STUDY OF A RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

解淀粉芽孢杆菌核糖核酸酶及其抑制剂的研究

• 批准号: 6289714
• 负责人: ROBERT W HARTLEY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289714
• 关键词: Bacillus; bacterial proteins; chemical stability; enzyme activity; enzyme biosynthesis; enzyme inhibitors; enzyme mechanism; enzyme structure; esterase inhibitor; exoribonucleases; gene expression; gene mutation; genetic regulatory element; intermolecular interaction; microorganism metabolism; molecular cloning; nucleic acid sequence; protein engineering; protein folding; protein structure function; recombinant DNA

Two proteins, barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens, and barstar, its intracellular inhibitor, are used as a model system for the study of protein folding and protein-protein interactions. Barnase is one of an homologous group of ribonucleases occurring in both prokaryotes and eukaryotes. Recombinant DNA techniques are being applied with three major aims: (1) to facilitate production of wild type and mutant proteins; (2) to examine the structural and control sequences of the genes; and (3) to make specific changes in the sequences to test theories of folding and to probe the barnase-barstar interaction. Both proteins can now be obtained from recombinant genes in E. coli where expression of barstar counters the lethal effect of barnase expression. The structures of both proteins and their complex are known, barnase at 1.5 angstrom resolution. Crystal structures of several barnase-barstar pairs having complementary mutations in the interface, obtained by an in vivo selective technique, have been solved, providing insight into the mechanisms that determine the strength of the bond. Barstar also inhibits a group of RNases from Streptomyces strains. These enzymes are distantly related to barnase with a sequence identity of only 25%. Among the four such enzymes in hand, identities ranges from 40% to 70%. The structures of two, RNases Sa and St, are known from work on nonrecombinant material and a third, RNase Sa2, from our recombinant material. The structure of recombinant RNase Sa in complex with barstar has also been solved.A phage display system has been developed for selection of varieties or homologs of barstar that bind tightly to barnase or its mutants. A procedure has also been developed for total synthesis of the barstar gene with randomization of selected residues and a multiplicity (the number of independently randomized sequences)on the order of 10exp9. We are now screening a synthetic barstar library with randomized hydrophobic cores. The phage display is also being applied to the cloning of barstar homologs from Streptomyces, two of which, for Sa2, have been cloned by conventional methods. The gene yrdF, identified in the the complete sequence of B. subtilis 168 as similar to that of barstar, has been cloned and, after several codon modifications, expressed in E. coli. Although there is no recognizable homolog of barnase in B. subtilis 168, yrdF is an effective inhibitor, in vitro and in vivo, of barnase. An improved system for in vivo selection and testing of barstars has also been developed, using a two plasmid system. - ribonuclease, protein folding, barnase, barstar, Bacillus, Streptomyces, directed mutagenesis, phage display

两种蛋白质，barnase，解淀粉芽孢杆菌的胞外核糖核酸酶，和barstar，其胞内抑制剂，被用作研究蛋白质折叠和蛋白质-蛋白质相互作用的模型系统。芽孢杆菌RNA酶是原核生物和真核生物中都存在的一类同源核糖核酸酶。重组DNA技术的应用有三个主要目的：（1）促进野生型和突变蛋白的生产;（2）检测基因的结构和控制序列;（3）对序列进行特异性改变，以检验折叠理论并探测芽孢杆菌RNA酶-芽孢杆菌RNA酶的相互作用。这两种蛋白质现在都可以从大肠杆菌中的重组基因获得。其中芽孢杆菌RNA酶抑制剂的表达抵消芽孢杆菌RNA酶表达的致死效应。这两种蛋白质及其复合物的结构是已知的，芽孢杆菌RNA酶的分辨率为1.5埃。通过体内选择性技术获得的在界面中具有互补突变的几个barnase-barstar对的晶体结构已经得到解决，提供了对确定键强度的机制的深入了解。Barstar还抑制来自链霉菌菌株的一组RNA酶。这些酶与芽孢杆菌RNA酶的亲缘关系较远，序列同一性仅为25%。在手头的四种这样的酶中，同一性范围从40%到70%。两种RNA酶Sa和St的结构是从非重组材料的工作中已知的，第三种RNA酶Sa 2来自我们的重组材料。重组RNA酶Sa与芽孢杆菌RNA酶的复合物的结构也已得到解决，并建立了噬菌体展示系统，用于筛选与芽孢杆菌RNA酶紧密结合的芽孢杆菌RNA酶Sa及其突变体。还开发了一种用于全合成芽孢杆菌RNA酶抑制剂基因的方法，其具有所选残基的随机化和10 exp 9数量级的多重性（独立随机化序列的数量）。我们现在正在筛选具有随机疏水核的合成barstar文库。噬菌体展示也被应用于从链霉菌中克隆芽孢杆菌RNA酶抑制剂同源物，其中两个，对于Sa 2，已经通过常规方法克隆。在B的全序列中鉴定了yrdF基因。subtilis 168克隆了一个与芽孢杆菌属芽孢杆菌相似的基因，并在经过几个密码子修饰后，在E.杆菌虽然在B中没有可识别的芽孢杆菌RNA酶同系物。在枯草芽孢杆菌168中，yrdF是芽孢杆菌RNA酶的有效抑制剂，在体外和体内。使用双质粒系统，还开发了用于芽孢杆菌RNA酶抑制剂的体内选择和测试的改进系统。- 核糖核酸酶，蛋白质折叠，芽孢杆菌RNA酶，芽孢杆菌，链霉菌，定向诱变，噬菌体展示