Studies on molecular structure of soybean glycinin by protein engineering.

丰富大豆贮藏蛋白的功能特性和营养价值是食品工业的主要目标之一。大豆蛋白中蛋氨酸含量较高，是大豆蛋白的限制性氨基酸。因此，大豆球蛋白是针对大豆蛋白的营养和功能性质进行改良的。为了从理论上实现这一目标，需要阐明大豆球蛋白的分子结构。本研究的目的是用蛋白质工程的方法阐明大豆球蛋白的分子结构。作者尝试在微生物(大肠杆菌和酿酒酵母)中表达编码大豆球蛋白亚基的cDNA。大豆球蛋白基因在大肠杆菌中的表达需要删除与信号序列对应的核苷酸序列。在此基础上，通过控制表达载体的培养条件，建立了高效表达系统。在酿酒酵母中，表达的Iycinin亚基的信号序列在正确的加工位点被切割。将大豆球蛋白基因在大肠杆菌中表达的蛋白纯化为均一蛋白。纯化后的蛋白自组装，二级结构和基本性质与天然大豆球蛋白相似。这表明大豆球蛋白基因的原核表达系统可用于球蛋白分子结构的研究。构建了携带改变球蛋白分子结构的球蛋白cDNA的表达载体。目前，修饰后的蛋白质正在进行纯化。修饰后的蛋白质及其高级结构的自组装能力将被研究，以阐明球蛋白的分子结构。

One of the major objectives of the food industries is the enrichment of the functional properties and nutritional value of soybean storage proteins. Of the storage proteins, glycinin contains more methionines, which is limiting amino acid of soybean proteins. Therefore, glycinin is targeted to improve the nutritional and functional properties of soybean proteins. To attain this goal theoretically, molecular structure of glycinin should be elucidated. In this research, the object is the elucidation of glycinin molecular structure by means of protein engineering.1. The author attempted to express cDNAs encoding glycinin subunits in microorganisms (Escherichia coli and Saccharomyces cerevisiae). It was necessary for the expression of glycinin cDNA in E. coil to delete nucleotide sequences corresponding to the signal sequence. Then, a high-level expression system was established by controlling the cultivation conditions of E. coli cells harboring the expression plasmids. In Saccharomyces cerevisiae, the signal sequence of the expressed Iycinin subunit was el cleaved at the right processing site.2. The expressed protein from glycinin cDNA in E. coil was purified to homogeneity. The purified protein self-assembled and its secondary structure and fundamental properties were similar to those of the native glycinin. This indicated that the E. coli expression system of glycinin cDNA may be employed for the studies of glycinin molecular structure.3. Expression plasmids carrying the glycinin cDNAs modified to change the molecular structure of glycinin were constructed. At present, the modified proteins are being purified. The ability of the self-assembly of the modified proteins and their higher structures will be investigated to elucidate the molecular structure of glycinin.