Kochinyan Samvel, Sun Luo, Ghosh Inca, Barshevsky Tanya, Xu Jie, Xu Ming-Qun
New England Biolabs, Inc., Ipswich, MA 01938-2723, USA.
Biotechniques. 2007 Jan;42(1):63-9. doi: 10.2144/000112311.
Synthetic peptides incorporating various chemical moieties, for example, phosphate groups, are convenient tools for investigating protein modification enzymes, such as protein phosphatases (PPs). However, short peptides are sometimes poor substrates, and their binding to commonly used matrices is unpredictable and variable. In general, protein substrates for PPs are superior for enzymatic assays, binding to various matrices, and Western blot analysis. The preparation and characterization of phosphoproteins, however can be difficult and technically demanding. In this study, the intein-mediated protein ligation (IPL) technique was used to readily generate phosphorylated protein substrates by ligating a synthetic phosphopeptide to an intein-generated carrier protein (CP) possessing a carboxyl-terminal thioester with a one-to-one stoichiometry. The ligated phosphoprotein (LPP) substrate was treated with a PP and subsequently subjected to array or Western blot analysis with a phospho-specific antibody. This approach is highly effective in producing arrays of protein substrates containing phosphorylated amino acid residues and has been applied for screening of PPs with specificity toward phosphorylated tyrosine, serine, or threonine residues, resulting in an approximately 240-fold increase in sensitivity in dot blot analysis compared with the use of synthetic peptides. The IPL technique overcomes the disadvantages of current methods and is a versatile system for the facile production of protein substrates containing well-defined structural motifs for the study of protein modification enzymes.
包含各种化学基团（例如磷酸基团）的合成肽是研究蛋白质修饰酶（如蛋白磷酸酶，PPs）的便捷工具。然而，短肽有时是较差的底物，并且它们与常用基质的结合是不可预测且可变的。一般而言，PPs的蛋白质底物在酶促测定、与各种基质的结合以及蛋白质印迹分析方面表现更优。然而，磷蛋白的制备和表征可能具有难度且对技术要求较高。在本研究中，内含肽介导的蛋白质连接（IPL）技术被用于通过将合成的磷酸肽与内含肽产生的具有羧基末端硫酯的载体蛋白（CP）以一对一的化学计量比连接，从而轻松生成磷酸化的蛋白质底物。将连接后的磷蛋白（LPP）底物用PP处理，随后用磷酸特异性抗体进行阵列分析或蛋白质印迹分析。这种方法在产生含有磷酸化氨基酸残基的蛋白质底物阵列方面非常有效，并且已被用于筛选对磷酸化酪氨酸、丝氨酸或苏氨酸残基具有特异性的PPs，与使用合成肽相比，在斑点印迹分析中灵敏度提高了约240倍。IPL技术克服了现有方法的缺点，是一种通用系统，可轻松生产含有明确结构基序的蛋白质底物，用于研究蛋白质修饰酶。
