Preparation of Phosphorylated Proteins for NMR Spectroscopy

Phosphorylation is a ubiquitous posttranslational modification that is essential for the regulation of many cellular processes. The human genome consists of more than 200,000 phosphorylation sites, whose phosphorylation is tightly controlled by ≥500 kinases and ~200 phosphatases. Given the large number of phosphorylation sites and the key role phosphorylation plays in regulating cellular processes, it is essential to characterize the impact of phosphorylation on substrate structure, dynamics, and function. However, a major challenge is the large-scale production of phosphorylated proteins in vitro for these structural, functional, and dynamic studies. Here, we describe an efficient protocol used routinely in our laboratory for the production of phosphorylated proteins. We also describe the methods used for identifying, characterizing, and separating the resulting phosphorylated proteins for subsequent studies.