权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Multi-disciplinary Studies with Protein Engineering Approaches on Proteinaceous Proteinase Inhibitors of Microbial Origin

利用蛋白质工程方法对微生物来源的蛋白质蛋白酶抑制剂进行多学科研究

基本信息

批准号：
62300011
负责人：
HIROMI Keitaro
金额：
$ 6.27万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Co-operative Research (A)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1989
项目状态：
已结题

项目摘要

SSI denotes here Streptomyces subtilisin inhibitor and SMPI, Streptomyces metallo-proteinase inhibitor. SSI was found to inhibit also Streptomyces metallo-proteinases, SGMPA & SGMPB, and Ki was determined with a novel fluorogenic substrate; SSI gene was cloned, site-directed mutagenesis was applied on it, and characters of the mutant SSI's and of SSI-expression system were elucidated; Clones producing mondclonal antibodies, IgG1 & IgM, against SSI were established, and the epitopes were analyzed by NMR; X-ray diffraction data on the crystals of SSI (wild and mutant)-subtilisin BPN' complexes were obtained with syncrotron radiation, and structural refinement was made; Base sequence of the gene of a thermophilic proteinase, Aqualysine I, was determined, and the structure and function of the enzyme was studied; Optimal conditions were searched for extra-cellular production of SMPI by making use of a cloned gene; Methodology of Orotein NMR by using stable isotope labelling was established and the effects of site-specific amino acid replacement on the higher structure of SSI was analyzed; Fluctuation in SSI molecular structure was analyzed by deuterium NMR; A sensitive electro-chemical method for micro-analysis of SH/SS compounds was established; Interactions of SSI mutants-subtilisin BPN' and of SMPI-thermolysin were analyzed statistically and kinetically; The primary structure of Marinostatin D, a novel serine-proteinase inhibitor isolated from a marine micro-organism, was determined; The primary structure of Tyrostatin, a novel inhibitor of Pepstatin-insensitive carboxyl-proteinases, was determined; Unique properties of a novel, thermophilic carboxyl-proteinase and its strict substrate specificity were shown.

SSI 在此表示链霉菌枯草杆菌蛋白酶抑制剂，SMPI 表示链霉菌金属蛋白酶抑制剂。发现 SSI 还可以抑制链霉菌金属蛋白酶、SGMPA 和 SGMPB，并且用新型荧光底物测定 Ki；克隆了SSI基因，对其进行定点诱变，阐明了突变体SSI和SSI表达系统的特征；建立了产生抗 SSI 蒙克隆抗体 IgG1 和 IgM 的克隆，并通过 NMR 分析了表位；利用同步辐射获得了SSI（野生型和突变型）-枯草杆菌蛋白酶BPN'复合物晶体的X射线衍射数据，并进行了结构精修；确定了嗜热蛋白酶Aqualysine I基因的碱基序列，并研究了该酶的结构和功能；利用克隆基因寻找细胞外生产SMPI的最佳条件；建立了稳定同位素标记的Orotein NMR方法，分析了位点特异性氨基酸取代对SSI高级结构的影响；通过氘核磁共振分析SSI分子结构的波动；建立了SH/SS化合物微量分析的灵敏电化学方法；对SSI突变体-枯草杆菌蛋白酶BPN'和SMPI-嗜热菌蛋白酶的相互作用进行了统计和动力学分析；确定了从海洋微生物中分离出的新型丝氨酸蛋白酶抑制剂 Marinostatin D 的一级结构；确定了酪抑素（一种对胃酶抑素不敏感的羧基蛋白酶的新型抑制剂）的一级结构；显示了一种新型嗜热羧基蛋白酶的独特性质及其严格的底物特异性。