Xu Jie, Sun Luo, Ghosh Inca, Xu Ming-Qun
New England Biolabs, Beverly, MA, USA.
Biotechniques. 2004 Jun;36(6):976-8, 980-1. doi: 10.2144/04366ST02.
We have applied intein-mediated peptide ligation (IPL) to the use of peptide substrates for kinase assays and subsequent Western blot analysis. IPL allows for the efficient ligation of a synthetic peptide with an N-terminal cysteine residue to an intein-generated carrier protein containing a cysteine reactive C-terminal thioester through a native peptide bond. A distinct advantage of this procedure is that each carrier protein molecule ligates only one peptide, ensuring that the ligation product forms a sharp band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrate the effectiveness of this approach by mutational analysis of peptide substrates derived from human cyclin-dependent kinase, Cdc2, which contains a phosphorylation site of human c-Src protein tyrosine kinase.
我们已将内含肽介导的肽连接（IPL）应用于激酶分析及后续蛋白质免疫印迹分析的肽底物使用中。IPL能够通过天然肽键将具有N端半胱氨酸残基的合成肽与含有半胱氨酸反应性C端硫酯的内含肽生成的载体蛋白进行高效连接。该方法的一个显著优点是每个载体蛋白分子仅连接一个肽，确保连接产物在十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）上形成一条清晰的条带。我们通过对源自人类细胞周期蛋白依赖性激酶Cdc2的肽底物进行突变分析，证明了这种方法的有效性，该底物包含人类c-Src蛋白酪氨酸激酶的一个磷酸化位点。
