一种基于酵母的磷酸二酯酶活性调节剂高通量筛选方法。

A yeast-based high-throughput screen for modulators of phosphodiesterase activity.

作者信息

de Medeiros Ana Santos, Hoffman Charles S

机构信息

Biology Department, Boston College, Higgins Hall 401b, 140 Commonwealth Avenue, Chestnut Hill, MA, 02467, USA.

出版信息

Methods Mol Biol. 2015;1294:181-90. doi: 10.1007/978-1-4939-2537-7_14.

DOI:10.1007/978-1-4939-2537-7_14

PMID:25783886

Abstract

Cell-based high-throughput screens (HTSs) targeting heterologously expressed proteins in yeast identify compounds that often display relevant biological activity when tested in cell culture. We developed a fission yeast-based HTS to detect small-molecule inhibitors of mammalian cyclic nucleotide phosphodiesterases (PDEs). These screens are carried out in Schizosaccharomyces pombe using a PKA-repressed fbp1-ura4 reporter whose expression due to low PKA activity prevents cells from growing in medium containing the pyrimidine analog 5-fluoro orotic acid (5FOA). We describe here the steps required to construct strains for screening and to optimize conditions for successful screens.

摘要

针对酵母中异源表达蛋白的基于细胞的高通量筛选（HTS）可鉴定出在细胞培养测试中通常显示出相关生物活性的化合物。我们开发了一种基于裂殖酵母的高通量筛选方法，用于检测哺乳动物环核苷酸磷酸二酯酶（PDE）的小分子抑制剂。这些筛选在粟酒裂殖酵母中进行，使用的是一种受PKA抑制的fbp1-ura4报告基因，由于PKA活性低导致其表达，从而阻止细胞在含有嘧啶类似物5-氟乳清酸（5FOA）的培养基中生长。我们在此描述构建用于筛选的菌株以及优化成功筛选条件所需的步骤。