DEFINING LPS INTERACTION WITH PROTEIN I

总的目标是检验假设， 特定的LPS结合蛋白与LPS的相互作用是 LPS插入β-螺旋的内部 蛋白质结构。 作为实现这一目标的第一步， 对尾穗上的氨基酸进行了研究 鼠伤寒沙门氏菌噬菌体P22的蛋白质（TSP），其结合并 水解细菌LPS。 这些研究可能会导致 开发用于诊断的LPS结合蛋白载体， 疗法 由于细菌对所有细菌的耐药性迅速上升， 已知的抗生素，探索非抗生素变得至关重要， 尽快制定诊断和治疗方案。 这些研究涉及噬菌体宿主范围突变体的选择， 诱变含有P22尾穗基因的质粒DNA。 含有这些突变尾穗基因的细胞提取物将被 筛选代表LPS相互作用的突变体 含有如反应所示的天然结构的突变体 针对天然三聚体TSP结构的单克隆抗体， 产生指示三聚体结构的SDS-PAGE图谱。 将测量与LPS的物理结合和酶活性， 将暂时确定活性或结合力有限的药物 作为LPS识别突变体。 在显示了P22和P23之间的密切结构相似性之后， epsilon 34 TSP，将继续努力在纯化的 ε 34 TSP。 TSP对识别LPS具有指导意义 结合位点

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.