Publication details

Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins


BRÁZDA Pavel ŠEDO Ondrej KUBÍČEK Karel ŠTEFL Richard

Year of publication 2019
Type Article in Periodical
Magazine / Source BioTechniques
MU Faculty or unit

Central European Institute of Technology

Keywords C-terminal domain; co-expression; CTD; IDP; intrinsically disordered proteins; phosphorylation; purification; RNA polymerase II; TRANSCRIPTION TERMINATION; LARGEST SUBUNIT; CELL-CYCLE; KINASE; STRATEGIES; SEMISYNTHESIS
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Description 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.
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