Study Of A Ribonuclease And Its Inhibitor From Bacillus

解淀粉芽孢杆菌的胞外核糖核酸酶barnase和胞内抑制剂barstar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。巴纳酶是存在于原核生物和真核生物中的同源核糖核酸酶群之一。重组DNA技术的应用主要有三个目的：(1)促进野生型和突变型蛋白质的生产；（二）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的表达可以对抗barnase表达的致命作用。这两种蛋白质及其复合物的结构都是已知的，在1.5埃的分辨率下。通过体内选择技术获得的几种在界面上具有互补突变的barnase-barstar对的晶体结构已经得到解决，为确定键强度的机制提供了见解。Barstar还抑制链霉菌菌株的一组rna酶。这些酶与藤壶酶有较远的亲缘关系，序列同源性只有25%。在这四种酶中，识别度从40%到70%不等。利用barstar在大肠杆菌中克隆和表达，其中一些已被很好地表征，以及三个barstar同源物，它们都能抑制藤壶酶。近年来，我们利用噬菌体展示技术，从S. erythus中发现并克隆了Sti基因，Sti是一种天然的RNase St抑制剂。该基因的有效表达需要通过沉默突变对其序列进行相当大的操作。它可以增加大肠杆菌中RNase St的产量，但仍不足以发挥作用。一种噬菌体展示系统已经被开发出来，用于选择与藤壶酶或其突变体紧密结合的barstar的品种或同源物。已经开发了barstar基因的全合成程序，选择残基随机化，多重性（独立随机序列的数量）为10exp9。我们筛选了3个合成barstar文库，其中疏水核心的8、11和11个残基部分空间紧凑，随机化，另一个文库随机化了核心的所有22个残基。每个取代位上可能的残基分别是Leu、Ile、Val、Met或Phe。未选择的基因转移到质粒barstar载体上，没有显示出功能barstar的迹象，而所有选择的基因在体内似乎都具有功能，允许它们的宿主被携带barnase的相容质粒转化。后者在体外也能产生可测量的barstar活性，但大多数水平很低。每一个部分随机的集合中都有一些产生了足够的物理研究数据。一个更大的完全随机的核心酒吧明星基因库正在准备中。所有22个核心残基的功能barstars随机序列表明，没有特定的氨基酸是不可缺少的，尽管在某些位置，野生型残基显然是首选。核心侧链占据的体积，由非氢原子的数量表示，比预期的变化要大得多，特别是在低的一边，这表明二级结构框架可以在不破坏barasse结合位点的情况下大幅崩溃。连续流电穿孔技术的发展使我们能够直接筛选相当大的大肠杆菌文库，以检测对藤蔓酶表达的抗性，从而使我们能够估计具有完全随机核心的活性barstars的产量约为1 / 10exp4或5。

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 a 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 range from 40% to 70%. Cloned and expressed in E.coli with the aid of barstar, several of these have been well characterized, along with three barstar homologs, all of which inhibit barnase. Recently, we have used phage display to find and clone the gene for Sti, a barstar homolog and natural inhibitor of RNase St from S. erythreus. Useful expression of this gene required considerable manipulation of its sequence with silent mutations. It allowed some increase in production of RNase St in E. coli but still not enough to be useful. A phage display system has been developed for selection of varieties or homologs of barstar that bind tightly to barnase or its mutants. Procedures have 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 have screened 3 synthetic barstar libraries with spatially compact 8, 11 and 11 residue portions of the hydrophobic core randomized and another with randomization of all 22 residues of the core. The possible residues in each substituted position were Leu, Ile, Val, Met or Phe. Unselected genes transferred to a plasmid barstar vector showed no indication of functional barstar, while all selected genes appear to be functional in vivo, allowing transformation of their host by a compatible plasmid carrying barnase. All of the latter also produce measurable barstar activity in vitro, most at a very low level. Some of each of the partially randomized sets produce enough for physical studies. A much larger library of genes for completely randomized core barstars is being prepared. Sequences of functional barstars with all 22 core residues randomized indicate that no particular amino acid is indispensable, although in some positions the wild type residue is clearly preferred. The volume occupied by the core sidechains, as represented by the number of non-hydrogen atoms, is much more variable than expected, especially on the low side, suggesting that the secondary structure framework can collapse considerably without destroying the barnase-binding site. Development of a continuous flow electroporation technique has allowed direct screening of fairly large E. coli libraries for resistance to barnase expression, allowing us to estimate the yield of active barstars with fully randomized cores at about one in 10exp4 or 5.