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Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins

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BRÁZDA Pavel ŠEDO Ondrej KUBÍČEK Karel ŠTEFL Richard

Druh Článek v odborném periodiku
Časopis / Zdroj BioTechniques
Fakulta / Pracoviště MU

Středoevropský technologický institut

Citace
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Doi http://dx.doi.org/10.2144/btn-2019-0033
Klíčová slova C-terminal domain; co-expression; CTD; IDP; intrinsically disordered proteins; phosphorylation; purification; RNA polymerase II; TRANSCRIPTION TERMINATION; LARGEST SUBUNIT; CELL-CYCLE; KINASE; STRATEGIES; SEMISYNTHESIS
Popis 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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