ELUCIDATION OF ACTIVE CENTER AND STEREO CHEMICAL STRUCTURE OF SWEET AND SWEET-INDUCED PROTEINS BY GENE TECHNIQUE AND X'RAY ANALYSIS

基因技术和X射线分析阐明甜味和甜味诱导蛋白的活性中心和立体化学结构

基本信息

批准号：
08458174
负责人：
KURIHARA Yoshie
金额：
$ 3.97万
依托单位：
YOKOHAMA NATIONAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458174/
关键词：
miraculin cDNA expression in yeast curculin dimer expression in E.coli クルクリンcDNA マビンリンcDNA

项目摘要

Miraculin, which exists in the pulp of Richadella dulcifica (miracle fruit), had the unusual property of modifying a sour taste into a sweet taste. cDNA clone for miraculin was isolated and sequenced. The DNA encoding matured miraculin was cloned into the expression vector, pGEX-4T-1 and pET-32A,respectively. The presence of expressed miraculin in the supernatant of cell extract was confirmed by SDS-PAGE and immunoblotting using anti-miraculin serum. The expressed miraculin had no sweetness-inducing activity. The DNA encoding pre-miraculin (MIR) and matured miraculin (MIRPCR) were cloned into the expression vector, pYPR3831 and pYERNAP-Rh. The presence of miraculin in the yeast EH13-15/pYMI-1 extract was confirmed by immunoblotting using anti-miraculin serum. The yeast extracts which were purified by ammonium sulfate fractionation showed no sweetness-inducing activity. Curculin, which exists in the pulp of Curculigo latifolia, has a sweet taste and the ability to induce sweetness. Expression of curculin in E.coli and yeast was performed by the same procedure as that of miraculin Expressed protein showed no activity at present. cDNA for mabinlin, a heat-stable sweet protein, was cloned and the structure of its prepro-protein was determined. Mabinlin expressed in E.coli showed a sweet activity.

奇迹蛋白存在于Richadella Dulcifica（Miracle Fruit）的果肉中，具有将酸味变成甜味的异常特性。分离并测序奇迹蛋白的cDNA克隆。分别将编码成熟奇迹素的DNA克隆到表达载体PGEX-4T-1和PET-32a中。通过SDS-PAGE证实了细胞提取物上清液中表达的奇迹素的存在，并使用抗髓样血清进行免疫印迹。表达的奇迹蛋白没有诱导甜度的活性。将编码前弹药蛋白（miR）和成熟奇迹蛋白（miRPCR）的DNA克隆到表达载体PypR3831和Pyernap-RH中。酵母EH13-15/PYMI-1提取物中奇迹素的存在通过使用抗髓样血清进行免疫印迹证实。通过硫酸铵分馏纯化的酵母提取物没有诱导甜度的活性。姜黄素存在于姜黄的果肉中，具有甜味和诱导甜味的能力。姜黄素在大肠杆菌和酵母中的表达是通过与奇迹素表达的蛋白质相同的过程进行的，目前尚无活性。克隆了一种热稳定的甜蛋白Mabinlin的cDNA，并确定其前蛋白的结构。在大肠杆菌中表达的Mabinlin表现出甜蜜的活性。