de Medeiros Ana Santos, Kwak Grace, Vanderhooft Jordan, Rivera Sam, Gottlieb Rachel, Hoffman Charles S
Biology Department, Boston College, 140 Commonwealth Avenue, Higgins Hall 401b, Chestnut Hill, MA, 02467, USA.
Methods Mol Biol. 2015;1263:77-91. doi: 10.1007/978-1-4939-2269-7_6.
Features of the fission yeast Schizosaccharomyces pombe cAMP/PKA pathway make S. pombe particularly amenable for heterologous expression of cAMP pathway proteins such as GαS subunits and their cognate adenylyl cyclases, PKA catalytic and regulatory subunits, and cyclic nucleotide phosphodiesterases. We have constructed two PKA-repressed reporters for use in high-throughput screens to detect compounds that elevate or reduce PKA activity, thus facilitating the discovery of both inhibitors and activators of these target proteins. Here, we describe steps to construct screening strains and to optimize and conduct these screens.
粟酒裂殖酵母cAMP/PKA信号通路的特性使得粟酒裂殖酵母特别适合用于cAMP信号通路蛋白的异源表达，如GαS亚基及其同源腺苷酸环化酶、PKA催化亚基和调节亚基以及环核苷酸磷酸二酯酶。我们构建了两种用于高通量筛选的PKA抑制型报告基因，以检测能提高或降低PKA活性的化合物，从而有助于发现这些靶蛋白的抑制剂和激活剂。在此，我们描述构建筛选菌株以及优化和进行这些筛选的步骤。
