WO2024057361A1 - Solubilized protein production method - Google Patents

Abstract

The present invention develops a method involving: solubilizing, without using a modifying agent, an insoluble protein included in an inclusion body, which is aggregate of a recombinant protein expressed in a microorganism; separating same as a solubilized protein without using a column or the like; and purifying same.　Provided is a method for producing a solubilized target protein from an inclusion body by mixing an insoluble fraction and a lithium solution, and subsequently separating a soluble fraction including the solubilized target protein from the insoluble fraction.

Description

Solubilized protein production method

The present invention relates to a method for producing a solubilized protein from an insoluble protein in an inclusion body, and a method for separating and purifying the solubilized protein from the inclusion body according to its molecular weight.

When a desired recombinant protein derived from a eukaryote is expressed in E. coli using gene recombination technology, the recombinant protein often forms an abnormal three-dimensional structure within the cell and becomes an insoluble protein, causing them to interact with each other. They aggregate to form insoluble protein aggregates called inclusion bodies.

Proteins in inclusion bodies are generally not easily degraded by proteases, and can be recovered as aggregates containing almost no other contaminants. On the other hand, proteins in inclusion bodies cannot be purified unless they are solubilized, and biological activity is often lost in inclusion bodies, so they often require refolding after solubilization. .

When purifying insoluble proteins contained in inclusion bodies, the conventional method was to solubilize the insoluble proteins by adding a denaturing agent such as urea or guanidine, and then purify them by column (Patent Documents 1 and 2, Non-patent document 1). However, this method requires complicated operations, increases production costs, and is expected to reduce yield. Therefore, there has been a need for a new technique for purifying insoluble proteins contained in inclusion bodies in a simple and inexpensive manner without using columns.

JP2011-088922 Special table 2017-526649

In the present invention, insoluble proteins contained in inclusion bodies, which are aggregates of recombinant proteins expressed in microorganisms, are solubilized without using a denaturing agent, and the solubilization obtained without using a column etc. Develop methods to isolate and purify proteins.

In order to solve the above problems, the present inventors have conducted extensive research and found that by mixing an insoluble fraction containing inclusion bodies with a lithium solution of a predetermined concentration, the desired target protein can be isolated according to its molecular weight. We developed a method to solubilize, separate and purify. The present invention is based on this new knowledge and provides the following.

(1) A method for producing a solubilized target protein from an inclusion body, comprising a mixing step of mixing an insoluble fraction with a lithium solution, and a step of mixing the soluble fraction containing the solubilized target protein and the insoluble fraction after the mixing step. The method includes a separation step of separating.
(2) The method according to (1), which includes, before the mixing step, an obtaining step of obtaining an insoluble fraction containing inclusion bodies containing the target protein from a cell lysate of a microorganism expressing the target protein.
(3) The method according to (1) or (2), comprising a recovery step of recovering the soluble fraction after the separation step.
(4) The concentration of the lithium solution is more than 1.25M and less than 5M when the molecular weight of the target protein is less than 30,000, and more than 5M and less than 8M when it is more than 30,000 and less than 50,000, and less than 8M when it is more than 50,000. The method according to any one of (1) to (3), wherein the concentration exceeds 9M.
(5) The method according to any one of (1) to (4), wherein the lithium solution is a lithium bromide solution.
(6) The method according to any one of (1) to (5), wherein the target protein is a fibroin protein or a derived protein thereof.
(7) The production method according to any one of (2) to (6), wherein the microorganism is Escherichia coli.

(8) A method for separating and purifying a target protein derived from inclusion bodies as a solubilized protein, the method comprising: a first mixing step of mixing an insoluble fraction with a first lithium solution; a first separation step of separating the soluble fraction from the first separation step; a removal step of removing the soluble fraction after the first separation step; a second mixing step of mixing the insoluble fraction after the removal step with a second lithium solution; a second separation step of separating an insoluble fraction and a soluble fraction after the second mixing step, wherein the concentration of the first lithium solution is 6M or more when the molecular weight of the target solubilized protein is less than 30,000. 5M or less when the molecular weight is 30,000 or more and less than 50,000, and less than 8M when the molecular weight is 50,000 or more, and the concentration of the second lithium solution is 1.25M when the molecular weight of the target solubilized protein is less than 30,000. and less than 6M, exceeds 5M and less than 8M when it is 30,000 or more and less than 50,000, and is 8M or more when it is 50,000 or more.
(9) The method according to (8), wherein the lithium solution is a lithium bromide solution.
(10) The method according to (8) or (9), wherein the target solubilized protein is an exogenous protein expressed in a microorganism.

(11) A method for solubilizing insoluble proteins in inclusion bodies, comprising a mixing step of mixing an insoluble fraction with a lithium solution, and a soluble fraction containing a solubilized target protein and an insoluble fraction after the mixing step. The method includes a separation step of separating.
(12) The method according to (11), which includes, before the mixing step, an obtaining step of obtaining an insoluble fraction containing inclusion bodies containing the target protein from a cell lysate of a microorganism expressing the target protein.
(13) The method according to (11) or (12), which includes a recovery step of recovering the soluble fraction after the separation step.
(14) The concentration of the lithium solution is more than 1.25M and less than 6M when the molecular weight of the target protein is less than 30,000, and more than 6M and less than 8M when it is more than 30,000 and less than 50,000, and less than 8M when it is more than 50,000. The method according to any one of (11) to (13), wherein the concentration exceeds 9M.

According to the method for producing a solubilized protein of the present invention, an insoluble exogenous protein contained in an inclusion body can be made into a solubilized protein without using a denaturing agent or the like.

According to the solubilized protein separation and purification method of the present invention, the target protein contained in the inclusion body can be separated and purified as a solubilized target protein according to its molecular weight without using a denaturing agent.

It is a diagram showing a production flow in the solubilized protein production method of the present invention. FIG. 2 is a diagram showing a production flow in the method for separating and purifying solubilized proteins of the present invention. FIG. 3 is an SDS polyacrylamide gel electrophoresis (SDS-PAGE) diagram showing the results of solubilizing the target protein (bw753ΔC protein) in the insoluble fraction with various candidate solubilization solutions. In the figure, the numbers on the horizontal axis correspond to the numbers shown in Table 1. In the figure, two arrows indicate the band positions of the solubilized bw753ΔC protein. FIG. 3 is an SDS-PAGE diagram showing the results of verifying the solubilization conditions of the solubilization solution. A 10M lithium bromide solution was used as the solubilization solution. The insoluble fraction used in FIG. 3 was solubilized using combinations of treatment temperatures of 30°C, 60°C, and 80°C and treatment times of 30 minutes, 60 minutes, and 120 minutes. In the figure, two arrows indicate the band positions of the solubilized bw753ΔC protein. FIG. 2 is an SDS-PAGE diagram showing the correlation between the concentration of the solubilization solution and the molecular weight of the protein to be solubilized. FIG. 2 is an SDS-PAGE diagram showing the results of removal of contaminant proteins and separation and purification of target proteins by two-step solubilization treatment according to molecular weight. In each electropherogram, 1: 6M lithium bromide soluble fraction, 2: 8M lithium bromide soluble fraction, and 3: 8M lithium bromide soluble fraction derived from 6M lithium bromide insoluble fraction are shown. A shows the band position of the solubilized bw753ΔC protein, and B shows the band position of the contaminant protein contained in the inclusion body.

1. Method for producing solubilized protein derived from inclusion bodies 1-1. Overview The first aspect of the present invention is a method for solubilizing insoluble proteins in inclusion bodies and producing solubilized proteins. The method for producing a solubilized protein of the present invention is characterized in that the insoluble fraction containing the inclusion bodies is mixed with a lithium solution to solubilize the protein in the inclusion bodies to form a solubilized protein. According to the present invention, a solubilized target protein can be obtained by efficiently solubilizing an insoluble target protein synthesized in a microorganism such as E. coli by genetic recombination technology.

1-2. Definitions The following terms used in this specification are defined.
In this specification, the term "inclusion body" refers to a protein formed by exposing the hydrophobic residues of amino acids constituting the protein to the molecular surface due to overexpression or abnormal three-dimensional structure of a recombinant protein that is not originally produced in the host. refers to an insoluble aggregate formed due to hydrophobic interactions between The proteins that constitute inclusion bodies are, in principle, insoluble proteins. In this specification, the term particularly refers to the above-mentioned insoluble aggregate formed by expression of a foreign gene or the like within the cells of a genetically modified microorganism.

"Soluble fraction" refers to the aqueous phase fraction obtained by fractionating a sample. In this specification, the supernatant or filtrate obtained after fractionating a cell lysate by centrifugation or filtration is particularly applicable.

"Insoluble fraction" refers to a hydrophobic fraction obtained by fractionating a sample. In this specification, it particularly refers to precipitates or residues (filtrate) obtained after fractionating a cell lysate by centrifugation or filtration.

As used herein, the term "target protein" refers to a protein that is intended to be produced and obtained in the production method of the present invention. The target proteins herein are in principle exogenous proteins expressed in microorganisms and contained as insoluble proteins in inclusion bodies. As used herein, "exogenous protein" refers to a protein expressed from a foreign gene introduced into a host by artificial techniques such as genetic recombination technology. Although not limited, fibroin protein or its derivative proteins are preferred.

"Fibroin protein" (herein often referred to as "Fib protein") is a protein that constitutes fibroin, which is a fiber component of silk thread. Fib proteins include fibroin H chain protein (herein often referred to as "Fib H protein") and fibroin L chain protein (herein often referred to as "Fib L protein"). However, it can be either. Preferably, it is Fib H protein, which is a major constituent protein of fibroin. The Fib protein may be a wild type fibroin protein (wild type Fib protein) or a mutant fibroin protein.

"Mutant fibroin protein" (mutant Fib protein) is a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence of wild-type Fib protein, or the above-mentioned amino acid sequence. Examples include proteins consisting of an amino acid sequence having an amino acid identity of 90% or more, preferably 95% or more, more preferably 96% or more, 97% or more, 98% or more, or 99% or more to. As used herein, "plurality" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. "Amino acid identity" is when two amino acid sequences are aligned and gaps are introduced into one or both amino acid sequences as necessary to maximize the amino acid identity between the two. , refers to the ratio (%) of amino acids identical to the wild-type amino acid sequence to all amino acid residues in the mutant amino acid sequence.

As used herein, the term "derived protein thereof" refers to a recombinant fibroin protein or a modified recombinant fibroin protein.

As used herein, "recombinant fibroin protein" (herein often referred to as "rFib protein") refers to a recombinant fibroin H chain gene (herein often referred to as "rFib protein") that has been cloned using gene cloning technology. Fib H protein encoded by the recombinant fibroin light chain gene (often referred to herein as the rFib L gene); Refers to protein. The rFib protein does not need to be composed of the same amino acid sequence as the wild-type full-length Fib protein, as long as it contains the basic components of the Fib protein. Furthermore, the rFib protein does not have to be a Fib protein derived from a single organism, but may be a chimeric fibroin protein (chimeric Fib protein) composed of polypeptides derived from two or more biological species. An example is a chimeric Fib H protein composed of Fib H proteins from bagworms and silkworms. The rFib protein also includes a mutant rFib protein in which mutations similar to those of the mutant Fib protein are introduced into the rFib protein.

The biological species from which the fibroin protein is derived in this specification is not particularly limited. Examples include silkworms or species belonging to organisms belonging to the order Araneae.

As used herein, "silkworm" is a general term for insects that have silk glands and can spin silk threads. Specifically, it refers to the types of Lepidoptera, Hymenoptera, Paleoptera, Chamoptera, etc. that are capable of spinning silk for nesting, cocooning, or movement during the larval stage. The growth stage, such as larva or adult, does not matter. For example, among the Lepidoptera, there are the Bombycidae, Saturnidae, Psychidae, Brahmaeidae, Eupterotidae, and Lasiocampidae, which can spin large amounts of silk. , species belonging to the family Archtiidae, Noctuidae, etc. Specifically, for example, silk moths (B. mori and B. mandarina) belonging to the genus Bombyx, S. cynthia and S. cynthia ricini belonging to the genus Samia, and S. cynthia ricini belonging to the genus Antheraea. (A. yamamai) and A. pernyi, S. japonica belonging to the genus Saturnia, and the genera Acanthopsyche, Anatolopsyche, Bacotia, Bambalina, Canephora, Chalioides, Dahlica, Species belonging to the genus Diplodoma, Eumeta, Eumasia, Kozhantshikovia, Mahasena, Nipponopsyche, Paranarychia, Proutia, Psyche, Pteroma, Siederia, Striglocyrbasia, Taleporia, Theriodopteryx, and Trigonodoma, etc. Applicable. In particular, silkworms, which are the larvae of silkworm moths, and bagworms, which are the larvae of moths belonging to the family Minogatidae, are suitable as silkworms. Specific examples of minoga include Eumeta japonica, Eumeta minuscula, and Nipponopsyche fuscescens.

Examples of organisms belonging to the order Araneae include species belonging to the families Araneidae, Nephilidae, Tetragnathidae, Theridiidae, and Linyphiidae. Specific examples include A. ventricosus, A. uyemurai, and A. maccacus, which belong to the genus Araneus; A. amoena, which belongs to the genus Argiope; and A. ventricosus, which belongs to the genus Nephila. N. clavata) etc.

As used herein, the term "water-soluble protein" refers to a protein that has hydrophilic amino acid residues exposed on its molecular surface and is soluble in water or an aqueous solution.

As used herein, "insoluble protein" refers to a protein that does not dissolve in water or an aqueous solution. Specifically, in this specification, as a result of abnormal folding of an originally water-soluble exogenous protein expressed in a microorganism to form a three-dimensional structure within the cell, hydrophobic residues of amino acids are exposed on the protein surface, resulting in water-soluble It refers to a protein that has become insoluble in water. Insoluble proteins aggregate within cells by hydrophobic interactions with each other and/or by electrostatic interactions with nucleic acids, forming the inclusion bodies.

As used herein, "solubilization" refers to converting an insoluble protein into a water-soluble protein by solubilization treatment.

As used herein, "solubilized protein" refers to a protein that has become water-soluble by solubilizing an insoluble protein.

As used herein, the term "solubilized target protein" refers to the final product of the production method of this embodiment, in which the target protein contained in the inclusion body as an insoluble protein is converted to a solubilized protein by a solubilization treatment. say something

As used herein, the term "microorganism" refers to a unicellular organism that can accumulate inclusion bodies within its cells, and in principle includes prokaryotes. The type of microorganism is not limited. In genetic recombination technology, any microorganism commonly used in the field may be used. For example, Escherichia coli is suitable. In this specification, unless otherwise specified, the term "microorganism" refers to a transformant (genetically modified microorganism) that expresses a target protein through genetic recombination.

1-3. Manufacturing method The flow of the manufacturing method of the present invention is shown in FIG. The production method of the present invention includes a mixing step (S0103) and a separation step (S0104) as essential steps, and an acquisition step (S0101), a washing step (S0102), and a recovery step (S0105) as optional steps. . Each step will be specifically explained below.

1-3-1. Acquisition Step The “acquisition step” (S0101) is a step of acquiring an insoluble fraction from the cell lysate. This step is an optional step that is performed as necessary before the mixing step described below. This insoluble fraction contains inclusion bodies containing the target protein.

The "cell disruption solution" used in this step refers to a cell extract obtained by physically and/or chemically disrupting the cell walls of microorganisms in an aqueous solution. The microorganism is derived from a genetically modified microorganism that expresses the target protein. Genetically modified microorganisms may be produced by transformation methods known in the art. Examples include, but are not limited to, methods in which an expression vector such as a plasmid containing a gene encoding a target protein is introduced into microbial cells by heat shock or electroporation. The transformed microorganism may be cultured under conditions that maintain the introduced expression vector and the like. After culturing, the microorganisms are collected by centrifugation, and the cells are disrupted to prepare a cell disruption solution.

The cell disruption method may be performed according to a cell disruption method known in the art. For example, physical crushing methods include the French press method and the ultrasonic crushing method. These physical crushing methods are preferably carried out at low temperatures, such as on ice, in order to prevent protein denaturation due to heat and oxidation generated during processing. Furthermore, examples of chemical disruption methods include decomposition of cell walls using enzymes such as lysozyme and surfactants.

The method for obtaining the insoluble fraction from the cell lysate may be performed according to a method known in the art for separating a water-soluble fraction and an insoluble fraction. Examples include centrifugation and filtration.

In the centrifugation method, the cell disruption solution is centrifuged at an appropriate centrifugal acceleration to separate the supernatant, which is a water-soluble fraction, and the precipitate, which is an insoluble fraction, and then the insoluble fraction can be obtained by removing the supernatant. Although the centrifugal acceleration is not limited, for example, the centrifugation time (sedimentation time) may be within the range of 10 to 30 minutes depending on the centrifugal acceleration.

The filtration method is a method in which the cell disruption solution is passed through a filter (membrane filter) to separate the filtrate, which is a water-soluble fraction, and the residue, which is an insoluble fraction, and the residue captured on the filter surface is recovered. be. The filter pore size may be in the range of 0.05 μm to 10 μm.

The acquisition step can be performed multiple times. In this case, the supernatant or filtrate obtained in the previous acquisition step is used again as a cell disruption solution. The acquisition conditions each time may be the same or different. Generally, it is preferable to make the conditions more stringent as the number of times the test is repeated. For example, if centrifugation is used, the rotational speed should be higher or the centrifugation should be performed for a longer period of time.

1-3-2. Washing Step The "washing step" (S0102) is a step of washing the insoluble fraction after the acquisition step (S0101), and is an optional step in the production method of this embodiment. By this step, impurities mixed in the insoluble fraction can be easily removed.

The cleaning liquid used for cleaning is not particularly limited. Examples include water, physiological saline, phosphate buffer, HEPES buffer, NaHCO ₃ /CO ₂ buffer, Tris-HCl buffer, glycine buffer, and the like. Further, Triton X-100 (polyethylene glycol mono-p-isooctylphenyl ether), urea, EDTA, etc. may be added as necessary.

The cleaning method is not limited. For example, there is a method in which the insoluble fraction is suspended in the washing solution, centrifuged, and the insoluble fraction is collected again as a precipitate, or the suspension is passed through a filter and the insoluble fraction is collected again as a filtrate. One example is a method of recovering. The washing solution may simply be passed through the insoluble fraction without suspension.

1-3-3. Mixing Step The “mixing step” (S0103) is a step of mixing the insoluble fraction with the lithium solution. This step is a central step in the manufacturing method of this embodiment. By this method, target proteins that were contained as insoluble proteins in inclusion bodies are converted to solubilized proteins.

The insoluble fraction used in this step can be the insoluble fraction obtained in the obtaining step (S0101).

A "lithium solution" is a solution containing lithium ions. Usually, lithium salt can be obtained by dissolving it in water or an aqueous solution. The lithium salt is not particularly limited as long as it can be ionized in an aqueous solution to generate lithium ions. Examples include lithium chloride, lithium bromide, lithium thiocyanate, and the like. Preferred is lithium bromide.

Solubilization of insoluble proteins contained in inclusion bodies is based on the correlation between the molecular weight of the protein and the concentration of the lithium solution. Therefore, the concentration of the lithium solution used depends on the molecular weight of the target protein to be solubilized. For example, when the molecular weight of the target protein is less than 30,000, the lower limit of the concentration of lithium solution is above 1.25M, for example, 1.3M or more, 1.4M or more, 1.5M or more, 1.6M or more, 1.7M or more, 1.8M or more. , 1.9M or more, 2M or more, 2.1M or more, 2.2M or more, 2.3M or more, 2.4M or more, or 2.5M or more. The upper limit is less than 6M, for example, 5.9M or less, 5.8M or less, 5.7M or less, 5.6M or less, 5.5M or less, 5.4M or less, 5.3M or less, 5.2M or less, 5.1M or less, or 5M or less. In addition, when the molecular weight of the target protein is 30,000 or more and less than 50,000, the lower limit of the concentration of the lithium solution is more than 5M, for example, 5.1M or more, 5.2M or more, 5.3M or more, 5.4M or more, 5.5M or more, 5.6M or more. , 5.7M or more, 5.8M or more, 5.9M or more, or 6M or more. The upper limit is less than 8M, for example, 7.9M or less, 7.8M or less, 7.7M or less, 7.6M or less, 7.5M or less, 7.4M or less, 7.3M or less, 7.2M or less, 7.1M or less, or 7M or less. Furthermore, when the molecular weight of the target protein is 50,000 or more, the concentration of the lithium solution should be adjusted to a concentration higher than 9M, such as 9.1M or higher, 9.2M or higher, 93M or higher, 9.4M or higher, 9.5M or higher, 9.6M or higher, 9.7M or higher. , 9.8M or more, 9.9M or more, or 10M or more. At this time, the upper limit may be below the saturation concentration.

The volume mixing ratio of the insoluble fraction and the lithium solution is not particularly limited as long as it is 1 mL or more per 1 g of the insoluble fraction. For example, 1.5 mL or more, 2 mL or more, 2.5 mL or more, 3 mL or more, 3.5 mL or more, 4 mL or more, 4.5 mL or more, 5 mL or more, 6 mL or more, 7 mL or more, 8 mL or more, 9 mL or more, or 10 mL or more per gram of insoluble fraction. That's fine. Usually, a range of 2 mL to 7 mL is suitable.

After mixing, the mixed liquid may be stirred if necessary.
The mixing temperature is not particularly limited as long as it is within the range of 10 to 85°C. For example, the temperature may be 15 to 82°C, 18 to 80°C, 20 to 78°C, 25 to 75°C, 28 to 70°C, or 30 to 65°C. In order to prevent protein denaturation and inactivation, the temperature is preferably 60°C or lower, or 50°C or lower.
The mixing time is not particularly limited. For example, it may be carried out for 5 minutes to 180 minutes, 10 minutes to 150 minutes, 15 minutes to 120 minutes, 20 minutes to 90 minutes, or 30 minutes to 60 minutes.

1-3-4. Separation Step The “separation step” (S0104) is a step of separating the soluble fraction and insoluble fraction after the mixing step, and is an essential step in the production method of this embodiment. Since the target protein migrates to the soluble fraction, it can be obtained as a solubilized protein.

This step may be performed according to a method known in the art for separating a water-soluble fraction and an insoluble fraction. As described in "1-3-1. Obtaining step" above, examples of methods for separating the water-soluble fraction and insoluble fraction include centrifugation and filtration. The basic procedure is based on the method described in "1-3-1. Acquisition step" above, so a detailed description will be omitted.
The solubilized target protein is included in the soluble fraction obtained in this step.

1-3-5. Recovery Step The “recovery step” (S0105) is a step of recovering the soluble fraction after the separation step, and is an optional step in the production method of this embodiment.

As described in "1-3-4. Separation step" above, the soluble fraction obtained after the separation step contains the solubilized target protein of interest. By collecting this soluble fraction, the insoluble target protein contained in the inclusion bodies can be obtained as a solubilized target protein contained in an aqueous solution.

The recovery method is performed according to the separation method performed in the separation step. For example, when a water-soluble fraction and an insoluble fraction are separated by centrifugation in the separation step, the supernatant may be collected. When a water-soluble fraction and an insoluble fraction are separated by a filtration method in the separation step, the filtrate may be collected. The collected supernatant and filtrate can be repeatedly subjected to separation steps as necessary. In this case, in the separation step, the same method may be used for separation, or different methods may be combined each time for separation. For example, after separation by centrifugation, the collected supernatant is separated again by filtration and the filtrate is collected to obtain a soluble fraction that does not contain insoluble impurities.

The target protein may be purified from the collected soluble fraction, if necessary. Proteins may be purified by methods known in the art. For example, gel electrophoresis, gel filtration chromatography, ion exchange chromatography, reversed phase chromatography, affinity chromatography, salting out method, solvent precipitation method, solvent extraction method, dialysis method, ultrafiltration method, etc. alone, Alternatively, they may be used in combination as appropriate.

1-4. Effects According to the method for producing solubilized proteins of the present invention, insoluble exogenous proteins expressed in host microorganisms and contained in inclusion bodies can be easily soluble without using denaturants etc. as in conventional methods. Solubilized proteins can be produced by solubilization.

2. Method for separating and purifying solubilized proteins derived from inclusion bodies 2-1. Overview The second aspect of the present invention is a method for separating and purifying an insoluble target protein in an inclusion body as a solubilized target protein. The method of this embodiment is a method that applies the phenomenon that the molecular weight of the insoluble protein that is solubilized differs depending on the concentration of the lithium solution. According to the separation and purification method of this embodiment, insoluble proteins in inclusion bodies are solubilized according to their molecular weights, and other proteins are separated and removed from the sample, without using a denaturing agent as in conventional methods. It can be purified by

2-2. Method The flow of the method of this embodiment is shown in FIG. The method of this embodiment includes a first mixing step (S0203), a first separation step (S0204), a removal step (S0205), a second mixing step (S0206), and a second separation step (S0207) as essential steps, It also includes an acquisition step (S0201), a washing step (S0202), and a collection step (S0208) as optional steps. Each step will be specifically explained below.

2-2-1. Acquisition Step The “acquisition step” (S0201) is a step of acquiring an insoluble fraction from the cell disruption solution. This is an optional step that is performed as necessary before the first mixing step, which will be described later. This step corresponds to the acquisition step (S0101) described in the manufacturing method of the first embodiment. Therefore, a detailed explanation will be omitted here.

2-2-2. Washing process The "washing process" (S0202) is a process of washing the insoluble fraction after the acquisition process (S0201) and/or the removal process (S0205) described below, and is a selective process performed as necessary. be. This step corresponds to the cleaning step (S0102) described in the manufacturing method of the first embodiment. Therefore, a detailed explanation will be omitted here.

2-2-3. First Mixing Step The "first mixing step" (S0203) is a step of mixing an insoluble fraction containing an inclusion body containing an insoluble target protein with a first lithium solution, and is an essential step in this method.

The insoluble fraction used in this step contains inclusion bodies containing the insoluble target protein. As this insoluble fraction, the insoluble fraction obtained in the obtaining step (S0201) can be used.

The basic steps and operations of this step are in accordance with the mixing step (S0103) described in the manufacturing method of the first embodiment. Therefore, the characteristic method of this step will be explained below.

This step is characterized by maintaining the insoluble target protein as insoluble and solubilizing other insoluble proteins contained in the inclusion bodies. As described in "1-3-3. Mixing step" in the production method of the first embodiment, solubilization of the insoluble protein contained in the inclusion body is based on the correlation between the molecular weight of the protein and the concentration of the lithium solution. In order to remove contaminant proteins other than the target protein contained in the inclusion bodies, a lithium solution having a concentration other than that which solubilizes the target protein may be mixed with the insoluble fraction in this step. Therefore, the first lithium solution used in this step has a concentration that does not solubilize an insoluble protein having the molecular weight of the target protein.

For example, when the molecular weight of the target protein is less than 30,000, the concentration of the first lithium solution is set to a concentration exceeding 5M, such as 5.1M or more, 5.2M or more, 5.3M or more, 5.4M or more, 5.5M or more, 5.6M or more, By increasing the concentration to 5.7M or more, 5.8M or more, 5.9M or more, or 6M or more, insoluble proteins having a molecular weight of 30,000 or more contained in the inclusion bodies are solubilized. On the other hand, proteins in less than 30,000 inclusion bodies containing the target protein remain insoluble. Similarly, when the molecular weight of the target protein is 30,000 or more and less than 50,000, the concentration of the first lithium solution is less than 6M, 5.9M or less, 5.8M or less, 5.7M or less, 5.6M or less, 5.5M or less, 5.4M or less, 5.3 By lowering M or lower, 5.2M or lower, 5.1M or lower, or 5M or lower, insoluble proteins with a molecular weight of less than 30,000 contained in inclusion bodies are solubilized, but in inclusion bodies of 30,000 or more containing the target protein. of proteins remain insoluble. Furthermore, when the molecular weight of the target protein is 50,000 or more, the concentration of the first lithium solution is less than 8M, 7.9M or less, 7.8M or less, 7.7M or less, 7.6M or less, 7.5M or less, 7.4M or less, 7.3M or less, By reducing the concentration to 7.2M or less, 7.1M or less, or 7M or less, insoluble proteins with a molecular weight of less than 50,000 contained in inclusion bodies are solubilized, but proteins in inclusion bodies with a molecular weight of 50,000 or more containing the target protein are solubilized. Remains insoluble. After this step, by separating into an insoluble fraction and a soluble fraction in a first separation step described below, it becomes possible to separate and remove a part of the contaminant proteins contained in the inclusion bodies as a soluble fraction.

2-2-4. First separation step The “first separation step” (S0204) separates the insoluble fraction (herein referred to as “first insoluble fraction”) and the soluble fraction (herein referred to as “first insoluble fraction”) after the first mixing step (S0203). In this specification, this step is an essential step in the method of this embodiment.

The basic steps and operations of this step are similar to the separation step (S0104) described in the manufacturing method of the first embodiment. Therefore, here, the difference from the separation step (S0104) will be explained below.

The difference from the separation step (S0104) described in the production method of the first embodiment is that in the separation step of the first embodiment, the target protein was included as a solubilized protein in the soluble fraction after separation. In this step, the target protein is included as an insoluble protein in the first insoluble fraction. In other words, the objects to be recovered after separation are different from each other. Specifically, in the separation step (S0104), the precipitate or filtrate after separation is unnecessary, whereas in this step (S0204), the first soluble fraction, which is the supernatant or filtrate after separation, is unnecessary. becomes.

2-2-5. Removal Step The “removal step” (S0205) is a step of removing the first soluble fraction after the first separation step (S0204), and is an essential step in the method of this embodiment.

The basic procedures and operations of this step are similar to the recovery step (S0105) described in the manufacturing method of the first embodiment. Therefore, descriptions of common procedures and operations will be omitted here. However, as explained in the above section "2-2-4. First separation step," the recovery targets in the first embodiment and the main removal step are opposite to each other, and the recovery targets in the first embodiment are opposite to each other. In this step, the soluble fraction is collected, whereas in the removal step of this step, the first soluble fraction is removed and the first insoluble fraction is collected. This is because some of the insoluble proteins contained in the inclusion bodies, which have a molecular weight other than the target protein, are solubilized and separated from the target protein.
The first separation step to removal step can be repeated multiple times as necessary.

2-2-6. Second Mixing Step The “second mixing step” (S0206) is a step of mixing the first insoluble fraction obtained after the removal step with the second lithium solution, and is an essential step in this method.

This step corresponds to the mixing step (S0103) described in the manufacturing method of the first embodiment. Therefore, here, the difference from the mixing step (S0103) will be explained below.

The insoluble fraction used in the mixing step (S0103) of the first embodiment and the first insoluble fraction used in this step both contain the target protein in the form of an insoluble protein. However, in the first insoluble fraction of this step, a part of insoluble proteins having a different molecular weight from the target protein has been removed in the first mixing step (S0203) to removal step (S0205), and furthermore, the first insoluble fraction is recovered. The difference is that the first unnecessary fraction obtained is treated in this step, the target protein is solubilized, and the rest remains as an insoluble fraction. For the rest, basically the same procedures and operations as in the mixing step (S0103) of the first embodiment may be used.

The concentration of the second lithium solution used in this step may be determined depending on the molecular weight of the target protein to be solubilized. For example, if the molecular weight of the target protein is less than 30,000, the lower limit for the second lithium solution is a concentration exceeding 1.25M, for example, 1.3M or more, 1.4M or more, 1.5M or more, 1.6M or more, 1.7M or more, 1.8M. 1.9M or more, 2M or more, 2.1M or more, 2.2M or more, 2.3M or more, 2.4M or more, or 2.5M or more, and the upper limit is less than 6M, such as 5.9M or less, 5.8M or less, 5.7M or less, By setting the concentration to 5.6M or less, 5.5M or less, 5.4M or less, 5.3M or less, 5.2M or less, 5.1M or less, or 5M or less, proteins with a molecular weight of less than 30,000 are solubilized. On the other hand, other proteins remain insoluble. Similarly, if the molecular weight of the target protein is 30,000 or more and less than 50,000, use a second lithium solution with a lower limit of concentration exceeding 5M, such as 5.1M or more, 5.2M or more, 5.3M or more, 5.4M or more, 5.5M or more. Set to 5.6M or more, 5.7M or more, 5.8M or more, 5.9M or more, or 6M or more, and the upper limit is less than 8M, 7.9M or less, 7.8M or less, 7.7M or less, 7.6M or less, 7.5M or less, 7.4 Set to M or less, 7.3M or less, 7.2M or less, 7.1M or less, or 7M or less. As a result, proteins with a molecular weight of 30,000 or more and less than 50,000 are solubilized, but other proteins remain insoluble. Here, since proteins with a molecular weight of less than 30,000 have already been solubilized and removed in the first mixing step (S0203) to removal step (S0205), proteins with a molecular weight of 30,000 or more and less than 50,000 are substantially solubilized here. That will happen. Furthermore, if the molecular weight of the target protein is 50,000 or more, the lower limit of the second lithium solution is 8M or more, such as 9M or more, 9.1M or more, 9.2M or more, 9.3M or more, 9.4M or more, 9.5M or more, 9.6M or more. , 9.7M or more, 9.8M or more, 9.9M or more, or 10M or more, and by setting the upper limit below the saturation concentration, proteins with a molecular weight of 50,000 or more can be solubilized.

2-2-7. Second separation step "Second separation step" (S0207) separates the insoluble fraction (herein referred to as "second insoluble fraction") after the second mixing step (S0206) and the soluble fraction ( In this specification, this step is an essential step in the method of this embodiment. The second soluble fraction contains the solubilized target protein.

The basic procedures and operations of this step are the same as the separation step (S0104) described in the manufacturing method of the first embodiment. Therefore, a detailed explanation will be omitted here.

2-2-8. Recovery Step The “recovery step” (S0207) is a step of recovering the second soluble fraction. As described above, the second soluble fraction obtained after the second separation step (S0206) contains the solubilized target protein of interest. By collecting this second soluble fraction, the insoluble target compound contained in the inclusion body can be separated and purified as a soluble target protein that does not contain insoluble proteins of other molecular weights.

The basic steps and operations of this step are the same as the recovery step (S0105) described in the manufacturing method of the first embodiment. Therefore, a detailed explanation will be omitted here.

The collected second soluble fraction can be repeatedly subjected to the second separation step as necessary. In this case, the separation may be performed using the same method, or may be performed using a combination of different methods each time. For example, after separation by centrifugation, the collected supernatant is separated again by filtration and the filtrate is collected to obtain a soluble fraction that does not contain insoluble impurities.

Although the soluble target protein contained in the soluble fraction collected by the separation and purification method of this embodiment is already in a purified state, it may be further purified if necessary. The protein may be purified by the known method described in the recovery step (S0105) in the production method of the first embodiment.

2-3. Effects According to the solubilized protein separation and purification method of the present invention, target proteins contained in inclusion bodies can be solubilized without using denaturing agents, and solubilized target proteins can be separated according to their molecular weights. , can be purified.

According to the solubilized protein separation and purification method of the present invention, it is possible to reduce the number of purification steps and obtain highly pure solubilized target proteins easily and at low cost.

<Example 1: Examination of solubilizing agents for insoluble target proteins contained in inclusion bodies>
(the purpose)
We search for nondenaturing solubilizing agents that solubilize insoluble proteins expressed in E. coli and contained in inclusion bodies.

(Method)
(1) Preparation of target protein "bw753ΔC protein" consisting of a 721 amino acid sequence shown in SEQ ID NO: 2 was used as a target protein expressed in E. coli. The bw753ΔC protein is encoded by the bw753ΔC gene consisting of the base sequence shown in SEQ ID NO: 1. The bw753ΔC gene is a modified recombinant bagworm fibroin H chain gene (rbFib H gene) that was newly constructed in WO2020/235692 based on the base information of the Fib H gene of Fib H gene identified in JP-A-2018-074403. As described in the example of patent application 2019-097154, pET-22b-bw753ΔC was constructed as a gene expression vector containing this bw753ΔC gene in an expressible state, and used in cells of Escherichia coli BL21 (DE3) strain (Novagen). A transformant, bw753ΔC strain, was obtained using a conventional method.

(2) Induction of target protein expression The bw753ΔC strain was inoculated into LB medium and cultured with shaking at 37°C. When the OD ₆₀₀ reached 0.7, IPTG was added to a final concentration of 1mM, and further incubated at 20°C. The cells were cultured for 22 hours to induce the expression of bw753ΔC protein. After the induced culture, the bacterial cells were collected as a precipitate by centrifugation.

(3) Isolation of insoluble fraction The collected cells were suspended in purified water of the original culture solution volume, and then the cells were disrupted using a French press. Centrifuge the cell suspension at 12,000 x G for 30 minutes at 15°C, suspend 10 g of the resulting precipitate in 10 g of purified water, and centrifuge three bottles of 2 g suspension at 10,600 x G for 20 minutes for 20 minutes. Centrifuged again at ℃. Add 1 mL of washing buffer [100 mM Tris-HCl (pH 7.0), 10 mM EDTA・2Na, 2M urea, 2% (w/v) Triton X-100] to each precipitate and mix with a vortex mixer. Resuspend. The suspension was centrifuged at 10,600×G for 20 minutes at 20°C, the supernatant was removed, and the precipitate was collected. The above-mentioned operations of suspension in a washing buffer, centrifugation, and precipitate collection by removing the supernatant were repeated four times for washing, and the precipitates were again suspended in the washing buffer, collected, and dispensed into 1.5 mL tubes. Finally, after centrifugation at 16,000 x G for 20 minutes at 20°C, the supernatant was removed and the precipitate was collected as an insoluble fraction containing inclusion bodies.

(4) Solubilization of insoluble proteins in inclusion bodies using candidate solubilization solutions Aqueous solutions of 10 types of compounds at the concentrations shown in Table 1 were prepared as candidate solubilization solutions.

500 μL of each candidate solubilization solution was added to the insoluble fraction obtained in (3) and suspended using a vortex mixer. Thereafter, the mixture was mixed overnight using a rotator (manufactured by TAITEC, RT-50) by inversion, and then centrifuged at 16,000×G for 20 minutes at 20° C. to collect the supernatant, which is a soluble fraction.

The collected supernatant was fractionated by 10% SDS-PAGE, and the gel after electrophoresis was stained for total protein with Coomassie brilliant blue (CBB). The insoluble target protein (bw753ΔC protein) in the inclusion bodies contained in the insoluble fraction is solubilized with a solubilization solution and can be fractionated by SDS-PAGE if it exists as a solubilized protein in the supernatant (solubilized fraction). Therefore, the presence of the target protein (bw753ΔC protein) in each solubilized fraction was confirmed by moving within the gel by electrophoresis and forming a band at its own molecular weight position.

(result)
The results are shown in Figure 3. The position of the band corresponding to the molecular weight of bw753ΔC protein is indicated by two arrows. Saturated lithium thiocyanate solution (No. 1), 14.5M lithium chloride solution (No. 6), and 12.3M lithium bromide solution (No. 7), all only when lithium solution is used as the candidate solubilization solution. , solubilized bw753ΔC protein was confirmed. These results revealed that the lithium solution is effective as a solubilization solution for solubilizing the insoluble target protein in the inclusion bodies in the insoluble fraction.

In addition, each supernatant of No. 1, No. 6, and No. 7 was subjected to SDS-PAGE in parallel with known concentrations of bovine serum albumin (BSA), and after the gel was stained with CBB, the BSA band was detected. As a result of calculating the solubilized amount of bw753ΔC protein in each supernatant as the relative amount of 2.8mg, 14.5M lithium chloride solution was 0.3mg, and 12.3M lithium bromide solution had the highest solubilization rate. Furthermore, the amounts of contaminant proteins other than bw753ΔC protein were all small, but in this case as well, the 12.3M lithium bromide solution had the least amount of contaminant proteins. Based on the above results, in the following examples, a lithium bromide solution was used as the solubilization solution.

<Example 2: Verification of conditions for lithium solution as a solubilization solution>
(the purpose)
In Example 1, a lithium solution was discovered as a novel solubilizing solution capable of solubilizing insoluble target proteins in inclusion bodies. Therefore, in this example, the optimal solubilization conditions for insoluble proteins in inclusion bodies using a lithium solution will be verified from the treatment time and treatment temperature.

(Method)
To the nine insoluble fractions isolated in "(3) Isolation of insoluble fractions" of Example 1, 500 μL of 10M lithium bromide solution was added and suspended using a vortex mixer. After that, the cells were incubated for 30 minutes, 60 minutes, and 120 minutes in a block thermostat set at 30°C, 60°C, and 80°C, and then centrifuged at 16,000 x G for 20 minutes at 20°C. A supernatant fraction was collected. The collected supernatant was subjected to SDS-PAGE under the same conditions as in Example 1, and the gel after electrophoresis was stained for total protein with CBB staining.

(result)
The results are shown in Figure 4. In this figure, only the band corresponding to the molecular weight of bw753ΔC protein is shown. The figure shows that when a 10M lithium bromide solution was used as the solubilization solution, there was no significant difference in solubilization rate depending on treatment temperature or treatment time.

<Example 3: Concentration of lithium solution and solubilized amount of insoluble protein>
(the purpose)
In this example, the relationship between the concentration of lithium solution and the amount of solubilized insoluble protein in inclusion bodies will be verified.

(Method)
1.25M, 2.5M, 5M, 6M, 7M, 8M, 9M, and 10M lithium bromide solutions were added to the 8 insoluble fractions isolated in "(3) Isolation of insoluble fractions" in Example 1. 500 μL of each was added and suspended using a vortex mixer. Thereafter, the mixture was incubated for 120 minutes in a thermostatic water bath set at 30°C, and then centrifuged at 16,000×G for 20 minutes at 20°C to collect the supernatant, which is a soluble fraction. The collected supernatant was subjected to SDS-PAGE under the same conditions as in Example 1, and the gel after electrophoresis was stained for total protein with CBB staining.

(result)
The results are shown in Figure 5. The band position corresponding to the molecular weight of bw753ΔC protein is indicated by an arrow. Very interestingly, among contaminant proteins other than the bw753ΔC protein, proteins with molecular weights less than 30,000 were solubilized by 2.5M and 5M lithium bromide solutions, but hardly solubilized by 6M or higher lithium bromide solutions. . In addition, contaminant proteins with molecular weights of 30,000 to 50,000 were efficiently solubilized by 6M to 8M lithium bromide solutions, but were hardly solubilized or the amount solubilized was very low at less than 6M or 10M. On the other hand, bw753ΔC protein with a molecular weight of 60,000 to 70,000 was efficiently solubilized using 8M to 10M lithium bromide solution. The above results revealed that there is a correlation between the concentration of the lithium solution and the molecular weight of the protein in the solubilization of insoluble proteins by the lithium solution.

<Example 4: Establishment of a method for separating and purifying solubilized target protein by multi-step solubilization treatment based on the molecular weight of target protein>
(the purpose)
The results of Example 3 revealed that insoluble proteins in inclusion bodies contained in the insoluble fraction can be efficiently solubilized by using a lithium solution with a concentration based on its molecular weight as a solubilization solution.

Utilizing the above correlation, insoluble contaminant proteins other than the target protein contained in the inclusion bodies are solubilized using a lithium solution with a concentration that can solubilize insoluble proteins of those molecular weights, removed, and then recovered. The target protein remaining in the insoluble fraction is suspended and centrifuged by solubilizing it with a lithium solution at a concentration other than that used to solubilize contaminant proteins that can solubilize insoluble proteins of that molecular weight. We will verify that it is possible to separate and purify the solubilized target protein to a high degree of purity by simply performing the following steps.

(Method)
(1) Recovery of soluble fraction by treatment with 6M lithium bromide solution 500 μL of 6M lithium bromide solution was added to the insoluble fraction isolated in “(3) Isolation of insoluble fraction” in Example 1, and vortexed. Suspended using a mixer. Then, after incubation for 120 minutes in a thermostatic water bath set at 30℃, centrifugation was performed at 16,000×G for 20 minutes at 20℃, and the soluble fraction, the supernatant, was collected as the "6M lithium bromide solubilized fraction". did.
The collected supernatant (6M lithium bromide solubilized fraction) was subjected to SDS-PAGE, and the gel after electrophoresis was stained with total protein by CBB staining.

(2) Recovery of soluble fraction by treatment with 8M lithium bromide solution 500 μL of 8M lithium bromide solution was added to the insoluble fraction isolated in “(3) Isolation of insoluble fraction” in Example 1, and vortexed. Suspended using a mixer. Then, after incubation for 120 minutes in a thermostatic water bath set at 30℃, centrifugation was performed at 16,000 x G for 20 minutes at 20℃, and the soluble fraction, the supernatant, was collected as the "8M lithium bromide solubilized fraction". did. The collected supernatant (8M lithium bromide solubilized fraction) was subjected to SDS-PAGE, and the gel after electrophoresis was stained with total protein by CBB staining.

(3) Recovery of soluble fraction by two-step solubilization using 6M and 8M lithium bromide solutions. 500 μL of lithium bromide solution was added and suspended using a vortex mixer. Thereafter, it was incubated for 120 minutes in a thermostatic water bath set at 30°C, and then centrifuged at 16,000×G for 20 minutes at 20°C. 500 μL of purified water was added to the “6M lithium bromide insoluble fraction” obtained as a precipitate and suspended. After recentrifugation at 16,000 x G for 20 minutes at 20°C, the supernatant was removed and the precipitate was collected again. This series of operations consisting of the three steps of suspension in purified water, centrifugation, and supernatant removal (sediment collection) was repeated three times to wash the "6M lithium bromide insoluble fraction." Add 500 μL of 8M lithium bromide solution to the washed 6M lithium bromide insoluble fraction, suspend, incubate at 30℃ for 120 minutes, and centrifuge at 16,000×G for 20 minutes at 20℃ to remove the soluble fraction. The supernatant (8M lithium bromide solubilized fraction) was collected. The supernatant (6M/8M lithium bromide solubilized fraction) collected after the two-step solubilization treatment was subjected to SDS-PAGE, and the gel after electrophoresis was stained with total protein by CBB staining.

(result)
The results are shown in Figure 6. In the figure, lanes 1, 2, and 3 listed below each SDS-PAGE image represent the solubilized fractions collected after each treatment described in (1), (2), and (3) of the above method, respectively. FIG. In the 6M lithium bromide solubilized fraction (lane 1), in which the insoluble fraction was solubilized with a 6M lithium bromide solution, the target protein bw753ΔC protein (A) was not solubilized, but contaminants with a molecular weight of less than 50,000 were detected. Protein (B) was solubilized. In addition, in the 8M lithium bromide solubilized fraction (lane 2), which was obtained by solubilizing the insoluble fraction with 8M lithium bromide solution, the target protein bw753ΔC protein (A) was solubilized, but the contaminant protein ( B) was also soluble at the same time. On the other hand, the insoluble fraction was solubilized with a 6M lithium bromide solution, and the resulting 6M lithium bromide insoluble fraction was solubilized with an 8M lithium bromide solution. In the 6M/8M lithium bromide solubilized fraction (lane 3) resulting from the treatment, the target protein bw753ΔC protein (A) was also solubilized, but almost no contaminant protein (B) was detected. From the above results, it was confirmed that the target protein can be efficiently solubilized with high purity by performing the solubilization treatment in multiple stages using lithium solutions with different concentrations.
All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims (12)

1. A method for producing a solubilized target protein from inclusion bodies, the method comprising:
   The method described above, comprising: a mixing step of mixing the insoluble fraction with a lithium solution; and a separation step of separating the soluble fraction containing the solubilized target protein and the insoluble fraction after the mixing step.
2. The method according to claim 1, further comprising, before the mixing step, an obtaining step of obtaining an insoluble fraction containing inclusion bodies containing the target protein from a cell lysate of a microorganism expressing the target protein.
3. The method according to claim 1 or 2, comprising a recovery step of recovering the soluble fraction after the separation step.
4. The concentration of the lithium solution is such that the molecular weight of the target protein is
   is less than 30,000, exceeds 1.25M, and is less than 5M,
   5M or more and less than 8M when the number is 30,000 or more and less than 50,000,
   50,000 or more when the concentration exceeds 9M,
   The method according to any one of claims 1 to 3.
5. The method according to any one of claims 1 to 4, wherein the lithium solution is a lithium bromide solution.
6. The method according to any one of claims 1 to 5, wherein the target protein is a fibroin protein or a derived protein thereof.
7. The method according to any one of claims 2 to 6, wherein the microorganism is Escherichia coli.
8. A method for separating and purifying a target protein derived from inclusion bodies as a solubilized protein, the method comprising:
   a first mixing step of mixing the insoluble fraction with a first lithium solution;
   a first separation step of separating an insoluble fraction and a soluble fraction after the first mixing step;
   a removal step of removing the soluble fraction after the first separation step;
   a second mixing step of mixing the insoluble fraction obtained after the removal step with a second lithium solution; and a second separation step of separating the insoluble fraction and soluble fraction after the second mixing step.
   including;
   The concentration of the first lithium solution is such that the molecular weight of the target solubilized protein is
   6M or more when less than 30,000,
   5M or less when 30,000 or more and less than 50,000,
   50,000 or more but less than 8M,
   The concentration of the second lithium solution is such that the molecular weight of the target solubilized protein is
   is less than 30,000, exceeds 1.25M, and is less than 6M,
   30,000 or more and less than 50,000, exceeding 5M and less than 8M,
   is more than 8M when more than 50,000,
   Said method.
9. 9. The method according to claim 8, further comprising, before the mixing step, an obtaining step of obtaining an insoluble fraction containing inclusion bodies containing the target protein from a cell lysate of a microorganism expressing the target protein.
10. The method according to claim 8 or 9, comprising a recovery step of recovering the soluble fraction after the second separation step.
11. The method according to any one of claims 8 to 10, wherein the lithium solution is a lithium bromide solution.
12. The method according to any one of claims 9 to 11, wherein the target solubilized protein is an exogenous protein expressed in the microorganism.

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