DEFINING LPS INTERACTION WITH PROTEIN I

总的目标是测试假设的方法 一种特定的内毒素结合蛋白与内毒素的相互作用是 内毒素在β-螺旋内的插入 这种蛋白质的结构。作为迈向这一目标的第一步， 建议对尾穗上的氨基酸进行定义的研究 鼠伤寒沙门氏菌P22噬菌体结合蛋白(TSP) 分解细菌内毒素。这些研究可能会导致 细菌脂多糖结合蛋白诊断载体的研制 心理治疗。因为细菌对所有细菌的抗药性迅速上升 已知的抗生素，探索非抗生素变得至关重要- 尽快建立以诊断和治疗为基础的制度。 这些研究涉及噬菌体寄主范围突变体的选择和 含有P22尾钉基因的质粒DNA的诱变。 从含有这些突变尾钉基因的细胞中提取出来的 筛选具有内毒素相互作用代表性的突变体 包含由反应指示的天然结构的突变体 对天然三聚体TSP结构的单抗和通过 生产指示三聚体结构的十二烷基硫酸钠-PAGE图谱。 将测量与内毒素的物理结合和酶活性 具有有限活性或约束力的将暂时确定 作为内毒素识别突变体。 显示了P22和P22之间的结构相似之处 在34个TSP中，将继续努力净化 约34茶匙。TSP将在识别内毒素方面具有指导意义 结合部位。

The overall goal is to test the hypothesis that the method of interaction of a particular LPS-binding protein with LPS is the insertion of the LPS within the interior of the beta-helical structure of that protein. As a first step towards this goal, studies are proposed to define the amino acids on the tailspike protein (TSP) of Salmonella typhimurium phage P22 which bind and hydrolyze the bacterial LPS. These studies may lead to the development of LPS binding protein vehicles for diagnosis and therapy. Because of the rapid rise in bacteria resistance to all known antibiotics, it becomes critical to explore non-antibiotic- based diagnosis and treatment regimes as soon as possible. These studies involve the selection of phage host range mutants and the mutagenesis of a plasmid DNA containing the P22 tailspike gene. Extracts fromcells containing these mutant tailspike genes will be screened for mutants which are representative of LPS interaction mutants which contain a native structure as indicated by the reaction to monoclonal antibodies to the native trimeric TSP structure and by production of SDS-PAGE profile indicative of a trimeric structure. Physcial binding to LPS and enzymatic activity will be measured and those with limited activity or binding will be tentatively identified as LPS recognition mutants. Having shown the close structural similarity between the P22 and epsilon34 TSPs, efforts will be continued at the purification of the epsilon34 TSP. The TSPs will be instructive in identifying the LPS binding site.