Gregan Juraj, Zhang Chao, Rumpf Cornelia, Cipak Lubos, Li Zhang, Uluocak Pelin, Nasmyth Kim, Shokat Kevan M
Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 1, 1030 Vienna, Austria.
Nat Protoc. 2007;2(11):2996-3000. doi: 10.1038/nprot.2007.447.
Reversible protein phosphorylation is a major regulatory mechanism in a cell. A chemical-genetic strategy to conditionally inactivate protein kinases has been developed recently. Mutating a single residue in the ATP-binding pocket confers sensitivity to small-molecule inhibitors. The inhibitor can only bind to the mutant kinase and not to any other wild-type kinase, allowing specific inactivation of the modified kinase. Here, we describe a protocol to construct conditional analog-sensitive kinase alleles in the fission yeast Schizosaccharomyces pombe. This protocol can be completed in about 3 weeks and should be applicable to other organisms as well.
可逆性蛋白质磷酸化是细胞中的一种主要调节机制。最近开发了一种用于有条件地使蛋白激酶失活的化学遗传学策略。在ATP结合口袋中突变单个残基可赋予对小分子抑制剂的敏感性。该抑制剂只能与突变型激酶结合，而不能与任何其他野生型激酶结合，从而实现对修饰激酶的特异性失活。在此，我们描述了一种在裂殖酵母粟酒裂殖酵母中构建条件性类似物敏感激酶等位基因的方案。该方案大约可在3周内完成，并且应该也适用于其他生物体。
