ISOLATION AND IDENTIFICATION OF THE LPS BINDING PROTEINS ON HUMAN MONOCYTES

人单核细胞LPS结合蛋白的分离和鉴定

• 批准号: 3792488
• 负责人: D S WEBB
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3792488
• 关键词: affinity chromatography; binding proteins; human tissue; immunoprecipitation; interferons; leukocyte activation /transformation; lipopolysaccharides; monocyte; protein structure function; western blottings

By utilizing two different approaches, we have isolated several proteins which may serve as LPS binding proteins on human monocytes. One method used was to add LPS to 35-S-methionine labeled monocytes, lyse the cells and then, using an anti-LPS antibody, immunoprecipitate the LPS along with any bound monocyte proteins. We have found two proteins, one greater than 200 kd and one approximately a 100 kd in site. The greater than 200 kd protein has been identified by Western blot analysis as CD45. The 110 kd protein has not yet been identified, but we have found that its expression is enhanced by pretreatment with IFN-gamma. The second approach used involved covalently linking LPS to CNBr-activated sepharose beads. The beads are then added to a lysate of 35-S-methionine labeled monocytes and washed several times to remove proteins which do not bind the LPS. Using this technique, we have found two additional proteins which are approximately 70 and 90 kd. Work is currently in progress to identify all these proteins and to verify their role in the LPS induced activation of human monocytes.

通过利用两种不同的方法，我们已经分离出几种蛋白质 其可以作为人单核细胞上的LPS结合蛋白。 一种方法 将LPS加入到35-S-蛋氨酸标记的单核细胞中，裂解细胞， 然后，使用抗LPS抗体，使LPS沿着 与任何结合的单核细胞蛋白质结合。 我们发现了两种蛋白质， 大于200 kd，一个在位点中约为100 kd。 越大 通过Western印迹分析，已将大于200 kd的蛋白鉴定为CD 45。 110 kd的蛋白质尚未被鉴定，但我们发现， 其表达通过用IFN-γ预处理而增强。 第二 所使用的方法涉及将LPS共价连接到CNBr活化的琼脂糖凝胶 珠 然后将珠子加入到35-S-蛋氨酸标记的裂解物中， 单核细胞并洗涤几次以除去不结合的蛋白质 LPS。 利用这项技术，我们发现了另外两种蛋白质 其约为70和90 kD。 目前正在开展工作， 鉴定所有这些蛋白质，并验证它们在LPS诱导的细胞凋亡中的作用。 激活人单核细胞。