WO2024057361A1 - Procédé de production de protéine solubilisée - Google Patents

Procédé de production de protéine solubilisée Download PDF

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WO2024057361A1
WO2024057361A1 PCT/JP2022/034058 JP2022034058W WO2024057361A1 WO 2024057361 A1 WO2024057361 A1 WO 2024057361A1 JP 2022034058 W JP2022034058 W JP 2022034058W WO 2024057361 A1 WO2024057361 A1 WO 2024057361A1
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protein
less
solubilized
insoluble
fraction
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PCT/JP2022/034058
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Japanese (ja)
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雅尚 渡辺
武 土肥
学 柴▲崎▼
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興和株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

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  • 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.
  • the recombinant protein 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. .
  • Patent Documents 1 and 2 Non-patent document 1
  • 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.
  • 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.
  • 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.
  • the present invention is based on this new knowledge and provides the following.
  • 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.
  • 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.
  • the method according to (1) or (2) comprising a recovery step of recovering the soluble fraction after the separation step.
  • 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.
  • a method for separating and purifying a target protein derived from inclusion bodies as a solubilized protein 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.
  • 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.
  • the lithium solution is a lithium bromide solution.
  • the target solubilized protein is an exogenous protein expressed in a microorganism.
  • 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.
  • 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.
  • the method according to (11) or (12) which includes a recovery step of recovering the soluble fraction after the separation step.
  • 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.
  • an insoluble exogenous protein contained in an inclusion body can be made into a solubilized protein without using a denaturing agent or the like.
  • 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.
  • 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 (bw753 ⁇ C protein) in the insoluble fraction with various candidate solubilization solutions.
  • the numbers on the horizontal axis correspond to the numbers shown in Table 1.
  • two arrows indicate the band positions of the solubilized bw753 ⁇ C protein.
  • FIG. 3 is an SDS-PAGE diagram showing the results of verifying the solubilization conditions of the solubilization solution.
  • 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.
  • 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.
  • 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.
  • 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.
  • solubility fraction refers to the aqueous phase fraction obtained by fractionating a sample.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • plural 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.
  • derived protein thereof refers to a recombinant fibroin protein or a modified recombinant fibroin protein.
  • recombinant fibroin 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.
  • 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.
  • 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.
  • insect 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.
  • 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.
  • silkworms which are the larvae of silkworm moths
  • bagworms which are the larvae of moths belonging to the family Minogatidae
  • minoga include Eumeta japonica, Eumeta minuscula, and Nipponopsyche fuscescens.
  • 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.
  • 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.
  • 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.
  • solubilization refers to converting an insoluble protein into a water-soluble protein by solubilization treatment.
  • solubilized protein refers to a protein that has become water-soluble by solubilizing an insoluble protein.
  • 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
  • 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.
  • microorganism refers to a transformant (genetically modified microorganism) that expresses a target protein through genetic recombination.
  • 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. .
  • S0103 mixing step
  • S0104 separation step
  • S0101 acquisition step
  • S0102 washing step
  • S0105 recovery step
  • the “acquisition step” 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a filter membrane filter
  • the filter pore size may be in the range of 0.05 ⁇ m to 10 ⁇ m.
  • 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.
  • 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.
  • One example is a method of recovering.
  • the washing solution may simply be passed through the insoluble fraction without suspension.
  • 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).
  • lithium solution is a solution containing lithium ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • a range of 2 mL to 7 mL is suitable.
  • the mixing temperature is not particularly limited as long as it is within the range of 10 to 85°C.
  • 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.
  • 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.
  • 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.
  • Recovery Step is a step of recovering the soluble fraction after the separation step, and is an optional step in the production method of this embodiment.
  • the soluble fraction obtained after the separation step contains the solubilized target protein of interest.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the insoluble fraction obtained in the obtaining step (S0201) can be used.
  • This step is characterized by maintaining the insoluble target protein as insoluble and solubilizing other insoluble proteins contained in the inclusion bodies.
  • 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.
  • 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.
  • 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,
  • 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
  • concentration 5.7M or more, 5.8M or more, 5.9M or more, or 6M or more
  • proteins in less than 30,000 inclusion bodies containing the target protein remain insoluble.
  • 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
  • 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.
  • 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.
  • concentration 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.
  • first separation step Separats 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).
  • first insoluble fraction the insoluble fraction
  • first insoluble fraction the soluble fraction
  • this step is an essential step in the method of this embodiment.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • concentration 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.
  • 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.
  • 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.
  • proteins with a molecular weight of 30,000 or more and less than 50,000 are solubilized, but other proteins remain insoluble.
  • 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.
  • 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.
  • Second separation step Separats 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 “recovery step” (S0207) is a step of recovering the second soluble fraction.
  • 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 collected second soluble fraction can be repeatedly subjected to the second separation step as necessary.
  • 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.
  • 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.
  • 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.
  • 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.
  • Target protein "bw753 ⁇ C 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 bw753 ⁇ C protein is encoded by the bw753 ⁇ C gene consisting of the base sequence shown in SEQ ID NO: 1.
  • the bw753 ⁇ C 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.
  • pET-22b-bw753 ⁇ C was constructed as a gene expression vector containing this bw753 ⁇ C gene in an expressible state, and used in cells of Escherichia coli BL21 (DE3) strain (Novagen).
  • a transformant, bw753 ⁇ C strain was obtained using a conventional method.
  • 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.
  • the collected supernatant was fractionated by 10% SDS-PAGE, and the gel after electrophoresis was stained for total protein with Coomassie brilliant blue (CBB).
  • CBB Coomassie brilliant blue
  • the insoluble target protein (bw753 ⁇ C 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 (bw753 ⁇ C protein) in each solubilized fraction was confirmed by moving within the gel by electrophoresis and forming a band at its own molecular weight position.
  • 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.
  • BSA bovine serum albumin
  • 14.5M lithium chloride solution was 0.3mg
  • 12.3M lithium bromide solution had the highest solubilization rate.
  • the amounts of contaminant proteins other than bw753 ⁇ C protein were all small, but in this case as well, the 12.3M lithium bromide solution had the least amount of contaminant proteins.
  • a lithium bromide solution was used as the solubilization solution.
  • Example 2 Verification of conditions for lithium solution as a solubilization solution> (the purpose)
  • 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.
  • 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.
  • 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.
  • 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.
  • the target protein bw753 ⁇ C protein (A) was solubilized, but the contaminant protein ( B) was also soluble at the same time.
  • 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.
  • the target protein bw753 ⁇ C 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.

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Abstract

La présente invention développe un procédé impliquant : la solubilisation, sans utiliser d'agent de modification, d'une protéine insoluble incluse dans un corps d'inclusion, qui est un agrégat d'une protéine recombinante exprimée dans un micro-organisme ; la séparation de celle-ci en tant que protéine solubilisée sans utiliser de colonne ou similaire ; et la purification de celle-ci.　L'invention concerne un procédé de production d'une protéine cible solubilisée à partir d'un corps d'inclusion par mélange d'une fraction insoluble et d'une solution de lithium, puis séparation d'une fraction soluble comprenant la protéine cible solubilisée à partir de la fraction insoluble.
PCT/JP2022/034058 2022-09-12 2022-09-12 Procédé de production de protéine solubilisée WO2024057361A1 (fr)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120214199A1 (en) * 2011-02-23 2012-08-23 Elona Biotechnologies Lis-pro proinsulin compositions and methods of producing lis-pro insulin analogs therefrom
JP2012182995A (ja) * 2011-03-03 2012-09-27 Nippon Flour Mills Co Ltd フィブリノゲンを産生するトランスジェニックカイコ
JP2014502140A (ja) * 2010-09-28 2014-01-30 ザ ユニバーシティー オブ ノートルダム キメラスパイダーシルクおよびその使用
WO2016153072A1 (fr) * 2015-03-26 2016-09-29 国立大学法人信州大学 Solution d'agent de coagulation sanguine, son procédé de production, et procédé de production d'une composition e2p intrinsèque de protéine d'embolie sanguine liquide
WO2018030499A1 (fr) * 2016-08-10 2018-02-15 Ｓｐｉｂｅｒ株式会社 Procédé de production d'un agrégat insoluble de protéines recombinantes
WO2021011431A1 (fr) * 2019-07-12 2021-01-21 Bolt Threads, Inc. Formulations d'extrudat de soie d'araignée de recombinaison
WO2021035184A1 (fr) * 2019-08-22 2021-02-25 Bolt Threads, Inc. Procédés d'extraction améliorée de polymères de protéines de soie d'araignée
JP2022145193A (ja) * 2021-03-19 2022-10-03 興和株式会社 可溶化タンパク質製造方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014502140A (ja) * 2010-09-28 2014-01-30 ザ ユニバーシティー オブ ノートルダム キメラスパイダーシルクおよびその使用
US20120214199A1 (en) * 2011-02-23 2012-08-23 Elona Biotechnologies Lis-pro proinsulin compositions and methods of producing lis-pro insulin analogs therefrom
JP2012182995A (ja) * 2011-03-03 2012-09-27 Nippon Flour Mills Co Ltd フィブリノゲンを産生するトランスジェニックカイコ
WO2016153072A1 (fr) * 2015-03-26 2016-09-29 国立大学法人信州大学 Solution d'agent de coagulation sanguine, son procédé de production, et procédé de production d'une composition e2p intrinsèque de protéine d'embolie sanguine liquide
WO2018030499A1 (fr) * 2016-08-10 2018-02-15 Ｓｐｉｂｅｒ株式会社 Procédé de production d'un agrégat insoluble de protéines recombinantes
WO2021011431A1 (fr) * 2019-07-12 2021-01-21 Bolt Threads, Inc. Formulations d'extrudat de soie d'araignée de recombinaison
WO2021035184A1 (fr) * 2019-08-22 2021-02-25 Bolt Threads, Inc. Procédés d'extraction améliorée de polymères de protéines de soie d'araignée
JP2022145193A (ja) * 2021-03-19 2022-10-03 興和株式会社 可溶化タンパク質製造方法

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