Miller Chad J, Turk Benjamin E
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, 208066, New Haven, CT, 06520, USA.
Methods Mol Biol. 2016;1360:203-16. doi: 10.1007/978-1-4939-3073-9_15.
Eukaryotic protein kinases phosphorylate substrates at serine, threonine, and tyrosine residues that fall within the context of short sequence motifs. Knowing the phosphorylation site motif for a protein kinase facilitates designing substrates for kinase assays and mapping phosphorylation sites in protein substrates. Here, we describe an arrayed peptide library protocol for rapidly determining kinase phosphorylation consensus sequences. This method uses a set of peptide mixtures in which each of the 20 amino acid residues is systematically substituted at nine positions surrounding a central site of phosphorylation. Peptide mixtures are arrayed in multiwell plates and analyzed by radiolabel assay with the kinase of interest. The preferred sequence is determined from the relative rate of phosphorylation of each peptide in the array. Consensus peptides based on these sequences typically serve as efficient and specific kinase substrates for high-throughput screening or incorporation into biosensors.
真核蛋白激酶在短序列基序背景下的丝氨酸、苏氨酸和酪氨酸残基上对底物进行磷酸化。了解蛋白激酶的磷酸化位点基序有助于设计用于激酶测定的底物，并绘制蛋白底物中的磷酸化位点。在此，我们描述了一种用于快速确定激酶磷酸化共有序列的阵列肽库方案。该方法使用一组肽混合物，其中20种氨基酸残基中的每一种都在围绕磷酸化中心位点的九个位置上进行系统取代。肽混合物被排列在多孔板中，并通过用感兴趣的激酶进行放射性标记测定来分析。从阵列中每个肽的相对磷酸化速率确定优选序列。基于这些序列的共有肽通常用作高通量筛选或整合到生物传感器中的高效且特异性的激酶底物。
