JP2020041861A - DETERMINATION METHOD OF DETERMINING EXISTENCE STATE ON LIPID DOUBLE MEMBRANE OF PROTEIN ALLOWING AMYLOID FIBER TO BE PRODUCED, DETERMINATION DEVICE USING THE SAME, SCREENING METHOD, SYNTHESIS METHOD OF OLIGOMER OF PROTEIN ALLOWING AMYLOID FIBER TO BE PRODUCED, AND ARTIFICIAL β SHEET PROTEIN - Google Patents

Abstract

To provide a determination method of determining the existence state on a lipid double membrane in a protein allowing an amyloid fiber to be produced.SOLUTION: A determination method of determining the existence state on a lipid double membrane in a protein allowing an amyloid fiber to be produced includes: a first process of applying a voltage between a first solution and a second solution containing a measurement sample that are separated from each other using a lipid double membrane, measuring, with time, the current intensity of the current flowing between the first solution and the second solution over time, and acquiring current secular change data; and a second process of determining the existence state on a lipid double membrane in a protein allowing an amyloid fiber contained in the measurement sample to be produced on the basis of the waveform of the current secular change data.SELECTED DRAWING: Figure 1

Description

  The present invention provides a method for determining the presence of a protein capable of forming amyloid fibrils on a lipid bilayer membrane, a determination apparatus and a screening method using the same, a method for synthesizing an oligomer of a protein capable of forming amyloid fibrils, and an artificial method. Regarding β-sheet protein.

  A protein is a polymer chain in which amino acids are linked by peptide bonds. This polymer chain is spontaneously folded into a predetermined shape to form a three-dimensional structure. The three-dimensional structure of the protein often determines the function and properties of the protein.

  An example of the three-dimensional structure of a protein includes amyloid fibrils. Amyloid fibers have a three-dimensional structure formed into a fibrous shape by a protein having a β-sheet structure (hereinafter, also referred to as “β-sheet protein”). Amyloid fibrils are fibers formed by aggregation of β-sheet proteins that change their conformation in response to some stimulus. This fiber morphology is formed via oligomer morphology. It has been suggested that the oligomer serving as a precursor of amyloid fibrils may exhibit cytotoxicity to nerve cells.

  It is necessary to detect oligomers appearing in the process of amyloid fiber formation in order to elucidate the cause of cytotoxicity. In addition, if the oligomer formation behavior can be grasped, it is effective for prevention and diagnosis.

  For this reason, various techniques have been developed for specifically detecting the oligomer form of the protein that forms amyloid fibrils. For example, Patent Literature 1 and Patent Literature 2 disclose antibodies against amyloid β peptide.

  Non-Patent Document 1 discloses that amyloid β protein monomers, oligomers, and fibers are each formed into micelles in advance, and the interaction between these and the lipid bilayer, which is an artificial cell membrane, is electrochemically determined. A method for detecting is disclosed.

JP 2010-531992 A JP 2010-530227 A

Montserrat Serra-Batiste et al., Proceedings of the National Academy of Sciences of the United States of America, (America) September 27, 2016.113 (39) p.10866-10871

  However, no attempt has been made so far to discriminate various existing states of proteins capable of forming amyloid fibrils on the lipid bilayer membrane by electrochemical measurement. Therefore, an object of the present invention is to provide a determination method for determining the presence state of a protein capable of forming amyloid fibrils on a lipid bilayer.

  The above problem is solved by the following means.

[1] A voltage is applied between a first solution and a second solution containing a measurement sample, which are separated from each other by a lipid bilayer, and flow between the first solution and the second solution. A first step of measuring the current intensity of the current over time and obtaining current temporal change data,
A second step of determining the presence state of a protein capable of forming amyloid fibrils contained in the measurement sample on the lipid bilayer membrane from the waveform of the current temporal change data,
including,
A method for determining the presence of a protein capable of forming amyloid fibrils on a lipid bilayer membrane.

[2] The determination method according to [1], wherein the lipid bilayer is a lipid bilayer obtained by a droplet contact method.

[3] The determination method according to [1] or [2], wherein in the first step, the measurement sample is contained in both the first solution and the second solution.

[4] The determination method according to any one of [1] to [3], wherein the protein capable of forming amyloid fibrils is amyloid β.

[5] An existence state of a protein capable of forming amyloid fibrils by a process including the determination method according to any one of [1] to [4], comprising a CPU and a memory, on a lipid bilayer membrane. A determining device configured to determine

[6] An amyloid fiber, which comprises a step of confirming the presence or absence of formation of an aggregate in a β-sheet protein forming an amyloid fiber by using the determination method according to any one of the above [1] to [4]. A method for screening an aggregation controlling agent which controls the formation of an aggregate in a β-sheet protein to be formed.

[7] In the first solution and the second solution separated from each other by the lipid bilayer, a mixing step of mixing a protein monomer capable of forming amyloid fibrils with the first solution is included. A method for synthesizing a protein oligomer capable of forming amyloid fibrils.

[8] The synthesis method according to [7], wherein the lipid bilayer is a lipid bilayer obtained by a droplet contact method.

[9] The synthesis method according to [7] or [8], further including a step of applying a voltage between the first solution and the second solution after the mixing step.

[10] An artificial β-sheet protein that is a protein having 80% or more sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, and having oligomer-forming ability.

  According to one embodiment of the present invention, there is provided a determination method for determining the presence state of a protein capable of forming amyloid fibrils on a lipid bilayer membrane.

FIG. 1 is a schematic cross-sectional view illustrating an example of an analyzer used for a determination method according to the present disclosure. It is an example of the waveform on the lipid bilayer membrane of the specific protein according to the present disclosure. 5 is a flowchart illustrating an example of a determination method for determining the presence state of a specific protein on a lipid bilayer membrane according to the present disclosure. 1 is a block diagram illustrating an example of a determination device including a determination device according to the present disclosure in a configuration. 2 is a circular dichroism spectrum of the protein of SEQ ID NO: 1 (SV14). It is a circular dichroism spectrum of the protein of sequence number 2 (SV28). 4 is data of a change over time in current in Example 1. 10 is data of current change with time in Example 2. 13 is data of current change with time in Example 4. 13 is data of current change with time in Example 5.

Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments. The following embodiments are exemplifications, and the components (including element steps and the like) are not essential unless otherwise specified. The same applies to numerical values and their ranges, and does not limit the present invention. Further, various changes and modifications by those skilled in the art are possible within the scope of the technical idea according to the present disclosure.
In the present disclosure, the numerical ranges indicated by using “to” include the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
In the numerical ranges described in stages in the present disclosure, the upper limit or lower limit described in one numerical range may be replaced with the upper limit or lower limit of the numerical range described in other stages. . Further, in the numerical range described in the present disclosure, the upper limit or the lower limit of the numerical range may be replaced with the value shown in the embodiment.
In the present disclosure, the term "step" includes, in addition to a step independent of other steps, even if the purpose of the step is achieved even if it cannot be clearly distinguished from the other steps, the step is also included. .
In the present disclosure, each component may include a plurality of corresponding substances. When there are a plurality of substances corresponding to each component, the content of each component means a total content of the plurality of substances unless otherwise specified.
It is apparent to those skilled in the art that the present invention can be implemented without using some or all of the specific and detailed contents described in the present disclosure. In other instances, well-known points are not described or illustrated in detail in order to avoid obscuring aspects of the present invention.
Also, the size of the members in the drawings is conceptual, and the relative relationship between the sizes of the members is not limited to this. In addition, members having substantially the same function are denoted by the same reference numerals throughout the drawings, and redundant description may be omitted.

[Determination Method for Determining Existence State of Specific Protein on Lipid Bilayer Membrane]
The determination method for determining the presence state of the protein capable of forming amyloid fibrils according to the present disclosure on a lipid bilayer membrane is separated from each other by a lipid bilayer membrane, a first solution and a second solution each containing a measurement sample. A first step of applying a voltage between the first solution and the current solution flowing between the first solution and the second solution and measuring the current intensity over time to obtain current time-dependent change data; and A second step of determining the presence state of the protein capable of forming amyloid fibrils contained in the measurement sample on the lipid bilayer from the waveform of the data. The determination method according to the present disclosure may include other steps.

  The “protein capable of forming amyloid fibrils” according to the present disclosure is also referred to as “specific protein”.

  According to the findings of the present inventors, a specific protein tends to oligomerize in the presence of a lipid bilayer. In addition, various existing states of the specific protein (for example, the state of interaction between branches) in the presence of the lipid bilayer membrane can be identified by electrochemical measurement. The above determination method is provided based on these findings.

An analyzer used for the determination method according to the present disclosure will be described with reference to the drawings.
FIG. 1 is a schematic cross-sectional view illustrating an example of an analyzer used for the determination method according to the present disclosure.
As shown in FIG. 1, for example, the analyzer used in the determination method according to the present disclosure includes a first solution 10, a second solution 20, lipid monolayers 30ML1 and 30ML2, and a lipid bilayer 30BL. And voltage application means 40, and specific protein oligomers 50 and 60 contained in the measurement sample. Further, the first solution 10 and the second solution 20 contain an electrolyte. The first solution and the second solution are contained in a container. Note that examples of the analyzer used in the determination method according to the present disclosure include analyzers disclosed in JP-A-2012-81405, JP-A-2014-100672, and the like.

  The lipid monolayer 30ML1 is contained in the first solution 10. The lipid monolayer 30ML2 is contained in the second solution 20. A lipid bilayer membrane 30BL is formed on the contact surface where the lipid monolayers 30ML1 and 30ML2 contact each other. The first solution 10 and the second solution 20 are separated from each other by the lipid bilayer 30BL.

  The voltage applying means 40 is connected to each of the first solution 10 and the second solution 20, and applies a voltage between the first solution 10 and the second solution 20 (that is, via the lipid bilayer 30BL). It can be applied. Although not shown, an electrode is provided at each tip of the voltage applying means 40 connected to the first solution 10 and the second solution 20. Further, the voltage applying unit 40 may be electrically connected to a power supply. The voltage applying means 40 may be electrically connected to a voltmeter.

  Hereinafter, each step will be described in detail.

(First step)
The determination method according to the present disclosure includes a first step.
In the first step, a voltage is applied between the first solution and the second solution containing the measurement sample, which are separated from each other by the lipid bilayer, and the first solution and the second solution The current intensity of the current flowing therethrough is measured over time to obtain current time-dependent change data.

The measurement sample according to the present disclosure includes a specific protein.
When the determination method according to the present disclosure is used for a measurement sample, the presence state of the specific protein on the lipid bilayer in the measurement sample can be determined.

  The measurement sample may be a solid or a liquid. When the measurement sample is a solid, for example, it is preferable to use a diluent obtained by suspending, dispersing, or dissolving the solid in a solution medium. The solution medium is not particularly limited, and includes, for example, water, a buffer and the like.

Examples of the measurement sample include a sample derived from a test object such as a human or a non-human animal (a mammal other than a human).
Examples of the sample derived from the test object include a specimen derived from a living body. The specimen derived from a living body is not particularly limited, and includes urine, blood, saliva, and the like. Blood samples include, for example, red blood cells, whole blood, serum, plasma, and the like.
The sample obtained from the test object may be a liquid or a solid. For example, an undiluted liquid of a sample obtained from a test object may be used as it is, or a diluent obtained by suspending, dispersing, or dissolving the sample in a medium may be used. When the sample obtained from the test object is a solid, for example, it is preferable to use a diluent obtained by suspending, dispersing, or dissolving the sample in a solution medium as the liquid sample. The solution medium is not particularly limited, and includes, for example, water, a buffer and the like. The liquid sample thus obtained can be used as the first solution containing the measurement sample or added to the first solution in the above-described determination method. As described later, a solution containing a measurement sample similar to the first solution may be used as the second solution.

  The concentration of the specific protein contained in the first solution is not particularly limited, and may be appropriately set. For example, when the specific protein is contained in the measurement sample, the concentration of the specific protein is preferably 100 nmol / L or more and 1000 μmol / L or less, from the viewpoint of suitably forming the oligomer of the specific protein, and 100 nmol / L or less. It is more preferably at least L and at most 500 μmol / L, even more preferably at least 1 μmol / L and at most 100 μmol / L.

  The form of the specific protein may be a monomer, an oligomer, or a fiber.

  The type of the specific protein is not particularly limited as long as it can form amyloid fibrils. Specific proteins include, for example, proteins having a β-sheet structure, such as amyloid β, tau, prion, polyglutamine peptide, huntingtin, α-synuclein, insulin, and TDP-43 (TAR DNA-binding protein 43).

  For example, insulin is an example of a specific protein that forms amyloid fibrils by a treatment such as heating. In addition, TDP-43 is a protein that is accumulated in the brain of patients suffering from amyotrophic lateral sclerosis (ALS), and is one of the proteins that are known to be capable of forming amyloid fibrils in recent years. It is.

  The protein having a β-sheet structure is a protein having a sheet-like secondary structure in which hydrogen bonds are formed between adjacent peptide chains. When the protein is formed from a plurality of subunits, any part of the subunit may have a β-sheet structure. The direction between β-sheet proteins, that is, the direction of adjacent polypeptide chains is not particularly limited.

  Among the above, the specific protein is preferably amyloid β.

The specific protein may be an artificial β-sheet protein.
The artificial β-sheet protein as a specific protein can be used as a model protein in oligomerization or amyloid fibril formation to determine the presence state of the specific protein on the lipid bilayer. The artificial β-sheet protein as the specific protein can be used for analysis such as oligomerization.

The artificial β-sheet protein may be a protein having oligomer-forming ability and having a sequence identity of 80% or more with SEQ ID NO: 1 or SEQ ID NO: 2.
When the artificial β-sheet protein has a sequence identity of 80% or more with SEQ ID NO: 1 or SEQ ID NO: 2, it tends to form oligomers stably.

As used herein, the term "sequence identity of an amino acid sequence" refers to the identity of the amino acid sequence between two proteins of interest, and the optimality of an amino acid sequence created using a mathematical algorithm known in the art. It is represented by the percentage (%) of amino acid residues that are identical in a perfect alignment.
The sequence identity of an amino acid sequence can be determined by visual inspection and mathematical calculation in the same manner as the identity, and a sequence identity search program (for example, BLAST, PSI-BLAST, HMMER) well known to those skilled in the art, It can be calculated using genetic information processing software (for example, GENETYX (registered trademark)) or the like. Specifically, the sequence identity of the amino acid sequence in the present specification can be determined by using GENETYX (registered trademark) network version ver. 11.1.3 (Genetics Co., Ltd.) and setting Protein vs Protein Global Homology to the initial setting conditions (Unit size to compare is set to 2).

  The lipid bilayer membrane according to the present disclosure is not particularly limited, and a lipid bilayer membrane appropriately manufactured by a known manufacturing method may be applied. For example, the lipid bilayer is preferably a lipid bilayer formed by a droplet contact method. As a method for producing a lipid bilayer membrane by a droplet contact method, a production method described in JP-A-2012-81405, JP-A-2014-100672, or the like may be applied.

  The droplet contact method is a method in which lipid-containing droplets are brought into contact with each other to form a lipid bilayer on the contact surface between the droplets. In addition, a lipid monomolecular film is formed on a surface where the droplets are not in contact with each other. For this reason, lipid bilayers formed by contact with droplets can be made into lipid monolayers even if oligomers of a specific protein enter the lipid bilayer by applying voltage and form pores on the lipid bilayer. Because of the support of the membrane, the lipid bilayer tends to be more stably present than the lipid bilayer produced by another manufacturing method. As a result, even if a voltage is applied to the lipid bilayer membrane for a long time (for example, 120 minutes or more), the lipid bilayer membrane is not easily broken, and longer time-lapse measurement can be performed. For example, determining the process over time from a monomeric form to an oligomeric form, which may require a long-term measurement; It is considered that the process can be determined.

  The lipid forming the lipid bilayer is not particularly limited as long as it can form a lipid bilayer stably at room temperature, and a suitable lipid may be used as appropriate. Examples of lipids forming a lipid bilayer membrane include diphthanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dimyristoyl. -Sn-glycero-3-phosphocholine (DMPC), diphytanoylphosphatidylethanolamine (DPhPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero And phospholipids such as -3-phosphoethanolamine (DPPE) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Among the above, as the lipid forming the lipid bilayer, diphthanoyl-sn-glycero-3-phosphocholine (DPhPC) having a high phase transition temperature is preferable from the viewpoint of obtaining a more stable lipid bilayer at room temperature. In addition, the lipid bilayer membrane may be configured to include a steroid compound such as cholesterol, if necessary.

  The first solution contains a measurement sample. From the viewpoint of observing oligomerization more efficiently, the second solution may also include a measurement sample.

The first solution and the second solution are not particularly limited as long as a current flowing between the first solution and the second solution can be observed, and a suitable electrolytic solution may be applied as appropriate. Examples of the type of the electrolyte contained in the electrolyte include KCl, NaCl, and the like. The electrolyte may be used alone or in combination of two or more.
When an electrolytic solution is used, the concentration of the electrolyte is, for example, preferably 10 μM or more and 10 M or less, more preferably 50 μM or more and 1 M or less, and more preferably 100 μM or more and 300 mM or less from the viewpoint of obtaining a sufficient current intensity. Is more preferred. When two or more electrolytes are present, the concentration of the electrolyte indicates the sum of the concentrations of the two or more electrolytes.

  The type of the first solution and the second solution is not particularly limited as long as the specific protein can be analyzed, and a suitable solution may be used as appropriate. The first solution and the second solution are preferably the same solution except for the presence or absence of the measurement sample, and more preferably both are the same solution containing the measurement sample.

  For example, when the specific protein is amyloid β, a pH buffer solution that adjusts the pH to 6.0 or more and 8.0 or less may be used as the first solution and the second solution. The pH buffer contained in the pH buffer solution is not particularly limited, and examples thereof include phosphoric acid (eg, disodium hydrogen phosphate and potassium hydrogen phosphate, PBS), 2-morpholinoethanesulfonic acid (MES), and 3-morpholino. Propanesulfonic acid (MOPS), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES), 2 -Hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (HEPPSO) and 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (EPPS) Is mentioned. Among them, a PBS buffer solution using phosphoric acid such as disodium hydrogen phosphate and potassium hydrogen phosphate is preferable.

  It is preferable that the first solution and the second solution are contained in a container or a well.

  The method of applying a voltage between the first solution and the second solution is not particularly limited as long as a voltage can be applied with time between the first solution and the second solution. For example, an electrode may be provided for each of the first solution and the second solution, and a voltage may be applied through these two electrodes. Further, the electrode may be electrically connected to a control device that controls a power supply and a voltage. The electrode may be electrically connected to a monitoring device that obtains current aging data.

  The voltage applied between the first solution and the second solution is not particularly limited as long as a specific protein can be analyzed. For example, from the viewpoint of shortening the process of oligomerization of a monomer of a specific protein (particularly, a monomer of an artificial β-sheet protein), it is easy to determine the oligomerization. Is preferably 10 mV or more and 400 mV or less, more preferably 30 mV or more and 300 mV or less, further preferably 50 mV or more and 250 mV or less.

(Second step)
The determination method according to the present disclosure includes a second step.
In the second step, the existence state of the protein capable of forming amyloid fibrils contained in the measurement sample on the lipid bilayer is determined from the waveform of the current temporal change data.

  "Determining the presence state of a specific protein on a lipid bilayer contained in the measurement sample from the waveform of the current temporal change data" means that the specific protein is a monomeric form, an oligomer on the lipid bilayer. (Hereinafter, simply referred to as “determination of specific protein morphology”).

  In the determination method according to the present disclosure, a morphology of a monomer, a morphology of an oligomer, and a morphology of a fiber can be determined based on a waveform of current temporal change data. Further, specific forms of the monomer can be distinguished from each other.

  FIG. 2 shows examples of waveforms of the specific protein according to the present disclosure in the form of a monomer, the form of an oligomer, and the form of a fiber on a lipid bilayer membrane. When the specific protein takes the form of a monomer on the lipid bilayer membrane, the waveform of the current temporal change data includes, for example, Erratic, Spike, Take at least 5 types of waveforms: Multi-level, Flicker, and Square-top.

  The current intensity observed when a voltage is applied between the first solution and the second solution before containing the specific protein is referred to as a baseline.

  As shown in FIG. 2A, the waveform that is Erratic means that the current intensity gradually increases while repeating fine oscillation from the current intensity at the baseline, and then gradually decreases while repeating fine oscillation. Represents a moving waveform.

  As shown in FIG. 2A, a waveform that is Spike is a waveform in which the current intensity sharply rises from the current intensity at the baseline, and then immediately decreases to near the baseline. Represent.

  As shown in FIG. 2A, a multi-level waveform means that the current intensity sharply increases from the current intensity at the baseline, and the current value fluctuates in an irregular noise state while the current intensity is increased. Thereafter, a waveform that sharply decreases to the same range as the current intensity at the baseline is shown.

  "The current intensity sharply increases or decreases" means, for example, that when the amount of change in the current intensity exceeds or falls below a predetermined threshold with respect to the current intensity of the baseline, the current intensity sharply increases. Alternatively, it may be determined that the temperature has decreased. The threshold value may be an arbitrary value, and is preferably 50% or more, more preferably 100% or more of the current intensity at the baseline.

  As shown in FIG. 2A, the Flicker waveform represents a plurality of rectangular waveforms in which the magnitude of the current intensity is in the same range.

  "The magnitude of the current intensity in the waveforms on the plurality of rectangles is in the same range" means, for example, the variation width in the absolute value of the difference between the maximum value and the minimum value of the current intensity between the respective rectangular waveforms. Is smaller than a predetermined threshold value, it may be determined that the magnitudes of the current intensities in the plurality of rectangular waveforms are in the same range. The threshold value may be any value, and is preferably ± 10% or less, more preferably ± 5% or less, of the arithmetic mean value of the fluctuation range in the absolute value of the difference between the maximum value and the minimum value of the current intensity.

  As shown in FIG. 2A, the square-top waveform refers to a plurality of rectangular waveforms in which the magnitude of the current intensity is random.

  `` The magnitude of the current intensity in the plurality of rectangular waveforms is random '' means, for example, that in each of the rectangular waveforms, the fluctuation range in the absolute value of the difference between the maximum value and the minimum value of the current intensity is If the value exceeds a predetermined threshold, it may be determined that the magnitude of the current intensity in the plurality of rectangular waveforms is random. The threshold value may be any value, and is preferably ± 10% or less, more preferably ± 5% or less, of the arithmetic mean value of the fluctuation range in the absolute value of the difference between the maximum value and the minimum value of the current intensity.

  When the specific protein takes the form of an oligomer on the lipid bilayer membrane, the waveform of the current temporal change data has a Step-up waveform as shown in FIG. 2 (2). The step-up waveform indicates a waveform in which the current intensity rises stepwise.

  When the specific protein takes the form of a fiber on the lipid bilayer membrane, the waveform of the current temporal change data has a No-signal waveform as shown in FIG. 2 (3). The waveform that is a No-signal indicates a waveform in which the current intensity is in the same range as the current intensity in the baseline.

  Hereinafter, an example of a method for determining the presence state of the specific protein on the lipid bilayer will be described based on the flowchart shown in FIG. As shown in FIG. 3, first, current time-dependent change data is obtained in step S00. Next, in step S10, it is determined whether or not the current intensity increases or decreases rapidly. When step S10 is denied, it progresses to step S11. When step S10 is affirmed, it progresses to step S12.

  In step S11, it is determined whether or not the current intensity is in the same range as the baseline current intensity. If step S11 is affirmative, it is determined that the waveform has a baseline waveform. If step S11 is negative, it may be determined that the monomer has an Erratic waveform that is one of the waveforms characteristic of the first embodiment.

  In step S12, it is determined whether “in the graph representing the relationship between the current intensity and the measurement time, the slope of the approximate straight line changes from positive to negative within 20 ms”. When step S12 is affirmed, it may be determined that the monomer has a Spike waveform which is one of the waveforms characteristic of the second embodiment. If step S12 is denied, the process proceeds to step S13.

  In step S13, the current intensity rises sharply, and the current value fluctuates in an irregular noise state while maintaining the increased state, and thereafter, the temporal change in current until the current intensity sharply falls to the same range as the current intensity in the baseline. In the data, it is determined whether or not the current value distribution indicates a Gaussian distribution. If step S13 is negative, it may be determined that the monomer has a multi-level waveform which is one of the waveforms characteristic of the third embodiment. When step S13 is affirmed, it progresses to step S14.

  In step S14, it is determined whether or not the current intensity does not decrease even after 1 s has elapsed. When step S14 is affirmed, it may be determined to have a characteristic step-up waveform in the oligomer form. When step S14 is denied, it progresses to step S15.

  In step S15, it is determined whether or not the two-state transition of the current intensity is repeated. If step S15 is affirmative, it may be determined that the monomer has a Flicker waveform, which is one of the waveforms characteristic of the fourth embodiment. When step S15 is denied, it may be determined that the monomer has a Square-top waveform which is one of the waveforms characteristic of the fifth embodiment.

  In the current aging data, for example, after the waveform of the step-up derived from the oligomer form is observed, the current intensity in the waveform of the step-up is not changed even after a certain period of time (for example, 300 s or more). If does not change, it may be determined that the specific protein is in the form of a fiber on the lipid bilayer.

  It is considered that the first to fifth forms of the monomer are different in the state of the presence of the monomer on the lipid bilayer. According to the method for determining a specific protein according to the present disclosure, it is possible to determine which form the monomer takes on the lipid bilayer membrane based on the waveform of the current temporal change data.

  In the present disclosure, the “monomer form” also refers to a form in which a plurality of monomers are unstablely aggregated to form a dimer or more in the process of becoming an “oligomer form”. include.

  For example, from the viewpoint of contributing to the mechanism of cytotoxicity to nerve cells, a monomer of a specific protein is selected as a measurement sample, and the form of the oligomer is determined from waveforms characteristic of the first to fifth forms of the monomer. A process until the waveform changes to a characteristic waveform over time may be monitored. For such monitoring, it is preferable to use a lipid bilayer by the droplet contact method as the lipid bilayer. This is because the lipid bilayer membrane is more stable even when voltage is applied for a long time.

  The method of determining the form of the specific protein from the waveform of the current temporal change data is not particularly limited, and may be a method of directly comparing the shape of the waveform using a specific protein whose form is known in advance as a standard sample. Alternatively, a method of assigning waveforms in a flowchart manner may be used.

  The method for determining the form of the specific protein is not particularly limited as long as it can determine each of the monomer, oligomer, and fiber of the specific protein. The waveform may be identified by a determination device provided.

[Determination device configured to determine presence state of specific protein on lipid bilayer membrane]
A determination device configured to determine the presence state of a specific protein on a lipid bilayer according to the present disclosure includes a measurement sample, which includes a CPU and a memory, and is separated from each other by the lipid bilayer. A voltage is applied between the first solution and the second solution, and current temporal change data obtained by measuring the current intensity of the current flowing between the first solution and the second solution over time. Determining the state of presence of a protein capable of forming amyloid fibrils on the lipid bilayer from the waveform in the above.

  FIG. 4 is a block diagram of a determination device 100 as an example of the determination device according to the present disclosure. As shown in FIG. 4, the determination device 100 includes a CPU (Central Processing Unit) 101, a ROM (Read Only Memory) 102, a RAM (Random Access Memory) 103, a nonvolatile memory 104, and an input / output interface (I / O). 106 are connected to each other via a bus 105. The I / O 106 is connected via the intranet 110 to a current intensity change measuring device 120.

  A program for executing the process of determining the existence state of the specific protein on the lipid bilayer can be stored in advance in, for example, the nonvolatile memory 104. In this case, the CPU 101 reads and executes a program for executing the process of determining the presence state of the specific protein on the lipid bilayer stored in the nonvolatile memory 104. Further, a program for executing the process of determining the presence state of the specific protein on the lipid bilayer may be recorded in a storage medium such as a CD-ROM, and the program may be read and executed by a CD-ROM drive or the like.

The analyzer shown in FIG. 1 can be used as the current intensity change measuring device 120. In addition, an analyzer described in JP-A-2012-81405, JP-A-2014-100672, or the like can also be used.
The current intensity temporal change data obtained by the current intensity temporal change measuring device 120 is transmitted to the determination device 100 via the intranet 110. The CPU 101 determines the presence state of the specific protein on the lipid bilayer membrane in accordance with the flow chart shown in FIG. Note that the branch contents and determination criteria in the flowchart shown in FIG. 3 are merely examples, and may be appropriately determined based on the type and concentration of the specific protein, the concentration of the electrolyte contained in the first solution and the second solution, and the like. May be set.

  The waveform of the obtained current aging data is compared with given data indicating a specific form of the specific protein, which is stored in the nonvolatile memory 104 in advance, so that lipids of the specific protein present in the measurement sample can be obtained. The existence state on the multilayer film may be determined.

  The above determination result may be output to and displayed on a display device 130 such as a monitor connected to the I / O 106. The target to be determined is not limited to the state of the specific protein existing on the lipid bilayer membrane, and may include the amount of the specific protein. For example, such a determination can be made based on the frequency of a waveform characteristic of each mode.

  In FIG. 4, the current intensity change data is transmitted via the intranet 110, but the determination device 100 may be directly connected to the current intensity change measurement device 120 to form one device. Alternatively, the determination device 100 may constitute a determination system that determines the presence state of the specific protein on the lipid bilayer together with the current intensity temporal change measurement device 120.

Further, according to the present disclosure, there is provided a computer program for causing a computer to execute a process of determining a presence state of a specific protein on a lipid bilayer membrane, wherein the process includes the following:
A voltage is applied between the first solution and the second solution containing the measurement sample, which are separated from each other by the lipid bilayer, and the current of the current flowing between the first solution and the second solution A step of determining the presence state of a protein capable of forming amyloid fibrils on a lipid bilayer membrane from a waveform in current temporal change data obtained by measuring intensity over time.

  The memory and CPU in the determination device according to the present disclosure are not particularly limited, and a suitable known memory may be applied as appropriate.

[Screening Method for Aggregation Control Agent that Controls Aggregate Formation in Specific Protein]
The screening method for an aggregation controlling agent that controls the formation of an aggregate in a specific protein according to the present disclosure is performed by using the above-described determination method for determining the presence state of a specific protein on a lipid bilayer membrane. Confirming the formation of aggregates in the sheet protein. The method for screening an aggregation controlling agent according to the present disclosure may include other steps.

  By using the determination method according to the present disclosure in a screening method for an aggregation controlling agent, the formation of an aggregate in a specific protein can be tracked over time from the form of the monomer, so that the aggregation controlling ability is high. The screening of the control agent can be performed efficiently.

  As a method for screening the aggregation controlling agent, a known method may be applied as it is, except that the determination method according to the present disclosure is used.

The screening method for the aggregation controlling agent can include, for example, the following steps.
(A) a contacting step of bringing a candidate compound into contact with a specific protein;
(B) after the contacting step, a determining step of determining the aggregation state of the specific protein by the determining method according to the present disclosure;
(C) a selection step of selecting the candidate compound as a specific protein aggregation controlling agent, based on the result of the determination step, when aggregation of the specific protein is suppressed as compared with the case where the candidate compound is not present.

  The candidate compound may be any of a naturally occurring compound, a derivative of a naturally occurring compound, an organic synthetic compound, an inorganic compound, and the like.

  In addition, the screening method of the aggregation controlling agent for controlling the formation of the aggregate in the specific protein according to the present disclosure is not limited to oligomerization, but may be based on a monomer form (first to fifth forms). The state of aggregation may be determined. Further, in this case, a determination of a pre-aggregation state which is a stage before the aggregation may be included.

  In the step (c), for example, the frequency at which a waveform derived from the oligomer form is observed within a predetermined time (for example, 60 minutes) is equal to or less than a predetermined ratio as compared with the case where the candidate compound is not present. In this case, it may be determined that the aggregation of the specific protein is suppressed as compared with the case where the candidate compound is not present. The ratio may be any value less than 100%, preferably 90% or less, more preferably 80% or less.

[Artificial β-sheet protein]
(Design method of artificial β-sheet protein)
According to the present disclosure, there is provided an artificial β-sheet protein having a sequence identity of 80% or more with SEQ ID NO: 1 “DGSYSVSVSVSYGR” or SEQ ID NO: 2 “RGSYSVSVSSYDSDGSYSVSVSVSYGR” and having oligomer-forming ability.

  Hereinafter, an artificial β-sheet protein having a sequence identity of 80% or more with SEQ ID NO: 1 or SEQ ID NO: 2 and having an oligomer-forming ability is also simply referred to as “artificial β-sheet protein”.

  The artificial β-sheet protein can be used, for example, in the following determination method. That is, a voltage is applied between the first solution and the second solution containing the measurement sample, which are separated from each other by the lipid bilayer, and the current flowing between the first solution and the second solution is applied. A first step of measuring current intensity over time to obtain current temporal change data, and the presence of a protein capable of forming amyloid fibrils contained in the measurement sample from the waveform of the current temporal change data on a lipid bilayer membrane. And a second step of determining the state. A method for determining the presence state of a protein capable of forming an amyloid oligomer on a lipid bilayer membrane. By such a determination method, it is also possible to determine the morphology up to the formation of the oligomer, which is the stage before the formation of amyloid fibrils.

  The sequence identity of SEQ ID NO: 1 to an artificial β-sheet protein (hereinafter also referred to as “SV14”) is 80% or more, preferably 90% or more, more preferably 95% or more, and more preferably 100% (Ie, the protein of SEQ ID NO: 1).

  The sequence identity of SEQ ID NO: 2 to the artificial β-sheet protein (hereinafter also referred to as “SV28”) is 80% or more, preferably 90% or more, more preferably 95% or more, and more preferably 100% (Ie, the protein of SEQ ID NO: 2).

  The artificial β-sheet protein can be used as a model protein instead of, for example, a natural protein that forms amyloid fibrils such as amyloid β.

  The method for designing the artificial β-sheet protein is not particularly limited as long as the sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 is 80% or more, and may have a known amino acid sequence as appropriate.

The method for designing an artificial β-sheet protein is, for example, from the viewpoint of designing to efficiently form an oligomer,
(1) one terminal site containing an aromatic amino acid residue and a positively charged amino acid residue;
(2) the other terminal site containing an aromatic amino acid residue and a negatively charged amino acid residue;
(3) a main skeleton in which hydrophilic amino acid residues and hydrophobic amino acid residues are alternately arranged;
Is preferably designed to have

  When the artificial β-sheet protein has aromatic amino acid residues at both ends, an interaction such as π-π stacking between the artificial β-sheet proteins tends to occur easily. As a result, it is considered that when the artificial β-sheet protein aggregates, the β-barrel structure is easily formed stably.

  When the artificial β-sheet protein has a positively charged amino acid residue at one end and a negatively charged amino acid residue at the other end, a voltage is applied to the solution system containing the artificial β-sheet protein. In this case, the orientation of the artificial β-sheet protein tends to be aligned. As a result, it is considered that the β barrel structure is easily formed stably.

  When the artificial β-sheet protein has a main skeleton in which hydrophilic amino acid residues and hydrophobic amino acid residues are alternately arranged, it becomes easier for the designed protein to efficiently form a β-sheet structure by intramolecular hydrogen bonding. Conceivable. Further, in the molecular structure of the artificial β-sheet protein, the surface where hydrophobic amino acid residues are exposed on the surface is oriented inward, and the surface where hydrophilic amino acid residues are exposed on the surface is easily oriented outward. There is a tendency. As a result, it is considered that the β barrel structure is easily formed.

  The terminal site in the artificial β-sheet protein refers to 5 or less residues from the 3 ′ end or 5 ′ end in the primary structure of the protein.

  The main skeleton in the artificial β-sheet protein refers to an amino acid sequence obtained by removing 5 or less amino acid residues from the 3 ′ terminal or 5 ′ terminal in the primary structure of the protein.

  The number of aromatic amino acid residues is not particularly limited as long as interaction such as π-π stacking occurs between the artificial β-sheet proteins, and may be appropriately designed according to the length of the artificial β-sheet to be designed. For example, when designing an artificial β-sheet protein having 10 to 30 amino acid residues, the number of aromatic amino acid residues at the terminal site is preferably 1 to 3 and more preferably 1 to 2. More preferably, it is more preferably 1.

  The positional relationship between the aromatic amino acid residue and the charged amino acid residue is not particularly limited. For example, from the viewpoint of appropriately aligning the orientation of the artificial β-sheet protein by applying a voltage, between the aromatic amino acid residue and the charged amino acid residue, the charged amino acid residue is located at a more terminal site. Is preferred.

  Examples of the type of aromatic amino acid include tyrosine, phenylalanine, tryptophan, histidine and the like. Among them, tyrosine is preferable as the kind of the aromatic amino acid.

The positively charged amino acid may be any amino acid having a higher isoelectric point (basic side) than the pH of the solution for producing the β-sheet structure. For example, when the solution for preparing the β-sheet structure is neutral (pH 7), examples of the positively charged amino acid include arginine, histidine, and lysine. Among the above, arginine is preferable as the type of the positively charged amino acid.

  The negatively charged amino acid may be any amino acid whose isoelectric point is lower (on the acidic side) than the pH of the solution for producing the β-sheet structure. For example, when the solution for producing the β-sheet structure is neutral (pH 7), examples of the negatively charged amino acid include aspartic acid and glutamic acid. Among the above, aspartic acid is preferable as the kind of the negatively charged amino acid.

  Examples of hydrophobic amino acids include valine, methionine, tryptophan, proline, leucine, phenylalanine, isoleucine, alanine, glycine, and the like. Among the above, valine is preferable as the type of the hydrophobic amino acid.

  Examples of the types of hydrophilic amino acids include serine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, threonine, histidine, tyrosine, cysteine, and the like. Among the above, serine is preferred as the type of hydrophilic amino acid.

(Method of synthesizing artificial β-sheet protein)
The method for synthesizing the artificial β-sheet protein according to the present disclosure is not particularly limited, and a public synthesis method may be appropriately selected depending on the amino acid sequence to be designed.

  For example, from the viewpoint of efficiently obtaining only a monomer without forming a fibrous state during synthesis, (1) a precursor of an artificial β-sheet protein containing one or more isoacyl dipeptide groups in the main chain of the amino acid sequence; The target artificial β-sheet protein may be synthesized by synthesizing a precursor, and then (2) transferring the ester bond of the precursor to a peptide bond under basic conditions.

  As a method for confirming the generation of the artificial β-sheet protein, a circular dichroism spectrum is used. The circular dichroism spectrum is measured in a MOPS solution (concentration: 1 mM, 25 ° C.) using a product number: J-820 manufactured by JASCO Corporation. At this time, the artificial β-sheet protein is contained in a liposome (phospholipid: DOPC) and measured. In the preparation of the liposome, the concentration of the artificial β-sheet protein having a sequence identity of 80% or more to SEQ ID NO: 1 is 30 μg / mL. In preparing a liposome, the concentration of the artificial β-sheet protein having a sequence identity of 80% or more with SEQ ID NO: 2 is 75 μg / mL.

  As a method for determining each of the form of the monomer, the form of the oligomer, and the form of the fiber in the artificial β-sheet protein, the determination method according to the present disclosure may be applied.

[Method of synthesizing oligomer in specific protein]
According to the present disclosure, in a first solution and a second solution separated from each other by a lipid bilayer, a mixing step of mixing a protein monomer capable of forming amyloid fibrils with the first solution. (Hereinafter, simply referred to as “mixing step”), the method for synthesizing an oligomer of a protein capable of forming amyloid fibrils is provided. The method for synthesizing the oligomer of the specific protein according to the present disclosure may include other steps.

  The first solution, the second solution, and the lipid bilayer in the method for synthesizing an oligomer according to the present disclosure are the first solution described in the method for determining the presence state of a specific protein on the lipid bilayer described above, Similar to the second solution and the lipid bilayer may be applied. In particular, the lipid bilayer is preferably a lipid bilayer formed by the above-described droplet contact method from the viewpoint of more efficiently synthesizing the oligomer.

  When a mixing step of mixing the monomer of the specific protein into the first solution is included, the monomer enters the lipid bilayer, and the monomer is aggregated with time in the lipid bilayer. There is a tendency. As a result, it is considered that an oligomer of the specific protein is formed.

  In the mixing step, a monomer of the specific protein is mixed into the solution.

  The monomer of the specific protein mixed in the solution may be a solid or a solution in which the monomer is dissolved in a buffer solution or the like.

  The concentration of the specific protein in the solution is not particularly limited, and may be appropriately set according to the amount of the oligomer in the specific protein to be synthesized. For example, from the viewpoint of efficiently synthesizing an oligomer of a specific protein, the concentration is preferably 1 μmol / L or more and 40 μmol / L or less, more preferably 5 μmol / L or more and 20 μmol / L or less, and 5 μmol / L or more and 15 μmol / L. More preferably, it is L or less.

  In the mixing step, from the viewpoint of synthesizing the oligomer more efficiently, the monomer of the specific protein may be mixed in the second solution in addition to the first solution.

  The method for synthesizing the oligomer in the specific protein according to the present disclosure includes, after the mixing step, a step of applying a voltage between the first solution and the second solution according to the type of the specific protein to be synthesized. Further, it may be included.

When a voltage is applied between the first solution and the second solution after the mixing step, monomers of the specific protein tend to aggregate efficiently and form oligomers.

  The voltage applied between the first solution and the second solution is preferably from 50 mV to 400 mV, more preferably from 50 mV to 300 mV, even more preferably from 50 mV to 250 mV.

  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples. Unless otherwise specified, “parts” means “parts by mass”.

-Judgment method / Synthesis method of oligomer of specific protein-

(Preparation of materials)
(1) Amyloid β1-42 protein capable of forming amyloid fibrils (Peptide Research Institute Co., Ltd., product number: 4307-v)
10 μM, pH 7.4 PBS buffer solution (Na ₂ HPO ₄ : 8.1 mM, KH ₂ PO ₄ : 1.4 mM, KCl: 2.7 mM, NaCl: 137 mM)
SEQ ID NO: 3: DAEFFRHDSGYEVHHQKLVFFAAEDVSNKGAIIGLMVGGVVIA

(2) Artificial β-sheet protein: Synthesis of protein of SEQ ID NO: 1 (SV14)
The protein of SEQ ID NO: 1 (hereinafter referred to as “SV14”) was synthesized as an artificial β-sheet protein.
First, by solid-phase synthesis, each amino acid whose N ′ terminal is protected with a 9-fluorenylmethyloxycarbonyl group (hereinafter, referred to as “Fmoc”) is sequentially added one by one from the amino acid on the C ′ terminal side, (A) Linkage is performed by a condensation reaction (step (A)). Next, (B) Fmoc at the N ′ end of the connected peptide chain is deprotected (step (B)). Steps (A) and (B) were sequentially repeated to synthesize a peptide chain from the C ′ terminal to the fifth amino acid residue. Next, in order to insert an isoacyl dipeptide compound which is an amino acid dimer in which serine at the sixth residue and valine at the seventh residue from the C ′ terminal are inserted through an ester bond, After the step (B) of deprotecting the N'-terminal Fmoc at the fifth amino acid residue from (B), a step (C) of inserting an isoacyl dipeptide compound was performed. Thereafter, after the step (B), the steps (A) and (B) are sequentially repeated again to synthesize the remaining peptide chains. After synthesizing all peptide chains, finally, the precursor of SV14 is subjected to the steps of (D) acetylation, step (B), and (E) cleavage of the synthesized peptide chain from the resin. Obtained.
The obtained SV14 precursor was subjected to (F) a step of forming a peptide bond under basic conditions to synthesize the desired SV14. Details of each of the steps (A) to (F) are shown below.

(A) Step of Linking by Condensation Reaction 10 equivalents of an amino acid whose N ′ terminal is protected by a 9-fluorenylmethyloxycarbonyl group (hereinafter referred to as “Fmoc”) and 1- [bis (dimethylamino) methylene] 10 equivalents of -1H-benzotriazolium 3-oxidehexafluorophosphate (HBTU), 10 equivalents of 1-hydroxybenzotriazole (HOBt), and 15 equivalents of N, N-diisopropylethylamine (DIPEA) were added to the solvent NMP. After dissolving, the solution was mixed with 681.82 mg of a resin having a polyethylene glycol chain (Fmoc-NH-SAL-PEG Resin, manufactured by Watanabe Chemical Co., Ltd.), and reacted at room temperature (25 ° C.) for 30 minutes. Thereafter, the resin was washed with a solvent NMP. The step of linking amino acids protected by Fmoc by this condensation reaction was repeated twice.
For the second and subsequent residues, Fmoc-protected amino acids were similarly linked using a resin having the polyethylene glycol chain to which an amino acid or peptide chain had already been bonded.

(B) Step of Deprotecting Fmoc at N ′ Terminal in Linked Peptide Chain In the deprotection of Fmoc in peptide chain synthesis from the C ′ terminal to the fifth residue, step (B-1) was used. Deprotection of Fmoc in the isoacyl dipeptide compound linked to the resin-peptide chain in step (C) used step (B-2). In the step of deprotecting Fmoc at amino acid residues other than those described above, the above step (B-3) was used.

(B-1): The resin was washed with NMP, the resin was suspended in an NMP solution of 25% piperidine, and reacted at room temperature (25 ° C.) for 4 minutes to remove Fmoc. This step of Fmoc deprotection was repeated twice.

(B-2): The resin was washed with a 1/1 solution of NMP / DMSO, and the resin was washed with 2 v / v% hexamethyleneimine, 25% 1-methylpiperidine and 3 w / v% HOBt in NMP / DMSO. The Fmoc was removed by suspending in a 1/1 solution of the above and reacting at room temperature (25 ° C.) for 4 minutes. This step of Fmoc deprotection was repeated twice.

(B-3): washing the resin with an NMP solution, suspending the resin in an NMP solution of 25% 1-methylpiperidine and 1 w / v% HOBt, and reacting at room temperature (25 ° C.) for 4 minutes. Then, Fmoc was removed. This step of Fmoc deprotection was repeated twice.

(C) Step of inserting an isoacyl dipeptide bond In SV14, an isoacyl dipeptide which is an amino acid dimer in which serine at the sixth residue and valine at the seventh residue from the C 'terminal side are linked via an ester bond compound _{"NH 2 -CH (i-C 3} H 7) -C (= O) -O-CH 2 -CH (NH 2) -C (= O) -OH " were inserts. Specifically, the resin is suspended in a mixed solution of 4 equivalents of an isoacyl dipeptide compound, 4 equivalents of a HOBt solution in chloroform, 4.4 equivalents of N, N'-diisopropylcarbodiimide (DIPCDI) and chloroform, and room temperature (25 ° C.). ) For 2 hours. The DIPCDI used was dehydrated with a molecular sieve (3 °).

(D) Acetylation Step The resin obtained through the above step (B) or (C) is suspended in a mixed solution obtained by mixing 20 equivalents of an acetic anhydride NMP solution and 10 equivalents of N, N-diisopropylethylamine (DIPEA). It was made cloudy and reacted at room temperature (25 ° C.) for 30 minutes.

(E) Step of Cleaving Synthesized Peptide Chain from Resin The resin having undergone the above steps was treated with 12 mL of trifluoroacetic acid (TFA), 0.3 mL of m-cresol, 0.3 mL of thioanisol, and 0.3 mL of distilled water (milliQ). The mixture was suspended in the mixed liquid and reacted at room temperature (25 ° C.) for 30 minutes. Thereafter, the resin was removed by filtration, and the filtrate was concentrated to obtain a precursor of SV14.

(F) Step of Forming a Peptide Bond Under Basic Conditions The precursor of SV14 obtained above and an aqueous solution of 100 mM final sodium hydroxide were mixed and reacted at room temperature (25 ° C.) for 5 minutes. Then, the target SV14 was obtained by neutralizing the reaction solution with HCl.

  The obtained SV14 was confirmed from the circular dichroism spectrum that the secondary structure of the peptide was a β-sheet structure in the presence of a liposome (phospholipid: DOPC) MOPS solution (concentration 1 mM, 25 ° C.). (See FIG. 5). The concentration of SV14 in the liposome solution was 30 μg / mL.

(3) Artificial β-sheet protein: Synthesis of protein of SEQ ID NO: 2 (SV28)
The above steps (A) and (B) were sequentially repeated to synthesize a peptide chain from the C ′ terminal to the fifth amino acid residue. Next, in order to insert the isoacyl dipeptide compound which is the amino acid dimer described above as the valine at the sixth residue and the serine at the seventh residue from the C ′ terminal, the fifth residue from the C ′ terminal was inserted. After the step (B) of deprotecting the N'-terminal Fmoc in the amino acid residue of (a), the step (C) was performed. Then, after the step (B), the steps (A) and (B) were sequentially repeated again to synthesize a peptide chain from the C ′ terminal to the 19th amino acid residue.
Next, in order to insert the above-mentioned isoacyl dipeptide compound which is an amino acid dimer as serine at the 20th residue and valine at the 21st residue from the C 'terminal, the 19th residue from the C' terminal was inserted. After the step (B) of deprotecting the N'-terminal Fmoc in the amino acid residue of (a), the step (C) was performed. Then, after the step (B), the steps (A) and (B) were sequentially repeated again to synthesize all the peptide chains. After synthesizing all the peptide chains, finally, the precursor of SV28 is subjected to the steps of (D) acetylation, step (B), and (E) cleavage of the synthesized peptide chain from the resin. Obtained.
The obtained SV28 precursor was subjected to (F) a step of forming a peptide bond under basic conditions to synthesize a target SV28. Details of each of the steps (A) to (F) are as described above.

  In the step (B) in the solid phase synthesis of the precursor of SV28, the step (B-1) was used in the deprotection of Fmoc in the synthesis of the peptide chain from the C 'terminal to the fifth residue. The step (B-2) was used in the step of deprotecting Fmoc of the amino acid dimer at the 6th to 7th residues and the 20th to 21st residues from the C 'terminal side. In the step of deprotecting Fmoc at amino acid residues other than those described above, the above step (B-3) was used.

In the first step (C) in the solid phase synthesis of the precursor of SV28, first, the aminoacyl residue at the 6th to 7th residues from the C ′ terminal is replaced with the above-mentioned isoacyl dipeptide compound which is an amino acid dimer. Inserted.
In the second step (C) in the solid phase synthesis of the precursor of SV28, the above-mentioned isoacyl dipeptide compound, which is an amino acid dimer, is inserted as the 20th to 21st amino acid residues from the C 'terminal side. Was.

  The obtained SV28 was confirmed from the circular dichroism spectrum that the secondary structure of the peptide was a β-sheet structure in the presence of a MOPS solution (concentration: 1 mM, 25 ° C.) of liposome (phospholipid: DOPC). (See FIG. 6). The concentration of SV28 in the liposome solution was 75 μg / mL.

(Preparation of lipid bilayer membrane by droplet contact method)
With reference to Japanese Patent Application Laid-Open No. 2012-81405, a chamber in which two wells are connected by a minute hole was prepared. Next, 2.3 μL of a decane solution (20 mg / mL) in which diphthanoyl-sn-glycero-3-phosphocholine (DPhPC, Avanti Polar Lipids, Inc.) as a lipid was dissolved was added to each well. Then, to each well, phosphate buffer 4.7μL (PBS buffer _{solution, Na 2 HPO 4: 8.1mM,} KH 2 PO 4: 1.4mM, KCl: 2.7mM, NaCl: 137mM) Micro the Pipette added to form droplets. By leaving it in this state, a lipid bilayer membrane was formed in the pores. The solutions in the wells separated from each other by the formed lipid bilayer were used as a first solution and a second solution, respectively.

[Example 1]
As a measurement sample, 4.7 μL of a solution containing 10 μL of an oligomer of amyloid β1-42 was added to both the first solution and the second solution. Next, a constant voltage of +100 mV was applied in the direction from the first solution to the second solution, and the current aging data for 120 minutes was converted to Clampex 9.0 (Molecular) equipped with a Digidata 1440A analog-digital converter (Molecular Devices). Devices). The sample rate was 40 Hz, the Bessel filter was 7.9 kHz, the gain value was 3.3 G, and the applied current was recorded using an Axopatch 200B Amplifier (Molecular Devices, San Jose, CA, USA). As the solution containing the amyloid β oligomer, a solution obtained by incubating the solution containing the amyloid β monomer at 37 ° C. for 4 hours was used (FIG. 7). In addition, it was previously confirmed that the incubated amyloid β was oligomerized by Western blotting.

[Example 2]
Current time-dependent change data was obtained by the same operation as in Example 1 except that the measurement was continued using a solution containing a monomer of amyloid β1-42 as a measurement sample until the oligomer form was recognized. (FIG. 8).

[Example 3]
The current time-dependent change data was obtained by the same operation as in Example 2 except that the measurement was continued until the fiber morphology was recognized.

[Example 4]
The measurement sample was 10 μL of a solution containing a monomer of the protein of SEQ ID NO: 1 (artificial β-sheet protein: SV14), and the voltage applied from the first solution to the second solution was +200 mV. In the same manner as in Example 1, current time-dependent change data was obtained (FIG. 9).

[Example 5]
Except that the measurement sample was 10 μL of a solution containing a monomer of the protein of SEQ ID NO: 2 (artificial β-sheet protein: SV28) and the voltage applied from the first solution to the second solution was +200 mV. Obtained current aging data by the same operation as in Example 2 (FIG. 10).

  As shown in FIG. 7, in the current aging data in Example 1, a waveform in which the current intensity rises in a stepwise manner, that is, a waveform that is Step-up, was observed from the point where the measurement had elapsed for about 2 minutes. Therefore, it was determined from this waveform that the measurement sample was in the form of an oligomer on the lipid bilayer membrane.

  As illustrated in FIG. 8, the current temporal change data in the second example is a plurality of rectangular waveforms in which the magnitude of the current intensity is in the same range from the point where the measurement has elapsed for about 4 minutes, that is, Flicker. A waveform was observed. Therefore, it was determined from this waveform that the measurement sample contained a monomer form on the lipid bilayer membrane. Thereafter, from the point where the measurement has been performed for about 14 minutes, a waveform in which the current intensity rises stepwise, that is, a waveform of Step-up is observed. Therefore, from this waveform, it was determined that the measurement sample changed from the monomer form to the oligomer form on the lipid bilayer membrane.

  Although not shown, waveforms of Erratic, Spike, and Multi-level were observed immediately after the start of measurement in the current temporal change data in Example 3. Therefore, from this waveform, it was determined that the measurement sample was in the form of a plurality of monomers on the lipid bilayer. In addition, at the point where the measurement has been performed for about 18 minutes, in addition to the waveforms derived from the forms of the plurality of monomers, a waveform that is a step-up was also observed. Therefore, it was determined that the present measurement sample changed over time from the form of a plurality of monomers to the form of an oligomer on the lipid bilayer membrane. Thereafter, at a point where the measurement has been performed for about 23 minutes, a state where the current intensity does not change even after 300 seconds or more has been observed from the current intensity in the waveform of Step-up. Therefore, it was determined that the measurement sample further changed from the oligomer form to the fiber form on the lipid bilayer membrane.

  Further, as shown in FIG. 9, in the current time-dependent change data in Example 4 in which SV14 which is an artificial β-sheet protein was used as a measurement sample, the current intensity rapidly increased from the current intensity in the baseline immediately after the start of measurement. A waveform in which the current intensity sharply decreased, and immediately thereafter increased to the vicinity of the baseline, that is, a waveform of Spike was observed. Furthermore, from the point where the measurement has been performed for about 9 minutes, the current intensity gradually increases while repeating fine oscillation from the current intensity at the baseline, and then gradually decreases while repeating fine oscillation, That is, an Erratic waveform was observed. Therefore, it was determined from this waveform that the measurement sample was in the form of a monomer on the lipid bilayer membrane.

  Similarly, as shown in FIG. 10, in the current time-dependent change data in Example 5 using SV28, which is an artificial β-sheet protein, as a measurement sample, the current intensity changes stepwise at a point where the measurement has elapsed for about 3 minutes. A rising waveform, that is, a step-up waveform was observed. Therefore, it was determined from this waveform that the measurement sample was in the form of an oligomer on the lipid bilayer membrane.

  As described above, according to the determination method according to the present disclosure, as shown in FIGS. 7 to 10, it is possible to determine the presence state of the specific protein on the lipid bilayer from the current temporal change data. In particular, by using a lipid bilayer membrane by the droplet contact method, it is possible to apply voltage more stably for a long time, and the process of aggregation from the monomer form to the oligomer form on the lipid bilayer membrane, And the process of aggregation from the oligomer form to the fiber form could be observed over time.

Reference Signs List 10 first solution 20 second solution 30BL lipid bilayer 30ML1, 30ML2 lipid monolayer 40 voltage application means 50, 60 oligomer of specific protein 100 determination device 101 CPU
102 ROM
103 RAM
104 Non-volatile memory 105 Bus 106 Input / output interface (I / O)
110 Intranet 120 Current intensity temporal change measuring instrument 130 Display device

Claims (10)

A voltage is applied between the first solution and the second solution containing the measurement sample, which are separated from each other by the lipid bilayer, and the current of the current flowing between the first solution and the second solution A first step of measuring the intensity over time and obtaining current temporal change data,
A second step of determining the presence state of a protein capable of forming amyloid fibrils contained in the measurement sample on the lipid bilayer membrane from the waveform of the current temporal change data,
including,
A method for determining the presence of a protein capable of forming amyloid fibrils on a lipid bilayer membrane.   The determination method according to claim 1, wherein the lipid bilayer is a lipid bilayer obtained by a droplet contact method.   The method according to claim 1, wherein in the first step, the measurement sample is included in both the first solution and the second solution.   The method according to any one of claims 1 to 3, wherein the protein capable of forming amyloid fibrils is amyloid β.   It is provided with a CPU and a memory, and a presence state of a protein capable of forming amyloid fibrils on a lipid bilayer membrane is determined by a process including the determination method according to any one of claims 1 to 4. The determination device configured as described above.   A β-sheet forming an amyloid fiber, comprising a step of confirming the presence or absence of formation of an aggregate in a β-sheet protein forming an amyloid fiber using the determination method according to any one of claims 1 to 4. A method for screening an aggregation controlling agent that controls formation of an aggregate in a protein.   In the first solution and the second solution separated from each other by the lipid bilayer, the first solution, a monomer of a protein capable of forming amyloid fibrils, including a mixing step of mixing, amyloid fibrils, A method for synthesizing a formable protein oligomer.   The synthesis method according to claim 7, wherein the lipid bilayer is a lipid bilayer formed by a droplet contact method.   The method according to claim 7 or 8, further comprising a step of applying a voltage between the first solution and the second solution after the mixing step.   An artificial β-sheet protein having a sequence identity of 80% or more with SEQ ID NO: 1 or SEQ ID NO: 2 and having oligomer formation ability.