Szewczuk Lawrence M, Tarrant Mary Katherine, Cole Philip A
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Methods Enzymol. 2009;462:1-24. doi: 10.1016/S0076-6879(09)62001-2.
Deconvolution of specific phosphorylation events can be complicated by the reversibility of modification. Protein semisynthesis with phosphonate analogues offers an attractive approach to functional analysis of signaling pathways. In this technique, N- and C-terminal synthetic peptides containing nonhydrolyzable phosphonates at target residues can be ligated to recombinant proteins of interest. The resultant semisynthetic proteins contain site specific, stoichiometric phosphonate modifications and are completely resistant to phosphatases. Control of stoichiometry, specificity, and reversibility allows for complex signaling systems to be broken down into individual events and discretely examined. This chapter outlines the general methods and considerations for designing and carrying out phosphoprotein semisynthetic projects.
特定磷酸化事件的反卷积可能会因修饰的可逆性而变得复杂。使用膦酸酯类似物进行蛋白质半合成提供了一种用于信号通路功能分析的有吸引力的方法。在该技术中，在目标残基处含有不可水解膦酸酯的N端和C端合成肽可以与感兴趣的重组蛋白连接。所得的半合成蛋白含有位点特异性、化学计量的膦酸酯修饰，并且对磷酸酶完全抗性。对化学计量、特异性和可逆性的控制使得复杂的信号系统能够分解为单个事件并进行离散研究。本章概述了设计和实施磷蛋白半合成项目的一般方法和注意事项。
