JP2018178296A - Solvent for dissolving protein, protein solution, method for producing protein fiber, and method for refining objective protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a solvent for dissolving protein capable of further reducing cost.SOLUTION: Provided is a solvent for dissolving protein comprising dimethyl sulfoxide and calcium chloride, and in which the concentration of the calcium chloride is 0.001 mol/L or more and 0.5 mol/L or less.SELECTED DRAWING: None

Description

  The present invention relates to a solvent for protein dissolution, a protein solution, a method for producing a protein fiber, and a method for purifying a target protein.

  Polar solvents such as dimethylsulfoxide (DMSO) are used as solvents for proteins such as spider silk proteins and silk proteins. Moreover, it is known to add an inorganic salt as a solubility promoter to these polar solvents (for example, patent documents 1-3).

Patent No. 5427322 Patent No. 5584932 International Publication No. 2016/163337

  A solvent obtained by adding an inorganic salt (eg, lithium chloride) to DMSO is extremely excellent as a solvent for dissolving a protein. On the other hand, from the viewpoint of industrial use, a solvent that can further reduce the cost is required.

  An object of the present invention is to provide a solvent for protein dissolution which can further reduce the cost.

  The present inventors have found that a solvent in which calcium chloride is added to DMSO as an inorganic salt is excellent in protein solubility even at a low calcium chloride concentration. The solvent has a low calcium chloride concentration, so that the raw material cost can be reduced, and in the process of removing inorganic salts from proteins, the number of washings can be significantly reduced, thereby further reducing the cost. be able to. The present invention is based on this novel finding.

The present invention relates to, for example, the following inventions.
[1]
Contains dimethyl sulfoxide and calcium chloride,
The solvent for protein solution whose density | concentration of the said calcium chloride is 0.001 mol / L or more and 0.5 mol / L or less.
[2]
The protein dissolving solvent according to [1], wherein the concentration of calcium chloride is 0.025 mol / L or more and 0.5 mol / L or less.
[3]
The solvent for protein dissolution as described in [1] or [2] whose density | concentration of the said calcium chloride is 0.025 mol / L or more and 0.1 mol / L or less.
[4]
The solvent for protein dissolution in any one of [1]-[3] whose temperature is 20 degreeC or more and 100 degrees C or less.
[5]
The solvent for protein dissolution in any one of [1]-[4] whose temperature is 50 degreeC or more and 70 degrees C or less.
[6]
A protein solution in which a protein is dissolved in the protein dissolving solvent according to any one of [1] to [5].
[7]
The protein solution according to [6], which is a dope solution.
[8]
The protein solution according to [6] or [7], wherein the protein is a structural protein.
[9]
The protein solution according to [8], wherein the structural protein is spider silk protein.
[10]
Using the protein solution according to any one of [6] to [9] as a doping solution,
A method for producing a protein fiber, comprising the steps of: extruding the dope solution into a coagulating solution to obtain an undrawn yarn.
[11]
The method for producing a protein fiber according to [10], further comprising the step of drawing the undrawn yarn.
[12]
A method for purifying a target protein, comprising the step of dissolving a host cell expressing the target protein in the protein dissolving solvent according to any one of [1] to [5].
[13]
The method for purifying a target protein according to [12], wherein the target protein is a structural protein.
[14]
The method for purifying a protein of interest according to [13], wherein the structural protein is spider silk protein.

  According to the present invention, it is possible to provide a solvent for protein dissolution which can further reduce the cost. The solvent for protein dissolution of the present invention is excellent in protein solubility even at a low calcium chloride concentration, so the amount of inorganic salt (calcium chloride) used can be significantly reduced. As a result, it is possible to reduce the cost of raw materials and also to reduce the number of times of washing significantly in the process of removing inorganic salts from proteins, thereby further reducing costs. In addition, since the amount of use of the inorganic salt (calcium chloride) can be small, it is possible to suppress the deterioration of the production facility and the like.

  Furthermore, according to the present invention, the gelation of the protein solution can be suppressed by adjusting the calcium chloride concentration to a predetermined range (0.025 mol / L or more and 0.5 mol / L or less).

It is explanatory drawing which shows an example of the manufacturing apparatus of protein fiber. It is explanatory drawing which shows an example of the manufacturing apparatus of protein fiber. FIG. 2 (A) shows an undrawn yarn manufacturing apparatus-first-stage drawing apparatus, and FIG. 2 (B) shows a second-stage drawing apparatus. It is explanatory drawing which shows the other example of the manufacturing apparatus of protein fiber. It is explanatory drawing which shows the other example of the manufacturing apparatus of protein fiber. Fig. 4 (A) shows an undrawn yarn production apparatus, and Fig. 4 (B) shows a dry heat drawing apparatus. It is a figure which shows the result of SDS (Sodium dodecyl sulfate) -polyacrylamide gel electrophoresis (SDS-PAGE) in an Example. It is a figure which shows the result of SDS-PAGE in an Example. It is a figure which shows the result of the gelation test of the modified fibroin extract in an Example. It is a figure which shows the powder recovery of the modified fibroin under each temperature and reaction time conditions in an Example. It is a figure which shows the result of SDS-PAGE in an Example.

  Hereinafter, modes for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[1. Protein Dissolution Solvent]
The solvent for dissolving a protein according to the present embodiment contains dimethylsulfoxide (DMSO) and calcium chloride (CaCl ₂ ) at a predetermined concentration.

  The concentration of calcium chloride may be 0.001 mol / L or more and 0.5 mol / L or less. The solvent for protein dissolution according to the present embodiment is excellent in the solubility of protein even if the concentration of calcium chloride is low, so that the cost of raw materials can be reduced, and in the process of removing inorganic salts from protein The cost can be further reduced because it becomes possible to reduce the number of times.

  The solvent for protein dissolution according to the present embodiment can also further reduce the calcium chloride concentration because it is excellent in protein solubility even if the calcium chloride concentration is low. That is, the concentration of calcium chloride may be 0.25 mol / L or less, 0.1 mol / L or less, or 0.09 mol / L or less, 0.08 mol / L or less It may be 0.07 mol / L or less and may be 0.06 mol / L or less. By adjusting the calcium chloride concentration to such a reduced range, the above-described cost reduction effect can be more significantly exhibited. If the lower limit of the calcium chloride concentration is 0.001 mol / L or more, sufficiently high protein solubility can be exhibited. The lower limit of the calcium chloride concentration is preferably 0.025 mol / L or more from the viewpoint of suppressing the gelation of the protein solution in addition to the solubility of the protein.

  The protein dissolving solvent according to this embodiment may further contain alcohol and / or water. When the solvent for protein dissolution is 100% by mass, the ratio of DMSO and calcium chloride is 22% by mass or more and 100% by mass or less, and the balance may contain alcohol. As the alcohol, lower alcohols having 1 to 6 carbon atoms are preferable, and one or more alcohols selected from the group consisting of methanol, ethanol and 2-propanol are more preferable. Moreover, when the solvent for protein solution is made into 100 mass%, the ratio of DMSO and calcium chloride is 10 mass% or more and 100 mass% or less, and the remainder may contain water. The remainder may mix water and alcohol.

  The temperature of the protein dissolving solvent according to this embodiment is preferably 20 ° C. or more and 100 ° C. or less. When the temperature of the protein dissolving solvent is 20 ° C. or more, the solubility of the protein is further improved. From the same viewpoint, the temperature of the protein dissolving solvent is more preferably 30 ° C. or more, still more preferably 40 ° C. or more, and still more preferably 50 ° C. or more. In addition, when the temperature of the protein dissolving solvent is 100 ° C. or less, the degradation of the dissolved protein can be further suppressed. From the same viewpoint, the temperature of the protein dissolving solvent is more preferably 90 ° C. or less, still more preferably 80 ° C. or less, and still more preferably 70 ° C. or less.

[2. Protein solution]
The protein solution according to the present embodiment is one in which a protein is dissolved in the above-described solvent for dissolving a protein. The protein solution according to the present embodiment may contain unavoidable components, such as contaminants contained in proteins.

<Target protein>
Examples of the protein to be dissolved (hereinafter sometimes referred to as "target protein") include any protein whose production on an industrial scale is preferable. Examples of the target protein include proteins that can be used for industrial use, proteins that can be used for medical use, structural proteins, and the like. Specific examples of proteins that can be used for industrial or medical use include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens used for vaccination, vaccines, antigen binding proteins, immunostimulatory proteins, Mention may be made of allergens, full-length antibodies or antibody fragments or derivatives. Specific examples of structural proteins include spider silk proteins, silk proteins, collagen, resilin and elastin, and proteins derived therefrom.

Examples of fibroin-like proteins, such as spider silk protein and silk protein, and proteins derived therefrom, include proteins containing a domain sequence represented by Formula 1: [(A) _n Motif-REP] _m . Here, in Formula 1, (A) _n motif indicates an amino acid sequence mainly comprising an alanine residue, n is 2 to 20, preferably 4 to 20, more preferably 8 to 20, and still more preferably 10 to 10 It may be an integer of 20, still more preferably 4 to 16, still more preferably 8 to 16, particularly preferably 10 to 16. The ratio of the number of alanine residues to the total number of amino acid residues in (A) _n motif may be 40% or more, preferably 60% or more, and more preferably 70% or more. % Or more is more preferable, 90% or more is still more preferable, and 100% (meaning it is composed of only alanine residues) may be used. REP shows the amino acid sequence comprised from 2-200 amino acid residues. m shows the integer of 2-300. The plurality of (A) _n motifs may be identical to each other or different from each other. The plurality of REPs may be identical amino acid sequences to each other or different amino acid sequences. Specific examples of spider silk proteins and silk proteins, and proteins derived therefrom can include proteins including the amino acid sequences shown in SEQ ID NO: 1 (PRT410) and SEQ ID NO: 2 (PRT587).

As a weft protein which is a kind of spider silk protein, and a protein derived therefrom, for example, a protein comprising a domain sequence represented by the formula 2: [REP2] _o can be mentioned. Here, in Formula 2, REP2 represents an amino acid sequence composed of Gly-Pro-Gly-Gly-X, and X is composed of alanine (Ala), serine (Ser), tyrosine (Tyr) and valine (Val) It shows one amino acid selected from the group. o shows the integer of 8-300. Specific examples of the weft protein and the protein derived therefrom include a protein comprising an amino acid sequence represented by SEQ ID NO: 3 (Recombinant spider silk protein Flag _ 92 — short 2). The amino acid sequence shown by SEQ ID NO: 3 is from the N-terminus corresponding to the repeat portion and motif of the partial sequence (NCBI accession number: AAF36090, GI: 7106224) of the flagellar silk protein of American jergent spider obtained from the NCBI database The amino acid sequence from residues 1220 to 1659 (referred to as the PR1 sequence) and a partial sequence of the flagella-like silk protein of the American spider spider obtained from the NCBI database (NCBI accession numbers: AAC38847, GI: 2833649) A C-terminal amino acid sequence from the C-terminal to the residues 816 to 907 is linked, and the amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 7 is added to the N-terminus of the linked sequence is there.

Examples of collagen and proteins derived therefrom include proteins containing a domain sequence represented by Formula 3: [REP3] _p . Here, in Formula 3, p shows the integer of 5-300. REP3 shows the amino acid sequence comprised from Gly-X1-Y, and X and Y show arbitrary amino acid residues other than Gly. The plurality of REP3 may be identical to each other or different from each other. Specific examples of collagen and a protein derived therefrom can include a protein comprising an amino acid sequence represented by SEQ ID NO: 4 (Collagen-type 4-Kai). The amino acid sequence shown by SEQ ID NO: 4 corresponds to the repeat portion and motif of a partial sequence of human collagen type 4 (NCBI GenBank accession numbers: CAA56335.1, GI: 3702452) obtained from the NCBI database. The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 7 is added to the N-terminal of the amino acid sequence from residue 301 to residue 540.

Examples of resilin and proteins derived therefrom include proteins containing a domain sequence represented by Formula 4: [REP4] _q . Here, in Formula 4, q shows the integer of 4-300. REP4 shows the amino acid sequence comprised from Ser 1 J 1 J 1 Tyr 1 Gly 1 U-Pro. J represents any amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Asp, Ser and Thr. U is any amino acid residue, and is particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr and Ser. The plurality of REP4 may be identical to each other or different from each other. Specific examples of resilin and proteins derived therefrom can include a protein comprising the amino acid sequence shown by SEQ ID NO: 5 (Resilin-Kai). The amino acid sequence shown by SEQ ID NO: 5 is such that in the amino acid sequence of resilin (NCBI GenBank accession numbers NP 611157, Gl: 24654243), Thr at position 87 is substituted with Ser, and Asn at position 95 The amino acid sequence (tag sequence and hinge sequence) shown in SEQ ID NO: 7 is added to the N-terminal of the amino acid sequence from the 19th residue to the 321st residue of the sequence in which

  Examples of elastin and proteins derived therefrom include proteins having amino acid sequences such as NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), NP786966 (bovine) and the like. Specific examples of elastin and proteins derived therefrom can include a protein comprising the amino acid sequence shown in SEQ ID NO: 6 (elastin short). The amino acid sequence represented by SEQ ID NO: 6 is the amino acid sequence represented by SEQ ID NO: 7 at the N-terminus of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence of NCBI GenBank accession number AAC 98 395 (tag sequence And hinge arrangement) are added.

  From the viewpoint of better solubility, the target protein is preferably spider silk protein and silk protein, and fibroin-like proteins such as proteins derived therefrom, and spider silk protein and a protein derived therefrom are more preferred.

  The target protein can be obtained, for example, by a general method using genetic engineering techniques. Specifically, for example, a target protein (eg, recombinant spider silk protein) is produced using a host transformed with an expression vector containing a gene encoding a natural spider silk protein to be recombinantly be able to. The gene production method is not particularly limited. For example, a gene encoding a natural spider silk protein is amplified from cells of spider origin by polymerase chain reaction (PCR) and cloned, and chemically synthesized The method is mentioned. The chemical synthesis method of the gene is not particularly limited, but, for example, AKTA oligopilot plus 10/100 (GE Healthcare) based on the amino acid sequence information of natural spider protein obtained from the NCBI web database etc. There is a method of linking and synthesizing oligonucleotides automatically synthesized by Japan Co., Ltd. etc. by PCR etc. To the target protein, an amino acid sequence consisting of an initiation codon and a His10 tag may be added to the N-terminus of the amino acid sequence to facilitate purification and confirmation of the protein. As an expression vector, an expression vector such as a plasmid, phage or virus capable of expressing a protein from a DNA sequence can be used. The plasmid-type expression vector is not particularly limited as long as it can express a target gene in a host cell and can amplify itself. Specifically, for example, when E. coli Rosetta (DE3) is used as a host, pET22b (+) plasmid vector, pCold plasmid vector, etc. can be used. From the viewpoint of excellent protein productivity, it is preferable to use a pET22b (+) plasmid vector as the plasmid type expression vector. As a host, for example, animal cells, plant cells, microorganisms and the like can be used.

<Method of preparing protein solution>
The protein solution according to the present embodiment can be obtained by dissolving the target protein in the protein dissolving solvent according to the present invention. The method for dissolving the target protein is not particularly limited, and examples thereof include a method in which a solvent for protein dissolution is added to a host expressing the target protein or the target protein itself. The method may optionally include separating insolubles. The separation of the insolubles is not particularly limited as long as the precipitate (aggregates) can be separated. It is preferable to carry out separation of insolubles by separation by filtration and / or centrifugation for the convenience of handling. The separation by filtration can be performed using, for example, filter paper, a filtration membrane, and the like. The conditions for centrifugation are not particularly limited. For example, it can be carried out at room temperature (20 ± 5 ° C.), 8000 × g to 15,000 × g for 5 to 20 minutes. The insolubles may be separated twice or more.

<Doping solution>
The protein solution according to the present embodiment can be used, for example, as a dope. The dope liquid which concerns on this embodiment can be used suitably for wet spinning, and is useful as a cast film liquid etc. The dope solution according to the present embodiment is manufactured, for example, by adjusting the viscosity of the protein solution to a viscosity at which it can be spun. The viscosity of the dope solution is, for example, 100 to 10,000 cP (centipoise). Adjustment of the viscosity of the dope solution can be performed, for example, by adjusting the concentration of protein in the solution. The viscosity of the dope solution can be measured, for example, using a trade name "EMS viscometer" manufactured by Kyoto Electronics Industry Co., Ltd.

[3. Method for producing protein fiber]
The method for producing a protein fiber according to the present embodiment is a step of obtaining the undrawn yarn by using the protein solution according to the present invention as a dope solution, extruding the dope solution into a coagulating solution (hereinafter also referred to as a "spinning step"). Including. The manufacturing method according to the present embodiment may further include the step of drawing the undrawn yarn (hereinafter, also referred to as “drawing step”).

<Spinning process>
The manufacturing method according to the present embodiment employs wet spinning. In the spinning step, the solvent in which the target protein is dissolved is removed from the dope solution (also referred to as desolvation or coagulation) to form fibers to obtain an undrawn yarn.

  The concentration of the target protein in the dope solution is preferably 3% by mass or more, more preferably 10% by mass or more, and still more preferably 20% by mass or more, based on the total amount of the dope solution. From the viewpoint of maintaining good solubility of the target protein, the concentration of the target protein in the dope solution is preferably 60% by mass or less, more preferably 50% by mass or less, and 40% by mass or less Is more preferred.

  The coagulation liquid is not particularly limited as long as it can remove the solvent, and, for example, lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, and acetone can be used. Water may be added to the coagulating solution as appropriate. The temperature of the coagulating liquid is, for example, 5 to 30 ° C. If the temperature of the coagulating liquid is in this range, spinning can be performed more stably.

  In the spinning step, for example, an undrawn yarn can be obtained by extruding the dope solution from a spinneret into a coagulated solution in a solvent removal tank. When the dope solution is extruded from a syringe pump having a nozzle with a diameter of 0.1 to 0.6 mm, the extrusion speed may be, for example, 0.2 to 6.0 ml / hour per hole, 1.4 to 4 It may be .0 ml / hour. If the extrusion speed is in this range, spinning can be performed more stably.

  The length of the desolvation tank (coagulation liquid tank) may be, for example, 200 to 500 mm. The withdrawal speed of the undrawn yarn from the solvent removal tank may be, for example, 1 to 14 m / min, or 1 to 3 m / min. The residence time in the desolvation tank may be, for example, 0.01 to 0.15 minutes. If the conditions for desolvation or coagulation fall within these ranges, desolvation or coagulation can be performed more efficiently. Further, the undrawn yarn may be stretched (pre-stretching) in the coagulating solution, but the coagulating solution is maintained at a low temperature in consideration of the ease of evaporation of the coagulating solution (for example, lower alcohol), It is preferable to pick up in the undrawn yarn state.

<Stretching process>
The undrawn yarn may be drawn by one-step drawing or may be performed by two or more multi-step drawing. When the target protein is a protein in which a molecule such as a protein derived from a natural spider silk protein is difficult to orient, by performing multistage stretching, the molecules can be oriented in multiple stages, and the total stretching magnification can also be increased. As a result, fibers with high toughness can be obtained.

  FIG.1 and FIG.2 is explanatory drawing which shows an example of the manufacturing apparatus of protein fiber. The manufacturing apparatus shown in FIG. 1 is a spinning and drawing apparatus that continuously performs a spinning process and a drawing process (two-stage drawing). The spinning and drawing apparatus 10 includes an extrusion apparatus 1, an undrawn yarn production apparatus 2, a wet heat drawing apparatus 3, and a dry heat drawing apparatus 4. The dope solution 6 is stored in the storage tank 7 and is pushed out of the base 9 by the gear pump 8. In the lab scale, the dope solution may be filled into a cylinder and pushed out of the nozzle using a syringe pump. The extruded dope 6 is supplied into the coagulation liquid 11 of the coagulation tank 20 directly through the air gap 19 or not through the air gap 19. The solvent is removed from the dope solution 6 in the coagulating solution 11 to form an undrawn yarn. Next, the undrawn yarn is supplied into the warm water 12 in the drawing bath 21 and the first stage drawing is performed. The draw ratio is determined by the speed ratio between the feed nip roller 13 and the take-up nip roller 14. The drawn yarn that has undergone the first-stage drawing is then supplied to the dry-heat drawing device 17, and the second-stage drawing is performed in the yarn path 22. The drawn yarn that has undergone the second-step drawing is taken up to form a wound yarn body 5. The draw ratio in the second stage drawing is determined by the speed ratio between the feed nip roller 15 and the take-up nip roller 16. 18a to 18f are yarn guides.

  The manufacturing apparatus shown in FIG. 2 is a spinning and drawing apparatus that performs a spinning process and a drawing process (two-step drawing). FIG. 2A shows the undrawn yarn production device 30 and the first-stage drawing device 40, and FIG. 2B shows the second-stage drawing device 50. For each device, the yarn may be wound up or stored in a container without being wound up. In the unstretched yarn manufacturing apparatus 30, the dope solution 32 is placed in an extrusion device (micro syringe) 31 and moved in the direction of arrow P by using a syringe pump to extrude the dope solution 32 from the nozzle 33 and coagulating solution tank The dope solution 32 is supplied to the coagulating solution 35 in 34 to form an undrawn yarn 36. Subsequently, in the first-stage drawing apparatus 40, the undrawn yarn 36 is supplied into the warm water 38 in the drawing bath 37 and drawn for the first stage to form a wound yarn body 39 of the first-stage drawn yarn. The draw ratio is determined by the speed ratio between the feed nip roller 41 and the take-up nip roller 42. Next, the first-step drawn yarn is drawn out from the wound body 39, supplied to the dry-heat drawing device 43, and drawn in the yarn path 47 for the second step. The draw ratio is determined by the speed ratio between the feed nip roller 45 and the take-up nip roller 46. Then, the drawn yarn is wound around the winding body 44.

  FIG.3 and FIG.4 is explanatory drawing which shows the other example of the manufacturing apparatus of protein fiber. The manufacturing apparatus shown in FIG. 3 is a spinning and drawing apparatus which continuously performs a spinning process and a drawing process (one-stage drawing). The spinning and drawing device 60 includes an extrusion device 61, an undrawn yarn production device 62, and a dry heat drawing device 63. The dope 66 is stored in the reservoir 67 and is pushed out of the base 69 by the gear pump 68. In the lab scale, the spinning solution may be filled into a cylinder and pushed out of the nozzle using a syringe pump. The extruded dope 66 is supplied into the coagulation liquid 71 of the coagulation tank 72 directly via the air gap 73 or not via the air gap 73. The solvent is removed from the dope solution 66 in the coagulating solution 71 to form an undrawn yarn. Next, the undrawn yarn is supplied to a dry heat drawing apparatus 77, and drawing is performed in the yarn path 78. The drawn yarn which has undergone drawing is wound to form a wound yarn body 64. The draw ratio is determined by the speed ratio between the feed nip roller 75 and the take-up nip roller 76. 74a to 74f are yarn guides.

  The manufacturing apparatus shown in FIG. 4 is a spinning and drawing apparatus that performs a spinning process and a drawing process (one-stage drawing). FIG. 4A shows an undrawn yarn production apparatus 80, and FIG. 4B shows a dry heat drawing apparatus 90. For each device, the yarn may be wound up or stored in a container without being wound up. In the unstretched yarn manufacturing apparatus 80, the dope solution 82 is placed in an extrusion device (micro syringe) 81, moved in the direction of arrow P using a syringe pump, the dope solution 82 is extruded from the nozzle 83, and the coagulation solution tank The solution is supplied to the coagulating solution 85 in 84 to form an undrawn yarn wound body 86. Next, in the dry heat drawing apparatus 90, the undrawn yarn is drawn out from the winding body 86, supplied to the dry heat drawing apparatus 89, and drawn in the yarn path 91. The draw ratio is determined by the speed ratio between the feed nip roller 87 and the take-up nip roller 88. Then, the drawn yarn is wound around a winding body 92. Thus, a drawn yarn is obtained.

[4. Purification method of target protein]
The method for purifying a target protein according to the present embodiment includes the step of dissolving a host cell expressing the target protein in the protein dissolving solvent according to the present invention (hereinafter, also referred to as “dissolution step”).

  For example, a method of screening host cells that naturally express a target protein using the expression amount of the target protein as an indicator, and transforming the host cell that expresses the target protein with an expression vector containing a gene encoding the target protein It can be obtained by a method of obtaining a host cell (for example, a recombinant microorganism) that expresses a protein (recombinant protein). The target protein can be expressed in the host cell by culturing the host cell that expresses the target protein by a conventional method.

  The amount of the solvent for dissolving a protein according to the present invention to be added to dissolve the target protein from host cells expressing the target protein is not particularly limited, but from the viewpoint of enhancing the solubility of the target protein, The ratio (volume (mL) / weight (g)) of the volume (mL) of the solvent for protein solubilization to the weight (g) of the host cell is preferably 5 or more, more preferably 7 or more Preferably, it is more preferably 10 times or more. The host cells at the time of addition of the protein dissolving solvent may be in a dry state or in a wet state.

  Although the temperature at the time of implementing a melt | dissolution process is not specifically limited, For example, 20 degreeC or more and 100 degrees C or less may be sufficient. The temperature is preferably 30 ° C. or higher, more preferably 40 ° C. or higher, and still more preferably 50 ° C. or higher, from the viewpoint of further enhancing the solubility of the target protein. The temperature is more preferably 90 ° C. or less, still more preferably 80 ° C. or less, and still more preferably 70 ° C. or less from the viewpoint of further suppressing the degradation of the target protein. The time for carrying out the dissolution step is not particularly limited, and may be, for example, 10 to 300 minutes, and preferably 30 to 180 minutes.

  The method for purifying a target protein according to the present embodiment may further include a step of separating insolubles from the host cell lysate obtained in the lysis step (hereinafter, also referred to as “separation step”). The separation of the insolubles is not particularly limited as long as the precipitate (aggregates) can be separated. It is preferable to carry out separation of insolubles by separation by filtration and / or centrifugation for the convenience of handling. The separation by filtration can be performed using, for example, filter paper, a filtration membrane, and the like. The conditions for centrifugation are not particularly limited. For example, it can be carried out at room temperature (20 ± 5 ° C.), 8000 × g to 15,000 × g for 5 to 20 minutes. The insolubles may be separated twice or more.

  The purification method of the target protein according to the present embodiment may further comprise the step of adding a dilution solvent to the host cell lysate obtained in the lysis step or the solution from which insolubles have been separated in the separation step. As a solvent for dilution, for example, a solvent for protein dissolution used in the dissolution step, DMSO and the like can be mentioned.

  The protein solution in which the target protein obtained by the purification method according to this embodiment is dissolved may be used, for example, for purification of the target protein, or may be used as it is as a dope solution.

  Hereinafter, the present invention will be more specifically described based on examples. However, the present invention is not limited to the following examples.

[(1) Preparation of Target Protein Expression Strain and Expression of Target Protein]
Based on the nucleotide sequence and amino acid sequence of fibroin (GenBank accession number: P46804.1, GI: 1174415) derived from Nephila clavipes as a target protein, a modified fibroin (amino acid sequence shown in SEQ ID NO: 2) Also called "PRT 587." The amino acid sequence shown by SEQ ID NO: 2 has an amino acid sequence obtained by substituting, inserting and deleting amino acid residues for the purpose of improving productivity with respect to the amino acid sequence of fibroin derived from Nephila clavipes, further A tag sequence and a hinge sequence are added to the N terminus.

  Next, the nucleic acid encoding PRT587 was synthesized. The NdeI site at the 5 'end and the EcoRI site downstream of the stop codon were added to the nucleic acid. The nucleic acid was cloned into a cloning vector (pUC118). Thereafter, the same nucleic acid was digested with NdeI and EcoRI, cut out, and then recombined into a protein expression vector pET-22b (+) to obtain an expression vector.

E. coli BLR (DE3) was transformed with the obtained pET-22b (+) expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 1) containing ampicillin so that the OD ₆₀₀ was 0.005. The culture solution temperature was maintained at 30 ° C., and flask culture was performed until the OD ₆₀₀ reached 5 (about 15 hours) to obtain a seed culture solution.

The seed culture solution was added to a jar fermenter to which 500 ml of production medium (Table 2) was added so that the OD ₆₀₀ was 0.05. The temperature of the culture solution was maintained at 37 ° C., and the culture was controlled at a constant pH of 6.9. Also, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

  Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 ml / min. The temperature of the culture solution was maintained at 37 ° C., and the culture was controlled at a constant pH of 6.9. Further, the culture was carried out for 20 hours while maintaining the dissolved oxygen concentration in the culture solution at 20% of the dissolved oxygen saturation concentration. Thereafter, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce expression of the target protein (modified fibroin). Twenty hours after the addition of IPTG, the culture solution was centrifuged to recover the cells. The collected cells were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were dried to obtain a predetermined amount of dried cells of E. coli expressing the target protein. In addition, SDS-PAGE was performed using cells prepared from the culture solution before IPTG addition and after IPTG addition, and expression of the target protein was confirmed by appearance of a band of the target protein size depending on IPTG addition.

[(2) Purification of target protein-examination of solution for protein dissolution]
10 g of the dry cells of recombinant E. coli expressing the target protein (modified fibroin) obtained as described above were weighed out, and a solvent was added. As a solvent, DMSO (100 mL), 0.5 M Lithium chloride (LiCl) in DMSO (100 mL), 0.001 M, 0.005 M, 0.01 M, 0.025 M, 0.05 M, 0.075 M, 0.1 M Alternatively, DMSO (100 mL each) containing 0.15 M calcium chloride (CaCl ₂ ) was used. After addition of the solvent, it was thermally dissolved while stirring at 60 ° C. for 1 hour. After completion of thermal dissolution, 100 mL of RO water is added with stirring, and after stirring for about 5 minutes in this state, the reaction solution is poured into a bottle filtration filter, and vacuum filtration is performed using a vacuum pump to remove contaminants and target protein solution. The (modified fibroin extract) was separated. Two to three hours after the start of filtration, it was confirmed that the extraction of the filtrate was completed.

  After separating a sample for SDS-PAGE analysis and a sample for gelation confirmation from the modified fibroin extract, the modified fibroin extract was added to 1.5 volumes of ethanol and allowed to stand for 10 minutes. After standing, the solution of the modified fibroin extract solution-ethanol was stirred (200 rpm), and the precipitation of the modified fibroin by the poor solvent was confirmed. After confirming the precipitation of the modified fibroin, it was allowed to stand still for 1 hour and 50 minutes to advance the precipitation. After completion of the precipitation, the precipitate was collected by centrifugation (11,000 g, 30 minutes, 20 ° C.), the supernatant was removed, and RO water equivalent to the supernatant was added. After addition of RO water, the precipitate was dispersed by a disperser to wash the precipitate. After washing, the conductivity of the solution was measured, and centrifugation and washing were repeated until the conductivity of the solution became 100 μs / cm or less. After washing, the filtrate was lyophilized to recover a powder of modified fibroin. Table 3 shows the recovery of the modified fibroin powder.

[(3) Analysis by SDS-PAGE]
The resulting modified fibroin powder, the modified fibroin extract, and the filtration residue were analyzed by SDS-PAGE.

<Quantification of protein (BCA method)>
After DMSO was added to the modified fibroin powder, the modified fibroin extract, and the filtration residue, each sample was dissolved by reacting at 85 ° C. for 30 minutes while stirring at 1500 rpm. After dissolution, it was diluted appropriately, and necessary amount of BCA Reagent A was mixed and aliquoted into an Eppendorf tube. BCA Reagent B was added to the separated Eppendorf tube, and allowed to react at 37 ° C. for 30 minutes. After completion of the reaction, the absorbance intensity of each sample was measured.

<SDS-PAGE>
Based on the results obtained from the measurement by BCA method for modified fibroin powder (Purified sample), modified fibroin extract (Extraction solution), and filtration residue (Residual dross), the protein concentration is adjusted to 10 mg / mL. -A sample for PAGE was prepared. The Mini-PROTEAN (registered trademark) Tetra System was set to Mini-PROTEAN (registered trademark) TGX (registered trademark) Gels, and each prepared sample for SDS-PAGE was loaded and subjected to electrophoresis. After the electrophoresis was completed, analysis was performed with Gel DOC (registered trademark) EZ Imager. The results are shown in FIG. 5 and FIG.

  The photograph on the left of FIG. 5 and the photograph on the left of FIG. 6 show that after electrophoresis, all proteins are stained with Striable Oriole (trademark) fluorescent gel stain (manufactured by Bio-Rad), the right of FIG. The photograph and the photograph on the right of FIG. 6 are those after electrophoresis and stained with InVisionTM His-tagged in-gel staining reagent (manufactured by Thermo Fisher Scientific) that reacts to the His-tag region of PRT410. PRT 587 with a theoretical molecular weight of 105.7 kDa was detected as a band between molecular weight markers of 100 kDa to 150 kDa.

[(4) Confirmation of gelation]
The modified fibroin extract was allowed to stand at room temperature for 1 week, and then the extract was checked for gelation. An example of the result is shown in FIG. When DMSO containing 0.025 M or more of CaCl ₂ was used as a solvent for protein dissolution, gelation was not confirmed (DMSO containing 0.05 M, 0.075 M, 0.1 M or 0.15 M of CaCl ₂ is shown in FIG. Not show.).

[(5) Purification of target protein-examination of temperature and time]
DMSO containing 0.025 M CaCl ₂ was used as a solvent for protein dissolution. First, 10 g of dry cells of recombinant E. coli expressing the target protein (modified fibroin) obtained in the above (1) were weighed out, and 100 mL of a solvent for dissolving the protein was added. While stirring under each temperature condition (50 ° C., 60 ° C., 70 ° C. or 80 ° C.) and each reaction time condition (0.5 hours, 1 hour, 2 hours or 6 hours for each temperature condition) It was melted by heat. After completion of thermal dissolution, 100 mL of RO water is added while stirring, and after stirring for about 5 minutes in this state, the reaction solution is poured into a bottle filtration filter, and vacuum filtration is performed using a vacuum pump to remove contaminants and target protein solution. The (modified fibroin extract) was separated. Two to three hours after the start of filtration, it was confirmed that the extraction of the filtrate was completed.

  After separating a sample for SDS-PAGE analysis from the modified fibroin extract, the modified fibroin extract was added to 1.5 volumes of ethanol and allowed to stand for 10 minutes. After standing, the solution of the modified fibroin extract solution-ethanol was stirred (200 rpm), and the precipitation of the modified fibroin by the poor solvent was confirmed. After confirming the precipitation of the modified fibroin, it was allowed to stand still for 1 hour and 50 minutes to advance the precipitation. After completion of the precipitation, the precipitate was collected by centrifugation (11,000 g, 30 minutes, 20 ° C.), the supernatant was removed, and RO water equivalent to the supernatant was added. After addition of RO water, the precipitate was dispersed by a disperser to wash the precipitate. After washing, the conductivity of the solution was measured, and centrifugation and washing were repeated until the conductivity of the solution became 100 μs / cm or less. After washing, the filtrate was lyophilized to recover a powder of modified fibroin. Analysis by SDS-PAGE was performed in the same manner as in (3) above. The results are shown in FIGS. 8 and 9 and Table 4.

  FIG. 8 and Table 4 show the powder recovery rate (%) of the modified fibroin under each condition. The powder recovery rate is calculated from the recovery amount of the modified fibroin powder obtained under each condition as 100% recovery amount under the conditions of reaction at 80 ° C. for 6 hours.

  FIG. 9 shows the result of staining of all proteins with Oriole (trademark) fluorescent gel stain (manufactured by Bio-Rad) after electrophoresis.

[(6) Comparison of the number of times of cleaning]
As a solvent for protein dissolution, DMSO containing 0.5 M LiCl and DMSO containing 0.025 M CaCl ₂ were used. First, 10 g of dry cells of recombinant E. coli expressing the target protein (modified fibroin) obtained in the above (1) were weighed out, and 100 mL of a solvent for protein dissolution was added. After addition, it was thermally dissolved at 60 ° C. for 1 hour while stirring. After completion of thermal dissolution, 100 mL of RO water is added with stirring, and after stirring for about 5 minutes in this state, the reaction solution is poured into a bottle filtration filter, and vacuum filtration is performed by a vacuum pump to remove impurities and objects. The protein solution (modified fibroin extract) was separated. Two to three hours after the start of filtration, it was confirmed that the extraction of the filtrate was completed.

  The modified fibroin extract was added to 1.5 volumes of ethanol and allowed to stand for 10 minutes. After standing, the solution of the modified fibroin extract solution-ethanol was stirred (200 rpm), and the precipitation of the modified fibroin by the poor solvent was confirmed. After confirming the precipitation of the modified fibroin, it was allowed to stand still for 1 hour and 50 minutes to advance the precipitation. After completion of precipitation, the precipitate was collected by centrifugation (11,000 g, 30 minutes, 20 ° C.), and after removing the supernatant, about 10 times the amount of RO water of the precipitate was added. After addition of RO water, the precipitate was dispersed by a disperser to wash the precipitate. After washing, the conductivity of the solution was measured. This operation was repeated four times. The results are shown in Table 5.

When the DMSO containing 0.025 M CaCl ₂ is used as a solvent for protein dissolution, the conductivity can be reduced with a smaller number of washings than when the DMSO containing 0.5 M LiCl is used (inorganic salt Was able to remove

  1, 31, 61, 81: extrusion device, 2, 30, 62, 80: undrawn yarn production device, 3, 40: wet heat stretching device (first stage stretching device), 4, 50, 63, 90: dry heat Stretching device (second stage stretching device), 5, 39, 44, 64, 86, 92 ... Winding body, 6, 32, 66, 82 ... Doping solution, 7, 67 ... Storage tank, 8, 68 ... Gear pump, 9 , 69, 83: spinneret, 10, 60: spinning and drawing apparatus, 11, 35, 71, 85: coagulating solution, 36: undrawn yarn, 12, 38: hot water, 13, 15, 41, 45, 75, 87 ... Supply nip roller, 14, 16, 42, 46, 76, 88: take-up nip roller, 17, 43, 77, 89: dry heat stretching device, 18a to 18f, 74a to 74f, yarn guide, 19, 73, air gap, 20 , 34, 72, 84 ... Coagulation liquid tank, 21, 37 ... Stretching Bath, 22,47,78,91 ... yarn path.

Claims (14)

Contains dimethyl sulfoxide and calcium chloride,
The protein dissolving solvent, wherein the concentration of calcium chloride is 0.001 mol / L or more and 0.5 mol / L or less.   The protein dissolving solvent according to claim 1, wherein the concentration of the calcium chloride is 0.025 mol / L or more and 0.5 mol / L or less.   The solvent for protein dissolution according to claim 1 or 2, wherein the concentration of calcium chloride is 0.025 mol / L or more and 0.1 mol / L or less.   The solvent for protein dissolution as described in any one of Claims 1-3 whose temperature is 20 degreeC or more and 100 degrees C or less.   The solvent for protein dissolution as described in any one of Claims 1-4 whose temperature is 50 degreeC or more and 70 degrees C or less.   A protein solution in which a protein is dissolved in the protein dissolving solvent according to any one of claims 1 to 5.   The protein solution according to claim 6, which is a dope solution.   The protein solution according to claim 6 or 7, wherein the protein is a structural protein.   The protein solution according to claim 8, wherein the structural protein is spider silk protein. A protein solution according to any one of claims 6 to 9 is used as a dope.
A method for producing a protein fiber, comprising the steps of: extruding the dope solution into a coagulating solution to obtain an undrawn yarn.   The method for producing a protein fiber according to claim 10, further comprising the step of drawing the undrawn yarn.   A method for purifying a recombinant protein, comprising the step of dissolving a host cell expressing the recombinant protein in the protein dissolving solvent according to any one of claims 1 to 5.   The method for purifying a recombinant protein according to claim 12, wherein the recombinant protein is a structural protein.   The method for purifying a recombinant protein according to claim 13, wherein the structural protein is spider silk protein.