CALMODULIN ACTIVATED ADENYLATE CYCLASE OF B. PERTUSSIS

我们的目标是研究波氏杆菌的腺苷环化酶。 百日咳，克隆其基因，并利用该基因生产更多的 可以从细菌中回收的蛋白质。我们发现这个 酶比之前认为的要复杂得多。我们的生化研究 允许我们开发亲和力程序，我们将使用它来净化 酵素。我们的初步实验也确定了 钙调蛋白是直接的，我们已经开发了筛选该基因的方法 在基于腺苷环化酶能力的重组DNA文库中 与钙调蛋白结合的多肽。 克隆的序列将通过测序来表征，然后进行操纵 为了几个目的，包括在大肠杆菌中生产酶， 特异波氏杆菌腺苷环化酶突变体的建立 该基因在真核细胞中的表达。 在物理表征之后解决了一些相互冲突的结果 在文献中，我们将研究腺苷环化案例对 哺乳动物的靶细胞。 最后，我们将利用基因的不同寻常的进化 尝试使用它长期诱导腺苷环化酶和cAMP。 哺乳动物细胞。

Our objective is to characterize the adenylate cyclase of Bordetella pertussis, to clone its gene, and to use the gene to produce more of the protein than can be recovered from the bacterium. We have found that this enzyme is more complex than previously thought. Our biochemical studies allowed us to develop affinity procedures which we will use to purify this enzyme. Our initial experiments have also determined that binding of calmodulin is direct and we have developed methods to screen for the gene in recombinant DNA libraries based on the ability of the adenylate cyclase peptide to bind calmodulin. Cloned sequences will be characterized by sequencing and then manipulated for several purposes which include the production of enzyme in E. coli, the creation of specific Bordetella adenylate cyclase mutants, and the expression of the gene in eucaryotic cells. After physical characterization to resolve some of the conflicting results in the literature, we will study action of the adenylate cyclcase on mammalian target cells. Finally we will take advantage of the unusual evolution of the gene and attempt to use it for long term induction of adenylate cyclase and cAMP in mammalian cells.