STUDY OF A RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

两种蛋白质，芽孢杆菌RNA酶，芽孢杆菌的胞外核糖核酸酶 解淀粉菌及其细胞内抑制剂芽孢杆菌RNA酶抑制剂 作为研究蛋白质折叠和蛋白质-蛋白质的模型系统 交互. 芽孢杆菌RNA酶是核糖核酸酶的同源群之一 在原核生物和真核生物中都存在。 重组dna 技术的应用有三个主要目标：（1）促进 野生型和突变蛋白的生产;（2）检查 基因的结构和控制序列;以及（3）使 序列中的特定变化来测试折叠理论， 探索巴恩斯和巴斯塔之间的互动 这两种蛋白质现在可以 从E.在大肠杆菌中表达芽孢杆菌RNA酶抑制剂 对抗芽孢杆菌RNA酶表达的致死作用。 的结构 这两种蛋白质及其复合物都是已知的。 一个快速和相对 精确的分析使得研究 络合物形成的动力学和稳定性。 巴纳斯-巴斯塔 通过体内技术正在探索界面， 抑制突变，将系统从致命突变中拯救出来 干扰芽孢杆菌RNA酶抑制作用。 例如，芽孢杆菌RNA酶（H102K） 与野生型芽孢杆菌RNA酶抑制剂在适当的条件下是条件致死的， vector. 芽孢杆菌RNA酶抑制剂Tyr29和Tyr30处的几个突变允许更大的 产生突变体芽孢杆菌RNA酶，并与突变体更紧密地结合 芽孢杆菌RNA酶体外研究 几种突变体的结构研究 组合正在进行中。 Barstar还抑制一组RNA酶 从链霉菌菌株中分离。这些酶与 芽孢杆菌RNA酶，序列同一性仅为25%。 在他们中间， 同一性范围为40%至70%。 其中两种酶的结构 已经从对非重组材料的研究中得知。 一 目前正在开展协作努力，沿着 Barnase及其近亲，结构和折叠研究， 单独和与Barstar组合。 我们有四种基因 这些酶在E.大肠杆菌中表达。 对于其中三种，产率已经在50 - 100 mg/ml范围内。 为 RNase St，需要对酶合成进行更严格的控制， 可以低水平产生酶。 的分离与克隆 芽孢杆菌RNA酶抑制剂的同源物及其体外进化 基因正在被追踪。 在其他地方工作，其中芽孢杆菌RNA酶基因 当附着在真核生物上时， 发起人，已经引起了相当大的兴趣，在其使用中， 发展研究和作为关键的各种抗病毒 战略布局

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. A fast and relatively precise assay has allowed the development of techniques for studying the kinetics and stability of complex formation. The barnase-barstar interface is being explored by an in vivo technique which selects suppressor mutations which rescue the system from lethal mutations that interfere with barnase inhibition. For example, barnase (H102K) with wild-type barstar is conditionally lethal in the appropriate vector. Several mutations at Tyr29 and Tyr30 of barstar allow greater production of the mutant barnase and bind more tightly to the mutant barnase in vitro. Structural studies of several such mutant combinations are under way. 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 themselves, identity ranges from 40% to 70%. The structures of two of the enzymes are already known from work on nonrecombinant material. A collaborative effort is under way to apply this group, along with barnase and its closer relatives, to structural and folding studies, alone and in combination with barstar. We have the genes for four such enzymes expressed in E. coli with the aid of the barstar gene. For three of these, yields are already in the 50-100 mg/ml range. For RNase St, tighter control of enzyme synthesis is required but the enzyme can be produced at a low level. Isolation and cloning of putative homologs of barstar and in vitro evolution of the barstar gene are being pursued. Work elsewhere, in which the barnase gene becomes a tissue-specific killer when attached to eukaryotic promoters, has aroused considerable interest in its use in developmental studies and as the key to a variety of anti-viral strategies.