KR20230080915A - Ligand efficacy analysis method for taste substances of ligand binding domain of human sweet receptor using tryptophan fluorescence assay - Google Patents

Abstract

The present invention relates to a method for analyzing ligand efficacy of a ligand binding domain of a human sweet taste receptor for a taste substance, comprising the steps of: producing a ligand binding domain consisting of a human sweet taste receptor T1R2 venus fly trap (VFT); and treating the ligand binding domain with a taste substance, and then analyzing fluorescence changes in tryptophan in a protein. According to the present invention, a dose-dependent and selective functionality of a sweet taste substance can be verified by tryptophan fluorescence assay, which is a relatively simple method, using the T1R2 VFTs produced to a high standard.

Description

Ligand efficacy analysis method for taste substances of ligand binding domain of human sweet receptor using tryptophan fluorescence assay}

The present invention relates to a method for analyzing ligand efficacy for taste substances of a ligand binding domain of a human sweet taste receptor using tryptophan fluorescence analysis.

Taste influences the taste of food and receives information about nutrients and harmful substances necessary for the body through it. Among the five basic tastes that humans feel, sweetness helps us recognize carbohydrates, which are the main energy source, and contributes to regulating calorie intake and food preference. However, as the association with diseases such as obesity and diabetes due to excessive intake of sugar by modern people has attracted attention, interest in developing sweet sugar substitutes or sweetness improvers has increased. Therefore, measurement of sweetness can be very important in the food, beverage and pharmaceutical industries.

Sensory evaluation, a representative existing method for measuring taste, has the advantage of being able to provide information on emotions when the panel feels the taste, but the disadvantage is that objectivity and accuracy are low because each individual has a different sensitivity to taste there is As methods for objective measurement of sweetness, the use of high performance liquid chromatography or the development of an electronic tongue have been developed, but all of them can identify the components that make up sweetness, but the actual human tongue feels it. The disadvantage and limitation of not being able to measure the degree of taste has been pointed out. Recently, as interest in taste measurement and standardization through simulation of human taste has increased, the use of biomaterials that contribute to detecting sweetness on the human tongue is attracting attention.

Sweet taste receptors, which are Class C GPCRs, are known to be difficult to bacterial expression due to their large size and complex structure. Therefore, the inventors of the present invention completed the present invention by producing T1R2 VFT, which is known as a major ligand binding site for sweetness, and analyzing the efficacy of sweetness by analyzing the fluorescence change of tryptophan in the protein as an alternative.

KR 10-1860097 B1

The present invention is to provide a method for verifying the dose-dependent and selective functionality of the T1R2 VFT of the human sweet taste receptor for various sweet substances.

In addition, the present invention is a biomaterial in which the T1R2 VFT of the human sweetness receptor can measure human sweetness, and is intended to suggest the possibility of objective measurement of taste substances that mimic human taste by mobilizing various analysis methods or biosensors in the future. .

In order to solve the above problems, an embodiment of the present invention produces a ligand binding domain consisting of human sweet receptor T1R2 VFT (Venus fly trap); and

Analyzing a change in fluorescence of tryptophan in the protein after treating the ligand-binding domain with a taste substance;

It provides a ligand efficacy analysis method for the taste material of the ligand binding domain of the human sweet receptor comprising a.

In addition, another embodiment of the present invention provides a sweetness measurement method using the T1R2 VFT of the human sweetness receptor as a biological material capable of measuring human sweetness.

According to the present invention, it is possible to verify the dose-dependent and selective functionality of sweet substances through tryptophan fluorescence analysis, which is a relatively simple method, by utilizing the high-level produced T1R2 VFT.

In addition, the T1R2 VFT of the sweetness receptor according to the present invention is a biomaterial capable of measuring human sweetness, and can be effectively used for objective measurement of taste substances that mimic human taste by mobilizing various analysis methods or biosensors in the future.

1 is a schematic diagram of a pET-DEST 42 vector including a T1R2 VFT according to an embodiment of the present invention.
Figure 2 is an SDS-PAGE result after expression, solubilization and purification of T1R2 VFT according to an embodiment of the present invention.
3 is an SDS-PAGE result after purification of T1R2 VFT protein according to an embodiment of the present invention.
4 is an SDS-PAGE result after refolding of the T1R2 VFT according to an embodiment of the present invention.
5 shows the results of tryptophan fluorescence analysis of T1R2 VFT for sweet substances (natural sweeteners) at various concentrations according to an embodiment of the present invention.
6 shows the results of tryptophan fluorescence analysis of T1R2 VFT for sweet substances (artificial sweeteners) at various concentrations according to an embodiment of the present invention.
7 shows the results of tryptophan fluorescence analysis of T1R2 VFT for various taste substances (taste molecules) according to an embodiment of the present invention.

Hereinafter, the present invention will be described.

All terms (including technical and scientific terms) used in this specification, unless otherwise defined, may be used in a meaning commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, terms defined in commonly used dictionaries are not interpreted ideally or excessively unless explicitly specifically defined.

As used herein, the term “sweet taste receptor” refers to a Class C G-protein coupled receptor (GPCR) in which T1R2 and T1R3 exist as heterodimers. T1R2 (TAS1R2) and T1R3 (TAS1R3) consist of an N-terminal Venus fly trap (VFT) region, a transmembrane region, and a cysteine-rich domain (CRD) connecting the VFT region and the transmembrane region. Consists of. The VFT of the T1R2 subunit provides binding sites for the sweetest substances such as sucrose, glucose and saccharin.

According to one embodiment of the present invention,

producing a ligand binding domain consisting of human sweet taste receptor T1R2 VFT (Venus fly trap); and

Analyzing a change in fluorescence of tryptophan in the protein after treating the ligand-binding domain with a taste substance;

A ligand efficacy analysis method for taste substances of a ligand binding domain of a human sweet receptor comprising a is disclosed.

The ligand binding domain consists of VFT (Venus fly trap) of human sweet taste receptor T1R2. Preferably, the VFT of the human sweet taste receptor T1R2 is a sequence from which the 1st to 20th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 is removed, that is, the 21st to 472nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 includes

In one embodiment of the present invention, the human sweet taste receptor T1R2 VFT (Venus fly trap) gene was amplified by polymerase chain reaction (PCR) of human genomic DNA, cloned into a bacterial expression vector, and then transformed into an E. coli strain. Overexpression of the T1R2 VFT gene was induced by culturing the transformed cells.

When a sample containing the ligand-binding domain and a sweet ligand is added, a structural change of the receptor protein occurs as the sweet ligand binds to the receptor protein. Tryptophan residues in proteins show intrinsic fluorescence at an emission wavelength of about 350 nm. As the ligand binds to the receptor protein, the intrinsic fluorescence is quenched by structural changes in the protein. That is, stimulation of the T1R2 VFT by a sample containing a sweet ligand causes a change in the fluorescence of tryptophan in the protein. In the present invention, the sweetness effect of the substance was analyzed by analyzing the fluorescence change of tryptophan in the protein.

The sweet ligand may be derived from natural sweeteners or artificial sweeteners. For example, sucrose, glucose, fructose, stevioside, aspartame, acesulfameK, saccharin, sucralose, and neotame are selected from the group consisting of It may be derived from one or more species, but is not limited thereto.

According to the present invention, it is possible to verify the dose-dependent and selective functionality of sweet substances through tryptophan fluorescence analysis, which is a relatively simple method, by utilizing the high-level produced T1R2 VFT.

The T1R2 VFT of the sweetness receptor according to the present invention is a biomaterial that can measure human sweetness, and can be effectively used for objective measurement of taste substances that mimic human taste by mobilizing various analysis methods or biosensors in the future.

Another embodiment of the present invention provides a method for measuring sweetness using the T1R2 VFT of human sweetness receptor as a biological material capable of measuring human sweetness.

The human sweet taste receptor T1R2 VFT includes the 21st to 472nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only for exemplifying the present invention, and the scope of the present invention is not limited to the examples.

<Example>

preparation of materials

Human cDNA, pCMV6-ENTRY-hTAS1R2, was purchased from Origene (USA). Semiconductor single-wall CNTs of 99% purity were purchased from NanoIngetris (Canada) and used as received. Sucrose, glucose, fructose, stevioside, aspartame, acesulfame K, saccharin, sucralose and neotame, sodium cyclamate, cellobiose, monosodium glutamate, and denatonium benzoate were purchased from Sigma-Aldrich (USA).

Example 1. T1R2 VFT gene cloning, expression and purification

1-1. T1R2 VFT gene cloning

The T1R2 VFT gene with the signal peptide (1-20 amino acids) deleted was transcribed into the human genome using primers (5' CAC CAG GAG ATA TAC ATA TGG CTG AGA ACT CG 3', 5' GAC ATA GGG ATC GTG TTG 3'). DNA was amplified by polymerase chain reaction (PCR). The amplified PCR product was inserted into the pENTR vector by directional TOPO cloning, and then cloned into the pET-DEST 42 bacterial expression vector having the 6xHis gene at the C-terminal multi-cloning site (MCS). The structure of the pET-DEST 42 bacterial expression vector is shown in FIG. 1 .

The primers used at this time are shown in Table 1 below:

Sense primer CAC CAG GAG ATA TAC ATA TGG CTG AGA ACT CG Anti-sense primer GAC ATA GGG ATC GTG TTG

1-2. Expression, solubilization and purification of T1R2 VFT

The pET-DEST 42 bacterial expression vector was transformed into an E. coli Rosetta ^TM (DE3) strain, and cultured at 37° C. for one day in LB (Luria-Broth) medium supplemented with 50 μg/Ml ampicillin. After growth to an OD ₆₀₀ value of 0.5, T1R2 VFT expression was induced using 0.5 mM isopropyl β-D-thiogalactoside (IPTG). Cells were cultured for 4 hours and harvested by centrifugation (7000 g, 20 min, 4 °C), and the resulting pellet was resuspended in phosphate buffered saline (PBS) containing 2 mM EDTA (pH 8.0) and sonicated (5 sec). on/off, 25% amplitude, 5 min). Samples were then collected by centrifugation (12000g, 30 min, 4°C). The expression of the T1R2 VFT was confirmed by SDS-PAGE, and the results are shown in FIG. 2 .

The insoluble fraction containing the T1R2 VFT was solubilized in solubilization buffer (0.1 M Tris-HCl, 20 mM sodium dodecyl sulfate (SDS), 100 mM dithiothreitol (DTT), 1 mM EDTA, pH 8.0) at 30 °C overnight. Solubilized samples were centrifuged (12000g, 30 min, 30°C) and collected. Samples were dialyzed in dialysis buffer (0.1 M sodium phosphate, 10 mM SDS, pH 8.0) using a MEMBRA-CEL® dialysis membrane (Viskase) with a 14 kilodalton molecular weight cutoff (MWCO). After filtering the dialyzed sample with a 0.45 μm bottle-top filter (Thermo Fisher Scientific), the sample was loaded onto a 5 ml HisTrap affinity column (GE Healthcare) equilibrated with binding buffer (0.1 M sodium phosphate, 10 mM SDS, pH 8.0). . The column was then gradually washed with wash buffer (0.1 M sodium phosphate, 10 mM SDS, pH 7.0). Finally, the T1R2 VFT was eluted with elution buffer (0.1 M sodium phosphate, 10 mM SDS, pH 6.0) and stored at -80 °C for later use. Solubilization and purification of the T1R2 VFT were confirmed by SDS-PAGE, and the results are shown in FIGS. 2 and 3 .

Gel staining and Western blot analysis showed a band at 54 kDa, which corresponds to the molecular weight of T1R2 VFT calculated by the online ExPASy bioinformatics tool. These results indicate that the T1R2 VFT was successfully overexpressed and purified.

1-3. Refolding of the T1R2 VFT

In order to reproduce the folding of the T1R2 VFT, 4-(2-hydroxyethyl)- It was dialyzed in 1-piperazineethanesulfonic acid (HEPES) buffer I (20 mM HEPES, 10 mM SDS, 0.5 mM EDTA, pH 8.0). The dialysis buffer was then changed to HEPES buffer II (20 mM HEPES, 5 mM SDS, 0.5 mM EDTA, pH 7.5) at room temperature. The buffer was then replaced with HEPES buffer III (20 mM HEPES, 3 mM SDS, 0.5 mM EDTA, pH 7.5) at room temperature. 10 mM methyl-β-cyclodextrin was added to the dialyzed protein sample and stirred overnight at 4°C. The mixed samples were finally dialyzed at 4° C. against refolding buffer (20 mM HEPES, 1 mM EDTA and 150 mM NaCl, pH 7.5) to allow refolding. The refolding results were confirmed through SDS-PAGE and Western blot analysis, and the results are shown in FIG. 4 .

Example 2. Tryptophan fluorescence assay

The ligand binding function of the refolded T1R2 VFT was analyzed by tryptophan fluorescence quenching assay using an LS 55 luminescence spectrometer (Perkin Elmer). Intrinsic tryptophan fluorescence of the T1R2 VFT was measured with excitation at 290 nm and emission at 350 nm.

Relative fluorescence intensity was calculated by Equation 1 below:

[Equation 1]

(Δ F/F ₀ (%) = [( F ₀ - F )/ F ₀ ] X 100).

F ₀ and F represent the fluorescence intensity of T1R2 VFT before and after treatment with taste molecules, respectively.

The function of T1R2 VFT for various taste substances listed in Table 2 below was evaluated by tryptophan fluorescence assay.

sample note Sucrose Sweetness (natural sweetener) Glucose Sweetness (natural sweetener) Fructose Sweetness (natural sweetener) Stevioside Sweetness (artificial sweeteners) Aspartame Sweetness (artificial sweeteners) Acesulfame K Sweetness (artificial sweeteners) Saccharin Sweetness (artificial sweeteners) Sucralose Sweetness (artificial sweeteners) Neotame Sweetness (artificial sweeteners) Sodium cyclamate Sweetness (artificial sweeteners) Cellobiose Tasteless reducing sugar Monosodium glutamate (L-Glutamic acid monosodium salt monohydrate (MSG)) Umami Denatonium benzoate bitter

2-1. Verification of dose-dependent functionality of refolded T1R2 VFT for various sweet substances using tryptophan fluorescence assay

The function of T1R2 VFT was evaluated by tryptophan fluorescence analysis at various concentrations for the natural and artificial sweeteners described in Table 2 above.

As shown in FIGS. 5 and 6, the higher the concentration, the higher the normalized fluorescence intensity.

It can be seen that when various sweet substances bind to the refolded T1R2 VFT, structural changes in the protein are induced, resulting in changes in tryptophan fluorescence quenching.

From this, it can be seen that the refolded T1R2 VFT according to the present invention has a dose-dependent functionality with respect to various sweet substances.

2-2. Verification of selective functionality for various taste substances of refolded T1R2 VFT using tryptophan fluorescence analysis

The function of T1R2 VFT for the taste substances listed in Table 2 was evaluated by tryptophan fluorescence analysis.

As shown in Figure 7, the relative fluorescence intensity change value when cellobiose, monosodium glutamate (MSG) and denatonium benzoate (denatonium, bitter taste) were introduced was less than 3%, whereas relative fluorescence intensity change for natural and artificial sweeteners The value was much greater. In particular, the relative fluorescence intensity change value for artificial sweeteners exceeded 7%. And no significant fluorescence change was detected using cyclamate, an artificial sweetener known to interact with the transmembrane domain of T1R3. From this, it can be seen that the refolded T1R2 VFT according to the present invention can selectively recognize sweet substances.

<110> Seoul National University R&DB Foundation <120> Ligand efficacy analysis method for taste substances of ligand binding domain of human sweet receptor using tryptophan fluorescence assay <130> DP210601 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 472 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 1 Met Ala Glu Asn Ser Asp Phe Tyr Leu Pro Gly Asp Tyr Leu Leu Gly 1 5 10 15 Gly Leu Phe Ser Leu His Ala Asn Met Lys Gly Ile Val His Leu Asn 20 25 30 Phe Leu Gln Val Pro Met Cys Lys Glu Tyr Glu Val Lys Val Ile Gly 35 40 45 Tyr Asn Leu Met Gln Ala Met Arg Phe Ala Val Glu Glu Ile Asn Asn 50 55 60 Asp Ser Ser Leu Leu Pro Gly Val Leu Leu Gly Tyr Glu Ile Val Asp 65 70 75 80 Val Cys Tyr Ile Ser Asn Asn Val Gln Pro Val Leu Tyr Phe Leu Ala 85 90 95 His Glu Asp Asn Leu Leu Pro Ile Gln Glu Asp Tyr Ser Asn Tyr Ile 100 105 110 Ser Arg Val Val Ala Val Ile Gly Pro Asp Asn Ser Glu Ser Val Met 115 120 125 Thr Val Ala Asn Phe Leu Ser Leu Phe Leu Leu Pro Gln Ile Thr Tyr 130 135 140 Ser Ala Ile Ser Asp Glu Leu Arg Asp Lys Val Arg Phe Pro Ala Leu 145 150 155 160 Leu Arg Thr Thr Pro Ser Ala Asp His His Ile Glu Ala Met Val Gln 165 170 175 Leu Met Leu His Phe Arg Trp Asn Trp Ile Ile Val Leu Val Ser Ser 180 185 190 Asp Thr Tyr Gly Arg Asp Asn Gly Gln Leu Leu Gly Glu Arg Val Ala 195 200 205 Arg Arg Asp Ile Cys Ile Ala Phe Gln Glu Thr Leu Pro Thr Leu Gln 210 215 220 Pro Asn Gln Asn Met Thr Ser Glu Glu Arg Gln Arg Leu Val Thr Ile 225 230 235 240 Val Asp Lys Leu Gln Gln Ser Thr Ala Arg Val Val Val Val Phe Ser 245 250 255 Pro Asp Leu Thr Leu Tyr His Phe Phe Asn Glu Val Leu Arg Gln Asn 260 265 270 Phe Thr Gly Ala Val Trp Ile Ala Ser Glu Ser Trp Ala Ile Asp Pro 275 280 285 Val Leu His Asn Leu Thr Glu Leu Arg His Leu Gly Thr Phe Leu Gly 290 295 300 Ile Thr Ile Gln Ser Val Pro Ile Pro Gly Phe Ser Glu Phe Arg Glu 305 310 315 320 Trp Gly Pro Gln Ala Gly Pro Pro Pro Leu Ser Arg Thr Ser Gln Ser 325 330 335 Tyr Thr Cys Asn Gln Glu Cys Asp Asn Cys Leu Asn Ala Thr Leu Ser 340 345 350 Phe Asn Thr Ile Leu Arg Leu Ser Gly Glu Arg Val Val Tyr Ser Val 355 360 365 Tyr Ser Ala Val Tyr Ala Val Ala His Ala Leu His Ser Leu Leu Gly 370 375 380 Cys Asp Lys Ser Thr Cys Thr Lys Arg Val Val Tyr Pro Trp Gln Leu 385 390 395 400 Leu Glu Glu Ile Trp Lys Val Asn Phe Thr Leu Leu Asp His Gln Ile 405 410 415 Phe Phe Asp Pro Gln Gly Asp Val Ala Leu His Leu Glu Ile Val Gln 420 425 430 Trp Gln Trp Asp Arg Ser Gln Asn Pro Phe Gln Ser Val Ala Ser Tyr 435 440 445 Tyr Pro Leu Gln Arg Gln Leu Lys Asn Ile Gln Asp Ile Ser Trp His 450 455 460 Thr Ile Asn Asn Thr Ile Pro Met 465 470

Claims (5)

producing a ligand binding domain consisting of human sweet taste receptor T1R2 VFT (Venus fly trap); and
Analyzing a change in fluorescence of tryptophan in the protein after treating the ligand-binding domain with a taste substance;
A method for analyzing ligand efficacy for taste substances in the ligand binding domain of a human sweet receptor comprising a. According to claim 1,
The human sweet receptor T1R2 VFT comprises the 21st to 472nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1, the ligand efficacy analysis method for the taste material of the ligand binding domain of the human sweet taste receptor. According to claim 1,
The step of analyzing the fluorescence change of tryptophan in the protein is to measure the unique tryptophan fluorescence of T1R2 VFT by excitation at 290 nm and emission at 350 nm, and then calculate the relative fluorescence intensity by the following formula 1, of human sweet taste receptor. Method for Assaying Ligand Efficacy of Ligand Binding Domains to Taste Substances:
[Equation 1]
(Δ F/F ₀ (%) = [( F ₀ - F )/ F ₀ ] X 100)
( F ₀ and F represent the fluorescence intensity of T1R2 VFT before and after treatment with taste molecules, respectively.) A method for measuring sweetness using T1R2 VFT of human sweetness receptor as a biological material capable of measuring human sweetness. According to claim 4,
The human sweet taste receptor T1R2 VFT comprises the 21st to 472nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1, sweetness measuring method.

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