1. Field of the Invention
The present invention relates to a method for transporting potassium ions from a front side of a lipid bilayer membrane to a back side thereof.
2. Description of the Background Art
G protein-coupled receptors (hereinafter, abbreviated as “GPCR”) existing in lipid bilayer membranes of cells functions in association with G proteins existing in the lipid bilayer membranes of cells. As shown in
As shown in
If the Gα subunit is a chimeric Gα subunit, the potassium ions move from the front side to the back side more efficiently in some cases. It is suggested to use, as a chemical substance sensor, a lipid bilayer membrane where a G protein comprising a chimeric Gα subunit has been expressed.
The following Non-patent Literatures 1 to 6 are relevant to the present invention.
An object of the present invention is to provide a method for transporting potassium ions from a front side of a lipid bilayer membrane to a back side thereof in a further highly sensitive manner in response to a chemical substance.
The present inventors have discovered that a chimeric G protein comprising a specific chimeric Gα subunit solves the above described problem, and have established the present invention.
Thus, the present invention provides the following items 1 to 9.
Item 1:
A method for transporting potassium ions from a front side of a lipid bilayer membrane to a back side thereof, the method comprising the following steps (a) and (b):
step (a) of preparing the lipid bilayer membrane, wherein
step (b) of supplying the chemical substance and the potassium ions to the front side to release the chimeric Gα subunit and Gβγ subunit complex, and to allow the Gβγ subunit complex to bind to the potassium ion channel, and transporting the potassium ions from the front side to the back side.
Item 2:
The method according to item 1, wherein the chemical substance is an adrenergic receptor agonist.
Item 3:
The method according to item 1, wherein the potassium ion channel is a G protein-coupled inwardly rectifying potassium ion channel.
Item 4:
A method for detecting or quantifying a chemical substance, the method comprising the following steps (c), (d), and (e):
step (c) of preparing a lipid bilayer membrane, a first liquid located on a front side of the lipid bilayer membrane, and a second liquid located on a back side of the lipid bilayer membrane, wherein
step (d) of supplying the chemical substance to the first liquid; and
step (e) of measuring an amount of potassium ions in at least one of the first and second liquids to detect or quantify the chemical substance based on the amount of the potassium ions.
Item 5:
The method according to item 4, wherein the chemical substance is an adrenergic receptor agonist.
Item 6:
The method according to item 4, wherein the potassium ion channel is a G protein-coupled inwardly rectifying potassium ion channel.
Item 7:
A method for detecting or quantifying a chemical substance, the method comprising the following steps (f), (g), and (h):
step (f) of preparing a lipid bilayer membrane, wherein
step (g) of supplying a first liquid and a second liquid located respectively on a front side and a back side of the lipid bilayer membrane such that the lipid bilayer membrane is interposed between the first liquid and second liquid, wherein
step (h) of measuring an amount of potassium ions in at least one of the first and second liquids to detect or quantify the chemical substance based on the amount of the potassium ions.
Item 8:
The method according to item 7, wherein the chemical substance is an adrenergic receptor agonist.
Item 9:
The method according to item 7, wherein the potassium ion channel is a G protein-coupled inwardly-rectifying ion channel.
The present invention increases an amount of potassium ions transported from a front side of a lipid bilayer membrane to a back side thereof. Thus, a target chemical substance is detected with higher sensitivity.
The terms used in the present specification are defined as follows:
The term “lipid bilayer membrane” refers to a membrane forming a surface of a cell, i.e., a cell membrane.
The term “chimeric Gα subunit” refers to a Gα subunit in which a region of the original Gα subunit (e.g., Gαi) is substituted with a corresponding region of a different Gα protein (e.g., Gαolf).
The term “chimeric G protein” refers to a G protein in which the α subunit thereof is a chimeric Gα subunit.
The lipid bilayer membrane used in the present invention comprises a chemical substance receptor, a chimeric G protein, and a potassium ion channel.
An example of cells having the lipid bilayer membrane is an established cell line derived from a human cell.
(Chemical Substance Receptor)
A G protein-coupled receptor is employed as a chemical substance receptor. G protein-coupled receptors include hormone receptors, neurotransmitter receptors, pheromone receptors, olfactory receptors and gustatory receptors, as well as various orphan G protein-coupled receptors. Examples of the hormone receptors are adrenergic receptors.
(Chimeric G Protein)
A chimeric G protein comprises a chimeric Gα subunit and a Gβγ subunit complex.
In the present invention, the chimeric Gα subunit is selected from the group consisting of Gi/olf13, Gi/olf28, Gi/olf94, Gi/olf113, Gi/olfα3-β5,C, and Gi/olfα4-β6,C. Subscripts included in the names of the chimeric Gα subunits indicate Gαi protein domains in which amino acid sequences thereof are substituted with corresponding amino acid sequences of Gαolf. When these chimeric Gα subunits are used, a larger amount of potassium ions are transported from a front side of the lipid bilayer membrane to a back side thereof in response to contact with the chemical substance. The amino acid sequences of each of the chimeric Gα subunits are shown below.
As shown in
As shown in
As shown in
As shown in
As shown in
As shown in
As shown in
(Potassium Ion Channel)
As potassium ion channels, G protein-coupled inwardly rectifying potassium channels (GIRKs) are used. The GIRKs are classified into four subtypes, Kir3.1, Kir3.2, Kir3.3 and Kir3.4. In the present invention, preferably used is a mutated form of Kir3.1, i.e., Kir3.1 (F137S) in which phenylalanine at position 137 of Kir3.1 is substituted with serine. The reason is described below. Each of the subtypes Kir3.1 to Kir3.4 assembles with a different subtype to form a heteromultimer. In other words, a GIRK includes two types of the subtypes. However, the mutated form Kir3.1 (F137S) assembles into a homotetramer, and the tetramer constitutes a potassium ion channel. Therefore, when the mutated form Kir3.1 (F137S) is used, the single type of protein is required for forming a potassium ion channel.
The lipid bilayer membrane comprising the chemical substance receptor, the chimeric G protein and the potassium ion channel, is prepared, for example, by a method described below. An expression plasmid coding for the chemical substance receptor, an expression plasmid coding for the chimeric G protein, and an expression plasmid coding for the potassium ion channel are constructed. Each of the constructed plasmids is introduced into cells and is expressed. Specific procedure of the method is described in detail in the examples.
(Potassium Ion Transport)
A front side and a back side of the lipid bilayer membrane comprising the chemical substance receptor, the chimeric G protein and the potassium ion channel, are each in contact with appropriate buffer solutions.
When the chemical substance is supplied to the front side of the lipid bilayer membrane and binds to the chemical substance receptor, the chimeric G protein bound to the chemical substance receptor on the back side of the lipid bilayer membrane is divided into and released as a chimeric Gα subunit and a Gβγ subunit complex. Contact of the Gβγ subunit complex to the potassium ion channel leads to opening of a gate of the potassium ion channel. When the gate of the potassium ion channel is opened, potassium ions are transported from the front side to the back side of the lipid bilayer membrane.
(Detection or Quantification of Chemical Substance)
The chemical substance can be detected or quantified by using the above described method of transporting potassium ions from the front side to the back side of the lipid bilayer membrane. A change in ionic current caused by the potassium ion transport is measured to detect or quantify the chemical substance existing on the front side of the lipid bilayer membrane. More particularly, the chemical substance is detected or quantified as described below. First, potassium ions are supplied to the front side of the lipid bilayer membrane comprising the chemical substance receptor, the chimeric G protein and the potassium ion channel described above. More particularly, for example, a first liquid is supplied to the front side of the lipid bilayer membrane, and a second liquid is supplied to the back side of the lipid bilayer membrane. Thus, the lipid bilayer membrane is interposed between the first liquid and the second liquid. Generally, the first liquid and the second liquid are buffer solutions having a pH of around 7. The first liquid contains potassium ions. The second liquid may also contain potassium ions.
A target chemical substance is also supplied on the front side of the lipid bilayer membrane. More particularly, the target chemical substance is supplied to the first liquid in a state where the lipid bilayer membrane is interposed between the first liquid and the second liquid. The target chemical substance is supplied to the chemical substance receptor. Supply of the target chemical substance to the chemical substance receptor causes the subunits of the chimeric G protein to be released. This gives rise to the transport of potassium ions (contained in the first liquid) locating in the front side of the lipid bilayer membrane to the back side (the second liquid) thereof. The change in ionic current generated by the potassium ion transport is measured to detect or quantify the chemical substance on the basis of the measurement result. The above-described supply of the chemical substance may be conducted with a solution instead of the first fluid, where the solution has the same composition as that of the first fluid except for containing an additional chemical substance.
Generally, a standard curve is used to quantify a chemical substance.
(Chemical Substance)
Any chemical substance capable of functioning as an agonist of the chemical substance receptor can be used as the chemical substance, and there is no particular limitation in the chemical substance. If an adrenergic receptor is used as the chemical substance receptor, the chemical substance includes, for example, isoproterenol, dopamine, and dobutamine.
Methods for constructing plasmids used in the Examples and Comparative Examples are shown below.
(Construction of Adrenergic Receptor Expression Plasmid)
Table 1 shows sequences of the used primers.
(Construction of Mutated Potassium Ion Channel Kir3.1 (F137S) Expression Plasmid)
The gene of a mutated potassium ion channel Kir3.1 (F137S) was constructed by partially mutating the mouse Kir3.1 gene.
(Construction of Wild Type G Protein (Gαolf) Expression Plasmid)
RNA was isolated from mouse olfactory bulb and was then reverse transcribed by using reverse transcriptase to obtain total cDNA in mouse olfactory cells. The obtained cDNA was used as template, and the Gαolf gene was amplified by PCR. Primer 17 (SEQ ID NO: 33) and primer 18 (SEQ ID NO: 34) were used. The obtained gene fragment was ligated into a plasmid, and thereby the Gαolf gene (GenBank Accession Number: AY179168.1) was cloned. The obtained Gαolf fragment was further amplified by PCR. Primer 19 (SEQ ID NO: 35) and primer 20 (SEQ ID NO: 36) were used. As a result, restriction enzyme sites were added to the ends of the amplified fragment. The amplified fragment having the restriction enzyme sites was ligated into an expression plasmid to obtain a wild type G protein (Gαolf) expression plasmid (hereinafter, referred to as “plasmid (Gαolf”).
(Construction of G Protein (Gαi (C351G)) Expression Plasmid)
(Construction of Chimeric G Protein (Gi/olf5) Expression Plasmid)
A chimeric G protein (Gi/olf5) expression plasmid (hereinafter, referred to as “plasmid (Gi/olf5)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (Gai), except for using primer 29 (SEQ ID NO: 45) instead of primer 28. The construction procedure is shown in
(Construction of Chimeric G Protein (Gi/s13) Expression Plasmid)
A chimeric G protein (Gi/s13) expression plasmid (hereinafter, referred to as “plasmid (Gi/s13)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (Gαi), except for using primer 30 (SEQ ID NO: 46) instead of primer 28. The construction procedure is shown in
(Construction of Chimeric G Protein (Gi/olf13) Expression Plasmid)
A chimeric G protein (Gi/olf13) expression plasmid (hereinafter, referred to as “plasmid (Gi/olf13)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (Gαi), except for using primer 31 (SEQ ID NO: 47) instead of primer 28. The construction procedure is shown in
(Construction of Chimeric G Protein (Gi/olf28) Expression Plasmid)
A chimeric G protein (Gi/olf28) expression plasmid (hereinafter, referred to as “plasmid (Gi/olf28)”) was constructed through overlap extension PCR by using the plasmid (Gi/olf13) as template I and the plasmid (Gαolf) as template II. As shown in
(Construction of Chimeric G Protein (Gi/olf45) Expression Plasmid)
A chimeric G protein (Gi/olf48) expression plasmid (hereinafter, referred to as “plasmid (Gi/olf48)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (Gi/olf28), except for using primers shown in Table 5.
(Construction of Chimeric G Protein (Gi/olf94) Expression Plasmid)
A chimeric G protein (Gi/olf94) expression plasmid (hereinafter, referred to as “plasmid (Gi/olf94)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (Gi/olf28), except for using primers shown in Table 5.
(Construction of Chimeric G Protein (Gi/olf113) Expression Plasmid)
A chimeric G protein (Gi/olf113) expression plasmid (hereinafter, referred to as “plasmid (Gi/olf113)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (Gi/olf28), except for using primers shown in Table 5.
(Construction of Chimeric G Protein (Gi/olf156) Expression Plasmid)
A chimeric G protein (Gi/olf156) expression plasmid (hereinafter, referred to as “plasmid (Gi/olf156)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (Gi/olf28), except for using primers shown in Table 5.
(Construction of Chimeric G Protein (Gi/olf195) Expression Plasmid)
A chimeric G protein (Gi/olf195) expression plasmid (hereinafter, referred to as “plasmid (Gi/olf195)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (Gi/olf28), except for using primers shown in Table 5.
Table 6 shows regions of amino acid sequences amplified by respective primers.
(Construction of Chimeric G Protein (Gi/olfα3-β5) Expression Plasmid)
A chimeric G protein (Gi/olfα3-β5) expression plasmid (hereinafter, referred to as “plasmid (Gi/olfα3-β5)”) was constructed through overlap extension PCR by using the plasmid (Gi/olf13) as template I and the plasmid (Gαi) as template II. As shown in
(Construction of Chimeric G Protein (Gi/olfα3-β5,C) Expression Plasmid)
A chimeric G protein (Gi/olfα3-β5,C) expression plasmid (hereinafter, referred to as “plasmid (Gi/olfα3-β5,C)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (Golfα3-β5), except for using templates and restriction enzymes described in Table 7.
(Construction of Chimeric G Protein (Gi/olfα4-β6) Expression Plasmid)
A chimeric G protein (Gi/olfα4-β6) expression plasmid (hereinafter, referred to as “plasmid (Gi/olfα4-β6)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (Golfα3-β5), except for using templates, E-PRIMER, F-PRIMER, and a restriction enzyme described in Table 7.
(Construction of Chimeric G Protein (Gi/olfα4-β6,C) Expression Plasmid)
A chimeric G protein (Gi/olfα4-β6,C) expression plasmid (hereinafter, referred to as “plasmid (Gi/olfα4-β6,C)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (Golfα3-β5), except for using templates, E-PRIMER, F-PRIMER, and restriction enzymes described in Table 7.
(Construction of Chimeric G Protein (Gi/olfα3-β5,α4-β6) Expression Plasmid)
A chimeric G protein (Gi/olfα3-β5,α4-β6) expression plasmid (hereinafter, referred to as “plasmid (Gi/olfα3-β5,α4-β6)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (Golfα3-β5), except for using templates, E-PRIMER, F-PRIMER, and a restriction enzyme described in Table 7.
(Construction of Chimeric G Protein (Gi/olfα3-β5,α4-β6,c) Expression Plasmid)
A chimeric G protein (Gi/olfα3-β5,α4-β6,C) expression plasmid (hereinafter, referred to as “plasmid (Gi/olfα3-β5,α4-β6,C)”) was constructed in accordance with a procedure same as that used for constructing the plasmid (Golfα3-β5), except for using templates, E-PRIMER, F-PRIMER, and restriction enzymes described in Table 7.
[Measurement of Electrophysiological Activities]
A cell was obtained that expressed a chimeric G protein, a potassium ion channel, and a chemical substance receptor (hereinafter, the cell is referred to as “chimeric G protein expressing cell”). An electrophysiological activity of the chimeric G protein expressing cell was measured by using patch-clamp method. The chimeric G protein expressing cell was prepared in accordance with the following procedure. First, a chimeric G protein expression plasmid, a potassium ion channel expression plasmid, and a chemical substance receptor expression plasmid were prepared. Next, the three expression plasmids were transfected into HEK293T cells, and were then expressed inside the cells.
A chimeric G protein comprising Gi/olf13 as a chimeric Gα subunit was employed. The mutated potassium ion channel Kir3.1 (F137S) was employed as a potassium ion channel. The β1 adrenergic receptor was employed as a chemical substance receptor.
(Preparation of Chimeric G Protein Expressing Cells)
The plasmid (βAR), the plasmid Kir3.1 (F137S), and the plasmid (Gi/olf13) were expressed in HEK293T cells. The procedure for the expression is shown below. Approximately 80% confluent cultured HEK293T cells were collected, and plated on a new culture petri dish. The passage number of the used cells was not more than ten generations. DMEM (supplemented with 10% FBS (fetal bovine serum) and streptomycin) was used. After culturing the cells for 24 hours, the cells were transfected with the plasmids by using a transfection reagent. The cells were cultured for 48 hours after being transfected with the plasmids, and thereby chimeric G protein expressing cells were prepared.
(Measurement of Current Change Levels)
Membrane current of the chimeric G protein expressing cell was measured by using a patch-clamp technique. Measurements were conducted with and without supplying isoproterenol (hereinafter, referred to as “ISO”) which was a β1 adrenergic receptor agonist. Procedure for the measurements is shown below.
(Samples for Measurements)
The chimeric G protein expressing cells were plated on coverslips (3 mm×10 mm; Matsunami Glass Ind., Ltd.,) treated with PLL (poly-L-lysine) and kept still for four hours to obtain samples for the measurements.
(Glass Electrodes)
Procedure for preparing glass electrodes used for the measurements is described below. By using a glass electrode manufacturing device (“Laser Puller P-2000” manufactured by Sutter Instrument Co.,), glass pipettes having a 1 μm diameter at one end were prepared from glass tubes (outer diameter 1.5 mm, internal diameter 0.86 mm, length 100 mm). Silver/silver chloride (Ag/AgCl) electrodes were inserted in the glass pipettes. The glass pipettes were filled with buffer A. The composition of the buffer A was similar to the composition of an intracellular fluid. The composition of the buffer A is shown in Table 8
(Measurements)
Measurement operations were conducted under a microscope (“1X71” manufactured by Olympus, Inc.). A liquid circulation chamber was mounted on the silver/silver chloride (Ag/AgCl) electrode. A sample for measurement was placed in the liquid circulation chamber filled with Tyroad's buffer. At a state where a single cell was in contact with the tip of a glass electrode, negative pressure was applied in the glass electrode. The portion of a cell membrane in contact with the tip of the glass electrode was torn to form an equivalent circuit (Whole-cell mode). The electric potential difference between inside and outside a cell was kept at 0 mV by using a patch clamp amplifier (“EPC10” manufactured by HEKA Instrument Inc.). Under this mode, the Tyroad's buffer in the liquid circulation chamber was substituted with a GIRK buffer. Pulse potentials (electric potential: −50 mV; duration time: 100 mS) were applied every 10 seconds, and membrane currents obtained upon the application were measured. Table 8 shows the compositions of the Tyroad's buffer and the GIRK buffer.
Next, membrane currents obtained when isoproterenol (hereinafter, abbreviated as “ISO”) which is a β1 adrenergic receptor agonist was brought into contact with the cell were measured in accordance with the following procedure. The GIRK buffer in the liquid circulation chamber was substituted with a sample solution. Under a condition in which the electric potential difference between inside and outside a cell was kept at 0 mV, pulse potentials (electric potential: −50 mV; duration time: 100 mS) were applied every 10 seconds, and membrane currents obtained upon the application were measured. The composition of the sample solution is shown in Table 8.
Chimeric G protein expressing cells were prepared and membrane currents were measured similarly to Example 1, except for employing chimeric G proteins comprising respective Gα subunits described in Table 9 instead of the chimeric G protein comprising Gi/olf13.
Chimeric G protein expressing cells were prepared and membrane currents were measured similarly to Example 1, except for employing chimeric G proteins comprising respective Gα subunits described in Table 10 instead of the chimeric G protein comprising Gi/olf13.
When the chimeric G protein expressing cells was prepared, the cells were prepared similarly to Example 1, except for transfecting the cells with the plasmid (Gi/olf13). Then, membrane currents of the prepared cells were measured.
The amino acid sequence of Gα subunit (Gαi (C351G) used in Comparative Example 1 is shown below.
The amino acid sequence of Gα subunit (Gαolf) used in Comparative Example 2 is shown below.
The amino acid sequence of a chimeric Gα subunit (Gi/olf5) used in Comparative Example 3 is shown below. Gi/olf5 is a chimeric Gα subunit obtained by substituting five amino acids on the C-terminal side of Gαi protein with corresponding amino acids of Gαolf.
The amino acid sequence of a chimeric Gα subunit (Gi/olf45) used in Comparative Example 4 is shown below.
The amino acid sequence of Gα subunit (Gi/olf156) used in Comparative Example 5 is shown below.
The amino acid sequence of a chimeric Gα subunit (Gi/olf195) used in Comparative Example 6 is shown below.
The amino acid sequence of a chimeric Gα subunit (Gi/olfα3-β5) used in Comparative Example 7 is shown below.
The amino acid sequence of a chimeric Gα subunit (Gi/olfα4-β6) used in Comparative Example 8 is shown below.
The amino acid sequence of a chimeric Gα subunit (Gi/olfα3-β5,α4-β6) used in Comparative Example 9 is shown below.
The amino acid sequence of a chimeric Gα subunit (Gi/olfα3-β5,α4-β6,C) used in Comparative Example 10 is shown below.
Table 11 and
Table 12 and
(Comparison to Camp Technique)
Chemical substance detection sensitivity according to the present invention was compared to detection sensitivity of cAMP technique, which is a commonly used chemical substance detection technique. The cAMP technique is a technique for measuring an increase in cAMP concentration in an intracellular fluid caused by an interaction between an agonist and a receptor, and quantifying the concentration of the agonist based on the measured value. More particularly, cells expressing β1 adrenergic receptor and potassium ion channel Kir3.1 (F137S) were placed in contact with a buffer solution containing ISO, and then were homogenized. The concentration of cAMP contained in the obtained homogenate liquid was measured. The concentration measurement was performed by using a measurement kit (cyclic AMP EIA kit; Assay Designs, Inc.). The kit measures cAMP concentration by utilizing competitive EIA (competitive enzyme immunoassay). A cAMP concentration obtained when the ISO concentration inside of a buffer solution was 1000 nM was represented as 100 as a standard, and cAMP concentrations at respective ISO concentrations were calculated as ratios with regard to this standard cAMP concentration. On the other hand, measurements were conducted similarly to Example 3, except that the measurements were conducted by changing the ISO concentration. A change value in current density obtained when the ISO concentration was 30 nM was represented as 100 as a standard, and change values in current density at respective ISO concentrations were calculated as ratios with regard to the standard change value. Table 13 and
The following describes what is understood from Table 13 and
(Detection of β Adrenergic Receptor Agonist)
Detections of β1 agonists other than isoproterenol were conducted. Measurements were conducted similarly to Example 3, except for using, in addition to ISO as chemical substances, dobutamine, which is a selective agonist of β1 adrenergic receptor, and dopamine which is an agonist of β adrenergic receptor. Table 14 and
With every agonist used, the change values in current density increased in association with increases in agonist concentrations. The EC50 values for dopamine and dobutamine were 1000 nM and 50 nM, respectively. It is reasonably presumed that a change in current density is generated by agonists activating chimeric G proteins via chemical substance receptors and opening gates of ion channels. This is because the EC50 values are different depending on the type of agonist. Therefore, agonists are not directly but indirectly activating chimeric G proteins.
The present inventors examined the following in advance of the present invention, and determined a design plan for the chimeric Gα subunits. Gαi (C351G), Gαolf, and Gi/olf13 were used as Gα subunits to measure ionic current generated upon contact with ISO. The measurements were conducted in accordance with a method similar to that in Example 1.
As shown in
As shown in
The present inventors derived the following conclusion from the above described measurement results. Gαi (C351G) can activate potassium ion channels but cannot bind to chemical substance receptors. Gαolf can bind to chemical substance receptors but cannot activate potassium ion channels. Thus, the current change level caused by the addition of ISO was small. On the other hand, when Gi/olf13 was used, a large current change was observed caused by the addition of ISO, since the chimeric Gα subunit can bind to chemical substance receptors via the Gαolf region at the C-terminal and the Gαi region activates potassium ion channels. Therefore, the chimeraization of the Gα subunit increases the current change level caused by the addition of ISO.
Furthermore, a current change level obtained when Gi/s13 was used as the chimeric Gα subunit was compared to a current change level obtained when Gi/olf13 was used as the chimeric Gα subunit. Table 15 and
The current change level obtained when Gi/olf13 was used was larger than the current change level obtained when Gi/s13 was used. Therefore, it was determined that larger current changes can be expected when a chimera of Gαi and Gαolf were used. Hence, it was determined to create a chimeric Gα subunit in which a region generally considered to participate in the coupling with a chemical substance receptor in the amino acid sequence of Gαi was substituted with a corresponding amino acid sequence of Gαolf.
According to a chimeric G protein of the present invention, a detection of a chemical substance that binds to a chemical substance receptor is enabled.
Number | Date | Country | Kind |
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2010-223737 | Oct 2010 | JP | national |
Number | Date | Country | |
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Parent | PCT/JP2011/000375 | Jan 2011 | US |
Child | 13248880 | US |