JP7312429B2 - Gel composition and dried gel - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act February 20, 2019 Released on page 87 and 88 of "Abstracts for Master's Thesis" issued by Nara Institute of Science and Technology, Graduate School of Materials Science and Technology, 2018

Application of Article 30, Paragraph 2 of the Patent Law February 22, 2019 Presented at the master's thesis presentation at the Graduate School of Materials Science and Technology, Nara Institute of Science and Technology, 2018

TECHNICAL FIELD The present invention relates to a gel composition and a dried gel.

Ferritin is an iron-storage protein widely present in organisms such as prokaryotes, archaea, plants, and higher eukaryotes. Ferritin is a globular protein in which 24 self-assembled subunits are formed from a single polypeptide chain (Fig. 1). Ferritin has a molecular weight of 450 kDa and a diameter of approximately 12 nm. A subunit of ferritin forms a four-helix bundle structure in which four α-helices are bundled (Fig. 1).

R. Tsukamoto, K.; Iwahori, M.; Muraoka, I.; Yamashita, Bull. Chem. Soc. Jpn. , 2005, 78, 2075-2081 K. Iwahori, I.; Yamashita, Nanotechnology, 2008, 19, 495601 I. Yamashita, K.; Iwahori, S.; Kumagai, Biochim. Biophys. Acta, 2010, 1800, 846-857

Ferritin has a spherical shell shape and exhibits higher thermal stability and pH stability than ordinary proteins. Therefore, ferritin is used in drug delivery system research. In addition to iron, ferritin can incorporate chromium or cobalt as oxides into the spherical shell (R. Tsukamoto, K. Iwahori, M. Muraoka, I. Yamashita, Bull. Chem. Soc. Jpn., 2005, 78, 2075-2081 (Non-Patent Document 1)). In addition, an example of synthesizing cadmium sulfide or zinc sulfide, which is a semiconductor material, in the spherical shell of ferritin has been reported (K. Iwahori, I. Yamashita, Nanotechnology, 2008, 19, 495601 (Non-Patent Document 2), I. Yamashita, K. Iwahori, S. Kumagai, Biochim. B. iophys.Acta, 2010, 1800, 846-857 (Non-Patent Document 3)). Due to the properties described above, ferritin is also expected to be applied to quantum dots or internal memories.

As described above, ferritin is one of the proteins for which various studies are being conducted as functional materials. However, most of them are ferritin 24-mers (ferritins consisting of 24 subunits). The production of ferritin gel is expected to promote the research and development of ferritin and its industrial application.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a gel composition and a dried gel product containing a polypeptide derived from a ferritin-like subunit as a main component.

As a result of intensive research, the present inventors have found that a gel composition can be obtained by making a solution containing ferritin subunits at a predetermined concentration into an acidic state and returning it to the original pH, and completed the present invention. That is, the present invention is as follows.

[1] A gel composition comprising a polypeptide having an amino acid sequence of a ferritin-like subunit and a liquid substance.
[2] The amino acid sequence of the ferritin-like subunit is
(A) an amino acid sequence set forth in any one of SEQ ID NOS: 1 to 4 in the sequence listing;
(B) an amino acid sequence in which at least one amino acid is substituted, deleted, inserted or added in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 in the sequence listing;
The gel composition according to [1], which consists of an amino acid sequence selected from the group consisting of:
[3] The gel composition according to [1] or [2], which has metal-trapping ability for Fe, Co, Ni, Cu, Pd or Pt.
[4] The gel composition according to any one of [1] to [3], wherein the liquid substance is water or alcohol.
[5] A dried gel obtained by removing the liquid substance from the gel composition according to any one of [1] to [4].

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a gel composition and a dried gel product containing a polypeptide derived from a ferritin-like subunit as a main component.

FIG. 2 is a schematic diagram showing the structures of a ferritin 24-mer and subunits of ferritin. It is a photograph explaining the procedure of gelation of ferritin. It is a photograph showing a study of water absorption in the gel composition according to the present embodiment. 4 is a photograph showing a study of the iron-trapping ability of the gel composition according to the present embodiment. 4 is a photograph (before addition of metal) showing a study of the metal-capturing ability of the gel composition according to the present embodiment. 4 is a photograph (after addition of metal) showing a study of the metal-capturing ability of the gel composition according to the present embodiment. 4 is a photograph (after washing with water) showing a study of the metal-capturing ability of the gel composition according to the present embodiment. 4 is a photograph (after addition of EDTA) showing a study of the metal-trapping ability of the gel composition according to the present embodiment.

Embodiments of the present invention will be described below, but the present invention is not limited to these. Here, in the present specification, the notation of the format "A to B" means the upper and lower limits of the range (that is, A to B or less), and when no unit is described in A and only a unit is described in B, the unit of A and the unit of B are the same.

<<Gel composition>>
The gel composition according to this embodiment contains a polypeptide having a ferritin-like subunit amino acid sequence (hereinafter sometimes referred to as "ferritin-like polypeptide") and a liquid substance. Since the gel composition contains a ferritin-like polypeptide, it has metal-capturing ability for Fe and the like. "Metal-capturing ability" will be described later.
In the present embodiment, the term "gel composition" refers to a colloid in which solid dispersoids are dispersed in a liquid dispersion medium, and which has lost fluidity and solidified.
In the present embodiment, "ferritin" means ferritin derived from any known biological species. Examples of such ferritin include horse-derived ferritin (ferritin composed of a subunit having the amino acid sequence set forth in SEQ ID NO: 2), human-derived ferritin (ferritin composed of a subunit having the amino acid sequence set forth in SEQ ID NO: 3), mouse-derived ferritin (ferritin composed of a subunit having the amino acid sequence set forth in SEQ ID NO: 4), and the like.

<Polypeptide having ferritin-like subunit amino acid sequence>
In the present embodiment, the "ferritin-like subunit" includes subunits that constitute ferritin and variants thereof. In other words, the “ferritin-like subunit” in this embodiment includes both wild-type ferritin subunits and mutant ferritin subunits. A subunit of wild-type ferritin includes a subunit having an amino acid sequence set forth in any one of SEQ ID NOS: 2-4. Subunits of mutant ferritin include subunits having the amino acid sequence set forth in SEQ ID NO:1.

In this embodiment, "polypeptide" means a molecule formed by peptide bonding of two or more amino acids. In the present embodiment, the ferritin-like polypeptide preferably contains a polypeptide consisting of an amino acid sequence of 160 to 190 amino acid residues, more preferably a polypeptide consisting of an amino acid sequence of 165 to 185 amino acid residues, even more preferably a polypeptide consisting of an amino acid sequence of 168 to 183 amino acid residues.

The amino acid sequence of the ferritin-like subunit is
(A) an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 in the sequence listing;
(B) an amino acid sequence in which at least one amino acid is substituted, deleted, inserted or added in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 in the sequence listing;
It preferably consists of an amino acid sequence selected from the group consisting of

Regarding the amino acid sequence (B) above, the number of substituted, deleted, inserted or added amino acid residues (mutated amino acid residues) in the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 4 in the sequence listing is at least 1, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5. The mutated amino acid sequence may be a naturally occurring amino acid sequence, that is, an amino acid sequence having spontaneous mutations, or an amino acid sequence artificially mutated at a desired site. When artificially mutating a desired site, it may be artificially mutated according to an amino acid sequence having naturally occurring mutations.

In the amino acid sequence (C) above, the sequence identity is a value calculated by appropriately aligning the query sequence (amino acid sequence to be evaluated) with the reference sequence (for example, the amino acid sequences set forth in SEQ ID NOs: 1 to 4 in the sequence listing). Specifically, sequence identity can be calculated with the CLUSTAL algorithm.

In this embodiment, the higher the sequence identity, the better, specifically 90% or more, 95% or more, or 98% or more. Although the upper limit of sequence identity is not particularly limited, it is, for example, less than 100%.

The content of the ferritin-like polypeptide may be 10% by mass or more and less than 100% by mass, may be 20% by mass or more and less than 100% by mass, or may be 20% by mass or more and 50% by mass or less.

In addition, the ferritin-like polypeptide is not particularly limited in terms of method of acquisition or production, and may be, for example, a polypeptide derived from wild-type ferritin, a recombinant polypeptide, or a synthesized polypeptide.

"Wild-type ferritin" includes, for example, the above-described horse-derived ferritin, human-derived ferritin, mouse-derived ferritin, and the like. These can be obtained from the blood of the mammals mentioned above. Specifically, a plasma component is collected from the blood, and ferritin is purified from the collected plasma component by a known method.

A "recombinant polypeptide" means a polypeptide artificially produced using genetic recombination technology using Escherichia coli, yeast, cultured cells, or the like as a host. As a method for producing a recombinant polypeptide, a conventionally known method may be used. Specifically, the following methods are mentioned, for example. First, a vector having a nucleotide sequence encoding a desired polypeptide is introduced into E. coli as a host for transformation. By culturing the transformed E. coli in a predetermined medium, the E. coli is allowed to synthesize the desired polypeptide.

A "synthetic polypeptide" means a polypeptide produced by sequentially linking starting amino acids. Methods of synthesizing from amino acids include, for example, solid-phase synthesis and liquid-phase synthesis. Solid-phase synthesis methods include, for example, the Fmoc method and the Boc method. The polypeptide according to this embodiment may be synthesized by any method.

For example, the above polypeptide can be synthesized by a known solid-phase synthesis method in which proline is immobilized on a polystyrene carrier and a fluorenyl-methoxy-carbonyl group (Fmoc group) or tert-Butyl OxyCarbonyl group (Boc group) is used as amino group protection.

<Liquid substance>
In the present embodiment, the "liquid substance" is not particularly limited as long as the gel composition can exist stably. Preferably, the liquid substance is water or alcohol. Examples of the alcohol include methanol, ethanol, propanol and butanol. From the viewpoint of biocompatibility, the liquid substance is preferably water.

The content of the liquid substance may be greater than 0% by mass and 90% by mass or less, may be greater than 0% by mass and may be 80% by mass or less, or may be 50% by mass or more and 80% by mass or less.

<Other ingredients>
The gel composition in this embodiment may contain other components as long as the gel composition can exist stably. Other components include, for example, buffers, pH adjusters, inorganic salts and the like.

<<Physical properties of the gel composition>>
<Metal capturing ability>
The gel composition according to the present embodiment preferably has a metal trapping ability for Fe, Co, Ni, Cu, Pd or Pt. Here, the term "metal trapping ability" means the ability to keep metal atoms or metal ions in the gel composition and prevent them from diffusing out of the gel composition. Since the gel composition contains a ferritin-like polypeptide, it is particularly excellent in trapping Fe and Co. Since the gel composition has a metal-trapping ability for a given metal, it can be used, for example, to remove toxic metals from industrial wastewater.

<Thermal stability>
The gel composition according to this embodiment is preferably stable at 25-60°C. Here, "stable at 25 to 60°C" means that the gel composition maintains its gel state at 25 to 60°C without causing decomposition, denaturation, or the like.

≪Gel dried product and its physical properties≫
The dried gel according to the present embodiment is obtained by removing the liquid substance from the gel composition. In one aspect of the present embodiment, it is preferable that the dried gel material has absorbability for the liquid substance. That is, the dried gel can be suitably used, for example, as a water absorbing material.

<<Method for producing gel composition>>
The method for producing a gel composition according to this embodiment includes:
a step of preparing a ferritin solution containing a polypeptide having an amino acid sequence of a ferritin-like subunit (hereinafter sometimes referred to as "first step");
a step of lowering the pH of the ferritin solution (hereinafter sometimes referred to as a “second step”);
and a step of increasing the pH of the ferritin solution (hereinafter sometimes referred to as a “third step”). Each step will be described below.

<First step>
In the first step, a ferritin solution containing a polypeptide having an amino acid sequence of ferritin-like subunits (ferritin-like polypeptide) is prepared. A ferritin-like polypeptide may be produced by a known method as described above.

Solvents in the ferritin solution include, for example, water, physiological saline, Tris-HCl buffer, other buffers, inorganic salt water, and the like. "Tris" is an abbreviation for trishydroxymethylaminomethane.

The concentration of the ferritin-like polypeptide (subunit unit concentration) is preferably above 6 mM, more preferably above 6 mM and 24 mM or less. By setting the concentration of the ferritin-like polypeptide within the above range, gelation tends to proceed easily in the subsequent steps.

The pH of the ferritin solution is preferably 5 or more, more preferably 7 or more and 9 or less.

<Second step>
In the second step, the pH of the ferritin solution is lowered. Although the method for lowering the pH of the ferritin solution is not particularly limited, for example, a method of adding a predetermined amount of 1M hydrochloric acid to the ferritin solution can be used. By lowering the pH, the ferritin-like polypeptide in the ferritin solution is denatured and becomes cloudy (for example, (A) in FIG. 2).

In the second step, the pH of the ferritin solution is preferably 1 or less, more preferably 0.5 or more and less than 0.7, and even more preferably 0.5 or more and 0.6 or less. By lowering the pH to the range described above, there is a tendency for gelation to proceed more easily in subsequent steps.

<Third step>
In the third step, the pH of the ferritin solution is raised. A method for increasing the pH of the ferritin solution is not particularly limited, but an example thereof includes a method of adding a predetermined amount of 1M sodium hydroxide aqueous solution to the ferritin solution. By raising the pH, the ferritin-like polypeptide in the ferritin solution is refolded, gelation proceeds, and a gel composition is obtained (eg, (B) in FIG. 2).

In the third step, the pH of the ferritin solution is preferably 5 or more, more preferably 7 or more and 9 or less. By increasing the pH to the above range, gelation tends to proceed.

The gel composition obtained as described above may be used for the purpose as it is. Alternatively, the gel composition can be immersed in a target liquid substance to replace the solvent of the ferritin solution with the liquid substance, and then used for the target application.

Furthermore, after the third step, the gel composition is dried by a known method to remove the liquid substance, thereby obtaining a dried gel.

EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these Examples. Unless otherwise stated, various manipulations were performed according to the Molecular Cloning A Laboratory Manual, Second Edition (Sambrook, J. et al., 1989).

<<Preparation of gel composition containing horse-derived ferritin-like polypeptide>>
<Preparation of horse-derived ferritin-like polypeptide>
(Preparation of LB medium)
LB Broth (manufactured by Nacalai Tesque Co., Ltd., trade name, 50 g) was dissolved in tap water (2 L) to obtain a pre-sterilized Luria-Bertani medium (LB medium). The obtained LB medium was sterilized by autoclaving (120° C., 1.2 atm, 20 minutes). The sterilized LB medium was allowed to cool to room temperature. Thereafter, 1 mL of an ampicillin solution (Amp, Wako Pure Chemical Industries, Ltd., 100 μg/mL) that had been passed through a sterilizing filter (pore size 0.22 μm, Merck Millipore) was added per 1 L of the LB medium. An LB medium containing ampicillin was prepared by the above procedure.

(Preparation of Escherichia coli containing a horse-derived ferritin-like gene)
A gene encoding the amino acid sequence of the horse-derived ferritin-like subunit (SEQ ID NO: 1) (hereinafter sometimes referred to as "horse-derived ferritin-like gene") was inserted into the multicloning site of the plasmid (pMK2) by a known genetic engineering method to construct a plasmid having the horse-derived ferritin-like gene. Then, the constructed plasmid was introduced into Escherichia coli (trade name: NovaBlue, manufactured by Merck Millipore) by a known genetic engineering technique. E. coli into which a plasmid containing a horse-derived ferritin-like gene was introduced was prepared by the above procedure. The E. coli was stored at -80°C as a glycerol stock. The amino acid sequence set forth in SEQ ID NO: 1 is an amino acid sequence obtained by deleting the N-terminal 8 amino acid residues in the amino acid sequence of the horse-derived ferritin subunit (SEQ ID NO: 2).

(Escherichia coli culture)
(1) Pre-preculture Escherichia coli into which a plasmid having a horse-derived ferritin-like gene was introduced was inoculated from a glycerol stock into 2 mL of LB medium and cultured with shaking (37° C., 200 rpm, 14 hours).
(2) Pre-Culture 2 mL of the pre-pre-cultured culture solution was added to 300 mL of LB medium and cultured with shaking (37° C., 170 rpm, 5 hours).
(3) Main Culture 50 mL of the precultured culture solution was added to 2 L of LB medium and cultured with shaking (37° C., 110 rpm, 24 hours). E. coli containing a polypeptide having an amino acid sequence of a horse-derived ferritin-like subunit (ferritin-like polypeptide) was cultured according to the above procedure.

(Preparation of crude product of horse-derived ferritin-like polypeptide)
(1) The E. coli-containing culture solution obtained in the above step was centrifuged (4° C., 8000 rpm, 6 minutes) to recover the E. coli as a precipitate (cell mass: about 50 g).
(2) The obtained cells were frozen with liquid nitrogen. Thereafter, 10 mL of 50 mM Tris-HCl buffer (pH 8.0) was added to 1 g of the cells to suspend the cells. The suspension containing the cells was stirred at 4°C for 10 minutes.
(3) The resulting suspension (10 mL, 10 g of cells) was sonicated on ice (output 40%, 1 second crush-2 second stop cycle, total 10 minutes).
(4) The suspension after sonication was centrifuged (4°C, 15000 rpm, 15 minutes). The supernatant was then collected.
(5) The recovered supernatant was incubated (60°C, 20 minutes), centrifuged (4°C, 17000 rpm, 10 minutes), and the supernatant was recovered. A crude ferritin-like polypeptide was obtained by the above procedure.

(Purification of horse-derived ferritin-like polypeptide)
(1) First, the anion exchange column carrier Q-Sepharose (GE Healthcare) was equilibrated with 50 mM Tris-HCl buffer (pH 8.5). Thereafter, the extracted crude ferritin-like polypeptide was adsorbed onto the anion exchange column carrier. Subsequently, 50 mM Tris-HCl buffer (pH 8.5) containing 200 mM NaCl was run through the anion exchange column. After that, a 50 mM Tris-HCl buffer (pH 8.5) containing 300 mM NaCl was passed through the anion exchange column to elute the ferritin-like polypeptide.
(2) A solution of the extracted ferritin-like polypeptide (hereinafter sometimes referred to as "ferritin solution") was diluted twice with 50 mM Tris-HCl buffer (pH 8.5), and the solution was passed through a filter (0.22 μm pore size, manufactured by Merck Millipore). The filtered ferritin solution was purified by anion exchange column chromatography using FPLC (Fast Protein Liquid Chromatography) (BioLogic DuoFlow system, manufactured by BIO-RAD). FPLC conditions are shown below.
[FPLC conditions]
Column: HiTrap Q HP (manufactured by GE Healthcare)
Flow rate: 3.0 mL/min
Buffer A: 50 mM Tris-HCl buffer (pH 8.5)
Buffer B: 50 mM Tris-HCl buffer (pH 8.5) containing 1.0 M NaCl
Gradient: 0→50% B (80 mL)
Detection wavelength: 280 nm

(3) Amicon Ultra-15 (MWCO 10000, manufactured by Merck Millipore) was used to concentrate the purified ferritin solution (about 30 mL) to about 10 mL. The concentrated ferritin solution was passed through a filter to remove impurities such as precipitates. After that, the ferritin solution was purified by gel filtration column chromatography using FPLC (AKTA prime plus, GE Healthcare). FPLC conditions are shown below.
[FPLC conditions]
Column: HiPrep 26/60 Sephacryl S-300 HR (manufactured by GE Healthcare)
Flow rate: 1.0 mL/min Buffer: 50 mM Tris-HCl buffer (pH 8.5) containing 150 mM NaCl
Detection wavelength: 280 nm

(4) The purity of ferritin in the obtained ferritin solution was confirmed by SDS-PAGE and found to be 95% or more.
Horse-derived ferritin-like polypeptides were prepared by the above procedure.

<Gelling of ferritin-like polypeptide>
(Generation of gel composition containing horse-derived ferritin-like polypeptide)
(1) The purified ferritin solution was concentrated to approximately 12 mM (molar concentration per subunit) using Amicon Ultra-15 (MWCO 10000, Merck Millipore).
(2) 10 μL of 1.0 M hydrochloric acid was added to a trace amount (about 10 μL) of the concentrated ferritin solution to denature the ferritin-like polypeptide to a white color (pH 0.5) ((A) in FIG. 2).
(3) After that, 10 μL of 1.0 M sodium hydroxide aqueous solution was added to the solution containing the denatured ferritin-like polypeptide to raise the pH of the solution to 8.5. A gel composition containing the horse-derived ferritin-like polypeptide was produced by the above procedure (FIG. 2(B)).

(Examination of gelation conditions)
(1) Ferritin solutions were prepared to 3 mM, 6 mM, 12 mM and 24 mM (all subunit molar concentrations).
(2) Hydrochloric acid was added to 10 μL of each ferritin solution so that the pH at the time of acid denaturation was 0.5 (about 10 μL of 1.0 M hydrochloric acid), 0.6 (about 5.5 μL of 1.0 M hydrochloric acid), 0.7 (about 3.5 μL of 1.0 M hydrochloric acid) and 0.8 (about 2 μL of 1.0 M hydrochloric acid). Thereafter, the pH of the ferritin solution was raised (pH 8.5) with an aqueous sodium hydroxide solution having the same amount and concentration as the added hydrochloric acid. In the above procedure, it was verified whether or not the ferritin-like polypeptide was gelled under different conditions of different molar concentrations of the ferritin-like polypeptide and pH during acid denaturation (pH in the second step). Table 1 shows the results. From the results in Table 1, it was found that gelation occurs when the molar concentration of ferritin-like polypeptide in the ferritin solution in the first step is 12 mM or higher and the pH in the second step is 0.6 or lower. It was found that the gelation depends on the pH of the solution at the time of acid denaturation and the molar concentration of the ferritin-like polypeptide.

<<Physical properties of the gel composition>>
(solubility)
The solubility of the produced gel composition was verified by the following procedure. First, about 1 mL of pure water, 1.0 M hydrochloric acid, 1.0 M sodium hydroxide aqueous solution, methanol (manufactured by Wako), 2-mercaptoethanol (manufactured by Wako) (liquid), or 43 μM trypsin (manufactured by Sigma-Aldrich) The gel composition was immersed in an aqueous solution and allowed to stand at room temperature for 1 week. After that, it was verified whether the gel composition was dissolved. As a result, the gel composition was insoluble in hydrochloric acid, sodium hydroxide aqueous solution, methanol and 2-mercaptoethanol solution, and maintained its gel form. On the other hand, when an aqueous trypsin solution was added, the gel composition gradually decomposed and finally dissolved. This result suggested that the gel composition had protease decomposability and that the gel composition was formed of a ferritin-like polypeptide.

(Thermal stability)
In order to obtain information on the thermal stability of the gel composition produced, the gel composition was immersed in pure water at 20° C. to 100° C. for 30 minutes, and the shape was observed with the naked eye. As a result, the gel composition gradually decomposed at 80°C and quickly decomposed at 100°C. On the other hand, even when incubated at 60° C. or lower, the shape of the gel composition hardly changed and was stable. These results suggested that the gel composition had thermal stability similar to that of wild-type ferritin.

(composition)
Ultrapure water was added to a portion of the prepared gel composition and suspended by vortexing to obtain a suspension. Subsequently, a mixed solution of 0.1% TFA/MilliQ (25 μL) and 0.1% TFA/MeCN (50 μL) was saturated with about 5 mg of sinapinic acid to prepare a matrix solution. The resulting suspension (1 μL) and the above matrix solution (8 μL) were mixed to obtain a mixed solution. The resulting mixed solution was placed on a plate for mass spectrometry and analyzed with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS). As a result, a peak corresponding to the molecular weight of the ferritin subunit (theoretical value: [ferritin subunit+H] ⁺ =19148 Da) was detected. These results revealed that the obtained gel composition was formed of a ferritin-like polypeptide.

(water absorption)
The gel composition was dehydrated by heat drying at 100° C. for 10 minutes to obtain a dried gel. After that, ultrapure water was added to the resulting dried gel, and the mixture was incubated at room temperature for 1 hour to obtain a gel composition again. As a result of adding pure water to the dried gel after dehydration ((A) in FIG. 3), the gel composition swelled about 3 times in length and about 5 times in mass ((B) in FIG. 3). When the moisture content was calculated by mass contrast in this series of operations, it was found to be about 80% on average. When the water-swollen gel composition was heat-dried in the same manner as above, a dried gel was obtained again ((C) in FIG. 3). From the above results, it was found that the dried gel product has water absorption ability (water absorption).

(Iron scavenging ability)
(Experiment 1) An aqueous solution of 30 mM ammonium iron(II) sulfate (manufactured by Nacalai Tesque) was added to the prepared gel composition and incubated at room temperature for 24 hours. Observation of the gel composition after incubation revealed a color change from light yellow to reddish brown. After the incubation, the gel composition was washed with ultrapure water and observed to remain reddish brown. The mass of the gel composition increased by about 30% compared to before immersion in the ammonium iron(II) sulfate aqueous solution.

(Experiment 2) The same operation as in (Experiment 1) was performed using an aqueous solution of 30 mM iron (III) chloride (manufactured by Kanto Kagaku Co., Ltd.). Observation of the obtained gel composition revealed that the color had changed from pale yellow ((A) in FIG. 4) to reddish brown ((B) in FIG. 4). After the incubation, the gel composition was washed with ultrapure water and observed to remain reddish brown. The mass of the gel composition increased by about 20% compared to before immersion in the iron (III) chloride aqueous solution.

(Experiment 3) An aqueous solution of 30 mM ammonium iron(II) sulfate (manufactured by Nacalai Tesque) was added to the prepared gel composition and incubated at room temperature for 1.5 hours. Observation of the gel composition after incubation revealed a change from pale yellow to dark green. After the incubation, the gel composition was washed with ultrapure water and incubated at room temperature for 24 hours, whereupon the gel composition turned reddish brown.

From the results of (Experiment 1) to (Experiment 3), it was found that the gel composition retains the iron-trapping ability similar to ferritin. It was found that the above gel composition binds to divalent iron ions and trivalent iron ions, and the divalently bound iron ions become trivalent. In addition, adding a chelating agent to the gel composition caused no change, suggesting that the iron ions remained inside the gel composition.

(Other metal trapping abilities)
In order to examine the ability to capture metals other than iron, gel compositions were prepared by the same procedure as described above ((A) to (D) in FIG. 5). An aqueous solution (30 mM) of metal salt was added to the prepared gel composition and incubated at room temperature for 2 hours. The metal salts used at this time were iron(III) chloride, copper chloride, cobalt chloride and nickel chloride. Observation of the gel composition after incubation revealed a change in color from light yellow to a color corresponding to each metal salt. Specifically, in the case of iron (III) chloride, the color of the gel composition changed from light yellow to reddish brown ((A) in FIG. 6). In the case of copper chloride, the color of the gel composition changed from light yellow to greenish blue ((C) in FIG. 6). In the case of cobalt chloride, the gel composition changed color from light yellow to pale purple ((B) in FIG. 6). In the case of nickel chloride, the gel composition changed color from pale yellow to pale orange ((D) in FIG. 6). Each of the gel compositions after incubation was washed with ultrapure water (at room temperature for 18 hours), and was observed to remain discolored ((A) to (D) in FIG. 7). From the above results, it was found that the gel composition retains the ability to capture metals other than iron as well as ferritin.

The metals (Fe, Ni, Co, and Cu) captured by the gel composition in the above experiments are all transition metals, and ferritin is known to capture transition metals other than Fe, such as Ni, Co, Cu, Pd (palladium), and Pt (platinum). Therefore, the present inventors believe that the gel composition has a metal capturing ability even for Pd and Pt, which are the same transition metals.

When the gel composition washed with ultrapure water was immersed in a 100 mM ethylenediaminetetraacetic acid aqueous solution (EDTA aqueous solution) and incubated for 3 hours, metals other than iron decolorized, and it was confirmed that the metal was liberated from the gel composition ((A) to (D) in FIG. 8).

Although the embodiments and examples of the present invention have been described as above, it is planned from the beginning to appropriately combine the configurations of the above-described embodiments and examples.

It should be considered that the embodiments and examples disclosed this time are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the scope of the claims rather than the above-described embodiments and examples, and is intended to include meanings equivalent to the scope of the claims and all modifications within the scope.

Claims (5)

A gel composition comprising a polypeptide having a ferritin-like subunit amino acid sequence and a liquid substance,
The gel composition is a gel composition, wherein the polypeptide forms a gel. The amino acid sequence of the ferritin-like subunit is
(A) an amino acid sequence set forth in any one of SEQ ID NOS: 1 to 4 in the sequence listing;
(B) an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, inserted or added to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 in the sequence listing;
2. The gel composition of claim 1, which is an amino acid sequence selected from the group consisting of: 3. The gel composition according to claim 1 or 2, which has metal scavenging ability for Fe, Co, Ni, Cu, Pd or Pt. The gel composition according to any one of claims 1 to 3, wherein said liquid substance is water or alcohol. A gel dried product obtained by removing the liquid substance from the gel composition according to any one of claims 1 to 4.