JP5458299B2 - Method for purifying sweetness-inducing substance miraculin - Google Patents

Description

  The present invention relates to a method for purifying miraculin protein.

  Miracrine contained in miracle fruit is known as a protein having a taste-modifying activity that induces sweetness by acidity (Non-patent Document 1). The total amino acid sequence of miraculin protein has been determined (Non-patent document 2), and the full-length cDNA sequence of the gene has also been reported (Non-patent document 3; GenBank accession number D38598).

  There have been several reports on refining miraculin from miracle fruit. Japanese Patent Laid-Open No. 62-242700 (Patent Document 1) discloses a method for extracting miraculin from miracle fruit pulp using a neutral salt solution around pH 6-9 as an extraction solvent. Further, according to the method of JP-A-63-185349 (Patent Document 2) and Theeracilp et al. (Non-Patent Document 4), after washing the miracle fruit pulp powder with water, a neutral sodium chloride aqueous solution is added. Extract miraculin and then purify using ammonium sulfate precipitation, CM Sepharose ion exchange column and affinity column. Furthermore, JP-A-6-17288 (Patent Document 3) efficiently produces miraculin with less colored impurities by extracting miraculin from a miraculin-containing raw material with a solution of an acidic buffer salt solution pH 3.5 to 5.5. A method is described.

  However, the method of Patent Document 1 has a problem that many impurities are mixed. On the other hand, in the methods of Patent Document 2 and Non-Patent Document 4, although high-purity miraculin is purified, since there are many purification steps, purification takes 1 to 2 weeks and a recovery loss tends to occur. Furthermore, there is a problem that the purification cost is increased. In addition, with respect to the method of Patent Document 3, since this step alone has a problem in terms of purity, it is actually necessary to combine another purification method. For this reason, a method that can recover miraculin with high purity and high efficiency is still desired.

JP-A-62-242700 JP-A 63-185349 JP-A-6-172388 Kurihara and Beidler, Science (1968) 161 (847), p.1241-1243 Theerasilp et al., J. Biol. Chem., (1989) 264 (12), p.6655-6659 Masuda et al., Gene (1995) 161, p.175-177 Theeracilp et al., J. Biol. Chem., (1988) 23, p.11536-11539

  An object of the present invention is to provide a highly efficient method for purifying miraculin.

  As a result of intensive studies to solve the above problems, the present inventors have found that miraculin can be purified with high efficiency by a method using a metal chelate carrier, and have completed the present invention.

That is, the present invention includes the following.
[1] A method for purifying miraculin, which comprises contacting a miraculin-containing liquid with a carrier chelated with metal ions and eluting the adsorbate on the carrier by pH gradient elution.
[2] The method according to [1] above, wherein the adsorbate is eluted by pH gradient elution using an eluate containing no imidazole.
[3] The method according to [1] or [2] above, wherein the pH gradient elution is elution by a gradient from pH 6.6 to pH 6.0.
[4] The method according to [1] to [3] above, wherein the pH gradient elution is pH linear gradient elution or pH stepwise gradient elution.
[5] The method of [1] to [4] above, wherein the metal ion is a divalent metal ion.
[6] The method of [5] above, wherein the metal ion is nickel ion.
[7] The method according to [1] to [6] above, wherein the miraculin protein is monomeric and / or multimeric miraculin.

  According to the method of the present invention, it is possible to purify miraculin with high purity and high efficiency.

Hereinafter, the present invention will be described in detail.
The present invention provides a method for purifying miraculin with high purity and high efficiency by affinity chromatography using a carrier (metal chelate carrier) in which a metal ion is chelate-bound using the characteristic three-dimensional structure of miraculin. provide.

  In the method of the present invention, miraculin is purified with high purity and a high recovery rate by contacting the miraculin-containing solution with a carrier chelated with metal ions and eluting the adsorbate on the carrier by pH gradient elution. .

  In the present invention, “milacrine-containing liquid” means any aqueous liquid containing miraculin protein to be purified by the method of the present invention. The aqueous liquid may be an aqueous solution, a suspension, a mixed solution, or the like. It is preferable that the miraculin-containing liquid does not contain a protein denaturant such as urea, a surfactant, and alcohol at least in an amount that causes protein denaturation, and more preferably does not contain such a protein denaturant. The miraculin-containing liquid in the present invention may preferably be an extract with a neutral buffer salt solution from the fruit of miracle fruit. For example, miraculin extracted with an extraction buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 7.2). It may be a crude extract.

  The miraculin purified by the method of the present invention may be a natural product contained in miracle fruit pulp or the like, or may be artificially produced by a genetic recombination technique known to those skilled in the art. Good. The miraculin in the present invention is typically composed of a mature miraculin protein, particularly a mature miraculin protein having a known amino acid sequence (shown in the sequence information of GenBank Accession No. D38598). It may be composed of a precursor protein in which a signal peptide is added to a miraculin protein, a fusion protein in which a labeled peptide is added, or a mutant protein as long as it has sweetness-inducing activity.

  In the method of the present invention, monomeric miraculin or multimeric miraculin (more preferably dimeric miraculin) can be suitably purified. In the present invention, “monomer miraculin”, “dimer miraculin” and “multimeric miraculin” are monomers, dimers each composed of one, two or plural mature miraculin proteins, respectively. It means miraculin, which is a body and multimer. Multimeric miraculins such as dimers or tetramers are known to be active miraculins having sweetness-inducing activity (taste-modifying activity) (Paladino et al., Biochem Biophys Res Commun (2008) 367, p.26-32). Further, miraculin that can be purified by the method of the present invention is preferably non-denatured.

  In the method of the present invention, miraculin contained in the miraculin-containing liquid is adsorbed to the carrier by bringing the above-mentioned miraculin-containing liquid into contact with a carrier chelated with (charged) metal ions. As the metal ion, a divalent metal ion is more preferable. For example, copper ion (Cu (II)), zinc ion (Zn (II)), nickel ion (Ni (II)), cobalt ion (Co (II)) , Iron (II) ion (Fe (II)), magnesium ion (Mg (II)), manganese ion (Mn (II)), cadmium ion (Cd (II)), lead ion (Pb (II)), calcium Examples thereof include, but are not limited to, ions (Ca (II)).

  The carrier for chelating the metal ion is not particularly limited, and various carriers used for metal chelate affinity chromatography (IMAC) can be used. Preferable specific examples of the carrier include Chelating Sepharose Fast Flow (GE Healthcare), IMAC Sepharose High Performance (GE Healthcare), IMAC Sepharose 6 Fast Flow (GE Healthcare), IMAC Profinity (Bio-Rad) and the like. It is also preferable to use a carrier packed with a metal ion in a form packed in a column, and various columns packed with such a carrier are commercially available (for example, HiTrap IMAC HP (GE Healthcare), HiTrap IMAC FF (GE Healthcare), Bio-Scale Mini Ni-IMAC Profinity Cartridge (Bio-Rad), etc.).

  A carrier to which a metal ion is chelate-bonded can be prepared by introducing the metal ion into the carrier or a column packed with the carrier by a conventional method. For example, when nickel ions are bound to the carrier, as an example, a nickel sulfate solution may be added to the carrier. It is preferable to equilibrate the carrier chelated with metal ions with an appropriate buffer before use.

  Protein extracts from miracle fruits tend to tend to acidic pH due to components contained in miracle fruit fruits. Therefore, when the miraculin-containing liquid shows an acidic pH or is highly likely, the liquid is adjusted to near neutral (preferably pH 6.7 to 7.5, more preferably pH 7.2) before contacting with the metal chelate carrier. It is more preferable to adjust to). The pH adjustment may be performed by adding 1N NaOH, for example.

  In the method of the present invention, the adsorbate such as miraculin adsorbed on the metal chelate carrier from the miraculin-containing liquid as described above is eluted using the eluate. It is preferable to wash the carrier before elution of the adsorbate. A person skilled in the art can select and use an appropriate washing solution, but it has the same composition as the eluate used to elute the adsorbate and is adjusted to a pH equal to or higher than the maximum pH of the eluate. It is preferable to use what was done.

  The elution of the adsorbate on the carrier is preferably performed by pH gradient elution. “PH gradient elution” refers to a technique in which elution is carried out using an eluate whose pH is changed so as to draw an increase or decrease gradient. The pH gradient elution in the present invention is preferably performed by a method of decreasing the pH of the eluate continuously (linearly) or stepwise (steps). The pH gradient elution which changes pH continuously is called “pH linear gradient elution”, and the pH gradient elution which changes pH stepwise is called “pH stepwise gradient elution”. In the method of the present invention, the pH gradient elution is preferably elution using a pH decrease gradient near neutrality, for example, elution using a pH decrease gradient in an arbitrary range of pH 7.0 to pH 4.0 is more preferable. Elution using a pH decrease gradient within a predetermined range of pH 7.0 to pH 5.6 is particularly preferable, and elution by a gradient (gradient) from pH 6.6 to pH 6.0 is more preferable. In the method of the present invention, miraculin can be eluted more specifically from the metal chelate carrier while minimizing the contamination of impurities by such pH gradient elution.

  In the present invention, the eluent used for pH gradient elution should be a buffer solution having a high buffer capacity in a relatively acidic region, such as a citrate buffer solution, an acetate buffer solution, a succinate buffer solution, and a phthalate buffer solution. preferable. Usually, an imidazole solution is used when a protein having a His tag bound to a metal chelate column is eluted. However, in the method of the present invention, it is preferable to use an eluate containing no imidazole. This is because when the carrier adsorbate is eluted using the imidazole solution in the method of the present invention, all the protein adsorbed on the carrier is eluted, and the purity of the eluted miraculin is reduced. As a suitable specific example of the eluate used for pH gradient elution, for example, when a carrier chelate-bonded with nickel ions is used, an elution buffer (50 mM acetate buffer solution) used as a linear gradient of pH 6.6 to pH 6.0. , 0.5M NaCl).

  The eluate from the carrier obtained as described above contains miraculin with high purity and high recovery rate. This can be confirmed by subjecting the eluate from the carrier to SDS-PAGE and performing Western blot analysis using an anti-miraculin antibody on the gel after electrophoresis.

  By the method of the present invention, purified miraculin can be obtained with a purity of generally 70% or more, usually 80% or more, preferably 90% or more, and particularly preferably 95% or more.

  Further, according to the method of the present invention, it is generally 60% or more, usually 70% or more, preferably 80% or more, compared to the miraculin content contained in the miraculin-containing solution subjected to the method. Particularly preferably, purified miraculin can be recovered at a recovery rate of 85% or more.

  So far, two histidine residues in the miraculin monomer and a total of four histidine residues in the dimer (the histidine residues at positions 30 and 60 of the mature miraculin protein) have faced the outside of the conformation. It has been reported by structure prediction (Paladino et al., Biochem Biophys Res Commun (2008) 367, p.26-32). The sweetness-inducing active form of miraculin is usually a dimer, but recent studies have lost sweetness-inducing activity in miraculin, in which the histidine residue outside this conformation of dimeric miraculin is mutated to an alanine residue (Ito et al., Biochem. Biophys. Res. Commun., (2007) 360, p.407-411). In view of the fact that amino acids such as histidine are known to form complexes with metal ions chelated in the neutral pH range (pH 6-8), the method of the present invention allows for the second in miraculin protein. The four histidine residues facing outward when forming a mer are thought to be involved in the activity outside the three-dimensional structure of miraculin, and the histidine residues enable efficient adsorption to the metal chelate column. It was considered possible. Such a method of purifying miraculin as in the present invention using the three-dimensional characteristics of miraculin is a method based on a new idea that is not found in conventional methods.

  In the conventional miraculin purification method, the miracle fruit pulp powder is extracted with distilled water and 0.5M NaCl solution, and the extract is subjected to ammonium sulfate precipitation, CM sepharose ion exchange chromatography, and Con-A sepharose affinity chromatography to produce miraculin. It was purified. This method requires considerable time and cost because it requires ammonium sulfate precipitation and chromatography followed by dialysis and ultrafiltration. On the other hand, in the method of the present invention, miraculin is adsorbed on a metal chelate carrier by a method such as immobilized metal chelate affinity chromatography (IMAC) and eluted by pH gradient elution. As described above, even when a miraculin-containing liquid containing many other proteins is used as a raw material, it is possible to eliminate impurities such as other proteins to the maximum and perform purification with higher purity. In the method of the present invention, miraculin can be purified with high purity by such a simple procedure, so that the number of purification steps can be reduced as compared with the conventional method, thereby suppressing the recovery loss occurring at each step and reducing the recovery rate. In addition to being able to improve, it is also possible to obtain a shortening effect of the purification process, in which it can be purified to the same purity as that of the conventional method in 1 day, preferably from 1 to 2 weeks in the conventional method. Furthermore, since the method of the present invention does not require the use of a salt, imidazole or the like at the time of elution, the need for additional treatment such as desalting on miraculin after purification is reduced, and handling is relatively easy. The advantage that it becomes possible can also be obtained.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

1. Preparation of anti-miracrine antibody Anti-miracrine antibody used for detection of miraculin protein was prepared by the following method. A DNA fragment encoding the full-length amino acid sequence of a mature miraculin protein (a 191 amino acid long mature protein sequence obtained by removing the signal sequence of positions 1 to 29 from the 220 amino acid precursor sequence shown in GenBank accession number D38598) A recombinant expression vector was prepared by incorporating into plasmid pQE (Qiagen), and the vector was introduced into Escherichia coli by a conventional method to produce miraculin protein by a recombinant method. The obtained protein was administered as an antigen to a rabbit to produce a polyclonal antibody (rabbit anti-miracrine antibody) against miraculin protein (consigned to Scrum Inc., Tokyo, Japan). The obtained rabbit anti-miracrine antibody was prepared by ammonium sulfate precipitation (40% saturation), and then left the 89 amino acid residues at the N-terminal of mature miraculin protein recombinantly produced in E. coli using the expression vector pGEX-4T-1 (Amersham Biosciences). Purification was performed by immunoaffinity chromatography using a column Sepharose 4 Fast Flow (Amercham Biosciences) to which a polypeptide consisting of a group (amino acid residues at positions 1 to 89) was coupled. Peroxidase labeling was added to the obtained anti-miraclein antibody using Peroxidase Labeling Kit-SH (Dojindo, Japan).

2. Crude extraction of miraculin from miracle fruit The pulp of miracle fruit (Richadella dulcifica) was frozen in liquid nitrogen and then ground using a pestle and mortar. About 25 g of the obtained powdered pulp sample was suspended by adding 100 ml of distilled water, and centrifuged at 12,000 g for 20 minutes at 4 ° C. The supernatant (without miraculin) was discarded, suspended again with 100 ml of distilled water, and centrifuged at 12,000 g for 20 minutes at 4 ° C. The supernatant was discarded, and the precipitate was suspended in 50 ml of extraction buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 7.2), and centrifuged at 12,000 g for 20 minutes at 4 ° C. The supernatant was collected and used as a crude miraculin extract (protein crude extract).

3. The miraculin purification column was prepared by the following method using Immobilized Metal Affinity Chromatography (IMAC) . First, a 0.1M nickel sulfate solution was prepared and filtered through a 0.45 μm filter. The stopper was removed from the IMAC column (1 ml, HiTrap IMAC HP, GE Healthcare; using IMAC Sepharose High Performance as a carrier), connected to a syringe filled with distilled water, and the column was washed with 5 ml distilled water. Pour 0.5 ml of the 0.1M nickel sulfate solution prepared above onto the washed IMAC column, wash with 5 ml of distilled water, and then add 5 ml or more of binding buffer (50 mM Tris-HCl, 0.5 M NaCl, pH 7.2). A nickel-IMAC column was prepared by equilibrating the column with. The column was then connected to an AKTAdesign chromatography system (Amersham Pharmacia Biotech).

  Immediately after adjusting 10 mL of the above prepared miraculin crude extract (acidic) to pH 7.2 with 1N NaOH, it was applied to a nickel-IMAC column equilibrated with the binding buffer prepared as described above. The column is then washed with the same binding buffer as above to remove weakly binding proteins (flow rate 1 ml / min, volume 30 ml), and then 20 ml wash buffer (50 mM acetate buffer, 0.5 M). NaCl, pH 6.6) was added for washing.

  The protein adsorbed on the IMAC column was eluted with a linear gradient from pH 6.6 to pH 6.0 of about 30 ml of elution buffer (50 mM acetate buffer, 0.5 M NaCl). Further, the adsorbed protein remaining on the column was eluted with 20 ml of an elution buffer (50 mM acetate buffer, 0.5 M NaCl) at pH 4.0. The elution solution was collected at a flow rate of 1 ml / min and a fraction of 3 ml.

  The protein in the elution solution was monitored by absorbance at 280 nm (FIG. 1). Flow through fraction (0-20 ml), wash fraction 1 (42-60 ml), target fraction (60-75 ml), wash fraction 2 (102-120 ml) Each was collected.

4). Assay for recovery of miraculin 20 μl each of the fraction collected above and the crude extract of miraculin were electrophoresed on a 12% SDS-PAGE gel and transferred to a Hybond-P membrane (Amersham Biosciences). The transferred membrane was blocked overnight at 4 ° C in a TBS-T solution containing 5% skim milk (3 g / L Tris, 8 g / L NaCl, 0.2 g / L KCL, 0.05% Tween-20). The peroxidase-labeled anti-miraculin antibody diluted with a PBS-T solution was added, and incubated at room temperature for 30 minutes. Subsequently, the protein that showed immunoreactivity with the anti-miraculin antibody (miraculin protein) was developed using an immunoblotting system based on Western blotting (Peroxidase Stain Kit for Immuno-blotting, Nuclease tested, nacalai tesque inc. Kyoto, Japan) It was detected by reaction (FIG. 2B). In addition, a 12% SDS-PAGE gel obtained by subjecting the fraction collected above and miraculin crude extract to electrophoresis in the same manner was silver-stained using Silver Staining Kit (Wako) to detect the total protein (FIG. 2A). ).

  As a result, the crude extract of miraculin contained many proteins of various sizes (lane 1 in FIG. 2A), but the column was washed with a pH 6.6 buffer by passing the crude extract of miraculin through a nickel-IMAC column. As a result, most of the proteins other than miraculin could be highly removed (lanes 2 and 3 in FIGS. 1, 2A and B). Further, it was confirmed that miraculin having high purity could be purified by gradient elution at pH 6.6 to 6.0 (the peak of the target fraction in FIG. 1, lane 4 in FIGS. 2A and B).

5). Purity test by reverse phase HPLC Milacrine purified from the crude miraculin extract as described above was subjected to reverse phase HPLC to examine its purity. A SOURCE RPC column (0.46 x 15 cm, GE Healthcare) was used for reverse phase HPLC. 1 ml of the target fraction with sweetness-inducing activity collected above was applied to a SOURCE RPC column and then eluted with a 20-70% linear gradient of acetonitrile containing 1% trifluoroacetic acid at a flow rate of 1 ml / min. The protein in the eluate was monitored by absorbance at 280 nm. As a result of the measurement, the purity of this purified miraculin was 95% or more (FIG. 3, Table 1).

  Furthermore, based on the total protein content in the solution before and after purification by the nickel-IMAC column and the value of the purity of miraculin, the miraculin recovery rate by the purification method of the present invention was calculated. As a result, the miraculin recovery rate (purification efficiency) in the above purification was about 87% (Table 1).

  The numerical values in Table 1 show the average value and standard error of three replicate experiments. The total protein content and the purity of miraculin in 10 ml of crude extract of miraculin before purification of nickel-IMAC column (amount applied to the column) and in the purified miraculin solution after purification of nickel-IMAC column (target fraction: about 15 ml) Quantified from the results of Bradford method (using Quick start protein assay kit, Bio-Rad) and reverse phase HPLC, respectively. The amount of miraculin in each solution was calculated from the total protein amount and the purity value of miraculin, and the miraculin recovery rate was calculated based on this result.

  As shown in the above results, according to the method of the present invention, it was shown that the purification of miraculin protein with a purity of 95% or more from the miracle fruit pulp is possible in one day.

6). Confirmation of miraculin protein by N-terminal amino acid sequencing In order to confirm that the purified protein is miraculin, the N-terminal amino acid sequence was examined. Analysis was performed by Edman method using a Model 490 amino acid sequencer (Procise ^™ , Applied Biosystems).

The collected protein of the target fraction is subjected to SDS-PAGE electrophoresis, then transferred to Hybond-P membrane (Amersham Bioscience) by electroblotting, and brilliant blue staining (using Brilliant Blue R Staining, Sigma, USA). went. The membrane of the stained protein portion was cut out and the amino acid sequence on the N-terminal side was determined using a Model 490 amino acid sequencer (Procise ^™ ).

  As a result, the N-terminal amino acid sequence of the purified protein in the target fraction is Asp-Ser-Ala-Pro, which is completely identical to the N-terminal 4 amino acid residue sequence of the mature miraculin protein used. I did it. From this, it was confirmed that the protein purified by metal chelate affinity chromatography as described above was miraculin.

7). Cutting in highly purified miraculin with purified metal chelate IMAC column miraculin denatured the three-dimensional structure, in order to examine the influence of the conformation of miraculin, urea acts to denature protein conformation are known (hydrophobic bond The modified miraculin was subjected to metal chelate IMAC column chromatography in the same manner as described above. Specifically, urea was added to the purified miraculin solution to a concentration of 8M before being applied to the nickel-IMAC column. Urea was also added to the binding buffer to a concentration of 8M before being added to the nickel-IMAC column. After applying the purified miraculin solution to which urea was added to the nickel-IMAC column, washing and elution were performed in the same manner as described above, and the protein was monitored at an absorbance of 280 nm. The result is shown in FIG. By modifying the three-dimensional structure of miraculin with 8M urea, it was shown that miraculin could not be adsorbed on the nickel-IMAC column and flowed into the flow-through fraction (FIGS. 4A and B). This indicates that the three-dimensional structure of miraculin is important in the miraculin purification method using metal chelate affinity chromatography used in this example. This is because the high purity and high recovery achieved by this miraculin purification method are based on its characteristic conformation, including the histidine residues exposed outside in the conformation of active miraculin. It is a result which shows.

  The method of the present invention can be used for simple, efficient and high-purity purification of miraculin.

FIG. 1 shows the elution profile of miraculin on a nickel-IMAC column. FIG. 2 is a photograph showing the results of SDS-PAGE showing the protein in each eluted fraction. FIG. 2A shows the result of silver staining of SDS-PAGE gel, and FIG. 2B shows the result of Western blot analysis. XL ladder marker (APRO) was used for the marker (M). 20 μl was run in each lane. In the figure, the respective sizes of dimer or monomer miraculin are indicated by arrows. “Dimer ^* ” and “monomer ^* ” are bands in which miraculin is not sufficiently modified by three-dimensional structure by SDS. These bands were enhanced when the heat denaturation treatment prior to sample application was omitted. M: XL ladder marker, lane 1: crude extract of miraculin, lane 2: flow-through fraction, lane 3: wash fraction 1, lane 4: target fraction, lane 5: wash fraction 2 FIG. 3 shows the reverse phase HPLC profile of the target fraction. 1 ml of the target fraction obtained from the nickel-IMAC column (fraction containing miraculin) was applied. FIG. 4 is an elution profile and a photograph showing the purification results of miraculin denatured with 8 M urea. FIG. 4A shows the elution profile of miraculin denatured with 8 M urea on a nickel-IMAC column. FIG. 4B shows the results of Western blot analysis of the flow-through fraction (FL). It was shown that the majority of miraculin was recovered as a flow-through fraction.

Claims (5)

A method for purifying miraculin, which comprises contacting a miraculin-containing solution with a carrier chelated with a divalent metal ion, and eluting the adsorbate on the carrier by pH gradient elution. -tag) all SANYO is not added, and a dimer miraculin method.   The method according to claim 1, wherein the adsorbate is eluted by pH gradient elution using an eluate containing no imidazole.   The method according to claim 1 or 2, wherein the pH gradient elution is a gradient elution from pH 6.6 to pH 6.0.   The method according to any one of claims 1 to 3, wherein the pH gradient elution is pH linear gradient elution or pH stepwise gradient elution. The method according to any one of claims 1 to 4 , wherein the divalent metal ion is a nickel ion.

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