Miraculin (1-20)
目录号 : GA23201
Miraculin (MCL) 是 R 的活性成分。
Cas No.:198694-37-0
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Cell experiment [1]: | |
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Cell lines |
HEK293 cells |
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Preparation Method |
Miraculin was dissolved in HBSS containing 10 mM HEPES, HEK293 cells were treated with Miraculin (10 µg/ml) for 3 minutes. |
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Reaction Conditions |
10 µg/ml Miraculin for 3 minutes |
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Applications |
Miraculin-applied cells displayed a pH dependence with citric acid (weak acid) being right shifted to that with hydrochloric acid (strong acid). When histidine residues in both the intracellular and extracellular region of hTAS1R2 were exchanged for alanine, taste-modifying effect of Miraculin was reduced or abolished. Stronger intracellular acidification of HEK293 cells was induced by citric acid than by HCl and taste-modifying effect of Miraculin was proportional to intracellular pH regardless of types of acids. |
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References: [1]: Sanematsu K, Kitagawa M, Yoshida R, Nirasawa S, Shigemura N, Ninomiya Y. Intracellular acidification is required for full activation of the sweet taste receptor by miraculin. Sci Rep. 2016 Mar 10;6:22807. doi: 10.1038/srep22807. PMID: 26960429; PMCID: PMC4785348. |
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Miraculin (MCL) is active component of R. dulcifica that modifies or converts sourness to sweetness. Miraculin is a taste-modifying protein that exhibits extremely unusual properties and is famous for its unique taste characteristics [2]. Taste-modifying effect of Miraculin is specific to humans but not to rodents,he role of Miraculin varies among different types of acids[4,7].
The TAS1Rs belong to the class C G-protein-coupled receptor (GPCR) family and consist of three principal domains: an amino-terminal domain (ATD) and a cysteine-rich domain (CRD) located in the extracellular region and a transmembrane domain (TMD) [6].
Miraculin interacts with the human sweet receptor subunit hTAS1R2. Miraculin-applied cells displayed a pH dependence with citric acid (weak acid) being right shifted to that with hydrochloric acid (strong acid). When histidine residues in both the intracellular and extracellular region of hTAS1R2 were exchanged for alanine, taste-modifying effect of Miraculin was reduced or abolished. Stronger intracellular acidification of HEK293 cells was induced by citric acid than by HCl and taste-modifying effect of Miraculin was proportional to intracellular pH regardless of types of acids. Intracellular acidity is required for full activation of the sweet taste receptor by Miraculin [1，3].
Recombinant Miraculin resembled native Miraculin in the secondary structure and the taste-modifying activity to generate sweetness at acidic pH. Since the observed pH-sweetness relation seemed to reflect the imidazole titration curve, suggesting that histidine residues might be involved in the taste-modifying activity.Both H30A and H30,60A mutants have lost the taste-modifying activity. Histidine-30 residue is important for the taste-modifying activity of Miraculin [5].
References:
[1]: Misaka T. Molecular mechanisms of the action of miraculin, a taste-modifying protein. Semin Cell Dev Biol. 2013 Mar;24(3):222-5. doi: 10.1016/j.semcdb.2013.02.008. Epub 2013 Mar 4. PMID: 23466289.
[2]: Kurihara K, Beidler LM. Taste-modifying protein from miracle fruit. Science. 1968 Sep 20;161(3847):1241-3. doi: 10.1126/science.161.3847.1241. PMID: 5673432.
[3]: Sanematsu K, Kitagawa M, et,al. Intracellular acidification is required for full activation of the sweet taste receptor by miraculin. Sci Rep. 2016 Mar 10;6:22807. doi: 10.1038/srep22807. PMID: 26960429; PMCID: PMC4785348.
[4]: Brouwer JN, Glaser D, et,al. The sweetness-inducing effect of miraculin; behavioural and neurophysiological experiments in the rhesus monkey Macaca mulatta. J Physiol. 1983 Apr;337:221-40. doi: 10.1113/jphysiol.1983.sp014621. PMID: 6875928; PMCID: PMC1199104.
[5]: Ito K, Asakura T, et,al. Microbial production of sensory-active miraculin. Biochem Biophys Res Commun. 2007 Aug 24;360(2):407-11. doi: 10.1016/j.bbrc.2007.06.064. Epub 2007 Jun 19. PMID: 17592723.
[6]: Kunishima N, Shimada Y, et,al. Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature. 2000 Oct 26;407(6807):971-7. doi: 10.1038/35039564. PMID: 11069170.
[7]: KURIHARA, K., BEIDLER, L. Mechanism of the Action of Taste-modifying Protein. Nature 222, 1176-1179 (1969). https://doi.org/10.1038/2221176a0
Miraculin (MCL) 是 R. dulcifica 的活性成分，可改变酸味或将酸味转化为甜味。 Miraculin 是一种味觉修饰蛋白，具有极其不寻常的特性，并以其独特的味觉特征而闻名[2]。 Miraculin 的味觉调节作用对人类有特异性，但对啮齿动物没有，Miraculin 的作用因酸的不同而异[4,7]。
TAS1R 属于 C 类 G 蛋白偶联受体 (GPCR) 家族，由三个主要结构域组成:氨基末端结构域 (ATD) 和位于细胞外区域的富含半胱氨酸结构域 (CRD) 和跨膜结构域 (TMD) [6]。
奇迹蛋白与人类甜味受体亚基 hTAS1R2 相互作用。应用 Miraculin 的细胞表现出 pH 依赖性，柠檬酸（弱酸）右移到盐酸（强酸）。当 hTAS1R2 细胞内和细胞外区域的组氨酸残基都被丙氨酸交换时，Miraculin 的味觉调节作用减弱或消失。柠檬酸比 HCl 诱导的 HEK293 细胞内酸化更强，而无论酸的类型如何，Miraculin 的味觉调节作用都与细胞内 pH 值成正比。 Miraculin [1，3] 完全激活甜味受体需要细胞内酸度。
重组的 Miraculin 在二级结构和在酸性 pH 下产生甜味的味道调节活性方面与天然 Miraculin 相似。由于观察到的pH-甜度关系似乎反映了咪唑滴定曲线，表明组氨酸残基可能参与了味觉修饰活性。H30A和H30,60A突变体都失去了味觉修饰活性。组氨酸 30 残基对 Miraculin 的味觉修饰活性很重要[5]。
| Cas No. | 198694-37-0 | SDF | |
| 分子式 | C88H146N26O34 | 分子量 | 2112.28 |
| 溶解度 | Soluble in DMSO | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 0.4734 mL | 2.3671 mL | 4.7342 mL |
| 5 mM | 0.0947 mL | 0.4734 mL | 0.9468 mL |
| 10 mM | 0.0473 mL | 0.2367 mL | 0.4734 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Safety assessment of miraculin using in silico and in vitro digestibility analyses
Food Chem Toxicol2019 Nov;133:110762.PMID: 31421212DOI: 10.1016/j.fct.2019.110762
Miraculin is a glycoprotein with the ability to make sour substances taste sweet. The safety of miraculin has been evaluated using an approach proposed by the Food and Agriculture Organization of the United Nations and the World Health Organization for assessing the safety of novel proteins. Miraculin was shown to be fully and rapidly digested by pepsin in an in vitro digestibility assay. The proteomic analysis of miraculin's pepsin digests further corroborated that it is highly unlikely that any of the protein will remain intact within the gastrointestinal tract for potential absorption. The potential allergenicity and toxigenicity of miraculin, investigated using in silico bioinformatic analyses, demonstrated that miraculin does not represent a risk of allergy or toxicity to humans with low potential for cross-reactivity with other allergens. The results of a sensory study, characterizing the taste receptor activity of miraculin, showed that the taste-modifying effect of miraculin at the concentration intended for product development has a rapid onset and disappearance with no desensitizing impact on the receptor. Overall, the results of this study demonstrate that the use of miraculin to impact the sensory qualities of orally administered products with a bitter/sour taste profile is not associated with any safety concerns.
The accumulation of recombinant miraculin is independent of fruit size in tomato
Plant Biotechnol (Tokyo)2021 Mar 25;38(1):161-165.PMID: 34177337DOI: 10.5511/plantbiotechnology.20.0904a
The taste-modifying protein miraculin (MIR) has received increasing interest as a new low-calorie sweetener. In our previous study using the tomato variety 'Micro-Tom,' it was shown that in transgenic tomatoes in which MIR was expressed by using the cauliflower mosaic virus 35S promoter (p35S) and a heat shock protein terminator (tHSP) cassette (p35S-MIR-tHSP), higher levels of miraculin accumulated than when MIR was driven by the nopaline synthase terminator (tNOS) cassette (p35S-MIR-tNOS). 'Micro-Tom' is a dwarf tomato used for research and shows a low yield. To achieve high productivity of MIR, it is essential to improve the MIR accumulation potential by using high-yielding cultivars. In this study, we evaluate whether the high MIR accumulation trait mediated by the tHSP appears even when fruit size increases. A line in which the p35S-MIR-tHSP cassette was introduced into a high-yielding variety was bred by backcrossing. The line homozygous for MIR showed higher accumulation of MIR than the heterozygous line. Despite large differences in fruit size, the MIR level in the backcross line was similar to that in the p35S-MIR-tHSP line (background 'Micro-Tom'). It was approximately 3.1 times and 4.0 times higher than those in miracle fruits and the p35S-MIR-tNOS tomato line 5B ('Moneymaker' background, which exhibits the highest miraculin productivity achieved thus far), respectively. These results demonstrate that the high MIR accumulation trait mediated by the tHSP appears even when fruit size is increased.
Structural and functional analysis of miraculin-like protein from Vitis vinifera
Biochim Biophys Acta Proteins Proteom2018 Nov;1866(11):1125-1130.PMID: 30282610DOI: 10.1016/j.bbapap.2018.08.009
The so-called miraculin-like proteins (MLPs) are homologous to miraculin, a homodimeric protein with taste-modifying activity that converts sourness into sweetness. The identity between MLPs and miraculin generally ranges from 30% to 55%, and both MLPs and miraculin are categorized into the Kunitz-type soybean trypsin inhibitor (STI) family. MLP from grape (Vitis vinifera; vvMLP) exhibits significant homology to miraculin (61% identity), suggesting that vvMLP possesses miraculin-like properties. The results of size-exclusion chromatography and sensory analysis illustrated that vvMLP exists as a monomer in solution with no detectable taste-modifying activity. Crystal structure determination revealed that vvMLP exists as a β-trefoil fold, similarly as other MLPs and Kunitz-type protein inhibitors. The conformation of the loops, including the so-called reactive loop in the STI family, was substantially different between vvMLP and STI. Recombinant vvMLP had inhibitory activity against trypsin (Ki = 13.7 μM), indicating that the protein can act as a moderate trypsin inhibitor.
Large-scale production of recombinant miraculin protein in transgenic carrot callus suspension cultures using air-lift bioreactors
AMB Express2020 Aug 13;10(1):140.PMID: 32789704DOI: 10.1186/s13568-020-01079-3
Miraculin, derived from the miracle fruit (Synsepalum dulcificum), is a taste-regulating protein that interacts with human sweet-taste receptors and transforms sourness into sweet taste. Since miracle fruit is cultivated in West Africa, mass production of miraculin is limited by regional and seasonal constraints. Here, we investigated mass production of recombinant miraculin in carrot (Daucus carota L.) callus cultures using an air-lift bioreactor. To increase miraculin expression, the oxidative stress-inducible SWPA2 promoter was used to drive the expression of miraculin gene under various stress treatments. An 8 h treatment of hydrogen peroxide (H2O2) and salt (NaCl) increased the expression of miraculin gene by fivefold compared with the untreated control. On the other hand, abscisic acid, salicylic acid, and methyl jasmonate treatments showed no significant impact on miraculin gene expression compared with the control. This shows that since H2O2 and NaCl treatments induce oxidative stress, they activate the SWPA2 promoter and consequently up-regulate miraculin gene expression. Thus, the results of this study provide a foundation for industrial-scale production of recombinant miraculin protein using transgenic carrot cells as a heterologous host.