STUDY OF A RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

芽孢杆菌的胞外核糖核酸酶Barnase 淀粉分解酶和其胞内抑制物Barstar被使用 作为蛋白质折叠和蛋白质-蛋白质研究的模型系统 互动。Barnase是核糖核酸酶的同源基团之一 存在于原核生物和真核生物中。重组DNA 应用这些技术有三个主要目的：(1)促进 野生型和突变型蛋白的生产；(2)检测 基因的结构和控制序列；以及(3)制作 序列中的具体变化以检验折叠理论和 探索藤壶与杆星的相互作用。这两种蛋白质现在都可以 从表达barstar的大肠杆菌中获得重组基因 对抗藤壶酶表达的致死作用。的结构 这两种蛋白质及其复合体都是已知的。一个快速而相对的 精确的分析使研究技术的发展成为可能。 络合物形成的动力学和稳定性。Barnase-Barstar 一种体内技术正在探索界面，这种技术选择了 将系统从致命突变中拯救出来的抑制子突变 它们干扰了藤壶酶的抑制。例如，Barnase(H102K) 具有野生型的巴斯塔尔在适当的条件下是致命的 向量。Barstar的Tyr29和Tyr30上的几个突变允许更大的 突变的Barnase的产生并与突变的更紧密地结合 体外培养的藤本酶。几个这样的突变体的结构研究 合并正在进行中。Barstar还抑制了一组核糖核酸酶 来自链霉菌菌株。这些酶与它们有远亲关系 Barnase的序列同源性仅为25%。在他们中间， 认同度从40%到70%不等。其中两种酶的结构 已经从非重组材料的研究中获知。一个 合作努力正在进行中，以应用该小组，以及 藤壶及其近亲，对结构和折叠的研究， 单独和巴斯塔尔联合使用。我们有四个人的基因 这种酶在巴斯塔尔基因的帮助下在大肠杆菌中表达。 其中三种的产量已经在50-100毫克/毫升范围内。为 RNase ST，需要更严格地控制酶的合成，但 酶可以在低水平下产生。新城疫病毒的分离与克隆 Barstar的推测同系物及Barstar的体外进化 吉恩正在被追捕。在其他地方工作，在那里，barnase基因 当与真核生物结合时成为组织特异性杀手 发起人，已经引起了人们对其在 作为发育研究的关键和多种抗病毒药物 战略。

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.