STUDY OF A RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

两种蛋白质，芽孢杆菌RNA酶，细胞外核糖核酸酶 解淀粉芽孢杆菌，和芽孢杆菌毒素，它的细胞内抑制剂， 被用作研究蛋白质折叠的模型系统， 蛋白质相互作用芽孢杆菌RNA酶是一种 核糖核酸酶：一组核糖核酸酶，存在于原核生物和 真核生物重组DNA技术正在应用于 三个主要目标：（1）促进野生型的生产， 突变蛋白;（2）检查结构和控制 基因序列;和（3）使特定的变化， 序列来测试折叠理论，并探索 barnase-barstar相互作用这两种蛋白质现在都可以获得 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，芽孢杆菌RNA酶在1.5 埃分辨率。几种barnase-barstar的晶体结构 在界面中具有互补突变的对，通过以下获得： 一种体内选择性技术，已经得到解决， 决定结合强度的机制。 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酶有远亲关系， 只有25%的认同。在这四种酶中， 同一性在40%到70%之间。核糖核酸酶的结构 Sa和St从非重组材料的工作中已知， 第三，RNase Sa 2，来自我们的重组材料。的结构 与芽孢杆菌RNA酶抑制剂复合的重组RNA酶Sa也已被用于 解决了噬菌体展示系统已被开发用于选择 与芽孢杆菌RNA酶或其 变种人它现在被用来筛选合成的barstar文库 带有随机的疏水核。它也可能有助于 从链霉菌中克隆芽孢杆菌RNA酶抑制剂同系物，其中之一， Sa 2已通过常规方法克隆。一种改进 用于体内选择和测试杆状病毒的系统也已经 使用双质粒系统开发。

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. It is now being used to screen a synthetic barstar library with randomized hydrophobic cores. It might also be useful for cloning barstar homologs from Streptomyces, one of which, for Sa2, has been cloned by conventional methods. An improved system for in vivo selection and testing of barstars has also been developed, using a two plasmid system.