Efficient and Robust Preparation of Tyrosine Phosphorylated Intrinsically Disordered Proteins

Abstract

Intrinsically disordered proteins (IDPs) are subject to post-translational modifications. This allows the same polypeptide to be involved in different interaction networks with different consequences, ranging from regulatory signalling networks to the formation of membrane-less organelles. We report a robust method for co-expression of modification enzyme and SUMO-tagged IDPs with a subsequent purification procedure that allows for the production of modified IDP. The robustness of our protocol is demonstrated using a challenging system: RNA polymerase II C-terminal domain (CTD); that is, a low-complexity repetitive region with multiple phosphorylation sites. In vitro phosphorylation approaches fail to yield multiple-site phosphorylated CTD, whereas our in vivo protocol allows the rapid production of near homogeneous phosphorylated CTD at a low cost. These samples can be used in functional and structural studies.