For washing, the absorbance at 280 nm of the eluate from the column was measured, and the same buffer solution was continued to flow (approximately 150 ml) until the absorbance became 0.1 or less.
Enzyme elution contains 1M KCl and 3mM folic acid
It was carried out using 10 mM potassium phosphate buffer, pH 9.0, and a fixed amount (about 5 ml) of the eluate was fractionated using a fraction collector. DHFR activity was measured for the fractionated eluate, and fractions containing enzyme activity were collected (approximately 25 ml). The obtained enzyme solution was
Dialysis was performed three times against buffer 1. When the dialyzed sample was examined by SDS-PAGE, it was shown to be a homogeneous protein sample. This specimen is
It contained 450 units of DHFR activity (66% recovery) and approximately 23 mg of fusion protein.

[Effects of the Invention] According to the present invention, not only is it possible to solubilize a fusion protein with DHFR that has become insoluble, but also DHFR in the amino terminal region of the fusion protein can be solubilized.
Activation of the enzyme moiety was achieved, making it easy to highly purify and homogenize the solubilized protein.

Claims (1)

[Claims] 1. Separation and purification of a fusion protein accumulated as an insoluble protein in Escherichia coli by expressing a gene encoding a fusion protein in which a heterologous protein is bound to the carboxy-terminal side of dihydrofolate reductase of Escherichia coli. In the method, after disrupting Escherichia coli cells expressing and producing an insoluble protein, the precipitated fraction obtained by centrifugation is solubilized with acetic acid, and the solubilized fusion protein is highly purified by reversed-phase high performance liquid chromatography. A method for separating and purifying a fusion protein, characterized by: 2. In a method for separating and purifying fusion proteins accumulated as insoluble proteins in E. coli, insoluble proteins can be isolated by expressing a gene encoding a fusion protein in which a heterologous protein is bound to the carboxy terminus of E. coli dihydrofolate reductase. After disrupting the expressed and produced E. coli cells, the precipitated fraction obtained by centrifugation is solubilized with a protein denaturant, and the solubilized fusion protein is diluted with a buffer to activate dihydrofolate reductase, and dihydrofolate reductase is activated. A method for separating and purifying a fusion protein, which comprises highly purifying the fusion protein by mesotrixate-linked affinity chromatography using folate reductase activity as a guide.