STUDY OF A RIBONUCLEASE AND ITS INHIBITOR FROM BACILLUS AMYLOLIQUEFACIENS

以淀粉解冻芽孢杆菌胞外核糖核酸酶barnase和胞内核糖核酸酶抑制物barstar两种蛋白质作为蛋白质折叠和蛋白质相互作用研究的模型体系。Barnase是存在于原核生物和真核生物中的一组同源核糖核酸酶之一。重组DNA技术的应用有三个主要目的：(1)促进野生型和突变型蛋白质的生产；(2)检查基因的结构和控制序列；(3)对序列进行特定的改变，以检验折叠理论和探索Barnase-barstar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中获得，其中barstar的表达抵消了barnase的表达的致死作用。这两种蛋白质及其复合体的结构都是已知的，Barnase的分辨率为1.5埃。通过体内选择技术获得的几个在界面上具有互补突变的barnase-barstar对的晶体结构已经被解决，这为决定键强度的机制提供了洞察。Barstar还抑制链霉菌菌株的一组核糖核酸酶。这些酶与Barnase有较远的亲缘关系，序列同源性仅为25%。在手头的四种这样的酶中，同一性从40%到70%不等。两个RNase Sa和St的结构是从非重组材料上得知的，第三个RNase SA2是从我们的重组材料中得知的。已建立了一种噬菌体展示系统，用于筛选与Barstar或其突变体紧密结合的Barstar的变种或同源物。还开发了一种全合成barstar基因的方法，选择的残基随机化，重复性(独立随机化序列的数量)约为10exp9。我们现在正在筛选具有随机疏水核心的合成Barstar文库。噬菌体展示也被应用于链霉菌Barstar同源物的克隆，其中两个是SA2，已经用传统方法克隆了。枯草杆菌YrdF基因在枯草芽孢杆菌168的全序列中被鉴定为与barstar相似的基因，经过几次密码子修改后，已在大肠杆菌中表达。虽然在枯草芽孢杆菌168中没有可识别的Barnase同源物，但yrdF在体外和体内都是一种有效的Barnase抑制剂。还开发了一种改进的Barstar体内选择和检测系统，使用双质粒系统。-核糖核酸酶、蛋白质折叠、Barnase、Barstar、芽孢杆菌、链霉菌、定向突变、噬菌体展示

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