Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins
|Year of publication
|Article in Periodical
|Magazine / Source
|MU Faculty or unit
|C-terminal domain; co-expression; CTD; IDP; intrinsically disordered proteins; phosphorylation; purification; RNA polymerase II; TRANSCRIPTION TERMINATION; LARGEST SUBUNIT; CELL-CYCLE; KINASE; STRATEGIES; SEMISYNTHESIS
|Intrinsically disordered proteins (IDPs) are subject to post-translational modifications. This allows the same polypeptide to undertake different interaction networks with different consequences, ranging from regulatory signalling networks to formation of membraneless organelles. We report a robust method for co-expression of modification enzyme and SUMO-tagged IDP with subsequent purification procedure allowing production of modified IDP. The robustness of our protocol is demonstrated on a challenging system, RNA polymerase II C-terminal domain (CM), 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 to rapidly produce near homogeneous phosphorylated CTD at a low cost. These samples can be used in functional and structural studies.