Peptide sequence

Abstract

Provided are: a peptide having good binding properties with isovaleric acid; and a composition for isovaleric acid measurement and an odor sensor each using the peptide. The peptide comprises an amino acid sequence represented by any one of SEQ ID NOS: 1-13. The composition for isovaleric acid measurement and the odor sensor contain a peptide comprising an amino acid sequence represented by any one of SEQ ID NOS: 1-13.

Description

Peptide sequence

[0001] The present invention relates to a peptide sequence, particularly a peptide, and a composition for measuring isovaleric acid and an odor sensor using the same.

[0002] Olfaction is a sensory mechanism for detecting various odor substances represented by volatile organic compounds and the like, and is caused by the binding of an odor substance to an olfactory receptor. Olfactory receptors (ORs) are present in olfactory nerve cells in the olfactory organ and are G protein-coupled receptors having a seven-transmembrane structure. There are hundreds to thousands of types of olfactory receptors depending on the biological species, and they can bind to odor substances in various patterns to detect countless odors. Thus, since the relationship between olfactory receptors and odor substances is a many-to-many relationship, detailed research on olfactory receptors is being advanced.

[0003] Incidentally, isovaleric acid is cited as a typical odor substance. Isovaleric acid is a pungent odor accompanied by unpleasant sensations such as body odor and foot odor, and is the cause of malodor pollution generated from livestock manure and the like. As human olfactory receptors that bind to this isovaleric acid, OR11H4, OR11H6, OR11H7 and OR51E1 are known (see Non-Patent Documents 1 to 3).

[0004] Mainland, J., et.al., Human olfactory receptor responses to odorants., Scientific Data, 2:150001(2015).Menashe, I., et al., Genetic Elucidation of Human Hyperosmia to Isovaleric Acid. PLoS Biology, Volume5, 11: e284(2007)Bushdid, C., et al., Mammalian class I odorant receptors exhibit a conserved vestibular-binding pocket., Cell Mol Life Sci., 76:995-1004(2019)

[0005] Olfactory receptors such as OR11H4 have very long amino acid sequences, making them impractical for use in odor sensors from the perspectives of manufacturing costs and quality control. Therefore, there is a need for peptides that specifically bind to odor substances such as isovaleric acid and have shorter sequences.

[0006] The present invention aims to provide a peptide with good binding affinity to isovaleric acid, as well as a composition for measuring isovaleric acid and an odor sensor using the same.

[0007] The peptide according to the first aspect of the present invention consists of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 13. The isovaleric acid measuring composition and odor sensor according to the first aspect of the present invention contain a peptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 13.

[0008] The peptide, isovaleric acid measurement composition, and odor sensor according to the first embodiment exhibit good binding affinity to isovaleric acid. Furthermore, because the amino acid sequence structure of the peptide used is relatively short, it is advantageous from the viewpoint of manufacturing cost and quality control.

[0009] 1. First Embodiment [Peptide] The peptide of the first embodiment of the present invention (the peptide) is a peptide consisting of an amino acid sequence shown in any one of the following SEQ ID NOs: 1 to 13. Hereinafter, the peptide consisting of an amino acid sequence shown in SEQ ID NO: n will be referred to as "peptide n". These peptides have good binding affinity to isovaleric acid.

[0010]

[0011] Preferably, the peptide is peptide 1, peptide 2, peptide 3, peptide 4, peptide 5, peptide 6, or peptide 7; more preferably, peptide 1, peptide 2, peptide 3, peptide 4, or peptide 5; even more preferably, peptide 1, peptide 2, or peptide 3; and most preferably, peptide 1. This further improves the binding affinity with isovaleric acid.

[0012] The N-terminus of this peptide may be protected by a protecting group such as a t-butoxycarbonyl group or a benzyloxycarbonyl group, or it may be modified by other functional groups. The C-terminus of this peptide may be protected by a protecting group such as a methyl ester group, an ethyl ester group or a benzyl ester group, or it may be modified by other functional groups.

[0013] This polypeptide may also be in the form of a salt with an acid or a base. The salt may be either an acidic salt or a basic salt. Examples of acidic salts include hydrochloride, sulfate, acetate, propionate, fumarate, maleate, aspartate, and glutamate. Examples of basic salts include sodium salt, potassium salt, and calcium salt.

[0014] This peptide also includes peptides in which one amino acid residue in its amino acid sequence is substituted, deleted, inserted, and / or added, within the range that allows it to bind to isovaleric acid.

[0015] This peptide can be synthesized using commercially available or known peptide synthesis equipment. Furthermore, since this peptide is a peptide fragment of one of the olfactory receptors OR11H4, OR11H6, OR11H7, and OR51E1, it may also be synthesized by cleaving the peptide bonds of these olfactory receptors using a desired proteolytic enzyme.

[0016] [Composition for measuring isovaleric acid] The composition for measuring isovaleric acid according to the first embodiment of the present invention (this composition) contains at least one peptide selected from the group consisting of peptides 1 to 13. Preferably, it contains at least one peptide selected from the group consisting of peptides 1 to 7, more preferably, it contains at least one peptide selected from the group consisting of peptides 1 to 5, even more preferably, it contains at least one peptide selected from the group consisting of peptides 1 to 3, and particularly preferably, it contains peptide 1. As a result, this composition has even better binding affinity to isovaleric acid.

[0017] Furthermore, this composition preferably contains all of peptides 1 to 3, more preferably all of peptides 1 to 5, even more preferably all of peptides 1 to 7, and particularly preferably all of peptides 1 to 13. By using two or more of these peptides in combination, this composition can more reliably detect isovaleric acid and measure it with high sensitivity.

[0018] This composition may contain other peptides or known additives in addition to the peptide described above. Furthermore, the peptide may be supported on a carrier or the like.

[0019] The composition may also be in the form of a solvate. The solvent is not particularly limited and includes, for example, water, buffer solutions (such as phosphate buffer, Tris buffer, HEPES buffer), ethanol, glycerol, and acetic acid.

[0020] This peptide and this composition (this peptide, etc.) can be suitably used as an odor sensor that includes isovaleric acid as the target for odor measurement.

[0021] [Odor Sensor] The odor sensor of the first embodiment of the present invention is equipped with the peptide, etc., as a sensing part (adsorption part) that detects or adsorbs odor molecules (i.e., isovaleric acid). Examples of such odor sensors include field-effect transistor type sensors, semiconductor type sensors, quartz oscillator type sensors, film-type surface applied force sensors, etc., and a field-effect transistor type sensor is preferred. Examples of field-effect transistor type sensors include graphene field-effect transistors (GFETs) and molybdenum disulfide field-effect transistors, and a GFET is preferred.

[0022] A graphene field-effect transistor sensor is a peptide-modified graphene field-effect transistor comprising a peptide or the like as a sensing part and a transistor part connected thereto. The transistor part is a field-effect transistor equipped with a graphene electrode, and known or commercially available ones can be used. The sensing part may be equipped with an immobilization part for fixing the peptide or the like to the graphene electrode of the transistor part. Examples of immobilization parts include compounds having functional groups that can bind to the peptide and graphene, respectively. Examples of such graphene field-effect transistors and their configurations are disclosed, for example, in Japanese Patent Application Publication No. 2020-46196 and Japanese Patent Application Publication No. 2022-144547.

[0023] [Method for measuring isovaleric acid] The method for measuring isovaleric acid according to the first embodiment of the present invention involves contacting the sample to be measured with the peptide, etc.

[0024] One example of this measurement method involves bringing the sample to be measured into contact with the odor sensor. For example, if the odor sensor is a GFET, the isovaleric acid in the sample to be measured is brought into contact with the sensing part of the GFET, causing the isovaleric acid to be captured by the sensing part. The amount of isovaleric acid is then calculated by measuring the change in the electrical signal caused by this capture.

[0025] Another example involves preparing a peptide carrier in which the peptide is immobilized on a support, and contacting the peptide carrier with isovaleric acid from the sample to be measured, thereby capturing the isovaleric acid on the peptide carrier. The isovaleric acid is then released from the peptide carrier, and the amount of released isovaleric acid is detected using an analytical instrument. Examples of support materials include filter paper, plastic substrates, and particles, and examples of analytical instruments include gas chromatographs and mass spectrometers.

[0026] The peptide, isovaleric acid measurement composition, and odor sensor of the first embodiment exhibit good binding affinity to isovaleric acid. Furthermore, the peptide used has approximately 12 amino acid residues and a short sequence structure, which is advantageous from the viewpoint of manufacturing cost and quality control. Moreover, because these molecules bind specifically and readily to isovaleric acid, they can measure only isovaleric acid with high accuracy.

[0027] 2. Embodiments The exemplary embodiments described above will be understood by those skilled in the art to be specific examples of the following embodiments.

[0028] (Paragraph 1) A peptide according to one embodiment may consist of an amino acid sequence shown in any of SEQ ID NOs: 1 to 13.

[0029] (Section 2) The peptide described in Section 1 may have an amino acid sequence represented by any of SEQ ID NOs: 1 to 7.

[0030] (Section 3) A composition for measuring isovaleric acid according to one embodiment may contain the peptide described in Section 1.

[0031] (Section 4) The isovaleric acid measurement composition described in Section 3 may contain at least one peptide consisting of an amino acid sequence shown in any of Sequence ID No. 1 to 7.

[0032] (Section 5) The isovaleric acid measurement composition described in Section 3 or Section 4 may contain all of the peptides consisting of the amino acid sequences shown in Sequence ID No. 1 to 7.

[0033] (Section 6) The isovaleric acid measurement composition described in any one of Sections 3 to 5 may contain all of the peptides consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 13.

[0034] (Section 7) An odor sensor according to one embodiment may contain the peptide described in Section 1 or Section 2, or the isovaleric acid measuring composition described in any one of Sections 3 to 6.

[0035] The present invention will now be described in detail with reference to examples and comparative examples, but the scope of the present invention is not limited thereto.

[0036] <Example 1> The peptide shown in Sequence ID No. 1 was synthesized on a piece of filter paper using a peptide synthesizer ("MultiPep RSi", manufactured by Intavis AG). This peptide-coated filter paper was placed in phosphate-buffered saline containing 10 μM isovaleric acid and left at room temperature for 2 hours to allow isovaleric acid to bind to the peptide. After that, the peptide-coated filter paper was removed, washed with phosphate-buffered saline, and then placed in an acetone solution. This allowed the isovaleric acid bound to the peptide to be eluted into the acetone solution.

[0037] Next, the content of eluted isovaleric acid was measured using a gas chromatograph-mass spectrometer ("GCMS-TQ8050NX," manufactured by Shimadzu Corporation). The isovaleric acid content was determined based on a calibration curve that was prepared in advance using isovaleric acid standard solutions of known concentration.

[0038] <Examples 2-13> The peptides shown in SEQ ID NOs: 2-13 were synthesized in the same manner as described above, and the amount of isovaleric acid that was bound to and eluted was measured. These results are shown in Table 2.

[0039]

[0040] <Comparative Examples 1-3> The peptides shown in SEQ ID NOs. 14-16 in Table 2 were synthesized in the same manner as described above, and the amount of isovaleric acid bound to and eluted was measured. These results are shown in Table 2.

[0041] <Screening Experiment Using Reference Compounds> Based on four types of olfactory receptors known to bind isovaleric acid—OR11H4, OR11H6, OR11H7, and OR51E1—various peptide fragments of these olfactory receptors were synthesized. The binding affinity of isovaleric acid to these peptide fragments was screened using reference compounds. As reference compounds, fluorescein-labeled isovaleric acid compounds possessing the isovaleric acid main skeleton and labeled with a fluorescent dye were adopted. Specifically, the screening experiment was conducted as follows.

[0042] (Preparation of reference compound) Valine and fluorescein 5-isocyanate (isomer 1) were dissolved in 10% acetonitrile carbonate-bicarbonate buffer (pH 9.25), and this solution was stirred at room temperature for 24 hours, and then lyophilized. The obtained powder was purified using a high-performance liquid chromatograph ("LC-20AR", manufactured by Shimadzu Corporation). A TSKgel (20 mm I.D., 25 cm, 5 μm, "ODS-80TS", manufactured by Tosoh Corporation) was used for the column. Thereby, as a reference compound, a fluorescein-labeled isovaleric acid-based compound represented by the following formula (1) was obtained. In addition, using a mass spectrometer (MALDI-TOFMS: "AXIMA Performance", manufactured by Shimadzu Corporation), it was confirmed that the obtained powder was a fluorescein-labeled isovaleric acid-based compound.

[0043]

[0044] (Peptide synthesis) Referring to the amino acid sequences of four types of olfactory receptors (OR11H4, OR11H6, OR11H7, and OR51E1), peptides composed of 12 amino acid residues were directly synthesized on filter paper using an automatic microwave peptide synthesizer (for high-speed synthesis, manufactured by Biotage AB Initiator), for a total of 223 types. Specifically, based on the N-terminus of each olfactory receptor, peptide groups consisting of amino acid sequences from the 1st to the 12th, from the 5th to the 16th, and so on, that is, amino acid sequences from the (4n + 1)-th to the (4n + 12)-th were synthesized in order. Among these 223 types, the peptides with the above-mentioned SEQ ID NOs: 1 to 16 are included.

[0045] (Screening) A phosphate-buffered saline containing the above fluorescein-labeled isovaleric acid-based compound was dropped onto each peptide region (223 locations) on the filter paper on which these peptide groups were synthesized, so that each peptide was brought into contact with the fluorescein-labeled isovaleric acid-based compound. Then, this filter paper was washed with phosphate-buffered saline.

[0046] Next, using a fluorescence scanner device ("Tyhoon FLA 9500", manufactured by GE Healthcare Japan), light with a wavelength of 494 nm was irradiated onto each peptide region (223 locations) on the filter paper, and the fluorescence intensity at an emission wavelength of 520 nm was measured.

[0047] (Results and Discussion) Among the 223 types of peptides, the peptides of SEQ ID NOs: 1 to 13 showed particularly high fluorescence intensity. All of the other peptides including the peptides of SEQ ID NOs: 14 to 17 showed lower fluorescence intensity than the peptides of SEQ ID NOs: 1 to 13. This result was consistent with the results of <Examples 1 to 13> and <Comparative Examples 1 to 3>. Therefore, it can be seen that the peptides of SEQ ID NOs: 1 to 13 have high binding affinity for isovaleric acid as compared with many peptides including SEQ ID NOs: 14 to 16.

[0048] <Examples 14 to 25> In the peptide of SEQ ID NO: 7, 11 types of alanine monosubstituted peptides of peptide 7 in which the 2nd to 12th amino acids from the n-terminus were each substituted with A (alanine) were also synthesized in the same manner as in Example 1, and the content of isovaleric acid bound and eluted with isovaleric acid was measured. Also, 1 type of serine monosubstituted peptide of peptide 7 in which the 2nd amino acid from the n-terminus was substituted with S (serine) was also measured for the content of isovaleric acid in the same manner as above. As a result, these 12 types of peptides also had high binding affinity for isovaleric acid, similar to the peptide of SEQ ID NO: 7.

[0049] <Examples 26 to 38> In the peptide of SEQ ID NO: 13, 12 types of alanine monosubstituted peptides of peptide 13 in which the 1st to 12th amino acids from the n-terminus were each substituted with A (alanine) were also synthesized in the same manner as in Example 1, and the content of isovaleric acid bound and eluted with isovaleric acid was measured. Also, 1 type of serine monosubstituted peptide of peptide 13 in which the 2nd amino acid from the n-terminus was substituted with S (serine) was also measured for the content of isovaleric acid in the same manner as above. As a result, these 13 types of peptides also had high binding affinity for isovaleric acid, similar to the peptide of SEQ ID NO: 13.

Claims

1. A peptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 1 to 13.

2. The peptide according to claim 1, wherein the amino acid sequence is represented by any of SEQ ID NOs: 1 to 7.

3. A composition for measuring isovaleric acid, containing the peptide described in claim 1.

4. The isovaleric acid measurement composition according to claim 3, comprising at least one peptide consisting of an amino acid sequence represented by any of Sequence IDs 1 to 7.

5. The isovaleric acid measurement composition according to claim 3, comprising all of the peptides consisting of the amino acid sequences shown in Sequence IDs 1 to 7.

6. The isovaleric acid measurement composition according to claim 3, comprising all of the peptides consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 13.

7. A scent sensor containing the peptide described in claim 1.

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